BIOEQUIVALENCE AND PHARMACOKINETIC STUDY OF RANAZOLINE IN HEALTHY MALE VOLUNTEERS: AN OPEN LABEL, RANDOMIZED, SINGLE-DOSE, TWO-WAY CROSSOVER STUDY

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ABOUT AUTHORS:
Suresh VV Babu¹, Talasila EGK Murthy²*, Chimakurthy Jithendra^3
1Dept. of Pharmaceutics, Natco Pharma Limited, Hyderabad, Telangana, India.
²Dept. of Pharmaceutics, Bapatla College of Pharmacy, Bapatla, Andhra Pradesh, India.
³Dept. of Pharmacology, Bapatla College of Pharmacy, Bapatla, Andhra Pradesh, India.
*drgkm@bcop.net

ABSTRACT
The present study was to assess the relative bioavailability and pharmacokinetic properties of extended release formulations of Ranolazine 1000 mg in healthy male volunteers usinga randomized, open-label, balanced, two-treatment, two-period, two sequence, single dose, crossover, bioequivalence study under fasting conditions. Bioavailability of the test product of Ranolazine extended release tablets 1000 mg was compared with that of the reference product of Ranexa® (Ranolazine extended release tablets 1000mg) of CV Therapeutics Inc., California. The plasma samples were collected at 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, 4.50, 5.00, 5.50, 6.00, 6.50, 7.00, 8.00, 10.00, 12.00, 16.00, 24.00 and 48.00 hours post dose after single administration of Ranolazine 1000mg. The plasma Ranolazine concentrations were estimated by using a validated bioanalytical method by LC-MS/MS. A ten day washout period is followed between two treatments. The formulations were considered to be bioequivalent if the 90% CIs for the log-transformed values were within the predetermined equivalence range 80%–125% for AUC and C_max. For Ranolazine, at 90% confidence intervals C_max, AUC_0-tand AUC _0-∞ were 83.43-113.29, 82.10-102.87 and 80.94-101.85 for log-transformed data respectively.The present results show that the formulation of Ranolazine was bioequivalent to the reference in fasting, healthy, male volunteers.

REFERENCE ID: PHARMATUTOR-ART-2336

INTRODUCTION
Ranolazine, a piperazine derivative, used for the treatment of angina and also for its anti-ischemic effects, is an inhibitor of late sodium channel current and thus decreases sodium entry into ischemic myocardial cells Fig 1. As a consequence, ranolazine is proposed to reduce calcium uptake indirectly via the sodium/calcium exchanger. Ranolazine reduces the frequency of anginal attacks and increases exercise capacity in patients with chronic angina ^[1],[2],[3]. The drug can be used in combination with other antianginal drugs, which are ineffective individually^[4],[5]. Recently extended-release ranolazine was also approved in the United States for the treatment of chronic angina.

Figure 1: Structure of Ranolazine

Ranolazine was patented in 1986 and is available in an oral form having both immediate release and extended release. But the immediate-release ranolazine is not in current use. Immediate release dosage form had an average terminal elimination half-life ranging from 1.4 to 1.9 hours and a 10-fold peak/trough difference with dosing of 240 to 400 mg 3 times per day ^[6]. For the extended release dosage forms the average terminal elimination half-life is 7 hours after multiple dosing to steady state, and the peak/trough difference is 1.6-fold with dosing of 500 to 1000 mg twice daily. The aim of this study was to evaluate the pharmacokinetic characteristics and the bioequivalence of the test formulation (Extended release tablet 1000mg) and the reference formulation Ranexa® (Ranolazine extended release tablets 1000mg) of CV Therapeutics Inc., Californiain healthy human male volunteers, in fasted state in order to determine whether any observed differences and exceeded regulatory guidelines for bioequivalence.

