Abstract
Objective:
To assess the bioequivalence of generic formulation of rivastigmine (test) and Exelon (reference).
Methods:
This randomized, open-label, 2-period, single-dose, 2-treatment, 2-sequence, crossover study was conducted in 40 healthy men under fed condition. Participants were randomized to receive a single dose of Exelon or rivastigmine capsule.
Results:
A total of 31 participants completed the study. Area under the concentration–time curve from time zero to time t (AUC0-t) and area under the concentration–time curve from time zero to infinity (AUC0-∞) for Exelon (mean [standard deviation], h·ng/mL) were 126.40 (56.95) and 129.46 (59.94), respectively, while they were 122.73 (43.46) and 125.08 (45.39) for rivastigmine. Geometric mean ratios of rivastigmine/Exelon were 99.17% for AUC0-t, 98.81% for AUC0-∞, and 105% for maximum observed plasma concentration (Cmax). The 90% confidence intervals (CIs) were 94.14% to 104.46%, 93.77% to 104.12%, and 93.08% to 118.44%, respectively. Both formulations were well tolerated.
Conclusion:
The generic and reference formulations were bioequivalent, as the 90% CIs for Cmax, AUC0-t, and AUC0-∞ were within the range of 80% to 125%.
Introduction
Dementia is a brain disease, which is very common among older people. The most common cause of dementia is Alzheimer’s disease. Dementia causes gradual decrease in the ability to remember, think, communicate, and perform usual daily activities. 1 Alzheimer’s disease is characterized by impaired neuronal signaling, which leads to a slow, progressive decline in cognitive and behavioral function. 2 The symptoms are caused by loss of cholinergic neurons. Currently, Alzheimer’s disease is incurable, but a few treatment options are available to relieve symptoms. One of the treatment strategies focuses on amelioration of the clinical manifestations of Alzheimer’s disease by enhancing cholinergic neurotransmission in the brain using cholinesterase inhibitors to delay the breakdown of acetylcholine released into synaptic clefts. 1
Rivastigmine is a cholinesterase inhibitor available as 1.5, 3, 4.5, and 6 mg hard capsules and 2 mg/mL oral solution; it is approved for the treatment of mild to moderate dementia caused by Alzheimer’s disease and Parkinson disease. 3 It is also available as a transdermal patch (Exelon patch), which is approved for the treatment of mild, moderate, and severe dementia of the Alzheimer’s disease and mild to moderate dementia associated with Parkinson disease in the United States 4 and for mild to moderately severe Alzheimer’s dementia in Europe. 5 Rivastigmine selectively increases the availability of acetylcholine in the brain, which allows cholinergic neurotransmission and improves the symptoms of dementia. 1
Rivastigmine has a time to maximum plasma concentration (Tmax) ranging from 0.8 to1.2 hours following oral administration in healthy human volunteers. 6 The compound is quickly detected in the cerebrospinal fluid (CSF) following oral twice daily administration (Tmax: 1.4-3.8 hours) and is quickly eliminated from the CSF (t1/2: 0.31-2.95 hours). 7 It is rapidly and extensively metabolized by its target enzyme (plasma elimination half-life of 1 hour), and metabolites are eliminated primarily through the renal system; hepatic microsomal enzymes are not involved to any significant extent. 6 Although rivastigmine has a short plasma half-life, its pseudo-irreversible mechanism of action allows it to inactivate acetylcholine esterase for prolonged periods of up to 10 hours. Thus, while rivastigmine has been virtually eliminated at this time, substantial acetylcholinesterase (AChE) inactivation in the CSF is still observed. 7
The medications for Alzheimer’s disease are expensive, and generic drugs offer an economically viable option owing to their affordability and availability. 8,9 According to the European Medicines Agency (EMA) guidelines, 2 different drug products with the same active substance, strength, and dosage form are considered equivalent if they have similar pharmacokinetic (PK) and safety profiles. 10 Rivastigmine exhibits nonlinear kinetics with more than proportional increase in area under the curve (AUC) and maximum observed plasma concentration (Cmax) with rising doses. 11 Therefore, a bioequivalence study on the highest dose of 6 mg was conducted. The selected dose of single rivastigmine 6 mg capsule is expected to provide adequate plasma concentration to be measured for assay. The objective of the present study was to assess the bioequivalence of rivastigmine 6 mg capsule (test formulation) developed by Cipla (Mumbai, India) and Exelon capsule (reference formulation) of Novartis (Basel, Switzerland) in healthy, adult male human participants under fed condition.
