1

Bioavailability and Bioequivalence study of an

Antidiabetic drug in healthy human volunteers

Industrial Guide Dr A T Bapuji, M. Pharm Phd.,

CPD-PK Dept, Aurobindo pharma ltd, Hyderabad.

Submitted by

K. SUJATHA(Reg. No.95501035)

Pharmacology.

Institutional Guide B. Kishore kumar,

M.Pharm, Dept. of Pharmacology, SK University.

2

Contents• Introduction

• Literature review

• Aim and objective

• Plan of work

• Clinical Phase

• Bioanalytical phase

• Results

• Conclusion

• References

3

INTRODUCTION

In recent years, generic drug products, which are those manufactured

by firms other than the innovator, have become very popular.

For the approval of a generic drug product, the FDA usually Require

ANDA Submission.

The generic drug companies can provide the evidence of

bioequivalence between the generic drug products and the innovator

Products in ANDA.

BIOAVAILABILITY:

The bioavailability of a drug is defined as the rate and extent to

which the active ingredient or therapeutic moiety is absorbed and

becomes available at the site of action.

4

BIOEQUIVALENCE

Definition: “The absence of a significant difference in the rate and extent to

which the active ingredient or active moiety in pharmaceutical equivalents becomes

available at the site of drug action when administered at the same molar dose under

similar conditions in an appropriately designed study.”

Three situations where Bioequivalence studies are required:

When the proposed dosage form is different from that used in pivotal clinical trails,

When significant changes are made in the manufacture of the marketed formulation

and,

When a new generic formulation is tested against the innovator marketed product

5

Overview of the disease

Diabetes Mellitus is a group of metabolic diseases in which a person has high

blood sugar, either because the body does not produce enough insulin, or because

cells do not respond to the insulin that is produced.

Two major types of Diabetes:

Type 1 diabetes

Type 2 diabetes

Diabetes mellitus type 1: (Insulin dependent diabetes) is a form of

diabetes mellitus that results from autoimmune destruction of insulin-producing

beta cells of the pancreas. The subsequent lack of insulin leads to increased blood

and urine glucose.

6

Diabetes mellitus type 2

7

TREATMENT

Type 1 Diabetes: Insulin: All patients with type1 diabetes require treatment with

insulin in order to survive. Exogenous insulin is used to mimic the normal physiological

pattern of insulin secretion as closely as possible, for each individual patient.

Type 2 Diabetes: oral hypoglycemic agents

Sulfonylureas: 1st&2nd Generations

Meglitinides: Repaglinide,Neteglinide

Biguanides: Metformin,Fenformin

Thiazolidinedions: Rosiglitazone,Pioglitazone

α-Glycosidase inhibitors: acarbose,miglitol

8

Drug profile Glyburide (5mg): Antidiabetic drug (sulfonylurea)

The molecular weight is 494.003520 [g/mol]

Mechanism of action:

High affinity receptors for sulfonylureas are present on the kATP channels in β-cell

plasma membranes, and the binding of sulfonylureas paralles their potency in

stimulating insulin release. The drugs reduce the k+ permeability of β-cells by

blocking kATP channnels, causing depolarisation, ca2+ entry and insulin secretion.

9

Pharmacokinetics

Absorption: Significant absorption with in one hour, Tmax is 4 hrs.

Half life is 10hrs.

Distribution: Volume of distribution of Glyburide is 0.14-0.16 l/kg. It is

extensively bound to serum proteins.

Metabolism: Major metabolite is 4-trans hydroxy derivative and

second metabolite is 3-cis hydroxy derivarive.

Excretion: It excreted as metabolites in bile and urine.

Dosage: Starting dose is 2.5 to 5 mg daily, Maintenance dose is 1.25

to 20 mg daily and Maximum dose is 20 mg.

10

Adverse reactions:

Hypoglycemia

GIT Reactions

Hemotological reactions

Drug interactions: It interacts with Antibiotics, Rifampicin,

Cyclosporine, etc.

