Impact of Crystal Habit on Biopharmaceutical Performance of Celecoxib

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Article

Impact of Crystal Habit on Biopharmaceutical Performance of CelecoxibSameer R Modi, Ajay K Dantuluri, Vibha Puri, Yogesh B Pawar, Prajwal Nandekar, AbhayT. Sangamwar, Sathyanarayana R Perumalla, Changquan C. Sun, and Arvind K. Bansal

Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/cg400140a • Publication Date (Web): 21 May 2013

Downloaded from http://pubs.acs.org on May 22, 2013

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Impact of Crystal Habit on Biopharmaceutical

Performance of Celecoxib

Sameer R. Modi a, Ajay K. R. Dantuluri

a, Vibha Puri

a, Yogesh B. Pawar

a, Prajwal Nandekar

b,

Abhay T. Sangamwar b, Sathyanarayana R. Perumalla

c, Changquan Calvin Sun

c, and Arvind K.

Bansal a*

a Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research

(NIPER), SAS Nagar, Punjab 160062, India

b Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and

Research (NIPER), SAS Nagar, Punjab 160062, India

c Department of Pharmaceutics, College of Pharmacy, University of Minnesota, 308 Harvard

Street S.E., Minneapolis, Minnesota 55455-0343, USA

*CORRESPONDING AUTHOR’S AFFILIATION

Department of Pharmaceutics

National Institute of Pharmaceutical Education and Research (NIPER)

Sector 67, SAS Nagar, Punjab 160 062, INDIA

Tel: +91-172-2214682-86. Fax: +91-172-2214692.

E- mail: [email protected]

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ABSTRACT. Poor biopharmaceutical performance of Biopharmaceutical Classification System

(BCS) class II drug molecules is a major hurdle in the design and development of pharmaceutical

formulations. Anisotropic surface chemistry of different facets in crystalline material affects

physicochemical properties, such as wettability, of drugs. In present investigation, a molecule-

centered approach is presented towards crystal habit modification of celecoxib (CEL) and its

effect on oral bioavailability. Two crystal habits of CEL, acicular crystal habit (CEL-A) and

plate shaped crystal habit (CEL-P), were obtained by recrystallization from toluene at 25 °C and

60 °C, respectively. Compared to CEL-A, CEL-P exhibited significantly faster dissolution

kinetics in aqueous media and significantly higher Cmax and shorter Tmax in an oral bioavailability

study. The significant enhancement in dissolution and biopharmaceutical performance of CEL-P

was attributed to its more abundant hydrophilic surfaces than CEL-A. This conclusion was

supported by wettability and surface free energy determination from contact angle

measurements, and surface chemistry determination by X-ray photoelectron spectroscopy (XPS),

crystal structure modeling, and crystal face indexation.

KEYWORDS. Celecoxib, crystal habits, Wettability, Solubility, Anisotropic surface chemistry,

Face indexation, Oral bioavailability

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1. INTRODUCTION

Variability in biopharmaceutical performance of active pharmaceutical ingredients

(APIs), can affect their bioavailability, safety, and efficacy.1, 2 Solid form of a chemical

compound is usually defined in terms of their internal structure. The ability of a compound to

pack into different crystal lattice arrangements is termed ‘polymorphism’. On the other hand, the

external appearance of crystal is termed as ‘crystal habit’. Both polymorphs and crystal habits of

an API can have considerable influence on physicochemical properties and, therefore, product

performance.3-5

Solvent of crystallization, degree of supersaturation, temperature, rate of change of

temperature, additives, and stirring rate can all influence the outcome of crystallization in terms

of both polymorphism6 and crystal habits.7-13 Crystallization variables that affect growth, either

promotion or inhibition, of different crystal facets subsequently affect the crystal habit.7, 11, 14

Studies have shown differential surface energetics, wetting behavior, and dissolution

kinetics between individual crystalline facets.15-21 Since the surface properties of crystal facets

are directly related to the localized chemical functionality22, a clear understanding of differential

pharmaceutical performance of crystal habits relies on an understanding of the molecular

arrangement on the surface of pharmaceutical solids. It has been shown that variations in

pharmaceutical processes involving interfaces, such as dissolution, of bulk crystalline materials

can be attributed to anisotropic surface chemistry of the crystals.19, 23, 24 This is because that

particle wetting, governed by powder surface energetics, is a prerequisite for interfacial

phenomena.19, 21, 23-31 However, the impact of crystal habits on biopharmaceutical properties of

an API remains poorly understood.

