Pharmaceutical Co-crystals: A New Paradigm of Crystal ...

Pharmaceutical Co-crystals: A New Paradigm of

Crystal Engineering to modify Physicochemical and

Pharmaceutical Properties of Paracetamol and

Naproxen

A THESIS SUBMITTED TO UNIVERSITY OF THE PUNJAB IN

PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE

DEGREE OF

Doctor of Philosophy in Pharmacy

(Pharmaceutics)

By

Sumera Latif (M. Phil.)

(December 2018)

Punjab University College of Pharmacy,

University of the Punjab, Lahore, Pakistan.

CERTIFICATE OF APPROVAL

The thesis entitled ―Pharmaceutical Co-crystals: A New Paradigm of Crystal

Engineering to modify Physicochemical and Pharmaceutical Properties of

Paracetamol and Naproxen” prepared by Sumera Latif under my guidance in partial

fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacy

(Pharmaceutics) is hereby approved for submission.

Dr. Nasir Abbas (Supervisor)

Assistant Professor


University of the Punjab,

Lahore, Pakistan.

Prof. Dr. Nadeem Irfan Bukhari

Professor of Pharmaceutics and Principal



Lahore, Pakistan.

Dr. Amjad Hussain

Assistant Professor



Lahore, Pakistan.

Pharmaceutical Co-crystals: A New Paradigm of Crystal Engineering to modify

Physicochemical and Pharmaceutical Properties of Paracetamol and Naproxen

Abstract

Co-crystals are of considerable relevance to the drug development as they offer

the ability of optimizing the physicochemical properties of an active pharmaceutical

ingredient by incorporation of a second component (a co-former), while retaining the

biological function of the parent material. Co-crystals have been emerged as an

alternative approach when salt or polymorph formation does not meet the required

targets. Furthermore co-crystals also represent a broad patent space because of the

availability of a large number of co-formers. The aim of the present study was to

synthesize and delineate co-crystals of paracetamol (a frequently used antipyretic and

analgesic drug) and naproxen (a nonsteroidal anti inflammatory drug). Both paracetamol

and naproxen had dissolution limited absorption and poor compressional behavior owing

to low plasticity.

A screening process, employing various methods namely dry grinding, liquid

assisted grinding, solvent evaporation and anti solvent addition, was carried out with

different ratios of paracetamol and caffeine. Implementation of the screen resulted in

paracetamol-caffeine co-crystals by liquid assisted grinding and solvent evaporation

methods with 1:1 and 2:1 ratios of paracetamol and caffeine respectively. Naproxen-

nicotinamide co-crystal was generated by liquid assisted grinding in 2:1 molar ratio. Co-

crystals gave characteristic PXRD patterns and DSC endotherms that were distinctive

from the starting materials. Mechanical properties of paracetamol-caffeine and naproxen-

nicotinamide co-crystals were analyzed by in-die Heckel model and tabletability curves

respectively.

Mean yield pressure, an inverse measure of plasticity, obtained from the Heckel

plots decreased significantly for paracetamol-caffeine co-crystals than pure paracetamol.

Over the entire range of compaction pressure used, tensile strength of naproxen was poor

and lamination and sticking occurred in some tablets. Tensile strength of naproxen-

nicotinamide co-crystal gradually increased with pressure achieving a desirable value of

more than 2 MPa which was ~ 1.80 times that of naproxen at 5000 psi. Moreover, co-

crystal pellets did not show any signs of cracking.

Intrinsic dissolution rates of co-crystals of both drugs showed more than 2 fold

faster dissolution compared to that of the respective pure drugs. Co-crystals were

successfully formulated into tablets by direct compression which was not possible to

employ for pure paracetamol and naproxen without extensive chipping. In-vitro

dissolution studies of paracetamol-caffeine and naproxen-nicotinamide co-crystals based

formulations also showed enhanced dissolution profiles in comparison to reference

formulations. In a single dose oral exposure study conducted in sheep model both co-

crystals revealed a significant increase (p < 0.05) in peak plasma concentration and area

under the curve. For selected paracetamol-caffeine and naproxen-nicotinamide co-

crystals, peak plasma concentrations were 2.45 and 1.61 times higher corresponding to

2.47 and 1.63 times higher area under the curve as compared to pure paracetamol and

naproxen. Relative bioavailability was also found to be ~ 2 and 1.6 times enhanced for

PCM and NAP co-crystals, respectively.

In conclusion, liquid assisted grinding was found to be a robust and easy method

and caffeine and nicotinamide as suitable co-formers for the synthesis of co-crystals of

paracetamol and naproxen, respectively. Co-crystals illustrated improved

physicochemical and mechanical properties. An enhancement in formulation

performance and in-vivo oral bioavailability was also shown by co-crystals.

Key words: Paracetamol, Naproxen, Co-crystals, Intrinsic dissolution rate, Plasticity,

Tensile strength, Direct compression, In-vitro dissolution, Bioavailability

DEDICATED TO

My Parents and Daughter

ACKNOWLEDGEMENTS

I would like to sincerely thank my supervisor Dr. Nasir Abbas, Assistant

Professor, Punjab University College of Pharmacy, University of the Punjab, for his

enthusiasm, encouragement and continued support throughout this course of study.

Working with him over the past few years has been a privilege. I would like to thank him

for his patience and endless advice. I thank him for his readiness to sit and talk even

during his busiest moments. His inputs on my research and his genuine concern for thesis

writing were a beacon light for me. May Allah always keep him in good health (Ameen).

My gratitude also goes to Professor. Dr. Nadeem Irfan Bukhari, Principal and Dr.

Amjad Hussain, Assistant Professor, Punjab University College of Pharmacy, University

of the Punjab, the co-supervisors for the study. Thank you very much for being such a

central part of my doctoral studies and for sharing your time and expertise. My sincere

thanks to Dr. Khalid Hussain, Dean, Punjab University College of Pharmacy, University

of the Punjab, for provision of the facilities to complete this work. I would also thank Dr.

Sohail Arshad, Associate Professor, Department of Pharmaceutics, Bahauddin Zakariya

University, Multan for his contribution in the preparation and technical review of the

manuscripts. My special thanks to Assistant Professor Dr. Sauleha Riffat and Mr. Abdul

Muqeet Khan, University of veterinary and animal sciences (UVAS). Dr. Sauleha

managed to provide a work place for me at animal shed of the Pattoki campus, UVAS. I

really valued Mr. Abdul Muqeet for his expertise in HPLC, method validation and data

interpretation.

I would also thank higher Education Commission (HEC) of Pakistan for provision

of funds (NRPU-3535) for the current study to Punjab University College of Pharmacy,

University of the Punjab, Lahore.

Then I would like to thank my parents for their love, guidance and

encouragement. They have both been superb and I hope this makes them proud. Thanks

to my little princess Meerab for her kind understanding and support. She learnt to work

independently when I was busy in my research work and thesis writing. A special thanks

to my sister Saima and friends Hafsa Afzal and Saleha Sadeeqa, their warmth and support

was always there when I needed it most.

I would also wish to extend my gratitude to technical staff, Lahore College for

Women University, Punjab University College of pharmacy, University of the Punjab,

UVAS and to everyone from whom I learnt even a single word. Foremost, thanks to

Allah for everything.

Sumera Latif

I

TABLE OF CONTENTS

1. Introduction ................................................................................................. 2

Prevalence of solid forms in pharmaceutical development process ........... 2 1.1.

Drug dissolution and bioavailability ........................................................... 4 1.2.

Strategies to alter properties of APIs .......................................................... 5 1.3.

Pharmaceutical particle technologies .......................................................... 5 1.3.1.

Solid forms of APIs .................................................................................... 6 1.3.2.

1.3.2.1. Solvates/Hydrates ....................................................................................... 6

1.3.2.2. Polymorphs ................................................................................................. 7

1.3.2.3. Salts ............................................................................................................. 8

1.3.2.4. Co-crystals .................................................................................................. 9

Crystal Engineering .................................................................................... 9 1.4.

Required features of components for co-crystallization ........................... 13 1.4.1.

Structural diversity of co-crystals ............................................................. 14 1.4.2.

Pharmaceutical co-crystals (PCs) ............................................................. 16 1.4.3.

Multidrug co-crystals (MDCs) .................................................................. 16 1.4.4.

Impact of co-crystallization on drug properties and performance ............ 18 1.4.5.

1.4.5.1. Mechanical and micromeritic properties .................................................. 18

1.4.5.2. Biopharmaceutical properties ................................................................... 19

1.4.5.3. Physical and chemical stability ................................................................. 21

Regulatory views and patent portfolios .................................................... 22 1.4.6.

Methodologies for co-crystal synthesis and screening ............................. 23 1.5.

Solution based methods ............................................................................ 23 1.5.1.

1.5.1.1. Evaporation or cooling methods ............................................................... 24

1.5.1.2. Anti solvent crystallization ....................................................................... 25

II

1.5.1.3. Supercritical carbon dioxide (scCO2) assisted processes.......................... 26

1.5.1.4. Freeze and spray drying ............................................................................ 26

Solid based methods ................................................................................. 26 1.5.2.

1.5.2.1. Mechanochemical grinding ....................................................................... 27

1.5.2.2. Hot melt extrusion (HME) ........................................................................ 29

1.5.2.3. Thermomicroscopy ................................................................................... 30

Characterization of co-crystal ................................................................... 30 1.6.

Single-crystal X ray diffraction ................................................................ 31 1.6.1.

Thermogravimetric analysis (TGA) .......................................................... 32 1.6.2.

Hot stage microscopy (HSM) ................................................................... 33 1.6.3.

Spectroscopic techniques .......................................................................... 33 1.6.4.

Problem statement ..................................................................................... 37 1.7.

Aim and objectives ................................................................................... 38 1.8.

2. Materials and methods .............................................................................. 40

Materials ................................................................................................... 40 2.1.

Solids......................................................................................................... 40 2.1.1.

Solvents ..................................................................................................... 40 2.1.2.

Preparation of phosphate buffer pH 6.8 .................................................... 40 2.1.3.

Preparation of phosphate buffer pH 7.4 .................................................... 40 2.1.4.

Preparation of 0.1M HCl pH 1.2 ............................................................... 41 2.1.5.

Methods..................................................................................................... 41 2.2.

Powder X-ray diffraction (XRD) .............................................................. 41 2.2.1.

2.2.1.1. Experimental PXRD method .................................................................... 42

Thermal characterization .......................................................................... 42 2.2.2.

2.2.2.1. Experimental DSC method ....................................................................... 43

III

Infrared (IR) spectroscopy ........................................................................ 43 2.2.3.

2.2.3.1. Experimental FTIR method ...................................................................... 44

Scanning electron microscopy (SEM) ...................................................... 44 2.2.4.

2.2.4.1. Experimental SEM method ....................................................................... 44

Intrinsic dissolution ................................................................................... 44 2.2.5.

UV spectrophotometry .............................................................................. 45 2.2.6.

High performance liquid chromatography (HPLC) .................................. 46 2.2.7.

Hirshfeld surface analysis ......................................................................... 47 2.2.8.

2.2.8.1. Features to be mapped on a Hirshfeld surface .......................................... 47

a) de and di 47

b) Fingerprint plots .................................................................................................... 48

3. Development of Paracetamol-Caffeine co-crystals to improve

compressional, formulation and in-vivo performance ...................................................... 51

Background ............................................................................................... 51 3.1.

Materials and Methods .............................................................................. 53 3.2.

Screening of co-crystals ............................................................................ 53 3.3.

Dry and liquid assisted grinding (LAG) ................................................... 53 3.3.1.

Solvent evaporation (SE) method ............................................................. 53 3.3.2.

Anti solvent addition (ASA) ..................................................................... 54 3.3.3.

Characterization ........................................................................................ 54 3.4.

Intrinsic dissolution studies....................................................................... 54 3.5.

Standard curve of PCM ............................................................................. 54 3.5.1.

Intrinsic dissolution rate (IDR) ................................................................. 55 3.5.2.

Drug content analysis ................................................................................ 55 3.6.

Compressional behavior............................................................................ 55 3.7.

IV

Plastic deformation measurement ............................................................. 57 3.7.1.

3.7.1.1. Particle density .......................................................................................... 57

Carr‘s compressibility index (CI) ............................................................. 58 3.7.2.

Bulk density .............................................................................................. 58 3.7.3.

Tapped density .......................................................................................... 58 3.7.4.

Angle of repose ......................................................................................... 59 3.7.5.

Preparation of tablets ................................................................................ 59 3.8.

Evaluation of tablets ................................................................................. 60 3.9.

Hardness (Breaking force) ........................................................................ 60 3.9.1.

Tensile strength ......................................................................................... 61 3.9.2.

Disintegration test ..................................................................................... 61 3.9.3.

In- vitro dissolution study ......................................................................... 61 3.9.4.

Release kinetic models .............................................................................. 62 3.10.

Zero order model....................................................................................... 62 3.10.1.

First order model ....................................................................................... 63 3.10.2.

Higuchi model ........................................................................................... 63 3.10.3.

Power law or Korsmeyer-Peppas model ................................................... 63 3.10.4.

Hixon-Crowell model ............................................................................... 64 3.10.5.

In-vivo studies ........................................................................................... 64 3.11.

Experimental animals................................................................................ 64 3.11.1.

Drug administration and collection of blood samples .............................. 65 3.11.2.

Extraction of the drug from the plasma .................................................... 65 3.11.3.

PCM Assay and HPLC conditions ............................................................ 66 3.11.4.

Preparation of stock and standard solutions.............................................. 66 3.11.5.

Pharmacokinetic (PK) analysis ................................................................. 67 3.11.6.

V

Statistical analysis ..................................................................................... 68 3.11.7.

Results and discussion .............................................................................. 68 3.12.

Characterization ........................................................................................ 70 3.12.1.

3.12.1.1. Powder X-ray diffraction (PXRD) ............................................................ 71

3.12.1.2. Differential scanning calorimetry (DSC) .................................................. 72

3.12.1.3. Fourier transform infrared spectroscopy (FTIR) ...................................... 74

3.12.1.4. Intrinsic dissolution rate (IDR) ................................................................. 76

Compressional and mechanical properties................................................ 80 3.12.2.

Pharmacotechnical evaluation of the formulated tablets .......................... 86 3.12.3.

In- vitro drug release study of the formulated tablets ............................... 88 3.12.4.

In-vivo performance of PCM and co-crystals ........................................... 92 3.12.5.

3.12.5.1. Standard curve and percent recovery ........................................................ 92

3.12.5.2. Pharmacokinetic (PK) parameters ............................................................ 96

a) Maximum plasma concentration (Cmax) ................................................................ 99

b) Area under the curve (AUC) ............................................................................... 101

c) Mean Residence Time (MRT) ............................................................................ 101

d) Elimination rate constant and half life (t1/2) ........................................................ 102

e) Total body clearance (CL) .................................................................................. 103

f) Volume of distribution (Vd) ............................................................................... 103

g) Relative bioavailability (R.B) ............................................................................. 104

Conclusion .............................................................................................. 105 3.13.

4. Simultaneously improving mechanical, formulation and in-vivo

performance of Naproxen by co-crystallization ............................................................. 107

Background ............................................................................................. 107 4.1.

Materials and methods ............................................................................ 109 4.2.

VI

Synthesis of co- crystals.......................................................................... 109 4.3.

Characterization ...................................................................................... 110 4.4.

Intrinsic dissolution studies..................................................................... 110 4.5.

Standard curve of NAP ........................................................................... 110 4.5.1.

Intrinsic dissolution rate (IDR) ............................................................... 110 4.5.2.

Drug content analysis .............................................................................. 111 4.6.

Powder compaction/Mechanical properties ............................................ 111 4.7.

Tabletability curves ................................................................................. 111 4.7.1.

Micromeritic properties .......................................................................... 111 4.7.2.

4.7.2.1. Hausner‘s ratio (HR) ............................................................................... 112

Formulation of tablets ............................................................................. 112 4.8.

Evaluation of the tablets.......................................................................... 112 4.9.

Hardness and disintegration test ............................................................. 113 4.9.1.

In-vitro drug release study ...................................................................... 113 4.9.2.

In-vivo studies in sheep ........................................................................... 113 4.10.

Drug administration and collection of blood samples ............................ 114 4.10.1.

Extraction of NAP from sheep plasma ................................................... 114 4.10.2.

Analysis of NAP and HPLC conditions.................................................. 114 4.10.3.

Preparation of stock and standard solutions............................................ 115 4.10.4.

Pharmacokinetic (PK) analysis ............................................................... 116 4.10.5.

Statistical analysis ................................................................................... 116 4.10.6.

Results and discussion ............................................................................ 116 4.11.

Characterization ...................................................................................... 117 4.11.1.

4.11.1.1. Powder X-ray diffraction (PXRD) .......................................................... 117

4.11.1.2. Differential scanning calorimetry (DSC) ................................................ 119

VII

4.11.1.3. Hirshfeld analysis.................................................................................... 121

4.11.1.4. Intrinsic dissolution rate (IDR) ............................................................... 122

Compaction/Micromeritic behavior of powders ..................................... 125 4.11.2.

4.11.2.1. Tabletability profiles ............................................................................... 126

4.11.2.2. Material flow behavior ............................................................................ 127

In-vitro performance of tablets ............................................................... 128 4.11.3.

In-vivo performance of pure NAP and NAP-NIC co-crystal .................. 131 4.11.4.

4.11.4.1. Standard curve and percent recovery ...................................................... 131

4.11.4.2. Pharmacokinetic (PK) metrics ................................................................ 136

a) Maximum plasma concentration (Cmax) .............................................................. 139

b) Area under the curve (AUC) ............................................................................... 140

c) Mean residence time (MRT) ............................................................................... 140

d) Disposition parameters........................................................................................ 140

e) Relative bioavailability ....................................................................................... 141

Conclusion .............................................................................................. 142 4.12.

5. Conclusion and Future Perspective ......................................................... 144

Conclusion .............................................................................................. 144 5.1.

Future perspective ................................................................................... 145 5.2.

Bulk manufacturing ................................................................................ 145 5.2.1.

Product Stability...................................................................................... 145 5.2.2.

Structure property relationship ............................................................... 146 5.2.3.

Intellectual property ................................................................................ 146 5.2.4.

6. References ............................................................................................... 148

Annexure A: Crystallographic information file of NAP –NIC co-crystal ...................... 176

Annexure B: Ethical approval ......................................................................................... 177

VIII

Annexure C: Individual pharmacokinetic profile of all sheep ........................................ 178

Annexure D: Publications ............................................................................................... 198

IX

LIST OF TABLES

Table 1.1: Pharmaceutical particle technologies and their limitations ............................... 6

Table 1.2: Comparison of the mechanochemical grinding and solution based methods .. 28

Table 1.3: Literature review on pharmaceutical co-crystals ............................................. 34

Table 3.1: Preparation of PCM standards in mobile phase and blank sheep plasma ........ 67

Table 3.2: Screening of PCM-CAF co-crystals by using various methods (―+― represents

formation of co-crystal, characterized by DSC and PXRD results, ―- ―represents

re-crystallization of starting material or no crystallization) (co-crystals from

successful techniques are coded A, B, C, D and E) .............................................. 70

Table 3.3: Onset, mid and off set temperatures of DSC thermograms of PCM, CAF and

PCM-CAF co-crystals ........................................................................................... 74

Table 3.4: Mechanical parameters derived for pure PCM and PCM-CAF co-crystals .... 84

Table 3.5: Physical properties of tablet formulations prepared from pure PCM (direct

compression (F1) and wet granulation (F2)) and PCM-CAF co-crystals (FA, FB,

FC, FD and FE) ..................................................................................................... 87

Table 3.6: Drug release kinetics from various formulations of PCM (F1 and F2), and

PCM-CAF co-crystals (FA, FB, FC, FD and FE)................................................. 91

Table 3.7: Recovery of PCM from spiked sheep plasma.................................................. 93

Table 3.8: Plasma concentration of PCM and PCM-CAF co-crystals (C and E) in sheep at

different time intervals, obtained after single oral administration at a dose of 30

mg/Kg body weight (n= 6) .................................................................................... 99

Table 3.9: Pharmacokinetic parameters of PCM and PCM-CAF co-crystals in sheep,

obtained after single oral administration at a dose of 30 mg/Kg body weight (n=

6). *R.B relative to PCM .................................................................................... 102

Table 4.1: Unit cell parameters of NAP-NIC co-crystal (a) obtained from CIF CCDC

904098 (Ando et al., 2012) (b) calculated from experimental PXRD ................ 119

Table 4.2: Micromeritic properties of NAP and NAP-NIC co-crystal ........................... 128

Table 4.3: Physical parameters and % drug released of NAP in 0.1 MHCl and phosphate

buffer pH 7.4 from commercial (F0) and NAP-NIC co-crystal tablets (F1) ...... 129

Table 4.4: Drug release kinetics from Neoprox (F0) and NAP-NIC co-crystal formulation

(F1) in 0.1 M HCl and phosphate buffer pH 7.4 ................................................. 131

X

Table 4.5: Recovery of NAP from sheep plasma (n= 3) ................................................ 133

Table 4.6: Plasma concentration of NAP and NAP-NIC co-crystal in sheep at different

time intervals following oral administration at a single dose of 10 mg/Kg body

weight (n= 6) ....................................................................................................... 138

Table 4.7: Pharmacokinetic parameters of NAP and NAP-NIC co-crystal in sheep

following oral administration at a single dose of 10 mg/Kg body weight (n= 6).

*R.B relative to NAP .......................................................................................... 139

XI

LIST OF FIGURES

Figure 1.1: Various solid forms of an API based on internal structure and composition ... 3

Figure 1.2: Crystal packing diagram of mefenamic acid form I (A) and form II (B),

(viewed on b-axis)................................................................................................... 8

Figure 1.3: Crystal Packing diagram of naproxen-alanine co-crystal (viewed on c-axis) 11

Figure 1.4: Supramolecular homosynthons (A and B) and supramolecular heterosynthons

(C and D)............................................................................................................... 12

Figure 1.5: Trend in the number of co-crystal hits ........................................................... 14

Figure 1.6: Structural diversity outcomes of co-crystals .................................................. 15

Figure 1.7: Methods for synthesis of co-crystals .............................................................. 24

Figure 1.8: Solid state characterization tools for co-crystals ............................................ 31

Figure 2.1: Representative figure to depict de and di of Hirshfeld surface ....................... 47

Figure 2.2: Hirshfeld surfaces and finger print plot with front (top) and back (bottom)

view. The molecule is shown with the Hirshfeld surface mapped with (a)

Curvedness, (b) shape index and (c) de ................................................................ 49

Figure 3.1: Molecular structures of Paracetamol (I) and Caffeine (II) ............................. 52

Figure 3.2: PXRD patterns of PCM, CAF and PCM-CAF co-crystals prepared by LAG

(A, B and C) and SE (D and E) ............................................................................. 72

Figure 3.3: DSC thermograms of PCM, CAF and PCM-CAF co-crystals prepared by

LAG (A, B and C) and SE (D and E) ................................................................... 73

Figure 3.4: Full ATR-FTIR spectra of PCM, CAF, physical mixture and PCM-CAF co-

crystals prepared by LAG (A, B and C) and SE (D and E) .................................. 75

Figure 3.5: ATR-FTIR spectra of PCM, physical mixture and PCM-CAF co-crystals

highlighting the N-H amide stretch and phenolic OH stretch regions for PCM ... 76

Figure 3.6: Calibration curve of PCM in phosphate buffer pH 6.8 .................................. 78

Figure 3.7: Intrinsic dissolution rate of PCM alone and from co-crystals A, B, C, D and E

in phosphate buffer pH 6.8 (5.5, 8.72, 9.53, 12.23, 11.11 and 14.40 mg/cm2-min

respectively) (n= 3) ............................................................................................... 79

Figure 3.8: SEM images of pure PCM and PCM-CAF co-crystals prepared by LAG (A, B

and C) and SE (D and E)....................................................................................... 80

XII

Figure 3.9: Heckel plots for PCM and PCM-CAF co-crystals prepared by LAG (A, B and

C) and SE (D and E) ............................................................................................. 83

Figure 3.10: In-vitro drug release comparison of various formulations of PCM (F1 and

F2), PCM-CAF co-crystals (FA, FB, FC, FD and FE) and physical mixtures (n=

3) ........................................................................................................................... 89

Figure 3.11: Standard curve of PCM in drug free sheep plasma ...................................... 92

Figure 3.12: HPLC chromatogram of PCM (10 µg/ml) in mobile phase ......................... 94

Figure 3.13: Representative chromatogram of drug free sheep plasma ............................ 94

Figure 3.14: Representative chromatogram of sheep plasma spiked with pure PCM (0.1

µg/ml) .................................................................................................................... 95

Figure 3.15: Representative chromatogram of sheep plasma spiked with pure PCM (10

µg/ml) .................................................................................................................... 95

Figure 3.16: Chromatogram from plasma of sheep administered orally at a dose of 30

mg/Kg body weight of pure PCM (30 min) .......................................................... 96


mg/Kg body weight of equivalent co-crystal (1.5 h) ............................................ 96

Figure 3.18: Plasma concentration profiles with time for PCM and PCM-CAF co-crystals

(C and E) in sheep model (n= 6) ......................................................................... 100

Figure 3.19: Pictorial representation showing the synthesis and characterization of PCM-

CAF co-crystal and improvement in mechanical, in-vitro and in-vivo performance

............................................................................................................................. 105

Figure 4.1: Molecular structures of Naproxen (I) and Nicotinamide (II) ....................... 108

Figure 4.2: PXRD patterns of NAP, NIC, NAP-NIC co-crystal and calculated pattern 118

Figure 4.3: Comparison of PXRD patterns of experimental and calculated data by Dash.

Experimental (black line), calculated (red line) and difference (blue line) ........ 118

Figure 4.4: DSC thermograms of NAP, NIC and NAP-NIC co-crystal ......................... 121

Figure 4.5: Two dimensional fingerprint plots of NAP (I) and NAP-NIC co-crystal (II)

............................................................................................................................. 122

Figure 4.6: Calibration curve of NAP in 0.1M HCl ....................................................... 123

Figure 4.7: Calibration curve of NAP in phosphate buffer pH 7.4 ................................. 123

XIII

Figure 4.8: Intrinsic dissolution rate of NAP alone and from NAP-NIC co-crystal in 0.1M

HCl (7.2 and 51.0 µg/cm2-min respectively) (n= 3) ........................................... 124

Figure 4.9: Intrinsic dissolution rate of NAP alone and from NAP-NIC co-crystal in

phosphate buffer pH 7.4 (552 and 1520 µg/cm2-min respectively) (n= 3) ......... 124

Figure 4.10: Tabletability plots of NAP, NIC and NAP-NIC co-crystal ........................ 127

Figure 4.11: Dissolution profiles of commercial (F0) and NAP-NIC co-crystal tablets

(F1) in 0.1M HCl ................................................................................................ 130


(F1) in phosphate buffer pH 7.4 .......................................................................... 130

Figure 4.13: Calibration curve of NAP in drug free sheep plasma ................................. 132

Figure 4.14: HPLC chromatogram of NAP (10 µg/ml) in mobile phase ....................... 133

Figure 4.15: Representative chromatogram of drug free sheep plasma .......................... 134

Figure 4.16: Representative chromatogram of sheep plasma spiked with pure NAP (0.5

µg/ml) .................................................................................................................. 134


µg/ml) .................................................................................................................. 135

Figure 4.18: Chromatogram from plasma of sheep administered at an oral dose of 10

mg/Kg body weight of pure NAP (2 h) .............................................................. 135


mg/Kg body weight of equivalent NAP-NIC co-crystal (30 min) ...................... 136

Figure 4.20: Sheep plasma concentration with time for NAP and NAP-NIC co-crystal (n=

6) ......................................................................................................................... 138

Figure 4.21: Pictorial representation showing the synthesis and characterization of NAP-

NIC co-crystal and enhancement in in-vitro and in-vivo performance ............... 142

XIV

LIST OF ABBREVIATIONS

API Active pharmaceutical ingredient

AUC Area under the curve

BCS Biopharmaceutic classification system

CAF Caffeine

CI Carr‘s index

CL Total body clearance

Cmax Maximum plasma concentration

DSC Differential scanning calorimetry

FDA Food and drug administration

FTIR Fourier transform infrared spectroscopy

GIT Gastro intestinal tract

GRAS Generally recognized as safe

HME Hot melt extrusion

HPLC High pressure liquid chromatography

HR Hausner‘s ratio

IDR Intrinsic dissolution rate

LAG Liquid assisted grinding

MDCs Multi drug co-crystals

MRT Mean residence time

NAP Naproxen

NIC Nicotinamide

PCM Paracetamol

PCs Pharmaceutical co-crystals

PDA Photodiode array

PM Physical mixture

PK Pharmacokinetics

PXRD Powder X-ray diffraction

RB Relative bioavailability

SE Solvent evaporation

SEM Scanning electron microscopy

XV

t1/2 Elimination half life

tmax Time for maximum plasma concentration

UV Ultra violet

Vd Volume of distribution

WG Wet granulation

1

Chapter 1

2

1. Introduction

Prevalence of solid forms in pharmaceutical development process 1.1.

The vast majority of active pharmaceutical ingredients (APIs) are isolated in solid

form and the selection of crystalline/amorphous form is invariably considered to be the

first step in formulation development. Solid forms offer a lot of advantages over other

forms, but often face the problem of physical and chemical properties such as solubility,

melting point, chemical interaction, stability and bioavailability. Moreover, most of the

drugs are administered orally in solid form, which is generally considered as convenient,

stable and safest dosage form offering accurate dose as well (Najar and Azim, 2014).

Drug molecules can exist in solid forms in either crystalline or amorphous phase.

Because of the instability of many amorphous materials, most drugs are formulated in the

crystalline state due to the intrinsic stability, lower surface energy, lower hygroscopicity

and the well established impact of crystallization processes on purification and isolation

of chemical substances (Paul et al., 2005). The presence of several hydrogen bonding

sites in drugs makes them inherently prone to form multiple crystal forms. These crystal

forms include but are not limited to salts, hydrates, solvates, polymorphs and co-crystals.

Figure 1.1 shows the possible solid forms of single and multicomponent crystalline

systems. The diversity of crystal forms of an API creates opportunities to customize its

physicochemical properties. For this reason, crystalline forms of an API have been

explored using high throughput screening (HTS). HTS has a greater potential leading to

the discovery of diverse crystal forms and providing an understanding of the factors that

direct the crystallization process (Gardner et al., 2004). However, in-vitro screening in

non aqueous solvents in order to identify the possible hits resulted in new drug candidates

3

exhibiting poor aqueous solubility and /or dissolution rate (Lipinski, 2000; Babu and

Nangia, 2011). As bioavailability is a function of solubility, poor bioavailability has

become characteristic of many new drug candidates (Serajuddin, 2007). In fact, drug

candidates with poor bioavailability are the primary reasons why > 40% of new drug

candidates fail in preclinical and clinical development with a resultant loss in terms of

time and cost (Van De Waterbeemd et al., 2001) and ~ 90% of new drug candidate fall

into class II and class IV of Biopharmaceutic Classification System (BCS). Therefore,

from a crystal engineering perspective, the design of pharmaceutical solids with desired

physicochemical properties becomes relevant (Mahata et al., 2014).

Figure 1.1: Various solid forms of an API based on internal structure and composition

4

In the above context, the control of the crystal form and the size and shape of the

crystal become of utmost importance. The processes of solid dosage form development

(such as filtration, drying, milling, granulation and tableting) are also required to be

simplified for better control of the product quality and performance (Sheth and Grant,

2005). This needs a thorough understanding of the inter relationship between solid state

structures, properties, performance and processes at the fundamental level (Sun, 2009).

Drug dissolution and bioavailability 1.2.

The number of new molecular entities (NMEs) approved by the Food and Drug

Administration (FDA) is increasing each year. To reduce the unnecessary testing, BCS

developed by Amidon et al is used by FDA to show that the bioavailability of the API is a

function of solubility and membrane permeability (Amidon et al., 1995). According to

this system, APIs are categorized in to four classes: Class I (highly soluble, highly

permeable), Class II (low soluble, highly permeable), Class III (highly soluble, low

permeable) and Class IV (low soluble, low permeable). When combined with dissolution

studies, a biowaiver for in-vivo bioavailability and bioequivalence can be applied for

class I and III drugs. A drug substance is considered highly soluble when the highest dose

strength is soluble in < 250 ml of aqueous medium over a pH range of 1-7.5; otherwise

drug substance is reflected as poorly soluble. A drug is regarded as highly permeable

when the extent of absorption in humans is determined to be > 90 % of an administered

dose (Shargel et al., 2015)

Intrinsic solubility and dissolution are prerequisite for oral drug delivery (Di et al.,

2012). Poor solubility and low dissolution rate lead to consequences such as higher dose

of drugs required to reach therapeutic level and lesser fraction absorbed in small intestine.

5

Low water solubility, therefore, continues to be a consistent challenge to successful drug

development and needs to be addressed.

Strategies to alter properties of APIs 1.3.

New strategies are being introduced including pharmaceutical particle

technologies (Hu et al., 2004) and solid forms of API (Censi and Di Martino, 2015;

Ticehurst and Marziano, 2015) to alter the properties of APIs for drug product

development and delivery (Williams et al., 2013). These technologies have been

described below.

Pharmaceutical particle technologies 1.3.1.

Various particle technologies have been adopted for improving the aqueous

solubility, dissolution rate and in turn bioavailability of drugs (Khadka et al., 2014).

These include reduction of particle size to increase surface area (Rawat et al., 2011), use

of water soluble carriers to form inclusion complexes (Brewster and Loftsson, 2007),

polymeric micelles (Kwon and Okano, 1996), solid-self emulsifying drug delivery

systems (Yi et al., 2008), solid lipid nanoparticles (Hu et al., 2004), and liposomes

(Mohammed et al., 2004) for the delivery of lipophilic drugs. Although these approaches

are very effective in enhancing the oral bioavailability and processing properties of the

drugs, there are practical limitations with these techniques (Khadka et al., 2014) which

are summarized in Table 1.1.

6

Table 1.1: Pharmaceutical particle technologies and their limitations

Solid forms of APIs 1.3.2.

Various solid phase modifications have been reported to improve the

physicochemical properties of APIs (Censi and Di Martino, 2015). These solid forms

include amorphous state as well as alternate crystalline structures such as polymorphs,

salts, solvates, hydrates, and co-crystals.

1.3.2.1. Solvates/Hydrates

Solvates are crystal structures containing stoichiometric proportions of one or

more solvent molecules incorporated within the crystal lattice. A solvate is called a

hydrate when a water molecule constitutes the solvent of crystallization. Prevalence of

organic compounds to exist as hydrates is more (~ 33%), due to the easy incorporation of

the water molecule within the crystal lattice governed by its versatile hydrogen bonding

capabilities (Gillon et al., 2003) as compared to form solvates (~ 10%) which are also

known to impart toxicity. As APIs have many proton donors and acceptors, they are

Particle Technologies Limitations

Micronization (Milling) Agglomeration, poor flowability, loss of expected

large surface area, low bulk density

Solid-self emulsifying drug

delivery systems

Oxidation of vegetable oils, physical aging

associated with glycerides, interaction between

drugs and excipients

Complexation with cyclodextrins Low extent of drug complexation and interaction

with complexing agent, increased toxicity, high

cost.

Liposomes and solid lipid

nanoparticles

Poor stability during storage

7

susceptible to hydrate formation and it is estimated that approximately one third of the

APIs are capable of forming hydrates during pharmaceutical manufacturing (Karagianni

et al., 2018). But stability is a matter of concern for pharmaceutical hydrates because they

can undergo solid phase transitions under storage conditions and therefore alter the

physicochemical properties (Khankari and Grant, 1995; Tong et al., 2010).

For the hydrated and solvated APIs, the crystal structure may or may not remain

intact after desolvation and are identified as pseudopolymorphic and polymorphic

solvates respectively. Powder X-ray diffraction (PXRD) patterns are similar for the

former forms whereas later have different PXRD patterns owing to differences in the

crystal structure (Healy et al., 2017).

1.3.2.2. Polymorphs

Polymorphs, having the same chemical composition but different

physicochemical properties, are characterized by variations in unit cell structure arising

from packing or conformational differences as shown in Figure 1.2. Their solid state

properties generally differ as a consequence. Properties such as solubility,

hygroscopicity, stability, compaction and bioavailability of an API are dependent on the

polymorphic state (Davey, 2002; Threlfall, 1995). The phenomenon of polymorphism is a

scientific challenge and its adverse effects are well established in pharmaceutical industry

leading to a difference in the bioavailability (Morissette et al., 2003).

