ABSTRACT: Aim: The study aimed to assess and compare the bioavailability and bioequivalence of two formulations of Gliclazide 80 mg in 14 healthy male volunteers along with the safety profile. Methods: Subject were selected and enrolled in study after successful screening and assessment of health status. After an overnight fasting, Gliclazide tablets, test and reference, were orally administered to 14 volunteers as per randomization schedule with 200 ml of 20% glucose solution and 50 ml of drinking water in each of two periods.
Total of 16 blood samples were collected, and the concentration determined for Gliclazide up to 72 hours after the administration. Gliclazide was measured using HPLC. The standard statistical analysis was carried out after the Bioanalytical results obtained. Results: The mean age of 14 volunteers was 26. 79, mean BMI was 20. 87 and clinical laboratory parameters were within normal limits. The Bioanalytical results show that the Test formulation exhibited mean peak plasma concentration (Cmax) at 4. 39±1. 1 µg/ml, the time to reach Cmax (Tmax) was 4.
21 ±1. 1 hr. The AUC0-t and AUC0-? had mean values of 65. 91±41. 4 µg. hr/ml and 1. 77±59. 5 µg. hr/ml, respectively. The corresponding Cmax, Tmax, AUC0-t and AUC0-? values for the Reference formulation were 4. 29 ±1. 26 µg/ml, 4. 71±1. 1 hr, 64. 53±34. 37 µg. hr/ml and 78. 38±38. 78 µg. hr/ml respectively. ANOVA was carried out using logarithmic transformations of Cmax, AUC0-t and AUC0-? and the mean (90% CI) of the Cmax, AUC0-t and AUC0-? ratios (test/reference) were 1. 04 (0. 94-1. 13); 0. 99 (0. 87-1. 12) and 1. 06 (0. 90-1. 23) respectively.
Conclusions: The results showed that the 90% confidence interval was within the acceptable limit of 0. 8-1. 25. Based on the statistical results the test formulation of Gliclazide is recommended to be considered as bioequivalent to the reference formulation. Both formulations were equally tolerated and safe. S093568 © Cranfield University 2009. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner 3 ACKNOWLEDGEMENT: The author acknowledged the help and guidance provided by the seniors and colleagues at BV Patel PERD Centre, Ahmedabad.
This work is a result of a clinical study which was collaborative team effort put forth by various departments at this centre and other support staff. In particular, sincere thanks to Dr Shah, Dr Kapadia and Dr Padh, Director of the centre, for their help and guidance, Dr V Kumar from the sponsor company for the permission of producing this work; and Dr. Gajabhiye, Supervisor and principal ICRI, Mumbai, for her inputs in drafting this work. S093568 © Cranfield University 2009. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner 4.
No part of this publication may be reproduced without the written permission of the copyright owner 5 List of Tables and Figures Page No. Table – 4. 1: Randomization schedule Table – 4. 2: Demographic Data Table – 4. 3a: Result from Haematology and Clinical Chemistry – Pre-Study Table – 4. 3b: Result from Haematology and Clinical Chemistry – PostStudy Table – 4. 4: Individual plasma concentration (µg/ml) of Gliclazide after oral administration of the REFERENCE formulation Table – 4. 5: Individual plasma concentration (µg/ml) of Gliclazide after oral administration of the TEST formulation Table 4.
6 – Comparative evaluation of Cmax (mg/ml) Table 4. 7 – Comparative evaluation of Tmax (Hr) Figure – 1: Linear plot of mean plasma Gliclazide concentrations versus time n 14 healthy male subjects under fasting conditions Figure – 2: Semi-log plot of mean plasma Gliclazide concentration versus time in healthy male subjects under fasting conditions Table – 4. 8: Comparative evaluation of AUC 0-t and AUC0-? (µg. hr/ml) Table – 4. 9: Comparative evaluation of kel (hr-1) and t? (hr-1) Table – 5. 1: Comparative data of ratios of test and reference formulations‘ pharmacokinetic parameters of Gliclazide in plasma Table – 5.