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The goal of this work was to systematically investigate how crystal habit can impact

biopharmaceutical properties of celecoxib (CEL). Two crystal habits of CEL were obtained by

controlled crystallization32 and evaluated for their surface molecular environment and in vivo

biopharmaceutical performance. The differences in biopharmaceutical performance were

correlated to the anisotropic surface properties of CEL crystals. Surface energetics of both

crystal habits were characterized using sessile drop contact angle technique. Surface chemistry

was determined using X-ray photoelectron spectroscopy (XPS) and MOLCAD® software in an

attempt to rationalize the observed differences in surface energetics. The findings were

confirmed by crystal face indexation experiments.

2. EXPERIMENTAL SECTION

2.1. Materials

The room temperature stable Form III of CEL, chemically designated as 4-[5-(4-methyl-

phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulphonamide (assay value > 99%), was

received as a gift from Zydus Cadila Healthcare Ltd. (Ahmadabad, India). Toluene (Merck,

India), ethylene glycol (EG, Merck, India) and diiodomethane (DIM, Sigma–Aldrich, Steinheim,

Germany) were of > 99.0% purity. The solvents used were of high-performance liquid

chromatography (HPLC) grade. All other chemicals used were of analytical grade.

2.2. High-Performance Liquid Chromatography (HPLC)

All the samples from solubility and oral bioavailability experiments were analyzed for

drug content using a validated HPLC method with minor modifications.33, 34 The HPLC system

(Shimadzu Corporation, Kyoto, Japan) included a system 210 controller (SCL-10A), a pump

(LC-10AT), a degasser (DGU-14A), an autosampler (SIL-10AD), a column oven (CTO-10AS)

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and a UV detector (SPD-10AP) with Class-VP (Release 6.10) software. The analytical column

used was LiChrospher®

100 RP-18e (250 mm x 4.6 mm, 5 μm), attached with a LiChroCART®

100 RP-18e guard column (4mm x 4 mm, 5 μm) (Merck, Darmstadt, Germany). The mobile

phase, acetonitrile (ACN): phosphate buffer (pH 3, 10 mmol) (55:45 v/v), was pumped in

isocratic mode at a flow rate of 1.0 mL/min at ambient temperature. Indomethacin was used as

internal standard for all plasma samples to nullify any processing errors during extraction. The

injection volume was 40 µL. The PDA detector was set at a wavelength of 252 nm.

2.3. Solubility studies

The equilibrium solubility of ‘as received’ CEL in toluene at different temperatures (28,

32, 36 and 40 °C) were determined by adding an excess amount of the drug in 20 mL of toluene

in 25 mL screw capped glass vials. These vials were then shaken mechanically in a shaker water

bath (Julabo Labortechnik GmbH, Seelbach, Germany), at 100 rpm maintained at required

temperature (± 0.2 ºC). Samples were withdrawn after 1, 2, 4, 8, 16, 24, 36, 48 and 72 h, filtered

using 0.22 µm nylon filters, and analyzed by HPLC after appropriate dilution. The equilibrium

solubility values at different temperatures were analyzed by the means of a van’t Hoff plot,

which was used to determine the equilibrium solubility at a different temperature, from which

the degree of supersaturation during crystallization experiments was calculated.

2.4. Crystallization experiments

Different CEL crystal habits were generated by controlling the degree of supersaturation

and crystallization temperature. Accurately weighed amount (about 200 mg) of drug was

dissolved in 10 mL of toluene by heating to 72 ºC. The hot drug solution was immediately

filtered into a glass beaker using 0.22 µm nylon filters and cooled to a predetermined

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temperature, 25 ºC or 60 ºC, to achieve a desired degree of supersaturation of 190% and 102%,

respectively. The crystals were collected after 72 h by filtration and dried under vacuum at room

temperature sieved, through British sieve size (BSS) No. 50 and retained on BSS No. 72, before

all further experiments.

2.5. Characterization of crystallized solid forms

2.5.1. Optical and polarized light microscopy

Crystals of CEL were observed at a magnification of 500X, under optical and polarized

light microscope (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) with and without

silicon oil. The birefringence pattern was viewed under cross polarizer. Aspect ratio was

determined using a pre-calibrated stage micrometer. For both crystal habits, the distribution of

particle size, taken as the length along the longest axis of individual crystal, was plotted using

100 particles. D90, i.e., length corresponding to 90% of cumulative undersize particles, was

determined from the size distribution plot.

2.5.2. Scanning electron microscopy (SEM)

SEM photographs of the crystals were captured using scanning electron microscope (S-

3400, Hitachi Ltd., Tokyo, Japan) operated at an excitation voltage of 25 kV. Samples were

prepared by laying particles on to a double-sided adhesive tape pasted over sample stubs and

sputter coated with gold using ion sputter (E-1010, Hitachi Ltd., Tokyo, Japan), before analysis.