High energy polymorphs and amorphous formulations have improved solubility

but the system is at serious risk of being crystallized to the thermodynamically stable

form, even in the solid state (Rodr guez-Spong et al., 2004; Seefeldt et al., 2007; Yu,

2001) compromising the performance of the formulation. Hence, it is essential that the

8

desired form be reproducible and that it can remain stable during production and

marketing.

A

B

Figure 1.2: Crystal packing diagram of mefenamic acid form I (A) and form II (B),

(viewed on b-axis)

1.3.2.3. Salts

Salts are multicomponent solid forms consisting of stoichiometric ratio of

negatively charged anion and positively charged cation. Currently, salt formation is a

primary solid state approach to alter physical properties of APIs and approximately over

half of the medicines on the market are administered as salts (Serajuddin, 2007).

Generally salts have a high bioavailability than single component forms of API or its

hydrates, and are biologically safer than solvates (Gillon et al., 2003). However a major

9

limitation with this approach is that the API must possess a suitable ionizable (acidic or

basic) site for proton transfer. Moreover due to toxicological reason only 12 or so acidic

or basic counterions are explored in a typical API salt screen (Swarbrick and Boylan,

2000).

1.3.2.4. Co-crystals

Co-crystals have been emerged as an alternative approach when salt or polymorph

formation does not meet the required targets. The design and synthesis of co-crystals

have received considerable attention partly because of fundamental interest in molecular

recognition driven assembly processes (Aakeröy et al., 2005) and partly because of

potential applications of co-crystals to many areas of functional solids including

pharmaceuticals (Almarsson and Zaworotko, 2004).

Co-crystals can be formed regardless of the API‘s ionizable status (Friščić and

Jones, 2010). Rather their formation relies on complementary functional groups between

the API and a biologically safe partner molecule termed the co-former, to allow for

hydrogen bonding or other forms of interactions in the solid. The number of biologically

safe co-formers that can be incorporated into a co-crystal screen is more extensive than

the number of acids or bases used in the production of salts. Thus co-crystals offer greater

crystal form diversity than salts.

Crystal Engineering 1.4.

Crystal engineering is defined as ―the understanding of intermolecular

interactions in the context of crystal packing and in the utilization of such understanding

in the design of new solids with desired physical and chemical properties‖ (Desiraju and

Parshall, 1989). Various disciplines exploring the crystal engineering include nonlinear

10

optics (Panunto et al., 1987), porous materials (Russell et al., 1997), photographic

materials and coordination polymers (McManus et al., 2007). However over the last two

decades, crystal engineering gained high impetus on APIs to alter their properties

(Blagden et al., 2007; Dunitz, 2003).

Co-crystallization is a flourishing research field with direct applications to

pharmaceutical industry and represents a new paradigm of crystal engineering for the

synthesis of new crystalline phases. This technique has expanded the landscape for solid

forms of APIs and increased the likelihood to enhance the specific physical, mechanical

and in-vivo properties. Co-crystallization has been successfully applied for a number of

drug molecules without changing their chemical nature, thereby maintaining their

therapeutic activity.

Generally, a co-crystal is a multicomponent molecular crystal, i.e., a crystalline

substance comprising two or more chemically different molecules (Dunitz, 2003). This

perspective also considers hydrates, solvates, inclusion compounds, clathrates as co-

crystals. Zaworotko and Aakeroy precisely differentiated co-crystals from the other

multicomponent compounds. Accordingly, a co-crystal is a stoichiometric multiple

component crystal where all components, a target molecule or ion and a co-former, are

solid under ambient conditions and coexist within a single crystalline lattice as shown in

Figure 1.3 (Aakery and Salmon, 2005; Vishweshwar et al., 2006). Other terms used for

the co-crystals are molecular compounds, organic molecular compounds, addition

compounds, molecular complexes and solid state complexes.

11

Figure 1.3: Crystal Packing diagram of naproxen-alanine co-crystal (viewed on c-axis)

Rational design of co-crystal has been based on crystal engineering approach of

identification and utilization of supramolecular synthon (Lehn, 2002). Synthons are non

covalent bonds which reliably form repetitive patterns of intermolecular interactions in a

crystal structure when forming between complementary functional groups. Directional

nature of these interactions makes predictions with reliable accuracy about the orientation

of molecules within a crystal structure. This dictates placement of the necessary

functional groups in the correct area of the molecule to produce the desired synthon

(Nangia, 2010) and hence, in theory, crystal structure designed.

Hydrogen bonding is the lead intermolecular interaction in synthon based designs

(Steiner, 2002) and involves conventional hydrogen bonds of the type, N-H…O, N-H…N

and O-H…N. Other weaker intermolecular interactions (C-H…O and C-H…N) can also

serve to stabilize the resulting structures (Li et al., 2008).

Hence for the successful design of co-crystals, the selected co-former should

have complimentary functional groups to those of the API to form the intermolecular

12

interactions which thereby constitute supramolecular synthons. The design of a co-crystal

commonly utilizes acid-acid and amide-amide homosynthons (Etter, 1982) and acid-

amide (Aaker y et al., 200 Fleischman et al., 2003) and acid-pyridine heterosynthons

(Olenik et al., 2003; Walsh et al., 2003). Figure 1.4 represents supramolecular

homosynthons (A and B) and heterosynthons (C and D).

Figure 1.4: Supramolecular homosynthons (A and B) and supramolecular heterosynthons

(C and D)

It is not always possible to produce a co-crystal experimentally designed on

supramolecular synthon approach because this method does not take into account all the

weaker interactions that may be present between the drug and co-former.

The Cambridge structural Database (CSD) is an established repository for small

molecule organic and metalorganic crystal structures. The large number of data in the

CSD can be statistically analyzed. Analysis of the data relating to co-crystal specific

subset of CSD has been reported to identify factors, beyond synthon matching, indicative

of the ability to form a co-crystal. Polarity and shape of the co-crystal components were

found to have a strong correlation whereas no obvious statistical relation was found

13

between hydrogen bond donors and acceptors. This statistical analysis can be used to

predict the likeliness of the components to form a co-crystal and thereby can provide a

base to reduce the number of co-formers in a physical screen (Fábián, 2009; Hofmann

and Kuleshova, 2005).

It is important to distinct salts and co-crystals with respect to both preformulation

activities and chemical/pharmaceutical development aspects. Indeed salts and co-crystals

constitute the opposite ends of multicomponent structures (Aakeröy et al., 2007; Childs et

al., 2007). A salt is formed as a result of transfer of a proton from an acid to base (acid

base reaction). On the other hand, in co-crystals proton transfer does not take place due to

the lack of ionizable sites in the substance. The pKa values can forecast the salt or co-

crystal formation. If the difference between the pKa base and pKa acid (ΔpKa) is > 3, a

salt forms (Bhogala et al., 2005). A co-crystal is formed when the ΔpKa < 0 where as for

a ΔpKa of 0-3 proton transfer is ambiguous and can result in formation of salt/co-crystal

hybrid containing proton sharing or intermediate ionization states (Sanphui et al., 2014;

Suresh et al., 2015).

Required features of components for co-crystallization 1.4.1.

Co-crystals can be generated from two or more molecules of any shape and size

having complementary functional groups. For co-crystallization, API should be evaluated

for number and arrangement of hydrogen bond donors and acceptors (Aakeröy et al.,

2010), salt forming ability (pKa‘s) (Aakeröy et al., 2005; Childs et al., 2007),

conformational flexibility and solubility requirements (Alhalaweh et al., 2012; Chadwick

et al., 2009). Usually APIs that are rigid, highly symmetrical, possess strong non bonded

interactions and low molecular weight are more appropriate to co-crystallize with co-

14

formers (Stahly, 2007). To be suitable, the non API component of the co-crystal should

be safe with no serious adverse effects. Co-formers are generally limited to the

substances included in the FDA‘s generally recognized as safe (GRAS) or Everything

Added to Food in the United States (EAFUS) lists (Steed, 2013).

The increasing significance of co-crystals can be inferred from the fact that

literature reveals a 500 fold increase in the number of co-crystal hits from 1951 to todate

as taken from google scholar (Figure 1.5).

Figure 1.5: Trend in the number of co-crystal hits

Structural diversity of co-crystals 1.4.2.

Studies regarding crystal form diversity of the co-crystals (Figure 1.6) have

shown an increasing trend recently (Healy et al., 2017). Unlike salts, weakly acidic, basic

or neutral substances have tendency to form co-crystals (Friščić and Jones, 2010).

Availability of an extensive list of co-formers also dictates a large structural diversity for

co-crystals. A single drug can form several co-crystals and more than 50 co-crystals are

known for few drugs (Childs and Hardcastle, 2007; Childs et al., 2008).

0

2000

4000

6000

8000

10000

12000

14000

Nu

mb

er o

f co

-cry

sta

l h

its

Year

15

As per the literature, co-crystals have shown a similar tendency as that of single

component crystalline systems to exist as polymorphs (Aitipamula et al., 2014), solvates

(Basavoju et al., 2006), hydrates (Clarke et al., 2010), and salts (Chen et al., 2007). Salt

co-crystals enclose three components in the lattice, i.e., free acid/base, counterion and

neutral guest. In this context co-crystals of fluoxetine HCl with carboxylic acids (Childs

et al., 2004) and norfloxacin saccharinate dehydrate with saccharine (Velaga et al., 2008)

have been reported. Hydrates and solvates are also possible for salt co-crystals. Co-

crystal polymorphs show significantly different structural and physical properties. Co-

crystals with two or more polymorphs include urea/glutaric acid, ethenzamide/3, 5-

dinitrobenzoic acid, carbamazepine/saccharine and carbamazepine/nicotinamide.

Figure 1.6: Structural diversity outcomes of co-crystals

16

Co-crystals have also been reported to show different stoichiometric ratios of the

API and co-former, e.g., 1:1, 1:2 and 2:1 urea/maleic acid co-crystals (Videnova-

Adrabinska et al., 1993), 1:1 and 2:1 carbamazepine/4-aminobenzoic acid co-crystals

(Jayasankar et al., 2009), and 1:2 and 2:1 caffeine/4-hydroxybenzoic acid co-crystals

( učar et al., 200 ). However their formation mechanisms are not fully understood.

The structural diversity of co-crystals has been proposed as an added advantage to

expand the diversity of drug solid forms. This feature can offer opportunities for fine

tuning the properties of a drug. Knowledge of co-crystal diversity is also important for

better solid form control during scale up operations.

Pharmaceutical co-crystals (PCs) 1.4.3.

Pharmaceutical co-crystals (PCs), a striking sub set of co-crystals, are the co-

crystals in which the target molecule or ion is an API which bonds to the co-former

through hydrogen bonds (Almarsson and Zaworotko, 2004). PCs have demonstrated to

have significant potential in their ability to modify the physicochemical (solubility and

dissolution rate, particle morphology, tableting and compaction, melting point, physical

form, biochemical and hydration stability, and permeability) and pharmacokinetic

properties of drugs (Bolla and Nangia, 2016). Recently, FDA has released a draft

guidance regarding the definition and the regulatory classification of PCs in lieu of the

significant impact of PCs to address properties of the drugs (FDA, 2016).

Multidrug co-crystals (MDCs) 1.4.4.

In a multidrug co-crystal (MDC), the co-former is replaced by another API; where

both APIs interact predominantly via nonionic interactions and rarely through hybrid

interactions, i.e., a combination of ionic and nonionic interactions involving partial

17

proton transfer and hydrogen bonding resulting in salt/co-crystal hybrids and ionic co-

crystals (Elacqua et al., 2012; Jacobs and Noa, 2014).

MDCs could offer potential advantages of synergistic and /or additive effects,

mitigating side effects of drugs, enhanced solubility and dissolution rate of at least one

component (Aitipamula et al., 2009; Chandel et al., 2011), enhanced bioavailability

(Cheney et al., 2011), and possible stabilization of unstable components through

intermolecular interactions (Jiang et al., 2014). MDC of two drugs ethenzamide and

gentisic acid with anti inflammatory activities was reported with enhanced intrinsic

dissolution rate (IDR) and could be used in the management of pain (Aitipamula et al.,

2009). MDC of meloxicam/aspirin was developed and showed a decrease in the time

required to reach the therapeutic concentrations with a 4 fold increase in bioavailability

(Cheney et al., 2011). An improved IDR for MDC of sildenafil/aspirin was observed in

comparison to a marketed sildenafil citrate salt. The dual therapeutic effects displayed by

this MDC might result from the antiplatelet activity of aspirin suggesting its potential use

in the treatment of erectile dysfunction in patients with cardiovascular disorders (Ţegarac

et al., 2014). MDCs of diflunisal and diclofenac with theophylline showed an

enhancement in apparent solubility in comparison to pure APIs while the IDRs were

comparable. A resistance to hydrate formation was also found for both MDCs at different

relative humidity (Surov et al., 2014).

Many neutraceuticals used as potential APIs have poor aqueous solubility and

hence less bioavailability (McClements et al., 2015). Studies have been reported

comprising co-crystals of neutraceuticals with GRAS compounds, e.g., co-crystals of

curcumin (an anticancer neutraceutical) with resorcinol and pyrogallol resulting in

18

improved solubility. Dissolution rates were also found to be 5 and 12 times enhanced for

curcumin/resorcinol and curcumin/pyrogallol co-crystals respectively as compared to the

pure drug (Sanphui et al., 2011).

Impact of co-crystallization on drug properties and performance 1.4.5.

1.4.5.1. Mechanical and micromeritic properties

The development and manufacturing of promising drugs find a major hindrance

on account of deficient mechanical and micromeritic properties which also present

difficulties during the key industrial processes including capsule filling, milling and

compaction in tablet formulation and in scale up operations. Poor tabletability of a

powder generally reveals a lack of plastic deformation as a result of development of very

small bonding area during compaction often accompanied by high elastic recovery.

Subsequently, the tablets are porous and weak in strength. Crystal engineering finds wide

applications to produce co-crystals with improved flow properties (Sansone et al., 2014)

as well as different intrinsic mechanical and plastic behavior (Sun and Hou, 2008). The

influence of co-crystallization on crystal mechanical properties can be assessed by

structure-property interrelation, e.g., 1:1caffeine/methyl gallate co-crystal has been

reported to reveal improved compaction owing to the presence of slip planes in its crystal

structure imparting plasticity also (Sun and Hou, 2008). Paracetamol (PCM), a poorly

compressible API, was co-crystallized with a number of co-formers including oxalic acid,

theophylline, naphthalene and phenazine that were found to promote intended

crystallographic features. PCM co-crystals showed better tabletability and improvement

in mechanical behavior was assessed from enhanced breaking force and tensile strength

(Karki et al., 2009). The better tabletability was credited to the successful development of

19

layered crystal structure. A 1:1 co-crystal embodying PCM and a new co-former

trimethyl glycine not only improved the compression moldability by producing a

sufficiently hard tablet but also found to mask the bitter taste of PCM (Maeno et al.,

2014). Mechanical properties of 1:1 flurbiprofen/nicotinamide and ibuprofen/

nicotinamide co-crystals were evaluated by drawing the tabletability curves (Chow et al.,

2012). Both co-crystals displayed better tabletability evident from the increasing tensile

strength as a function of compaction pressure.

1.4.5.2. Biopharmaceutical properties

A key challenge in drug delivery is the poor bioavailability which depends on

several factors including aqueous solubility, dissolution rate and drug permeability

(Williams et al., 2013). Present studies regarding PCs have mainly focused on increasing

bioavailability for BCS class II and IV drugs by enhancing their solubility and dissolution

rate. Co-crystallization is thought as a promising alternative over several other dissolution

supporting techniques involving amorphous solids, liposomes, micelles and salt crystals

(Aitipamula et al., 2012).

Co-crystals of itraconazole with succinic acid, malic acid and tartaric acid

achieved a 4-20 fold solubility increase in 0.1N HCl over the crystalline itraconazole

(Remenar et al., 2003). The dissolution rate of itraconzole/succinic acid co-crystal

prepared by gas anti solvent method was significantly enhanced and achieved a 90 %

release in 2 h than the pure drug which, regardless of present in micronized form, showed

less than 30 % release in 2 h (Ober and Gupta, 2012).

The dissolution profiles of co-crystal incorporating fenofibrate and nicotinamide

prepared by various methods were appeared to show more than 95 % release after 75 min

20

as compared to 50 % release of pure drug after 90 min. Co-crystal produced by anti

solvent addition method gave 90 % release in 45 min and this enhancement was

attributed to the prevention of transformation of pure drug (Shewale et al., 2015).

A 1:1 co-crystal of a poorly soluble development API 2-[4-(4-chloro-2-

fluorophenoxy) phenyl] pyrimidine-4-carboxamide with glutaric acid showed a

significant enhancement in maximum plasma concentration and area under the curve in

direct comparison with crystalline API at two different doses (5 and 50 mg/kg) in dogs.

Intrinsic dissolution experiments also showed the co-crystal 18 times more soluble than

the parent drug (McNamara et al., 2006). A 2:1 carbamazepine/succinic acid co-crystal,

which was stabilized by Polymers (HPMSAS, Soluplus and Kollidon VA 64), showed a 3

fold increase in IDR in phosphate buffer pH 6.8. When formulated into tablets and

compared with the commercial product Epitol, co-crystal based tablet formulations

resulted in significantly improved in-vitro and in-vivo performance. This improvement

was credited to the combined effect of the higher solubility of co-crystal and inhibition of

the crystallization of carbamazepine by polymers (Ullah et al., 2016). For 1:1 co-crystal

of AMG517 with sorbic acid, a 30 mg/kg dose of the co-crystal gave a comparable

exposure to 500 mg/kg dose of the free base in rats (Bak et al., 2008).

Co-crystallization may not bring a positive change in bioavailability in spite of

increase in the physiochemical properties, e.g., a 1:1 carbamazepine/saccharine co-crystal

was found suitable for addressing low solubility, stability and dissolution rate but when

compared with marketed immediate release carbamazepine tablets, pharmacokinetic

metrics were appeared comparable (Hickey et al., 2007).

21

Co-crystal formation does not necessarily enhance API solubility and dissolution

rate but in certain instances PCs have been shown to lower the solubility of the parent

drug, e.g., a sulfacetamide/caffeine co-crystal has demonstrated a lower solubility than

parent drug (Goud et al., 2014). This lower solubility of co-crystal, which is attributed to

stronger hydrogen bonds and a denser crystal packing in co-crystal, may possibly address

the poor retention and faster elimination of sulfacetamide from the eyes.

In agrochemicals, where low solubility is desirable to avoid rapid leaching of the

applied substance in runoff, decreased solubility of highly soluble active ingredient by

co-crystallization is of utmost concern (Aaker y et al., 200 ).

1.4.5.3. Physical and chemical stability

Physical and chemical stability of an API in the presence of atmospheric moisture

is of great importance in drug development. Various manufacturing techniques (such as

wet granulation, spray drying, aqueous film coating and crystallization), use of the

hydrated excipients and storage in a humid environment are prone to result into a hydrate

formation or a new polymorphic form (Khankari and Grant, 1995). This conversion can

adversely affect the physicochemical properties especially the bioavailability and can also

implicate the processing, formulation, packaging and shelf life of the API. Co-

crystallization is a suitable and feasible alternative to resolve the stability issues.

Caffeine and theophylline are well known hygroscopic drugs but their co-crystals

with oxalic acid were found to be non hygroscopic and stable at high relative humidity

for 7 days (Trask et al., 2005; Trask et al., 2006). A 1:1 co-crystal embodiment of

carbamazepine and saccharine reduced the incidence of polymorphism (Hickey et al.,

2007) compared to pure carbamazepine which has four known polymorphs. When

22

carbamazepine (Form III) and co-crystal were subjected to a physical stability study at 25

°C /60 % RH and 40

°C /75 % RH, both materials not only appeared to be physically

stable but also showed a comparable physical stability.

Co-crystals have also been explored for their ability to enhance the chemical and

photo stability of APIs. Chemical stability of adefovir dipivoxil in a sealed glass vial was

inferior to its co-crystal with saccharine and nicotinamide. The co-crystal did not show a

significant change in content during storage for 30 days at 60 °C whereas only 3.8 % of

the pure drug content remained on the 20th

day (Gao et al., 2012). On irradiation with

light, nitrofurantoin undergoes degradation. But a co-crystal of nitrofurantoin with 4-

hydroxy benzoic acid was disclosed to be more photostable than the parent drug when

exposed to a 315-400 nm UV lamp for 168 h (Vangala et al., 2011).

Regulatory views and patent portfolios 1.4.6.

The increasing use of co-crystals for drug product development has led to the

release of draft guidance by the United States Food and Drug Administration (USFDA,

2016) and European Medicines Agency (EMA, 2015) regarding the regulatory

classification of co-crystals. FDA classifies co-crystal analogous to new polymorph of

API while the reflection paper released by EMA has regarded co-crystals as new active

substances provided their safety and efficacy is demonstrated.

Currently, there are few marketed co-crystal products like Entresto (sacubitril-

valsartan co-crystal approved for the management of heart failure) (Fala, 2015), Lexapro

(escitalopram oxalate co-crystal approved for the management of anxiety disorders)

(Harrison et al., 2007), Suglat (ipragliflozin- L proline co-crystal) (Poole and Dungo,

2014), and Abilify (aripiprazole-fumaric acid co-crystal approved for the treatment of

23

schizophrenia) (Devarakonda et al., 2009). Some are under clinical investigation like

phase III clinical study of celecoxib-tramadol co-crystal in pain management (Almansa et

al., 2017).

The criteria for the patentability of PCs are novelty, utility and non obviousness.

Patent filing of co-crystals is associated with their distinct chemical composition,

supramolecular frameworks in crystal structure and evaluation of their pharmaceutical

and biopharmaceutical properties. As part of a comprehensive solid form patent portfolio,

PCs can offer a distinctive commercial advantage with respect to market exclusivity.

Methodologies for co-crystal synthesis and screening 1.5.

A number of variables including API/co-former ratio, types of co-former,

crystallization technique, solvents, pressure and temperature etc. direct the API to form a

co-crystal. Various traditional and advanced methods useful for achieving the targeted

quality of co-crystals have been summarized in Figure 1.7.

Solution based methods 1.5.1.

Traditional solution crystallization based approaches including solvent evaporation,

cooling, anti solvent addition, supercritical fluid crystallization and spray drying are

frequently used to prepare co-crystals and have a number of reasons for their popularity

(Blagden et al., 2007). Solution crystallization can yield large, well formed single crystals

which may help in evaluation of crystal habit and surface features. Single crystal

diffraction pattern is the best means to obtain an absolute crystal structure determination.

Further, solution crystallization offers a well known and effective purification step (Trask

and Jones, 2005). This technique, however, is associated with certain limitations

(Barikah, 2018; Trask and Jones, 2005) which have been summarized in Table 1.2.

24

Seeding is a valid way to improve the success rate of solution based co-crystallization.

Solid state grinding is one of the means to prepare seeds (Trask et al., 2005).

Figure 1.7: Methods for synthesis of co-crystals

1.5.1.1. Evaporation or cooling methods

Using these methods, a saturated solution is prepared with a stoichiometric ratio

of drug and co-former in a solvent (or a solvent mixture) in which both components have

comparable solubility. To predict the miscibility of a drug substance and a potential co-

former, the use of Hansen solubility parameters can be investigated (Mohammad et al.,

2011).

25

In slow evaporation methods, the solvent is allowed to evaporate slowly under

ambient conditions causing the solution to become supersaturated which induces

crystallization of possible co-crystals. Slow evaporation can also be performed at low

temperature in an attempt to increase product yield (Cains, 2009). Moreover a kinetically

controlled crystallization process involving rapid evaporation of the solvent from a

drug/co-former solution has been suggested as a rapid mean for the screening of novel

co-crystals.

In slow cooling methods, the saturated solution is stored at low temperature

higher than the freezing point of the solvent inducing the supersaturation followed by

crystallization of the co-crystal.

1.5.1.2. Anti solvent crystallization

In this technique, a second liquid miscible with the first solvent but in which co-

crystal is insoluble or sparingly soluble is added to a drug/co-former solution to achieve

supersaturation. Though, in many cases, co-crystallization can also be facilitated by

adding a co-former solution to drug solution. Synthesis of high quality co-crystals can be

achieved by anti solvent crystallization. The first study employing this method was

reported by Chun et al who synthesized indomethacin/saccharine co-crystal by adding

water to drug/co-former solution (Chun et al., 2013). The effect of five solvents with

different relative polarity, permittivity, water solubility, boiling point and vapour pressure

was explored on the co-crystal quality and methanol was found to result in best co-

crystal. Co-crystal synthesis was attributed to markedly reduced solubility of drug/co-

former on addition of anti solvent and to solution complexation due to the interaction of

drug/co-former in methanol (Chun et al., 2013).

26

1.5.1.3. Supercritical carbon dioxide (scCO2) assisted processes

Supercritical fluid technology may have a potential to enhance the rate of co-

crystal formation and is associated with certain advantages, e.g., a low environmental

impact, operative at low temperature, solvent free product and a single step generation of

microparticles for substances with thermal sensitivity or structural instability (Padrela et

al., 2010). The role of fluid changes from one method to another, e.g., scCO2 may act as

solvent (Müllers et al., 2015), anti solvent or spray enhancer (Cuadra et al., 2016).

1.5.1.4. Freeze and spray drying

Freeze drying has been suggested as a suitable method for large scale production

of co-crystals than other methods as this process has previously been used on industrial

scale. This method avoids problems caused by differences in the solubility of drug and

co-formers. In freeze drying, a solution of the compound is prepared, rapidly frozen and

then held under high vacuum causing the solvent to sublime and the solute is liable to

crystallization (Eddleston et al., 2013).

Formation of co-crystals by spray drying has been reported (Alhalaweh and

Velaga, 2010; Patil et al., 2014). Speed, a continuous one step process and an excellent

control on the process variables are the factors which facilitate the large scale

manufacture of co-crystals by spray drying. In this process, a feed is transformed from

liquid phase to a dried particulate form by allowing the feed to spray through a gaseous

drying medium under elevated temperature.

Solid based methods 1.5.2.

Solid state generation of PCs has received considerable importance due to the

additional advantages such as co-crystals preparation in the absence of solvents or by

27

using negligible amounts, excellent purity and quality, high throughputs and fast

processing times in some occasions. Solid based methods include solid state or

mechanochemical grinding, hot stage microscopy and hot melt extrusion.

1.5.2.1. Mechanochemical grinding

By this technique, co-crystals are formed via a mechanochemical reaction,

induced solely by mechanical energy ( aláţ et al., 2013). In this method, stoichiometric

ratios of components are agitated either manually (by mortar and pestle) or mechanically

(by Ball milling) without (neat grinding) or with few drops of solvent (solvent drop or

liquid assisted grinding). Literature has described mechanochemical technique as an

efficient method to achieve the highest efficiency of co-crystal screening and to provide

improved results compared to solution based crystallization (Karki et al., 2009).

Moreover, the fact that experimental design is free from solubility considerations,

increased interest in solid state grinding has increased (Friščić et al., 200 ). Finally, while

the exact mechanism is not fully understood, there are cases in which certain structures or

stoichiometry of co-crystals unobtainable from solution based experiments can be

generated by mechanochemical grinding. One such example is synthesis of 1:1.5 Bis-β-

napthol/benzoquinone co-crystal (Imai et al., 2002) which could not be produced through

solution methods. A comparison of the mechanochemical grinding with solution based

methods in terms of advantages and disadvantages has been given in Table 1.2.

28

Table 1.2: Comparison of the mechanochemical grinding and solution based methods

There are many reported cases when the energy needed to complete the co-

crystallization of substances is insufficient with neat grinding due to absence of heating

Methodology Advantages Limitations

Solution based

methods

Large well formed single

crystals

Effective purification

method

A suitable solvent in which

starting materials must have

comparable solubility

Large volume of the solvent

required

Undesired solvate formation

Slow processing time

For polymorphic co-crystal

system, formation of a

kinetically controlled

metastable polymorph

Mechanochemical

grinding

Environmental friendly due

to absence or least amount

of solvent

Short reaction time

Effective method for

screening of APIs with low

solubility

Co-crystals not generated by

solution based methods can

be obtained by solid state

grinding

Temperature independent

Avoidance of undesired

solvate formation

Microcrystalline sample

unsuitable for single crystal

XRD

Less purification process

Increased crystalline

disorders leading to changes

in physicochemical

properties

29

stage in the process (Ross et al., 2016). This problem can be overcome by introduction of

a small amount of a solvent during grinding thus facilitating the process in a process

known as liquid assisted grinding (LAG). The liquid phase, assisting the LAG, acts

purely as a catalyst but is depleted during the course of co-crystallization (Braga et al.,

2007).

LAG has been shown to be superior to neat grinding and other solvent free

methods, e.g., five new co-crystals of piroxicam with different carboxylic acid were

generated through LAG after failure of the preceding attempts with solvent free methods

(Childs and Hardcastle, 2007). Their formation was contributed to the inclusion of few

drops of solvent improving the diffusion rate of components at interfaces, as well as

enhancing the opportunity for molecular collision.

1.5.2.2. Hot melt extrusion (HME)

High quality co-crystals can be synthesized employing hot melt extrusion (HME).

In this process, raw materials are pumped with a rotating screw at elevated temperature

through a die ensuring a product of uniform shape. HME was first used to produce

caffeine/AMG517 co-crystals (Medina et al., 2010). Extrusion offers shear, close material

packing and high intensive mixing improving surface contact between drug/co-former

blends and resulting in the formation of co-crystals without use of any solvents. In a

study the effect of processing parameters including screw configuration, screw speed and

barrel temperature were determined on extruded co-crystal agglomerates (Dhumal et al.,

2010). A change in the screw configuration was appeared to improve the mixing

intensity. Likewise, extended residence times and processing temperatures above the

eutectic point resulted in high quality ibuprofen/nicotinamide co-crystals.

30

1.5.2.3. Thermomicroscopy

This technique is accompanied by advantages like rapidly obtainable results,

avoidance of formation of undesired product and environmental friendly method.

However, its use is limited to co-formers with comparable melting points, to avoid

molecular decomposition of one co-former before the other has melted.

Thermomicroscopy is based on contact or mixed fusion approach (Kofler and

Kofler, 1952; McCrone, 1957) involving heating of the higher melting component and

allowing it to solidify. Second component is then heated between the same microscope

slides consequently dissolving a part of the first co-former at the regions where they

meet. This region is known as zone of mixing. The system is then cooled down to

ambient temperature and subsequently heated again until all the components melt and

monitored with a microscope with crossed polarizing filters. If co-crystallization occurs,

a distinct new morphology with a melting point different to the starting components

would be recognized within the zone of mixing. A 1:1 nembutal/phenobarbital co-crystal

has been reported by this method (Rossi et al., 2012). Contact preparation has also been

used as a co-crystal screening scheme, e.g., a physical screen employing various APIs

resulted in the isolation of three novel co-crystals of nicotinamide with salicylic acid and

racemic and S-ibuprofen (Berry et al., 2008).

Characterization of co-crystal 1.6.

Under certain circumstances, co-crystal formation is evident from the physical

properties of resultant product. For example paracetamol (PCM) and 2, 4-pyridine

dicarboxylic acids are white solids but the co-crystal screening by solution mediated

phase transformation resulted in a red colored product. The red color was observed due to

31

the conversion of co-former to the zwitterion form in the co-crystal as part of the overall

hydrogen bonded crystal packing arrangement (Sander et al., 2010). However, in

majority of the reported cases, co-crystals have been investigated by various

characterization tools as specified in Figure 1.8.

Figure 1.8: Solid state characterization tools for co-crystals

A detailed description of the characterization techniques, including PXRD, DSC,

FTIR, SEM and Hirshfeld analysis, used in the present work has been given in Chapter 2.

Other techniques are briefly described here.

Single-crystal X ray diffraction 1.6.1.

X-rays, having the ideal wavelength for reflecting off of atoms, can be directed at

a crystal causing scattering of the radiation in a certain way called a diffraction pattern.

32

For single crystal and powder techniques, following diffraction condition must be

satisfied as given by ragg‘s law (Equation 1.1)

Eq 1.1

Where

λ= wavelength of radiation, d= lattice spacing of parallel planes in a crystal and

θ= the diffraction angle

Single-crystal XRD gives an insight into the crystal lattice, stoichiometric ratio

and intermolecular bonding of the molecular structures. This technique alone is sufficient

to declare formation of co-crystal. It finds particular use in validating crystal engineering

strategies based on supramolecular synthons (Desiraju, 1995; Vangala et al., 2016).

In this technique, crystals of a suitable size and quality are exposed to a beam of

monochromatic X-ray radiation with a wavelength comparable to the interatomic

distances. As a result of interaction with the electrons in the crystal, radiation is diffracted

and recorded by an X-ray detector. The diffracted data is collected in the form of a series

of images for different crystal orientations and these are analyzed to solve 3D structure of

the crystal.

Thermogravimetric analysis (TGA) 1.6.2.

Thermogravimetric analysis (TGA) is a tool used to study the thermal behavior of

materials, facilitating the solvate/hydrate characterization as well. In this technique, the

mass change of a sample is measured as temperature is altered at a constant rate. TGA is

an excellent mean for determining the onset temperature of co-crystal decomposition and

loss of a volatile component. In case of volatile co-formers, stoichiometry can be

confirmed by quantifying the mass loss (Steed, 2013).

33

Hot stage microscopy (HSM) 1.6.3.

Hot stage microscopy (HSM) is based on direct optical observation of the crystal

or co-crystal as a function of temperature by using a polarizing lens (Chadha and

Bhandari, 2014). HSM is sensitive to solid phase transitions such as melting,

recrystallization, and dehydration and desolvation events. A description of the HSM has

already been given under thermomicroscopy Section 1.5.2.3.

Spectroscopic techniques 1.6.4.

Raman spectroscopy is a fast investigation tool to identify solid forms in drug

substances and drug products (Sheng et al., 2016). In this technique, an unprocessed

sample contained in stainless steel or glass holder interfaces with the laser beam and

Raman scattering is directly related to the concentration of the scattering material,

making it a significant technique for qualitative and quantitative analyses (Zakharov et

al., 2012). This technique becomes ineffective if the concentration of sample under

analysis is below 1% of the bulk material where FTIR finds considerable importance.

An overview of the PCs along with the methods of preparation, characterization

techniques and ultimate outcomes in terms of pharmaceutical properties has been given in

Table 1.3.

