18 journal of applied biopharmaceutics and ......20 journal of applied biopharmaceutics and...

18 Journal of Applied Biopharmaceutics and Pharmacokinetics, 2015, 3, 18-26

E-ISSN: 2309-4435/15 © 2015 Pharma Publisher

Comparative Bioavailability Study of Two Antiretroviral FDC Containing Abacavir 600mg and Lamivudine 300mg in Healthy Human Indian Volunteers

Shubhasis Dan1, Dhiman Halder1, Anwesha Barik1, Easha Biswas1, Pragnya Chakraborty1, Pradipta Sarkar1, Murari Mohan Pal2, Chinmoy Das1, Rubina Bose3, Balaram Ghosh4 and Tapan Kumar Pal1,*

1Bioequivalence Study Centre, Dept of Pharm Tech, Jadavpur University, Kol-32, India 2TAAB Biostudy Services, 27 Central Road, Kolkata 700 032, India 3Central Drugs Standard Control Organization (CDSCO), Govt. Of India, FDA Bhawan, New Delhi, India 4Midnapore Medical College and Hospital, Govt. of West Bengal, paschim Midnapore-721101, India

Abstract: Introduction: Abacavir and Lamivudine both are nucleoside analogs acting as reverse transcriptase inhibitors (nRTI), widely used for the treatment of HIV infection. Both the drugs are in the World Health Organization's List of Essential Medicines, which contains the most important medications needed for a basic health system.

Materials and Methods: In this present investigation, a randomized, two period, two treatment cross over study of test preparation of FDC tablet containing abacavir 600mg and lamivudine 300mg of Lok-Beta Pharmaceuticals Pvt. Ltd. Mumbai and reference preparation Abamune-L 600/300mg Tablet (containing abacavir 600mg and lamivudine 300mg) of Cipla Laboratories Ltd, India have been carried out in 24+2 healthy male, adult, human volunteers under fasting condition to establish the bioequivalence of the test formulation with comparison to the reference formulation. The clinical study of two tablet formulations, one as reference and other one as test FDC tablet containing abacavir 600mg and lamivudine 300mg was performed in 24 healthy male Indian volunteers. This was a single dose, two periods and randomized crossover study with a washout period of one week. The formulations were compared using the parameters such as Area under the plasma concentration-time curve (AUC0-t), Area under the plasma concentration-time curve from zero to infinity (AUC0-∞), Peak plasma concentration (Cmax), and time to reach peak plasma concentration (tmax).

Results: Plasma samples for pharmacokinetic analysis were collected before dosing as well as at the pre-specified time points after dosing. The concentrations of the analytes in plasma were determined by a validated LC-MS/MS method using nevirapine as an internal standard. The 90% confidence intervals of the mean values for the test/reference ratios for AUC and Cmax to compare these results with the bioequivalence acceptance are ranged 0.80-1.25 .The relative bioavailability of the test preparation were found to be 104.00% and 101.65% for abacavir and lamivudine respectively Conclusion: On the basis of comparison of the AUC0-t and the relative bioavailability of the test and reference preparation this can be concluded that the test preparation is found to be bioequivalent with the reference preparation.

Keywords: Abacavir, lamivudine, fixed dose combinations (FDC), LC-MS/MS.

1. INTRODUCTION

Abacavir and lamivudine as a combined formulation is widely used for the treatment of HIV infection. Both the drugs are nucleoside reverse transcriptase inhibitors (NRTI).

Abacavir (CAS No. 136470-78-5) is a nucleoside analog (guanosine) reverse transcriptase inhibitor (NRTI) used to treat HIV infection. Abacavir is given orally and has a high bioavailability (83%). It is metabolized primarily by the hepatic enzyme alcohol dehydrogenase or glucuronyl transferase. It is capable of crossing the blood–brain barrier.