2.5.3. Thermogravimetric analysis (TGA)

Presence of solvent or any degradation during heating was examined using Mettler

Toledo 851e TGA/SDTA (Mettler Toledo, Switzerland) operating with Stare software (version

Solaris 2.5.1). Accurately weighed (5-10 mg) samples were loaded in alumina crucibles and

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heated at a rate of 20 ºC/min over a temperature range of 35 to 200 ºC, under nitrogen purge (50

mL/min), to determine loss in weight.

2.5.4. Hot stage microscopy (HSM)

HSM was carried out to observe thermal transitions using Leica DMLP polarized

microscope (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) equipped with Linkam

LTS 350 hot stage. Photomicrographs were captured using JVS color video camera and analyzed

using Linksys32 software. Samples were mounted in silicon oil and heated from 35 to 200 ºC, at

a heating rate of 20 ºC /min.

2.5.5. X-Ray powder diffraction (XRPD)

XRPD patterns of samples were recorded at room temperature on Bruker’s D8 Advance

X-ray diffractometer (Karlsruhe, Germany) using Cu-Kα radiation (λ = 1.54 Ǻ) at 35 kV, 30 mA

passing through a nickel filter. Data was collected in a continuous scan mode with a step size of

0.01o and dwell time of 1 s over an angular range of 3° to 40° 2θ. Accurately weighed amount of

powder (about 300 mg) was loaded in a 25 mm poly-methyl methacrylate (PMMA) holder and

gently pressed by a clean glass slide to ensure coplanarity of the powder surface with the surface

of the holder. Obtained diffractograms were analyzed with DIFFRACplus EVA (version 9.0)

diffraction software.

2.5.6. Differential scanning calorimetry (DSC)

Conventional DSC experiments were conducted to determine melting point and heat of

fusion using DSC Q2000 (TA Instruments, Delware, USA) equipped with a refrigerated cooling

system and operating with Universal Analysis 2000 software (version 4.5A). The sample cell

was purged with dry nitrogen at a flow rate of 50 mL/min. Accurately weighed samples (3–5mg)

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in aluminum crimped pans were scanned at a heating rate of 20 ºC/min over a temperature range

of 35-200 ºC. The DSC instrument was pre-calibrated for temperature and heat flow using high

purity indium. All measurements were performed in triplicate.

2.5.7. Specific surface area

Specific surface area was determined using nitrogen gas sorption (SMART SORB 91

Surface Area analyzer; Smart Instruments, Mumbai, India). The instrument was calibrated by

injecting a known quantity of nitrogen. The measured parameters were then used to calculate the

surface area of the sample by employing the adsorption theories of Brunauer, Emmett and Teller

(BET). The weighed samples (5 g) were degassed to remove moisture. Samples were dipped in

liquid nitrogen and the quantity of the adsorbed gas was measured using a thermal conductivity

detector. The obtained data were integrated using an electronic circuit in terms of counts. The

reported values were average of three measurements.

2.5.8. Solubility study

Solubility of CEL crystals was determined (n=3) in double distilled water and in pH 12

phosphate buffer. Accurately weighed sample (about 20 mg) was added to 20 mL of medium in

a tightly capped vial. The vial is placed in a shaker water bath (Julabo Labortechnik GmbH,

Seelbach, Germany) maintained at 100 rpm and 37 ± 0.5 °C. Samples were withdrawn at

appropriate intervals (up to 72 h), filtered through 0.22 µm nylon filter and analyzed for drug

content using HPLC. Residual solids were analyzed by XRPD for phase identification.

2.5.9. Contact angle measurement

Sessile drop contact angle is most commonly measured on surface of compacted disc.

However, compaction of the material can alter the particle morphology and surface free energy.

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Alternatively, contact angle may be measured on a powder layer adhered to an inert support.35, 36

The later method was adopted in the present work as it allows the study of ‘as is’ powder

properties.

Advancing and receding contact angles made by double distilled water, pH 12 phosphate

buffer, EG and DIM with powder samples were measured using sessile drop method on a Drop

Shape Analyzer (FTA 1000, First Ten Angstrom, Virginia, USA). Powder samples were

mounted on double sided adhesive tape adhered to a glass slide. Excess powder was removed by

tapping and a drop of liquid medium was dispensed on them. From a captured video, contact

angle was calculated as a function of time by fitting mathematical expression to the shape of the

drop and then calculating the slope of the tangent to the drop at the liquid-solid-vapor interface

line. All measurements were performed under ambient conditions of 25 ± 2 °C and 55 ± 5% RH,

with the reported values being an average of six measurements.