Methods
The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines 12 and other applicable national laws as well as regulations specified by Sitec Labs Pvt Ltd (Mumbai, India). The clinical study protocol was approved by the Dakshata independent ethics committee, Mumbai, India. All participants provided written informed consent before entering the study.
Study Design
This study was an open-label, randomized, 2-treatment, 2-period, 2-sequence, single-dose, balanced, crossover bioequivalence study to compare the rate and extent of absorption of rivastigmine from capsule developed by Cipla (test formulation) versus Exelon capsule developed by Novartis (reference formulation) under fed condition. This study was conducted to get marketing approval in Europe. As per Exelon summary of product characteristics, 3 rivastigmine should be administered with food. Therefore, the study was conducted under fed condition.
Participants
The number of participants to be included in the study was based on the PK parameters of in-house study data: intraparticipant coefficient of variation (CV) for Cmax and AUC of rivastigmine 1.5 mg is 28.6% and 21.2%, respectively; therefore, a minimum of 28 participants were required to get 90% CI of untransformed data within 80% to 125% for Cmax with 80% power at significant level of .05 when calculated using SAS version 9.1.3. Therefore, the study was planned with a target of 40 participants to allow for dropout or discontinued participants during the study.
A total of 40 healthy nonsmoking and nonalcoholic male participants (age: 18-45 years, body weight: 50-90 [10] kg) with normal vital signs, physical examination, and other laboratory parameters (blood pressure, pulse rate, respiration rate, and oral temperature), standard clinical laboratory tests (hematological, biochemistry, and urinalysis) were included in the study.
Participants were excluded if they fulfilled any of the following criteria: history of hypersensitivity to any of the components of the investigational product, ondansetron, or related class of drugs (eg, donepezil, granisetron); history or presence of cardiovascular, pulmonary, hepatic, renal, hematological, gastrointestinal, metabolic, immunologic, dermatologic, neurological, or psychiatric disease; positive HIV, hepatitis B surface antigen, plasma, or hepatitis C tests; had participated in any other clinical investigation of an investigational product; or had donated blood within 90 days prior to the screening of the study.
The participants were randomly assigned to either test or reference group in accordance with the computer-generated block randomization scheme using SAS version 9.1.3 software (block size = 4, random variable). The participants were enrolled by a qualified investigator and were assigned numbers corresponding to the randomization scheme. Volunteers were administered the test or reference formulation as per the randomization scheme. The bioanalytical division was blinded to the randomization codes until completion of the clinical and analytical phases. According to the randomization schedule, the 2 treatments were administered in a crossover design and separated by a washout period of 7 days.
Test and Reference Formulations
The test formulation was rivastigmine 6 mg capsule (lot number G86868; Cipla) and reference formulation was Exelon 6 mg capsule (lot number B8057; Novartis).
After an overnight fasting of at least 10 hours, standardized breakfast devoid of high fat content was given to the participants 30 minutes prior to rivastigmine dosing. Each participant received a single oral dose of rivastigmine capsule 6 mg as either the test or reference formulation with 240 mL of water at room temperature. About 15 minutes prior to the rivastigmine dosing, ondansetron 8 mg (2 mg/mL) injection was given intravenously as a prophylactic for nausea and vomiting associated with rivastigmine. Another dose of ondansetron 8 mg (2 mg/mL) was also administered post rivastigmine dosing at the discretion of the principal investigator. The participants were dosed as specified in the protocol and subsequently fasted for a period of at least 4 hours.
