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CLINICAL STUDY REPORT

EVALUATION OF THE BIOAVAILABILITY OF A 2 mg TABLET OF BUMET ANIDE

IN HEALTHY VOLUNTEERS

A Single-Centre, Randomized, Single-blind, Cross-over Study

INCLUDING: Appendix I: Appendix II:

Statistical Report Individual Subject Data

The clinical study report has be en re da cte d using the followmg principles: Where necessary. information is anonymise d to protect the privacy of study subjects and named persons associated ~ith the trial as well as to re.tain commercial confidential information. Summary data are included but data on individual study .subjects, including data listings, are remove d. This rna y result in page numbers not being consecutively numbered. Access to anonymise d data on indi\.idual study subject rna y be obtained up on a ppro"-al of a re.se,arch proposal by the Patient and Scientific Review Board. Appendices to the clinical s.tudy report are omitted. Further details and principles for anonymisationis available in the documentLEOPHARlviA PRINCIPLES FOR ANON"ll\\fiSATlON OF CLINICAL TRLtU. DATA

BU 9302 CAN STUDY Medical Department Leo Pharmaceutical Products

00155768 .

FINAL

February 28, 1994

BU 9302 CAN Study

COMPLIANCE WITH GOOD CLINICAL PRACTICE

This Study was designed to comply with the Canadian Health Protection Branch, Drugs Directorate Guidelines for the Conduct of Clinical Inv~stigations.

BU 9302 CAN Clinical Study Report

Pagel

BU 9302 CAN Study

BU 9302 CAN Study Page3

ABSTRACT

Objectives: The objective of the study was to compare the bioavailability of bumetanide

following a single oral dose of 2 mg bumetanide given as either one 2 mg tablet, or as

two 1 mg tablets (reference) of bumetanide in healthy subjects.

Methods: This was a single-centre, randomised, cross-over, bioequivalence study. The

subject's eligibility for the study was assessed at a pre-study visit, then subjects were

randomised to receive (following an overnight fast) 2 mg of bumetanide as either one

2 mg tablet or as two 1 mg tablets as a single dose. After a one week washout period

the patients received the alternative formulation of 2 mg bumetanide. Blood and urine

samples were collected from all subjects during the first 12 and 24 hours, respectively,

after each dose. Subjects had a follow-up assessment, including repeat laboratory

analyses performed at a visit which took place within seven days of the administration

of the second dose of bumetanide.

Results: Pharmacokinetic assessments were performed in 20 healthy subjects, 10 males

and 10 females. The mean Cmax and AUCO-oo were 106.54 ~g/1 and 224.31 hrs x J.lg/ 1,

respectively, following one 2 mg tablet of bumetanide vs 95.76 ~g/1 and

210.27 hrs x J.lg/ 1, respectively, following two 1 mg tablets (reference). The ratio of

AUCO-oo for the two dosages was 1.056, and the 90% confidence interval was 95.8% to

116.3%. Urinary recovery of bumetanide was 46.3% vs 45.1% following one 2 mg

tablet and two 1 mg tablets respectively.

Conclusion: From the pharmacokinetic results of this study it can be concluded that

one 2 mg bumetanide tablet is bioequivalent to two 1 mg bumetanide tablets (reference)

as 90% confidence interval for the ratio of AUC being 95.8-116.3% was within the limit

of 80-125% according to EEC guidelines. on bioavailability and bioequivalence and the

equivalent Canadian HPB Guidelines.

BU 9302 CAN Study

BU 9302 CAN Study

CLINICAL STUDY REPORT APPROVAL

Leo Pharmaceutical Products DK-2750 Ballerup DENMARK

DK-2750 Ballerup DENMARK

Signature:

Date:

Signature:

Date:

Signature:

Date:

Signature:

Date:

PageS

REPORT AUTHORS

Leo Laboratories Canada Ltd. 555 Kingston Road West Ajax, Ontario LlS 6Ml CANADA

M.Sc.Pharm.