2.5.10. X-Ray photoelectron spectroscopy (XPS)

X-Ray photoelectron spectra were recorded using an ESCA-3000 (VG Scientific Ltd,

England) with a 9 channeltron CLAM4 analyzer under a vacuum better than 1 x 10-8 Torr, using

Mg-Kα radiation (1253.6 eV) and a constant pass energy of 50 eV. Binding energy range was

from 0 to 1100 eV for regions of C 1s, N 1s, O 1s, F 1s, and S 2p with average peak binding

energy of 286.0, 400.9, 533.0, 688.7, and 170.2 eV, respectively. All spectra were corrected for

baseline and fitted using Gaussian function. Fitting was performed using PeakFit®

(V.4.12,

SeaSolve Software, Inc., MA, USA). Similar curve fitting treatments were given for both the

crystal habits. Surface atomic concentration was determined from integrated peak intensities and

the corresponding relative sensitivity factor.

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2.5.11. Molecular modelling

Molecular lipophilic surface potential (MLSP) analysis of CEL was carried out using the

MOLCAD program implemented in the SYBYL7.1 molecular modelling package. The

Gasteiger-Hückel charges were assigned to the atoms of CEL structure and surfaces were

generated and visualized. The color ramp for the MLSP ranges from deep blue color,

representing lower lipophilic potential (LP), to the deep red color, representing higher LP. This

analysis can provide LP surrounding each atom or group of atoms and the 3D spatial features of

the molecular interactions in crystal. Molecular arrangement on different crystal facets of CEL

form III was visualized using Mercury (version 2.3, Cambridge Crystallographic Data Centre,

Cambridge, UK).

2.5.12. Face indexation

A crystal was placed onto the tip of a 0.1 mm diameter glass capillary and mounted on a

Bruker Smart Apex 2 diffractometer (Karlsruhe, Germany) with CCD area detector for

determining unit cell parameters and orientation matrices at -100 °C. Cell constants were

determined from reflections harvested from three sets of 12 frames. These initial sets of frames

were oriented such that orthogonal wedges of reciprocal space were surveyed. This produced

initial orientation matrices determined from 89 and 104 reflections for CEL-A and CEL-P

respectively. The data collection was carried out using Mo-Kα radiation having λ = 0.71073 Å

(graphite monochromator) with a frame time of 30 seconds and a detector distance of 6 cm. A

series of images were taken with a video microscope as the crystal is rotated through 360° about

the ψ axis. Miller’s indices of various facets of the crystal were identified using T-tool, the face-

indexing plug-in of APEX 2.

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2.5.13. Oral bioavailability study

All animal experiments were performed in accordance with the Committee for Purpose of

Control and Supervision on Experiments on Animals (CPCSEA) guidelines and the experimental

protocols were approved by the Institutional Animals Ethics Committee (IAEC/12/40). Male

Sprague–Dawley rats ranging from 250 ± 25 g, were kept on fasting for 12 h before the

experiment and were allowed free access to water before and during the experiment. Both

CEL crystal habits were administered at a dose of 5 mg/kg of rat body weight via an oral gavage.

The powder was filled in wide bore, bulb tipped gastric gavage of sufficient length, to allow

intra-gastric administration. The gavage was attached to a syringe, filled with 1.0 mL double

distilled water, and the dose was delivered with the aid of a jet of water, which drained the

sample along with it. Blood samples were collected from retro-orbital plexus after 0.5, 1, 1.5, 2,

3, 4, 6, 8, 12, 24, and 48 h in heparinzed microcentrifuge tubes. Plasma was separated

immediately by centrifugation at 15000 rpm for 10 min at 4 °C and stored at -80 °C until

processed and analyzed. Plasma samples were extracted with acetonitrile and quantified using a

validated HPLC method.

Various pharmacokinetic parameters were calculated from the mean plasma CEL

concentration–time profiles using the Thermo Kinetica software (V5.0, Thermo Fischer

Scientific, USA). Statistical significance for pharmacokinetic parameters was compared using the

paired t-test assuming equal variances. The test was considered to be statistically significant, if p

< 0.05.

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3. RESULTS

3.1. Solubility studies

Equilibrium solubility of “as received” CEL in toluene, at different temperatures, is

captured in table 1. The data was used to generate van’t Hoff plot, which allowed extrapolation

of solubility to different temperatures for determining degree of supersaturation during

recrystallization experiments.

Table 1. Equilibrium solubility of CEL in toluene

Equilibrium Solubility (mg/mL)

28 °C 32 °C 36 °C 40 °C

11.56 ± 0.22 12.70 ± 0.16 13.88 ± 0.36 14.97 ± 0.20

3.2. Crystallization experiments

The degree of supersaturation was varied by controlling solution concentration and

temperature of crystallization. Polarized light microscopic images (Figure 1a, 1b) and SEM

photographs (Figure 1c, 1d) revealed that acicular crystals (CEL-A) were obtained from toluene

when saturated solution of CEL was cooled and allowed to crystallize at 25 °C (degree of

supersaturation was 190%). On the other hand, plate shaped crystals (CEL-P) were generated

when solvent was evaporated at 60 °C (degree of supersaturation was 102%). The aspect ratio for

CEL-A was 12-20 while that for CEL-P was 4–8.