34

Table 1.3: Literature review on pharmaceutical co-crystals

Reference Co-crystal Method of co-

crystallization

Techniques used for

characterization

Outcomes

(Gadade et al., 2017) Lornoxicam: Saccharin

sodium *(1:1)

Neat grinding PXRD, DSC, FTIR, SEM Increase in solubility

and in-vitro dissolution

rate

(Du et al., 2017) Lamotrigine: 4,4-

bipyridine (2:1)

Lamotrigine: 2,2-

bipyridine (1:1.5)

Neat grinding and LAG PXRD, DSC, TGA.FTIR Improvement in

solubility and

dissolution rate

(Zhou et al., 2016) Resveratrol: 4-

aminobenzamide (1:1)

Resveratrol : Isoniazid

(1:1)

Liquid assisted grinding and

Rapid solvent removal

PXRD, DSC, FTIR, NMR Solubility and

tabletability

improvement

(Li and Matzger,

2016)

Carbamazepine: 4-

amino benzoic acid

(2:1)

solvent evaporation PXRD, Raman and proton

NMR spectroscopy

solubility and stability

was not found to be

improved

(Bagde et al., 2016) Darunavir: Succinic

acid

Cooling crystallization PXRD, DSC, FTIR, SEM Improvement in

micromeritic properties

and dissolution rate

(Liu et al., 2016) Myricetin: Proline Solution crystallization PXRD, DSC, SEM Improvement in

dissolution and oral

bioavailability

(Hiendrawan et al.,

2016)

Paracetamol: 5-

nitroisophthalic acid

Solvent evaporation PXRD, DSC, TGA,FTIR,

SEM, Hot stage microscopy

Improvement in

stability and

mechanical properties

(Savjani and Pathak,

2016)

Acyclovir: Tartaric acid Solvent evaporation, wet

grinding, antisolvent addition

PXRD, DSC, FTIR Improvement in

dissolution rate

* represents the stoichiometry of the co-crystal

35

Table 1.3: Continued…


crystallization

Techniques used for

characterization

Outcomes

(Hoxha et al., 2015) Cytocine: 1,10

phenanthroline (1:1)

Ball milling and solvent

evaporation

Single crystal XRD,

PXRD, FTIR

Pharmaceutical

properties not

evaluated

(Évora et al., 2014) Diflunisal:

Nicotinamide (2:1)

Liquid assisted ball mill

grinding and solution

crystallization

DSC, FTIR, PXRD Improvement in IDR

by about 20%

(Soares and Carneiro,

2014)

Ibuprofen:

Nicotinamide (1:1)

Slow evaporation TGA, DSC, ATR-FTIR,

PXRD

Improvement in

solubility

(Maeno et al., 2014) Paracetamol:

Trimethylglycine (1:1)

Solution precipitation and

solid state grinding

PXRD, IR, DSC Improvement in

dissolution rate and

compressional

properties

(Nijhawan et al.,

2014)

Lornoxicam: Catechol

Lornoxicam:Resorcinol

Liquid assisted grinding Melting point, PXRD,

DSC, FTIR,

Improvement in

physicochemical

characteristics

(Pathak et al., 2013) Paracetamol:

Indomethacin

Paracetamol:

Mefenamic acid

Solvent evaporation and

Liquid assisted grinding

DSC, PXRD and

microscopic evaluation

Pharmaceutical

properties not

evaluated

(Chow et al., 2012) Ibuprofen :

Nicotinamide (1:1)

Flurbirofen :

Nicotinamide (1:1)

Rapid solvent removal by

rotary evaporation

PXRD, DSC, TGA, FTIR Improvement in

mechanical properties,

hygroscopicity and

dissolution

performance

(Ando et al., 2012) Naproxen:

Nicotinamide (2:1)

Liquid assisted grinding Solid state NMR, Single-

crystal XRD

Improvement in IDR

(Grossjohann et al.,

2012)

Benzamide:

Dibenzyloxide (1:1)

Solution precipitation and

solid state grinding

PXRD, ATR-FTIR, DSC Improvement in

solubility and IDR

36

Table 1.3: Continued…


crystallization

Techniques used for

characterization

Outcomes

(Elbagerma et al.,

2011)

Paracetamol: Citric

acid (2:1)

Slow evaporation PXRD, DSC, Raman

Spectroscopy

Improvement in

compressional

properties and

dissolution rate

(Cheney et al., 2011) Meloxicam: Aspirin

(1:1)

Solution phase

crystallization,slurring and

solvent assisted grinding

FTIR, SEM, DSC Improvement in

physicochemical and

pharmacokinetic

properties

(Chandel et al., 2011) Paracetamolc:

Aceclofenac

Solution crystallization and

LAG

PXRD, DSC Increase in solubility

of both drugs

(Vangala et al., 2011) Nitrofurantoin: 4-

hydroxy benzoic acid

(1:1)

Neat and solvent drop

grinding

PXRD, DSC, TGA. IR,

Raman

Improvement in

photostability

(Sun and Hou, 2008) Caffeine: Methyl

gallate (1:1)

Slow evaporation PXRD, DSC, TGA, Particle

size analysis

Improvement in

compaction properties

(Basavoju et al., 2008) Indomethcin: Saccharin

(1:1)

Slow evaporation PXRD, DSC, Raman

Spectroscopy

Improvement in

solubility and

dissolution rate

37

Problem statement 1.7.

High hydrophobicity, low aqueous solubility and dissolution rate, and poor

processing properties are common characteristics of the newly synthesized and

discovered chemical entities and ultimately marketed drugs. There are number of

strategies to address these issues, among them co-crystallization, a crystal engineering

technique is a relatively new and effective method. Depending on the selection of co-

former, co-crystals may be designed to address physicochemical and a number of other

pharmaceutical problems including processing properties, bulk properties, granulation

and compression behavior in the manufacturing of a dosage form. However, majority of

the studies on pharmaceutical co-crystals have focused on the determination of single

crystal structure and intrinsic dissolution rate. Bioavailability studies on co-crystals and

the use of this solid form in a drug product are rare. Paracetamol and naproxen have

dissolution limited absorption and poor compressional behavior due to low plasticity.

Lack of plastic deformation presents a challenge to formulate them in to a tablet by direct

compression. Hence the present study was designed to synthesize co-crystals of

paracetamol and naproxen to improve compressional and formulation behavior and in-

vivo oral bioavailability. To our knowledge this is the first bioavailability study

performed on the paracetamol and naproxen co-crystals.

38

Aim and objectives 1.8.

The overall aim of this study was the synthesis, screening and characterization of

co-crystals of model APIs Paracetamol and Naproxen using various ratios of drugs and

co-formers (caffeine and nicotinamide) to produce co-crystals with desired

physicochemical, mechanical and in-vivo performance.

The objectives of this work were

Physical co-crystal screening of paracetamol with caffeine by employing a

number of methods namely dry grinding, liquid assisted grinding, solvent

evaporation and anti solvent addition; and synthesis of naproxen-nicotinamide co-

crystal by liquid assisted grinding method.

Characterization of synthesized co-crystals, APIs and co-formers by solid state

investigation tools, e.g., PXRD, DSC, FTIR and intrinsic dissolution rate.

Investigation of properties related to processing of materials to determine the

potential improvements of the co-crystals.

Mechanical evaluation of the co-crystals, APIs and co-formers by Heckel model

and tabletability curves.

Formulation development based on co-crystals.

Pre and post formulation studies for synthesized co-crystals.

Study of in-vitro dissolution profiles of the formulated co-crystals in comparison

to physical mixtures and reference formulations.

Pharmacokinetic and bioavailability studies of the synthesized co-crystals in

comparison to the pure APIs.

39

Chapter 2

40

2. Materials and methods

Materials 2.1.

Solids 2.1.1.

Paracetamol (PCM) form I (monoclinic) and caffeine (CAF) were obtained as gift

samples from UNEXO Pharmaceuticals and Avicel PH 102, croscarmelose sodium and

magnesium stearate from Remington Pharmaceuticals, Lahore, Pakistan and were used as

received. Naproxen (NAP) and nicotinamide (NIC) were obtained from Sigma-Aldrich,

Germany and BDH, UK, respectively. Sodium hydroxide (NaOH), potassium dihydrogen

phosphate (KH2PO4), sodium dihydrogen phosphate (NaH2PO4) and anhydrous disodium

hydrogen phosphate (Na2HPO4) were of analytical grade.

Solvents 2.1.2.

Methanol, ethanol, acetone, and acetonitrile were used in the co-crystal screening

and were of analytical grade. Methanol, acetonitrile and trifluoroacetic acid used for the

mobile phase preparation were purchased from the Sigma Aldrich and were of HPLC

grade. Perchloric acid, phosphoric acid, ethyl acetate and hexane used for the extraction

of drugs were of HPLC grade and were obtained from the Sigma Aldrich.

Preparation of phosphate buffer pH 6.8 2.1.3.

Phosphate buffer pH 6.8 was prepared by mixing 250 ml of KH2PO4 (0.2 M) with

112 ml of NaOH (0.2 M) and diluting to 1000 ml with distilled water.

Preparation of phosphate buffer pH 7.4 2.1.4.

Phosphate buffer pH 7.4 was prepared by dissolving NaH2PO4 (2.62 g) and

Na2HPO4 (11.50 g) in quantity sufficient (q.s) of distilled water to give a final dilution of

1000 ml.

41

Preparation of 0.1M HCl pH 1.2 2.1.5.

0.1 M HCl pH 1.2 was prepared by diluting 8.3 ml of 37 % HCl (12 M) with q.s

of distilled water to give a final volume of 1000 ml.

Methods 2.2.

A general description of the methods including solvent evaporation (SE), anti

solvent addition (ASA) and mechanochemical grinding (dry grinding and LAG) used for

the screening and synthesis of co-crystals of PCM and NAP has been given in Chapter 1

Sections 1.5.1.1, 1.5.1.2 and 1.5.2.1. Specific conditions, e.g., drug/co-former ratio and

solvents have been given in the respective Chapters. Characteristic methods for the

investigation of co-crystals of both drugs along with the experimental conditions are

being described here.

Powder X-ray diffraction (XRD) 2.2.1.

While XRD is the most valuable, it is not always possible to obtain single

crystals. PXRD is regarded as the most frequently used method to characterize the co-

crystals in situations where single crystals are not attainable, e.g., when co-crystals are

generated by mechanochemical grinding. Co-crystal formation will change the crystal

lattice with a subsequent powder pattern that is distinctive from the starting materials.

Under favorable circumstances, PXRD is likely to give the full 3D crystal

structure from powder data with a precision not less than single crystal method (Desiraju,

1995). Using this technique, a rotated powdered sample is allowed to expose to a

monochromatic beam of X-ray radiation over a range of angles and the substantial

diffracted X-rays are detected in terms of intensity. A vacuum tube, in which a large

voltage is applied, is used to generate X-rays with a resulting beam of electrons from the

42

cathode. These electrons impinge on an anode (usually a metal plate of copper or

molybdenum) resulting in production of X-rays in the surface layers of the anode. A

powder pattern is obtained on processing the diffracted data.

In this study, PXRD was used to detect for new peaks during co-crystal screen

and for the identification of materials. Following parameters were used as the standard

method for collecting PXRD patterns during this work.

2.2.1.1. Experimental PXRD method

PXRD data were collected on a Pan Analytical diffractometer XPERT-PRO

(operating at 40 kV, 40 mA), using Cu-Kα radiation (λ = 1.541 Å) with a position

sensitive detector. Diffraction data were collected in the range 2θ = 5- 40 °

with a step size

of 0.02 °. Powder samples were used as such without any pretreatment.

Thermal characterization 2.2.2.

Materials can be thermally analyzed by differential scanning calorimetry (DSC).

By this technique, difference in the heat flow rate of a test and a reference sample is

measured by subjecting both samples to a controlled temperature (Höhne et al., 2003).

Measurement of the heat capacity of the materials, detection of the temperature at which

phase transitions (e.g., polymorphic transitions and melting) occur as well as

quantification of these phenomena are potential applications of DSC (Steed, 2013).

With respect to solid state characterization of co-crystals, thermal analysis plays a

critical role. An endothermic peak, above or below the melting temperature of the drug

and co-former can be detected in the thermograms of co-crystals. Following parameters

were used as the standard method for collecting DSC thermograms during this work.

43

2.2.2.1. Experimental DSC method

DSC thermograms were recorded using a TA instrument (model Q 600, USA).

With a heating rate of 10 °C /min. Samples (2-5 mg) contained in standard aluminium

pans were evaluated from room temperature (25±2 °C) to 300

°C under a nitrogen purge

(100 ml/min). The temperature axis and the cell constant of DSC were previously

calibrated with Indium.

Infrared (IR) spectroscopy 2.2.3.

Infrared (IR) spectroscopy, also known as vibrational spectroscopy, provides the

fingerprint of specific crystalline materials. IR spectroscopy measures absorption of IR

radiation (falling within the region of electromagnetic spectrum having wavelength

between 4000-400 cm-1

) at specific wavelength as a result of vibrational transitions in the

molecules (Mukherjee et al., 2013). Vibrational modes present in the molecules greatly

affect the frequency at which absorption takes place and are responsible for peaks

characteristic of specific functional groups (Porter Iii et al., 2008). IR spectra can be

recorded using an attenuated total internal reflectance (ATR) unit or a KBr pellet.

IR spectroscopy can be a useful and simple method in detecting co-crystal

formation when there is a significant shift in the bands related with vibration of hydrogen

bond donors and acceptors intricate in the intermolecular attachment that is prone to

stabilize and synthesize co-crystals. But less significant differences are found between IR

spectra of co-crystals and pure components, when molecule‘s solid state environment

slightly disturbs bond vibrational modes. Following parameters were used as the standard

method for collecting FTIR spectra during this work.

44

2.2.3.1. Experimental FTIR method

IR spectra were recorded using FTIR (Fourier transform infrared)

spectrophotometer (Alpha-P Bruker, Germany) equipped with an ATR unit across the

range of 4000-400 cm-1

. Samples were analyzed in transmittance mode based on 10 scans

and a resolution of 2 cm-1

.

Scanning electron microscopy (SEM) 2.2.4.

Scanning electron microscopy (SEM) is useful in determining crystal

habit/morphology. In this technique, a beam of high energy electrons is used to produce a

range of signals at the solid surface. Signals generated as a result of interactions between

electron and solid sample disclose the crystalline structure, external morphology and

sample composition etc. (Rajurkar et al., 2015; Sevukarajan et al., 2011).

2.2.4.1. Experimental SEM method

Powder samples were photographed by ZEISS EVO HD 15 scanning electron

microscope (Carl Zeiss, NTS Ltd. Cambridge, UK) with auto imaging system. The

sample was mounted on carbon adhesive tape fixed on aluminum stubs (Agar Scientific

Ltd., Stansted, UK), flushed with air and photographed at the voltage of 2 KV.

Intrinsic dissolution 2.2.5.

Intrinsic dissolution rate (IDR) is the rate of mass transfer per area of dissolving

surface. It is a valuable parameter to measure being independent of many factors directly

affecting the standard dissolution rate. By measuring IDR the effect of varying exposed

surface area, unavoidable due to disintegration in traditional dosage forms, can be

nullified (Giorgetti et al., 2014).

45

In the present work, IDR was determined by static disc method in the acidic and

basic buffers by compressing 300mg of the pure drug, physical mixture and co-crystal to

produce a constant surface area disc (13mm) using a hydraulic press (Carver, USA).

After allowing the disc to rotate at a fixed speed in the USP paddle apparatus, samples

were taken from the dissolution media, by observing the sink conditions, at

predetermined time intervals and analyzed by UV/Visible spectrophotometer to monitor

the release of the drug. The IDR was found from the slope of the curve obtained by

plotting the cumulative amount of the drug dissolved (mg/cm2) versus time (min).

UV spectrophotometry 2.2.6.

UV spectrometry is based on measuring absorbance/transmittance of UV

radiation by a sample at specific wavelengths due to electronic transitions in the

molecules, while UV radiation fall within the region of electromagnetic spectrum with

wavelengths between 200 and 400nm (Christian and O'Reilly, 1988). The solvents used

for making solutions must be transparent within the wavelength to be examined.

UV spectrometry is based on Beer-Lambert law and finds its large application in

quantitative analysis to determine the concentration (c) of an absorbing species in a

solution according to the equation 2.1

Eq. 2.1

Where

A= absorbance of the solution at the wavelength at which maximum absorption

occurs, ɛ= absorptivity or extinction coefficient and l= path length through the sample

solution

46

Throughout this work a double beam UV/Visible spectrophotometer (U-2800

BMS, UK) was used to measure the absorbance of the solutions using quartz cells of 1cm

path length.

High performance liquid chromatography (HPLC) 2.2.7.

Liquid chromatography uses a mechanical pump to deliver the mobile phase at a

constant flow rate through a column packed with stationary phase, mostly chemically

modified silica. A large range of solvents and their mixtures can be used as mobile phase.

HPLC is performed by achieving a constant flow of mobile phase prior to

injecting sample under investigation. The sample injected into the mobile phase passes

through the column and sample components will be separated according to their

differential affinity between the stationary phase and mobile phase defining their

retention times in the column. After elution from the column, sample components will be

presented to the detector for analysis. According to the chemical nature of the sample,

different types of detectors can be used including UV/Visible spectrophotometers,

fluorometers, mass spectrometers, electrochemical and photodiode array (PDA)

detectors.

In the present work, the HPLC system used (Shimadzu 20A, Japan) was provided

with a LC-20AT VP pump, a SIL-20AC HT auto sampler, CTO 20 AC column oven,

SPD-M20A detector (PDA), CBM 20A controller unit and a reversed phase symmetry

C18 (4.6 x 250 mm, 5-μm particle-size) column.

Description of the solvent system and detection wavelength has been given in the

relevant Chapters.

47

Hirshfeld surface analysis 2.2.8.

Hirshfeld surface technique has been regarded as a method to define molecules in

molecular crystals and is based on calculation of molecular surfaces. The Hirshfeld

surface was developed in an attempt to divide the crystal electron density into molecular

fragments (Spackman and Byrom, 1997).

2.2.8.1. Features to be mapped on a Hirshfeld surface

Curvedness and shape index are some of the properties that could be mapped on

Hirshfeld surface. The curvedness is a function of curvature of the surface where flat

areas of the surface and areas with sharp curvature are depiction of a low curvedness and

high curvedness, respectively. Shape index is a determinant of the shape of the surface

where concave regions are negative and convex regions are positive. It measures the

shape qualitatively and is proportional to variations in shape of the surface mainly the

areas with very low total curvature.

a) de and di

de and di represent the distance from the Hirshfeld surface to the nearest nucleus

outside and inside the surface respectively (Figure 2.1). Generally only de is recorded

because this feature can describe the crystal packing environment about the surface.

Figure 2.1: Representative figure to depict de and di of Hirshfeld surface

de

di

48

b) Fingerprint plots

Different crystal structures can be compared by fingerprint plots (2D) that result

from the respective Hirshfeld surfaces. The fingerprint plot provides a mean for

summarizing the features of a molecular crystal into a single distinctive coloured plot,

providing a characteristic pattern of the intermolecular interactions.

It is required by Hirshfeld surface analysis to determine both of de and di for every

single point. Every point and its colour on the 2D fingerprint plot links to a distinctive

(de, di) pair and the relative area of the surface with that pair respectively. Areas having

no impact on the surface remain uncoloured whereas points with an impact on the surface

are coded with blue colour showing a small contribution through green to red for areas

representing the maximum contribution (Figure 2.2). Equivalent relative scale is used to

give colour codes to fingerprint plots, so red points are not present for some of the

fingerprint plots. These plots are also useful for distinguishing between different densities

of polymorphic compounds, since values at large de and di correspond to those areas on

Hirshfeld surface that are not closely contacted to adjacent molecules while the

development of small cavities occurs as a result of close packing of surfaces.

Hirshfeld surfaces and respective fingerprint plots find considerable significance,

owing to their uniqueness, to explicate and compare crystal structures especially the

polymorphs, and also to recognize the common features and trends in the specific class of

compounds.

In the present work, Hirshfeld surfaces and fingerprint plots were generated by

the pragramme Crystal Explorer (Wolff et al., 2013) and de was mapped between 0.8 (

red) and 2.2 (blue).

49

Figure 2.2: Hirshfeld surfaces and finger print plot with front (top) and back (bottom)

view. The molecule is shown with the Hirshfeld surface mapped with (a) Curvedness, (b)

shape index and (c) de

a b c

50

Chapter 3

51

3. Development of Paracetamol-Caffeine co-crystals to improve

compressional, formulation and in-vivo performance

Background 3.1.

Paracetamol (PCM) is one of the examples of pharmaceutical which has been

studied by various co-crystallization techniques. It is a widely used analgesic and

antipyretic agent and has low dissolution rate. Its molecular structure is shown in Figure

3.1. Under ambient conditions, PCM exists in the following polymorphic forms;

thermodynamically stable form I (monoclinic) with poor compression moldability,

metastable form II (orthorhombic) with superior compaction and form III (André et al.,

2012; Thomas et al., 2011) the crystal structure of which has recently been solved (Perrin

et al., 2009). Though form II is, therefore, a preferred choice from an industrial tableting

perspective, however its lower thermodynamic stability precludes its commercial use. As

a result, marketed PCM tablets are a compromise involving form I but the literature has

documented the problem of capping due to its stiff nature and a relatively high elastic

deformation (Di Martino et al., 1996). Thus, PCM tablets are being prepared through wet

granulation (WG) process incorporating a large amount of binding agents intended to

prevent chipping and disintegration (Fachaux et al., 1995).

Development of thermodynamically stable and pharmaceutically acceptable form

of PCM having properties similar to form II is, consequently, a big challenge to

pharmaceutical industry. Lack of acidic or basic functional groups, preventing the

synthesis of salt as alternative solid form, make this challenge more difficult. Rational

design of powder particles and crystal engineering can offer viable potentials to address

this challenge (York, 1992). A very few studies have been reported concerning PCM

52

crystal aggregates by recrystallization or agglomeration with resultant improvements in

the compaction (Ettabia et al., 1997; Fachaux et al., 1995) but with a significant crystal

damage as well. Alternatively, co-crystallization is likely to be emerged as a feasible

practice to improve the mechanical behavior of PCM (Karki et al., 2009). Nevertheless

absence of ionizable groups (Trask et al., 2006) and the presence of both proton donors

(phenolic O-H and the amidic N-H groups) and acceptor (the amidic oxygen atom) make

PCM a good model drug for co-crystallization.

Caffeine (CAF), classified as a GRAS substance, has been shown to be an

effective co-former with many literature examples (Patil et al., 2018). It has functional

groups (keto-amide groups and basic nitrogen) which make it a suitable candidate to form

intermolecular hydrogen bonds in co-crystal formation (Figure 3.1) (Sun and Hou, 2008).

Moreover a ΔpKa of 0.9 between PCM (pKa=9.5) and CAF (pKa=10.4) may also dictate

the reactivity potential leading to co-crystal formation.

I II

Figure 3.1: Molecular structures of Paracetamol (I) and Caffeine (II)

Formerly, a number of co-formers including citric acid, trimethyl glycine, oxalic

acid, phenazine, naphthalene and theophylline etc. have been used for co-crystallization

of PCM and the focus of the majority of the reported data was the crystal structure

analyses and solid state characterization. In the present work, PCM has been co-

53

crystallized with CAF to get a more deep insight into the dissolution rate, promotion of

plastic deformation facilitating tablet formation by direct compression and in-vivo

bioavailability. To our knowledge, this is the first bioavailability study performed on the

PCM co-crystal and as a result of co-crystallization with CAF, PCM absorption and

disposition was greatly modified.

Materials and Methods 3.2.

A description of materials and methods has been given in chapter 2 Section 2.1.

Screening of co-crystals 3.3.

Dry and liquid assisted grinding (LAG) 3.3.1.

PCM and CAF in three different weight ratios (1:1, 1:2 and 2:1) were co ground

in a mortar and pestle without and with small amount of liquid (~50 µL for each of 100

mg of PCM and CAF) for 30 min. Solvents used were V/V mixture of methanol and

ethanol (1:1 and 1:2) and pure acetone. The resulting powders were collected in tight

containers and stored at room temperature.

Solvent evaporation (SE) method 3.3.2.

PCM and CAF in three different weight ratios (1:1, 1:2 and 2:1) were dissolved in

a V/V mixture of methanol and ethanol (1:1 and 1:2), mixture of distilled water and

ethanol (1:1 and 1:2) and pure acetone with aid of slight heating until a clear solution was

obtained. Each of these solutions was filtered and left for evaporation at room

temperature. The solid crystals obtained were collected in tight containers for further

analyses and stored at room temperature.

54

Anti solvent addition (ASA) 3.3.3.

Excess amounts of PCM and CAF (1:1, 1:2 and 2:1 weight ratio) were added to 5

ml of methanol and acetone separately to make turbid solutions, stirred for 15 min and

filtered through Whatman filter paper. Distilled water (2 ml) used as an anti solvent

(Lonare and Patel, 2013) was added to the filtrate to induce precipitation. Samples were

then centrifuged at 3000 rpm for 20 min to allow solids to deposit out.

Characterization 3.4.

The pure drug, co-former and co-crystals prepared by the above methods were

investigated by PXRD, DSC, ATR-FTIR and SEM. PXRD patterns, DSC thermograms,

ATR-FTIR spectra and SEM photographs were recorded for PCM, CAF and prepared co-

crystals using instruments as described in Chapter 2 under experimental conditions

specified in Sections 2.2.1.1, 2.2.2.1, 2.2.3.1 and 2.2.4.1.

Intrinsic dissolution studies 3.5.

Standard curve of PCM 3.5.1.

A stock solution containing 1 mg/ml of pure drug was prepared by dissolving 100

mg of PCM in 100 ml of phosphate buffer pH 6.8. The standard stock solution was

further diluted to obtain a working standard solution of 100 µg/ml. Working standard

solution was then diluted with phosphate buffer pH 6.8 to obtain a series of solutions with

the concentration range of 2-16 µg/ml. A calibration curve for PCM was drawn by

measuring the absorbance of each dilution at the λmax of 243 nm. Statistical parameters

like slope, intercept and coefficient of correlation were determined to model the linearity

of absorbance data.

55

Intrinsic dissolution rate (IDR) 3.5.2.

Intrinsic dissolution rate (IDR) of pure drug, physical mixtures (PMs) and co-

crystals was determined by static disc method using a constant surface area of 13 mm in

diameter. Discs were prepared by compressing 300 mg of powder samples by a hydraulic

press (Carver, USA) at a pressure of ~5000 psi for 60-90 sec. The compacts were coated

with hard paraffin leaving a surface available for dissolution (Healy et al., 2002;

Nicklasson et al., 1981) and placed to the base of the dissolution vessel.

The dissolution studies, performed in triplicate, were carried out in 500 ml of

phosphate buffer pH 6.8 maintained at 37 °C in USP Apparatus II with paddles rotating at

50 rpm. Aliquots (5 ml) were withdrawn at time intervals of 2, 4, 6, 8, 10, 14, 18 and 22

min. Samples were filtered through 0.45 μm filter and analyzed at 243 nm. Dissolution

medium was replenished with the same volume (5 ml) of blank (phosphate buffer pH 6.8)

in order to maintain the sink conditions. IDR was determined by plotting the cumulative

amount of drug dissolved per surface area (mg/cm2) versus time (min).

Drug content analysis 3.6.

For drug content analysis, 10 mg of co-crystal sample was weighed accurately and

dissolved in phosphate buffer pH 6.8 using volumetric flask to give a solution of 10

µg/ml concentration. Drug content of co-crystal was estimated from the standard curve.

Compressional behavior 3.7.

Compressional behavior of the pure PCM and synthesized co-crystals was studied

by Heckel model. Heckel equation is the most widely used and accepted compaction

equation (Heckel, 1961) because of ease and simplicity offered by it to differentiate the

plastic and brittle materials (Hooper et al., 2016). To ensure adequate tensile strength of a

56

formulation, it is critical to have the appropriate balance between plastic and brittle

substances.

The model given by Heckel (density pressure relationship) is illustrated by

equation 3.1

( )

Plastic deformation is described by integration of equation 3.1

( )

( )

( )

Where

D= relative density of the compact during compression at applied pressure P,

ɛ= porosity and K and A= Heckle constants

―A‖ describes die filling and particle rearrangement whereas the 1/ K is a measure

of Py, i.e., the mean yield pressure. Py, a determinant of the plasticity, is the stress

indicating the onset of plastic deformation of a particle, where plastically deforming

materials are characterized by low Py values (< 80) and high Py values (> 80) are

representative of brittle materials (Göran Alderborn and Nystrom, 1996).

The relative density, DA, can be calculated from the intercept, A, according to the

equation 3.4 and represents total precompression density at zero and low pressures

(Adedokun and Itiola, 2013)

57

Plastic deformation measurement 3.7.1.

In the present study, in-die Heckel model for powder deformation under pressure

(Equation 3.2) was used to evaluate the compressional behavior of pure PCM and

prepared co-crystals. Each of the powder material (300 mg) was individually compacted

at six different compressional pressures between 2 and 14 MPa through a hydraulic press

equipped with flat faced die 13 mm in diameter. Volume reduction was measured at each

of the applied compaction pressure. The relative density (D) for PCM and synthesized co-

crystals was calculated from the ratio of the density of each powdered sample at pressure

P to its particle density.

3.7.1.1. Particle density

Particle density of pure PCM and co-crystals was determined with liquid

displacement method using xylene (Ayorinde et al., 2013).Weight (W) of an empty

pycnometer (density bottle) of known volume (50 ml) was determined which was then

filled to overflow with xylene. Density bottle with inert solvent was weighed again (W1)

after wiping off the excess of fluid. Difference of the weights was noted (W2). Each of

the powder (2g) was transferred to the pycnometer (W3) and xylene was allowed to

overflow. Density bottle was reweighed (W4) after cleaning excess of the fluid. True

density ρ of each powder in g/cm3 was calculated by equation 3.5

( )

From the values of relative density obtained at various applied pressures, Heckel

plot was drawn between Ln (1)/1-D and compaction pressure (P) for each powder. Linear

regression was applied on each Heckel plot and the region which contained the highest

regression coefficient was selected. ―A‖ is the intercept of the extrapolated linear region

58

of the curve. Py, a measure of the material‘s resistance for deformation (Hersey and Rees,

1971), was obtained from the 1/K (reciprocal of the slope) of the linear portion of the

curve. Higher the value of slope K, more plastic is the material.

Carr’s compressibility index (CI) 3.7.2.

Carr‘s compressibility index (CI) measures inter particulate cohesive properties of

a powder. Changes in the density of a powder after compression can be determined by

measuring the CI by the following equation

Where

dB =bulk density of the powder and dT =tapped density of the powder

Standard methods were used to measure bulk and tapped density of the powders

(Lachman et al., 1976).

Bulk density 3.7.3.

To determine the bulk density (dB) of each powdered sample at zero pressure, a

known mass (M) of powder was introduced through a funnel into a graduated glass

cylinder to measure its volume (Vo). Bulk density was calculated from the ratio as given

in equation 3.7

Tapped density 3.7.4.

Tapping the cylinder containing the powder produces a new volume, called

tapped volume (Vt) which was used to calculate the tapped density (dT) according to

equation 3.8

59

Angle of repose 3.7.5.

Angle of repose is a determinant of the frictional force in powders and represents

the maximum angle between the heap formed by powder and the horizontal surface. A

powder having an angle of repose between 20-30° is considered suitable for compaction

because of its good flow characteristics. Powder surface attributes also affect the angle of

repose. Powders with rougher and more irregular surfaces usually have higher angle of

repose. For less spherical particles an increase in angle of repose is accompanied by a

decreased bulk density and flowability.

Angle of repose was measured by fixed funnel method (Lachman et al., 1976). In

this method, the height of a funnel attached to a stand was such adjusted that the lower tip

of the funnel touched the apex of the heap formed by the powder. A pre weighed quantity

(1 g) of each of the powder sample (PCM and co-crystals) was allowed to fall freely on a

paper through the funnel. The radius (r) of heap was noted and angle of repose was

determined using equation

Where

θ= angle of repose, h= height of the heap of powder and r= radius of the heap of

powder

Preparation of tablets 3.8.

Direct compression method was employed to prepare tablets from co-crystal

powders. For this purpose co-crystal equivalent to 250 mg of PCM was blended with

60

Avicel PH 102 (20 % of the weight of co-crystal) and magnesium stearate (0.5 % of the

weight of co-crystal) for about 15 min. The mass was compressed using a hydraulic press

fitted with 13 mm diameter die. As pure PCM cannot be directly compressed due to poor

compressional properties, it was moistened with small amount of water, dried at 50 °C for

10 min and then compacted (F1). Tablets from PMs (1:1 and 2:1 by weight ratio of PCM

and CAF) were prepared following the same procedure. Tablets of pure PCM (250 mg

equivalent) were also prepared by the standard WG method (F2) using starch paste (10 %

of the weight of PCM) as a binding agent and dry starch as disintegrating agent (10 % of

the weight of PCM).

Evaluation of tablets 3.9.

All the formulated tablets were evaluated for hardness, tensile strength, and

disintegration test and in-vitro dissolution.

Hardness (Breaking force) 3.9.1.

A definite amount of hardness is needed by tablets to tolerate mechanical shocks

during the processes of manufacturing and shipping. Additionally, tablet hardness has

also found to be related to tablet disintegration and more significantly to tablet

dissolution (Kitazawa et al., 2010). This parameter is particularly important for drug

products possessing a bioavailability problem or showing sensitivity to altered dissolution

profiles as a result of applied compression force.

Pharmatron Multitest 50H is a multipurpose instrument and was used to measure

diameter, hardness and thickness of formulated tablets. Hardness was expressed in

newton (N), and diameter and thickness in meter (m).

61

Tensile strength 3.9.2.

Tensile strength specifies powder cohesiveness and is defined as force/unit area

required to split a powder bed in tension and is greatly affected by particle size

distribution of the powder (Cheng, 1968). Tensile strength (σ) expressed in MPa was

determined by diametral compression test (Fell and Newton, 1970) and calculated by

equation 3.10

Where

σ= tensile strength (MPa), F= breaking force (N), D and T= representation of

tablet diameter and thickness (m)

Disintegration test 3.9.3.

Disintegration is the breakdown of any solid material into small fragments so as

to increase the surface area for drug release. Disintegration test was carried out by USP

tablet disintegration test apparatus (Basket rack assembly) using distilled water at 37 °C.

Six tablets were subjected to the test and time for complete disintegration was noted

when no palpable mass left on mesh of the assembly.

In- vitro dissolution study 3.9.4.

Quantity of a drug going into the solution, over a period of time under a set of

specified conditions, can be measured by dissolution testing. Dissolution studies for all

prepared tablets were performed in triplicate using 900 ml of phosphate buffer pH 6.8

maintained at 37 °C in USP Apparatus II with a paddle speed of 50 rpm. Aliquots (5 ml)

were withdrawn at time intervals of 3, 6, 9, 12, 17, 22, 27 and 32 min and replaced with

fresh dissolution medium in order to maintain the sink conditions. These aliquots were

62

analyzed for PCM concentration by UV/Visible spectrophotometer at 243 nm. Drug

release (%) was determined from the calibration curve.

Release kinetic models 3.10.

A number of kinetic models have been established to describe the drug release from

the formulations. These models provide basis to study mass transport mechanisms

controlling the drug release (Siepmann and Peppas, 2001). As the qualitative and

quantitative modifications in the dosage form affect drug release and bioavailability, it is

desired to develop tools that assist product development process by reducing the

necessity of biostudies. Moreover, It is a rational approach to predict in-vivo performance

on the basis of in- vitro drug dissolution data (Dressman et al., 1984)

The drug dissolution data obtained from all formulations was fitted to various

model dependent kinetic models such as zero order, first order, and Higuchi, Korsmeyer-

Peppas and Hixon-Crowell model using DD solver (Zhang et al., 2010). Cumulative drug

release results obtained were used for fitting kinetic models (Dash et al., 2010). Best fit

model was selected based on correlation coefficient, R2.

Zero order model 3.10.1.

This is a concentration independent model which describes the release of poorly

soluble drugs with constant rate. In this model, % cumulative drug release is plotted

verses time using the following equation (Costa and Lobo, 2001).

Where

Qt= quantity of drug released at any time t, Q0= drug concentration at time t=0

and Ko= zero order release rate constant expressed in units of concentration per time

63

First order model 3.10.2.

This is a concentration dependent model (Gibaldi and Feldman, 1967; Wagner,

1969) in which natural log of % drug remained in the formulation is plotted verses time t

and defined by the equation 3.12

Qt and Q0 have already been described above and K1= first order release rate

constant expressed in units of time-1

. Dissolution of the water soluble drugs from the

porous matrices can best be described by the first order kinetics.

Higuchi model 3.10.3.

Higuchi model, proposed by Higuchi in 1961, describes the drug release from

insoluble matrix based on Fickian diffusion. This model is represented by plotting %

cumulative drug release verses square root of time t (Higuchi, 1963) and illustrated by

equation 3.13

⁄

Where

Q= quantity of drug released at any time t and KH= Higuchi dissolution rate

constant

Power law or Korsmeyer-Peppas model 3.10.4.

In this model, fraction of drug released in time t is plotted against time t (Peppas,

1985) and is represented by equation 3.14

Where

64

Mt/M∞ = fraction of drug released at time t and time ∞, K= release rate constant

for power law and n= release exponent

The value of n is helpful in characterizing various mechanisms responsible for the

drug release, e.g., n < 0.45 describes Fickian release, n= 0.89 explains case II

(relaxational) transport, n > 0.89 illustrates super case II type drug release and 0.45 < n <

0.89 defines non Fickian (anamolous) drug release.

Hixon-Crowell model 3.10.5.

Hixon-Crowell model (Hixson and Crowell, 1931) of drug release delineates the

relation of the surface area of drug particle with cube root of its volume and is given by

the equation

⁄

⁄

Where

Wo = initial drug concentration, Wt

= remaining drug concentration at time t and

Ks= surface volume relation constant

By this model, drug release is described by dissolution and with the changes in the

surface area of the particles.

In-vivo studies 3.11.

Experimental animals 3.11.1.

