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.

Contents

Introduction

Literature review
Aim and objective
Plan of work
Clinical Phase
Bioanalytical phase
Results
Conclusion
References

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

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

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.

Diabetes mellitus type 2

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:

Meglitinides:

Biguanides:

1st&2nd Generations

Repaglinide,Neteglinide

Metformin,Fenformin

Thiazolidinedions:

-Glycosidase

Rosiglitazone,Pioglitazone

inhibitors: acarbose,miglitol

Drug profile
Glyburide
The

(5mg): Antidiabetic drug (sulfonylurea)

molecular weight is 494.003520 [g/mol]

Mechanism

of action:

High affinity receptors for sulfonylureas are present on the k ATP channels in -cell
plasma membranes, and the binding of sulfonylureas paralles their potency in
stimulating insulin release. The drugs reduce the k + permeability of -cells by
blocking kATP channnels, causing depolarisation, ca2+ entry and insulin secretion.
8

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:
Dosage:

It excreted as metabolites in bile and urine.

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

to 20 mg daily and Maximum dose is 20 mg.

9

Adverse reactions:
Hypoglycemia
GIT Reactions
Hemotological

Drug

reactions

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.
10

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 SemiEuglucon (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 10400 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. Hypoglycmic episodes were more common and severe in
the glibenclamide-treated patients.

11

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.

12

Plan of work

Preparation of Protocol
Submit IRB for approval
Registration of the volunteers
Screening
Subject selection (12+2)
Period 1
R

Washout
period
7days
T

Checkout process

Period 2
R

13

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.

14

 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.

15

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.
16

Demographic data of subjects

Subject No.

Sex

Age

Weight

(Yrs)

(Kg)

Height
(cm)

BMI

S(X)/

(kg/m2)

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

17

Randomization scheme
Subject
No

Sequence

Period I

Period II

TR

RT

TR

RT

RT

TR

TR

RT

10

TR

11

RT

12

TR

13

RT

14

TR

18

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 postdose.

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.

19

 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 10 o C.

Reporting

of Adverse Events:

Subject

Perio

Adverse

Action

Number

Events

Taken

S1

Mild
hypoglycemia

S8

Nausea

S11

Nausea

S12

II

Mild

Recovered
without
Medication

hypoglycemia
20

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.

21

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

Materials

and methods:

Name of the Analyte

Glyburide

Molecular Weight

494.003520 [g/mol]

Lower Limit of Quantification

2.08 ng/mL

Calibration Range

2.08 ng/mL to 303.55

ng/mL

Internal Standard

Glimepiride

Sample Processing Method

Liquid Liquid Extraction

Instrument Method

LC/MS/MS
22

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

All subjects mean linear plot

Linear Mean plasma Concentration Vs Time profile of Glyburide 5 mg tablets under fasting conditions
140
120
100

80
T
60
40
20
0
0

12

16

20

24

28

32

36

40

44

48

Time (hr)

23

All subjects mean semilog plot

Semilog Mean plasma Concentration Vs Time profile of Glyburide 5 mg tablets under fasting conditions
1000.0

100.0
R
T

10.0

1.0

0.1
0

12

16

20

24

28

32

36

40

44

48

Time (hr)

24

Comparative evaluation of PK parameters

Subj
ect
1
2
3
4
5
6
8
9
10
11
12
13
14

Sequ
ence
TR
RT
TR
RT
RT
TR
TR
RT
TR
RT
TR
RT
TR
N
Mean
SD
Min
Median
Max
CV%

Tmax

Cmax

AUClast

Treatment

Treatment

Treatment

R
10.00
4.00
5.00
6.00
8.00
12.00
8.00
6.00
6.00
4.00
6.00
6.00
8.00
13
6.85
2.304
4.00
6.00
12.00
33.65

T
4.50
6.00
6.00
8.00
8.00
8.00
8.00
6.00
4.00
8.00
6.00
6.00
6.00
13
6.50
1.384
4.00
6.00
8.00
21.30

R
68.31
127.86
158.28
121.38
107.26
164.70
299.94
171.07
129.76
142.35
134.31
203.83
127.90
13
150.53
55.615
68.31
134.31
299.94
36.95

T
94.60
121.07
218.51
115.00
89.44
153.30
346.51
89.02
158.33
115.12
186.97
195.79
96.93
13
152.35
72.647
89.02
121.07
346.51
47.68

R
761.03
1129.53
1531.50
598.17
836.05
1199.93
4972.65
897.42
998.61
750.37
645.87
1546.34
926.37
13
1291.83
1146.066
598.17
926.37
4972.65
88.72

T
747.34
1487.59
1658.26
826.77
873.23
1130.21
4959.50
832.74
1056.10
819.85
699.51
1399.83
780.53
13
1328.57
1134.092
699.51
873.23
4959.50
85.36
25

Comparative evaluation of PK
parameters
AUCINF_obs
Treatment

Lambda_z
Treatment

HL_Lambda_z
Treatment

Subj
ect

Sequ
ence

TR

822.12

825.54

0.0966

0.0509

7.18

13.62

2
3
4
5
6
8
9
10
11
12
13
14

RT
TR
RT
RT
TR
TR
RT
TR
RT
TR
RT
TR
N

1156.34
1617.44
611.89
863.59
1217.97
5614.36
925.03
1035.23
783.14
658.38
1594.83
954.54
13

1526.67
1707.06
840.12
895.52
1170.73
5109.14
855.86
1071.80
842.04
711.03
1420.89
794.64
13

0.1544
0.0524
0.1727
0.1227
0.1397
0.0582
0.1955
0.1213
0.1401
0.2198
0.0870
0.2041
13

0.1121
0.0826
0.2337
0.1306
0.1959
0.0780
0.1969
0.1414
0.2041
0.2404
0.1092
0.2267
13

4.49
13.24
4.01
5.65
4.96
11.92
3.54
5.72
4.95
3.15
7.96
3.40
13

6.18
8.39
2.97
5.31
3.54
8.89
3.52
4.90
3.40
2.88
6.35
3.06
13

Mean

1373.45

1367.00

0.1357

0.1540

6.17

5.62

1312.117
611.89

1167.658
711.03

0.05348
0.0524

0.06531
0.0509

3.186
3.15

3.144
2.88

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

SD
Min
Median

26

Statistical summary of the

Lntransformed PK parameters
Cmax

AUC0-t

AUC0-inf

(ng/mL)

(hr*ng/mL)

(hr*ng/mL)

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

Parameter
Lntransformed Data
Geometric Mean

90% Confidence
86.37 - 109.00 97.64 - 111.79 96.37 - 110.63
Interval
Intra subject CV
(%)

16.63

9.63

9.82

Power (%)

93.62

99.92

99.90
27

Conclusion
All

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.
28

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30

THANK
YOU

31