Effect on pregnancy: Abnormal blood glucose levels during

pregnancy are associated with a higher incidence of congenital

abnormalities.

Lactation: It is not known whether glyburide is excreted in human

milk or not.

11

LITERATURE REVIEW

Neugebauer G et al., (1985) evaluated the absolute bioavailability and bioequivalence of glibenclamide (Semi-Euglucon N) in healthy human volunteers. It could be experimentally confirmed on further 10 healthy volunteers that 1.75 mg HB 420 (identical with Semi-Euglucon N) and Semi-Euglucon (containing 2.5 mg HB 419) are bioequivalent with respect to the absorbed quantity of active agents.

Florin Albu et al., (2006) determined the glibenclamide in human plasma by liquid chromatography and atmospheric pressure chemical ionization/MS-MS detection.. Inter-sequence accuracy expressed as % bias from theoretical concentration values over the concentration interval of 10–400 ng/mL fall within −13.9% and +14.6%. The method was applied for evaluation of the bioequivalence between two formulations containing 3.5 mg glibenclamide per dose.

B. F. Clarke et al., (2003) conducted the long-term comparative trial of glibenclamide and chlorpropamide in diet-failed, maturity-onset diabetics: The final blood-glucose and change in body-weight were similar in patients from both treatment groups still taking the original sulphonylurea agent 2 years later. Hypoglycæmic episodes were more common and severe in the glibenclamide-treated patients.

12

Aim and Objective Aim:

The basic aim of this project was conductance of bioequivalence study, in accordance with the regulatory guidelines. The conductance of Bioequivalence study on a test product A, (Glyburide 5mg tablets of Aurobindo Pharma Ltd; India) and reference product B, (Micronase tablets containing Glyburide 5mg of Pharmacia &Upjohn; USA)

• Objectives:

Compare the rate and extent of absorption of Glyburide tablets (test) of Aurobindo Pharma Ltd; India with that of Reference tablets (Micronase of Pharmacia &Upjohn, USA) when given in equal doses of single oral dose containing 5mg of Glyburide in 14 healthy, adult, male, human subjects under fasting conditions.

• Monitoring of the adverse events and ensure safety of the subjects.

13

Plan of work

Preparation of Protocol

Submit IRB for approval

Registration of the volunteers

Screening

Subject selection (12+2)

Period 1 Period 2

R T T

R

Checkout process

Washout period 7days

14

Clinical phase ETHICAL CONSIDERATIONS:

Institutional Review Board:

The Institutional Review Board was reviewed this protocol and the informed consent form for the study. The study

procedure started only after the approval of the protocol by the institutional review board / independent ethics committee.

Written Informed Consent:

ICF is designed as per the ICH-GCP and local regulatory requirements. ICF is conducted in order to get the consent from

the volunteer to participate in the study.

Study Design:

Open label, randomized, two-treatment, two-sequence, two-period, cross-over, single-dose,

comparative oral bioavailability study of Glyburide 5mg tablets in 14 healthy, adult, male, human

subjects, under fasting condition.

15

Standby: In the present study, 14 subjects were included out of which 2 of them were standby.

Registration of volunteers: New people are informed and

registered in the database after they gave the written consent. The following

eligibility assessments were carried out before enrolling any volunteer into

study which include

• Screening:• Demographic data, including , height and weight, BMI, nutrition status,

diet, history of smoking, substances of abuse.

• Complete physical examination including recording of vital signs (B.P,

Pulse, Temperature and respiratory rate) and systemic examination.

• Urine test to determine the consumption of drugs like morphine, codeine or

any other drug of abuse.

16

Inclusion Criteria: Subjects who will provide written consent form. Healthy males within the age range of 18 to 45 years. Willingness to provide written informed consent to

participate in the study. Body mass index of 18.5 kg/m2 and 24.9 kg/ m2, with body

weight not less than 50 kg. Have a normal chest X-ray.Exclusion Criteria: Personal/Family history of allergy or hypersensitivity to the

drug or allied drugs. History of seizures, epilepsy or any kind of Neurological

disorders. Use of any recreational drug or a history of drug addiction. Presence of any clinically significant abnormal values during

screening e.g. significant abnormality of liver function test, renal (kidney) function test etc.

