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In Vivo Study of Macrolide

Antibiotics & Optimized

Recrystallized Agglomerates

Chapter 10: In vivo study of macrolide antibiotics and optimized recrystallized

agglomerates

Direct tabletting and BA improvement of MA by spherical crystallization tech. 286

10. In vivo study (Bioavailability) of MA and optimized recrystallized agglomerates:

10.1. Introduction:

During the past decade, formulation and delivery of Active Pharmaceutical Ingredients

(APIs) have played a critical role in the development and commercialization of new

pharmaceutical products. The major objective of formulation chemistry is to improve

bioavailability, stability and convenience to the patient. Bioavailability means the rate

and extent to which the active substance or therapeutic moiety is absorbed from a

pharmaceutical form and becomes available at the site of action. The bioavailability of an

orally administered drug depends on its solubility in aqueous media over the pH range of

1.0–7.5 and the rate of mass transfer across biological membranes. A limiting factor in

the oral bioavailability of poorly water soluble compounds is the inadequate dissolution

rate. Development of solid dosage forms for water insoluble drugs has been a major

challenge for pharmaceutical scientists for decades to improve BA. It is well known that

drug efficacy can be severely limited by poor aqueous solubility because the driving

force for absorption of most drugs across biological membranes is concentration of drug

in solution. Dosage forms that enter the stomach and travel down the gastrointestinal tract

must release the drug in solution to achieve good drug bioavailability. Consequences of

poor solubility include low bioavailability, large inter and intra subject variation, and

large variations in blood drug concentrations under fed versus fasted conditions.

Poor bioavailability can be also due to poor solubility, degradation in GI lumen, poor

membrane permeation and presystemic elimination (1, 2). By many estimates up to 40

percent of new chemical entities (NCEs) discovered by the pharmaceutical industry today

and many existing drugs are poorly soluble or lipophilic compounds which leads to poor

oral bioavailability, high intra- and inter-subject variability and lack of dose

proportionality (3). Thus, for such compounds, the absorption rate from the

gastrointestinal (GI) lumen is controlled by dissolution (4). The ability to deliver poorly

soluble drugs will grow in significance in the coming years as innovator companies rely

upon NCEs for a larger share of the revenue within the pharmaceutical market. Many

technological methods of enhancing the dissolution characteristics of slightly water-

soluble drugs have been reported in various literatures (5). These include reducing

particle size to increase surface area (6), solubilization in surfactant systems, formation of

water-soluble complexes, use of pro-drug, drug derivatization and manipulation of solid

state of drug substance to improve drug dissolution, i.e. by decreasing crystallinity of

drug substance (7). Recently, natural polymers such as polysaccharides and proteins have

received much attention in the pharmaceutical field owing to their good biocompatibility

and biodegradability (8).

The spherical crystallization technique has already been successfully applied to improve

the micromeritic properties of several drugs such as acebutolol hydrochloride, celecoxib,

and mefenamic acid etc [9-11]. Besides modifying the size and shape, flowability,

packability and bulk density of the particles, this technique can also be exploited to

increase solubility, dissolution rate and hence bioavailability of poorly soluble drugs (12).

In the spherical crystallization technique the manipulation of solid state of drug substance

to improve solubility and drug dissolution i.e. by decreasing crystallinity of drug

substances by recrystallizing the drug substances in different solvents by using different

pharmaceutical excipients. Hence the objective of present investigation is to evaluate the


agglomerates


in vivo performance of optimized recrystallized agglomerates of MA with respect to

pharmacokinetic parameters like Cmax, Tmas and AUC in animal models.

10.2. Bio-analytical method development (13-20):

HPLC equipment

The HPLC system consisted of PU – 2080 plus intelligent system pump, a sampler with

20μl loop, a UV detector UV– 2075 plus intelligent and an interface LC-Net II/ADC, all

from Jasco (Tokyo, Japan). The reverse-phase column was a HiQ Sil C18-W,

4.6mm×250mm column.

Sample preparation:

1 ml blood of rat was collected from Retro orbital vein and immediately transferred into

anti-coagulant free 1.5 ml centrifugation tube and subjected to centrifugation at room

temperature at 8000 rpm for 10 min. The serum obtained from plasma was mixed with 1

ml of known concentration of drug in acetonitrile and centrifuged at 8000 rpm for 2 min.