2: 90% CI range for the ratios bioavailability parameters 39 41 43 44 46 47 49 50 52 53 54 55 61 61 S093568 © Cranfield University 2009. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner 6 CHAPTER – 1: INTRODUCTION AND LITERATURE REVIEW In Indian pharmaceutical market, there are still many opportunities for the generic manufacturers. It is a regulatory requirement that the applicant has to prove the generic formulation equally effective in terms of pharmaceutical and therapeutical parameters [2].
The bioavailability and bioequivalence studies are prescribed and required to evaluate the comparative equivalence of the pharmacokinetic parameters of the generic formulation against the innovator’s formulation. In this work, the bioavailability and bioequivalence study of Gliclazide has been described. Gliclazide is an oral hypoglycaemic agent which is prescribed often to Indian patients. However, there are no sufficient evidences of evaluations of the pharmacokinetic parameters of Gliclazide in Indian population.
This study was aimed to evaluate the required pharmacokinetic parameters of the formulation in question (Test), and to compare the same with that of innovator formulation (Reference); in order to check its bioequivalence. This study was sponsored by industry and was carried out in the facility of an accredited research centre under supervision of qualified principal investigator. The author of this work was part of clinical evaluation team and managed the complete study progress as a capacity of project manager at the said facility.
The work is represented here as the author was involved in the all the aspects of S093568 © Cranfield University 2009. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner 7 this project and is experienced with the process of conducting such bioequivalence studies at this centre, as per the different regulatory requirements. This study was a double-blind, randomized, balanced, two-treatment, two-sequence, two-period, single dose, crossover bioequivalence study in healthy, adult, male, human subjects under fasting condition.
In this study total of fourteen healthy, adult, male, human subjects were enrolled. The Investigational product used in this study was a test formulation of Gliclazide 80 mg developed locally by the sponsor company and reference tablets from Innovator Company. The sequence of administration these formulations was as per the randomization scheme and each subject was exposed to both formulation in either period-I or period-II. The study was two period and total of sixteen blood samples were collected during each period. The venous blood samples were withdrawn at pre-dose (before dosing) and 1.
0, 2. 0, 3. 0, 4. 0, 5. 0, 6. 0, 7. 0, 8. 0, 12. 0, 16. 0, 20. 0, 24. 0, 36. 0, 48. 0 and 72. 0 hours following drug administration in each period. Washout window of 7 days was given between each period. The determination of Gliclazide in human serum was carried out using validated HPLC method by the Bioanalytical team. Each collected sample was analyzed and the results of the assessment were submitted for statistical analysis. The statistical interpretation was carried out by the qualified biostatistician and the comparative analysis of the pharmacokinetic parameters was done.
The result of this comparative analysis have demonstrated acceptable values by which, it can be concluded that the two formulations were having essentially similar behaviour pharmacokinetically. The statistical data has showed that the parameter falls within the limit as specified by the regulatory authorities and the standard values obtained in different other research work reported. On basis of the results it was interpreted and concluded that the test formulation is bioequivalent to the reference formulation. S093568 © Cranfield University 2009. All rights reserved.
No part of this publication may be reproduced without the written permission of the copyright owner 8 BACKGROUND INFORMATION: 1. Bioavailability and Bioequivalence studies: 1. 1. Definitions and requirements: To exert an optimal therapeutic action, an active substance or drug should be delivered at the site of its action in an effective concentration during the desired period. This is an absorption property of an active substance and first event of drug pharmacokinetics. The extent to which an active drug absorbed and thus delivered in to the systemic circulation will determine its therapeutic response in the body.
This characteristic value is generally depends on the nature of active substance and the dosage form it will be administered. It is essential that to predict the therapeutic effect of the pharmaceutical form containing the active substance, the value of its extent of absorption should be reproducible for any particular dosage form. Thus the bioavailability of an active substance from a pharmaceutical product should be known and be reproducible. [1, 2] This is especially the case if one product is substituted for another.