Leo Pharmaceutical Products DK-2750 Ballerup DENMARK

ceutical Products DK-2750 Ballerup DENMARK

BU 9302 CAN Study


TABLE OF CONTENTS

ABSTRACT . . . .. . . . . ....... ............ . .. . . . . ...... . ....... 3 CLINICAL STUDY REPORT APPROVAL . . . . . . . . . . . . . . . . . . . . . . . . . . 5 REPORT AUTHORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 INVESTIGATORS AND STUDY CENTRE ... ... .................... 9 COMPANY PERSONNEL .. . . . .......... ... . ..... ........... . . 10 EXPANDED SUMMARY . . .... ... .. . .... . .. . . .... .... ... . . . . . . 11

1 INTRODUCTION AND RATIONALE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2 INVESTIGATIONAL PLAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.1 STUDY OBJECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.2 STUDY DESIGN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.3 CRITERIA FOR SELECTION OF STIJDY SUBJECfS . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2.4 CRITERIA FOR EARLY WITHDRAWAL FROM TilE STUDY . . . . . . . . . . . . . . . . . . . 24 2.5 TREATMENT ASSIGNMENT METIIOD . . . . . .. . . . . .. . .. . . . . . . . . . . . . . . . . . . . . 25 2.6 BLJli.JDII'lG OF rnE STUDY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.7 BREAKING TilE TREATMENT CODE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 2.8 STUDY MEDICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 2.9 STORAGE OF STUDY MEDICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 2.10 ADMINISTRATION OF STUDY MEDICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 2.11 DRUG ACCOUNTABIUTY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2.12 CONCURRENT TREATMENT .. . . . .. .. .. .. .. .. . .. . . .. . .. . . . .. . .. . .. .. . .. 27 2.13 STUDY PROCEDURES .......... . .. . ...... ... .. . .... . .. . . . .. .. . . ... . . . . 28 2.14 PARAMETERS FOR ANALYSIS ...... . .. .. . . ...... . .. . . ...... . . . . ..... . .. 32 2.15 COMPLIANCE WITII ETHICAL RESPONSIDILmES. . . . . . . . . . . . . . . . . . . . . . . . . . 32

3 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 3.1 STUDY PERIOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 3.2 STUDY POPULATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 3.3 BASELINE CHARACTERISTICS OF SUBJECT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 3.4 PROTOCOL DEVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.5 PHARMACOKINETIC RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.6 SAFETY OF STUDY DRUGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

4 COMPLIANCE WITH GOOD CLINICAL PRACfiCE . .. ...... .... .. . 48

5 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

6 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

7 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

APPENDICES:

Appendix 1 -Appendix II -Appendix ill-Appendix N-Appendix V -Appendix VI -

Statistical Report Individual Subject Data Analytical Reports Analysis Certificates Study Protocol Case Record Form Book

BU 9302 CAN Study

INVESTIGATORS AND STUDY CENTRE

PRINCIPAL INVESTIGATOR

, Pharm.D.

INVESTIGATORS

- ' M.D., FRCP(C)

STUDY CENTRE

Page9

LABORATORY FOR HAEMATOLOGY AND CLINICAL BIOCHEMISTRY ASSAYS

LA BORA TORY FOR BUMETANIDE ASSAY

COMPANY PERSONNEL

PRINCIPAL CLINICAL PROJECT CO.ORDINATOR

-, M.Sc.Pharm. ~t Leo Pharmaceutical Products DK-2750 Ballerup DENMARK

LOCAL CLINICAL PROJECT CO.ORDINATOR

~ B.Sc.Phm., M.B.A. ~ent Leo Laboratories Canada Ltd. 555 Kingston Road West Ajax, Ontario LlS 6Ml CANADA

STATISTICIAN

DK-2750 Ballerup DENMARK

COMPUTERISATION OF DATA

Leo Pharmaceutical Products DK-2.750 Ballerup DENMARK

SECRETARIES OF CLINICAL STUDY REPORT

Canada Ltd. 555 l

BU 9302 CAN Study Page 11

EXPANDED SUMMARY

PROTOCOL SYNOPSIS

Study Objective: To compare the bioavailability of bumetanide following a single

oral dose of 2 mg bumetanide given as either one 2 mg tablet or as two 1 mg tablets

(reference) of burnetanide in healthy subjects. The assessment of bioequivalence

between the two dosages was based on the pharmacokinetic parameter, area under

serum concentration vs time curve (AUC).