*Address correspondence to this author at the Bioequivalence Study Centre, Dept of Pharm Tech, Jadavpur University, Kol-32, India; Tel: (+91) 033-2414 6967; Fax: (+91) 033-24146186; E-mail: [email protected]

Lamivudine is a potent nucleoside analog (cytidine) reverse transcriptase inhibitor (NRTI). Lamivudine (CAS No. 134678-17-4) has been used more widely for the treatment of chronic hepatitis B at a lower dose than for the treatment of HIV. Both the drugs are on the World Health Organization's List of Essential Medicines, a list which contains most important medication needed in a basic health system [1].

Abacavir sulfate/lamivudine (ABC/3TC) is widely used as a nucleoside reverse nucleoside transcriptase inhibitor combination drug for initial HIV-1 treatment. Numerous trials have been performed so far to study the antiretroviral efficacy, safety and tolerance of this fixed dose combination. In one of those trials, combination therapy containing abacavir–zidovudine–lamivudine had shown a highly effective antiretroviral regimen, resulting in substantial reductions in plasma HIV-1 RNA level which may be compared to the other

Comparative Bioavailability Study of Two Antiretroviral FDC Journal of Applied Biopharmaceutics and Pharmacokinetics, 2015, Vol. 3, No. 1 19

combined protease inhibitor drugs [2]. In another randomized, blind bioequivalence study involving 1858 eligible patients, comparison had been done between four once-daily antiretroviral regimens as initial therapy for HIV-1 infection: abacavir–lamivudine or tenofovir disoproxil fumarate (DF)–emtricitabine plus efavirenz or ritonavir-boosted atazanavir. As a result of which, the time required to virologic failure and the occurance of first adverse event were found to be significantly shorter in patients randomly assigned to abacavir–lamivudine than in those assigned to another regimens [3].

According to the literature survey no bioanalytical method were found to quantify abacavir and lamivudine simultaneously in human plasma or any other biological matrix till date. LC-MS/MS methods were found to be used for quantification of lamivudine and abacavir in human plasma either individually or in combination with other antiretroviral drugs [4-6].

In the present investigation, a randomized, two period, two treatment crossover study of two preparations of FDC tablets containing abacavir 600mg and lamivudine 300mg of Lok-Beta Pharmaceuticals Pvt. Ltd. Mumbai and Abamune-L 600/300mg Tablet (containing abacavir 600mg and lamivudine 300mg) of Cipla Laboratories Ltd, India had carried out in 24+2 healthy male, adult, human volunteers under fasting condition to establish the bioequivalence of the two formulations.

2. EXPERIMENTAL 2.1. Drug (Test and Reference) Information

2.1.1. Reference Preparation (A)

Abamune-L 600/300mg tablet (containing abacavir 600mg and lamivudine 300mg);

Mfg. by: Cipla Laboratories Ltd, India,

Batch No.: K31127, Mfg. Date: September, 2013 and Exp. Date: August, 2015.

2.1.2. Test Preparation (B)

FDC tablet containing abacavir 600mg + lamivudine 300mg;

Mfg. by: Lok-Beta Pharmaceuticals (I) Pvt. Ltd., Mumbai (India),

Batch No.: JPD924, Mfg. Date: July, 2013 and Exp. Date: June, 2015.

2.2. Chemicals and Reagents

Acetonitrile (ACN), Ethyl acetate, Ammonium formate were purchased from Merck (MERCK India Ltd., Mumbai). All solvents used during the analysis were of HPLC grade. Other chemicals and reagents of analytical grade were used throughout the study. HPLC grade water with a resistivity of 18 MΩ was obtained from a Milli-Q gradient system of Millipore (Elix, Milli-Q A10, USA).

2.3. Study Design

Present study was a single dose, random, two treatment and crossover study, with a washout period of 7 days between the two dosing sessions. In each dosing session, volunteers received either of the Test or the Reference preparation containing Abacavir 600mg and Lamivudine 300mg only on the day of study at a fixed time according to the approved protocol. The study was in accordance with the guidelines standardized by Central Drug Standard Control Organization (CDSCO), New Delhi, India set for BA/BE Studies [7].

