Pharmacokinetics of fixed-dose combination of tenofovir disoproxil fumarate,lamivudine,and efavirenz: results of a randomized,crossover,bioequivalence study

Abstract

The objective of this study was to assess the bioequivalence between a fixed-dose combination of tenofovir disoproxil fumarate/lamivudine/efavirenz 300/300/600 mg and the individual innovator products. A randomized, balanced, open-label, two-sequence, two-treatment, two-period, single dose, crossover study in 48 healthy adults was conducted. Dosing was separated by a washout period of 32 days. Twenty-seven blood samples were collected in each period from pre-dose to 72 h post-dose. The data of 45 subjects were analyzed for pharmacokinetics and safety. Ninety percent CIs of geometric mean ratio on C_max, AUC_0–t, and AUC_0-inf for tenofovir and lamivudine and on C_max and AUC_0-72 for efavirenz were within the acceptance criteria (80–125%). For tenofovir disoproxil fumarate, the T_max, K_el, and t_1/2 values for the test and reference products were 1.02 versus 0.91 h, 0.04 versus 0.04/h, 18.67 versus 18.46 h, respectively. For lamivudine, the T_max, K_el, and t_1/2 values were: 1.38 versus 1.30 h, 0.21 versus 0.19/h, 3.44 versus 3.91 h, respectively. For efavirenz, the T_max values for the test and reference products were 3.71 and 3.65 h, respectively. Both the treatments were well tolerated. Our findings suggest that the tested formulation is bioequivalent to the innovators’ formulations, and both treatments were well tolerated.

Keywords

Tenofovir disoproxil fumarate lamivudine efavirenz bioequivalence

Introduction

According to United Nations and acquired immunodeficiency syndrome (AIDS) (UNAIDS) 2014 global data, approximately 36 million people are living with human immunodeficiency virus (HIV), and 1.2 million people died in 2014 due to AIDS-related illnesses. Antiretroviral treatments (ARTs) have considerably reduced AIDS-related deaths.¹ Development of drug resistance was a major problem early in the HIV-1 epidemic, when antiretroviral monotherapy was commonly used. This is due to the emergence of resistant isolates during monotherapy. Therefore, antiretroviral drugs are usually administered in combination.²

According to the 2013 World Health Organization (WHO) HIV treatment guideline, first-line ART for adults should consist of two nucleoside reverse transcriptase inhibitors (NRTIs) plus a non-nucleoside reverse-transcriptase inhibitor (NNRTI). WHO strongly recommends tenofovir disoproxil fumarate (DF)/lamivudine/efavirenz fixed-dose combination (FDC) as the preferred regimen to initiate ART.³ Tenofovir DF and lamivudine are NRTIs,^4,5 whereas efavirenz is an NNRTI.⁶ Tenofovir DF is an oral prodrug of tenofovir.⁷ The recommended oral dose of tenofovir DF in adults and pediatric patients (≥12 years of age and weighing ≥35 kg) is 300 mg once-daily.⁴ The recommended oral dose of lamivudine in adults, adolescents, and children (weighing at least 25 kg) is 300 mg once-daily.⁵ The recommended oral dose of efavirenz in adults is 600 mg once-daily.⁶ When given together, it was found to be virologically and immunologically effective, and well tolerated in HIV-1 patients.^8,9 Further, no drug–drug interactions were reported with these drugs.^10–13

Treatment adherence is the primary predictor of viral suppression,^14–18 progression to AIDS, and death.^19–21 Antiretroviral regimens are complex and lifelong, and a large proportion of patients are not able to achieve the target adherence.^22–27 Adherence to ART is associated with many factors, including patient’s social situation and clinical condition, prescribed regimen, and patient–provider relationship.²⁸ Data suggest that the use of FDCs and single-tablet regimen (STR) can improve treatment adherence as compared to non-FDCs since it can reduce the pill burden. A retrospective analysis in 1435 HIV-infected patients, showed higher complete non-adherence (23%) in the non-FDC (p < 0.001 versus FDC and STR). Also, the partial non-adherence was higher in the non-FDC than FDC (4.2% versus 3.4%; p = 0.020).²⁹