Both CEL-A and CEL-P were confirmed to be anhydrous by HSM and TGA. Further, gas

chromatography and Karl Fischer analysis confirmed the absence of residual solvent and water

respectively in these samples (< 0.02%).

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Figure 1. Polarized microscopic and SEM images of (a, c) CEL-A and (b, d) CEL-P respectively

XRPD patterns of both CEL-A and CEL-P showed characteristic diffraction peaks at 2θ

values of 5.37°, 10.72°, 16.11°, 19.72° and 21.52° corresponding to CEL form III. The observed

variation in relative peak intensity (Figure 2) can be ascribed to preferred orientation of the

crystals during XRPD analysis.

Figure 2. Overlay of XRPD scans of CEL crystal habits

Lin (Counts)

0

100000

200000

3 10 20 30 40

CEL-P

CEL-A

2-Theta Scale

Lin

(C

ou

nts

)

2-Theta scale

Lin

(C

ou

nts

)

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DSC heating curves (Figure 3) of CEL-A and CEL-P showed melting point of 160.75 ±

1.00 °C and 161.09 ± 1.22 °C with melting enthalpy of 90.92 ± 1.76 J/g and 91.63 ± 1.50 J/g

respectively (Table 2).

Table 2. DSC results for CEL crystal habits

Crystal habit Melting point (οC) Heat of fusion (J/g)

CEL-A 160.75 ± 1.00 90.92 ± 1.76

CEL-P 161.09 ± 1.22 91.63 ± 1.50

Figure 3. Overlay of DSC heating curves of CEL crystal habits

Further, the D90 value and specific surface area of two sieved crystal habits were comparable (Table 3).

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Table 3. Particle size distribution and specific surface area of CEL crystal habits

Crystal

habits

D90

(µm)

Specific surface area (m2/g)

(n = 3)

CEL-A 252.8 0.92 ± 0.04

CEL-P 248.2 0.85 ± 0.02

3.3. Solubility study

The dissolution profiles of CEL-A and CEL-P, in double distilled water and pH 12

phosphate buffer, are presented in Figure 4. The CEL-P dissolves much faster than CEL-A in

both media. The solution concentration of CEL-A is considerably lower than that of CEL-P up to

24 h (p < 0.05) in both media. However, at 48 and 72 h, the difference in concentration is

statistically insignificant (p > 0.05) in both media. Table 4 summarizes the solubility values at 2

h, 4 h, and 12 h (S2, S4 and S12), respectively. There were no polymorphic transformations

observed at the end of experiment in both cases.

Figure 4. Solution concentration - time profiles of CEL crystal habits in (a) double distilled water and (b)

pH 12 phosphate buffer

(a) (b)

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Table 4. Solution concentration at different time points for two CEL crystal habits

Crystal habits Water (µg/mL) pH 12 buffer (µg/mL)

S2 S4 S12 S2 S4 S12

CEL-A 0.8 ± 0.2 1.8 ± 0.2 2.8 ± 0.1 166.6 ± 7.4 227.1 ± 16.5 365.7 ± 11.8

CEL-P 2.7 ± 0.2 3.4 ± 0.1 3.8 ± 0.1 283.6 ± 16.9 345.3 ± 11.6 396.7 ± 7.1

3.4. Contact angle and wettability

Wetting behavior of pharmaceutical solids was assessed by contact angle

measurements.35, 37 The advancing and receding contact angle of drop of different probe liquids,

deposited on powder surface, was determined and also characterized for loop of hysteresis (Table

5).

The advancing contact angles, using double distilled water, were 102.0° ± 0.7° and 91.5°

± 1.3° for CEL-A and CEL-P, respectively (p < 0.05). When using pH 12 phosphate buffer as

the probe liquid, advancing contact angle of CEL-A and CEL-P were 94.5° ± 1.2° and 85.3° ±

1.8°, respectively (p < 0.05). Further, with EG, CEL-A and CEL-P showed a contact angle of

72.1° ± 0.8° and 66.4° ± 2.2°, respectively (p < 0.05). Hence, CEL-P has a higher wetting

tendency with both polar and semipolar media.

When the nonpolar DIM was used as the probe liquid, CEL-A and CEL-P showed an

initial contact angle of 20.8° ± 2.0° and 27.8° ± 0.6°, respectively (p < 0.05). Hence, CEL-A has

a higher wetting tendency in nonpolar medium.