The resultant supernatant solution was further filtered through 0.2 µ filter and injected

manually in 20 µl loop.

10.2.1. Azithromycin:

Chromatographic conditions:

Mobile phase: phosphate buffer pH 6.5(0.02M): acetonitrile (25:75)

Flow rate: 0.9 ml/min

Retention time: 7.9 min

Sample volume: 20 µl

Detection wavelength: 215 nm

Concentration range: 50-1000 ng/ml.

HPLC analysis: Azithromycin:

Validation data was collected from three analytical runs. All rat serum lots have used to

prepare calibration standards. Retention time of approximately 7.9 min was consistently

observed for Azithromycin throughout all analytical runs. The obtained peak for ATM in

plasma was mentioned in figure 10.1. Calibration curve data (Figure 10.2) for

Azithromycin in rabbit serum demonstrate that the calibration curve was linear in the

concentration range from 50-1000 ng/ml. The correlation coefficient was found to be

0.9993.


agglomerates


Fig 10.1: HPLC chromatogram of Azithromycin.

Figure: 10.2 Standard calibration curve of Azithromycin by HPLC method.

Calibration Report

Component: ATM


agglomerates


User: Venkat, Group: MA, Model: Y = AX

Nb of Points: 7, A = 149.4062, B = 0.0000

Correlation = 0.99638

Standard Error Vy = 1971.2201

Mean %Error = 5.446

Equation: Y=151.0x-1157

Table: 10.1. Observation table for standard curve for Azithromycin by HPLC

method.

Sr. No. Concentration

(ng/ml) Area

1 0.0 0.000

2 50 7520.1867

3 100 14590.3537

4 200 33250.7174

5 400 52557.4249

6 600 85875.1324

7 800 116202.9881

8 1000 156565.5573

The linearity was performed with a 7 point calibration curve. The method was found to

be linear over the examined concentration range 50-1000 ng/ml. The average calibration

equation could be described by: Y=151.0x-1157, with a correlation coefficient of 0.9964.

Where the y is the ratio of the peak area of ATM and internal standard and x is the

concentration (ng/ml). The limit of detection (LOD) was 20 ng/ml with HQC

(1000ng/mL) and MQC (400ng/mL). The retention time is 7.9 min.

10.2.2. Roxithromycin:

Chromatographic conditions:

Mobile phase: phosphate buffer pH 6.5(0.02M): acetonitrile (24:76)

Flow rate: 0.5 ml/min

Retention time: 7.4 min

Sample volume: 20 µl

Detection wavelength: 205 nm

Concentration range: 2 –12 µg/ml

HPLC analysis of Roxithromycin:

The chromatographic peak due to RTM was observed at 7.4 min, which indicated

sensitivity and selectivity of the developed method. The calibration curve was plotted in

the range between 2-12 µg/ml. The obtained peak for RTM in plasma was mentioned in

figure 10.1. Correlation coefficient of 0.99996 indicated linearity of the method within

the calibration range. Calibration curve and statistical analysis of RTM was reported in

following figure: 10.4.


agglomerates


Figure: 10.3. HPLC chromatogram of Roxithromycin.

Figure: 10.4. Standard calibration curve of Roxithromycin by HPLC method.

Calibration Report.

Component: RTM


agglomerates


User: Venkat, Group: MA, Model: Y = AX

Nb of Points: 6, A =108021.0464, B = 0.0000

Correlation = 0.99946, Standard Error Vy = 6088.9780

Mean %Error = 2.323

Equation: Y=10757x+3905.

Table: 10.2. Observation table for standard curve for Roxithromycin by HPLC

method.

Sr. No. Concentration

(µg/ml) Area

1 0.0 0.000

2 2 230633.7500

3 4 421752.2800

4 6 655091.8387

5 8 853986.1200

6 10 1098521.7300

7 12 1285310.5800

Y=10757x+3905.