Study Design: This was a single-centre, randomised, cross-over, bioavailability

study. The two drug administrations consisted of single, 2 mg oral doses of

bumetanide with a one week washout period between doses: one 2 mg tablet and

two 1 mg tablets, respectively. Blood and urine samples were collected from all

subjects during the first 12 and 24 hours, respectively.

Study phases:

The study was divided into three phases summarized in the diagram below:

Days:

Visit{s):

Phase 1:

Phase 2:

-14 to -1 1 and 8 9 to 15

1 2and3 4

Pre-study assessment (visit 1)

Physical examination and haematology and clinical biochemistry

assessments to assess the subject's eligibility for the study.

Drug administration (visits 2 and 3)

The study included two drug administrations of a single oral2 mg

bumetanide dose: one 2 mg tablet or two 1 mg tablets administered

after an overnight fast. Subjects were stratified on the basis of

gender and randomised for order of treatment. There was an

interval of one week between these two study visits. Following

each dose of bumetanide 16 blood samples were taken over 12

Phase 3:

BU 9302 CAN Study

hours, and urine samples were collected in 6 fractions over 24

hours.

Follow-up assessment (visit 4)

Subjects had a follow-up assessment, including repeat haema-

tology and clinical biochemistry analyses performed within seven

days of the administration of the second dose of bumetanide.

Sample Size Calculation: Previous bioavailability studies have shown a 7-10%

intrasubject coefficient of variation for determination of bumetanide. Assuming an

intra-subject coefficient of variation of 15% for AUC, and a true difference between

formulations of less than 5%, 18 subjects will with 80% probability result in a 90%

confidence interval of the difference between formulations in respect of

bioavailability within :t 20%, or equivalently a 90% confidence interval of the ratio

of AUC's within 80 to 120%, thus establishing bioequivalence in accordance with

both the EEC Guidelines on bioavailability and bioequivalance and the equivalent

Canadian HPB Guidelines.

A total of 20 subjects, were to be entered into the study in order to ensure that the

study was balanced for the order of treatment within each gender.

Subject Eligibility Criteria: Healthy subjects of either sex, aged 18 to 55 and withi.n

15% of normal weight for height according to the Metropolitan Life Tables were

to be included in the study. Excluded were subjects i) known or suspected to be

hypersensitive to bumetanide, ii) with a history of significant gastrointestinal

disorders, iii) with pre-treatment haematology or biochemistry laboratory results

outside the reference range, iv) taking medications considered to be enzyme

inducers or inhibitors, and v) female subjects who were pregnant or breastfeed.ing.

All subjects gave their signed informed consent to join the study.

Treatment Assignment Method: Subjects were assigned a randomisation code

number in the order in which they presented for their first dose of bumetanide.

Randomisation was in balanced blocks of ten subjects of the same sex to allow the

inclusion of an equal number of subjects of each gender.


Study Medication: Bumetanide 2 mg tablets and bumetanide 1 mg tablets

(reference). Each dose was packaged separately. All medication was contained in

identical packages. The label on the medication bottle indicated only randomisation

code number and the treatment number (first or second). Each of the two

bumetanide doses (2 mg) was taken in the morning under supervision of the

investigator following an overnight fast. Subjects maintained an alcohol and

methylxanthin-free diet for 48 hours prior to each study day and fluid intake and

meals were restricted during the study day.

Assessments: The table summarizes the study procedures:

Day(s):

Informed Consent

Medical History

Physical Examination

Blood sampling for haematology and clinical biochemistry(l>

Drug Administration

Recording of Adverse Events

Blood SampJing

Urine Sampiing

-14 to -1 1

..

8

..

1) includes a pre-study pregnancy test in females of child-bearing ability. 2) Blood sampling time points for detennination of bumetanide:

9 to 15

0 (pre-dose) 025, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, and 12 hrs post dosing. Urine coUection intervals: Pre-dose void, 0-2, 2-4, 4-8, I> 12, and 12-24 hrs post dosing.