Generic drug products play an important role in the clinical practice owing to their affordability and ease of accessibility. To obtain marketing authorization, generic products should be therapeutically equivalent to the reference (innovator’s) product. Therapeutic equivalence is established if two products or formulations have the same beneficial and adverse effects, thereby resulting in the interchangeability of both the products.^30,31 To establish therapeutic equivalence, a pharmacokinetic equivalence study can be performed. According to the Food and Drug Administration (FDA) guidelines for industry, pharmacokinetic equivalence or bioequivalence is defined as the absence of a significant difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available at the site of drug action, when administered at the same molar dose under similar conditions in an appropriately designed study.³⁰ As per an FDA guidance (Statistical Approaches to Establishing Bioequivalence), to establish bioequivalence, 90% confidence interval (CI) values for the ratio of the geometric mean for test and reference products should fall within 80–125%.³²

Cipla Ltd. (India) has developed an FDC of tenofovir DF, lamivudine, and efavirenz to the corresponding innovator products, to reduce the pill burden and to improve the patients’ compliance. The objective of the present study was to determine whether the FDC of tenofovir DF/lamivudine/efavirenz 300/300/600 mg tablet, developed by Cipla, is bioequivalent to the three individual reference products, Viread® (Gilead Sciences, Inc., USA), Epivir® (GlaxoSmithKline, USA), and Sustiva® (Bristol-Myers Squibb Company, USA) in healthy, adult subjects under fasting condition.

Methods

Study design and subjects

The study was conducted according to the declaration of Helsinki,³³ International Conference on Harmonisation Good Clinical Practice (ICH GCP) guidelines,³⁴ and relevant national laws and regulations. All the subjects voluntarily gave written informed consent for their participation in the study. All study-related documents were reviewed and approved by the members of the Drushti Independent Ethics Committee on 11 January 2012. The present study was a randomized, balanced, open-label, two-sequence, two-treatment, two-period, single-dose, crossover, bioequivalence study under fasting conditions in healthy subjects. The duration of the study was 36 days, including the washout period of 32 days.

The inclusion criteria were as follows: male or non-pregnant female subject between 18 and 45 years; body mass index (BMI) ranging from 18.5 to 24.9 kg/m²; normal vital signs, physical examination, and other laboratory parameters; and non-smoker and non-alcoholics. Subjects were excluded if they had known history of hypersensitivity to tenofovir DF, lamivudine, efavirenz, or related drugs; had medication having enzyme-modifying activity during the previous 28 days, prior to dosing day; had prescription medications, over-the-counter products, or topical medication meant for systemic absorption within 14 days prior to administration of investigational product; had depot injection or an implant of any drug within the three months prior to administration of investigational product; had any medical or surgical conditions, which might significantly interfere with the functioning of gastrointestinal tract, blood-forming organs, etc.; history of cardiovascular, renal, hepatic, ophthalmic, pulmonary, neurological, metabolic, hematological, gastrointestinal, endocrine, immunological, psychiatric, malignancy, or other serious diseases; HIV-positive, HBsAg, serum β-hCG (for females), or hepatitis C tests; were lactating women; did not confirm the use of birth control measures; or menstruation cycle coincided with the study periods.

Study products

After an overnight fast of at least 10 h, a single oral dose of the test product (tenofovir DF/lamivudine/efavirenz 300/300/600 mg tablet) or the reference products (Viread®, tenofovir DF 300 mg; Epivir®, lamivudine 300 mg; and Sustiva®, efavirenz 600 mg) were randomly administered to the subjects, with 240 ml of water. Compliance for dosing was assessed by a thorough check of the oral cavity using a tongue depressor and torch immediately after dosing. The randomization schedule was generated in blocks using the PROC PLAN program in SAS® 9.2. A total of 48 subjects in period 1 and 45 subjects in period 2 were exposed to a single oral dose of the test product or the reference product as per the randomization schedule.

Pharmacokinetic analysis

A total of 27 blood samples (6 ml each) were collected in each period from pre-dose to 72 h post-dose. Maximum plasma concentration (C_max), AUC–time curve up to the last measurable concentration (AUC_0-t), and AUC–time curve to infinite time (AUC_0-inf) were taken as primary pharmacokinetic variables for establishing the bioequivalence for tenofovir DF and lamivudine, and C_max and AUC–time curve up to 72 h (AUC_0-72) for efavirenz. Other PK variables evaluated were T_max, t_1/2, and K_el. A non-compartmental method was used to calculate the pharmacokinetic parameters using drug concentration time profile.