Pharmacokinetic Sampling
Venous blood samples (6 mL) were collected in a suitable labeled precooled vacuum tube containing K2EDTA as anticoagulant and 60 µL of 0.1 mM physostigmine solution (to inhibit the process of in vitro hydrolysis of rivastigmine in plasma) at predose and 0.33, 0.67, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.50, 5.00, 6.00, 8.00, 10.00, and 12.00 hours postdose in each period following administration of rivastigmine capsule 6 mg. Blood samples were centrifuged to separate plasma as soon as possible after collection normally within 15 minutes and not more than 30 minutes after collection. Plasma was separated by centrifugation at 4°C and 3000 rpm for 10 minutes. Each plasma sample was divided into 2 aliquots stored in separate, stoppered suitably labeled tubes in a deep freezer set below −30°C until further analysis of the samples.
Analytical Procedure
Concentration of rivastigmine in plasma was determined by high-performance liquid chromatography coupled to mass spectrometry (LC-MS/MS) using tramadol hydrochloride as internal standard. Rivastigmine was extracted from human plasma using solid phase extraction procedure and injected into the liquid chromatography column coupled with tandem MS/MS detector (MDS Sciex API 4000). Physostigmine was added to study samples, and plasma was used for the preparation of standards and controls to avoid in vitro enzymatic hydrolysis of rivastigmine. The validated method was linear in the concentration range of 0.10 ng/mL to 60.20 ng/mL. BDS HYPERSIL C8 (4.6 × 100 mm, 5 µ) was used as analytical column with 5 mM ammonium acetate buffer as mobile phase. To avoid interassay variations, all samples from the same participants were analyzed in the same analytical run. The lower limit of quantitation for rivastigmine was 0.10 ng/mL. Calibration curves were found to be consistent and accurate over the range 0.10 to 60.20 ng/mL, with a determination coefficient (r) >0.99, accuracy (% nominal) of 97.63% to 102.79%, and precision (%CV) of 0.00% to 3.80%. Analysis of drug concentration was performed according to Good Laboratory Practice standards.
Pharmacokinetic and Statistical Analyses
Pharmacokinetic analysis was performed by a noncompartmental method using WinNonlin software (version 5.2) and SAS version 9.1.3. The elimination rate constant (Kel) was estimated by linear regression of points describing the elimination phase in a graphic log-linear model. Half-life (T1/2) was obtained from this constant (T1/2 = ln (2)/Kel). Maximum observed plasma concentration and Tmax were directly obtained from the curves. The areas under the plasma concentration versus time curve of rivastigmine 0 to 12 hours (AUC0-12 h) were calculated by applying the trapezoidal rule. Extrapolation of these areas to infinity (AUC0-∞) was carried out by adding the value of C12 (last measurable plasma concentration)/Kel calculated by the AUC0-12 h (where C12 = plasma concentration calculated from a log-linear regression equation obtained for estimating Kel 12 hours postdose). The bioequivalence between formulations was evaluated both through the average individual calculations of the ratios of Cmax, AUC0-12h, and AUC0-∞ and 90% CI after logarithmic transformation of the data. Analysis of variance was performed (α = .05) on the untransformed and log-transformed PK parameters Cmax, area under the concentration–time curve from time zero to time t (AUC0-t) and AUC0-∞ including sequence, subjects nested within sequence, period, and treatment as factors. Least square means of adjusted differences between formulation means and standard error associated with these differences were also calculated. The significance of the sequence effect was tested using the subjects nested within the sequence as the error term. In accordance with current EMA guidelines, the formulations were considered bioequivalent if the 90% CI for Cmax, AUC0-t, and AUC0-∞ fell within the range of 80% to 125%. 10
Concomitant Medication
To prevent nausea and vomiting that are very commonly associated with rivastigmine 6 mg, a single dose of ondansetron 8 mg (2 mg/mL) injection was given within 15 minutes prior to rivastigmine dosing by slow intravenous injection in period 1. A second dose of ondansetron 8 mg (2 mg/mL) was administered post rivastigmine dosing at the discretion of the principal investigator. In period 2, to further prevent nausea and vomiting, 1 dose of granisetron 1 mg (1 mg/mL) injection after diluting it to a volume of 5 mL and 1 dose of ranitidine 50 mg (25 mg/mL) injection were given within 15 minutes prior to rivastigmine dosing by slow intravenous injection instead of ondansetron 8 mg (2 mg/mL) injection. A second dose of granisetron 1 mg (1 mg/mL) was administered post rivastigmine dosing at the discretion of the principal investigator.