2.4. Informed Consent

Before recruitment and enrollment into the study, each prospective candidate was given a full explanation of the study. Once this essential information was provided to the subject and once the physician in charge had the conviction that he had understood the implications of participating in the study, the subject was asked to sign the informed consent form.

2.5. Ethical Clearance

Guidelines as drawn up by the Institutional Review Board were followed regarding the treatment of human volunteers in the study. These guidelines met the requirements of the U. S. Code of Federal Regulations (Title 21, Part 56) [8], the Declarations of Helsinki and the Canadian MRC Guidelines. The protocol and the informed consent form were submitted to the Ethical Committee prior to the initiation of the study. The approval of the Ethical Committee was taken in advance of the study commencement. This study protocol was also approved by the Drugs Controller General of India (DCGI), New Delhi, India and as well as by the HURIP Independent ethics Committee, Kolkata, India [9, 10].

20 Journal of Applied Biopharmaceutics and Pharmacokinetics, 2015, Vol. 3, No. 1 Tapan Kumar Pal

2.6. Subject Selection

Healthy adult men Indian human volunteers within 18 to 40 years of age were (Table 2) selected from the panel of volunteers recruited by CPU. The selected volunteers were screened before the commencement of the study and enrolled as per the study specific inclusion/ exclusion criteria.

2.7. Randomization and Dosing of the Volunteers

The volunteers were randomly sorted on the previous day of study initiation of study. During the study phase I and II, each volunteer received either the test or reference preparation as a single dose, only on the study day, at a fixed time as per the Table 2 with 240ml. of water after overnight fasting of at least 10hrs.

2.8. Sampling Schedule and Collection

A total of 14 blood samples were collected from antecubital vein of the volunteers at 0 hr. (before drug administration) 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 6.0, 8.0, 12.0, 16.0 and 24.0hrs in labeled, centrifuge tubes containing EDTA. Blood samples were centrifuged immediately, the plasma was separated into the duplicate polypropylene tubes and stored frozen at–20oC. The tubes were labeled with volunteer code number, sampling time and study date beforehand.

2.9. Instrumental Conditions

Mass spectrometric detection was performed by API 2000 triple quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Toronto, ON, Canada) equipped with an Turbo electrospray ionization (ESI) interface. The tandem mass spectrometry parameters for the detection of abacavir, lamivudine and the internal standard (IS, nevirapine) were optimized by carrying out full scans in positive ion detection mode using ESI as an ion source. The detection and quantification of the analytes and IS were performed in multiple reaction monitoring (MRM) mode. Quadrupoles Q1 and Q3 were set to unit resolution. Full Q3 scan positive ions Turbo Ionspray of analytes are presented in Figure 1 (A and B). Data acquisition and quantitation were carried out using Analyst software version 1.5 (Applied Biosystems/MD SCIEX). The mass spectrometry conditions optimized for lami-vudine, abacavir and IS are summarized at Table 1.

2.10. Liquid Chromatographic Method

Chromatographic analysis was performed using a Shimadzu HPLC system equipped with LC-20AD

Binary pump, SIL-20A Autosampler, CTO-10ASvp Oven and CBM-20A Lite System Control Compartment. Separation was carried out using an Agilent ZORBAX C18; 50x4.6mm column, having particle size-5µm. The mobile phase consisted of ACN: 10 mM Ammonium Formate in water :: 9 : 1 (v/v) at a flow rate of 0.5 mL/min. The auto-sampler was maintained at 4◦C. The total run time for each sample analysis was 3.0 min.