The linearity was performed with a 6 point calibration curve. The method was found to

be linear over the examined concentration range 2-12 µg/ml. The average calibration

equation could be described by: Y=10757x+3905, with a correlation coefficient of

0.9994. Where the y is the ratio of the peak area of RTM and internal standard and x is

the concentration (µg/ml). The limit of detection (LOD) was 1.0 µg/ml with HQC

(12µg/mL) and MQC (6µg/mL). The retention time is 7.4 min.

10.3. Comparative in vivo study of MA and recrystallized agglomerates in wistar

rats (21-27):

The protocol was approved by institutional animal ethics committee (IAEC). The

experiments were carried out as per CPCSEA (Committee for Prevention, Control and

Supervision of Experimental Animals) guidelines. The in-vivo bioavailability study of

macrolide antibiotics (Azithromycin and Roxithromycin) was carried out on Male Wistar

rats. A parallel design comprising of two groups with six rats in each group was selected.

To evaluate the effects of the optimized spherical agglomerates on the release profile in

vivo, the bioequivalence of macrolide antibiotics and optimized agglomerated crystals

were studied in rats. A single-dose, randomized study was conducted on 12 rats (Wistar

rats, male, weight 250–350 g). All animals had free access to tap water and pelleted diet.

The rats were divided in two groups and fasted for overnight. The API (Azithromycin

and Roxithromycin) were selected as reference formulation; however, the prepared

recrystallized agglomerates ATM-EudS and RTM-SSG were selected as test formulation

for azithromycin and roxithromycin respectively. The first group received API whereas

the second group received recrystallized agglomerates ATM-EudS and RTM-SSG.

1. Animal model: Rats


agglomerates


2. Study Design: Parllel

3. Study period: 1 week

4. Sample: Plasma

5. Sample collection: from marginal ear vein with out anticoagulant

6. Sampling points(hrs):

Azithromycin: 0.0,0.5,1.0,1.5,2.0,2.5,3.0,4.0,6.0,12,48,96,

Roxithromycin: 0.0,0.5,1.0, 1.5,2.0,2.5,3.0,4.0,6.0,12,24,48,

7. Number of sampling point:

Azithromycin:12

Roxithromycin:12

8. Number of animal in each group: 6

9. Animal selection: Randomization

The Macrolide antibiotics (Azithromycin and Roxithromycin) and optimized

recrystallized agglomerates ATM-EudS and RTM-SSG respectively were dispersed in

distilled water and immediately administered through oral gavage.Blood samples (≈1.0

mL) were then collected in eppindrop tube at time interval mentioned in protocol through

retro orbital vein. The eppindrop tube with blood sample was then subjected to

centrifugation at room temperature at 8000 rpm for 10 minutes to separate plasma

The separated plasma samples were taken after centrifugation in eppindrop tube and

frozen at −20 0C until HPLC analysis.

Analysis:

Took 100 µl of above stored plasma to it added 100 µl acetonitrile in eppindrop tube. The

mixture was vortexed for 5min and centrifuged at 8000 rpm for 5 min.The supernatant

(20µl) was then injected through micro syringe.

The pharmacokinetics and statistical analysis were computed using the different

statistical software. Cmax (the maximum plasma concentration) and Tmax (time point of

maximum plasma concentration) were obtained directly from the measured data; AUC

(area under the plasma concentration–time curve) was calculated according to the

trapezoidal rule.AUC from hour 0 to time t (AUC0-t) was calculated using the linear

trapezoidal method, whereas AUC from hour 0 to any time point ∞ (AUC0-∞) was

calculated as the sum of AUC0- t and the ratio of time of the last measurable

concentration over the elimination rate constant (Ct/Ke). Ke was obtained from the slope

of the terminal log-linear phase of the semilog plot of concentration versus time.

Tl/2 was estimated using the following equation:

T1/2 = 0.693/ke

Extrapolation of AUC0-∞ was calculated as follows:

AUC0-∞ = AUCo-t + (Ct/k e)


agglomerates


Table: 10.3. Plasma concentrations of Azithromycin from reference and test sample

at respective time interval.