The in-vivo study was conducted in sheep model in order to assess the

pharmacokinetic profiles of pure PCM and co-crystals. Sheep model was selected for

preclinical evaluation owing to its easy handling, more clinically precise and relevant

dose alignment. Moreover collection of multiple samples per animal is possible without

adding risk to animal‘s life. The protocols used for these studies were approved by

65

animal ethical committee, University College of Pharmacy, University of the Punjab,

Lahore, Pakistan (ref. no. AEC/PUCP/1047A). The studies were accomplished in

agreement with Helsinki Declaration and Animal Scientific Procedure Act 1986 (UK).

Eighteen healthy adult 2-3 years aged sheep with a weight ranging from 25-30 Kg

were obtained from and maintained at the animal shed of the Pattoki campus, University

of the Veterinary and Animal Sciences (UVAS), Lahore. Sheep were observed for two

weeks before the start of study and subjected to clinical examination to assess the

possibility of any disease. Animals were provided ad libitum water and standard feed.

Sheep were tagged by using bands for identification.

Drug administration and collection of blood samples 3.11.2.

Three groups (n= 6) of sheep were orally administered at a dose of 30 mg/kg body

weight with pure PCM and equivalent co-crystals in gelatin capsules after an overnight

fast. Collection of blood samples (3 ml) were undertaken from jugular vein, after being

cleaned and disinfected with methylated spirit, in heparinized tubes at 0, 0.25, 0.5, 1, 1.5,

2, 4, 6, 8 and 10 h post dose intervals. The whole blood was centrifuged at 3500 rpm for

5 min to separate the plasma and supernatant was collected in labeled Eppendorf tube and

stored at -20 °C for further studies.

Extraction of the drug from the plasma 3.11.3.

For drug analysis, the sheep plasma was allowed to thaw. 50 % perchloric acid

(60 µl) was added to 1 ml of plasma for proteins to precipitate out. This mixture was

vortex mixed for 5 min and subjected to centrifugation at 13500 rpm for 15 min.

Supernatant was filtered using 0.22 µm syringe filter. Clear filtrate (50 µl) was injected

66

into the HPLC and run time was kept 10 min and drug content was analyzed with a

previously reported method (Alswayeh et al., 2016).

PCM Assay and HPLC conditions 3.11.4.

The HPLC system (Shimadzu 20A, Japan) was used with specifications as

described in Chapter 2 Section 2.2.7. The mobile phase was composed of water,

methanol, and acetonitrile (80:10:10, V: V: V). Membrane filter (0.45 µm) and

ultrasonication were used respectively for removing any particulate matter and degassing

of the mobile phase. It was delivered at a flow rate of 1 ml/min and the analysis was

carried out under isocratic conditions maintaining column temperature at 40 °C.

Chromatograms were recorded using a PDA detector set at 245 nm.

Preparation of stock and standard solutions 3.11.5.

The standard stock solution of PCM (1.0 mg/ml) was prepared in methanol and

further diluted with mobile phase to obtain a working stock solution of 100 µg/ml.

Further dilution of the working stock solution was carried out with mobile phase to

produce a series of solutions as shown in Table 3.1. Calibration curve standards (n=3)

ranging 0.1–15 µg/ml were prepared by spiking drug free sheep plasma with appropriate

PCM standard solutions (Table 3.1). Each standard prepared in plasma was then treated

by procedure as described in Section 3.11.3 for extraction of PCM from the plasma. Each

of the standards prepared in plasma (50 µl) was injected in to the HPLC to know the

response in terms of peak area. A standard curve was plotted between peak areas and the

drug concentrations (0.1, 0.25, 0.5, 1.0, 5.0, 10.0 and 15.0 µg/ml).

The absolute recovery of PCM was estimated by comparing the peak areas of

PCM standards prepared in mobile phase and plasma containing equivalent

67

concentrations of the drug. Quantification of PCM in sheep plasma was done by

reference to the resultant standard curve.

Table 3.1: Preparation of PCM standards in mobile phase and blank sheep plasma

Mobile phase or drug free plasma + Final solution

Solution (concentration)

850 µl +150 µl working stock solution Standard A (15 µg/ml)

900 µl + 100 µl working stock solution Standard B (10 µg/ml)

500 µl + 500 µl Standard B Standard C (5 µg/ml)

900 µl + 100 µl Standard B Standard D (1 µg/ml)

500 µl + 500 µl Standard D Standard E (0.5 µg/ml)

500 µl + 500 µl Standard E Standard F (0.25 µg/ml)

900 µl + 100 µl Standard D Standard G (0.1 µg/ml)

Pharmacokinetic (PK) analysis 3.11.6.

Various pharmacokinetic (PK) parameters were calculated from plasma level time

curves of PCM and co-crystals. Cmax and tmax representing the peak plasma concentration

and the time to reach peak concentration were obtained directly from the plasma level

time curves. PK parameters including mean residence time (MRT), apparent volume of

distribution (Vd), area under the curve (AUC), area under the first moment curve

(AUMC), elimination rate constant (Kel), plasma clearance (CL) and half life (t1/2) were

computed using PK solver (Version 2.0) pharmacokinetic program (Zhang et al., 2010)

PK solver relies on the non compartmental approach to estimate the PK parameters. The

classical trapezoidal rule was used to compute the AUC. PK solver focuses on producing

graphs and tables representing model independent PK solutions rather than

compartmental equations.

68

Statistical analysis 3.11.7.

Statistical analysis was conducted by SPSS (IBM SPSS Statistics 22, Armonk, NY,

USA). PK parameters were compared using one way analysis of variance (ANOVA)

followed by post hoc comparisons. Statistical significance was set at p < 0.05.

Results and discussion 3.12.

In recent times, Interest in PCs, has been much increased due to their ability to

modify the physical and chemical properties of APIs. Literature reports modifications in

these attributes from orders of magnitude; solubility > dissolution > hygroscopicity >

photodegradation (Thakuria et al., 2013; Trask et al., 2005; Vangala et al., 2011). The

improvements in these properties is undertaken before any further development and

without any change in the molecular make up of APIs, which is another advantage of

employing co-crystallization in the development of pharmaceuticals and other solid

forms. On the contrary, there is no guarantee that formation of a co-crystal will result in

an improvement in a particular property of interest. There is also no perfect method to

forecast that which compounds will form co-crystals in spite of advancements in

computational prediction.

Even with perfect prediction, a method of physical screening is needed to evaluate

the ease of fabrication of the co-crystal phase. Thus the development process of co-

crystals of pharmaceutical relevance must involve a robust physical screening employing

potential co-formers and co-crystallization methods to produce a number of co-crystals.

Investigation of the co-crystals obtained by a variety of methods allows the determination

of the improvements or the failure to make a decision to take a co-crystal into further

development.

69

As the methodologies for co-crystal screening are concerned, various techniques

remained under investigation including dry or neat grinding (Friščić and Jones, 2009),

LAG (Trask and Jones, 2005), solution crystallization, microwave (Pagire et al., 2013),

hot stage microscopy (Berry et al., 2008), HME (Kelly et al., 2012), and freeze drying

(Eddleston et al., 2013). A co-crystal screen employing multiple methods to form co-

crystals is argued to be ideal so in the present work a physical co-crystal screening was

carried out by four different methods as described in Table 3.2. Solvents employed in the

screening process were methanol (MeOH), ethanol (EtOH), acetone and water. These

solvents were selected to cover a range of solvent properties and represent the main

classes of solvents, e.g., polar protic (water, MeOH and EtOH), polar aprotic (acetone)

etc. Parameters to be considered were solvent/solvent ratio, rate of evaporation,

temperature and grinding time.

PCM-CAF co crystals were successfully prepared by LAG and SE methods. LAG

and SE as methods of co-crystal screening have been reported previously demonstrating

the feasibility of the techniques (Karki et al., 2009). Formation of PCM co-crystals was

recorded by difference in melting points and PXRD patterns from the individual

components. In LAG, mixtures of PCM and CAF at 1:1 weight ratio ground with small

quantities of V/V solutions of MeOH and EtOH in 1:1 and 1:2 ratio (S1 and S2) and with

pure acetone (S3) were resulted in co-crystals, coded as A, B and C, respectively (Table

3.2). In SE method, solution of PCM and CAF in 2:1 weight ratio using solvent systems

S1 and S2 resulted in formation of co-crystals which were coded D and E. All other

mixtures/solutions in the screening produced intact crystals of the individual components,

i.e., PCM and CAF.

70

Table 3.2: Screening of PCM-CAF co-crystals by using various methods (―+― represents

formation of co-crystal, characterized by DSC and PXRD results, ―- ―represents re-

crystallization of starting material or no crystallization) (co-crystals from successful

techniques are coded A, B, C, D and E)

Method Used Solvent Used Solvent

ratio

Weight ratio

of

PCM/CAF

Observations

(co-crystal

formation)

Neat (dry)

grinding

N/A 1 : 1 - 1 : 2 -

2 : 1 -

Liquid assisted

grinding (LAG)

MeOH : EtOH 1:1 (S1) 1 : 1 +(A)

1 : 2 -

2 : 1 -

1:2 (S2) 1 : 1 + (B)

1 : 2 -

2 : 1 -

Acetone Alone (S3) 1 : 1 + (C)

1 : 2 -

2 : 1 -

Solvent

evaporation (SE)

MeOH : EtOH 1 : 1 (S1) 1 : 1 -

1 : 2 -

2 : 1 + (D)

1 : 2 (S2) 1 : 1 -

1 : 2 -

2 : 1 + (E)

Acetone Alone (S3) 1 : 1 -

1 : 2 -

2 : 1 -

Water : EtOH 1 : 1 (S4) 1 : 1 -

1 : 2 -

2 : 1 -

1 : 2 (S5) 1 : 1 -

1 : 2 -

2 : 1 -

Anti solvent

addition (ASA)

Solvents: MeOH &

Acetone

Anti solvent: Water

1 : 1 -

1 : 2 -

2 : 1 -

Characterization 3.12.1.

Characterization of newly formed co-crystals was performed by three different

techniques namely PXRD, DSC and FTIR.

71

3.12.1.1. Powder X-ray diffraction (PXRD)

PXRD patterns of co-crystals were compared with three polymorphs of PCM

(denoted as Form I, II and III) and two polymorphs of CAF (denoted as Form I and II) as

shown in Figure 3.2. Characteristics peaks of all the polymorphs of PCM and CAF were

not observed in the PXRD patterns of co-crystals. Moreover new peaks were emerged at

2θ value of 8.41°, 9.06

°, 11.39

° and 17.66

° suggesting the formation of a new phase. The

difference in the intensity of major peaks in PXRD parents of co-crystals (Figure 3.2)

were due to preferred orientation effect (instrument used had a flat sample stage), which

may be due to dissimilarity in the habit of particles produced by two different methods.

Co-crystals coded as A, B and C were prepared by LAG, where grinding resulted in fine

powders. However, co-crystals coded as D and E were prepared by SE method.

Recrystallization in this case resulted in small crystallites which created problem in the

packing of samples for PXRD analysis. DSC data has also shown that the resultant

powders are new phases as the melting point of co-crystals are significantly different

from all the polymorphs of starting materials. However, precise stoichiometry and

structural description of these co-crystals needs further analysis by single-crystal XRD,

which may form the subject of our future studies.

72

Figure 3.2: PXRD patterns of PCM, CAF and PCM-CAF co-crystals prepared by LAG

(A, B and C) and SE (D and E)

3.12.1.2. Differential scanning calorimetry (DSC)

DSC thermograms of co-crystals and pure PCM (identified as form I) and CAF

are shown in Figure 3.3. PCM showed a sharp endothermic peak with mid point at 172.37

°C representing the melting of PCM form I. Although not evident in this study, PCM also

exists as polymorphs II and III. Form II has melting point at 160 °C, while Form III is

most unstable form and converts back to form I. CAF thermogram exhibited two

endothermic peaks with mid points at 160.33 °C and 231.08

°C representing melting of its

polymorphic forms, i.e., form II and I, respectively. DSC curves of co-crystals prepared

by LAG and SE showed endothermic peaks with onset temperatures ~ 130 °C and 133

°C

5 10 15 20 25 30

0

1

2

3

4

5

6

7

8

9

10

11

12

E

D

C

B

A

CAF FORM II

CAF FORM I

PCM FORM III

PCM FORM II

PCM FORM I

Inte

nsi

ty (

arb

itra

ry s

cale

)

2 theta (degree)

73

respectively (Table 3.3), which were lower than the individual components (PCM and

CAF) and all their polymorphs. However sample D showed two peaks which indicated

lack of phase purity. With small exceptions, it can be presumed that the co-crystal

formation resulted in increased entropy evident as a broad endothermic peak at

temperatures lower than the individual counterparts.

Figure 3.3: DSC thermograms of PCM, CAF and PCM-CAF co-crystals prepared by

LAG (A, B and C) and SE (D and E)

50 100 150 200 250

-2

0

2

4

6

8

10

12

14

Temperature (°C)

E

D

C

B

A

CAF

PCM

Hea

t fl

ow

(w/g

)

74

Table 3.3: Onset, mid and off set temperatures of DSC thermograms of PCM, CAF and

PCM-CAF co-crystals

Material Onset temperature

(°C)

Mid temperature

(°C)

Offset temperature

(°C)

PCM 165.59 172.37 204.20

CAF Form I

Form II

216.99 231.08 247.45

146.49 160.33 175.91

A 130.31 141.02 156.57

B 128.21 140.92 174.19

C 131.93 142.5 165.87

D 133.58 160.92 194.16

E 133.58 148.68 172.51

3.12.1.3. Fourier transform infrared spectroscopy (FTIR)

Figure 3.4 illustrates the full IR spectrum for PCM, CAF, PM and PCM co-

crystals while Figure 3.5 shows an enlargement of FTIR spectra of prepared co-crystals

with PCM and its PM with caffeine in the functional group region of 3000-3400 cm-1

. For

PCM, peaks for N-H amide stretch and phenolic OH stretch were observed at 3323 and

3108 cm-1

respectively. These functional groups are involved in the hydrogen bonding of

PCM molecules in the crystal structure (Haisa et al., 1974). Overall FTIR spectra of co-

crystals in the range 400 to 3500 cm-1

showed almost similar pattern as that of PCM,

however a shift in the above described bands (i.e., to 3316 cm-1

and 3104 cm-1

respectively) was observed for the co-crystals. This indicated a slight change in the

hydrogen bonding pattern of PCM in the form of co-crystal. Moreover these shifts were

not observed for PM.

75

Figure 3.4: Full ATR-FTIR spectra of PCM, CAF, physical mixture and PCM-CAF co-

crystals prepared by LAG (A, B and C) and SE (D and E)

3500 3000 2500 2000 1500 1000

300

250

200

150

100

E

D

C

B

A

PM

CAF

PCM

Tra

nsm

itta

nce

(%

)

Wavenumber (cm-1

)

76

Figure 3.5: ATR-FTIR spectra of PCM, physical mixture and PCM-CAF co-crystals

highlighting the N-H amide stretch and phenolic OH stretch regions for PCM

3.12.1.4. Intrinsic dissolution rate (IDR)

Solubility measurement has a great importance in the drug development.

Solubility in aqueous medium is required for pharmacokinetic and stability studies and

also for drug formulation. An API with poor solubility and consequent low absorption is

unlikely to be developed into a formulation. Thus it becomes important to measure the

solubility of a drug to predict the pharmacokinetic metrics and to select a proper drug

candidate.

3400 3300 3200 3100 3000

0.80

0.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

E

D

C

B

A

PM

PCM

Tra

nsm

itta

nce

(%

)

Wavenumber (cm-1

)

77

Solubility can be measured by equilibrium and intrinsic dissolution methods.

Since solid state transitions in the solution make it difficult to measure equilibrium

solubility (Good and Rodr guez-Hornedo, 2009), intrinsic dissolution is an alternative to

define the solubility class of a drug by virtue of determining intrinsic dissolution rate

(IDR).

IDR was employed as it offers advantages over other powder dissolution methods,

e.g., the exposed area of the disc is constant thus minimizing the dissolution variability

(Wells, 1988). Moreover, provided the sink conditions are maintained, the IDR has been

found to be directly proportional to the solubility of drug in the dissolution media

(Aulton, 2013).

IDR can be carried out by compressing a pellet of the drug using rotating disc

method (Woods apparatus) or alternately by coating the pellet with hard paraffin leaving

only the top surface free for dissolution and affixing the pellet to the base of the

dissolution vessel (static disc method). The latter method finds preference for

multicomponent systems like co-crystals as compared to rotating disc method which is

more suitable for single component systems and is also associated with risks of air

bubbles forming on the disc surface and disintegration and disruption of constant surface

area (Healy and Corrigan, 1996). Considering these factors, static disc method was used

to determine IDR.

UV calibration curve for PCM in phosphate buffer pH 6.8 (Figure 3.6) gave R2

value of greater than 0.99 and thus found acceptable to determine the drug content, IDR

and dissolution rate from the formulations.

78

Intrinsic dissolution profiles of the prepared co-crystals compared with pure drug

and PMs (1:1 and 2:1 by weight ratio of PCM and CAF) have been shown in Figure 3.7.

IDR of PCM recorded in phosphate buffer pH 6.8 was 5.06 mg/cm2-min, while for co-

crystals A, B, C, D and E, IDR was 8.72, 9.53, 12.23, 11.11 and 14.40 mg/cm2-min

respectively. Based on the initial dissolution rate, co-crystals A, B, C, D and E showed

1.72, 1.88, 2.42, 2.19 and 2.84 fold increase in dissolution rate. In comparison the PMs

showed almost same IDR (~ 5.5 mg/cm2-min) as that of the pure drug. The high

dissolution rate might indicate the stability of prepared co-crystals to prevent dissociation

of drug and co-former from each other and achieve the solubility and dissolution

advantages of co-crystals (Shiraki et al., 2008).

Figure 3.6: Calibration curve of PCM in phosphate buffer pH 6.8

y = 0.0533x + 0.0178

R² = 0.9985

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20

Ab

sorb

an

ce

Concentration (µg/ml)

79

Figure 3.7: Intrinsic dissolution rate of PCM alone and from co-crystals A, B, C, D and

E in phosphate buffer pH 6.8 (5.5, 8.72, 9.53, 12.23, 11.11 and 14.40 mg/cm2-min

respectively) (n= 3)

IDR is the dissolution rate that is greatly influenced by the intrinsic factors

especially the crystal habit and particle size distribution. The variation among the IDR

values of co-crystals can be explained by the fact that the powders prepared by the two

different methods (LAG and SE) have different mechanical properties, cohesiveness,

particle size distribution and shape. This is evident from the SEM images of these

samples as shown in Figure 3.8. Cohesiveness of a powder increases with a decrease in

particle size because the tendency of the individual particles to stick together increases

(Cheng, 1968). Hence, mechanochemical method (LAG) produced fine powders with

high compressibility index (~ 31.12 %) while the co-crystals prepared by SE showed

larger particles with low compressibility index of ~ 20.55 %. This fact led to the

development of more compact discs from co-crystals prepared by LAG signifying lower

values of IDR. This is further supported from the higher breaking force of the pellets

prepared from co-crystals A, B and C as compared to the D and E samples which

produced discs with a low value of breaking force (Table 3.4).

0

20

40

60

80

100

120

140

160

0 2 4 6 8 10

Cu

mu

lati

ve

dru

g d

isso

lved

(m

g/c

m2)

Time (min)

PCM

A

B

C

D

E

PM (1:1)

PM (2:1)

80

Figure 3.8: SEM images of pure PCM and PCM-CAF co-crystals prepared by LAG (A,

B and C) and SE (D and E)

Compressional and mechanical properties 3.12.2.

Poor compressibility of PCM due to low plasticity is always a problem for the

formulation scientists. To overcome this problem, PCM is normally tableted by WG

method, which is a time consuming and laborious procedure and requires a large amount

A

B

C

D

E

PCM

81

of excipients to be added to the formulation. However, development of crystal structures

providing large inter particulate bonding area and plasticity has been recommended as a

mean of improving the tensile strength of materials (Sun, 2013).

On application of pressure to a powder, its bulk volume reduces. During this

process of volume reduction, which is often known as compression, particles move into

close proximity to each other and different types of bonds are formed between them as a

result of decrease in the energy of the system (Coffin-Beach and Hollenbeck, 1983).

Under low compaction pressure, the first thing happening on powder is

rearrangement of the particles to form a close packing structure. Regularly shaped

particles tend to rearrange more easily than the particles with irregular shape. Further

increase in pressure prevents further rearrangement and particles undergo plastic and

elastic deformation and /or fragmentation (Duberg and Nyström, 1986). Brittle particles

are likely to go through the fragmentation. Particle shape endures a permanent change as

a consequence of plastic deformation which is an irreversible process; however after

elastic deformation, regarded as a reversible phenomenon, original shape is resumed by

the particles. Mechanical properties and mechanisms involved in compression define the

degree of volume reduction of powders. Mechanical behavior of the materials is

influenced by particle size and speed of compression (Roberts and Rowe, 1987), e.g., a

reduced particle size results in a decreased tendency of powders to fragment (Alderborn

et al., 1985). Similarly for some materials, at an acute particle size, brittle behavior is

switched to ductile as the particles become smaller. Brittle materials undergoing a high

degree of fragmentation produce highly porous tablets with a large number of bonding

points preventing further volume reduction. Low porosity tablets will be produced by a

82

ductile material because of the ability of the particles to move closer to each other as a

result of extensive plastic deformation (Roberts and Rowe, 1987).

Literature describes many studies regarding the material deformation at the

crystalline level (Egart et al., 2014; Egart et al., 2015; Meier et al., 2009). Methods such

as nano indentation utilize small amounts of materials but have limitations in terms of

describing the relationship between crystal and bulk mechanical behavior. This limitation

has directed towards the well recognized compaction equations (Heckel, Kawakita and

Cooper-Eaten equations) to define the volume reduction of a powder bed quantitatively

and to classify materials based on their deformation.

Heckel equation finds its wide application to calculate the relative density of a

powder during the course of compression at each of the applied pressure and ultimately to

get density pressure relationship (Alebiowu and Itiola, 2001). In the present study,

density pressure plots for pure PCM and co-crystals were obtained using in-die method,

which measures the compact volume by computing its dimensions in the die. This

method was selected because of the speedy data collection and utilization of less material

in comparison to out-of-die method which requires a new compact at each of the applied

pressure.

Heckel plots between Ln (1)/ (1_D) and the applied pressure, P, were drawn for

pure PCM and co-crystal powders. Both PCM and co-crystals showed typical Heckel

plots, expressed initially by a curve and then showing linearity (Figure 3.9). The

curvilinear region indicates the initial movement followed by rearrangement of the

particles in the die. Co-crystals displayed improved compaction and hence high plasticity.

This was shown by low mean yield pressure (Py) values for co-crystals as compared to

83

pure drug (Table 3.4). The yield pressure, Py, is the stress at which particle deformation is

initiated reflecting the deformation of the particles during compression (Adetunji et al.,

2006). Py recorded for PCM was 133.33 MPa. The same parameter for co-crystals A, B,

C, D and E was found to be significantly decreased, i.e., 33.03, 80, 28.65, 33.03 and

47.62 MPa respectively. The ranking of the Py values was C < A&D < E < B < PCM.

Regions of Heckel plots presenting the highest correlation coefficient for linearity of >

0.989 were used to calculate Py values for all powder samples. Mechanisms namely

kinking, glide (slip) and twinning may be responsible for imparting the plastic

deformation to particles (Bandyopadhyay and Grant, 2002).

Figure 3.9: Heckel plots for PCM and PCM-CAF co-crystals prepared by LAG (A, B

and C) and SE (D and E)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 5 10 15

Ln

(1/1

-D)

Compaction pressure (Mpa)

PCM

A

B

C

D

E

84

Table 3.4: Mechanical parameters derived for pure PCM and PCM-CAF co-crystals

Parameters PCM A B C D E

Breaking force (N) 5±0.35 131±5.25 139±4.85 147±4.38 63±1.01 68±1.01

Carr‘s index (CI) 49.23±1.03 30.25±0.75 30.87±0.69 31.12±0.41 19.87±0.15 20.55±0.17

Angle of repose (°) 43.05±0.26 24.55±0.27 25.55±0.33 24.55±0.43 22.85±0.19 22.03±0.21

Particle

density(g/cm3)

1.264±0.02 1.373±0.01 1.361±0.012 1.391±0.024 1.380±0.018 1.385±0.033

Relative

density(g/cm3)

0.573±0.01 0.724±0.02 0.753±0.01 0.753±0.002 0.634±0.01 0.65±0.01

Porosity (%) 42.7 27.6 24.7 24.7 36.6 34.7

Mean yield pressure

(MPa)

133.33 33.03 80 28.65 33.03 47.62

% content 94.10±3.71 94.25±2.91 95.07±3.01 94.74±2.90 93.80±1.85

85

Better compressibility of the co-crystal powders was further sustained by porosity

measurements (Equation 3.3). The compressibility of the PCM was poor as indicated by

the higher porosity value (42.7%) as compared to the co-crystals which gave low values

for the porosity (Table 3.4). True density values for co-crystals were observed to be

higher indicating greater bond strength and more plasticity than PCM (Chang and Sun,

2017). Flow properties of co-crystals were also improved as demonstrated by low values

of CI and angle of repose (Table 3.4).

Furthermore, where pure PCM was not able to compress into a tablet without

extensive chipping, all co-crystal powders readily formed tablets and showed higher

breaking force values (Table 3.4). These enhanced values may be credited to increase

particle-particle contact points resulting in more solid bonds. Increase in relative density,

as shown in Table 3.4, is another reason for increase in breaking force of co-crystals due

to creation of more contact points leading to an enhancement in the degree of bonding

between the particles (Hancock et al., 2001).

Difference in compression properties among co-crystals produced by LAG and

SE is due to dissimilar crystal plasticity which may result from unique molecular packing

features in the respective crystal lattices. Co-crystals synthesized by LAG are more

plastic and have high tensile strength as evident from their low Py values (33.03 and

28.65). Previous literature also confirms mechanochemical synthesis as an efficient

approach to produce plastic and compressible forms of PCM using oxalic acid,

phenazine, theophylline and naphthalene as co-formers (Karki et al., 2009).

86

Pharmacotechnical evaluation of the formulated tablets 3.12.3.

Tablets are the preferred dosage forms for therapeutic substances as they are easy

to manufacture, convenient to use, provide an accurately measured dose of an API, stable

compared to oral liquids and safe compared to parenterals (Bello-Imam et al., 2008).

Direct compression is thought as a striking method for preparing tablets due to the

requirement of fewer unit operations, cost effectiveness, appropriateness for moisture and

heat sensitive materials and capability for producing consistent dissolution profiles in

tablets (Lachman et al., 1976). On storage, directly compressed tablets are less likely to

undergo changes in dissolution profiles than those formulated by granulation methods.

This is extremely important because of the inclusion of dissolution specifications in

compendia for most dosage forms (Banaker, 1994). Considering the above mentioned

reasons, direct compression method was used to prepare tablets from co-crystal powders

Results from the dosage form data (Table 3.5) disclosed that co-crystals can easily

be formulated by direct compression, with adequate hardness, to the tablet dosage form

without adding any binding agent. Tablet formulations based on co-crystals prepared by

LAG using solvent systems S1 and S2, and pure acetone as described in Table 3.2 were

coded FA, FB and FC whereas FD and FE are the codes given to the formulations based

on co-crystals prepared by SE using solvent systems S1and S2. A tensile strength higher

than 3MPa was exhibited by FA, FB and FC, while the tensile strength of FD and FE was

in the vicinity of > 1.8 MPa. This suggested that mechanochemical methods of co-

crystallization are more suitable to improve the pharmacotechnical properties of PCM.

87

Table 3.5: Physical properties of tablet formulations prepared from pure PCM (direct compression (F1) and wet granulation (F2)) and

PCM-CAF co-crystals (FA, FB, FC, FD and FE)

Properties F1 F2 FA FB FC FD FE

Tablet

diameter(mm)

13±0.01 13±0.01 13±0.01 13±0.01 13±0.01 13±0.01 13±0.01

Breaking

force(N)

18±1.0 66±1.0 270±5 285±5 309±5 90±2.5 94±2.5

Tensile

strength(MPa)

0.52±0.01 1.90±0.01 3.48±0.01 3.67±0.01 3.98±0.01 1.82±0.01 1.88±0.01

Disintegration

time(min)

1±0.5 0.67±0.5 8.0±0.5 7.5±0.5 7.5±0.5 5±0.5 5±0.5

88

In- vitro drug release study of the formulated tablets 3.12.4.

Dissolution testing is regarded a prerequisite as well as a quality control tool for

all solid dosage forms. It is used in drug development process for the product release and

stability testing to identify any physical change in the drug and formulation (FDA, 1997).

In R & D, dissolution study finds great significance to explore the impact of production

and formulation variables and in comparative studies for in-vitro/in-vivo correlation

(Zhang and Yu, 2004).

Drugs administered orally in the form of solid dosage forms, e.g., tablets and

capsules are not supposed to be instantaneously available to the body because of the

restrictions offered by disintegration and dissolution (Morrison and Campbell, 1965).

This means such drugs must dissolve in gastrointestinal fluids before absorption can

occur. Since dissolution precedes absorption, factors affecting the rate of solution

formation can also affect the rate of absorption. Consequently, dissolution rate may affect

the onset, intensity and duration of pharmacological response. Previously co-

crystallization has been shown to have a profound effect on the solubility and dissolution

of the poorly soluble drugs (Jung et al., 2010; Ullah et al., 2016).

In the above context, various tablet formulations prepared from pure drug, PMs

(1:1 and 2:1 by weight ratio of PCM and CAF) and co-crystals were subjected to the in-

vitro dissolution studies. Direct compression was employed for co-crystals (FA, FB, FC,

FD and FE) and PMs based formulations. However for pure drug formulations, both

liquid assisted direct compression (F1) and WG method (F2) were used as described in

Section 3.8. Figure 3.10 shows comparison of dissolution profiles of these formulations.

All the co-crystals formulations showed 90 - 97 % release within 20 min. The amount

89

released was greater than from the equivalent PMs based formulations (~ 55 %), F1 (72

%) and F2 (85 %).

Figure 3.10: In-vitro drug release comparison of various formulations of PCM (F1 and

F2), PCM-CAF co-crystals (FA, FB, FC, FD and FE) and physical mixtures (n= 3)

As already described before that the LAG method of co-crystallization produced

powders with small particle size (confirmed by SEM results Figure 3.8) leading to a large

surface area which instigated mechanochemical synthesis based formulations to give

better dissolution profiles as compared to SE based formulations. Furthermore, grinding

in the presence of acetone resulted in even more improved dissolution (FC) as compared

to grinding with V/V mixtures of MeOH and EtOH (FD and FE). This can be justified by

the fact that high solubility of PCM in MeOH/EtOH mixture as compared to pure acetone

(solubility of PCM in MeOH, EtOH and acetone is 371, 232 and 111 mg/g of the solvent)

resulted in solvent assisted recrystallization as demonstrated by spottiness (loss of peak

resolution, i.e., preferred orientation effect) of PXRD pattern (Figure 3.2).

0

20

40

60

80

100

0 5 10 15 20 25 30

Dru

g R

elea

sed

(%

)

Time (min)

F1

F2

PM(1:1)

PM(2:1)

FA

FB

FC

FD

FE

90

Release kinetic results have shown that the release of PCM from formulations F1

and F2 followed Korsmeyer-Peppas and Hixon-Crowell models, respectively. While

dissolution data of the co-crystal formulations FA and FB followed Korsemeyer-Peppas

and formulations FC, FD and FE followed Hixon-Crowell model for drug release (Table

3.6). Moreover all co-crystal formulations showed non Fickian drug release 0.45 < n <

0.89.

91

Table 3.6: Drug release kinetics from various formulations of PCM (F1 and F2), and PCM-CAF co-crystals (FA, FB, FC, FD and FE)

Release kinetics F1 F2 FA FB FC FD FE

Zero order R2 0.9847 0.8559 0.7634 0.7383 0.5443 0.8172 0.7257

First order R2 0.9821 0.9628 0.9693 0.9575 0.9531 0.9368 0.9688

Higuchi model R2 0.8926 0.9148 0.9340 0.9053 0.8891 0.8834 0.9327

Hixon-Crowell

model

R2 0.9904 0.9764 0.9775 0.9594 0.9712 0.9517 0.9810

Korsmeyer-

Peppas model

R2 0.9926 0.9421 0.9843 0.9791 0.9230 0.9077 0.9340

n 0.873 0.659 0.758 0.751 0.549 0.650 0.531

92

In-vivo performance of PCM and co-crystals 3.12.5.

HPLC method used for the determination of PCM was specific, accurate, simple

and rapid. The method utilized a simple protein precipitation procedure employing 50 %

perchloric acid and was successfully applied to quantify PCM in sheep plasma samples.

Peaks of PCM and CAF were well resolved with the mobile phase used consisting of

water, methanol, and acetonitrile (80:10:10, V: V: V) pumped at a flow rate of 1ml/min

by using the isocratic mode. The retention times of PCM and CAF were found to be ~ 5

and ~ 7 min, respectively. Moreover, the blank plasma undergoing the same extraction

procedure was appeared to have no peaks at the retention times of PCM and CAF (Figure

3.13).

3.12.5.1. Standard curve and percent recovery

Calibration curve was based on peak area measurements of plasma

samples spiked with various concentrations of PCM. The assay was found to be sensitive,

reproducible and linear over the concentration range of 0.1-15 µg/ml with mean

correlation coefficient R2= 0.997 (Figure 3.11).

Figure 3.11: Standard curve of PCM in drug free sheep plasma

y = 292622x + 122434

R² = 0.997

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

0 3 6 9 12 15

Pea

k a

rea


93

The mean % recovery values (Table 3.7) for PCM over the concentration range of

0.1-15 µg/ml were found to be 92.38-103.87 % with RSD < 5% claiming the accuracy of

method. The limits of detection and quantification were observed to be 0.03 and

0.1µg/ml, respectively.

Table 3.7: Recovery of PCM from spiked sheep plasma

Concentration

(μg/ml)

Peak area Recovery (%)

Standard solution Spiked plasma

0.1 42784 41789 97.67

0.25 172741 159588 92.38

0.5 367008 345177 94.05

1.0 403711 422197 104.58

5.0 1827423 1780353 97.42

10.0 3685549 3573693 96.96

15.0 4187427 4349398 103.87

Figures 3.12, 3.13, 3.14 and 3.15 show representative chromatograms of PCM in

mobile phase, blank sheep plasma and plasma spiked with various concentrations of

PCM (0.1 and 10 μg/ml) while Figures 3.16 and 3.17 are illustrative of chromatograms

from plasma of sheep administered orally at a dose of 30 mg/Kg body weight of pure

PCM and equivalent co-crystal at 30 min and 1.5 h, respectively.

94

Figure 3.12: HPLC chromatogram of PCM (10 µg/ml) in mobile phase

Figure 3.13: Representative chromatogram of drug free sheep plasma

0

10000

20000

30000

40000

50000

0 1 2 3 4 5 6 7 8 9 10

Inte

nsi

ty (

mA

u)

Time (min)

0

20000

40000

60000

80000

100000

0 1 2 3 4 5 6 7 8 9 10

Inte

nsi

ty (

mA

u)

Time (min)

95

Figure 3.14: Representative chromatogram of sheep plasma spiked with pure PCM (0.1

µg/ml)

Figure 3.15: Representative chromatogram of sheep plasma spiked with pure PCM (10

µg/ml)

0

50000

100000

150000

200000

250000

300000

0 1 2 3 4 5 6 7 8 9 10

Inte

nsi

ty (

mA

u)

Time (min)

0

50000

100000

150000

200000

250000

0 1 2 3 4 5 6 7 8 9

Inte

nsi

ty (

mA

u)

Time (min)

96


mg/Kg body weight of pure PCM (30 min)


mg/Kg body weight of equivalent co-crystal (1.5 h)

3.12.5.2. Pharmacokinetic (PK) parameters

Pharmacokinetics (PK) is the study of the kinetics of drug absorption, distribution

and elimination. These biological processes dictate the efficacy and toxicity of drugs. PK

is based on measuring drug concentration in various biologic samples (plasma, urine,

0

40000

80000

120000

160000

200000

0 1 2 3 4 5 6 7 8 9 10

Inte

nsi

ty (

mA

u)

Time (min)

0

100000

200000

300000

400000

500000

600000

700000

800000

0 1 2 3 4 5 6 7 8 9 10

Inte

nsi

ty (

mA

u)

Time (min)

97

saliva and milk). Drug concentration determined in plasma samples taken at various time

intervals is a mean to obtain plasma drug concentration time curve and to compute the

PK parameters. Moreover plasma level time curve is also related with various

pharmacologic parameters for the drug such as MEC and MTC, i.e., minimum effective

and minimum toxic concentration, onset time, duration of action and intensity of

pharmacologic effect. PK studies are important to recommend appropriate and rational

use of drug as well as to design a dosage regimen.