Analysis of plasma concentrations of tenofovir DF, lamivudine, and efavirenz was carried out using a validated LC–MS/MS analytical method. Tenofovir, lamivudine, and efavirenz were extracted from human plasma using solid phase extraction and injected into the liquid chromatograph coupled with tandem MS/MS detector. Tenofovir DF was used as an internal standard for the quantification of tenofovir. Tenofovir and the internal standard were monitored by LC–MS/MS in the MRM mode using the mass transitions 288.0→176.3 amu for tenofovir and 294.1→182.2 amu for internal standard. A weighted linear regression using weighting 1/concentration² was prepared to determine the concentration of tenofovir in the presence of lamivudine and efavirenz in human plasma. The lower limit of quantification for tenofovir was 9.9 ng/ml. An eight-point calibration curve in the range of 9.9–1387 ng/ml (for 16 subjects) and 9.9–594.6 ng/ml (for 32 subjects) was prepared for tenofovir. These calibration curves were used to determine concentration of tenofovir in presence of lamivudine and efavirenz in subject samples. The precision (% CV) and accuracy (% nominal) of the back-calculated concentrations for calibration curve levels having range 9.9–1387 ng/ml of tenofovir during the study, ranged from 1.41 to 5.04% and 95.96 to 102.39%, respectively. The precision (% CV) and accuracy (% nominal) of the back-calculated concentrations for calibration curve levels having range 9.9–594.6 ng/ml of tenofovir during the study, ranged from 1.65 to 6.06% and 96.97 to 101.54%, respectively.

Nevirapine was used as an internal standard for the quantification of lamivudine and efavirenz. Lamivudine, efavirenz, and the internal standard were monitored by LC–MS/MS in the MRM mode using the mass transitions 230.00→95.00, 316.10→244.10, and 267.00→226.00 amu, respectively. A weighted linear regression using weighting 1/concentration² was prepared to determine the concentration of lamivudine and efavirenz in human plasma. The lower limit of quantification was 49.6 ng/ml for lamivudine and 79.8 ng/ml for efavirenz. An eight-point calibration curve in the range of 49.6–7140.3 and 79.8–9974.0 ng/ml was used for lamivudine and efavirenz, respectively. The precision (% CV) and accuracy (% nominal) of the back-calculated concentrations for calibration curve of lamivudine during the study, ranged from 2.16 to 6.46% and 97.29 to 102.36%, respectively. The precision (% CV) and accuracy (% nominal) of the back-calculated concentrations for calibration curve of efavirenz during the study, ranged from 1.19 to 3.66% and 98.61 to 101.25%, respectively.

Safety analysis

Safety was evaluated by monitoring of adverse events (AEs), physical examination results, vital signs (including blood pressure, pulse rate, axillary temperature and respiratory rate), and clinical laboratory results during the study.

Statistical analysis

Based on the in-house study data, tenofovir was found to be the most variable of the three analytes. The maximum intra CV of 22.71% was observed for AUC_0-t for tenofovir. Assuming the ‘test to reference’ null ratio to be varying by 5% and from the observed intrasubject CV, and considering the power as 90% at 5% level of significance, approximately 31–32 subjects were required for the statistical evaluation of the study. Again expecting certain dropouts and/or withdrawals, a sample size of 48 subjects was suggested for the study.

Analysis of variance (ANOVA) was performed on log-transformed C_max, AUC_0-t, and AUC_0-inf for tenofovir DF and lamivudine, and C_max and AUC_0-72 for efavirenz at the α level of 0.05. The criteria for evaluating bioequivalence between the test and reference products were 90% CI based on two one sided t-test, for the test-to-reference ratio of geometric least square mean to be within the range of 80–125% for C_max, AUC_0-t, and AUC_0-inf for tenofovir and lamivudine; and C_max and AUC_0–72 for efavirenz. Median difference of T_max between the products was analyzed by non-parametric Wilcoxon test. All the pharmacokinetic procedures and statistical analysis were performed by SAS® 9.2.

Results

Study population

A total of 48 subjects (42 males and six females) were randomized and dosed. The mean age was 28.29 years (18–38 years), mean weight 59.83 kg, and mean BMI 22.19 kg/m². Forty-five subjects completed the study and were analyzed for PK and safety. Two subjects were withdrawn; one due to misbehavior and the other due to an adverse event. One subject dropped out due to personal reasons. All the subjects included in the study were healthy Indians, exhibiting normal vital signs and laboratory parameters. The evaluation of the plasma drug concentration of the samples confirmed 100% compliance of the subjects for whom the data were analyzed.