2.11. Bio-analytical Method Development and Validation

In a stoppered test tube, 100 µL of plasma was taken. To this 10 µL of Internal Standard (IS; nevirapine 2000 ng ml−1) was added and mixed for 30 s. Following this, 3ml of ethyl acetate was added and vortex for 10 min and centrifuged for 15 min at 5000 rpm. Approximately 2.5ml of the organic layer was separated and evaporated in presence of nitrogen atmosphere at low temperature (40°C-45°C). The residue was reconstituted with 200µL of acetonitrile and an aliquot of 20µL was injected in LC-MS/MS System. Peak areas of analytes and IS were recorded for statistical analysis and interpretation of data.

Following these steps, calibration samples were prepared by spiking the serial dilutions of the analytes in blank human plasma along with a double blank and blank sample. The concentration range for lamivudine and abacavir were (10-10000) ng mL-1 for both. The calibration curve for each analyte was drawn by plotting the ratios of areas of the analyte and IS against the ratios of the respective concentration of the analyte and IS.

The method was fully validated for accuracy, precision, sensitivity, recovery, linearity, and stability in accordance with the updated version of FDA guidelines [8], using nevirapine as IS. Linearity, precision, and accuracy were tested by analysis of five batches of spiked plasma quality-control (QC) samples. Each batch of spiked plasma samples included one complete set of calibration standards (comprising six different concentrations, blank and blank with internal standard) and QC samples at low (30 ng mL−1; LQC), middle (4000 ng mL−1; MQC), and high (8000 ng mL−1; HQC) concentrations.

2.12. Pharmacokinetic Parameters

The plasma concentration levels achieved by the administration of the studied drugs in each volunteer and collected for the measured content of the dosage form used to establish the pharmacokinetic profile of


(A)

(B)

Figure 1A: Abacavir Q3 Scan. B: Lamivudine Q3 Scan.

test and reference preparations. The following pharmacokinetic parameters were calculated for each subject [9] Cmax: Peak Plasma Concentration, tmax: Time

to reach Maximum Plasma Concentration, AUC (0-24): The area under plasma concentration time curve 0 to 24 hours, AUC(0-∝): The area under plasma


concentration time curve 0 to ∝, t1/2: Elimination half life and Kel: Elimination rate constant. Recovery after extraction was calculated to be 98.90-99.90% by comparing the peak areas of the plasma LQC, MQC, and HQC samples to the absolute peak area of the un-extracted standards containing the same concentrations of lamivudine.

2.13. Statistical Analysis

Usual descriptive analysis including the mean and standard deviation (SD) were used for the variables such as the height, weight and age. These statistical parameters including coefficient of variance are also used to describe plasma concentrations at each individual time point as well as the pharmacokinetic parameters. Following statistical tests were applied on untransformed (tmax, Cmax, AUC(o-t), AUC(o-α)) and log-transformed pharmacokinetic data (Cmax, AUC(0-t),

AUC(0-α)). Anova of tmax, Cmax, AUC(o-t), AUC(o-α) were subjected to a 3-way ANOVA accounting for subjects, period and treatment. 90% confidence interval (CI) consistent with two-one sided t-test with the significance level of 5% for untransformed and log transformed parameters (tmax, Cmax,, AUC(o-t), AUC(o-α)). Products will be considered as bioequivalent if the 90% confidence interval (CI) of difference in the average values of logarithmic AUC and Cmax between test and reference preparations is within the acceptable range of Log (0.8) to Log (1.25).

3. RESULTS AND DISCUSSION

3.1. Demographic Data and Randomization of Volunteers

The average age and BMI of the male subject were found to be 28.42 years and 21.36 kg/m2 respectively. Table 2 shows the details of the age, height, weight and BMI data of each volunteer. As it is a crossover two phase study, each volunteer was exposed to the reference and test drugs either in phase-I or phase-II.

3.2. Liquid Chromatographic Method Development and Validation

A simple, reproducible, sensitive, and specific LC-MS/MS method was developed and validated for the simultaneous determination of abacavir and lamivudine in human plasma.