17

Subject

No.

Sex Age

(Yrs)

Weight

(Kg)

Height

(cm)

BMI

(kg/m2)

S(X)/

NS

S1 Male 36 56 171 19.15 S(2)

S2 Male 25 58 162 22.10 NS

S3 Male 27 58 160 22.66 NS

S4 Male 22 56 166 20.32 NS

S5 Male 33 72 170 24.91 NS

S6 Male 28 50 160 19.53 NS

S7 Male 22 60 156 24.65 NS

S8 Male 41 64 173 21.38 NS

S9 Male 28 60 165 22.04 NS

S10 Male 22 58 174 19.16 NS

S11 Male 20 52 160 20.31 NS

S12 Male 27 70 172 23.66 NS

S13 Male 28 54 163 20.3 NS

S14 Male 24 66 165 24.2 NS

Demographic data of subjects

18

Subject No

Sequence Period I Period II

1 TR T R

2 RT R T

3 TR T R

4 RT R T

5 RT R T

6 TR T R

8 TR T R

9 RT R T

10 TR T R

11 RT R T

12 TR T R

13 RT R T

14 TR T R

Randomization scheme

19

Dietary plan:

Subjects were fasted overnight (at least 10 hours) before dosing and minimum of 4 hours

thereafter. Post dose meal on Day –1(Dosing Day) consisting of approx. 2200 Kcal as per the meal

plan provided by dietician was provided.

Dosing of Investigational product:

A single oral dose of 5 mg x 1 tablet of reference (R) or test (T) products were administered as

per the randomization schedule. Subjects were dosed with 240 ml of 20% glucose solution in water.

Sampling schedules:

The venous blood samples were withdrawn pre-dose and at 0.5, 1.0, 2.0,

3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 8.0, 10.0, 12.0, 16.0, 24.0, 36.0 and 48.0 hours post-

dose.

Blood loss:

For each subject the total number of blood draws were 34 (17x2 for 2

periods). The total volume of blood withdrawn was 255 mL.

20

Subject

Number

Perio

d

Adverse

Events

Action

Taken

S1 IMild

hypoglycemiaRecovered

without

Medication

S8 I Nausea

S11 I Nausea

S12 II Mild

hypoglycemia

Sampling procedure and handling of samples:

Blood samples were collected through an cannula placed in a

forearm vein using disposable syringe with needles. samples were

centrifuged with machine set at 4000 rpm, 10 minutes and 10o C.

Reporting of Adverse Events:

21

Study Conduct: All the 14 subjects were dosed and 13 subjects completed the

study. Subject No. S7 was tested positive for Benzodiazepines during the day of check-in of Period-II. Hence, that subject was withdrawn from the study.

SAFETY ASSESSMENT:

Safety assessment was carried out at the time of screening and during the course of study by conducting medical examination, recording vital signs and adverse event monitoring.

CHECK OUT PROCESS:

After the completion of the study the subjects were checked out.

Post-study Lab Investigations:

10 mL of blood was collected for post-study laboratory investigations (Hematological Investigations) at the time of 48.0 hour blood sample collection in Period-II.

22

Name of the Analyte Glyburide

Molecular Weight 494.003520 [g/mol]

Lower Limit of Quantification 2.08 ng/mL

Calibration Range2.08 ng/mL to 303.55

ng/mL

Internal Standard Glimepiride

Sample Processing Method Liquid Liquid Extraction

Instrument Method LC/MS/MS

Bioanalytical phase Before beginning of the analysis for a molecule a method for its analysis has to be

developed.