In that case the product should show the same therapeutic effect in the clinical situation. [1] Definitions: Bioavailability means the rate and extent to which the active substance or therapeutic moiety is absorbed from a pharmaceutical form and becomes available at the site of action. It may be useful to distinguish between the ? absolute bioavailability? of a given pharmaceutical form as compared with that (100%) following intravenous administration, and the ? relative bioavailability? as compared with another form administered by any route other than intravenous (e. g.tablets v. oral solution). [1, 3] Bioequivalence:
It is achieved if the extent and rate of absorption of a drug product are not statistically significantly different from those of the reference product when administered at the same molar dose [2] Bioequivalent: Two medicinal products are bioequivalent if they are pharmaceutical equivalents and if their bioavailability (rate and extent) after administration in the same S093568 © Cranfield University 2009. All rights reserved.
No part of this publication may be reproduced without the written permission of the copyright owner 9 molar dose are similar to such degree that their effects, with respect to both efficacy and safety, will be essentially the same. [1]
Pharmaceutical equivalents: Medicinal products are pharmaceutical equivalents if they contain the same amount of the same active substance(s) in the same dosage forms that meet the same or comparable standards. Both Bioavailability (BA) and bioequivalence (BE) focus on the release of a drug substance from its dosage form and subsequent absorption in to the systemic circulation.
For this reason, similar approaches to measuring the BA should generally be followed in demonstrating equivalence. [2] Bioequivalence studies should be conducted for the comparison of two medicinal products containing the same active substance and/or drug. The studies should provide an objective means of critically assessing the possibilities of alternative use of them. Two products marketed by different licenses, containing same active substance, must be shown to be therapeutically equivalent to one another.
Assuming that in the same subject an essentially similar plasma concentration time course will result in essentially similar concentrations at the site of action and thus in an essentially similar effect, pharmacokinetic data instead of therapeutic results may be used to establish equivalence. [1,2] Studies to establish BE between two products are important for certain changes before approval for a pioneer product in NDA and ANDA submissions and in the presence of certain post approval changes in NDAs and ANDAs.
[3] Both BA and BE studies are required by regulations, depending on the type of application being submitted. Under US 21 CFR part 314. 94, BE information is required to ensure therapeutic equivalence between a pharmaceutically equivalent test drug product and a reference listed drug. As per Schedule-Y of India, these studies are required to be submitted as part of license application for a therapeutic equivalent. BE studies are a critical component of abbreviated new drug application submissions for US-FDA.
The purpose of these studies is to demonstrate BE between a pharmaceutically equivalent generic drug product and the corresponding reference listed drug. Together with the determination of pharmaceutical equivalence, establishing BE allows a regulatory conclusion of therapeutic equivalence. [3] S093568 © Cranfield University 2009. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner 10 As noted in the definitions, both BE and BA focuses on the release of a drug substance from a drug product and subsequent absorption into the systemic circulation.
As a result, it is recommend by regulatory authorities that similar approaches to measuring BA in a drug application generally be followed in demonstrating BE for the license application of a therapeutic equivalent. Establishing product quality BA is a benchmarking effort with comparisons to an oral solution, oral suspension, or an intravenous formulation. In contrast, demonstrating BE is usually a more formal comparative test that uses specified criteria for comparisons and predetermined BE limits for such criteria.
[2, 3] 1. 2. Design [1-3]: The study should be designed in such a way that the treatment effect (formulation effect) can be distinguished from other effects. In order to reduce variability a Non-replicate, two-period, two-sequence cross-over design usually is the first choice for BE studies of immediate release and modified-release dosage forms. However, sponsors and/or applicants have the option of using replicate designs for BE studies for these drug products.