Results
Study Population
A total of 40 healthy male volunteers were randomized and dosed, of which, 9 participants were excluded from the final analysis as they vomited twice at or before median Tmax. Therefore, 31 participants were included in the statistical analysis of PK parameters. The mean age (standard deviation [SD]) of the participants was 27 (5.37) years (range: 19-39 years); mean weight, 65.66 (6.50) kg (range: 53.1-80.1 kg); and mean height, 169.29 (5.72) cm (range: 156.8-180 cm; Table 1).
Demographic Data at Baseline.a
Abbreviation: SD, standard deviation.
an = 31.
Pharmacokinetic and Statistical Analyses
Table 2 summarizes the PK parameters, and Figure 1 shows the mean plasma rivastigmine concentration profile versus time. The mean (SD) value of Cmax was 37.23 (15.81) ng/mL for reference formulation and 38.13 (11.83) ng/mL for the test formulation. The Tmax for reference formulation was 1.50 hours, compared with 1.25 hours for the test formulation. The mean (SD) AUC0-t and AUC0-∞ values were 126. 40 (56. 95) h·ng/mL and 129.46 (59. 94) h·ng/mL for the reference formulation compared to 122.73 (43.46) h·ng/mL and 125.08 (45.39) h·ng/mL for the test formulation, respectively. Pharmacokinetic parameters Cmax, AUC0-t, AUC0- ∞, and their corresponding log-transformed values showed no significant effects (P > .05) between the test and reference formulation.
Mean Pharmacokinetic Parameters.
Abbreviations: AUC0-∞, area under the concentration–time curve from time zero to infinity; AUC0-t, area under the concentration–time curve from time zero to time t; Cmax, maximum observed plasma concentration; Kel, the elimination rate constant; SD, standard deviation; T1/2, half-life; Tmax, time to maximum plasma concentration.
Comparative mean linear plasma concentration of rivastigmine versus time. T indicates test formulation; R, reference formulation.
Table 3 shows the statistical analysis for bioequivalence assessment. The mean ratios of Cmax, AUC0-t and, AUC0-∞ between test and reference formulations were found to be 105.00, 99.17, and 98.81, respectively, and the 90% CIs for Cmax and AUC0-t were within the bioequivalence acceptance criteria of 80% to 125%.
Comparative Bioequivalence Data of Test and Reference Formulations: Rivastigmine.a
an = 31.
Abbreviations: AUC0-∞, area under the concentration–time curve from time zero to infinity; AUC0-t, area under the concentration–time curve from time zero to time t; CI, confidence interval; Cmax, maximum observed plasma concentration.
Safety Analysis
All 40 participants were included in the safety analysis. Both formulations were reasonably well tolerated after a single oral dose in healthy adult male human participants. Tables 4 and 5 detail the adverse events reported by participants after administration of test and reference formulations.
Adverse Events.
Characterization of Postdose Adverse Events.a,b
aTotal number of participants: 40.
bIn period 1, participants received either test or reference formulation as per the randomization scheme. In period 2, participants who received the test were switched to the reference and vice versa.
Reported mild adverse events, probably related to treatments, were giddiness (2.50% each for test and reference formulations), headache (2.50% for test formulation), nausea (2.50% for reference formulation), and nausea and vomiting (12.50% for test formulation and 7.50% for reference formulation). Moderate adverse events, probably related to treatments, were nausea (5% each for test and reference formulations) and nausea and vomiting (2.50% for test and 15% for reference formulation). No serious adverse events were reported during the study. There was no significant deviation observed from the baseline values of vital signs, and no clinically significant changes were noted in poststudy laboratory data or results of physical examination.