It is therefore important to explore the co-crystals in terms of PK parameters

which may provide the basis for dose reduction and avoidance of adverse drug reactions.

Powder co-crystal samples from each of the successful method showing higher IDR, i.e.,

―C‖ and‖ E‖ were selected for oral bioavailability studies. Both pure drug and co-crystals

enclosed in gelatin capsules were administered to overnight fasted sheep to eliminate the

effect of food on drug absorption. Food was continued after 4 h. No toxic effects were

observed during the experimental period following the oral administration of a single

dose of 30 mg/kg body weight of pure PCM and equivalent co-crystal.

PK parameters were computed using non compartmental approach by PK solver

version 2.0. In non compartmental approach, body is not considered as a compartment

(Gibaldi, 1991) rather computation of PK parameters is based on statistical moment

theory, called the moments of random variables. The plasma concentration is regarded as

random variable because of its variability with the function of time. Statistically, a set of

plasma concentration time data may be taken as statistical distribution about a mean

value approximated by the first moments. AUC is the area under the zero moment curve

while area under the moment curve (AUMC) is the area under the first moment curve. A

98

parameter, called as mean residence time (MRT) can be calculated from the ratio of

AUMC0-∞ to AUC0-∞ (Shargel et al., 2015).

Figure 3.18 shows the mean plasma profiles (n= 6) for PCM and selected co-

crystals after a single dose oral administration (30 mg/kg) in sheep. The concentration of

drug at first and the last time intervals (0.25 and 10 h) after administration of pure PCM

could not be detected (Table 3.8). However, the drug was detectable for both co-crystals

of PCM from 0.25 h to 10 h.

Following oral administration of the co-crystals ―C‖ and ―E‖ drug absorption

started immediately reaching a concentration of 0.333+0.308 and 0.162+0.25 µg/ml

respectively at 0.25 h and suggesting a faster onset of action of co-crystals as compared

to pure PCM which showed very slow absorption (-0.107+0.129 µg/ml) at the same time

(Table 3.8). In fact plasma concentration of PCM from the co-crystals showed a

substantial increase than the pure drug up to 10 h and could be credited to greater

solubility and improved intrinsic dissolution profiles as compared to pure PCM.

The mean PK metrics calculated from the sheep model have been presented in

Table 3.9.

99

Table 3.8: Plasma concentration of PCM and PCM-CAF co-crystals (C and E) in sheep

at different time intervals, obtained after single oral administration at a dose of 30 mg/Kg

body weight (n= 6)

Time (h) PCM

concentration

(µg/ml)

Mean+SD

PCM concentration

from co-crystal “C”

(µg/ml)

Mean+SD

PCM concentration

from co-crystal “E”

(µg/ml)

Mean+SD

0.25 -0.107+0.129 0.333+0.308 0.162+0.253

0.5 0.265+0.201 0.924+0.528 0.554+0.451

1 0.661+0.236 1.53+0.566 1.04+0.549

1.5 0.975+0.296 2.38+0.639 1.71+0.772

2 0.907+0.251 2.26+0.513 1.61+0.422

4 0.761+0.298 1.34+0.319 1.25+0.359

6 0.498+0.368 0.916+0.193 0.879+0.435

8 0.203+0.212 0.599+0.150 0.587+0.196

10 -0.0085+0.184 0.313±0.174 0.292+0.136

a) Maximum plasma concentration (Cmax)

Maximum plasma concentration (Cmax) is the highest drug concentration attained

in the blood after drug administration. It gives information that the drug is sufficiently

systemically adsorbed to give a therapeutic action. Cmax depends on both the rate and

extent of drug absorption in plasma. So, it is a strong indicator of rate of absorption, i.e.,

higher the peak concentration, faster the rate of absorption. In addition, Cmax also gives

warning of possible toxic levels of drugs (Shargel et al., 2015).

Concentration for pure drug and co-crystals peaked at 1.67 h showing no change

in the time for maximum concentration. Cmax for co-crystals C (2.40 µg/ml) and E

100

(1.72µg/ml) was found to be 2.45 and 1.76 fold that of PCM (0.98 µg/ml) and again

credited to the improvement in physicochemical profile of PCM as a result of co-

crystallization. This significant increase (p= 0.001) in peak concentration for co-crystals

may also be attributed to decreased elimination because of higher accumulation of drug.

When the PCM co-crystals were compared, the Cmax of ―E‖ was less than ―C‖

suggesting a greater extent of drug absorption from ―C‖ as compared to from ―E‖.

However the difference between the ―C‖ and ―E‖ may not be statistically significant as

the inter-subject variability is very high with co-crystal ―C‖. The decreased Cmax for ―E‖

also caused a proportional decrease in AUC0-∞ value in comparison to ―C‖. Difference in

in-vivo behavior between the co-crystals (C and E) prepared by two different methods

(LAG and SE) can be attributed to the dissimilarity in the mechanical properties,

cohesiveness, particle size distribution and particularly the dissolution rate (Figure 3.7).

Figure 3.18: Plasma concentration profiles with time for PCM and PCM-CAF co-

crystals (C and E) in sheep model (n= 6)

0

0.5

1

1.5

2

2.5

3

0 2 4 6 8 10

Pla

sma

co

nce

ntr

ati

on

of

PC

M(µ

g/m

l)

Time (h)

Mean conc.

(PCM)

Mean conc. C

Mean conc. E

101

b) Area under the curve (AUC)

Area under the plasma concentration time curve (AUC) is one of the fundamental

parameters of PK studies having a direct relation with the dose of administered drug.

AUC finds its particular usage to estimate the extent of drug absorption that actually

appears in blood stream (Gibaldi, 1991). AUC is of utmost importance to compute

several other physiologically significant PK parameters like clearance, bioavailability and

Vd.

The mean AUC0-∞ values for co-crystals ―C‖ and ―E‖ were 13.01+1.22 and

10.44+1.93 µgh/ml respectively (Table 3.9) which were 2.48 and 1.98 fold enhanced that

of PCM (5.25+0.5 µgh/ml) and were also significant (p < 0.001). AUC is also inversely

related to clearance of drugs and proportional to fraction of drug that is present in

systemic circulation. Thus, the lower the clearance, the higher the AUC and vice versa.

c) Mean Residence Time (MRT)

Mean residence time (MRT) is termed as the time which is required by an intact

drug molecule to pass through body. Thus, MRT becomes an outstanding determinant,

just like half life and is used in the non compartmental modeling, to define the period for

which the drug persists in the body. The MRT computed following single dose oral

administration of pure PCM and co-crystals (C and E) was 3.86+0.87, 5.52+1.07 and

5.13+0.78 h respectively (Table 3.9). Though MRT values are arbitrary, higher value of

MRT for PCM co-crystals than pure drug may specify that PCM remained in the body for

quite longer period of time due to comparatively slower elimination of the drug.

102

Table 3.9: Pharmacokinetic parameters of PCM and PCM-CAF co-crystals in sheep,

obtained after single oral administration at a dose of 30 mg/Kg body weight (n= 6). *R.B

relative to PCM

Parameters PCM

Mean+SD

Co-crystal “C”

Mean+SD

Co-crystal “E”

Mean+SD

P-value

Cmax (μg/ml) 0.98+0.18 2.40+0.51 1.72+0.66 0.001

tmax (h) 1.67+0.25 1.67+0.25 1.67+0.25 1.00

AUC0-10h

(μgh/ml)

5.02+1.3 11.22+1.67 9.36+1.60 0.001

AUC0-∞ (μgh/ml) 5.25+0.5 13.01+1.22 10.44+1.93 P < 0.001

AUMC0-∞

(μgh/ml)

21.86+05.19 72.46+14.84 53.64+10.54 0.004

MRT (h) 3.86+0.87 5.52+1.07 5.13+0.79 0.124

Kel (h-1

) 0.33+0.15 0.26+0.13 0.27+0.03 0.001

t1/2 (h) 1.36+0.41 3.29+1.75 2.53+0.38 0.021

CL (μg/ml)/h 161.48+19.01 56.45±4.48 75.27+14.49 P < 0.001

Vd (μg/ml) 294.18+28.33 264.83+26.78 276.72+19.19 0.893

*Relative

bioavailability

(R.B)

2.47 1.98

d) Elimination rate constant and half life (t1/2)

PCM was shown to be eliminated fastly, as indicated by a higher value of Kel

(0.33+0.15 h-1

) as compared to that shown by co-crystals C and E (0.26+0.13 and

0.27+0.03 h-1

, respectively). A ~2.0 fold significant increase (p= 0.021) was observed in

the elimination half life (t1/2) of PCM as a result of co-crystallization with CAF. The high

values of t1/2 for the co-crystals ―C‖ and ―E‖ (3.29+1.75 and 2.53+038 h, respectively) in

103

comparison to pure PCM (1.36+0.41 h-1

) following single dose oral administration in

sheep could be attributed to a decrease in the hepatic metabolism of PCM. This resulted

in sufficient concentration of drug from co-crystals continued to be present even at the

last time interval with a consequent accumulation in the plasma. The increase in t1/2 is

further supported by low clearance of co-crystals, as discussed in the next Section, in

comparison to pure PCM.

e) Total body clearance (CL)

Total body clearance (CL) is a key PK parameter giving sum of clearances by

elimination organs without identifying the mechanism. Drug clearance is defined as the

volume of plasma fluid that is cleared of drug per unit time and is important to calculate

the maintenance dose to maintain the steady state. CL is also regarded as an indicator of

metabolism (Shargel et al., 2015). The CL values computed for co-crystals ―C‖ and ―E‖

were 56.45+4.48 and 75.27+14.49 μg/ml/h which were significantly less (p < 0.001) than

the CL of pure PCM (161.48+19.01). So the co-crystallization with CAF profoundly

altered the disposition profile of PCM. A low clearance of PCM as a result of co-

crystallization with CAF resulted in prolongation and potentiation of its effects in the

form of co-crystal. The low clearance of co-crystals is also a function of the low Vd for

the co-crystals (Table 3.9).

f) Volume of distribution (Vd)

Volume of distribution (Vd), being a disposition parameter, is majorly determined

by the physicochemical profile of the drug and physiologic characteristics of the body.

Clinically, Vd is important to estimate loading dose which is required to maintain a

104

desired blood concentration. Vd is also used to measure drug plasma concentration in

treatment of overdose (Mushtaq et al., 2017)

The mean Vd values computed after single dose oral administration of PCM and

co-crystals ―C‖ and ―E‖ were 294.18+28.33, 264.83+26.78 and 276.72+19.19 µg/ml,

respectively. The low Vd for co-crystal may also be responsible for the high plasma drug

concentration after a given dose.

g) Relative bioavailability (R.B)

Relative bioavailability (R.B) measures the fraction of a drug absorbed intact into

blood plasma from product under investigation relative to a recognized standard and is

determined by comparisons of the AUCs associated with test sample and reference

according to the equation 3.16

Co-crystal and the pure PCM were used as test sample and reference respectively.

Co-crystals showed more than two times enhancement in the relative oral bioavailability

than the pure PCM.

105

Conclusion 3.13.

PCM is a compound with low dissolution rate as well as poor tabletability. A co-

crystal incorporating PCM and CAF was successfully developed by liquid assisted

grinding and solvent evaporation. Co-crystals showed improved IDR and compressional

properties. In-vivo kinetic results from sheep model also confirmed a significant increase

in the oral bioavailability of PCM. This suggests co-crystallization a useful technique to

improve the compressional, physicochemical and pharmacokinetic properties of PCM

and proving caffeine a suitable co-former. A pictorial representation starting from the

screening of PCM-CAF co-crystals, their characterization and improvement in

mechanical, in-vitro and in-vivo performance has been given in Figure 3.19.

Figure 3.19: Pictorial representation showing the synthesis and characterization of PCM-

CAF co-crystal and improvement in mechanical, in-vitro and in-vivo performance

106

Chapter 4

107

4. Simultaneously improving mechanical, formulation and in-vivo

performance of Naproxen by co-crystallization

Background 4.1.

Naproxen (NAP) is a nonsteroidal anti inflammatory (NSAID) drug that belongs

to BCS class II (Low solubility, High permeability). Like other NSAIDs, NAP is

frequently used to reduce the inflammation, pain and fever (Boynton et al., 1988) owing

to their ability to block (Sun et al., 2009) the cyclooxygenase (COX) enzymes comprising

COX-1 and COX-2. COX enzymes produce prostaglandins, where the prostaglandins

intricate in promoting inflammation, fever and pain, COX-1 produced prostaglandins are

well known to protect the stomach and support platelets and blood clotting (Aresta et al.,

2005). Thus, after an injury or surgery occurs, NSAIDs are prone to produce ulcers in the

stomach and promote bleeding.

Hydrogen ion concentration (pH) of the surrounding medium greatly affects the

solubility of NAP (Chowhan, 1978). NAP has a high content of lipophilic aromatic

region which prevents wetting and interactions with water resulting in its poor solubility

in aqueous medium. On the other hand at higher pH, it ionizes forming favorable

interactions with water thus enhancing dissolution. Oral administration of NAP can result

in variable plasma concentration and ultimately low bioavailability because of its

restricted wettability and very limited water solubility (0.021 mg/ml at 25 °C). Low

solubility enhances the residence time of NAP in the mucosa thereby increasing its

irritating side effect on the gastrointestinal tract (GIT) (Akbari et al., 2015). Many

approaches have been investigated to modify NAP low solubility, e.g., complexation with

beta cyclodextrins (Junco et al., 2002), formulation with various excipients (Dahl et al.,

108

1990), liquisolid tablet formulation (Tiong and Elkordy, 2009), nanocrystals (Peltonen

and Hirvonen, 2010), microparticles (Montes et al., 2013), amorphization (Löbmann et

al., 2013), and co-precipitation with a polymer (Subra-Paternault et al., 2014). Currently,

NAP is marketed as free acid and also as sodium salt which is a mean to address the

solubility problem. Nevertheless to improve the solubility across all pH range, an

alternative solution such as co-crystallization is desired. NAP is considered a candidate

drug for this crystal engineering strategy owing to the presence of carboxylic acid

functional group (proton donor) (Figure 4.1).

Nicotinamide (NIC), a hydrophilic co-former, is a GRAS compound and forms

intermolecular hydrogen bonds by its effective functional groups (amide group and

pyridine ring) (Figure 4.1). Moreover a ΔpKa of 0.8 between NAP (pKa=4.15) and NIC

(pKa=3.35) may also dictate the reactivity potential leading to co-crystal formation.

I II

Figure 4.1: Molecular structures of Naproxen (I) and Nicotinamide (II)

There are a number of examples where NIC has been utilized as a potential co-

former for ibuprofen (Berry et al., 2008), lamotrigine (Cheney et al., 2009), theophylline

(Lu and Rohani, 2009), celocoxib (Remenar et al., 2007), and carbamazepine (Chieng et

al., 2009). Co-crystals of NAP have also been reported with various co-formers (Ando et

al., 2012; Neurohr et al., 2013; Tumanova et al., 2014). Majority of these studies have

109

focused only on the structural evaluation by single-crystal XRD and in few cases IDR

measurements of co-crystal have been investigated as pharmaceutical properties. None of

the study has grasped the mechanical, formulation and in-vivo behavior of co-crystals

from the perspective of pharmaceutical development. Solubility, dissolution and

bioavailability are important parameters in drug development process (Di et al., 2012).

Furthermore intrinsic solubility and dissolution of a drug are prerequisite for oral drug

delivery. Therefore, there was a need to evaluate the co-crystal in terms of their efficacy

in the improvement of these properties. Keeping these gaps in view, the current study was

designed to investigate the multiple utility of NAP-NIC co-crystal system to modify the

mechanical properties of NAP to make the production process more feasible and the

development of NAP-NIC co-crystal as alternative formulation to overcome the

dissolution issues particularly in acidic pH leading to a dose reduction and greater

fraction of dose absorbed in small intestine. On formulation into tablets by direct

compression method, NAP co-crystal illustrated a faster dissolution especially in acidic

medium as compared to commercial NAP tablets, Neoprox®. As per the literature review,

only a few co-crystal systems have gone through bioavailability studies. To the best of

our information, we are reporting the bioavailability studies of the NAP co-crystal for the

first time.

Materials and methods 4.2.

A description of materials and methods has been given in Chapter 2 Section 2.1.

Synthesis of co- crystals 4.3.

Liquid assisted grinding (LAG) was used to prepare NAP-NIC co-crystal. NAP

was co ground with NIC at 1:1 (1.0 g: 0.5 g), 1:2 (1.0 g: 1.0 g) and 2:1 (1.0 g: 0.25 g)

110

molar ratios in a mortar and pestle with small amount of acetonitrile (~10 µL for each of

100 mg of powder mixture) for 30 min. The powder was collected and stored in air tight

containers at room temperature.

Characterization 4.4.

The pure drug, co-former and co-crystal prepared by LAG were investigated by

PXRD, DSC and Hirshfeld surface analysis. PXRD patterns, DSC thermograms and

fingerprint plots were recorded for NAP, NIC and prepared co-crystal using experimental

conditions specified in Chapter 2 under Sections 2.2.1.1, 2.2.2.1 and 2.2.8.

Intrinsic dissolution studies 4.5.

Standard curve of NAP 4.5.1.

Accurately weighed 10 mg of pure drug powder was dissolved separately in 100

ml of 0.1M HCl pH 1.2 and phosphate buffer pH 7.4 to get NAP concentration of 100

µg/ml. Stock solution was further diluted with respective diluents to obtain a series of

standard solutions over the concentration range of 0.005-10 µg/ml for 0.1M HCl and 1.0-

40 µg/ml for phosphate buffer pH 7.4. Absorbance of each of the standard solution was

measured using a UV/Visible spectrophotometer at the λmax of 226 nm for 0.1 M HCl and

at 330 nm for phosphate buffer pH 7.4 to construct a calibration (standard) curve for

NAP. Linearity of the absorbance data was predicted by calculating the statistical

parameters like intercept, slope and correlation coefficient.

Intrinsic dissolution rate (IDR) 4.5.2.

Intrinsic dissolution rate (IDR) of NAP, physical mixture (PM) and NAP-NIC co-

crystal was determined using the method as described in Chapter 3 Section 3.5.2 except

for few changes as defined below.

111

IDR was explored using 900 ml each of phosphate buffer pH 7.4 and 0.1 M HCl

pH 1.2 at 37 °C and 50 rpm paddle speed. Aliquots (5 ml) were withdrawn from basic and

acidic buffers at predetermined time intervals up to 100 and 800 min, respectively. UV

absorptions were recorded at the λmax of drug in each of the medium.

Drug content analysis 4.6.

For analysis of drug content, 10 mg of co-crystal sample was weighed accurately

and dissolved in each of the medium using volumetric flask to give 10 µg/ml solutions.

Drug content of co-crystal was determined from the above described calibration curve.

Powder compaction/Mechanical properties 4.7.

Tabletability curves 4.7.1.

Three tablets of each pure substance, i.e., NAP, NIC and NAP-NIC co-crystal

(300 mg) were prepared (without excipients) using a hydraulic press fitted with flat faced

dies (13 mm in diameter) over the pressure range of 500–6000 Psi at ambient conditions,

i.e., 22 °C and 33 % RH. All powders were allowed to pass through a 425 µm sieve

before compaction. Tablets were stored at above prescribed conditions for 48 h.

Hardness, expressed in newton (N), was measured using Pharmatron Multitest 50H

hardness tester. Tensile strength was determined using diametral compression test and

calculated by the equation 3.10 described in Chapter 3 Section 3.9.2. Tabletability curves

were plotted between tensile strength and compaction pressure.

Micromeritic properties 4.7.2.

Micromeritic properties, e.g., Carr‘s index (CI), bulk density, tapped density and

angle of repose of NAP and synthesized co-crystal were investigated to evaluate the

112

impact of co-crystallization on the material flow by the methods specified in Chapter 3

Sections 3.7.2, 3.7.3, 3.7.4 and 3.7.5.

4.7.2.1. Hausner’s ratio (HR)

Hausner‘s ratio (HR), related to inter particle friction, is a good mean to predict

powder flow properties and can be calculated by using equation

Where

dT and dB = tapped and bulk density of the powder respectively

A powder with a HR < 1.25 is associated with low inter particle friction and is

indicative of good flow properties where as a HR > 1.6 represents more cohesive

powders with poor flowing properties.

Formulation of tablets 4.8.

Tablets of NAP-NIC co-crystal (F1) were prepared by direct compression

technique. Co-crystal powder equivalent to 250 mg of NAP, croscarmellose sodium (7 %

of the weight of co-crystal) and Avicel PH102 (10 % of the weight of co-crystal) were

passed through a 425 µm sieve and thoroughly mixed for 10 min. Finally the mixture was

blended with magnesium stearate (0.5 %) for another period of 1 min and compressed

with a hydraulic press fitted with a die 13 mm in diameter.

Evaluation of the tablets 4.9.

Co-crystal tablets and reference formulation Neoprox® were evaluated for

hardness, disintegration and in-vitro dissolution.

113

Hardness and disintegration test 4.9.1.

Hardness and disintegration test were carried out as per the methods given in

Chapter 3 Sections 3.9.1 and 3.9.3.

In-vitro drug release study 4.9.2.

In-vitro release of reference formulation, Neoprox® (F0) and co-crystal tablets (F1)

was conducted in 900 ml each of the dissolution medium as used in IDR section. These

dissolution media were used in order to simulate the pH change along the GIT. A USP

type II dissolution apparatus was used for dissolution studies with a rotation speed of 50

rpm. Aliquots (5 ml) were taken at predetermined intervals (0, 5, 10, 15, 30, 45, 60, 75,

90 and 120 min) from phosphate buffer pH 7.4 and (0, 30, 60, 90, 120, 180, 240 and 300

min) from 0.1M HCl and replaced with fresh media maintained at 37 °C. The samples

were filtered, diluted and analyzed for NAP by UV/Visible spectrophotometer at 330 and

226 nm. All the analyses were performed in triplicate. Percent drug release was

determined from the respective calibration curves. Various kinetic equations such as zero

order, first order, and Higuchi, Korsmeyer-Peppas and Hixon-Crowell models were

applied using DDsolver to determine the best fit of release data based on correlation

coefficient, R2 (Zhang et al., 2010).

In-vivo studies in sheep 4.10.

Pharmacokinetic (PK) profiles of pure NAP and co-crystal were determined by

carrying out a single dose sheep exposure study. The study was performed according to

the research protocols got approval by animal ethical committee, University College of

Pharmacy, University of the Punjab, Lahore, Pakistan (ref. no. AEC/PUCP/1047A). The

114

studies were executed in agreement with Helsinki Declaration and Animal Scientific

Procedure Act 1986 (UK).

Twelve healthy adult 2-3 years aged sheep with a weight of 25-30 Kg were used

for in-vivo studies. Animals were maintained, fed and tagged as described in Chapter 3

Section 3.11.1.

Drug administration and collection of blood samples 4.10.1.

A dose of 10 mg/kg body weight of pure NAP and equivalent co-crystal enclosed

in gelatin capsules was orally administered to two groups of sheep (n= 6) after an

overnight fast. From each sheep, blood sample (3 ml) was collected from jugular vein in

sterile heparinized vials at 0 (before drug administration), 0.5, 1, 2, 4, 6, 8, 12, 24 and 36

h post dose intervals. The Whole blood was subjected to centrifugation at 3500 rpm for 5

min to separate the plasma. Separated plasma samples were transferred to labeled

Eppendorf tubes and stored at -20 °C for further studies.

Extraction of NAP from sheep plasma 4.10.2.

After allowing the plasma to thaw, phosphoric acid (0.5 ml) was added to plasma

(0.5 ml). Then a V/V mixture (3 ml) of ethyl acetate and hexane (2:3) was added to above

mixture followed by vortex mixing for 2 min and centrifugation at 10,000 rpm for15 min.

The organic layer was allowed to dry under a stream of nitrogen. The solid was

reconstituted in acetonitrile (1ml) and was filtered using 0.22 µm syringe filter.

Analysis of NAP and HPLC conditions 4.10.3.

Chromatographic separation was achieved at room temperature using the HPLC

specifications as given in Chapter 2 Section 2.2.7. The solvent system was comprised of

20 Mm phosphate buffer (pH 7.4) containing 0.1 % trifluoroacetic acid (TFA)-

115

acetonitrile (65:35 V/V).The mobile phase was pumped at a flow rate of 1 ml/min after

being filtered through a 0.45μm membrane filter and degassed by ultrasonication. Clear

filtrate (20 µl) was injected into the HPLC with a run time of 15 min and a previously

reported method was used to determine the NAP content (Yilmaz et al., 2013) under

isocratic conditions. NAP was eluted at ~5.8 min under the prescribed conditions and

drug concentrations were determined using a PDA detector at 225 nm.

Preparation of stock and standard solutions 4.10.4.

A standard stock solution of NAP (1.0 mg/ml) was prepared in methanol and was

further diluted with mobile phase to get a working stock solution (100 µg/ml) which on

further dilution with mobile phase produced a series of solutions. Appropriate volumes of

NAP standard solutions were added to drug free sheep plasma to give calibration curve

standards (n= 3) in the range of 0.1–15µg/ml following the scheme given in Table 3.1

Chapter 3 Section 3.11.5. Each standard prepared in plasma was treated by procedure as

described in Section 4.10.2 for extraction of NAP from the plasma. Each of the standard

prepared in plasma and mobile phase (20 µl) was injected in to the HPLC to determine

the peak areas. Standard curve was plotted between peak areas of standards prepared in

plasma and the drug concentrations (0.1, 0.25, 0.5, 1.0, 5.0, 10.0 and 15.0 µg/ml).

Peak areas of NAP standards prepared in mobile phase and plasma containing

equivalent concentrations of the drug were compared to determine the absolute recovery

of NAP. Quantification of NAP in sheep plasma was done by reference to the resultant

standard curve.

116

Pharmacokinetic (PK) analysis 4.10.5.

Non compartmental pharmacokinetic metrics were calculated using PK solver

(Version 2.0) pharmacokinetic program (Zhang et al., 2010). Cmax and tmax were read

from the plasma level time curve. Trapezoidal rule was employed to get estimation of the

AUC.

Statistical analysis 4.10.6.

SPSS (IBM SPSS Statistics 22) was used for statistical analyses. To compare the

means of samples, PK data was subjected to independent samples t-test. Statistical

significance was set at p < 0.05.

Results and discussion 4.11.

Methods used for the co-crystallization of APIs can be classified as solution based

and solid state (mechanochemical) methods. A numbers of variables dictate the capacity

of an API to be fabricated into co-crystal including co-former type, API/co-former ratio,

solvent systems, pressure, temperature and crystallization method. Crystallization from

solution can yield well formed single crystals required for single-crystal XRD to obtain

an absolute crystal structure determination. However, these methods are limited by

selection of a suitable solvent for the starting materials and in some instances by the

solvate formation. Mechanochemical (dry and liquid assisted grinding) methods, on the

other hand, are more environment friendly due to the absence or least amount of solvent,

have a short reaction time and are particularly important when screening for co-crystals

of APIs with low solubility (Friščić et al., 200 ). However, with dry grinding the energy

required to complete the co-crystallization of compounds is lacking (Ross et al., 2016).

One method to overcome this limitation is introduction of a small amount of a solvent

117

during grinding (LAG) which acts as a catalyst and is depleted during the course of

grinding. Therefore interest in LAG as a method of co-crystal preparation has been

developed over past few years (Childs and Hardcastle, 2007; Ullah et al., 2016;

Douroumis et al., 2017). For the above described reasons, LAG was used for the

preparation of NAP co-crystal using a small amount of acetonitrile (~10 µL for each of

100 mg of powder mixture).

Characterization 4.11.1.

Difference in melting point, PXRD, DSC and Hirshfeld surface analysis were

taken as characterization tools to evaluate the co-crystal.

4.11.1.1. Powder X-ray diffraction (PXRD)

Mixture of NAP with NIC at 2:1 molar ratio was resulted in co-crystal. Other

ratios resulted either in PM or crystals of NAP or NIC. Co-crystal exhibited a PXRD

pattern that did not match the patterns of the starting materials (Figure 4.2).

Characteristics peaks at 2θ value of 6.6°, 12.7

°, 13.46

° and 19.01

° for NAP and 11.39

°,

14.86°, 22.19

°and 36.95

° for NIC were not observed in the PXRD pattern of co-crystal

(Figure 4.2). Moreover new peaks were appeared at 2θ value of 5.46°, 10.79

°, 12.08

°,

16.19°, 17.20

° and 18.90

° suggesting the existence of interactions between NAP and co-

former. These results are in agreement with the previously published data (Neurohr et al.,

2013). Comparison of experimental pattern with simulated pattern obtained from

Crystallographic information file (CIF) using MERCURY did not show any significant

differences in peak positions, confirming the production of NAP-NIC co-crystal (Figure

4.3).

118

Figure 4.2: PXRD patterns of NAP, NIC, NAP-NIC co-crystal and calculated pattern

Figure 4.3: Comparison of PXRD patterns of experimental and calculated data by Dash.

Experimental (black line), calculated (red line) and difference (blue line)

119

For lattice parameters, experimental pattern was indexed and analyzed. The

parameters implemented in the program DASH (David et al., 2006) were the background

subtraction, Pawley refinement, indexing and peak fitting. However because of the poor

quality of PXRD pattern full structure solution was not possible. Therefore only lattice

parameters were compared as shown in Table 4.1.

Table 4.1: Unit cell parameters of NAP-NIC co-crystal (a) obtained from CIF CCDC

904098 (Ando et al., 2012) (b) calculated from experimental PXRD

NAP-NIC co-crystala NAP-NIC co-crystal

b

Crystal System orthorhombic orthorhombic

Space group P 212121 P 212121

a (Å) 5.96338(11) 5.96440(15)

b (Å) 15.1283(3) 15.0868(4)

c (Å) 32.5411(6) 32.4857(4)

V (Å3

) 2935.72(9) 2918.045(5)

Z 4 4

Dc (g cm-3

) 1.318 -

T(K) 223(2) 298(2)

4.11.1.2. Differential scanning calorimetry (DSC)

DSC is another important technique for the characterization of co-crystals.

Comparison of thermal behavior of co-crystals with the starting materials is not only used

for identification purpose but also gives evidence about the stability (Pagire et al., 2017)

and dissolution of the prepared entities (Karagianni et al., 2018). Melting point in

specific, being a fundamental physical property, measures the energy needed to overcome

120

the attractive forces holding a crystal structure. It is influenced by chemical structure,

intermolecular interactions, crystal lattices, molecular symmetry and conformational

degree of freedom etc. (Katritzky et al., 2001; Simperler et al., 2006); therefore any

change in these parameters may result in relative change in thermodynamic stability. A

better molecular symmetry in crystals or strong hydrogen bond is prone to intensify the

solid state intermolecular interactions with a consequent enhanced melting temperature

(Simperler et al., 2006). On the other hand low melting point crystals can show better

dissolution properties. A review on melting points of fifty reported co-crystals has shown

that half of co-crystals had melting points between those of the API and co-former and

approximately 40 % were less than that of either starting material (Schultheiss and

Newman, 2009).

DSC thermograms of co-crystal and starting materials are shown in Figure 4.4.

Co-crystal indicated an endothermic melting event with mid temperature 113.41 °C which

was lower than the mid points of the individual components (NAP-156.84 °C and NIC

132.80 °C). This temperature was also different from the previously reported co-crystals

of NAP, i.e., NAP-Urea, NAP-Thiourea and NAP-Picolinamide at 121.43 °C, 150.27

°C

and 92.85 °C, respectively (Kerr et al., 2017; Rajurkar et al., 2015).

121

Figure 4.4: DSC thermograms of NAP, NIC and NAP-NIC co-crystal

4.11.1.3. Hirshfeld analysis

Fingerprint plots (for NAP and co-crystal) produced with CRYSTAL EXPLORER

(Wolff et al., 2013) are shown in Figure 4.5. Since these plots are distinct for any crystal

structure, they give a powerful visual tool to elucidate and compare intermolecular

interaction, and spotting common features and trends as well. Both fingerprint plots show

sharp "tails" and spikes. The outer two tails, correspond to N-H...O=C hydrogen bond

extending down to around de and di = 1.0 Ǻ, the upper one corresponds to the hydrogen

bond donor and lower one to the hydrogen bond acceptor. As evident from the Figure 4.5,

finger print plot obtained from pure NAP crystal structure showed sharp and well

40 80 120 160 200 240

-4

-2

0

2

4

6

8

10

Temperature (°C)

Co-crystal

NIC

NAP

Hea

t fl

ow

(w

/g)

122

resolved hydrogen bond peaks as compared to co-crystal owing to stronger hydrogen

bond. This resulted in more compressed plots representing a better packed structure.

I II

Figure 4.5: Two dimensional fingerprint plots of NAP (I) and NAP-NIC co-crystal (II)

4.11.1.4. Intrinsic dissolution rate (IDR)

UV calibration curves for NAP in each of the dissolution medium (Figures 4.6

and 4.7) gave R2 values of greater than 0.99 and thus found acceptable to determine the

drug content, IDR and dissolution rate from the formulations.

IDR minimizes the effect of particle surface area on dissolution kinetics and has

been extensively used to characterize dissolution behavior of APIs. In this study, IDR

was determined to evaluate the dissolution characteristics of the NAP-NIC co-crystal in

0.1M HCl and phosphate buffer pH 7.4. Figures 4.8 and 4.9 represent intrinsic dissolution

profiles for NAP, PM and co-crystal in 0.1M HCl and phosphate buffer pH 7.4.

Being a weakly acidic drug, NAP is only slightly soluble in acidic pH. But as a

result of co-crystallization, a promising increase in IDR was noticed in 0.1M HCl. The

IDR from 0 to 800 min in 0.1M HCl for the co-crystal was 51.0 µg/cm2-min.

This value

was 7.08 times higher than that for NAP (7.2 µg/cm2-min) and 2.76 times higher than PM

(14.4 µg/cm2-min). Similarly, the IDR from 0 to 100 min in phosphate buffer pH 7.4 for

123

the co-crystal was 1520 µg/cm2-min.

This value was 2.75 times higher than that for NAP

(552 µg/cm2-min) and 1.30 times higher than PM (720 µg/cm

2-min). NIC, being a

hydrophilic co-former, may be responsible for decreasing the interfacial tension acting

between the lipophilic drug particles and dissolution media and imparting a positive

effect on the dissolution profiles of PM.

Figure 4.6: Calibration curve of NAP in 0.1M HCl

Figure 4.7: Calibration curve of NAP in phosphate buffer pH 7.4

y = 0.2599x + 0.0136

R² = 0.9953

0

0.05

0.1

0.15

0.2

0.25

0.3

0 0.2 0.4 0.6 0.8 1

Ab

sorb

an

ce


y = 0.0068x + 0.0047

R² = 0.998

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5 10 15 20 25 30 35 40

Ab

sorb

an

ce


124


0.1M HCl (7.2 and 51.0 µg/cm2-min respectively) (n= 3)


phosphate buffer pH 7.4 (552 and 1520 µg/cm2-min respectively) (n= 3)

0

5

10

15

20

25

30

35

40

45

50

0 100 200 300 400 500 600 700 800

Cu

mu

lati

ve

dru

g d

isso

lved

(m

g/c

m2)

Time (min)

NAP

Co-crystal

PM

0

20

40

60

80

100

120

140

160

0 20 40 60 80 100

Cu

mu

lati

ve

dru

g d

isso

lved

(m

g/c

m2)

Time (min)

NAP

Co-crystal

PM

125

Compaction/Micromeritic behavior of powders 4.11.2.

Heckel equation and tensile strength determinations are popular means of

studying powder compaction. Heckel model can easily distinguish between plastic,

elastic and brittle materials based on mean yield pressure (Py) values derived from the

Heckel plot. Therefore materials can be classified using Heckel equation (Density

pressure relationship). However, Py values for the same material calculated by different

researchers, are greatly varied due to many experimental (in-die or out-of-die

compaction) (Ilić et al., 2013) and physical factors (compaction speed, compaction

pressure, and particle size) (Patel et al., 2007). On the other hand, tablet tensile strength

can be precisely measured during formulation development and is independent of

tableting speed (Tye et al., 2005). Moreover most of the published data evaluating

mechanical/compaction behavior of co-crystals has focused on tensile strength

measurement (Chow et al., 2012; Karki et al., 2009; Sun and Hou, 2008).