Scheduled

Time

(hours)

Concentration (ng/ml)

Standard

sample (ATM)

ATM-EudS

0.0 00 ± 0.000 00 ± 0.000

0.5 28.2 ±1.722 53.7 ±10.633

1.0 99.3 ±8.937 201.8 ±46.649

1.5 211.0 ±9.381 390.7 ±88.188

2.0 306.2 ±49.036 488.3 ±44.684

2.5 371.7 ±13.307 449.2 ±72.428

3.0 346.0 ±28.879 383.5 ±20.579

4.0 233.7 ±35.998 321.7 ±14.909

6.0 169.7 ±38.255 265.8 ±18.755

12 136.2 ±23.836 203.8 ±15.039

48 105.8 ±11.873 153.2 ±8.377

96 81.3 ±12.987 105.7 ±11.708

Table: 10.5. Plasma concentrations of Azithromycin from reference (ATM) and test

(ATM-EudS) sample at respective time interval in rats (n=6).


agglomerates


Table: 10.4. Pharmacokinetic parameters of reference (ATM) and test (ATM-EudS)

sample of azithromycin in rats (n=6)

Subject No Pharmacokinetic parameters

Cmax

(ng/mL)

Tmax

(hr)

AUC(0-t)

(ng.hr/mL)

AUC(0-∞)

(ng.hr/mL)

T1/2

(hrs)

Subject for Reference sample

R1 385 2.5 9544.2 13831.21 39.61

R2 405 2 9917.3 14400.10 39.52

R3 378 2.5 12049.1 18539.48 47.10

R4 366 2.5 10871.65 16933.44 46.42

R5 390 2.5 11829.1 17251.24 43.19

R6 377 3.0 12097.25 15133.32 35.07

Mean 383.5 2.5 11051.4 16014.8 41.8

SD 13.569 0.316 1121.086 1838.424 4.618

Subject for Test sample

T1 480 2 15413.25 20925.90 40.21

T2 550 2 16138.75 20812.50 35.99

T3 495 2 15234.00 22108.65 43.31

T4 562 1.5 15161.75 21076.66 39.04

T5 588 2.5 17393.00 24646.10 41.20

T6 525 2 15908.00 22722.68 42.17

Mean 533.3 2.0 15874.8 22048.7 40.3

SD 41.161 0.316 837.591 1479.058 2.590

P-Value ** P<0.01 * P<0.05 ** P<0.01 ** P<0.01 ns P>0.05

One-way Analysis of Variance (ANOVA)

The P value is 0.0217, considered significant.

The P value is 0.6720, considered not significant.


The P value is < 0.0001, considered extremely significant.

Table: 10.5. Single factor Anova treatment to Cmax (ng/mL) of ATM and ATM-

EudS.

Groups Count Sum Average Variance

ATM(Cmax) 6 35.69305 5.948842 0.001191

ATM-EudS(Cmax) 6 37.65989 6.276648 0.006017

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 0.322371 1 0.322371 89.45121 2.64E-6 4.964603

Within Groups 0.036039 10 0.003604

Total 0.35841 11

Anova: Single Factor


agglomerates


Table: 10.6. Single factor Anova treatment to Tmax (hr) of ATM and ATM-EudS.


ATM(Tmax) 6 5.456922 0.909487 0.016551

ATM-EudS(Tmax) 6 4.094345 0.682391 0.026372

ANOVA


Between Groups 0.154718 1 0.154718 7.209062 0.022897 4.964603

Within Groups 0.214616 10 0.021462

Total 0.369334 11


Table: 10.7. Single factor Anova treatment to AUC (0-∞) (ng.hr/mL) of ATM and

ATM-EudS.


ATM- AUC (0-∞) 6 58.05467 9.675778 0.013176

ATM-EudS- AUC(0-∞) 6 59.99519 9.999198 0.004278

ANOVA


Between Groups 0.313802 1 0.313802 35.95797 0.000133 4.964603

Within Groups 0.087269 10 0.008727

Total 0.401071 11


Table: 10.8. Single factor Anova treatment to T1/2 (hrs) of ATM and ATM-EudS.