Discussion
The study evaluated the bioequivalence of rivastigmine 6 mg capsule (Cipla) and the same dose of innovator formulation (Exelon capsule) in healthy human male volunteers. Our results showed no significant PK differences between the test and reference formulations, as indicated by Cmax and AUC comparisons and the plasma concentration–time curves. The sample size used in the study was found to be adequate to achieve a power of >90% for bioequivalence evaluation of rivastigmine capsule. As this was a crossover study, the participants served as their own control. Moreover, in order to avoid variability in the biomedical experimentation and to control factors that may influence the evaluation and comparison of primary efficacy variables, the population selected for this study was healthy adult male human participants.
Placebo-controlled clinical trials have demonstrated that rivastigmine is an effective treatment for the symptoms of Alzheimer’s disease. Cognitive improvement monitored by the Alzheimer’s Disease Assessment Scale—cognitive subscale (ADAS-Cog)—is the greatest for rivastigmine compared with other compounds of the same class. The doses are increased gradually over several weeks, and the 6 to 12 mg/d dose shows significant improvement in the global functioning and activities of daily life of ADAS-Cog scale. 13,14 Therefore, availability of generic formulations of the drug would benefit the growing population 15 of these patients. Usually, generic medicines apart from their cost-saving potential through reduced prices, increase access to pharmacotherapy, and have a positive impact on medication adherence. 16
Pharmacokinetic and bioequivalence studies require sensitive and selective methods for the quantitation of drugs and active metabolites in biological matrices without interference of endogenous compounds. 17 Plasma levels of rivastigmine analyzed using a validated LC-MS/MS method showed no interference from endogenous plasma components or other sources. The method had an accuracy of 97.63% to 102.79%, and the interassay variability ranged between 0.00% and 3.80%, suggesting a precise and accurate measurement of the drug compounds. Rivastigmine was not detected in plasma samples of any participant in the predose sample indicating the absence of carryover effects and ensuring a sufficient washout period.
The bioavailability of a pharmaceutical form refers to the extent of absorption of the active ingredient. Two dosage forms are considered bioequivalent when there are no significant differences in terms of their bioavailability. In our study, no significant differences in the plasma concentrations were found between the test and reference formulations of rivastigmine at any time points (P > .05). Furthermore, the statistical comparison of Cmax, AUC0-t, and AUC0-∞ also indicated no significant difference between the 2 formulations of rivastigmine 6 mg. The 90% CIs for the mean ratio test (T)/reference (R) Cmax, AUC0-t, and AUC0-∞ of log-transformed variables were also within the European guidelines for bioequivalence range (80%-125%), thereby suggesting that the 2 formulations were equivalent in their extent of absorption.
Adverse events observed during the study with rivastigmine were generally those expected from an AChE inhibitor. They were usually mild to moderate, of short duration, and usually resolved spontaneously. Adverse events were more frequently reported in clinical studies related to dose-titration phase of rivastigmine development, particularly in patients eligible to receive higher dosages (12 mg/d) of the drug. 18 In phase 2 trials, rivastigmine was given either before or after food, whereas in phase 2/3 trials the drug was given after food. 19 The tolerability of higher dosages was improved by administration of the drug after food and introduction of antiemetics to the treatment regimen. In our study, both the formulations of rivastigmine were well tolerated with mild and moderate adverse events. The occurrence of adverse events were comparable between the test and reference formulation suggesting that the test formulation has a similar safety profile as the reference drug.
During the study, a few comedications were used which were not planned in the study protocol. However, these comedications did not interfere with the measurements of rivastigmine in plasma (in-house study data on file). Therefore, the administration of these comedications was not considered to have altered the validity of the study.
Conclusion
The 90% CIs of the geometric mean ratio of log-transformed Cmax, AUC0-t, and AUC0-∞ for the test and reference formulations of rivastigmine were within the acceptance range of 80% to 125% specified by EMA. Based on the PK and statistical results of this study, the rivastigmine capsule 6 mg test formulation is bioequivalent to the reference formulation, both in terms of rate and extent of absorption. Thus, the test formulation can be considered interchangeable in clinical practice.
Footnotes
Acknowledgments
The authors thank all volunteers for participating in the study. The authors thank Sitec Labs Pvt Limited 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 authors are employees of Cipla Limited, India.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was sponsored by Cipla Limited, India.