Tensile strength is a measure of the powder cohesiveness which in turn influences

different aspects of powder processing including milling, mixing and storage and powder

compression to produce granules or compacts and hence has great industrial importance.

Tensile strength is defined as force per unit area required to split a powder bed in tension

and is influenced by particle size distribution (Cheng,1968) and moisture content of the

powder (Esezobo and Pilpel, 1977). Tensile test, being a fundamental mechanical test,

indicates the plasticity, modulus of elasticity and elastic limit etc. Powder particles

undergoing tablet processing must be of sufficient compressibility and be strong enough

to withstand undesired breakage during handling. Bond strength can be obtained from the

tensile strength of the compacts (Itiola and Pilpel, 1986a, 1986b).

126

4.11.2.1. Tabletability profiles

Tabletability is the ability of a powder to be converted into a tablet of definite

strength under the influence of compaction pressure (Joiris et al., 1998). A plot between

tensile strength and compaction pressure is representation of the tabletability of a powder.

Poor tabletability of a powder indicates plastic deformation during compaction followed

by high elastic recovery. NAP is unsuitable for direct tableting due to its poorly

compressible properties (Saritha et al., 2012). Improvement in mechanical properties of

paracetamol, carbamazepine and caffeine has been previously reported as a result of co-

crystallization (Karki et al., 2009; Rahman et al., 2011; Sun and Hou, 2008). In this

study, we have investigated the tableting properties of pure NAP and its co-crystal.

Tabletability profiles of pure NAP, NIC and co-crystal are shown in Figure 4.10.

Compaction properties of NAP and NIC were poor and tablets were low in strength. A

drop in tensile strength of NAP tablets was noticed at > 3000 psi as the compaction

pressure was increased (Figure 4.10). This could be due to over compaction where a

greater elastic recovery produced weaker tablets due to interruption of inter particulate

bonds (Sun, 2011). In contrast, the prepared co-crystal showed good tabletability and

exhibited a required tensile strength of more than 2 MPa. In fact tensile strength of co-

crystal tablets continued to increase with increasing pressure. This may be explained by

the fact that higher compaction pressure caused more permanent plastic deformation of

co-crystal particles leading to larger boding area between the adjacent particles (Chang

and Sun, 2017). But the tensile strength gradually leveled off which is a characteristic

behavior of plastic materials (Chow et al., 2012). Moreover no severe lamination was

127

observed for co-crystal. Thus, the co-crystal powder is more suitable for tableting than

the NAP.

4.11.2.2. Material flow behavior

Micromeritic properties of NAP-NIC co-crystal have been presented in Table 4.2.

These values disclosed that co-crystal had improved flowability as well as

compressibility. The high bulk and tapped densities for co-crystal (0.363 and 0.46 g/cm3)

as compared to pure drug (0.22 and 0.44 g/cm3) indicated a lower porosity leading to

improved compressibility of the co-crystal powder. CI for the co-crystal (9.7 %) was

observed to be lower in comparison to NAP (22 %) suggesting that the co-crystal powder

is suitable for direct tableting (Thenge et al., 2017). Better flow properties of co-crystal

were reflected by low values of HR and angle of repose (1.26 and 28.79°) as compared to

the pure drug which showed higher values for these parameters (2 and 41.48°).

Figure 4.10: Tabletability plots of NAP, NIC and NAP-NIC co-crystal

0

0.5

1

1.5

2

2.5

3

3.5

4

0 1000 2000 3000 4000 5000 6000

Ten

sile

str

en

gth

(M

pa

)

Compaction pressure (psi)

NAP

NIC

Co-crystal

128

Table 4.2: Micromeritic properties of NAP and NAP-NIC co-crystal

Parameters NAP

Mean+SD Co-Crystal

Mean+SD

Bulk density (g/cm3) 0.22+ 0.013 0.363+ 0.017

Tapped density (g/cm3) 0.44+0.02 0.46+0.02

Car‘s index (%) 22.0 9.7

Hausner‘s ratio 2.0 1.26

Angle of repose (°) 41.4+0.503 28.79+0.303

% content 97.88+6.13

In-vitro performance of tablets 4.11.3.

A comparison of the pharmaceutical properties and key parameters describing

dissolution profiles for commercial tablet, Neoprox® (F0) and co-crystal formulation (F1)

are shown in table 4.3. Dissolution was carried out in 0.1M HCl and phosphate buffer

pH 7.4 (Figures 4.11 and 4.12) as these solutions are used in official compendia in lieu of

gastric and intestinal environments, respectively. F1 showed enhanced dissolution

profiles in both media. In phosphate buffer pH 7.4, more than 90 % of NAP was released

from F1. In contrast only 80 % of drug was released from Neoprox® (Table 4.3).

Improved drug release from F1 might be attributed to higher dissolution rate leading to a

higher solution concentration at the same time point. Most promising effect was observed

in acidic medium. More than 70 % of drug was released from FI as compared to F0 (38

%). It is pertinent to state that IDR of NAP co-crystal in both acidic and basic medium

have shown enhanced profiles (Figures 4.8 and 4.9) without adding any surfactants,

proposing this increase as a result of change in intrinsic properties of NAP on co-

129

crystallization. Similar to that co-crystal formulation has also shown enhanced

dissolution rate as compared to the commercial formulation. This suggests that the

formulation difference may not have significant effect on the dissolution results as we

have kept co-crystal formulation as simple as possible and the main effect may be

credited to a change in intrinsic property due to co-crystallization. This enhanced

dissolution rate for F1 may decrease the irritating effect of NAP on GIT as the drug may

have less residence time in the stomach.

When dissolution data was fitted to various kinetic models, the release profiles of

F0 and F1 followed korsmeyer-Peppas model in both dissolution media based on R2

values (Table 4.4). Moreover both formulations exhibited Fickian release (n ˂ 0.45).

Table 4.3: Physical parameters and % drug released of NAP in 0.1 MHCl and phosphate

buffer pH 7.4 from commercial (F0) and NAP-NIC co-crystal tablets (F1)

Parameters F0 (Neoprox) F1

Diameter (mm) 10+0.01 13+0.01

Breaking force (N) 98.00+1.3 217.56+3.8

Dis. time (min) 2.5+0.8 4.5+1.4

Drug

released

(%)

0.1M HCL 60 min 26.31 44.65

180 min 38.70 69.03

phosphate

buffer pH

7.4

15 min 55.20 67.87

60 min 81.44 93.66

130


(F1) in 0.1M HCl


(F1) in phosphate buffer pH 7.4

0

10

20

30

40

50

60

70

80

0 50 100 150 200 250 300

Dru

g R

elea

sed

(%

)

Time (min)

F0

F1

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100 120

Dru

g R

elea

sed

(%

)

Time (min)

F0

F1

131

Table 4.4: Drug release kinetics from Neoprox (F0) and NAP-NIC co-crystal

formulation (F1) in 0.1 M HCl and phosphate buffer pH 7.4

Release kinetics F0 F1 F0 F1

0.1M HCl Phosphate buffer pH 7.4

Zero order R2 0.0762 0.1018 0.7383 0.5443

First order R2 0.3828 0.7229 0.7625 0.9495

Higuchi model R2 0.8475 0.8510 0.7026 0.6635

Hixon-

Crowell model

R2 0.2888 0.5833 0.6291 0.7515

Korsmeyer-

Peppas model

R2 0.9902 0.9839 0.9704 0.9638

n 0.260 0.266 0.235 0.224

In-vivo performance of pure NAP and NAP-NIC co-crystal 4.11.4.

For measurements of low amounts of drugs in biological samples, HPLC is a

powerful analytical tool with high specificity and precision. HPLC method employed to

detect NAP was found to be efficient in analyzing a large number of sheep plasma

samples for PK studies of NAP-NIC co-crystal. The method of extraction of NAP from

the sheep plasma was simple and appropriate as no peak was observed at the retention

time of the NAP (Figure 4.15). Peak of NAP was well resolved under isocratic conditions

with mobile phase consisting of 20 Mm phosphate buffer (pH 7.4) containing 0.1%

trifluoroacetic acid (TFA)-acetonitrile (65:35 V/V) delivered at a flow rate of 1ml/min.

Under the prescribed conditions, NAP was found to be eluted at ~5.8 min.

4.11.4.1. Standard curve and percent recovery

Peak areas of spiked plasma samples for NAP concentrations of 0.1, 0.25, 0.5,

1.0, 5.0, 10.0, and 15.0 µg/ml were measured and a calibration curve (Figure 4.13) was

132

generated. The assay was found to be linear over the above mentioned range of

concentration with correlation coefficient R2= 0.9987. The mean % recovery values for

NAP over the above concentration range were found to be 92.44-102.44 % (Table 4.5)

with RSD < 5 % claiming accuracy of the analytical method. The limits of detection and

quantification were 0.03 and 0.1 µg/ml, respectively indicating that the method was

sensitive.

Figure 4.13: Calibration curve of NAP in drug free sheep plasma

y = 402175x + 48537

R² = 0.9987

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

0 3 6 9 12 15

Pea

k a

rea


133

Table 4.5: Recovery of NAP from sheep plasma (n= 3)

Concentration

(μg/mL)

Peak area Recovery (%)

Standard solution Spiked plasma

0.1 46577 44151 94.79+2.39

0.5 241001 239821 96.31+3.02

1.0 396008 371160 93.72+4.77

2.5 1231213 1138121 92.44+2.87

5.0 2126324 2178230 102.44+3.01

10.0 4500050 4305994 95.68+4.89

15.0 6057013 5978991 98.71+3.45

Figures 4.14, 4.15, 4.16 and 4.17 show representative chromatograms of NAP in

mobile phase, from blank sheep plasma and plasma spiked with various concentrations of

NAP (0.5 and 2.5 µg/ml) while figures 4.18 and 4.19 are illustrative of chromatograms

from plasma of sheep administered at an oral dose of 10 mg/Kg body weight of pure

NAP and equivalent co-crystal at 2 h and 30 min respectively.

Figure 4.14: HPLC chromatogram of NAP (10 µg/ml) in mobile phase

0

20000

40000

60000

80000

100000

0 1 2 3 4 5 6 7 8 9 10 11 12

Inte

nsi

ty (

mA

u)

Time (min)

134

Figure 4.15: Representative chromatogram of drug free sheep plasma


µg/ml)

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3 4 5 6 7 8 9 10 11 12

Inte

nsi

ty (

mA

u)

Time (min)

0

10000

20000

30000

40000

50000

60000

70000

0 1 2 3 4 5 6 7 8 9 10 11 12

Inte

nsi

ty (

mm

Au

)

Time (min)

135


µg/ml)


mg/Kg body weight of pure NAP (2 h)

0

20000

40000

60000

80000

100000

120000

140000

0 1 2 3 4 5 6 7 8 9 10 11 12

Inte

nsi

ty (

mA

u)

Time (min)

0

200000

400000

600000

800000

1000000

0 1 2 3 4 5 6 7 8 9 10 11 12

Inte

nsi

ty (

mA

u)

Time (min)

136


mg/Kg body weight of equivalent NAP-NIC co-crystal (30 min)

4.11.4.2. Pharmacokinetic (PK) metrics

Co-crystallization is a useful technique for the enhancement of physicochemical

properties of the drugs. Poor physicochemical properties especially the solubility show

low oral bioavailability resulting in enhanced dose and adverse effects for the drugs. NAP

has low aqueous solubility and dissolution rate which limit its oral bioavailability. In the

present study, a 2:1 NAP-NIC co-crystal synthesized by LAG demonstrated a faster

dissolution in 0.1 M HCL and phosphate buffer pH 7.4 as compared to pure NAP and

PM. As the in-vitro dissolution rate, partially or completely controls the rate at which a

drug dissolves in GIT, the rate of appearance of drug in the blood will therefore be

controlled (Levy, 1961) hence, NAP-NIC co-crystal was further evaluated for

bioavailability and PK studies

Quantification of the drug absorption, distribution, biotransformation and

excretion in the animals or humans is the primary goal of PK studies. This information

can be used to forecast the effects of changes in the route of drug administration, dose

0

100000

200000

300000

400000

500000

0 1 2 3 4 5 6 7 8 9 10 11 12

Inte

nsi

ty (

mA

u)

Time (min)

137

and dosage regimen on the drug accumulation and disposition. The PK of a drug can be

studied by measuring the change in plasma concentration as a course of time action and

drawing the plasma level time curve.

In order to compare the bioavailability, pure NAP and NAP-NIC co-crystal

enclosed in gelatin capsules were administered to overnight fasted sheep to eliminate the

effect of food on drug absorption. Food was continued after 4 h. No adverse effects were

observed during the experimental period following the oral administration of a single

dose of 10 mg/kg body weight of pure NAP and equivalent co-crystal. PK parameters

were computed using non compartmental approach by PK solver version 2.0.

The plots for mean NAP and NAP-NIC co-crystal plasma concentration after a

single dose oral administration (10 mg/kg) in sheep have been represented in Figure 4.20.

On oral administration to sheep, drug absorption from NAP-NIC co-crystal was

considerably higher than pure NAP throughout the period of study, i.e., up to 36 h (Table

4.6). This rapid absorption can provide a rapid onset of activity and can be credited to the

improvement in physicochemical properties of NAP as a result of co-crystallization.

Table 4.7 shows mean PK metrics for NAP and co-crystal calculated from the sheep

model study data.

138

Table 4.6: Plasma concentration of NAP and NAP-NIC co-crystal in sheep at different

time intervals following oral administration at a single dose of 10 mg/Kg body weight

(n= 6)

Time (h) NAP concentration

(µg/ml)

Mean+SD

NAP concentration from co-

crystal

(µg/ml)

Mean+SD

0.5 15.29+3.92 25.05+6.57

1.0 18.06+4.55 36.78+5.10

2.0 25.18+5.52 40.99+6.59

4.0 25.21+5.75 41.19+7.20

6.0 21.62+5.92 37.85+4.78

8.0 20.14+5.96 34.27+5.02

12.0 18.19+5.62 31.17+4.98

24.0 14.22+2.81 23.80+7.31

36.0 11.98+3.32 19.77±5.73

Figure 4.20: Sheep plasma concentration with time for NAP and NAP-NIC co-crystal

(n= 6)

0

5

10

15

20

25

30

35

40

45

50

0 5 10 15 20 25 30 35

Co

nce

ntr

ati

on

(μ

g/m

l)

Time (h)

Mean conc. NAP

Mean Conc. Co-

crystal

139

Table 4.7: Pharmacokinetic parameters of NAP and NAP-NIC co-crystal in sheep

following oral administration at a single dose of 10 mg/Kg body weight (n= 6). *R.B

relative to NAP

Parameters NAP

Mean+SD Co-Crystal

Mean+SD P-value

Cmax (μg/ml) 26.08+5.13 42.03+ 8.31 0.042

tmax (h) 3.00+1.09 3.00+1.09 1.00

AUC0-36h (μgh/ml) 601.13+49.2 1017.09+80.53 0.018

AUC0-∞ (μgh/ml) 1302.57+215.32 2119.44+345.13 0.027

AUMC0-∞ (μgh/ml) 63900.05+1872.21 121423.0+2869.01 0.075

MRT (h) 50.05+7.89 57.88+6.13 0.245

Kel (h-1

) 0.023+ 0.004 0.018+0.004 0.296

t1/2 (h) 31.78+4.99 37.96+7.89 0.275

CL (μg/ml)/h 0.187+0.045 0.118±0.038 0.069

Vd (μg/ml) 6.28+0.37 5.35+0.45 0.855

*Relative

bioavailability(R.B) 1.62

a) Maximum plasma concentration (Cmax)

An analysis of the peak concentration and time to peak concentration provides an

evaluation of the rate of absorption (Gibaldi, 1991). Cmax for the co-crystal

(42.03+8.31µg/ml) was 1.62 times that of pure drug (26.08+ 5.13µg/ml) and was also

appeared to be significant (p= 0.042). Hence the NAP-NIC co-crystal can be taken as an

improved form of NAP as an analgesic agent due to its rapid availability in the blood.

However the time for maximum concentration for the NAP and co-crystal (3.0+1.09 h)

140

remained unaltered (Table 3.7). The increase in peak concentration can, therefore, be

attributed to a decreased elimination of drug from the co-crystal since the accumulation

of the drug will be higher when the output is reduced. Thus its effects may be prolonged

and potentiated.

b) Area under the curve (AUC)

A comparison of the AUCs gives an estimate of the extent of the absorption

(Shargel et al., 2015). NAP-NIC co-crystal showed a significantly better (p= 0.027)

AUC0-∞ (2119.44+345.13 µgh/ml) than the pure drug (1302.57+215.32 µgh/ml).

c) Mean residence time (MRT)

The MRT computed after single dose oral administration of co-crystal was found

to be extended to 57.88+6.13 h as compared to MRT for pure NAP (50.05+7.89 h). This

suggests the prolonged persistence of co-crystal most probably due to its slow elimination

which might be responsible for enhanced absorption of drug from the co-crystal.

d) Disposition parameters

Co-crystallization of NAP with NIC altered the disposition profile of the drug. A

higher value of Kel for the NAP (0.023+0.004 h-1

) showed its fast elimination as

compared to co-crystal which appeared to be eliminated relatively slowly as revealed by

its lower Kel value (0.018+0.004 h-1

). Prolongation of t1/2 (37.96+7.89 h) for the co-

crystal as compared to pure NAP (31.78+4.99 h) might be due to a reduction in hepatic

metabolism resulting in an increase in absorption of drug from the co-crystal. The

Prolonged half life can be of great significance for chronic anti inflammatory therapy.

Clearance is regarded as an indicator of metabolism. Hence, a lower value of clearance

for the co-crystal, i.e., 0.118+0.038 (μg/ml)/h also supports the above finding. The mean

141

Vd values computed after single dose oral administration of NAP and its co-crystals were

6.28+0.37 and 5.35+0.45 µg/ml (Table 4.7) respectively. The low Vd for co-crystal might

be responsible for the high plasma drug concentration after a given dose.

e) Relative bioavailability

Relative oral availability for the NAP co-crystal was estimated by comparisons of

the AUCs of the reference (pure NAP) and test sample (NAP-NIC co-crystal) according

to equation 3.16 given in Chapter 3 Section 3.12.5.2 and was observed to be 1.62 fold

than pure drug.

142

Conclusion 4.12.

The present study has illustrated that co-crystallization with NIC has improved

pharmaceutical properties including intrinsic and in-vitro dissolution rate particularly in

acidic medium, tableting properties and in-vivo profile for poorly water soluble drug

NAP. The results also suggest that NAP-NIC co-crystal can be further developed into

high quality drug products. A pictorial representation starting from the synthesis of NAP-

NIC co-crystal, its characterization and improvement in mechanical, in-vitro and in-vivo

performance has been given in Figure 4.21.

Figure 4.21: Pictorial representation showing the synthesis and characterization of NAP-

NIC co-crystal and enhancement in in-vitro and in-vivo performance

143

Chapter 5

144

5. Conclusion and Future Perspective

Conclusion 5.1.

The co-crystallization, one of the crystal engineering techniques, described in this

thesis, has been shown to be efficacious for the preparation of more soluble and

compressible forms of PCM and NAP. This work has also addressed important topics

related to the development of pharmaceutical co-crystal formulations.

A physical screening process has revealed LAG and SE as appropriate methods

demonstrating their potential use for discovering new co-crystals of PCM employing 1:1

and 2:1 ratios of the PCM and CAF, respectively. A key factor in contributing to this

success has been a combination of a range of techniques including PXRD, DSC, FTIR

and SEM to detect a form change in the co-crystal samples. The solubility behavior of

poorly soluble drugs PCM and NAP was improved significantly when the co-crystal form

of the drug was used. This was shown by enhanced intrinsic dissolution rates of co-

crystals in comparison to the starting components and physical mixtures. Pre formulation

(solubility, bulk density, and powder flow and compression properties) and post

formulation (Hardness, disintegration, compaction and in-vitro dissolution) studies

exhibited an improvement in the behavior of co-crystals. The presence of certain features

in the crystal structures of the co-crystals could be contributed to the differences in the

compressibility and hardness from the pure drugs. These findings led to the development

of improved formulations.

Co-crystals were successfully incorporated into tablets by direct compression

without any signs of capping and lamination credited to the improvement in plasticity and

145

mechanical properties of co-crystals. In-vitro dissolution profiles of the co-crystal

formulations were found to be enhanced in comparison to the reference formulations.

The oral bioavailability of both co-crystals measured in sheep model was also

shown to be improved in comparison to the pure drugs as a result of modifications in the

absorption and disposition parameters.

Future perspective 5.2.

Bulk manufacturing 5.2.1.

As LAG and SE methods have limitations with respect to scale up, for PCs to

move from small scale screening to a possible option for the development and

subsequently drug products, scalable processes ensuring continuous co-crystal

manufacturing and proposing high co-crystal purity and yield must be established.

Therefore a comprehensive study is required to develop or modify the crystallization

methods.

CMAC (Continuous manufacturing and advance crystallization) is a leading

project in the field of pharmaceutical manufacturing. Co-crystals can be a good candidate

for this project.

Product Stability 5.2.2.

Stability is an important parameter in formulation design. Although our study has

shown that the prepared co-crystal entities are stable over the test conditions used, the

detailed stability studies on formulations prepared from these co-crystals using varied

environmental conditions (according to ICH guidelines) can form the subject of further

studies.

146

Structure property relationship 5.2.3.

Single crystal structure for PCM-CAF co-crystal should be devised which will not

only provide the exact packing and conformation of molecules but also the relationship of

structure variation with various pharmaceutical properties.

Intellectual property 5.2.4.

Co-crystals represent a broad patent space because of the availability of a large

number of co-formers available based on the GRAS and EADUS lists. Thus co-crystals

find an opportunity to generate a broader range of intellectual property. By performing

above described studies on our established co-crystals, application for the grant of patent

can be filed to relevant authorities.

A reduced dose study (half the dose of co-crystal formulation) may further be

suggested in a group of similar sheep to compare the PK parameters. A reduced dose may

match the drug exposure (AUCs) with full dose standard PCM formulation and may

produce similar t1/2 and elimination rates. This could confirm the concentration dependent

clearance of the drug in the animal model.

147

Chapter 6

148

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175

Annexures

176

Annexure A: Crystallographic information file of NAP –NIC co-crystal

####################################################################

#

# This file contains crystal structure data downloaded from the

# Cambridge Structural Database (CSD) hosted by the Cambridge

# Crystallographic Data Centre (CCDC).

#

# Full information about CCDC data access policies and citation

# guidelines are available at http://www.ccdc.cam.ac.uk/access/V1

####################################################################

_chemical_name_common Naproxen/Nicotinamide Co-crystal

_chemical_melting_point 113.41 oC

_chemical_formula_moiety '2(C14 H14 O3), C6 H6 N2 O'

_chemical_formula_sum 'C34 H34 N2 O7'

_chemical_formula_weight 582.63

_chemical_absolute_configuration rm # known chiral centre

_symmetry_cell_setting orthorhombic

_symmetry_space_group_name_H-M 'P 21 21 21'

_symmetry_space_group_name_Hall 'P 2ac 2ab'

_symmetry_Int_Tables_number 19

_cell_length_a 5.96338(11)

_cell_length_b 15.1283(3)

_cell_length_c 32.5411(6)

_cell_angle_alpha 90.00

_cell_angle_beta 90.00

_cell_angle_gamma 90.00

_cell_volume 2935.72(9)

_cell_formula_units_Z 4

_cell_measurement_temperature 223(2)

_cell_measurement_reflns_used 5368

_cell_measurement_theta_min 0.928

_cell_measurement_theta_max 0.978

_exptl_crystal_description colorless

_exptl_crystal_colour platelet

_exptl_crystal_size_max 0.10

_exptl_crystal_size_mid 0.10

_exptl_crystal_size_min 0.03

_exptl_crystal_density_diffrn 1.318

_exptl_crystal_density_method 'not measured'

_exptl_crystal_F_000 1232

_exptl_absorpt_coefficient_mu 0.757

_exptl_absorpt_correction_type empirical

_exptl_absorpt_correction_T_min 0.9282

_exptl_absorpt_correction_T_max 0.9777

_refine_ls_number_reflns 5368

_refine_ls_number_parameters 401

_refine_ls_R_factor_all 0.0896

_refine_ls_R_factor_gt 0.0471

_refine_ls_wR_factor_ref 0.1487

_refine_ls_wR_factor_gt 0.1027

177

Annexure B: Ethical approval

178

Annexure C: Individual pharmacokinetic profile of all sheep

Table I: Pharmacokinetic parameters of co-crystal ―E‖ in individual sheep, obtained after

single oral administration at a dose of 30 mg/Kg body weight

Parameters Sheep 1 Sheep 2 Sheep 3 Sheep 4 Sheep 5 Sheep 6

Cmax (μg/ml) 1.76 2.04 1.8 0.86 2.85 1.38

tmax (h) 1.5 1.5 1.5 2 1.5 2

AUC0-10h

(μgh/ml)

11.74 9.31 12.37 5.74 10.03 7.0

AUC0-∞

(μgh/ml)

13.29 9.88 14.20 6.53 10.62 8.15

AUMC0-∞

(μgh/ml)

72.44 38.70 81.44 38.29 46.61 44.36

MRT (h) 5.45 3.92 5.74 5.86 4.39 5.44

Kel (h-1

) 0.28 0.33 0.27 0.28 0.3 0.21

t1/2 (h) 2.52 2.11 2.54 2.43 2.34 3.24

CL (μg/ml)/h 56.43 75.95 52.83 110.28 67.80 88.36

Vd (μg/ml) 205.18 231.55 193.67 387.12 229.22 413.58

179

Table II: Pharmacokinetic parameters of co-crystals ―C‖ in individual sheep, obtained

after single oral administration at a dose of 30 mg/Kg body weight


Cmax (μg/ml) 2.83 2.02 3.08 2.65 1.62 2.93

tmax (h) 1.5 2 1.5 2 1.5 1.5

AUC0-10h (μgh/ml) 11.75 9.20 11.31 13.96 9.75 11.37

AUC0-∞ (μgh/ml) 11.83 13.18 12.69 15.21 13.21 11.94

AUMC0-∞ (μgh/ml) 37.47 111.63 62.01 71.40 101.28 50.96

MRT (h) 3.17 8.47 4.89 4.69 7.67 4.27

Kel (h-1

) 0.47 0.12 0.25 0.30 0.14 0.31

t1/2 (h) 1.47 5.94 2.77 2.30 4.98 2.27

CL (μg/ml)/h 60.87 54.62 56.72 49.31 55.65 61.55

Vd (μg/ml) 129.25 467.90 226.99 163.47 399.85 201.56

180

Table III: Pharmacokinetic parameters of PCM in individual sheep, obtained after single

oral administration at a dose of 30 mg/Kg body weight


Cmax (μg/ml) 1.38 0.88 0.78 1.04 1.22 0.61

tmax (h) 1.5 1.5 2.0 1.5 1.5 2

AUC0-10h (μgh/ml) 9.26 3.97 5.16 3.17 5.56 3.12

AUC0-∞ (μgh/ml) 10.02 4.04 5.24 3.19 5.82 3.21

AUMC0-∞ (μgh/ml) 49.80 13.88 23.34 7.96 24.69 11.50

MRT (h) 4.97 3.43 4.46 2.49 4.24 3.58

Kel (h-1

) 0.35 0.67 0.64 0.69 0.38 0.54

t1/2 (h) 1.96 1.03 1.08 1.01 1.80 1.27

CL (μg/ml)/h 74.83 178.07 137.46 225.52 129.04 223.96

Vd (μg/ml) 211.18 264.20 215.02 327.42 335.63 411.68

181

Figure I: Plasma concentration profile with time for PCM in sheep 1

Figure II: Plasma concentration profile with time for PCM in sheep 2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 2 4 6 8 10

Pla

sma

co

nce

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ati

on

of

PC

M (

µg

/ml)

Time (h)

-0.4

-0.2

0

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0.8

1

0 2 4 6 8 10

Pla

sma

co

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M(µ

g/m

l)

Time (h)

182

Figure III: Plasma concentration profile with time for PCM in sheep 3

Figure IV: Plasma concentration profile with time for PCM in sheep 4

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 2 4 6 8 10

Pla

sma

co

nce

ntr

ati

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of

PC

M (

µg

/ml)

Time (h)

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10Pla

sma

co

nce

ntr

ati

on

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M (

µg

/ml)

Time (h)

183

Figure V: Plasma concentration profile with time for PCM in sheep 5

Figure VI: Plasma concentration profile with time for PCM in sheep 6

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10Pla

sma

co

nce

ntr

ati

on

of

PC

M (

µg

/ml)

Time (h)

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

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M (

µg

/ml)

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184

Figure VII: Plasma concentration profile with time for co-crystal E in sheep 1

Figure VIII: Plasma concentration profile with time for co-crystal E in sheep 2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

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M (

µg

/ml)

Time (h)

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Pla

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PC

M (

µg

/ml)

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185

Figure IX: Plasma concentration profile with time for co-crystal E in sheep 3

Figure X: Plasma concentration profile with time for co-crystal E in sheep 4

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

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0 2 4 6 8 10

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/ml)

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0.1

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0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 2 4 6 8 10

Pla

sma

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PC

M (

µg

/ml)

Time (h)

186

Figure XI: Plasma concentration profile with time for co-crystal E in sheep 5

Figure XII: Plasma concentration profile with time for co-crystal E in sheep 6

0

0.5

1

1.5

2

2.5

3

0 2 4 6 8 10

Pla

sma

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M (

µg

/ml)

Time (h)

0

0.2

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1

1.2

1.4

1.6

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Pla

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M (

µg

/ml)

Time (h)

187

Figure XIII: Plasma concentration profile with time for co-crystal C in sheep 1

Figure XIV: Plasma concentration profile with time for co-crystal C in sheep 2

0

0.5

1

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M (

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/ml)

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2

2.5

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M (

µg

/ml)

Time (h)

188

Figure XV: Plasma concentration profile with time for co-crystal C in sheep 3

Figure XVI: Plasma concentration profile with time for co-crystal C in sheep 4

0

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6 8 10

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M (

µg

/ml)

Time (h)

0

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1

1.5

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Pla

sma

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PC

M (

µg

/ml)

Time (h)

189

Figure XVII: Plasma concentration profile with time for co-crystal C in sheep 5

Figure XVIII: Plasma concentration profile with time for co-crystal C in sheep 6

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10

Pla

sma

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ati

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M (

µg

/ml)

Time (h)

0

0.5

1

1.5

2

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3

3.5

0 2 4 6 8 10

Pla

sma

co

nce

ntr

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of

PC

M (

µg

/ml)

Time (h)

190

Table IV: Pharmacokinetic parameters of NAP in individual sheep, obtained after single

oral administration at a dose of 10 mg/Kg body weight


Cmax (μg/ml) 27.46 34.79 22.11 23.68 27.76 20.66

tmax (h) 4 2 2 4 4 2

AUC0-36h

(μgh/ml)

593.36 556.35 558.73 591.97 700.01 528.43

AUC0-∞

(μgh/ml)

820.80 1485.89 1088.03 1192.16 1629.59 1598.96

AUMC0-∞

(μgh/ml)

52504.65 62458.7 61057.08 69024.56 75807.46 62552.97

MRT (h) 39.06 42.03 50.11 57.90 55.79 56.66

Kel (h-1

) 0.024 0.025 0.018 0.017 0.018 0.024

t1/2 (h) 28.82 28.03 39.19 35.19 33.37 26.06

CL

(μg/ml)/h

0.27 0.15 0.20 0.18 0.15 0.16

Vd (μg/ml) 4.14 5.99 5.43 7.70 8.71 5.75

191

Table V: Pharmacokinetic parameters of NAP-NIC co-crystal in individual sheep,

obtained after single oral administration at a dose of 10 mg/Kg body weight


Cmax (μg/ml) 63.71 53.59 34.19 44.13 29.13 27.42

tmax (h) 2 4 4 4 2 2

AUC0-36h

(μgh/ml)

1435.57 1180.10 944.14 1174.67 664.94 703.11

AUC0-∞

(μgh/ml)

2837.01 1791.84 2132.76 3019.92 1163.21 1771.91

AUMC0-∞

(μgh/ml)

143576.4 59474.0 131446.3 220240.7 46991.53 126811.8

MRT (h) 50.61 43.19 61.63 72.93 47.40 71.57

Kel (h-1

) 0.0197 0.02 0.0163 0.014 0.026 0.014

t1/2 (h) 35.20 22.85 42.65 50.53 26.57 49.98

CL

(μg/ml)/h

0.074 0.14 0.10 0.07 0.2 0.13

Vd (μg/ml) 3.76 4.60 6.35 4.83 7.58 9.36

192

Figure XIX: Plasma concentration profile with time for NAP in sheep 1

Figure XX: Plasma concentration profile with time for NAP in sheep 2

0

5

10

15

20

25

30

0 5 10 15 20 25 30 35 40

Pla

sma

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P (

µg

/ml)

Time (h)

0

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35

40

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Pla

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P (

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/ml)

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193

Figure XXI: Plasma concentration profile with time for NAP in sheep 3

Figure XXII: Plasma concentration profile with time for NAP in sheep 4

0

5

10

15

20

25

0 5 10 15 20 25 30 35 40

Pla

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NA

P (

µg

/ml)

Time (h)

0

5

10

15

20

25

0 5 10 15 20 25 30 35 40

Pla

sma

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NA

P (

µg

/ml)

Time (h)

194

Figure XXIII: Plasma concentration profile with time for NAP in sheep 5

Figure XXIV: Plasma concentration profile with time for NAP in sheep 6

0

5

10

15

20

25

30

0 5 10 15 20 25 30 35 40

Pla

sma

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NA

P (

µg

/ml)

Time (h)

0

5

10

15

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0 5 10 15 20 25 30 35 40

Pla

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NA

P (

µg

/ml)

Time (h)

195

Figure XXV: Plasma concentration profile with time for NAP-NIC co-crystal in sheep 1

Figure XXVI: Plasma concentration profile with time for NAP-NIC co-crystal in sheep 2

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30 35 40

Pla

sma

co

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NA

P (

µg

/ml)

Time (h)

0

10

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60

0 5 10 15 20 25 30 35 40

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NA

P (

µg

/ml)

Time (h)

196

Figure XXVII: Plasma concentration profile with time for NAP-NIC co-crystal in sheep

3

Figure XXVIII: Plasma concentration profile with time for NAP-NIC co-crystal in

sheep 4

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25 30 35 40

Pla

sma

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P (

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/ml)

Time (h)

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P (

µg

/ml)

Time (h)

197

Figure XXIX: Plasma concentration profile with time for NAP-NIC co-crystal in sheep 5

Figure XXX: Plasma concentration profile with time for NAP-NIC co-crystal in sheep 6

0

5

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15

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30

35

0 5 10 15 20 25 30 35 40

Pla

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/ml)

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198

Annexure D: Publications

Sumera Latif, Nasir Abbas, Amjad Hussain, Muhammad Sohail Arshad, Nadeem

Irfan Bukhari, Hafsa Afzal, Sualeha Riffat and Zeeshan Ahmad. Development of

paracetamol-caffeine co-crystals to improve compressional, formulation and in vivo

performance. Drug development and industrial pharmacy, (2018), 44, NO. 7, 1099–1108.

(IF 2.295)

Nasir Abbas, Sumera Latif, Hafsa Afzal, Muhammad Sohail Arshad, Amjad

Hussain, Saleha Sadeeqa, Nadeem Irfan Bukhari. ―Simultaneously improving

mechanical, formulation and in-vivo performance of Naproxen by co-crystallization‖.

AAPS PharmSciTech (2018), 1, 1-9, DOI: 10.1208/s12249-018-1152-7. (IF 2.666)

Conference Presentations

Sumera Latif, Nasir Abbas. Development of paracetamol-caffeine co-crystals to

improve formulation properties and in vivo performance. International Pharmaceutics

Conference (IPC), Bahaudin Zakiria University, Multan, Pakistan, 2018.

Nasir Abbas, Sumera Latif. Co-crystallization; an approach to improve

dissolution and compaction properties of pharmaceutical compounds. Riphah 2nd

International conference on recent innovations in pharmaceutical sciences Margala Hotel,

Islamabad, Pakistan, March, 2016

Sumera Latif, Nasir Abbas. Co-crystals of paracetamol with Caffeine. National

Conference on recent innovations in Pharmaceutical Sciences, Islamabad, 2016.