For lamivudine, Within-run and between-run precision values (%CV) were ranged from 2.15% to 4.79%. Within-run and between-run accuracy values (%nominal) were 99.20-102.20% for LQC, 102% for MQC, and 100.90-101.60% for HQC samples. The stability of LQC, MQC, and HQC samples were determined after three freeze thaw cycles comparing against freshly thawed samples of the same concentration. The stability of lamivudine ranged between 94.57-99.90% after three cycles.

Table 1: LC-MS/MS Instrumental Conditions

Parameter(s) Value

Source temperature (ºC) 400

Dwell time per transition (msec) 200

Curtain gas (psi) 15

CAD gas (psi) 4

Ion spray voltage (V) 5500

Ion source gas 1(psi) 55

Ion source gas 2(psi) 55

Focussing potential (V) 320

Declustering potential (V) 31(abacavir), 32 (lamivudine) and 31(IS) nevirapine

Entrance potential (V) 12 (abacavir), 4 (lamivudine) and 10 (IS) nevirapine

Cell entrance potential (V) 15.20 (abacavir), 16 (lamivudine) and 14.30 (IS) nevirapine

Collision energy (V) 32 (abacavir), 37 (lamivudine) and 36 (IS) nevirapine

Collision cell exit potential (V) 2 (abacavir), 6 (lamivudine) and 10(IS) nevirapine

Ionization mode MRM (+)

Transition pair of abacavir and lamivudine (analytes) 287.2/191.10 and 230/112

Transition pair of nevirapine (IS) 267.10/226.10


Table 2: Demographic Data and Randomization Chart of the 24+2 Volunteers

Drug Received Vol. No. Sex Age (Yrs) Height (cm) Weight (kg) BMI (Kg/m2)

Phase-I Phase-II

1 34 163 60 22.58 A B 2 20 166 50 18.14 A B 3 21 162 55 20.96 A B 4 30 165 52 19.10 B A 5 23 170 55 19.03 B A 6 29 162 52 19.81 A B 7 22 165 60 22.04 B A 8 36 161 53 20.45 B A 9 35 165 62 22.77 B A 10 31 164 64 23.80 B A 11 27 170 62 21.45 A B 12 32 167 66 23.67 A B 13 31 172 58 19.61 A B 14 30 170 60 20.76 A B 15 31 167 61 21.87 A B 16 32 181 65 19.84 B A 17 24 162 58 22.10 B A 18 26 178 72 22.72 A B 19 28 175 67 21.88 B A 20 30 172 60 20.28 B A 21 25 168 62 21.97 B A 22 28 171 66 22.57 B A 23 31 178 75 23.67 A B 24 22 180 72 22.22 A B 25 30 162 57 21.72 B A 26

Mal

e

31 175 62 20.24 B A

Mean ± SD 28.42 ± 4.37 168.88 ± 6.04 61.00 ± 6.36 21.36 ± 1.53 A – Reference B – Test

Table 3: Pharmacokinetic Parameters in 24 Volunteers

Abacavir 600 mg Lamivudinee 300 mg Pharmacokinetic

parameters Reference Preparation (A)

Test Preparation (B)

Reference Preparation (A)

Test Preparation (B)

Cmax (ng./ml.) 6250.000 ± 138.564 6184.583 ± 188.033 4309.583 ± 175.709 4306.250 ± 176.926

tmax (hr.) 1.354 ± 0.312 1.313 ± 0.247 2.021 ± 0.345 1.792 ± 0.509

AUC 0-t (ng. hr./ml.) 30849.038 ± 3173.232 32083.825 ± 4510.989 31579.417 ± 5986.620 32101.563 ± 6674.982