Materials and methods:

23

Results

All subjects mean linear plot

0

20

40

60

80

100

120

140

0 4 8 12 16 20 24 28 32 36 40 44 48

Time (hr)

R

T

Linear Mean plasma Concentration Vs Time profile of Glyburide 5 mg tablets under fasting conditions

Statistical analysis was performed on plasma glyburide using the SAS® system version 9.1.3..

24

0.1

1.0

10.0

100.0

1000.0

0 4 8 12 16 20 24 28 32 36 40 44 48

Time (hr)

R

T

Semilog Mean plasma Concentration Vs Time profile of Glyburide 5 mg tablets under fasting conditions

All subjects mean semilog plot

25

Subject

Sequence

Tmax  Cmax  AUClast 

Treatment  Treatment  Treatment 

R T R T R T1 TR 10.00 4.50 68.31 94.60 761.03 747.342 RT 4.00 6.00 127.86 121.07 1129.53 1487.593 TR 5.00 6.00 158.28 218.51 1531.50 1658.264 RT 6.00 8.00 121.38 115.00 598.17 826.775 RT 8.00 8.00 107.26 89.44 836.05 873.236 TR 12.00 8.00 164.70 153.30 1199.93 1130.218 TR 8.00 8.00 299.94 346.51 4972.65 4959.509 RT 6.00 6.00 171.07 89.02 897.42 832.74

10 TR 6.00 4.00 129.76 158.33 998.61 1056.1011 RT 4.00 8.00 142.35 115.12 750.37 819.8512 TR 6.00 6.00 134.31 186.97 645.87 699.5113 RT 6.00 6.00 203.83 195.79 1546.34 1399.8314 TR 8.00 6.00 127.90 96.93 926.37 780.53

  N 13 13 13 13 13 13  Mean 6.85 6.50 150.53 152.35 1291.83 1328.57  SD 2.304 1.384 55.615 72.647 1146.066 1134.092  Min 4.00 4.00 68.31 89.02 598.17 699.51  Median 6.00 6.00 134.31 121.07 926.37 873.23  Max 12.00 8.00 299.94 346.51 4972.65 4959.50  CV% 33.65 21.30 36.95 47.68 88.72 85.36

Comparative evaluation of PK parameters

26

Subject

Sequence

AUCINF_obs  Lambda_z  HL_Lambda_zTreatment  Treatment  Treatment 

R T R T R T

1 TR 822.12 825.54 0.0966 0.0509 7.18 13.62

2 RT 1156.34 1526.67 0.1544 0.1121 4.49 6.183 TR 1617.44 1707.06 0.0524 0.0826 13.24 8.394 RT 611.89 840.12 0.1727 0.2337 4.01 2.975 RT 863.59 895.52 0.1227 0.1306 5.65 5.316 TR 1217.97 1170.73 0.1397 0.1959 4.96 3.548 TR 5614.36 5109.14 0.0582 0.0780 11.92 8.899 RT 925.03 855.86 0.1955 0.1969 3.54 3.52

10 TR 1035.23 1071.80 0.1213 0.1414 5.72 4.9011 RT 783.14 842.04 0.1401 0.2041 4.95 3.4012 TR 658.38 711.03 0.2198 0.2404 3.15 2.8813 RT 1594.83 1420.89 0.0870 0.1092 7.96 6.3514 TR 954.54 794.64 0.2041 0.2267 3.40 3.06

  N 13 13 13 13 13 13

  Mean 1373.45 1367.00 0.1357 0.1540 6.17 5.62

  SD 1312.117 1167.658 0.05348 0.06531 3.186 3.144  Min 611.89 711.03 0.0524 0.0509 3.15 2.88

  Median 954.54 895.52 0.1397 0.1414 4.96 4.90

  Max 5614.36 5109.14 0.2198 0.2404 13.24 13.62

  CV% 95.53 85.42 39.40 42.40 51.66 55.98

Comparative evaluation of PK parameters

27

ParameterCmax AUC0-t AUC0-inf

(ng/mL) (hr*ng/mL) (hr*ng/mL)

Lntransformed Data

Geometric Mean

Test Formulation 138.09 1106.60 1138.78

Reference Formulation

142.32 1059.20 1102.91

T/R Ratio (%) 97.03 104.47 103.25

90% Confidence Interval

86.37 - 109.00 97.64 - 111.79 96.37 - 110.63

Intra subject CV (%)

16.63 9.63 9.82

Power (%) 93.62 99.92 99.90

Statistical summary of the Lntransformed PK parameters

28

ConclusionAll the study procedures followed were in

compliance with the protocol, Declaration of Helsinki and the ICH-GCP guidelines.