Poster Presentation

Sumera Latif, Amjad Hussain, Nadeem Irfan Bukhari, Nasir Abbas. Improvement

of formulation, in-vitro and in-vivo performance of naproxen by co-crystallization. 78th

FIP World Congress of Pharmacy and Pharmaceutical Sciences, Glasgow, UK,

September 2018.

Sumera Latif, Amjad Hussain, Nadeem Irfan Bukhari, Nasir Abbas. Improvement

of compressional, formulation and in-vitro performance of Naproxen by co-

crystallization. Proceedings of the APS@FIP Conference, Glasgow, UK 2018.

Sumera Latif, Nasir Abbas, Amjad Hussain, Muhammad Sohail Arshad.

Development of paracetamol-caffiene co-crystals to improve formulation properties and

in vivo performance. International Pharmaceutics Conference (IPC), Bahaudin Zakiria

University, Multan, Pakistan, 2018.

Sumera Latif, Nasir Abbas, Amjad Hussain. Co-crystallization; An approach to

improve dissolution and compaction properties of Paracetamol. European Medical and

Clinical Congress, Stockholm, Sweden, August 2017.

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Development of paracetamol-caffeine co-crystalsto improve compressional, formulation and in vivoperformance

Sumera Latif, Nasir Abbas, Amjad Hussain, Muhammad Sohail Arshad,Nadeem Irfan Bukhari, Hafsa Afzal, Sualeha Riffat & Zeeshan Ahmad

To cite this article: Sumera Latif, Nasir Abbas, Amjad Hussain, Muhammad SohailArshad, Nadeem Irfan Bukhari, Hafsa Afzal, Sualeha Riffat & Zeeshan Ahmad (2018)Development of paracetamol-caffeine co-crystals to improve compressional, formulation andin vivo performance, Drug Development and Industrial Pharmacy, 44:7, 1099-1108, DOI:10.1080/03639045.2018.1435687

To link to this article: https://doi.org/10.1080/03639045.2018.1435687

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RESEARCH ARTICLE

Development of paracetamol-caffeine co-crystals to improve compressional,formulation and in vivo performance

Sumera Latifa, Nasir Abbasa, Amjad Hussaina, Muhammad Sohail Arshadb, Nadeem Irfan Bukharia, Hafsa Afzala,Sualeha Riffatc and Zeeshan Ahmadd

aUniversity College of Pharmacy, University of the Punjab, Lahore, Pakistan; bDepartment of Pharmacy, Bahaudin Zakiria University, Multan,Pakistan; cUniversity of Veterinary and Animal Sciences, Lahore, Pakistan; dLeicester School of Pharmacy, De Montfort University, Leicester, UK

ABSTRACTParacetamol, a frequently used antipyretic and analgesic drug, has poor compression moldability owing toits low plasticity. In this study, new co-crystals of paracetamol (PCM) with caffeine (as a co-former) wereprepared and delineated. Co-crystals exhibited improved compaction and mechanical behavior. A screen-ing study was performed by utilizing a number of methods namely dry grinding, liquid assisted grinding(LAG), solvent evaporation (SE), and anti-solvent addition using various weight ratios of starting materials.LAG and SE were found successful in the screening study. Powders at 1:1 and 2:1 weight ratio of PCM/CAFby LAG and SE, respectively, resulted in the formation of co-crystals. Samples were characterized by PXRD,DSC, and ATR-FTIR techniques. Compressional properties of PCM and developed co-crystals were analyzedby in-die heckle model. Mean yield pressure (Py), an inverse measure of plasticity, obtained from theheckle plots decreased significantly (p< .05) for co-crystals than pure drug. Intrinsic dissolution profile ofco-crystals showed up to 2.84-fold faster dissolution than PCM and physical mixtures in phosphate bufferpH 6.8 at 37 �C. In addition, co-crystals formulated into tablets by direct compression method showed bet-ter mechanical properties like hardness and tensile strength. In vitro dissolution studies on tablets alsoshowed enhanced dissolution profiles (�90–97%) in comparison to the tablets of PCM prepared by directcompression (�55%) and wet granulation (�85%) methods. In a single dose sheep model study, co-crys-tals showed up to twofold increase in AUC and Cmax. A significant (p< .05) decrease in clearance as com-pared to pure drug was also recorded. In conclusion, new co-crystals of PCM were successfully preparedwith improved tabletability in vitro and in vivo profile. Enhancement in AUC and Cmax of PCM by co-crystal-lization might suggest the dose reduction and avoidance of side effects.

ARTICLE HISTORYReceived 18 October 2017Revised 27 December 2017Accepted 28 January 2018

KEYWORDSParacetamol; caffeine; co-crystals; intrinsic dissolutionrate; tabletability; in vitrodissolution; bioavailability

Introduction

The successful development of a pharmaceutical dosage formrequires adequate physical and mechanical properties of an activepharmaceutical ingredient (API). However, APIs with limited phys-ical properties are becoming prevalent during the research anddevelopment process in pharmaceutical companies. Some com-pounds have less aqueous solubility and slow in vivo dissolutionrate especially those belonging to the BCS class II (low solubilityand high permeability) and IV (low solubility and low permeability)[1–4] others have high hygroscopicity and exhibit poor compac-tion properties. The properties, for example, solubility and dissol-ution rate are directly related to the bioavailability of the drugs,particularly when delivered via oral route [5,6]. On the other hand,poor mechanical properties cause difficulties in the processing,for example, milling and compaction of a solid dosage form.Advancement in the pharmaceutical sciences has resulted in theestablishment of a number of approaches to address these issues.These approaches include reduction of particle size [7], salt forma-tion [8], prodrug formation, complexation [9,10], and liposomes forthe delivery of lipophilic drugs [11]. Although these techniquesare very effective in enhancing the oral bioavailability and process-ing properties of drugs, the success of these approaches isdependent on the specific physicochemical nature of the com-pound being studied. For example, salt formation which is only

limited to ionizable compounds, complexation with disadvantageslike low extent of interaction with complexing agent, increasedtoxicity, high cost and precipitation, prodrug which may not beadequately delivered at the site of action and particle size reduc-tion limited by particle size induction and amorphization.

In recent years, advancement in crystal engineering techniqueslike co-crystallization provides an alternative approach for improv-ing various physicochemical properties of drug substances [12–15].Control of molecular packing within a crystal structure/lattice pro-duces a solid that shows desirable characteristics including chem-ical stability [16–18], solid state stability [19], dissolution rate andbioavailability [20,21], and compaction behavior [22–24].Furthermore, co-crystals may have an advantage over the high-energy crystal forms of superior thermodynamic stability in thesolid-state [6,25]. Therefore, the interest in the co-crystallizationhas increased considerably over the past few years.

Pharmaceutical co-crystals are defined as supramolecular enti-ties consisting of two or more molecular moieties held togetherby weak non-covalent and nonionic intermolecular interactions(e.g. hydrogen bonding, p stacking, and VanderWaals forces) inwhich at least one or both constituents are active pharmaceuticalagents, that is, two different molecules in a single crystalline lat-tice [26,27]. Drugs having the ability to form hydrogen bondingwith co-formers are considered as potential candidates for

CONTACT Nasir Abbas [email protected] University College of Pharmacy, University of the Punjab, Lahore, Pakistan� 2018 Informa UK Limited, trading as Taylor & Francis Group

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co-crystallization technique. Co-crystals of many pharmaceuticaldrugs are reported in literature, for example, carbamazepine, indo-methacin, furosemide, and piroxicam [28]. Recently, various novelmethods for the efficient production and monitoring of co-crystalshave been proposed [29].

Paracetamol (PCM) is widely used analgesic and antipyreticagent. It has low aqueous solubility and poor mechanical and com-pressional properties and hence has poor tabletability. Many stud-ies have reported the problem of high capping which is related tostiff nature and a relatively high elastic deformation of PCM [30].Due to these reasons, PCM tablets are being prepared using largeamount of a binder to prevent chipping and disintegration of tab-let dosage form [24]. Similarly, a change in crystal structure, forexample, polymorphsim or crystal habit has been shown toimprove tableting properties of PCM [31]. Nevertheless lack ofacidic or basic functional groups and the presence of both protondonors (phenolic O–H and the amidic N–H groups) and acceptor(the amidic oxygen atom) make PCM a good model drug for co-crystal study. Caffeine (CAF) has already been successfully utilizedas a co-former, and is a generally recognized as safe (GRAS) statuscompound. It has functional groups (keto-amide groups and basicnitrogen) which make it as a good candidate to form intermolecu-lar hydrogen bonds in co-crystal formation (Figure 1) [22].Furthermore, PCM is also available as fixed dose combination withcaffeine to synergise the analgesic effect of PCM.

In the present work, attempts were made to produce co-crys-tals of PCM with CAF as a co-former, these co-crystals has notbeen reported previously. Four different co-crystallization methodswere employed in the screening studies, with the aim to improvethe compressional, mechanical behavior, and dissolution profile ofthe drug. Prepared co-crystals were evaluated for intrinsic dissol-ution rate (IDR) and compaction behavior. Previously, mechanicalproperties of co-crystals were determined by measuring the tensilestrength only, however our study involves the heckle analysis,which give more thorough insight about the plasticity.Pharmacokinetic studies of newly formed co-crystals were also per-formed on sheep model in order to highlight the potentialincrease in oral bioavailability. Literature review has shown thatonly a few bioavailability studies have been reported for the co-crystals [19,20]. To our knowledge, this is the first bioavailabilitystudy done one the PCM co-crystals. Lastly, tablet formulationsprepared from these co-crystals were also assessed for the phar-macotechnical properties.

Experimental

Materials and methods

PCM and CAF were obtained as gift samples from UNEXOPharmaceuticals and Avicel PH 102 and magnesium stearate from

Remington Pharmaceuticals, Lahore, Pakistan and were used asreceived. All organic solvents used were of HPLC or analyticalgrade. A phosphate buffer at pH 6.8 was prepared by mixing250ml of KH2PO4 (0.2M) and 112ml of NaOH (0.2M) with finaldilution to 1 l with distilled water.

Screening of co-crystals

Various methods like mechanochemical (dry grinding, liquid assistedgrinding (LAG)), solvent evaporation (SE), and anti-solvent addition(ASA) were used for the formation of co-crystals of PCM with CAF.

Dry and liquid assisted grindingPCM and CAF in three different weight ratios (1:1, 1:2, & 2:1) wereco-ground in a mortor and pestle without and with small amountof liquid (�50 ml for each of 100mg of PCM and CAF) for 30min.Solvents used were V/V mixture of methanol & ethanol (1:1 & 1:2)and pure acetone. The powder was collected in tight containersand stored at room temperature.

Solvent evaporation methodPCM and CAF in three different weight ratios (1:1, 1:2, & 2:1) weredissolved in a V/V mixture of methanol & ethanol (1:1 &1:2), mix-ture of distilled water & ethanol (1:1, 1:2), and pure acetone withaid of slight heating until a clear solution obtained. Each of thesesolutions was then filtered and left for evaporation at room tem-perature. The solid crystals obtained were collected in a tight con-tainer for further analysis and stored at room temperature.

Anti-solvent additionExcess amount of PCM and CAF (1:1, 1:2, & 2:1 weight ratio) wereadded to 5ml of methanol and acetone separately to make turbidsolutions, stirred for 15min, and filtered through Whatman filterpaper. Distilled water (2ml) (as an anti-solvent [32]) was added tothe filtrate to induce precipitation. Samples were then centrifugedat 3000 rpm for 20min to allow solids to deposit out.

Characterization

The pure drugs and co-crystals prepared by the above methodswere investigated by DSC, PXRD, ATR-FTIR, and IDR studies.

Differential scanning calorimetry (DSC)DSC studies were performed using a TA instrument (model Q 600).Samples (2–5mg) contained in alumina pan with an empty pan as

Figure 1. Molecular structures of paracetamol (left) and caffeine (right).

1100 S. LATIF ET AL.

a reference were analyzed from room temperature (25 ± 2 �C) to300 �C with a constant heating rate (10 �C/min) under a nitrogenpurge (100ml/min).

Powder X-ray diffraction (PXRD)PXRD patterns were obtained for PCM, CAF, and co-crystals syn-thesized by various methods. Powder samples were used as suchwithout any pre-treatment. PXRD data were collected on a PanAnalytical diffractometer XPERT-PRO (operating at 40 kV, 40mA),using Cu-Ka radiation (k¼ 1.5418 Å) with a position sensitivedetector. Diffraction data were collected in the range 2h¼ 5–30�

with a step size of 0.02� .

ATR-FTIR spectroscopyFourier Transform Infrared (FTIR) spectra were recorded for thePCM, CAF, and synthesized co-crystals using FTIR spectrophotom-eter (Alpha-P Bruker, Germany) equipped with an ATR unit acrossthe range of 4000–400 cm�1.

Scanning electron microscopy (SEM)Powder samples were photographed by ZEISS EVO HD 15 scan-ning electron microscope (Carl Zeiss, NTS Ltd. Cambridge, UK)with auto-imaging system. The samples were mounted on carbonadhesive tape fixed on aluminum stubs (Agar Scientific Ltd.,Stansted, UK), flushed with air and photographed at the voltage of2 kV for 25 s.

Drug content analysis

A stock solution containing 1mg/ml of pure drug was preparedby dissolving 100mg of PCM in 100ml of phosphate buffer (pH6.8) using a volumetric flask. The standard stock solution was fur-ther diluted to obtain a working standard solution of 100mg/ml.Working standard solution was then diluted with phosphate buffer(pH 6.8) to obtain a series of solutions with the concentrationrange of 2–16 mg/ml. A calibration curve for PCM was prepared bymeasuring the absorbance (using a UV spectrophotometer) at thekmax of 243 nm. Statistical parameters like slope, intercept, andcoefficient of correlation were determined to model the linearityof absorbance data.

For drug content analysis, each of the co-crystal samples wasaccurately weighed (10mg) and dissolved in phosphate buffer pH6.8 to give final concentration of 10 mg/ml. The drug content wascalculated from the calibration curve.

Intrinsic dissolution studies

The IDR of pure drug, physical mixtures and co-crystals was deter-mined by rotating disc method using a constant surface area of13mm in diameter. Discs were prepared by compressing 300mgof powder samples by a hydraulic press (Carver) at a pressure of�5000 psi for 60–90 s. The compacts were coated with hard paraf-fin leaving a surface available for dissolution [33,34] and thenplaced to the base of the dissolution vessel.

The dissolution studies, performed in triplicate, were carriedout in phosphate buffer pH 6.8 (volume: 500ml, temperature:37 �C) in a paddle apparatus (Apparatus 2, USP) at a rotationspeed of 50 rpm. Aliquots (5ml) were withdrawn at time intervalsof 2, 4, 6, 8, 10, 14, 18, and 22min. Samples were filtered through0.45mm filters and analyzed at 243 nm. Dissolution medium wasreplenished with the same volume (5ml) of blank (phosphate

buffer pH 6.8) in order to maintain the sink conditions. IDR wasdetermined by plotting the cumulative amount of drug dissolvedper surface area (mg/cm2) versus time (min).

Compressional behavior

In-die heckle model [35] for powder deformation under pressureEquation (1) was used to study the compressional behavior ofpure PCM and prepared co-crystals powders. Bulk density of eachpowder was determined by the standard method. Each of thematerial (300mg) was individually compacted under compres-sional force between 2 and 14MPa using a hydraulic press(Carver) equipped with flat faced dies 13mm in diameter. Volumereduction was measured at each compaction pressure used.

dDdP

¼ K 1� Dð Þ (1)

where D is the relative density of the compact at applied pressureP, and the constant K is a measure of the plasticity of the com-pressed material. Integration of Equation (1) gives Equation (2)

Lnð1Þð1� DÞ ¼ KPþ A (2)

A plot was drawn between Ln(1)/1�D and compaction pres-sure (P) for each powder. A is the intercept of the extrapolated lin-ear region of the curve. Mean yield pressure (Py), a measure of thematerial’s resistance for deformation [36], was obtained from thereciprocal of the slope (K) of the linear portion of the heckle plot.The higher the value of slope K is, more plastic is the material.

Preparation of tablets

Direct compression method was used to prepare tablets from allco-crystal powders. For this purpose, co-crystal equivalent to250mg of PCM was blended with Avicel PH 102 (20% of theweight of co-crystal) and magnesium stearate (0.5% of the weightof co-crystal) for about 15min. The mass was then compressedusing a hydraulic press fitted with 13mm diameter die. As purePCM cannot be directly compressed due to poor compressionalproperties, it was moistened with small amount of water and thendried at 50 �C for 10min to induce plasticity and then compacted(F1). Tablets from physical mixtures (1:1 & 2:1 by weight ratio ofPCM and CAF) were prepared following the same procedure.Tablets of pure PCM (F2) (250mg equivalent) were also preparedby the standard wet granulation (WG) method using starch paste(10% of the weight of PCM) as a binder and dry starch as disinte-grating agent (10% of the weight of PCM).

Evaluation of tablets

All the prepared tablets were evaluated for hardness, tensilestrength, disintegration time, and in vitro dissolution study.Hardness was determined using Pharmatron Multitest 50H andexpressed in newton. Tensile strength, r, in MPa was determinedby diametral compression test [37] and calculated by usingEquation (3)

r ¼ 2F

106pDT(3)

where F is the breaking force (N), D is the tablet diameter (m),and T is the thickness of tablet (m). Disintegration test wascarried out by tablet disintegration test apparatus using distilledwater at 37 �C.

DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 1101

In vitro dissolution study

Dissolution studies for all prepared tablets were performed usingphosphate buffer pH 6.8 (volume: 900ml, temperature: 37 �C) as adissolution medium in a paddle apparatus (Apparatus 2, USP) at arotation speed of 50 rpm. Aliquot (5ml) was withdrawn at timeintervals of 3, 6, 9, 12, 17, 22, 27, and 32min and replaced withfresh dissolution medium in order to maintain the sink conditions.These samples were analyzed for PCM concentration by UV Visiblespectrophotometer at 243 nm. All the measurements were done intriplicate. Percent drug release was determined from the calibra-tion curve (R2¼ 0.996). The drug release data were also fittedto various kinetic models such as zero order, first order, andHiguchi, Korsmeyer–Peppas, and Hixon–Crowell model using DDsolver [38].

In vivo studies in sheep

An in vivo study was conducted in sheep model in order assessthe pharmacokinetic profiles of pure PCM and co-crystals. Sheepmodel was selected for preclinical evaluation owing to its easyhandling, more clinically precise, and relevant dose alignment.Moreover, collection of multiple samples per animal is possiblewithout adding risk to animal’s life. The protocols used for thisstudies were approved by animal ethical committee, UniversityCollege of Pharmacy, University of the Punjab, Lahore, Pakistan(ref no. AEC/PUCP/1047A). Three groups (n¼ 6) of sheep wereorally administered at a dose of 30mg/kg body weight with purePCM and equivalent co-crystals in gelatin capsules after an over-night fast. Collection of blood samples (3ml) were undertakenfrom jugular vein in heparinized tubes at 0, 0.25, 0.5, 1, 1.5, 2, 4, 6,8, and 10 h intervals post-dose. From the whole blood, plasma wasseparated by centrifugation at 3500 rpm for 5min and supernatantwas collected in Eppendorf tube and stored at �20 �C for furtherstudies.

For drug analysis, to 1ml of plasma, 60 ml of 50% perchloricacid was added. This mixture was vortexed for 5min and centri-fuged at 13,500 rpm for 15min. Supernatant was filtered using0.22mm syringe filter. A 50ml of clear filtrate was injected into theHPLC with a run time of 10min and drug content was analyzedwith a previously reported method [39]. The HPLC system(Shimadzu 20A, Japan) was equipped with a LC-20AT VP pump, aSIL-20AC HT auto sampler, SPD-M20A detector (photodiode array),CTO 20 AC column oven, CBM 20A controller unit and a reversedphase symmetry C18 (4.6� 250mm, 5-mm particle-size) column.The mobile phase was composed of water, methanol, and aceto-nitrile (80:10:10, v:v:v), filtered through a 0.45 membrane filter andwas delivered at a flow rate of 1ml/min. The analysis was carriedout under isocratic conditions maintaining column temperature at40 �C. A photodiode array detector set at 245 nm was used forrecording chromatograms. Calibration curve (R2¼ 0.9974) preparedfor PCM was based on peak area measurements of standard solu-tions and spiked plasma samples for concentrations of 0.1, 0.5, 1.0,5.0, 10.0, and 15.0mg/ml. The limits of detection and quantificationwere 0.03 and 0.1mg/ml, respectively.

The maximum plasma concentration (Cmax) and the time toreach maximum concentration (Tmax) were directly obtained fromthe plasma concentration versus time curves. Pharmacokineticparameters including area under the curve (AUC), area underthe first moment curve (AUMC), mean residence time (MRT),plasma clearance (CL), apparent volume of distribution (Vd),elimination rate constant (Kel), and half-life (t1/2) were calculatedusing non-compartmental analysis and PK solver pharmacokineticprogram [40].

Statistical analysis was conducted by SPSS (IBM SPSS Statistics22, Armonk, NY, USA). Statistical data were obtained by using oneway ANOVA and post hoc comparisons. Statistical significance wasconsidered when p< .05.

Results and discussion

Co-crystal screening was carried out by four different methods asdescribed in Table 1. Formation of co-crystals was recorded by dif-ference in melting points and PXRD patterns from the individualcomponents. PCM-CAF co crystals were successfully prepared byLAG and SE methods. In LAG, mixtures of PCM and CAF at weightratio 1:1 with solvents S1, S2, and S3 were resulted in co-crystals(PXRD pattern were different from either of the two starting mate-rials, Figure 2). In SE method, the solution of PCM and CAF inweight ratio 2:1 using solvent systems (S1 and S2) resulted in for-mation of co-crystals. All the other mixtures/solutions in thescreening study produced intact crystals of the individual compo-nents, that is, PCM and CAF.

Characterization of newly formed co-crystals was performedusing three different techniques namely PXRD, DSC, and FTIR.PXRD patterns of co-crystals were compared with three poly-morphs of PCM (denoted as Forms I, II, and III) and two poly-morphs of CAF (denoted as Forms I and II) in Figure 2.Characteristics peaks of all the polymorphs of PCM and CAF werenot observed in the patterns of co-crystals. Moreover new peakswere emerged at 2h value of 8.41�, 9.06�, 11.39�, and 17.66� sug-gesting the formation of a new phase. The difference in the

Table 1. Screening of co-crystals by using various methods.

Method used Solvent usedSolventratio

Wt ratio ofPCM/CAF

Observations(co-crystalformation)

Neat (dry) grinding N/A 1:1 �1:2 �2:1 �

Liquid assisted MeOH:EtOH 1:1 (S1) 1:1 þ(A)grinding 1:2 �

2:1 �1:2 (S2) 1:1 þ(B)

1:2 �2:1 �

Acetone Alone (S3) 1:1 þ(C)1:2 �2:1 �

Solvent evaporation MeOH:EtOH 1:1 (S1) 1:1 �1:2 �2:1 þ(D)

1:2 (S2) 1:1 �1:2 �2:1 þ(E)

Acetone Alone (S3) 1:1 �1:2 �2:1 �

H2O:EtOH 1:1 (S4) 1:1 �1:2 �2:1 �

1:2 (S5) 1:1 �1:2 �2:1 �

Anti-solvent Solvents:MeOH & acetoneAnti-solvent: water

1:1 �

1:2 �2:1 �

‘þ’ represents formation of co-crystals, characterized by DSC and PXRD results,‘�‘ represents re-crystallization of starting material or no crystallization. Co-crys-tals from successful techniques are coded A, B, C, D & E.


intensity of major peaks in PXRD parents of co-crystals (Figure 2)was due to preferred orientation effect (instrument used had a flatsample stage), which may be due to dissimilarity in the habit ofparticles produced by two different methods. A, B, and C sampleswere prepared by the liquid assisted grinding (LAG) technique,where grinding resulted in fine powders. However, samples D andE were prepared by solvent evaporation (SE) technique.Recrystallization in this case resulted in small crystallites which cre-ate problem in the packing of samples for PXRD analysis. DSCdata also showed that the resultant powders are new phases asthe melting point of co-crystals are significantly different from allthe polymorphs of starting materials. However, precise stoichio-metric and structural description of these co-crystals needs furtheranalysis by single crystal XRD, which may form the subject of ourfuture studies.

DSC curves of newly formed co-crystals and pure PCM (identi-fied as Form I) and CAF are shown in Figure 3. PCM sampleshowed a sharp endothermic peak with mid point at 172.37 �Crepresenting the melting of PCM form I. Although not evident inthis study, paracetamol also exist as polymorphs II and III. Form IIhas melting point at 160 �C, while Form III is most unstable formand converts back to Form I. CAF thermogram showed two endo-thermic peaks with mid points at 160.33 and 231.08 �C

representing melting of its polymorphic forms, that is, Forms IIand I, respectively. DSC curves of co-crystals prepared by the LAGand SE techniques showed endothermic peaks with onset temper-atures �130 and 133 �C, respectively (Table 2), which are lowerthan the individual components (PCM and CAF) and all their poly-morphs. However, sample D showed two overlapping peaks whichindicate lack of phase purity. With small exceptions, it can be pre-sumed that the co-crystals formation resulted in increased entropyevident as a broad endothermic peak at temperatures lower thanthe individual counterparts.

Figure 4 shows comparison of FTIR spectra of prepared co-crys-tals with PCM and its physical mixture with caffeine in the func-tional group region of 3000–3400 cm�1. For PCM, peaks for N–Hamide stretch and phenolic OH stretch were observed at 3323 and3108 cm�1, respectively. These functional groups are involved inthe hydrogen bonding of PCM molecules in the crystal structure[41]. Overall, FTIR spectra of co-crystals in the range400–3500 cm�1 show almost similar pattern as of PCM, however ashift in the above described bands (i.e. to 3316 and 3104 cm�1,respectively) was observed. This indicates a slight change in thehydrogen bonding pattern of PCM in the form of co-crystal. Theseshifts were not observed for physical mixture.

Intrinsic dissolution rate (IDR)

IDR method was employed as it affords advantages over otherpowder dissolution methods because the exposed area of the diskis constant and thus dissolution variability can be minimized.

5 10 15 20 25 30

0

1

2

3

4

5

6

7

8

9

10

11

12

E

D

C

B

A

CAF FORM II

CAF FORM I

PCM FORM III

PCM FORM II

PCM FORM I

Inte

nsit

y (a

rbit

rary

sca

le)

2 theta (degree)

Figure 2. PXRD patterns of PCM, CAF, and PCM-CAF co-crystals.

50 100 150 200 250

-2

0

2

4

6

8

10

12

14E

D

CB

A

CAF

PCM

Hea

t flo

w(w

/g)

Temperature(°C)

Figure 3. DSC thermograms of PCM, CAF, and PCM-CAF co-crystals.

Table 2. Onset, mid, and off set temperatures of DSC thermograms of PCM,CAF, and PCM-CAF co-crystals.

MaterialOnset

temperature (�C)Mid

temperature (�C)Offset

temperature (�C)PCM 165.59 172.37 204.20CAF Form I 216.99 231.08 247.45Form II 146.49 160.33 175.91A 130.31 141.02 156.57B 128.21 140.92 174.19C 131.93 142.5 165.87D 133.58 160.92 194.16E 133.58 148.68 172.51

A, B, C, D, and E are codes given to co-crystals prepared by successful methodsas shown in Table 1.

3400 3300 3200 3100 30000.80

0.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

E

D

CB

A

PM

PCMT

rans

mit

tanc

e (%

)

Wavenumber (cm-1)

Figure 4. ATR-FTIR spectra of PCM, PM, and PCM-CAF co-crystals highlighting theN–H amide stretch and phenolic OH stretch regions for PCM.


Intrinsic dissolution profiles of the prepared co-crystals comparedwith pure drug are shown in Figure 5. IDR of PCM recorded inphosphate buffer pH 6.8 was 5.06mg/cm2min, while for co-crys-tals A, B, C, D, and E was 8.72, 9.53, 12.23, 11.11, and 14.40mg/cm2min, respectively. Based on the initial dissolution rate, co-crys-tals A, B, C, D, and E showed 1.72-, 1.88-, 2.42-, 2.19-, and 2.84-foldincrease in dissolution rate. In comparison, the physical mixtureshowed almost same IDR (5.5mg/cm2min) as of the pure drug.This high dissolution rate might indicate the stability of preparedco-crystals to prevent dissociation of drug and co-former fromeach other and achieve the solubility and dissolution advantagesof co-crystals [42].

IDR is the dissolution rate that is greatly influenced by theintrinsic factors especially the crystal habit and particle size distri-bution. The variation among the IDR values of co-crystals can beexplained by the fact that the powders prepared by the two differ-ent methods (LAG and SE) have different particle size distributionand shapes. This is evident from the SEM images of these samplesas shown in Figure 6. Mechanochemical method (LAG) producedfine powders with high compressibility index (�31.12%). On theother hand, the powder samples prepared by SE method showlarger particles with low compressibility index value of �20.55%.Owing to this fact more compact IDR discs were prepared fromLAG samples which resulted in lower values of IDR as comparedto samples of SE. This is also evident from the higher breakingforce (hardness) of the tablet formulations prepared from A, B,and C samples as compared to D and E samples (Table 3).

Compressional and mechanical properties

Poor compressibility of PCM due to low plasticity is always a prob-lem for the formulation scientists. To overcome this problem, PCMis normally tableted by wet granulation (WG) method, which is atime consuming and laborious procedure and required a largeamount of excipients to be added to the formulation.Development of crystal structures, which provide large inter-par-ticulate bonding area and plasticity, has been proposed as a strat-egy to improve the tensile strength of materials [43]. In thepresent study, heckle plots were drawn for pure PCM and co-crys-tals powders to assess the plastic behavior induced by co-crystal-lization (Figure 7). Co-crystals showed improved compaction andhence high plasticity. This was shown by low Py (mean yield pres-sure) value for co-crystals as compared to pure drug. Py recordedfor PCM was 133.33MPa. The same parameter for formed co-crys-tals A, B, C, D, and E was significantly decreased, that is, 33.03, 80,

28.65, 33.03, and 47.62MPa, respectively. The ranking of the Py val-ues was C<A&D< E< B< PCM (Table 3). These values were calcu-lated from the region of heckle plots showing the highestcorrelation coefficient for linearity of >0.989 for all the powdersamples. The yield pressure, Py, is the stress at which particledeformation is initiated reflecting the deformation of the particlesduring compression [44]. Difference in compression propertiesamong co-crystals produced by LAG and SE is due to dissimilarcrystal plasticity which may result from unique molecular packingfeatures in the respective crystal lattices. Mechanochemically syn-thesized co-crystals are more plastic and have high tensilestrength as evident from their low mean yield pressure (Py) value(33.03 & 28.65). Previous literature also confirms mechanochemicalsynthesis as an efficient approach to produce plastic and com-pressible forms of PCM using oxalic acid, phenazine, theophylline,and naphthalene as coformers [24].

Results from the compression data indicate that co-crystals caneasily be formulated, by direct compression with adequate hard-ness to the tablet dosage form without adding any binder. Thetensile strength higher than 3MPa was exhibited by tablets basedon co-crystals prepared by LAG (FA, FB, and FC) while the tabletsbased on co-crystals prepared by SE were in the vicinity of>1.8MPa (Table 3). This suggests that mechanochemical methodsof co-crystallization are more suitable to improve the pharmaco-technical properties of PCM.

In vitro drug release study of tablets

Various tablet formulations prepared from pure drug, physical mix-tures, and co-crystals were subjected to the in vitro dissolutionstudies for the release of PCM. Direct compression method wasemployed for the co-crystals formulations (FA, FB, FC, FD, and FE).However for pure drug, both liquid assisted direct compression(F1) and wet granulation method (F2) were used. Figure 8 showscomparison of dissolution profiles of these formulations. All theco-crystals formulations showed 90–97% release within 20min.This was greater than corresponding physical mixtures (55%), F1(72%) and F2 (85%).

As already explained before that the LAG method of co-crystal-lization produce powders with a large surface area so mechano-chemical synthesis based formulations were expected to give betterdissolution profiles as compared to SE-based formulations. This canalso be confirmed by SEM results. Furthermore, grinding in thepresence of acetone (FC) resulted in even more improved dissol-ution as compared to grinding with V/V mixtures of methanol andethanol (FD and FE). This can be explained by the fact that highsolubility of PCM in methanol/ethanol mixture as compare to pureacetone (solubility of PCM in methanol 371, in ethanol 232, and inacetone 111mg/g of the solvent) resulted in solvent assisted recrys-tallization as demonstrated by spottiness (loss of peak resolution,i.e. preferred orientation effect) of PXRD pattern.

Release kinetics results show that the release of PCM fromformulations F1 and F2 followed Korsemeyer–Peppas andHixon–Crowell models, respectively. While dissolution data of theco-crystal formulations FA &FB followed Korsemeyer–Peppas andformulations FC, FD, & FE followed Hixon–Crowell model for drugrelease (Table 4). Moreover, all the formulations showed non-Fickian (anomalous) drug release (0.45� n� 0.89).

In vivo performance of PCM and co-crystals

Powder co-crystal samples from each of the successful methodshowing higher IDR, that is, C and E were selected for oral

0

20

40

60

80

100

120

140

160

0 2 4 6 8 10

Cum

ulat

ive

drug

dis

solv

ed(m

g/cm

2 )

Time(minutes)

PCM

A

B

C

D

E

Figure 5. Intrinsic dissolution profiles of PCM alone and from co-crystals in phos-phate buffer pH 6.8 at 37 �C.


bioavailability studies. Figure 9 shows the plasma concentrationprofiles for PCM and selected co-crystals. The mean pharmacoki-netic metrics calculated from the sheep model study data are pre-sented in Table 5. The mean AUC values were 11.22 and 9.36mgh/ml for co-crystals C and E, respectively, which were almost twotimes enhanced that of PCM (5.02 mg h/ml). Cmax of C (2.40mg/ml)and E (1.72mg/ml) was 2.45- and 1.80-fold that of PCM (0.98 mg/ml)

without any change in the time for peak concentration. Theincrease in peak concentration for co-crystals may be attributed todecreased elimination because of higher accumulation of drug.Mean residence time of the co-crystals was observed to be highwith low clearance values suggesting enhanced absorption win-dow. Co-crystals also showed more than two times enhancementin the relative oral bioavailability than the pure PCM. The

Figure 6. SEM images of pure PCM and PCM-CAF co-crystals.


bioavailability of both co-crystals was significantly better thanpure drug based on one-way ANOVA followed by Tukey’s post hoctest (p< .05).

Difference in in vivo behavior between the co-crystal samplesprepared by two methods can be attributed to the dissimilarity inthe physicochemical properties especially dissolution rate (Figure 8).

Physiological variations among the subjects may also contribute tothese small differences. However, in comparison with each other(C with E and E with C) the pharmacokinetic parameters Cmax andAUC were insignificant (p> .05).

Conclusion

PCM is a low solubility compound with poor tabletability. A co-crystal incorporating PCM and CAF was successfully developed byliquid assisted grinding and solvent evaporation. Co-crystalsshowed improved IDR and compressional properties. In vivo kin-etic results from sheep model also confirmed a significant increasein the oral bioavailability of PCM. This suggests co-crystallization auseful technique to improve the compressional, physicochemical,

Table 3. Physical and mechanical properties of tablet formulations prepared from pure PCM (direct compression (F1) and wet granulation (F2)) and PCM-CAFco-crystals.

Properties F1 F2 FA FB FC FD FE

Tablet diameter (mm) 13 ± 0.01 13 ± 0.01 13 ± 0.01 13 ± 0.01 13 ± 0.01 13 ± 0.01 13 ± 0.01Breaking force (N) 18 ± 1.0 66 ± 1.0 270 ± 5 285 ± 5 309± 5 90 ± 2.5 94 ± 2.5Tensile strength (MPa) 0.52 ± 0.01 1.90 ± 0.01 3.48 ± 0.01 3.67 ± 0.01 3.98 ± 0.01 1.82 ± 0.01 1.88 ± 0.01Disintegration time (min) 1 ± 0.5 0.67 ± 0.5 8.0 ± 0.5 7.5 ± 0.5 7.5 ± 0.5 5 ± 0.5 5 ± 0.5Mean yield pressurea (MPa) 133.33 33.03 80 28.65 33.03 47.62aMean yield pressure values were derived from heckle plots only from pure PCM and PCM-CAF co-crystals powders.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 5 10 15

Ln(

1/1-

D)

Compaction pressure (Mpa)

PCM

A

B

C

D

E

Figure 7. Heckle plots for PCM and PCM-CAF co-crystals.