The loop of hysteresis (θHys) was determined by subtracting receding contact angle from

advancing contact angle for each probe liquid. Most liquid-solid interactions exhibit hysteresis

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because of differences in behavior of liquid, advancing over surface or receding from it.

Hysteresis is indicative of surface roughness and / or heterogeneity. Both CEL-A and CEL-P

showed similar hysteresis values (<8) for all four liquids suggesting their similar surface

roughness and / or heterogeneity38.

Table 5. Advancing and receding contact angles along with loop of hysteresis of CEL-A and CEL-P with

different probe liquids

Crystal

habits

Contact angle (°)

Water pH 12 buffer EG DIM

θA θR θHys θA θR θHys θA θR θHys θA θR θHys

CEL-A 102.0 ±

0.7

95.5 ±

1.1

6.5 ±

0.9

94.5 ±

1.2

86.2 ±

2.2

8.3 ±

0.8

72.1 ±

0.8

66.9 ±

2.8

5.2 ±

1.9

20.8 ±

2.0

13.9 ±

1.2

7.0 ±

1.3

CEL-P 91.5 ±

1.3

85.2 ±

1.6

6.3 ±

1.3

85.3 ±

1.8

77.6 ±

1.7

7.7 ±

0.9

66.4 ±

2.2

60.9 ±

2.3

5.5 ±

2.1

27.8 ±

0.6

21.9 ±

2.0

5.9 ±

2.0

θA: Advancing contact angle, θR: Receding contact angle, θHys: Loop of hysteresis

3.5. Determination of surface free energy

The measured advancing contact angle values were used to determine the total (γS),

dispersive (γLW), polar (γAB) components of surface energy of solids using van Oss Good

Choudhary theory39. The theory subdivides the polar component of surface energy of solids and

liquids into two specific components: the surface energy due to acidic interactions (γA) and due

to basic interactions (γB). The acid component theoretically describes propensity of a surface to

have polar interactions with a second surface that has basic characteristics by donating electrons.

Conversely, the base component of the surface energy describes the propensity of a surface to

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have polar interactions with another surface that has acidic characteristic by accepting

electrons40. The principle equation for this theory is:

( ) ][ 2 cos1 B

S

A

L

B

L

A

S

LW

L

LW

SLV γγγγγγγθ ++=+ Eq. (1)

The method requires use of at least three liquids –(i) liquid having only a dispersive

component to its surface energy, (ii) liquid having a dispersive and an acidic or basic component

and (iii) liquid having either a dispersive and a basic or acidic component (whichever the second

probe liquid did not have), or a liquid with all three components. In present study, DIM, EG and

water were selected as probe liquids. The dispersive component had a major contribution to total

surface free energy in both powders. However, CEL-P has relatively lower dispersive component

and higher acidic component that contribute to its higher polar component compared to CEL-A

(Table 6).

Table 6. The total (γS), dispersive (γLW ), polar (γAB ), basic (γB) and acidic (γA)surface energies of CEL

crystal habits

3.6. Surface chemistry by XPS

XPS spectra showed presence of carbon (C), nitrogen (N), oxygen (O), sulfur (S) and

fluorine (F) on the surface of both habits. Peak shape and chemical shift for these elements were

similar between the two powders, indicating no qualitative differences in CEL crystal habits.

Crystal habits γS (mJ/m2) γLW(mJ/m2) γAB (mJ/m2) γA(mJ/m2) γB(mJ/m2)

CEL-A 47.93 47.54 0.39 0.04 0.77

CEL-P 47.10 45.10 2.00 2.20 0.45

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However, the relative abundance of surface elements shows significant differences (Table 7).

The surface of CEL-A sample exhibited a relatively lower concentration of O, N, S and higher

concentration of C than the CEL-P. The surface polarity, expressed as (O + N + S)/(C + F), was

0.42 and 0.52 for CEL-A and CEL-P, respectively.

Table 7. Surface elemental composition of CEL crystal habits

Crystal habits % Elemental composition

O 1s N 1s S 2p F 1s C 1s

CEL-A 8.48 10.51 10.70 10.12 60.17

CEL-P 9.25 13.13 11.72 10.47 55.41

3.7. Molecular modelling studies

Hydrophilic and hydrophobic moieties of CEL were traced using MLSP of the molecule,

which shows sites of high and low LP (Figure 5). Clearly, the lower LP site (shown by blue

shade) is around polarisable sulphonamide group. In contrast, higher LP (shown by orange

shade) of methyl phenyl moiety contributes to hydrophobic domains of molecule.

