Bioequivalence Study of Metoclopramide Hydrochloride 10 mg Tablets in Healthy Male Volunteers

Metoclopramide (4-amino-5-chloro-2-methoxy-N-(2 diethylaminoethyl) benzamide) is one of the oldest true prokinetic drugs, that is its administration results in coordinated contractions that improve gastrointestinal transit. It is a competitive antagonist of dopamine that accelerates gastric emptying and gastrointestinal transit, inhibiting the relaxation of the upper gastric body, increasing antrum phasic activity, relaxing the superior part of the duodenum and increasing bowel peristalsis. It also increases the basal tone of the gastroesophagic sphincter and avoids gastroesophagic reflux [1-3].


Introduction
Metoclopramide (4-amino-5-chloro-2-methoxy-N-(2 diethylaminoethyl) benzamide) is one of the oldest true prokinetic drugs, that is its administration results in coordinated contractions that improve gastrointestinal transit. It is a competitive antagonist of dopamine that accelerates gastric emptying and gastrointestinal transit, inhibiting the relaxation of the upper gastric body, increasing antrum phasic activity, relaxing the superior part of the duodenum and increasing bowel peristalsis. It also increases the basal tone of the gastroesophagic sphincter and avoids gastroesophagic reflux [1][2][3].
Metoclopramide is readily absorbed from the gastrointestinal tract. Its distribution matches a bi-compartmental model, with low plasma protein binding (40 ± 4%) and wide tissue diffusion. The absolute oral bioavailability of metoclopramide is 80 ± 15.5 % and the peak plasma concentrations occur approximately 1-2 h after ingesting a single oral dose. Hepatic metabolism of metoclopramide is mediated primarily though the cytochrome P450 CYP2D6 pathway producing glucuronide and sulphate metabolites. The average terminal half-life of metoclopramide is 5-6 h in individuals with normal renal function. Enterohepatic circulation is present [4,5] Metoclopramide has linear pharmacokinetics over oral doses ranging from 5 to 20 mg [6].
As part of the evidence to support that two solid oral forms of metoclopramide hydrochloride are interchangeable, a bioequivalence study was performed; in which bioavailability of Carnotprim ® tablets, manufactured by Productos Científicos S. A. de C. V., was compared to a reference product: Plasil ® tablets, manufactured by Aventis Pharma S. A. de C. V.
In Mexico, the cost of brand-products tend to be higher compared to generic products; it is, therefore, important to demonstrate bioequivalence between generic and the established reference products. Bioequivalence studies are mandatory in Mexico to commercialize a generic product.

Subjects
Forty-four healthy male subjects between 18 and 55 years old were recruited for the study. Subjects were identified from Asociación Mexicana para la Investigación Clínica A.C. (AMIC) volunteer database and contacted to determine whether they were interested in participating. All subjects provided written informed consent prior to the realization of any study activity or procedure. The informed consent form was reviewed and authorized by an independent Institutional Review Board (Ethics Committee and Research Committee of the Pachuca General Hospital) and also by the National Ministry of Health [7].
Subjects were considered eligible through the realization of a complete clinical evaluation, clinical laboratory studies (complete blood count, blood chemistry, urinalysis, HBV serology and HIV ELISA), chest X-ray and 12-lead electrocardiography. Subjects with clinically significant abnormalities were not considered for the trial. Electrocardiograms were obtained with calibrated devices by trained nurses, and interpreted by a cardiologist. Blood samples for clinical laboratory tests were analyzed at Quest Diagnostics Inc. (Van Nuys, California, USA), a laboratory with Clinical Laboratory Improvement Amendments certification and College of American Pathologists accreditation.
Metoclopramide has notable fluctuations in plasma concentration after an oral dose, only male subjects were eligible to reduce intersubject plasma concentration variability for the study [5] Mexican regulation [8] does not require participation of female subjects or fed studies for this type of research.
Twenty-five healthy male subjects were randomized to study sequences.

Study design
This study was conducted in accordance with the principles established in the Declaration of Helsinki and its reviews, the Good Clinical Practice and national regulatory requirements [8][9][10].
Study design was an open-label, randomized, controlled, 2 x 2 cross-over, clinical trial; with 2 treatments, 2 study periods and 2 treatment sequence and wash-out period of 7 days between doses. On the first day, subjects were randomized [11] to one of the treatment sequences (Sequence 1: Treatment A followed by treatment B, or Sequence 2: Treatment B followed by treatment A). One dose of the corresponding investigational product (according to the sequence assigned) was administered to each subject for study period.