Pharmacokinetics and bioequivalence

The values of the primary pharmacokinetic parameters and 90% CIs for geometric mean ratios of the test/reference formulation for tenofovir DF, lamivudine, and efavirenz are presented in Table 1.

Table 1.

Pharmacokinetic parameters of tenofovir DF, lamivudine, and efavirenz.

Parameters	Test product	Reference product	% Ratio A/B	90% CI
Tenofovir DF
AUC_0-inf (ng h/ml)	2612.48	2572.26	101.56	97.31–106.01
AUC_0–t (ng h/ml)	2230.95	2210.69	100.92	96.31–105.74
C_max (ng/ml)	326.56	332.5	98.22	93.24–103.45
T_max (h)	1.02	0.91	–	–
K_el (h^-1)	0.04	0.04	–	–
t_1/2 (h)	18.67	18.46	–	–
Lamivudine
AUC_0-inf (ng h/ml)	13,029.45	12,442.40	104.72	99.48–110.23
AUC_0–t (ng h/ml)	12,641.72	12,064.59	104.78	99.36–110.51
C_max (ng/ml)	2790.96	2813.12	99.21	93.58–105.19
T_max (h)	1.38	1.30	–	–
K_el (h^-1)	0.21	0.19	–	–
t_1/2 (h)	3.44	3.91	–	–
Efavirenz
AUC_0–72 (ng h/ml)	59,177.07	54,475.80	108.63	102.27–115.38
C_max (ng/ml)	3015.82	2693.51	111.97	104.44–120.03
T_max (h)	3.71	3.65	–	–

ANOVA: analysis of covariance; AUC: area under the curve; CI: confidence interval.

The AUC and C_max values are in geometric mean. The T_max, K_el, and t_1/2 values are in arithmetic mean.

The geometric mean and 90% CI values were based on least squares mean obtained from ANOVA for the log–transformed pharmacokinetic parameters.

The mean log-transformed plasma concentration versus time profiles of tenofovir DF, lamivudine, and efavirenz are presented in Figures 1 to 3.

Figure 1.

Mean log-transformed plasma concentration versus time profile of tenofovir disoproxil fumarate.

Figure 2.

Mean log-transformed plasma concentration versus time profile of lamivudine.

Figure 3.

Mean log-transformed plasma concentration versus time profile of efavirenz.

The secondary PK parameters from the test and reference products were also similar for tenofovir DF, lamivudine, and efavirenz. For tenofovir DF, the T_max, K_el, and t_1/2 values from the test and reference products were 1.02 versus 0.91 h, 0.04 versus 0.04/h, 18.67 versus 18.46 h, respectively. For lamivudine, the T_max, K_el, and t_1/2 values from the test and reference products were 1.38 versus 1.30 h, 0.21 versus 0.19/h, 3.44 versus 3.91 h, respectively. For efavirenz, the T_max values for the test and reference products were 3.71 and 3.65 h, respectively.

Safety

A total of 21 AEs were reported during the clinical phase of the study; 13 AEs were reported in the test product-treated subjects, four in the reference product-treated subjects, and four during the post-study evaluation. All the AEs were mild and expected; 16 were definitely related to study drug and five probably related to study drug. All the AEs were resolved. No deaths or serious AEs were reported during the study.

The most commonly reported AE was giddiness (nine events with test products versus four events with reference product). Other reported AEs were headache, high blood pressure, itching, and vomiting; all with the test products (one event each). The AEs of high serum glutamic pyruvic transaminase, high serum glutamic oxaloacetic transaminase, and high eosinophil count were reported during the post-study evaluation; thus, these AEs were not attributed to any treatments. Both the treatments were well tolerated at the administered dose.

Discussion

The study evaluated bioequivalence of an FDC formulation (test) containing tenofovir DF/lamivudine/efavirenz 300/300/600 mg with the same doses of the individual innovators formulations. This bioequivalence study was conducted in healthy Indian subjects to minimize the pharmacokinetic variability. The sample size was determined based on in-house data on intrasubject CV and CI of the test drugs. The study was conducted in fasted state because efavirenz is recommended on an empty stomach.