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Figure 5. MLSP analysis of CEL using the MOLCAD program implemented in the SYBYL7.1 molecular

modeling package (encircled portion shows the hydrophilic moiety of CEL)

3.8. Face indexation

The unit cell parameters of both CEL-A and CEL-P were in agreement to the reported

cell parameters of CEL form III (CSD Reference code: DIBBUL), i.e., both are triclinic unit

cells with α = 97.62° (7), β = 100.62° (6), γ = 95.95° (4) and a = 10.136 Å (5), b = 16.778 Å (6),

c = 5.066 Å (6).41

Because of the structural anisotropy, the orientation of molecules on the surface of each

facet of a crystal is different. The different relative abundance of crystal facets between crystal

habits will lead to different surface properties of bulk powders. Table 8 shows the face

indexation data for CEL crystal habits. Both crystal habits have shown dominance of (0 0 1) and

(0 1 0) facets and a few minor facets (Figure 6). However, relative abundance of the main facets

differs significantly between the crystal habits. The (0 1 0) and its opposite facet (0 -1 0)

contribute more in CEL-P (32.48%) than CEL-A (21.39%). On the other hand, (0 0 1) and (0 0 -

1) contribute less in CEL-P (57.64%) than CEL-A (74.87%).

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Table 8. Percentage contribution of major facets in CEL crystal habits

Crystal habit Facets on the surface

(0 0 1) and (0 0 -1) (0 1 0) and (0 -1 0) Other

CEL-A 74.87 21.39 3.74

CEL-P 57.64 32.48 9.88

Figure 6. Face indexation data of (a) CEL-A and (b) CEL-P

3.9. Oral bioavailability

Figure 7 shows the mean plasma concentration-time profiles for CEL-A and CEL-P. The

pharmacokinetic parameters were summarized in Table 9. Although no significant differences

observed in AUC0-48, Cmax and Tmax differ significantly between the two crystal habits. The Cmax

and Tmax of CEL-A are 351.12 ± 31.00 ng/mL and 2.8 ± 0.4 h. On the other hand, CEL-P

exhibits higher Cmax (423.5 ± 33.4 ng/mL) and reduced Tmax (1.0 ± 0.5 h).

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Table 9. Pharmacokinetic parameters for CEL crystal habits in male Sprague-Dawley rats,

obtained after single oral administration of single dose (5 mg/kg)

Crystal habits AUC0-48 (ng.h/mL) Cmax (ng/mL) Tmax (h)

CEL-A 5399.30 ± 452.51 351.12 ± 31.00 2.8 ± 0.4

CEL-P 6304.00 ± 519.79 423.50 ± 33.40* 1.0 ± 0.5*

* p < 0.05, statistically significant difference in comparison with CEL-A

Figure 7. Mean plasma concentration–time profile for CEL-A and CEL-P

4. DISCUSSION

Crystal growth rate is determined by the rate at which growth units attach and / or remove

themselves from the growing surfaces.42, 43 Crystal habit is determined by the different

deposition kinetics of solute molecules on different crystal facets. 7, 11, 14, 44 Crystal growth rate

differs along various axes of the unit cell and is usually faster in directions associated with

shorter unit cell axis.43 For CEL form III, c axis (a = 10.136 Å, b = 16.778 Å, and c = 5.066 Å)

is the shortest axis. Hence, CEL form III crystal grows faster along ‘c’ direction, which leads to

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its propensity to form needle-like crystals. However, it is possible to alter the growth rate by

altering crystallization conditions, such as type of solvent, temperature, degree of

supersaturation, and stirring rate.1, 7 In fact, two crystal habits were successfully prepared by

varying degree of supersaturation and crystallization temperature in this study.

The two crystal habits of CEL form III exhibited significantly different dissolution behaviors,

with CEL-P dissolves faster in water and pH 12 phosphate buffer (Figure 4) despite the similar

powder surface area (Table 3). To rationalize the different dissolution behaviors, we consider

different wettability of the two crystal habits since surface wetting of the crystals precedes

dissolution. For the same liquid, a stronger interaction between solid and liquid will predictably

result in a smaller contact angle.22, 45-47 CEL-P possessed greater polar surface free energy and

exhibited better wettability with water and pH 12 phosphate buffer than CEL-A. In contrast,

CEL-A, with more dispersive surface free energy component, showed better wettability with

non-polar solvent DIM. Hence, the faster dissolution of CEL-P in polar solvents is a result of the

better wettability. Differences in wettability of CEL form III crystal habits reflect differences in

intermolecular interactions between surface groups and the liquid medium.48-50 We hypothesized

that these differences resulted from different relative abundance of various facets in the two

crystal habits. XPS have been successfully used for identification and quantification of the

surface elemental composition.16, 17, 51 Prestidge and Tsatouhas had established a correlation

between contact angle and intensity ratios of X-ray photoelectron signals from hydrophilic and

hydrophobic moieties of morphine molecule.52 In this work, XPS results showed that the relative

abundance of each element differed significantly between crystal habits of CEL form III (Table

7).