Investigational products
Two metoclopramide oral formulations were evaluated. Treatment A (test product) was: metoclopramide hydrochloride 10 mg tablets for oral administration, batch: BIO001, expiration date: SEP 2010, manufactured by Productos Científicos S. A. de C. V. (Carnotprim ® ). Treatment B (reference product) was: metoclopramide hydrochloride 10 mg tablets for oral administration, batch: B7E006, expiration date: OCT 2009, manufactured by Aventis Pharma S. A. de C. V. (Plasil ® ). The reference product is the product established by the Mexican authority. Mexican regulation provides that the reference product for pharmacokinetic comparisons shall be the first product registered with that active principle in the country. The Ministry of Health publishes and regularly updates this list.

Methods
During study periods, subjects remained in the clinical facilities for at least 12 hours before the dose, and at least 24 hours after it.
During these inpatient periods, subjects remained under standardized conditions regarding physical activity, environmental conditions and diet.
For logistic reasons, subjects were divided into seven groups (six groups of four subjects each and one group with one subject). Activities in each group were performed simultaneously, such as diet, doses and blood sampling. Activities from one group to another were separated by 5 minutes.
The first day of each study period (Day -1) a standard dinner was served 12 hours before dose administration. The second day of each period (Day 0) a standard breakfast, lunch and dinner were served 4, 8, and 12 hours respectively after dose administration. On the last day of each period (Day 1) a free breakfast was served after the last blood sample was obtained. Water consumption was not allowed from 10 hours prior to the dose administration, until 2 hours after it. Investigational product doses were administered PO with 250 mL of water.
During inpatient periods, vital signs (breathing, blood pressure, pulse and heart rate) were measured at admission, in the hour before dose administration, 3.5, 7.0 and 11.0 hours after the dose, and before discharge. Vital signs measurements were performed by qualified nurses, using calibrated instruments and results were analyzed by the study investigators.
On admission to and discharge from each study period, all subjects were given clinical exams to confirm their health status and eligibility for the study. Before the wash-out period, subjects were provided with written instructions for reporting adverse events, concomitant medication, alcohol and tobacco abstention, amongst others. Before admission to the second study period, the investigators questioned the study subjects about compliance with these instructions.

Safety evaluation of investigational products
Adverse events were spontaneously reported by study subjects and actively sought by study investigators, through interviews and physical examinations. Adverse events' treatment association was determined using Naranjo algorithm [12] by the principal investigator.
Subjects received economic compensation for the time spent in the study. The amount was previously reviewed and approved by the Ethics and Research Committees. The subjects were not eligible for participation in any other clinical trials at this site for 60 days after the study termination.

Blood sampling for pharmacokinetic study
Six milliliter blood samples were obtained for the pharmacokinetic characterization. Blood samples were obtained at baseline and at 0. Blood was drawn using an IV catheter or venipuncture, and collected in heparin sodium tubes (Vacutainer -BD, 1 Becton Drive, Franklin Lakes, NJ). Approximately 0.4 mL of blood was extracted and discarded from the catheter before each sample, and catheters were flushed with 0.08 mL of 1000 IU/mL heparin solution after each sample, to avoid its occlusion.
Blood samples were kept refrigerated (2 to 8°C) for a maximum of one hour before they were centrifuged. Centrifugation was performed for 15 minutes at 3000 ± 200 rpm (rcf: 1864 g), at 2 to 8°C. The plasma obtained from each sample was transferred to 2 cryotubes (two complete series to be safely transported separately to the analytical facilities), which were immediately stored at -40 ± 5°C until analysis.

Tolerability
Drug tolerability was clinically assessed by medical personnel who questioned subjects about symptoms of possible adverse event and through evaluation of spontaneous reports from subjects throughout the study period.

Analytical method
Plasma concentrations of unaltered metoclopramide was quantified with a validated High Performance Liquid Chromatography (HPLC) method. The method was validated in selectivity, precision, accuracy, recovery with a limit of detection of 2 ng/mL. Analysis was performed at CAFET -Centro A. F. de Estudios Tecnológicos, S. A., in Mexico D. F., Mexico; which is an analytical laboratory approved by the Mexican Authority.