Our results showed no significant differences between the test and reference products, as indicated by C_max and AUC comparisons and the plasma concentration–time curves. ANOVA analysis of tenofovir DF revealed no significant sequence, period, and treatment effects for log-transformed C_max, AUC_0-t, and AUC_0-inf. For lamivudine, the ANOVA analysis revealed no significant sequence and treatment effects for log-transformed C_max, AUC_0-t, and AUC_0-inf; however, a period effect was observed for log-transformed C_max. Clinical conditions were kept equivalent during both the periods, and no pre-dose concentrations were observed. Since the period effect was not coupled with the sequence effect and showed no impact on the power, it was considered insignificant. In ANOVA analysis of efavirenz, no significant period effect was observed for log-transformed C_max and AUC_0-72; however, sequence effect was observed for log-transformed AUC_0-72. The sequence effect can be caused because there may be (a) a difference between subjects assigned to the two sequences, (b) an unequal carryover effect of two formulations into the next period, and/or (c) a formulation-by-period interaction. However, difference between subjects is expected; therefore the first cause can be neglected. No pre-dose concentration was observed in the second period; therefore, there was a lack of unequal carryover or formulation-by-period interaction. Thus, sequence effect was considered insignificant. Treatment effect was observed for log-transformed C_max and AUC_0-72, which was attributed to low intrasubject variation. Considering that 90% CIs of the ratios of test and reference products for log-transformed C_max and AUCs were within the predetermined range (80–125%) and the Schuirmann’s two one-sided t-test showed all probability values < 0.05, both regimen tested were proved to be bioequivalent.

The PK profile of tenofovir DF 300 mg described in our study was similar to the previous PK studies in HIV adults with mean AUC_0-t and C_max values in the range of 2000–3000 ngh/ml^35,36 and 300–325 ng/ml,^4,35 respectively. The PK parameters of lamivudine 300 mg reported in our study were slightly higher than a previous study in 60 healthy volunteers, where the mean steady-state C_max and AUC_24h values were 2040 ng/ml and 8870 ngh/ml, respectively.³⁷ In another study in HIV patients, mean AUC_24h was 11,800 ngh/ml, which was similar to our data; however, C_max was lower (2230 ng/ml).³⁸ The difference in the PK parameters of lamivudine could be due to interindividual variability of lamivudine.³⁹ The C_max value of efavirenz 600 mg reported in our study was lower than a historical C_max (3015.82 versus 4072 ng/ml); however, the AUC values were similar (59,177.07 versus 58,085 ngh/ml).⁶ The mean half-life values for tenofovir DF and lamivudine test and reference formulations did not differ much from previously reported data.^4,5,39,40

Healthcare practitioners are encouraged to make clinical and policy decisions based on economic aspects as well as on clinical outcomes. Assessment of direct medical costs is one of the most fundamental pharmacoeconomical assessments, which includes drug cost.⁴¹ Availability of a generic medicinal product can provide pharmacoeconomic benefits because it assists both patient and healthcare provider for selection of the most rational medication.

The FDC of tenofovir DF/lamivudine/efavirenz, 300/300/600 mg tablets is a novel combination developed by Cipla. It would result in enhanced adherence of the patients to the therapy as compared to simultaneous intake of three drugs as separate tablets. This would lead to better management of the disease.

The limitation of this study is that it was conducted in healthy subjects instead of HIV patients. The pharmacokinetic profile of efavirenz may vary between healthy subjects and HIV-infected patients.⁴² In addition, healthy population may not adequately reflect the pharmacokinetics of antiretroviral drugs in HIV-infected patients.⁴³ Thus, the results of present study cannot be extrapolated to patients.

Conclusion

The 90% CI of the geometric mean ratio of log-transformed C_max, AUC_0–t, and AUC_0-inf for tenofovir DF and lamivudine and log-transformed C_max and AUC_0-72 for efavirenz were within the bioequivalence interval of 80–125%. Hence, it is concluded that the test product is bioequivalent to the reference products, in terms of rate and extent of absorption under fasting condition, and the test formulation met the regulatory criteria for bioequivalence.

Footnotes

Acknowledgements

This study was sponsored by Cipla Ltd (India). The authors thank all the volunteers for participating in the study. The authors thank Accutest Research Laboratories (I) Pvt. Ltd (Clinical laboratory and statistical facility), Sitec Labs Pvt. Limited (Bioanalytical facility) and their personnel for their support in conducting the study.

Declaration of conflicting interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: All the authors are employees of Cipla Limited, India.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

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