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CEL molecules between two adjacent layers, with the electron donating and accepting

groups oriented towards each other, form a hydrogen bonded bi-layered structure (Figure 8a).

The (0 1 0) facet has a bed of –SO2NH2- moieties exposed on its surface and thus making this

facet more hydrophilic (Figure 8b). In case of (0 0 1) facet, the exposure of methyl phenyl

moieties and fluorine makes it relatively more hydrophobic (Figure 8c).

Figure 8. Visualization of (a) hydrogen bonded bilayered structure of CEL form III (b) molecular surface

packing along with surface chemistry of (010) facet and (c) (001) facet using Mercury 2.3 software

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Face indexation data revealed that the relative abundance of different facets of CEL form

III crystal differs significantly amongst its crystal habits. Hence, we visualized the surface

molecular packing of two prominent facets of CEL crystal habits (Figure 8). As already

mentioned, the relative contribution of (0 1 0) facet in CEL-P is higher than CEL-A. These

observations are in line with the XPS data that indicated a higher presence of O, N, S and F on

the surface of CEL-P. A lower LP around –SO2NH2- group signifies molecular domain having

greater propensity for interactions with polar solvents. However, methyl phenyl moiety has a

higher LP, hence, weaker interaction with aqueous phase. The overall more abundant polar facets

in CEL-P explain its higher dissolution rate in aqueous media.

Importantly, we have found that the better in vitro dissolution performance of CEL-P

translates into in vivo oral bioavailability advantage. There was statistically significant

improvement in Cmax and Tmax of CEL-P (Figure 7). Cmax of CEL-P was approximately 20%

higher than CEL-A while Tmax of CEL-P was also significantly reduced (Table 9). This level of

improvement is relevant to biopharmaceutical performance of BCS class II drugs.

5. CONCLUSIONS

Acicular and plate shaped crystal habits of CEL form III were obtained by varying

crystallization conditions. Although these crystal habits have the identical crystal structure,

significant differences were observed in their dissolution kinetics and wettability. CEL-P

exhibited greater polar component of surface energy due to the higher percentage of crystal

facets richer in polar domains of CEL molecule. Thus differences in physicochemical properties

and oral bioavailability of CEL-A and CEL-P, could be ascribed to surface anisotropy and

different abundance of exposing crystal facets. This study establishes the potentially significant

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contribution of crystal habit on the performance of crystalline drug, which has special relevance

to BCS class II drugs that have solubility and/or dissolution limited oral bioavailability.

AUTHOR INFORMATION

Corresponding Author

*Arvind K. Bansal

Department of Pharmaceutics

National Institute of Pharmaceutical Education and Research (NIPER)

Sector 67, SAS Nagar, Punjab 160 062, INDIA

Tel: +91-172-2214682-86. Fax: +91-172-2214692.

E- mail: [email protected]

NOTES

The authors declare no competing financial interest

ACKNOWLEDGMENT

We thank the National Chemical Laboratory (NCL), Pune, India for providing facility of X-ray

photoelectron spectroscopy (XPS) and the X-Ray Crystallographic Laboratory, S146 Kolthoff

Hall, Department of Chemistry, University of Minnesota, USA for supporting the crystal face

indexing studies. We are also thankful to Dr. Dimitrios A. Lamprou, University of Strathclyde,

UK for his guidance on surface free energy calculations.

SUPPORTING INFORMATION AVAILABLE

Representative X-ray photoelectron (XP) spectra of different elements of celecoxib. This

material is available free of charge via the Internet at http://pubs.acs.org.

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ABBREVIATIONS

API, active pharmaceutical ingredient; BCS, biopharmaceutical classification system; CEL,

celecoxib; CEL-A, acicular crystals of celecoxib; CEL-P, plate shaped crystals of celecoxib; LP,

lipophilic potential; MLSP, molecular lipophilic surface potential; XPS, X-ray phaotoelectron

spectroscopy.

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For Table of Contents Use Only

TABLE OF CONTENTS GRAPHIC

SYNOPSIS

The goal of this work was to systematically investigate how different crystal habits and their

differential anisotropic surface properties can impact on biopharmaceutical performance of same

polymorphic form of pharmaceutical actives. This study establishes the potentially significant

contribution of crystal habit on the performance of crystalline drug, which has special relevance

to BCS class II drugs that have solubility and/or dissolution limited oral bioavailability.

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Impact of Crystal Habit on Biopharmaceutical Performance of Celecoxib

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