0

20

40

60

80

100

0 5 10 15 20 25 30

% d

rug

rele

ased

Time (minutes)

F1

F2

PM(1:1)

PM(2:1)

FA

FB

FC

FD

FE

Figure 8. In vitro drug release comparison of various formulations of PCM,PCM-CAF co-crystals, and physical mixtures (n¼ 3).

Table 4. Drug release kinetics from various formulations of PCM and PCM-CAFco-crystals.

Release kinetics F1 F2 FA FB FC FD FE

Zero orderR2 0.9847 0.8559 0.7634 0.7383 0.5443 0.8172 0.7257

First orderR2 0.9821 0.9628 0.9693 0.9575 0.9531 0.9368 0.9688

Higuchi modelR2 0.8926 0.9148 0.9340 0.9053 0.8891 0.8834 0.9327

Hixon–Crowell modelR2 0.9904 0.9764 0.9775 0.9594 0.9712 0.9517 0.9810

Korsemeyer–Peppas modelR2 0.9926 0.9421 0.9843 0.9791 0.9230 0.9077 0.9340n 0.873 0.659 0.758 0.751 0.549 0.650 0.531

0

0.5

1

1.5

2

2.5

3

0 2 4 6 8 10

Pla

sma

conc

entr

atio

n(µg

/ml)

Time (hours)

Mean conc. (PCM)

Mean conc. C

Mean conc. E

Figure 9. Sheep plasma concentration with time for PCM and PCM-CAFco-crystals.

Table 5. Pharmacokinetic parameters of PCM and PCM-CAF co-crystals in sheep,obtained after single oral administration at a dose of 30mg/kg body weight(n¼ 6).

Parameters PCM Mean ± SD C Mean ± SD E Mean ± SD

Cmax (lg/ml) 0.98 ± 0.18 2.40 ± 0.51 1.72 ± 0.66Tmax (h) 1.67 ± 0.25 1.67 ± 0.25 1.67 ± 0.25AUC0–10h (lg h/ml) 5.02 ± 1.3 11.22 ± 1.67 9.36 ± 1.60AUC0–1 (lg h/ml) 5.25 ± 0.5 13.01 ± 1.22 10.44 ± 1.93AUMC0–1 (lg h/ml) 21.86 ± 05.19 72.46 ± 14.84 53.64 ± 10.54MRT (h) 3.86 ± 0.87 5.52 ± 1.07 5.13 ± 0.79Kel (h

�1) 0.33 ± 0.15 0.26 ± 0.13 0.27 ± 0.03t1/2 (h) 1.36 ± 0.41 3.29 ± 1.75 2.53 ± 0.38CL/F (lg/ml)/h 161.48 ± 19.01 56.45 ± 4.48 75.27 ± 14.49Vd/F (lg/ml) 294.18 ± 28.33 264.83 ± 26.78 276.72 ± 19.19Relative bioavailabilitya (R.B) 2.47 1.98aR.B relative to PCM.


and pharmacokinetic properties of PCM and proving caffeine asuitable co-former.

Acknowledgements

We would also like to thank Mr. Abdul Muqeet Khan fromUniversity of Veterinary and Animal Sciences for helping in phar-macokinetic analysis.

Disclosure statement

Authors report no conflict of interest.

Funding

Authors would like to acknowledge Higher Education Commissionof Pakistan for providing funding under NRPU program toUniversity College of Pharmacy, University of the Punjab.

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[25] Fleischman SG, Kuduva SS, McMahon JA, et al. Crystalengineering of the composition of pharmaceutical phases:multiple-component crystalline solids involving carbamaze-pine. Cryst Growth Des. 2003;3:909–919.

[26] Arora KK, Zaworotko MJ. Pharmaceutical co-crystals: a newopportunity in pharmaceutical science for a long-knownbut little studied class of compounds. Polym Pharm Solids.2009;2:281–313.

[27] Vishweshwar P, Mcmahon JA, Bis JA, et al. Pharmaceuticalco-crystals. J Pharm Sci. 2006;95:499–516.

[28] Thakuria R, Delori A, Jones W, et al. Pharmaceutical cocrys-tals and poorly soluble drugs. Int J Pharm. 2013;453:101–125.

[29] Huski�c I, Christopherson JC, U�zarevi�c K, et al. In situ moni-toring of vapour-induced assembly of pharmaceutical coc-rystals using a benchtop powder X-ray diffractometer.Chem Commun. 2016;52:5120–5123.

[30] Di Martino P, Guyot-Hermann A, Conflant P, et al. A newpure paracetamol for direct compression: the orthorhombicform. Int J Pharm. 1996;128:1–8.

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Research Article

Simultaneously Improving Mechanical, Formulation, and In Vivo Performanceof Naproxen by Co-Crystallization

Nasir Abbas,1 Sumera Latif,1,2,4 Hafsa Afzal,2 Muhammad Sohail Arshad,3 Amjad Hussain,1

Saleha Sadeeqa,2 and Nadeem Irfan Bukhari1

Received 2 May 2018; accepted 10 August 2018

ABSTRACT. Naproxen (NAP), an anti-inflammatory drug belonging to class II ofBiopharmaceutic Classification System, has low aqueous solubility and dissolution rate whichlimit its oral bioavailability. The focus of this investigation was to assess the impact of co-crystallization in improving the physico-mechanical and in vivo performance of NAP. NAP wasco-crystallized using nicotinamide as a co-former employing liquid-assisted grinding method andcharacterized by intrinsic dissolution rate, DSC, and PXRD. Prepared co-crystal exhibitedimproved physicochemical and mechanical properties. Mechanical behavior of NAP anddeveloped co-crystal was analyzed by drawing tabletability curves. Over the entire range ofused compaction pressure, NAP showed poor tensile strength (< 2 MPa) which resulted inlamination and capping in some tablets. In contrast, tensile strength of co-crystal graduallyincreased with pressure and was ~ 1.80 times that of NAP at 5000 psi. Intrinsic dissolution profileof co-crystal showed a more than five and twofold faster dissolution than NAP in 0.1 MHCl andphosphate buffer pH 7.4 at 37°C. In addition, formulation of co-crystal powder into tablets bydirect compression demonstrated enhanced dissolution profiles (~ 43% in 0.1 MHCl and ~ 92%in phosphate buffer pH 7.4) in comparison to a marketed product, Neoprox (~ 25 and ~ 80%)after 60 min. In a single dose oral exposure study conducted in sheep, co-crystal showed morethan 1.5-fold increase in AUC and Cmax. In conclusion, co-crystals of NAP illustrated bettertabletability, in vitro and in vivo performance.

KEY WORDS: naproxen; co-crystal; intrinsic dissolution; tensile strength; bioavailability.

INTRODUCTION

Predictive and precise control of the physicochemicalproperties of drug substances is an important task in formulationdevelopment process with the ultimate aim of improving in vivobioavailability (1). Most of the active pharmaceutical ingredi-ents (APIs), which are being discovered during the course ofdrug development process, have low aqueous solubility andconsequent slow in vivo dissolution. Bioavailability of drugs,particularly on oral administration, is directly affected bysolubility and dissolution rate (2,3). Poor mechanical behavioris another persistent challenge that hinders the development ofpromising drugs. A thorough understanding of different me-chanical properties (e.g., elasticity, plasticity, fracture, or

fragmentation) and material deformation phenomena is criticalwith respect to powder compaction and material processing bymilling and filling. In this perspective, crystal engineeringapproaches find considerable significance (4). Multiple hydro-gen bonding sites in theAPIs result in crystallization into severalsolid forms, which include salts, polymorphs, hydrate, co-crystalsetc. These strategies confer to structural changes in the crystalstructures that may modify different physical, chemical, andcompressional properties, which finally affect formulation,manufacturing, and performance of the product (5). However,salt formation is limited by that API must possess an ionizablesite for proton transfer. Hence, this technique is not suitable forweakly ionized and neutral molecules which do not undergosuch transfer. Likewise, there are many examples in literaturewhere hydrates exhibit undesired phase transformations duringpharmaceutical production and storage (6,7). Under circum-stances when salt or hydrate formation is not possible, co-crystallization, by its structural diversity, may serve as a viablealternative to fine tune the solubility or stability problem andmodify mechanical properties of an API.

Pharmaceutical co-crystals are supramolecular entities whichconsist of two or more molecular moieties bonded together, in asingle crystalline lattice, by weak intermolecular interactions such

1University College of Pharmacy, University of the Punjab, Lahore,Pakistan.

2 Institute of Pharmacy, Lahore College for Women University,Lahore, Pakistan.

3 Department of Pharmaceutics, Bahauddin Zakariya University,Multan, Pakistan.

4 To whom correspondence should be addressed. (e–mail:[email protected])

AAPS PharmSciTech (# 2018)DOI: 10.1208/s12249-018-1152-7

1530-9932/18/0000-0001/0 # 2018 American Association of Pharmaceutical Scientists

http://orcid.org/0000-0003-1673-6234

http://crossmark.crossref.org/dialog/?doi=10.1208/s12249-018-1152-7&domain=pdf

as hydrogen bonding, van der Waals forces, and p stacking (8,9).Drugs having ability to interact through hydrogen bonds withcomplementary functional groups of co-formers are potentialcandidates for co-crystallization. There are many reported exam-ples of co-crystals of APIs, e.g., indomethacin, furosemide,piroxicam, carbamazepine, etc. (10). On the other hand, variousinert co-formers are also available for the formation of these co-crystals. Previously reported studies have shown that co-crystals ofdrugs are being used to address a specific pharmaceutical propertyincluding chemical stability (11,12), solid state stability (13),dissolution and bioavailability (14,15), and tabletability (16–18).

Naproxen (NAP) is a nonsteroidal anti-inflammatory(NSAID) drug belonging to class II (low solubility, highpermeability) of Biopharmaceutic Classification System. Hy-drogen ion concentration (pH) of the surrounding mediumgreatly affects the solubility of NAP (19). NAP has a highcontent of lipophilic aromatic region which prevents wettingand interactions with water resulting in its poor solubility inaqueous medium. Conversely, at higher pH, it ionizes formingfavorable interactions with water thus enhancing dissolution.Oral administration of NAP can result in variable plasmaconcentration because of its restricted wettability and verylimited water solubility (0.021 mg/ml at 25°C). Low solubilityenhances the residence time of NAP in the mucosa therebyincreasing its irritating side effect on the gastrointestinal tract(20). Currently, NAP is also marketed as a sodium salt to addressthe solubility problem associated with free acid form.However, inorder to to improve the solubility across all pH range, analternative solution is desired. NAP can be considered as a modeldrug for co-crystallization study owing to the presence ofcarboxylic acid functional group (proton donor). Nicotinamide(NIC), a hydrophilic co-former, is a GRAS compound and formsintermolecular hydrogen bonds by its effective functional groups(amide group and pyridine ring) (Fig. 1).

Co-crystals of NAP have been reported with variousco-formers (21–23). Majority of these studies have focusedonly on the structural evaluation by single crystal XRD, andin few cases, intrinsic dissolution rate (IDR) measurementsof co-crystal have been investigated as pharmaceuticalproperties. None of the study has grasped the mechanical,formulation, and in vivo behavior of co-crystals from theperspective of pharmaceutical development. Solubility, dis-solution, and bioavailability are important parameters indrug development process. Furthermore, intrinsic solubilityand dissolution of a drug are prerequisite for oral drugdelivery. Therefore, there was a need to evaluate the co-crystals in terms of their efficacy in the improvement ofthese properties. Keeping these gaps in mind, the currentstudy was aimed to investigate the multiple utility of NAP-NIC co-crystal system to modify the mechanical properties

of NAP to make the production process more feasible andthe development of NAP-NIC co-crystal as alternativeformulation to overcome the dissolution issues particularlyin acidic pH leading to a dose reduction and greater fractionof dose absorbed in small intestine. On formulating theNAP co-crystal into tablets by direct compression method, afaster dissolution was illustrated especially in acidic mediumas compared with commercial NAP tablets, Neoprox. Anenhancement in the plasma area under the curve and peakconcentration was also shown by NAP-NIC co-crystal whencompared to the parent compound in a single dose studyperformed on the sheep model. As per the literature review,only a few co-crystal systems have gone through bioavail-ability studies (14,15). To the best of our information, weare reporting the bioavailability studies of the NAP co-crystal for the first time.

EXPERIMENTAL

Materials

NAP and NIC were obtained from Sigma-Aldrich,Germany and BDH, UK, respectively. Avicel PH 102,croscarmellose sodium, and magnesium stearate were re-ceived as gift samples from Remington Pharmaceuticals,Pakistan. All the chemicals used were analytical/and orHPLC grade. Neoprox, a commercial formulation containingNAP as free acid, was used as a reference.

Preparation of Co-Crystal

Liquid-assisted grinding (LAG) was used to prepareNAP-NIC co-crystal. NAP was co-ground with NIC at 1:1(1.0 g:0.5 g), 1:2 (1.0 g:1.0 g), and 2:1 (1.0 g:0.25 g) molarratios in a mortar and pestle with small amount (~ 10 μl foreach of 100 mg of powder mixture) of acetonitrile for 30 min.The resultant powder was stored in an airtight container forfurther analysis.

Characterization

DSC, X-ray powder diffraction (XRPD), and intrinsicdissolution rate were used as characterization tools for theNAP, NIC, and NAP-NIC co-crystal prepared by LAG.

Thermal Analysis

Thermal analysis was performed by differential scan-ning calorimetry. Samples were gently ground and analyzedusing a thermo-analytical (TA) instrument (Q600, USA)

Fig. 1. Molecular structures of naproxen (a) and nicotinamide (b)

Abbas et al.

under nitrogen purge (100 ml/min). Sample powders (2–5)were placed in aluminum pans (using an empty pan asreference) and heated at a rate of 10°C/min in thetemperature range of 25–300 ± 2°C.

X-Ray Powder Diffraction

X-ray powder diffraction analysis was conducted for puredrug, co-former and prepared co-crystals powders withoutany pretreatment by PanAnalytical XPERT-PRO diffractom-eter using radiation (CuKα, λ = 1.5418 Å) with a positionsensitive detector (PSD). The tube voltage and tube currentwere 40 kV and 40 mA, respectively. Samples were scannedover a 2θ range of 5.0–40° with a step size of 0.02°.

Drug Content Analysis

Accurately weighed 10 mg of pure drug powder wasdissolved separately in 100 ml of phosphate buffer (pH 7.4)and 0.1 M HCl (pH 1.2) to get NAP concentration of100 μg/ml. Stock solution was then diluted with respectivebuffers to attain a series of standard solutions over theconcentration range 0.005–1.0 μg/ml for 0.1 M HCl and 1.0–40 μg/ml for phosphate buffer pH 7.4. Absorbance of each ofthe standard solution was measured using UV-Visible spec-trophotometer (UV-1900 BMS, Canada) at the λmax of226 nm for 0.1 M HCl and at 330 nm for phosphate bufferpH 7.4 to construct a calibration curve for NAP. Linearity ofthe absorbance data was predicted by calculating thestatistical parameters like intercept, slope, and correlationcoefficient.

For analysis of drug content, 10mg of co-crystal sample wasweighed accurately and dissolved in each of the medium usingvolumetric flask to give 10 μg/ml solutions. Drug content of co-crystal was estimated from the calibration curve.

Intrinsic Dissolution Rate

IDR of pure drug, physical mixture (PM), and co-crystalwas carried out by static disk method. Non-disintegratingcompacts of constant surface area were prepared by a hydraulicpress (Carver, USA) using a 13-mm die and flat-faced punches.Three hundred milligrams of each powder sample was com-pressed at a pressure of ~ 5000 psi for 60–90 s. The lower side ofthe compacts was covered with paraffin leaving the top side ofthe compact available for dissolution.

Each coated compact was then affixed to the base of thedissolution vessels (apparatus 2). IDR was determined using900ml of phosphate buffer (pH 7.4) and 0.1MHCl (pH 1.2) at37°C at 50 rpm paddle speed. Aliquots (5 ml) were withdrawnfrom basic and acidic buffers at predetermined time intervalsup to 100 and 800 min, respectively. UV absorption readingswere taken at the λmax of drug in each of the medium afterfiltering the aliquots through a 0.45-μm Millipore nylon filterand diluting with respective buffers. Sink conditions weremaintained by replenishing with the same volume (5 ml) ofthe diluents. A curve was drawn between cumulative amountof drug dissolved per surface area (mg/cm2) and time (min).IDR was determined from the slope of the curve.

Powder Compaction/Mechanical Properties

Three tablets of each pure substance (300 mg) wereprepared (without excipients) using a hydraulic press (Carver,USA) fitted with a 13-mm die over the pressure range of 500–6000 psi at ambient conditions, i.e., 22°C and 33% relativehumidity. All powders were passed through a 425-μm sievebefore compaction. The tablets were then stored at above-prescribed conditions for 48 h. Hardness, expressed in newton(N), was measured using Pharmatron MultiTest 50H hardnesstester. Tensile strength was determined using diametralcompression test and calculated by the equation

σ ¼ 2F

106πDT

where σ is tensile strength (MPa), F is the breaking force (N),andD and Tare representation of tablet diameter and thickness(m). Tabletability curves were plotted between tensile strengthand compaction pressure. Micromeritic properties of co-crystalwere also investigated to evaluate the impact of co-crystallization on the material flow. Standard methods wereused to determine the bulk and tapped densities, Carr’s index,Hausner’ ratio, and angle of repose (24).

Formulation of Tablets

Tablets of NAP-NIC co-crystal were prepared (F1) bydirect compression technique. Co-crystal powder equivalentto 250 mg of NAP, croscarmellose sodium (7% of the weightof co-crystal), and Avicel PH-102 (10% of the weight of co-crystal) were passed through a 425-μm sieve and thoroughlymixed for 10 min. Finally, the mixture was blended with 0.5%magnesium stearate for 1 min. A hydraulic press equippedwith a die of 13-mm diameter was used to compress the mass.

Hardness, disintegration, and in vitro dissolution testswere performed for the evaluation of the co-crystal tablets.Hardness (N) was measured by Pharmatron MultiTest 50H.Disintegration was carried out in USP basket rack assemblyusing distilled water at 37°C.

In Vitro Drug Release

In vitro drug release of tablets was conducted in 900 mlof 0.1 M HCl and phosphate buffer pH 7.4 at 37°C. Thesedissolution media were used in order to simulate the pHchange along the GIT. Commercial NAP tablets, Neoprox(250 mg), were used as a reference (F0). A USP type IIdissolution apparatus was used for dissolution studies at apaddle speed of 50 rpm. Samples (5 ml) were taken atpredetermined intervals (0, 5, 10, 15, 30, 45, 60, 75, 90, and120 min) from phosphate buffer pH 7.4 and (0, 30, 60, 90, 120,180, 240, and 3000 min) for 0.1 M HCl and replaced withfresh media maintained at 37°C. The samples (n = 3) werefiltered, diluted, and analyzed for NAP content by spectro-photometer at 330 and 226 nm. Drug release (%) wasdetermined from the standard curve (R2 = 0.996). Kineticanalysis was performed by model dependent methods(including zero order, first order, Korsmeyer-Peppas, Hixon-

Improving Physico-Mechanical and Pharmacokinetic Properties

Crowell, and Higuchi model) using DDsolver to determinethe best fit of release data (25).

In Vivo Studies in Sheep

A single dose sheep exposure study was conducted tocompare the bioavailability of pure NAP and co-crystal.Easy handling, more precise dose adjustment, and manifoldblood sample collections per animal without threatening itslife are the possible reasons for selecting the sheep model.Recently, bioavailability studies of paracetamol/caffeine co-crystals have also been carried out employing sheepindicating the suitability of this model for preclinicalevaluation of co-crystals (26). The study was conducted asper the protocols approved by animal ethical committee,University College of Pharmacy, University of the Punjab,Lahore, Pakistan (ref. no. AEC/PUCP/1065A). The studieswere executed in agreement with Helsinki Declaration andAnimal Scientific Procedure Act 1986 (UK). A dose of10 mg/kg body weight of pure NAP and equivalent co-crystal enclosed in gelatin capsules was orally administeredto two groups of sheep (n = 6) after an overnight fast. Fromeach sheep, 3 ml of blood sample was taken from jugularvein in heparinized tubes at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, and36 h post-dose intervals. Whole blood was centrifuged at3500 rpm for 5 min to collect the plasma which was stored inEppendorf tube at − 20°C for further analysis.

A previously reported HPLC method (27) was used forthe drug analysis in the plasma samples. The HPLC system(Shimadzu 20A, Japan) consisted of a pump (LC-20AT VP),an autosampler (SIL-20AC HT), a photodiode array detector(SPD-M20A), and a column oven and a controller unit (CBM20A). Chromatic separation was achieved using reversedphase C18 (4.6 × 250 mm, 5 μm particle size) column at roomtemperature. The mobile phase was made of 20 mM phos-phate buffer (pH 7.4) containing 0.1% trifluoroacetic acid(TFA)-acetonitrile (65:35 v/v) and was pumped at a flow rateof 1 ml/min. The total run time was 15 min and injectionvolume was 20 μl. NAP was eluted at ~ 5.8 min under theprescribed conditions and drug concentrations were deter-mined at 225 nm. The standard curve for NAP wasconstructed based on the peak area measurements of spikedplasma samples for concentrations 0.1, 0.5, 1.0, 2.5, 5.0, 10.0,and 15.0 μg/ml. The limit of detection and limit of quantifi-cation were 0.03 and 0.1 μg/ml, respectively.

Non-compartmental pharmacokinetic metrics were cal-culated using PK solver pharmacokinetic program version 2.0(28). Estimation of the area under the curve (AUC) wascarried out by applying linear trapezoidal rule. The maximumplasma concentration (Cmax) and the time of maximumconcentration (Tmax) were directly read from the plasmalevel time curve. SPSS (IBM SPSS Statistics 22) was used forstatistical analyses. To compare the means and standarddeviation of the samples, one-way analysis of variance wasused. Statistical significance was considered when p < 0.05.

RESULTS AND DISCUSSION

Methods used for the co-crystallization of APIs can beclassified as solution-based and solid-state (mechano-chemical) methods. A number of variables dictate the

capacity of an API to fabricate into co-crystal including co-former type, API/co-former ratio, solvent systems, pressuretemperature, and crystallization method. Crystallization fromsolution can yield well-formed single crystals required forsingle crystal XRD to obtain an absolute crystal structuredetermination. However, these methods are limited byselection of a suitable solvent for the starting materials andin some instances by the solvate formation. Mechanochemical(dry and liquid assisted grinding) methods, on the other hand,are more environmentally friendly due to the absence or leastamount of solvent, have a short reaction time, and areparticularly important when screening for co-crystals of APIswith low solubility (29). However, with dry grinding, theenergy required to complete the co-crystallization of com-pounds is lacking (30). One method to overcome this isintroduction of a small amount of a solvent during grinding(LAG) which acts as a catalyst and is depleted during thecourse of grinding. Therefore, interest in LAG as a method ofco-crystal preparation has been developed over past fewyears (31). For the above-described reasons, we have selectedLAG for the preparation of NAP co-crystal using a smallamount of acetonitrile. Previous literature also finds LAG asan effective method for the synthesis of co-crystals (16,32,33).

Difference in melting point and PXRD pattern weretaken as primary tools to assess the formation of co-crystal.Mixture of NAP with NIC at molar ratio 2:1 resulted in co-crystal. Co-crystal exhibited a PXRD pattern that does notmatch the patterns of the two starting materials (Fig. 2).Characteristics peaks at 2θ value of 6.6°, 12.7°, 13.46°, and19.01° for NAP and 11.39°, 14.86°, 22.19°, and 36.95° for NICwere not detected in the pattern of co-crystal (Fig. 2). Newpeaks appeared at a value of 2θ = 5.46°, 10.79°, 12.08°, 16.19°,17.20°, and 18.90° suggesting the existence of interactionsbetween NAP and co-former. These results are in agreementwith the previously published data (21).

DSC is another important technique for the characteri-zation of co-crystals. Comparison of thermal behavior of co-crystals with the starting materials is not only used foridentification purpose but also gives evidence about thestability (34) and dissolution of the prepared entities (35).Melting point in specific, being a fundamental physicalproperty, measures the energy needed to overcome theattractive forces holding a crystal structure. It is influencedby chemical structure, intermolecular interactions, crystallattices, molecular symmetry, conformational degree of free-dom, etc. (36,37); therefore, any change in these parametersmay result in relative change in thermodynamic stability.Crystals with a better molecular symmetry and or/strongerhydrogen bonding in solid state will result in an increasedmelting point (37). On the other hand low melting pointcrystals can show better dissolution properties. A review onmelting points of 50 reported co-crystals has shown that halfof co-crystals showed melting points in between of drug andco-former and approximately 40% had melting points lessthan that of starting materials (38).

DSC thermograms of co-crystal and starting materials areshown in Fig. 3. Co-crystal indicated an endothermic meltingevent with mid-temperature 113.41°C which was lower than themid-points of the individual components (NAP 156.84°C andNIC 132.80°C). This temperature was also different from thepreviously reported co-crystals of NAP, i.e., NAP-Urea, NAP-

Abbas et al.

Thiourea, and NAP-Picolinamide co-crystals at 121.43°C,150.27°C, and 92.85°C, respectively (39,40).

Fingerprint plots (for NAP and co-crystal) producedwith CRYSTAL EXPLORER (41) is shown in Fig. 4. Sincethese plots are distinct for any crystal structure, they give apowerful visual tool to elucidate and compare intermolec-ular interaction and spotting common features and trends aswell. Both fingerprint plots show sharp Btails^ and spikes.The outer two tails correspond to N-H...O=C hydrogenbond extending down to around de and di = 1.0 Ǻ, the upperone corresponds to the hydrogen bond donor, and lowerone to the hydrogen bond acceptor. As evident from theFig. 4, finger print plot obtained from pure NAP crystalstructure showed sharp and well-resolved hydrogen bondpeaks as compared to co-crystal owing to stronger hydrogenbond. This resulted in more compressed plots representing abetter packed structure.

Static Disk Intrinsic Dissolution Rate Studies

IDR minimizes the effect of particle surface area ondissolution kinetics and has been extensively used to charac-terize dissolution behavior of APIs. In this study, IDR wasdetermined to evaluate the dissolution characteristics of theNAP-NIC co-crystal in acidic (pH 1.2) and basic (pH 7.4)buffers. Figure 5a, b shows dissolution profiles for NAP, PM,and co-crystal.

Being a weakly acidic drug, NAP is only slightly solublein acidic medium. But as a result of co-crystallization, apromising increase in IDR was noticed in 0.1 M HCl. TheIDR from 0 to 800 min in 0.1 M HCl for the co-crystal was

Fig. 5. IDR profiles of NAP, co-crystal, and physical mixture in a0.1 M HCl and b phosphate buffer pH 7.4 at 37°C

Fig. 4. Two-dimensional fingerprint plots of NAP (a) and NAP-NICco-crystal (b)

40 80 120 160 200 240

-4

-2

0

2

4

6

8

10

Co-crystal

NIC

NAP

)g/w(

wolftaeH

Temperature (0C)

Fig. 3. DSC thermograms of NAP, NIC, and NAP-NIC co-crystal

Fig. 2. PXRD patterns of NAP, NIC, and NAP-NIC co-crystal andcalculated pattern


51.0 μg min/cm2. This value was 7.08 times higher than thatfor NAP (7.2 μg min/cm2) and 2.76 times higher than physicalmixture (14.4 μg min/cm2). Similarly, the IDR from 0 to100 min in phosphate buffer pH 7.4 for the co-crystal was1520 μg min/cm2. This value was 2.75 times higher than thatfor NAP (552 μg min/cm2) and 1.30 times higher thanphysical mixture (720 μg min/cm2). NIC, being a hydrophilicco-former, may be responsible for enhancing the wetting ofthe hydrophobic drug particles with dissolution media andimparting a positive effect on the dissolution profile ofphysical mixtures.

Compaction Behavior of Powders

Heckle equation and tensile strength determinations arepopular means of studying powder compaction behavior.Heckle model can easily distinguish between plastic, elastic,and brittle materials based on mean yield pressure (Py)values derived from the Heckle plot. Therefore, materials canbe classified using Heckle equation. However, Py values forthe same material calculated by different researchers aregreatly varied owing to many experimental (in-die or out-of-die compaction) (42) and physical factors (compaction speed,compaction pressure, and particle size) (43). On the otherhand, tensile strength is a parameter that can be preciselyused to predict tablet tensile strength during formulationdevelopment and is independent of tableting speed (44).Moreover, most of the published data evaluating mechanical/

compaction behavior of co-crystals has focused on tensilestrength measurement (16,18,45).

Tabletability is the ability of a powder to be convertedinto a tablet of definite strength under the influence ofcompaction pressure (46). A plot between tensile strengthand compaction pressure is a representation of thetabletability of a powder. Poor tabletability of a powderindicates plastic deformation during compaction followed byhigh elastic recovery. NAP is unsuitable for direct tabletingdue to its poorly compressible properties (47). Improvementin mechanical properties of a drug substance has beenpreviously reported as a result of co-crystallization(16,18,48). In this study, we have investigated the tabletingproperties of pure NAP and prepared co-crystal.Tabletability profiles of pure NAP, NIC, and co-crystal areshown in Fig. 6. Compaction properties of NAP and NICwere poor and tablets were low in strength. A drop intensile strength of tablets was noticed at > 3000 psi as thecompaction pressure was increased (Fig. 6). This could bedue to overcompaction where higher elastic recoveryproduced weaker tablets due to weakening of inter-particulate cohesive forces (49). In contrast, the preparedco-crystal showed good tabletability and exhibited a re-quired tensile strength of more than 2 MPa. In fact, tensilestrength of tablets continued to increase with pressure andgradually leveled off which is a characteristic behavior ofplastic materials (45). Moreover, no severe lamination wasobserved for co-crystal. Thus, the co-crystal powder is moresuitable for tableting than the NAP.

Table I. Physical Paramesters and Percent Drug Released of NAP in 0.1 M HCl and Phosphate Buffer pH 7.4 from Commercial and Co-Crystal Tablets

Parameters F0 (Neoprox) F1

Diameter (mm) 10 ± 0.01 13 ± 0.01Breaking force (N) 98.00 ± 1.3 217.56 ± 3.8Dis. time (min) 2.5 ± 0.8 4.5 ± 1.4Drug released (%) 0.1 M HCl 60 min 26.31 44.65

180 min 38.70 69.03Phosphate buffer pH 7.4 15 min 55.20 67.87

60 min 81.44 93.66

0

0.5

1

1.5

2

2.5

3

3.5

4

0 1000 2000 3000 4000 5000 6000

Ten

sile

str

eng

th (

Mp

a)

Compaction pressure (psi)

NAP

NIC

Co-crystal

Fig. 6. Tabletability plots of NAP, NIC, and co-crystal

0

10

20

30

40

50

60

70

80

0 50 100 150 200 250 300

Per

cen

t d

rug

rel

ease

d

Time (minutes)

F0

F1

Fig. 7. Dissolution profiles of commercial and co-crystal tablets in0.1 M HCl

Abbas et al.

Micromeritic properties of co-crystal were also im-proved. The high bulk and tapped densities for co-crystal(0.363 and 0.46 g/cm3) as compared to pure drug (0.22 and0.44 g/cm3) indicated a lower porosity leading to improvedcompressibility of the co-crystal powder. Carr’s index for theco-crystal (9.7%) was observed to be low in comparison toNAP (22%) suggesting that the co-crystal powder is suitablefor direct tableting (24). Better flow properties of co-crystalwere reflected by low values of Hausner’s ratio and angle ofrepose (1.26° and 28.79°) as compared to the values for thepure drug (2° and 41.48°).

In Vitro Performance of Tablets

A comparison of the pharmaceutical properties and keyparameters describing dissolution profiles for commercialtablet (F0) and co-crystal formulation (F1) are shown inTable I. Dissolution was conducted in 0.1 M HCl andphosphate buffer pH 7.4 (Figs. 7 and 8) as these solutionsare used in official compendia to simulate gastric andintestinal environments, respectively. F1 showed enhanceddissolution profile in both media. In phosphate buffer pH 7.4,more than 90% of NAP was released from F1. In contrast,only 80% of drug was released from Neoprox (Table I).Improved drug release from F1 may be attributed to higherdissolution rate leading to a higher solution concentration atthe same time point. Most promising effect was observed inacidic medium. More than 70% of drug was released from F1as compared to F0 (38%). It is pertinent to state that resultsfrom intrinsic dissolution rates of co-crystal in both acidic and

basic medium have shown enhanced profiles (Fig. 5a, b)without adding any surfactants, suggesting this increase as aresult of change in intrinsic property of NAP in the form ofco-crystal. Co-crystal formulations have also shown enhanceddissolution rate as compared to the commercial formulationThis suggests that the formulation difference may not havesignificant effect on the dissolution results as we have keptour formulation as simple as possible and the main effect maybe credited to a change in intrinsic property due to co-crystallization. This enhanced dissolution rate for F1 maydecrease the irritating effect of NAP on GIT as the drug mayhave less residence time in the stomach. The dissolutionprofiles of F0 and F1 followed Korsmeyer-Peppas model inboth dissolution media (Table II). Moreover, both formula-tions exhibited Fickian release (n 0.45).

In Vivo Performance of Pure NAP and NAP Co-Crystal

Plasma level time curves of NAP and co-crystal havebeen shown in Fig. 9. The mean AUC and Cmax values forthe co-crystal (1017.09 μg h/ml and 42.03 μg/ml, respec-tively) significantly enhanced based on one-way ANOVA(p < 0.05) and were 1.69 and 1.62 times that of NAP (AUC601.13 μg h/ml and Cmax 26.08 μg/ml). The time formaximum plasma concentration was not altered for bothNAP and co-crystal (3.0 ± 1.09 h). Reduced elimination ofNAP as a result of co-crystallization may account for theincrease in peak concentration of drug. Mean residence

0

20

40

60

80

100

0 20 40 60 80 100 120

Per

cen

t d

rug

rel

ease

d

Time (minutes)

F0

F1

Fig. 8. Dissolution profiles of commercial and co-crystal tablet inphosphate buffer pH 7.4

Table II. Drug Release Kinetics from Neoprox and Co-Crystal Formulation in 0.1 M HCl and Phosphate Buffer pH 7.4

Release kinetics F0 F1 F0 F10.1 M HCl Phosphate buffer pH 7.4

Zero order R2 0.0762 0.1018 0.7383 0.5443First order R2 0.3828 0.7229 0.7625 0.9495Higuchi model R2 0.8475 0.8510 0.7026 0.6635Hixon-Crowell model R2 0.2888 0.5833 0.6291 0.7515Korsmeyer-Peppas model R2 0.9902 0.9839 0.9704 0.9638

n 0.260 0.266 0.235 0.224

0

5

10

15

20

25

30

35

40

45

50

0 5 10 15 20 25 30 35

Co

nce

ntr

atio

n (µ

g/m

l)

Time (hours)

Mean conc. NAP

Mean Conc. Co-crystal

Fig. 9. Plasma level time curves for NAP and NAP-NIC co-crystal(n = 6)


time (MRT) of the co-crystal was found high and may beattributed to low clearance of the drug suggesting enhancedabsorption window. Relative oral availability for the NAPco-crystal was also observed to be 1.62-fold than pure drug.Table III shows the mean pharmacokinetic parametersderived from the plasma level time curve.

CONCLUSION

The present study has illustrated that co-crystallizationwith NIC has improved pharmaceutical properties includingintrinsic and in vitro dissolution rate particularly in acidicmedium, tableting properties, and in vivo profile for poorlywater-soluble drug NAP. The results also suggest that NAP-NIC co-crystal can be further developed into high-qualitydrug products.

ACKNOWLEDGEMENTS

The authors present their acknowledgement to HigherEducation Commission (HEC) of Pakistan for provision offunds (NRPU-3535) for the current study to PunjabUniversity College of Pharmacy, Punjab University, Lahore.We also acknowledge Abdul Muqeet Khan, LecturerUniversity of Veterinary and Animal Sciences for his helpin bioavailability studies.

COMPLIANCE WITH ETHICAL STANDARDS

Conflict of Interest We report no declaration of interest.

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