ATM- T1/2 6 22.36885 3.728141 0.012607

ATM-EudS- T1/2 6 22.17047 3.695079 0.004306

ANOVA


Between Groups 0.003279 1 0.003279 0.387774 0.547409 4.964603

Within Groups 0.084567 10 0.008457

Total 0.087847 11



agglomerates


In vivo performance (pharmacokinetic) data of ATM and their optimized recrystallized

agglomerate ATM-EudS are given in table: 10.3, 10.4 and figure: 10.5.In vivo results of

ATM shows mean (SD) Cmax was 383.5 (13.569) ng/mL and mean (SD) Tmax was 2.5

(0.316) hours; with ATM-EudS, mean (SD) Cmax was 533.3 (41.161) ng/mL and mean

(SD) Tmax was 2.0 (0.316) hours. Cmax and Tmax values were significantly (**

P<0.01/* P<0.05) improved in ATM-EudS comparative to ATM.

Mean (SD) T1/2 values of the test and reference formulations were 33.5 (2.418) and 41.8

(4.618) hours, respectively, shows non significant difference (ns P>0.05).

The mean (SD) AUC0-t and AUC0-∞ (as an index of extent of absorption) 15874.8

(837.591) and 22048.7 (1479.058) for test sample (ATM-EudS) where as 11051.4

(1121.086) and 16014.8 (1838.424) for reference sample (ATM).Both the area under

curve AUC0-t and AUC0-∞ were significantly improve (*P<0.05) in ATM-EudS

comparative to ATM.

Further to determine the level of significance these pharmacokinetics parameters were

treated by using single factor one way ANOVA.The p valve was < 0.001 for comparison

of Cmax(table:10.5) indicated extremely significant difference in Cmax and similar

results were observed with Tmax having p valve P<0.05 indicate their significant

difference (table: 10.6). The obtained data of Cmax and Tmax indicate significant

increase in rate of absorption of recrystallized agglomerates compared to raw crystals of

azithromycin. Similarly p valve for comparison of AUC0-∞ was < 0.001 indicated

extremely significant enhancement in extent of absorption following administration of

recrystallized agglomerates (table: 10.7).In contrast there is an insignificant changes in

half life (table:10.8).

Table: 10.9. Plasma concentrations of Roxithromycin from reference and test

sample at respective time interval.

Scheduled

Time

(hours)

Concentration (µg/mL)

Reference

sample (RTM)

Test sample

(RTM-SSG)

0.0 0.0 ±0.000 0.0 ±0.000

0.5 0.2 ±0.041 0.7 ±0.235

1.0 0.5 ±0.055 1.4 ±0.525

1.5 1.2 ±0.172 2.4 ±0.650

2.0 2.5 ±0.105 3.3 ±0.437

2.5 3.2 ±0.084 4.7 ±0.479

3.0 3.3 ±0.103 5.0 ±0.622

4.0 3.4 ±0.516 4.1 ±0.329

6.0 2.6 ±0.595 3.3 ±0.286

12 1.7 ±0.103 2.3 ±0.197

24 1.3 ±0.075 1.4 ±0.186

48 1.2 ±0.055 0.5 ±0.147


agglomerates


Table: 10.6. Plasma concentrations of Roxithromycin from reference (RTM) and

test (RTM-SSG) sample at respective time interval in rats (n=6).


agglomerates


Table: 10.10. Pharmacokinetic parameters of reference (RTM) and test (RTM-SSG)

sample of Roxithromycin in rats (n=6)

Subject No Pharmacokinetic parameters

Cmax

(µg/mL)

Tmax

(hr)

AUC(0-t)

(µg.hr/mL)

AUC(0-∞)

(µg.hr/mL)

T1/2

(hrs)

Subject for Reference sample

R1 3.8 4.0 130.95 149.16 18.02

R2 3.4 4.0 123.8 139.02 17.58

R3 3.8 6.0 128.3 141.95 15.77

R4 3.7 3.0 126.025 137.27 15.58

R5 3.2 4.0 111 122.85 16.43

R6 3.6 4.0 127.65 146.46 18.62

Mean 3.6 4.2 124.6 139.5 17.00

SD 0.240 0.983 7.084 9.275 1.253

Subject for Test sample

T1 4.9 2.5 145.75 162.12 16.20

T2 5.1 3.0 143.675 159.54 15.70

T3 5.2 3.0 155.45 174.68 16.66

T4 5.5 2.5 148.3 170.92 17.42

T5 5.8 3.0 192.85 231.97 20.85

T6 5.5 3.0 211.875 247.34 20.48

Mean 5.3 2.8 166.3 191.1 17.90

SD 0.327 0.258 28.837 38.330 2.230

P-Value ** P<0.01 ** P<0.01 *P<0.05 *P<0.05 ns P>0.05

One-way Analysis of Variance (ANOVA)


The P value is 0.6720, considered not significant.