In general, single dose studies will suffice, but there are situations in which steady-state studies may be required: i) if problems of sensitivity prevent precise plasma concentration measurement after single dose; ii) if the intra-individual variability in the plasma concentrations or disposition rate is inherently large; iii) in the case of dose- or time-dependent pharmacokinetics; iv) in the case of extended release products (in addition to single dose studies).
In such steady-state studies the administration scheme should follow the usual dosage recommendations. The allocation of the subjects to the treatment sequences should be randomized. The number of subjects required is determined by the error variance associated with the primary characteristic to be studied (as estimated from a pilot experiment, from previous studies or from published data), by the significance level desired: usually 0. 05, and by the deviation from the reference product compatible with bioequivalence and with safety and efficacy.
It should be calculated by appropriate methods and should not be smaller than 12. However, certain regulatory authorities asked for not less than 16.
[2] The power of the study should be >80% and thus the deviation allowable usually is ± 20%. The number of recruited subjects should always be ethically justified. The use of replicate design reduces the number of subjects participate in the study. S093568 © Cranfield University 2009. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner 11 Subsequent treatments should be separated by periods long enough to eliminate the previous dose before the next one (wash-out period).
The wash-out period should ideally be equal to or more than five half-lives of the drug moieties to be measured. In steadystate studies, wash-out of the last dose of the previous treatment can overlap with the build-up of the second treatment, provided the build-up period is sufficiently long (at least three times the dominating half-life). Sampling should be done long enough to cover at least 80% of the area under the plasma concentration curve as extrapolated to infinity. The extrapolation should be based on knowledge of the dominating elimination half-life.
In steady-state study, sampling should be carried out over a full 24 hours cycle, enabling the detection of circadian rhythms in bioavailability, unless these rhythms can be argued not to have practical importance. 1. 3. Subjects / Volunteers [1, 2]: Bioavailability studies generally will be performed with healthy volunteers. If feasible, taking into account reproduction toxicology, they should belong to both sexes and be between 18 and 55 years old. In the case of genetic polymorphism in clearance it is wise to take this into consideration in selecting subjects.
In some cases the toxic character of the active substance studied may be such that only patients – under suitable precautions and supervision – can be studied. In that case the applicant will have to justify his alternative. To minimise intra- and inter-individual variation subjects should be standardized as much as possible and acceptable. They should preferably be fasting at least during the night before administration of the products or they should take a standard meal at a specified time before the treatment. Time and preferably composition of meals taken after the treatment should be standardized.
Because fluid intake may profoundly influence gastric passage, it should be strictly standardized and specified. The subjects should not take other medicines during a suitable period before and during the study. They should preferably abstain from food and drinks which may interact with circulatory, gastro-intestinal, liver or renal function (e. g. alcoholic or xanthine-containing beverages). Preferably they should be non-smokers. If smokers are included they should S093568 © Cranfield University 2009. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner.
12 be identified as such. In some cases (e. g. study of high clearance substances) even posture or physical activity may have to be standardized. 1. 4. Parameters to be investigated [1, 2] In bioavailability studies the form of, and the area under the plasma concentration curve (AUC) or the cumulative renal excretion and excretion rate are mostly used to assess extent and rate of absorption. Sampling points or periods should be thus chosen, that a sufficiently detailed time course of the characteristics to be measured will be produced.
From the primary results the bioavailability parameters AUCt, AUC, Cmax, Ae, Ae? , dAe/dt desired are calculated along with any other justifiable characteristics. The method of calculating AUC values should be specified. For additional information t1/2 and Kel can be calculated. During studies in steady-state AUC and fluctuation can be calculated. If pharmacodynamic effects are used as characteristics the measurements should provide a sufficiently detailed time course and the initial values should be the same. Specificity, precision and reproducibility of the measurements should be sufficient.