AUC 0-∞ (ng. hr./ml.) 31058.074 ± 3178.882 32248.140 ± 4517.151 32558.140 ± 5984.255 33043.953 ± 6652.829

kel(hr.-1) 0.225 ± 0.007 0.239 ± 0.015 0.151 ± 0.009 0.151 ± 0.016

t1/2 (hr.) 3.084 ± 0.100 2.909 ± 0.176 4.607 ± 0.296 4.630 ± 0.461

Relative Bioavailability (%) 104.00% 101.65%

For abacavir, Within-run and between-run precision values (%CV) were ranged from 5.062% to 9.570%. Within-run and between-run accuracy values (%nominal) were 97.20-102.30% for LQC, 101.75-102.39% for MQC, and 101.74-102.95% for HQC samples. Recovery after extraction was calculated to

be 99.13-102.45% by comparing the peak areas of the plasma LQC, MQC, and HQC samples to the absolute peak area of the unextracted standards containing the same concentrations of abacavir. The stability of LQC, MQC, and HQC samples were determined after three freeze thaw cycles comparing against freshly thawed


samples of the same concentration. The stability of abacavir had been found to be ranged between 96.65-99.85% after three cycles. Chromatograms for Blank + IS, LLOQ are represented as Figure 2 and Figure 3 respectively.

3.3. Plasma Sample Analysis

Volunteer plasma samples were analysed by LCMS/MS after extracting the drug from plasma and

injecting it on the Agilent ZORBAX C18 column for chromatographic analysis. Abacavir and lamivudine were detected in the plasma from 0.5 hour to about 24.0 hours for both test and reference preparations. The time of peak plasma levels of abacavir in test and reference preparation were achieved between 1.0 to 2.0 hours and in case of lamivudine it was found to be between 1.0 to 3.0 hours. The mean peak plasma levels of abacavir and lamivudine with reference

Figure 2: Blank+ IS.

Figure 3: LLOQ.


preparation, ABAMUNE-L (containing abacavir 600mg and lamivudine 300mg) ranged between 5860 – 6450 ng/ml and 3920 – 4650 ng/ml respectively, while for the test preparation of FDC tablet containing abacavir (600mg) and lamivudine (300mg), it ranged between 5820 – 6470 ng/ml and 3880 – 4510 ng /ml for abacavir and lamivudine respectively. Chromatogram of extracted volunteer plasma taken at 1hr. after oral

admintration of the drug is represented as Figure 4. Mean plasma concentration-time curves for Abacavir and Lamivudine (n=24) following single-dose administration of test and reference (both FDC tablet Containing Abacavir 600mg and Lamivudine 300mg) tablets are represented as Figure 5A and Figure 5B respectively.

Figure 4: Volunteer plasma Sample.

A B Figure 5A and B: Abacavir and Lamivudine Mean Plasma Concentration.


4. CONCLUSION

In the present study attempts were made to develop a rapid, sensitive LC-MS/MS bioanalytical method for simultaneous determination of abacavir and lamivudine in human plasma. The developed method has been found to be at par to the standard industry guidelines for validation.

On the basis of comparison of the AUC0-t for abacavir and lamivudine after single dose administration, the relative bioavailability of the test and reference preparation were found to be 104.00% and 101.65% respectively. No occurrence of adverse event was reported by the volunteers throughout the study duration. Therefore it may be concluded that the test preparation was found to be bioequivalent with the reference preparation.

CONFLICT OF INTEREST

None

ACKNOWLEDGEMENT

Author, Shubhasis Dan is thankful to UGC, India for providing fellowship under BSR-Scheme. Authors are also thankful to M/S, TAAB Biostudy Services, KolKata and Sponsor of this study M/S, LOK-Beta Pharmaceuticals, Mumbai.

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[8] US Food and Drug Administration. Guidance for Industry: Bioavailability and Bioequivalence Studies for orally Administered Drug Products. 2000. www.fda.gov/downloads/ Drugs/.../Guidances/ucm070124.pdf.

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Received on 06-02-2015 Accepted on 03-07-2015 Published on 30-07-2015 DOI: http://dx.doi.org/10.14205/2309-4435.2015.03.01.3

© 2015 Tapan Kumar Pal; Licensee Pharma Publisher. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

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