The P value is < 0.0001, considered extremely significant.

Table: 10.11. Single factor Anova treatment to Cmax (µg/mL) of RTM and RTM-

SSG.


RTM 6 7.646195 1.274366 0.004696

RTM-SSG 6 10.03449 1.672415 0.003748

ANOVA


Between Groups 0.475329 1 0.475329 112.5919 9.21E-7 4.964603

Within Groups 0.042217 10 0.004222

Total 0.517546 11


agglomerates


Table: 10.12. Single factor Anova treatment to Tmax (hr) of RTM and RTM-SSG.


RTM 6 8.435549 1.405925 0.04897

RTM-SSG 6 6.227031 1.037838 0.008864

ANOVA


Between Groups 0.406463 1 0.406463 14.05608 0.003789 4.964603

Within Groups 0.289172 10 0.028917

Total 0.695635 11

Table: 10.13. Single factor Anova treatment to AUC (0-∞) (µg.hr/mL) of RTM and

RTM-SSG.


RTM 6 29.61478 4.935796 0.00471

RTM-SSG 6 31.42215 5.237026 0.036554

ANOVA


Between Groups 0.272217 1 0.272217 13.19371 0.004595 4.964603

Within Groups 0.206324 10 0.020632

Total 0.478541 11

Table: 10.14. Single factor Anova treatment to AUC (0-∞) (µg.hr/mL) of RTM and

RTM-SSG.


RTM 6 16.98569 2.830948 0.005437

RTM-SSG 6 17.2661 2.877684 0.014829

ANOVA


Between Groups 0.006553 1 0.006553 0.646656 0.440009 4.964603

Within Groups 0.101334 10 0.010133

Total 0.107887 11


agglomerates


In vivo performance data of RTM and their optimized recrystallized agglomerate RTM-

SSG were given in table: 10.9, 10.10 and figure: 10.6.For the test formulation, the mean

(SD) Cmax was 5.3 (0.327) µg/mL and the mean (SD) Tmax was 2.8 (0.258) hours; for

the reference formulation, the Cmax was 3.6 (0.240) µg/mL and the Tmax was 4.2

(0.983) hours. Cmax and Tmax values were significantly (** P<0.01) improved in RTM-

SSG comparative to RTM.Mean (SD) t1/2 values of the test and reference formulations

were 17.90 (2.230) and 17.00 (1.253) hours, respectively shows non significant

difference (ns P>0.05).

The mean (SD) AUC0-t and AUC0-∞ (as an index of extent of absorption) 166.3(28.837)

and 191.1(38.330) for test sample (RTM-SSG) where as 124.6(7.084) and 139.5(9.275)

for reference sample (RTM).Both the area under curve AUC0-t and AUC0-∞ were

significantly improved (*P<0.05) in RTM-SSG comparative to RTM.

Additionally to determine the level of significance these pharmacokinetics parameters

were treated by using single factor one way ANOVA.The p valve was < 0.001 for

comparison of Cmax(table:10.11) indicated extremely significant difference in Cmax and

similar results were observed with Tmax having p valve P<0.05 indicate their significant

difference (table: 10.12). The obtained data of Cmax and Tmax indicate significant

increase in rate of absorption of recrystallized agglomerates compared to raw crystals of

roxithromycin. Similarly p valve for comparison of AUC0-∞ was < 0.001 indicated

extremely significant enhancement in extent of absorption following administration of

recrystallized agglomerates (table: 10.13).In contrast there is an insignificant changes in

half life (table:10.14).

The enhancement in rate of absorption (Tmax, Cmax) and extent of absorption (AUC) of

recrystallized agglomerates may be due to improvement in wettability, solubility and

dissolution profile compared to raw crystal of macrolide antibiotics because the rate of

absorption and bioavailability of poorly water soluble drugs is often controlled by the rate

of dissolution of the drug in the gastrointestinal tract.


agglomerates


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