1. 5. Reporting The report of a bioavailability or bioequivalence study should give the complete documentation of its protocol, conduct and evaluation complying with GCP-rules. This implies that the authenticity of the whole of the report is attested by the signature of the study monitor. The responsible investigators should sign for their respective sections of the report. 2. Gliclazide: Pharmacology: [4-7] Gliclazide is a second generation oral antidiabetic agent of Sulfonylurea class and belongs to the group of intermediate acting hypoglycaemic agents.
It differs from other related compounds by the addition of an azabicyclo-octane ring. The primary mechanism of action of Gliclazide is dependent on stimulating the release of Insulin from functioning pancreatic beta cells. In addition, extra pancreatic effects may also play a S093568 © Cranfield University 2009. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner 13 role in the activity of sulfonylurea. Gliclazide administration can lead to increased sensitivity of peripheral tissues to insulin.
In man, apart from having similar hypoglycaemic effect to the other sulphonylureas, Gliclazide has been shown to reduce platelet adhesiveness and aggregation and increase fibrinolytic activity. These factors are thought to be implicated in the pathogenesis of long-term complications of diabetes mellitus. Hence the treatment of Gliclazide helps in treatment of NIDDM and probably prevention of its associated macro-vascular and micro-vascular complications. Pharmacokinetics: The drug is well absorbed and there is extensive binding of Gliclazide to plasma proteins. The single oral dose of Gliclazide, 40 to.
120 mg results in Cmax of 2. 2 to 8 mg/l within 2 to 8 hours (Tmax). Administration of Gliclazide with food reduces Cmax and delays Tmax. Half life (t? ) of Gliclazide in man is approximately 10-12 hours which can vary from 8. 1- 16. 5 hours after single dose administration. Gliclazide is extensively metabolized to seven metabolites in the liver. Gliclazide is predominantly excreted in the urine, the most abundant being the carboxylic acid derivative. 60-70% dose is excreted in urine and 10-20% in faeces and less than 5% of the dose is excreted unchanged in the urine.
Gliclazide is usually completely eliminated within 144 hours post dose. Adverse Drug Reactions: Some of the adverse effects of Gliclazide include hypoglycaemia, diarrhoea, nausea, anorexia, headache, dizziness, pereshtesia and weight gain. Some hypersensitivity reaction includes skin rashes and photosensitivity. Dosage: The recommended daily dosage of Gliclazide is 80 mg – 320 mg per day. Indications and Usage: Gliclazide 80 mg tablets are indicated in patients with Type 2 Diabetes Mellitus and impaired glucose tolerance.
Contraindication: Juvenile onset diabetes, Diabetes complicated by ketosis and acidosis, Pregnancy, Diabetics undergoing surgery, after severe trauma or during infections, Patients known to have hypersensitivity to other sulphonylureas and related drugs, Diabetic pre-coma and coma, severe renal or hepatic insufficiency S093568 © Cranfield University 2009. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner 14 Precautions: In patients with impaired renal or hepatic dysfunctions, glaucoma and in patients with known sensitivity of drug.
3. B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Ahmedabad, Gujarat, India. B V Patel PERD Centre is one of the unique education entities in Gujarat, affiliated with National Institute of Pharmaceutical Education and Research (NIPER) and Bhavnagar University. It provides opportunities for post-graduate and post-doctoral research in field of pharmaceutical sciences, herbal drug and biochemistry. The department of pharmacology of PERD centre is having dedicated facilities to carry out pharmacokinetics studies on human volunteers.
The centre is having 24 bed facility to perform a bioavailability and bioequivalence (BA and BE) studies. This department carries out the in-house research as well as the industry sponsored BA or BE studies. For this, the centre has qualified pharmacologist as principal investigator, physician incharge, large volunteer data base and qualified staff to perform the activities. The centre is having bio-analytical department with standardize equipments and bio-statistical department for complete evaluation of the data.