The 90% confidence intervals for Cmax, AUC (o-t) and AUC (o-) of Glyburide 5 mg tablets were well within the 80-125% regulatory acceptance range.

Hence the test product Glyburide 5 mg tablets (Test) of Aurobindo Pharma Ltd., India is bioequivalent with that of Micronase (Reference) of Pharmacia & Upjohn, USA under fasting conditions.

29

References Shein-Chung Chow, Jen Pei Liu. Design and Analysis of BA & BE Studies,

Marcel Dekker, 2nd Ed. Vol 133:1-30. Shargel, L.; Yu, A.B. 1999. Applied Biopharmaceutics & pharmacokinetics 4th

Ed. New York: McGraw-Hill. Food and drug administration (FDA), Guidance for Industry: Statistical

approaches to establishing bioequivalence 2001. Brahmankar D.M and Sunil B. Jaiswal, Biopharmaceutics and

Pharmacokinetics, A treatise published by M.K Jain for Wallabh Prakashan, printed by goyal offset works, Delhi 110088, first edition, page 282-305.

Center for Drug Evaluation and Research. "Guidance for Industry: Bioavailability and Bioequivalence Studies for Orally Administered Drug Products — General Considerations". United States Food and Drug Administration, 2003:1-23.

Code of Federal Regulations Title 21--Food and Drugs Chapter I--Food and Drug Administration, Department of Health and Human Services--Continued Part 320—Bioavailability and Bioequivalence Requirements [Title 21, Volume 5, Parts 300 to 499][Revised as of April 1, 2000][CITE: 21CFR320][Page 186].

Guidelines for Bioavailability and Bioequivalence Studies, Central Drugs Standard Control Organization, Directorate General of Health Services, Ministry of Health and Family welfare, Government of India, New Delhi, March 2005; 3-33.

30

S W Coppack, A F lant, C S M clntosh, and A V Rodgers: Phar macokinetic and pharmacodynamic studies of glibenclamide in non-insulin dependent diabetes mellitus. The British journal of Clinical Pharmacology 1990 June, 29[6]:673-684.

F. Tomai, F Crea, A Gaspardone, F Versaci, R De Paulis, A Penta de peppo, L Chiariello and PA Gioffre: Ischemic preconditioning during coronary angioplasty is prevented by glibenclamide, a selective ATP-Sensitive K+ channel blocker. Circulation1994: Vol 90: 700-705.

PH Marathe, ME Arnold, J Meeker: Pharmacokinetics and bioavailability of a metformin/glyburide tablet administered alone and with food.Journal of Clinical Pharmacology: Dec 1, 2001: Vol 40:1494-1502.

A. Green and P. Hougaard: Epidemiological studies of diabetes mellitus in Denmark: Mortality and causes of death among insulin-treated diabetic patients. Diabetologia, Vol 26:190-194, 1984.

Neugebauer G, Betzien G, Hrstka V, Kaufmann B, von Möllendorff E, Abshagen U.: Absolute bioavailability and bioequivalence of glibenclamide (Semi-Euglucon N). International journal of clinical pharmacology, therapy and toxicology; 1985 Sep; 23(9): 453-60.

Florin Albu, Cristina Georgiţă, Victor David and Andrei Medvedovici. Determination of glibenclamide in human plasma by liquid chromatography and atmospheric pressure chemical ionization/MS-MS detection. Journal of chromatography: 2007 Feb1, Vol 846: 222-229.

31

THANK YOU