Methods for Results Evaluation
Evaluations of results were performed via pharmacokinetic analysis, for where a following pharmacokinetic parameters were calculated: C max , t max , AUC 0-t , AUC 0-∞ , Ke and t ½ .

Pharmacokinetic variables
Statistical analysis and data processing were executed on WinNonlin ® Professional Version 5.0.1., Microsoft ® Office Excel ® 2007, SAS ® version 9.1 (September 2008) and Stat Graphics Plus version 5.0 [13,14]. Pharmacokinetic parameters were calculated by a noncompartmental method. Plasma concentrations versus time were averaged by administered dose and for each sampling time. For each parameter was calculated: Mean, geometric mean, standard deviation, variation quotient, minimum, maximum and number of determinations.
Individual and average pharmacokinetic profiles were characterized for both treatments in arithmetic and semilogarithmic scales.

Statistical analyses
Analysis of variance (ANOVA) was performed to assess the effects of unbalanced sequences, variability in the number of subjects, and extreme inter-and intrasubject variability.
Power analysis indicated that this sample of 25 subjects had adequate size to detect statistically significant differences between the trial and reference products in pharmacokinetic parameters converted to natural logarithms of C max , AUC 0-t , and AUC 0-∞ . Schüirmann's unilateral double t test (p ≤ 0.05) confidence intervals at 90% (80 -125%) were used, with a power of 80%.

Results
Fourty-four subjects signed informed consent form, 14 were not eligible due to clinically significant abnormalities in diagnostic tests. From the 30 eligible subjects, 25 were enrolled and randomized. All 25 subjects were compliant regarding inclusion and exclusion criteria, and satisfactorily completed the study.
Three adverse events not related to the investigational product were detected during the study. No related adverse event was detected; no serious or unexpected adverse events were detected.
Mean plasmatic concentrations in arithmetic and semilogarithmic scales are shown in Figures 1 and 2.
Mean C max was 36.4 and 37.28 ng/mL with Carnotprim ® and Plasil ® , respectively. This was achieved one hour after the administration of the dose (t max ). Elimination half-life for both products was approximately 5.0 hours.

Bioequivalence of investigational products
Results from pharmacokinetics and bioavailability for both investigational products show that their pharmacokinetic parameters (C max , AUC and t max ) are similar.
Schüirmann's unilateral double t-test and confidence intervals at 90% conclude that both oral forms of metoclopramide are bioequivalent on C max , AUC 0-t , and AUC 0-∞ .Statistical power was higher than 0.8, which indicates that the sample sized allowed adequate pharmacokinetic characterization and comparison of these metoclopramide tablets.

Discussion
Metoclopramide has been in the international market for a long time (more than 30 years), yet despite this fact, an important number of scientific articles about its comparative bioavailability in healthy subjects could not be found.
Metoclopramide shows a great inter-individual variability, which is explained by its great first step hepatic metabolism. Metoclopramide it is still a popular drug in medical practice.
Adverse reactions after prolonged use or with high plasma concentrations is frequent, especially in subjects with concomitant therapy (drug-drug interactions) with drugs metabolized by P450 cytochrome and in patients with liver cirrhosis [15].
Several reports in scientific literature mention pharmacokinetic differences between different populations, in drugs metabolized by P450 cytochrome [16,17] It would very interesting and useful to investigate what would be the case with metoclopramide; its inter-individual variability may increase within different populations, considering the fact that metoclopramide is metabolized mainly by the CYP2D6 [2,18], one of the enzyme families of P450 cytochrome.
The results of the study show that the study formulations are bioequivalent, despite great inter-individual variability in the Mexican population, evidenced by this study (see standard deviation in plasmatic concentration-time curves in Figures 1 and 2).
Metoclopramide has been available in the U.S. since 1979 and its use has increased in the last decade [2] however, in the literature there are few bioequivalence or bioavailability studies with metoclopramide available (none in Mexicans subjects), Drovev et al. [19] conducted a bioequivalence study of metoclopramide in 12 caucasian healthy volunteers (8 female and 4 male). They report a similar value for C max (35 ± 16.9 ng/mL), AUC 0-24 (234 ± 188.7 h*ng/mL) and T max (1.00 ± 0.40) for the reference product, compared with the values that we are

Conclusions
This single dose study found that the test and reference products met the regulatory criteria for bioequivalence in these fasting healthy male volunteers.
The study results are reliable and true. The results showed that both study formulations are bioequivalent. None of the study subjects presented serious or unexpected adverse events.