All research activities‘ proposals, involving human volunteers, are duly informed to independent ethics committee (IEC), which oversees the research at the centre and only after receiving the written approval from IEC, the activity commences. The centre follows the Declaration of Helsinki by WMA in all research activities involving human participants. The author of this literature has served as a capacity of Clinical Project Manager at the B V Patel PERD Centre, Bioavailability and Bioequivalence Unit from May-2008 to April2009.
The author was responsible of managing the start-up, execution and completion of various BA-BE projects at this centre during his tenure. The author is a qualified pharmacist and a trained clinical research professional. S093568 © Cranfield University 2009. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner 15 LITERATURE REVIEW: Gliclazide is a second-generation sulphonylurea class of oral hypoglycaemic agent that is used in the treatment of Type 2 diabetes.
Its effectiveness and safety are previously studied and it is having established safety and efficacy for which it has been marketed as oral hypoglycaemic agent [4, 6, 7]. Gliclazide improves defective insulin secretion by interacting with specific receptors on pancreatic ? -cells. This stimulation of insulin secretion leads to a gradual improvement of glycaemic control [4, 7]. There have been several studies of the pharmacokinetics and pharmacodynamics of Gliclazide in diabetic patients and healthy human volunteers.
Both pharmacokinetics and protein binding of Gliclazide has been studied and reported by Kobayashi [8]. Total and free Gliclazide has been estimated in the healthy (n = 12) and diabetic subjects (n = 11). This study was able to show that the pharmacokinetics of the total blood level of Gliclazide reflect the free Gliclazide level, moreover, Gliclazide predominantly binds with albumin in the blood and its binding ratio is not constant, but variable according to the dose-relation between the drug and the serum protein.
On the basis of these observations, Kobayshi [8] has studied the pharmacokinetics of total and free Gliclazide, in healthy (n = 12) and diabetic (n = 12) subjects as an extension [9]. The ratio of Gliclazide-protein binding remained constant at approximately 92% in serum after administration to healthy and diabetic subjects. The mean pharmacokinetic parameters of elimination rate (kel), time to reach the peak level (Tmax), elimination half-life (t1/2), and volume of distribution (Vd) were 0. 07 h-1, 2. 8 h, 12. 3 h, and 17. 4 L, respectively.
The parameters were not differing significantly between healthy and diabetic subjects or between single and successive administrations. The result of this study has showed that the pharmacokinetics of the total Gliclazide level reflects those of the free Gliclazide in serum. Since then there were numerous studies have been carried out on Gliclazide which has established this agent as an effective Oral Hypoglycaemic Agent and have shown the secondary benefits of its long term use. Various geographical regions have studied the S093568 © Cranfield University 2009.
All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner 16 pharmacokinetics properties of this agent. These efforts were to establish the therapeutic correlation between the pharmacokinetics properties and its therapeutic response. It has been reported that there was no appreciable difference in pharmacokinetics of Gliclazide between Japanese and Caucasian subjects. [7] The racial differences in the pharmacokinetics and pharmacodynamics of a variety of drugs have been well documented.
The pharmacokinetics and pharmacodynamics of Gliclazide in Caucasians and Australian Aborigines in western Australian regions has also been very well studied by Davis and coworkers and the results are published [10]. Gliclazide pharmacokinetics and pharmacodynamics were assessed in 9 Caucasians and 10 Australian Aborigines with uncomplicated type 2 diabetes. Subjects have been administered 160 mg Gliclazide. In this study a pioneer method of estimation of Gliclazide in human serum by high performance liquid chromatography was used.
All blood samples were centrifuged and sera separated within 1 hour of venipuncture. Sera were stored at –80 ? C until assayed. Serum glucose estimations were performed using the glucose oxidase technique and serum insulin was assayed by immunoenzyzmometric methods. Gliclazide concentrations in serum were measured by high performance liquid chromatography (HPLC). Serum (0. 25 ml) and internal standard (glibenclamide 1. 5 mg) were acidified and extracted with 5ml of 2% v/v isoamyl alcohol in hexane. The samples were centrifuged and the organ.