Subjects and Methods:

Subjects:
Twelve healthy, adult, male volunteers were selected for the study. Each volunteer was required to provide written informed consent for participation before the study. Medical history, physical examination, electrocardiography and various laboratory tests (hematology, blood biochemistry, hepatic function, and urinalysis) were carried out prior to the beginning of the study. Inclusion criteria included being within age 18 – 40 years and ideal body mass index. Exclusion criteria included smoking; the presence of heart, kidney, neurologic, or metabolic disease, a history of drug hypersensitivity and current pharmacologic treatment. Volunteers were instructed to adhere to a standard protocol and to refrain from administering any medication 1 week before and during the course of the study.

Study Design:
The study wascarried out in accordance with the provisions of the current version of the ICH ‘Guidance for Good Clinical Practices’, ICMR ‘Guidelines for Biomedical Research on Human Participants’ and the principles enunciated in the Declaration of Helsinki (WMA General Assembly, Seoul, October 2008)^[7],[8].  Study was conducted in a randomized, open-label, balanced, two-treatment, two-period, two sequence, single dose, crossover, bioequivalence study with 10 days washout period between the dose administrations. Subjects arrived at the study center the day before the study and fasted overnight before drug administration. According to the randomization, subjects were divided into 2 groups. During the first period, volunteers from group A received a single 1000 mg tablet of the reference product Ranexa® (Ranolazine extended release tablets 1000mg) of CV Therapeutics Inc., California while volunteers from group B received a single 1000 mg tablet of the test product (Ranolazine). The drug was administered with 250ml of water. After the 10 days washout, the alternate treatment was administered.

Blood Sampling Schedule:
In each period, 21, (1 x 5-mL) blood samples were collected. The predose blood sample (1 x 5-mL) was collected within 1 hour prior to dosing. The post-dose blood samples (1 x 5-ml each) were collected at 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, 4.50, 5.00, 5.50, 6.00, 6.50, 7.00, 8.00, 10.00, 12.00, 16.00, 24.00 and 48.00 hours post dose. 48.00 hour sample time point was collected on ambulatory basis. For each subject the total number of blood samples drawn during the study was 42. The total volume of blood drawn including 15-ml for screening and 18-ml of blood discarded (i.e., 0.5 ml of blood is discarded via an indwelling cannula up to 12 hours) was not exceed 243-ml for the entire study. Plasma was immediately separated by centrifugation at 3000 RPM for 10 minutes at 4°C ± 2°Cusing  Heareus Centrifuge.

Fasting/Meals:
All subjects were on fast overnight for a period of 10 hours before commencement of dosing. Drinking water was not allowed from one hour.  Uniform and low fat meals were provided to all the subjects. A standard meal (lunch), snacks, and dinner, at four, eight and twelve hours, respectively was given after the drug administration.

Tolerability:
Tolerability was assessed using vital signs (blood pressure, temperature, and heart rate) taken before dosing and approximately every 4 hours after administration in each study period. Subjects were interviewed during the study concerning the occurrence of adverse events.

Ranolazine quantification in Human Plasma:
The plasma Ranolazine concentrations were estimated by using a validated bioanalytical method LC-MS/MSusing gliclazide as an internal standard as described by^[9],[10]. The  analyte and internal standard gliclazide were extracted from plasma by liquid-liquid extraction, using ethyl acetate and separated on hypersil BDS column (C18 5µm; 50L x 2.1mm I.D) using acetonitrile : ammonium formate (80:20 v/v), at a flow rate of 1.0ml/min. Detection is carried out by reaction monitoring on a Qtrap TM LC-MS-MS system. (MDS Sciex API - 4000). The injector volume was 30 µl and coloumn oven temperature maintained was 40°C.

Pharmacokinetic and statistical analysis:
The pharmacokinetic parameters like AUC0–t(area under the plasma concentration-time curve measured at t hours)_,AUC_0–∞ (area under the plasma concentration-time curve measured at ∞hours)_,AUC_0–t/ AUC_0–∞,C _max (maximum observed drug concentration during the study)_, T_max (time to observe maximum drug concentration)_, K_el (apparent first-order terminal rate constant)_and T_½ (terminal half-life)for Ranolazine were calculated using WinNonlin Pro® software version 5.2.1 (Pharsight, USA). C_max and T_max were determined directly from the respectively observed plasma concentration-time data. Analysis of variance (ANOVA) was performed (∞=O.05) on the Untransformed Pharmacokinetic parameters AUC_0–t, AUC_0–∞,C_max and T_max. Additionally, Log-transformed data was used for analysis of AUC_0–t, AUC_0–∞and C_max. The analysis of variance model included sequences; subjects tested within sequence, period and drug formulation as factors. Ratio analysis was reported for untransformed and log-transformed AUC_1ast, AUC_inf and C_max. The geometric mean value was reported for log transformed data.For bioequivalence evaluation, in accordance with current FDA guidelines, the products were considered bioequivalent if the 90% Confidence Interval for Cmax, AUC_0–tand AUC_0–∞fell within the range of 80% to 125%.

RESULTS AND DISCUSSION:

Quantification of Ranolazine:
The linear range of Ranolazine was 10-5000 ng/mlwith correlation coefficient of 0.9937. The lower limit of quantification was found to be 10 ng/ml.  Intra-assay precision was found to be between 4.6% and 10.7% and inter assay precision was between 5.5% and 11.1%. Intra-assay accuracy ranged from 91.06% to 98.43% and interassay accuracy from 98.81% to 103.52%.

Pharmacokinetic parameters:
The results of pharmacokinetic parameters of ranolazine such as C_max (ng/ml), T_max (hr), AUC _0-t (ng.h/ml), AUC _0-∞ (ng.h/ml), t _1/2 (hr), K_el (h^-1), AUC _0-t/ AUC _0-∞ for test and reference formulations were reported in Table 1. The profile of log mean plasma concentrations of Ranolazine versus time in subjects (n=12) for test product and reference product were given in Fig 2.

Table 1: Pharmacokinetic parameters after administration of 1000mg of ranolazine in test and reference formulations in 12 healthy male volunteers

Figure 2: Log mean plasma concentrations of Ranolazine vs time profile for 12 subjects

Ranolazine least square mean ratios (C_max, AUC_0-t and AUC _0-∞) were 97.22, 91.90 and 90.79 for log-transformed data respectively indicating a comparable Bioequivalence of test formulation to the reference formulation. Ranolazine 90% confidence intervals (C_max, AUC_0-t and AUC _0-∞) were 83.43-113.29, 82.10-102.87 and 80.94-101.85 for log-transformed data respectively (Table 2). The 90% confidence intervals for the ratio of C_max, AUC_0-tand AUC _0-∞ values for the test and reference were withintheBioequivalance acceptable range 80-125% for the log-transformed data as per the established regulatory guidelines.

Table 2:Summary statistics of LOG-Transformed Pharmacokinetic Parameters for Ranolazine in 12 Healthy Male volunteers

CONCLUSION
In this small study of 12 healthy male volunteers, no statistically significant differences in Cmax, AUC_0–t, and AUC_0–∞ were found between the test and reference formulations of ranolazine 1000mg extended release tablets. The 90% CIs for the mean ratio values for the test and reference formulations of Cmax, AUC_0–t, and AUC_0–∞ indicated that the reported data were entirely within the bioequivalence acceptance range proposed by the FDA of 80% to 125% (using log-transformed data).

REFERENCES:
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8. ICMR - Ethical Guidelines for Biomedical Research on Human Participants. Indian Council of Medical Research (ICMR), New Delhi, (2006). Available from:<http://icmr.nic.in/ethical_guidelines.pdf> [9 January 2014]
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10. Jerling M, Huan BL, Leung K, Chu N, Abdallah H and Hussein Z; Studies to investigate the pharmacokinetic interactions between ranolazine and ketoconazole, diltiazem, or simvastatin during combined administration in healthy subjects; J. Clin. Pharmacol; 2005; 45(4); 422–433.

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