A medical history was taken and a physical examination performed at visit 1 to

assess the subject's suitability for the study with respect to the protocol eligibility

criteria.

BU 9302 CAN Study

Laboratory assessments including haematology and clinical biochemistry tests were

performed on all subjects at baseline and within 7 days after the last dose.

Pregnancy test was performed on all female subjects of child-bearing ability.

Bumetanide serum and urine concentrations were determined by HPLC-assay.

Primary evaluation parameters: Primary evaluations consisted of measurement of

bumetanide in serum and urine samples and subsequent calculations of

pharrnacokineti.c parameters, (AUC0_r, AUCO-oot Cmax, Tmax' Tw and urinary recovery).

Adverse events were elicited at post randomisation visits.

RESULT SYNOPSIS

The study started on June 14, 1993 and ended on August 9, 1993 and thus had a

duration of two months. A total of 22 subjects were recruited, but two subjects had

to be withdrawn on their first dosing day due to poor veins, making collection of

blood samples difficult. Only the blood and urine samples of the twenty patients

who completed the study were analysed for concentration of bumetanide. Thus,

analysis of bioavailability was performed on data for twenty subjects.

Study Population: Twenty healthy subjects, 10 males, and 10 females, aged 20-42

(mean: 30 years), completed the study according to the protocol.

Pharmacokinetic Parameters: Mean serum concentrations of bumetanide (left

panel) and recovery of bumetanide in urine {right panel) are shown below:


Serum concentration of bumetanide versus time following a 2 mg single dose in healthy, fasting subjects (n=20), mean values

100 90 80

70

60 50 40

so 20 10

1-One 2 mg tablet 1 -lWo , mg tablets

o~~~~~~~=r~~ 0 1 2 3 4 5 6

Hours

8 10 12

Serum concentration of bumetanide verus time following a 2 mg single dose in healthy, fasting subjects (n=20), mean values, log scale

l-One 2 mg tablet 1 -1Wo1 mg~

1

0.1

0.01

0123466 8 1012

Hours

BU 9302 CAN Study

Urinary recavery of bumetanide following a 2 mg single dose in healthy, fasting subjects (n=20), mean, cumulative values

t-'9

1000

900 800

, ....... ........... - --------. --............ ~ ... ~

700 . 600 !

r 500 0

400 aoo 200 100

0

0 2 4 8 12 24 Hours


Pharmacokinetic parameters for bumetanide following ingestion of single, oral doses

of bumetanide as either two 1 mg tablets or as one 2 mg tablets are given below.

Phannacokinetic parameters for bumetanide Jolluwing a 2 mg single dose in healthy, fasting subjects (n=20), mean values

Treatment

Ph.umacokinetic Treatmem: one 2 mg tablet, n = 20 Treatment: two 1 mg tablets, n = 20 parameter

Mean so Range Mean so Range

c:_ (~~og/1) 106.54 . 36.42 60.40 176.00 95.76 27.89 5730164.00

TDIAX (hrs) 1.08 0.64 0..50 3.00 1.23 0.89 0.50 3.98

AUCo-1) 224.31 62.52 133.32 354.43 210.27 49.54 135.07 319.84

(hrs X ~&g/J)

T~ (hrs) 1.13 OZJ 0.78 1.71 1.14 0.18 0.88 1.46

Kel (hrs"l) 0.64 0.14 0.41 0.89 0.62 0.09 0.48 0.79

1) AUCo... ::: AUCo.T, i.e. AUCo.12b

The mean 0 to 24 hour cumulative urinary excretion of bumetanide following

administration of bumetanide as i) one 2 mg tablet and ii) two 1 mg tablets

(reference) to fasting subjects were 925.72JA.g/l and 901.33 JA.g/ 1, respectively, which

corresponds to a 46.4% and 45.1% urinary recovery.

The difference in AUCo.oo following ingestion of two 1 mg tablets and that

following ingestion of one 2 mg tablets, both in the fasting state, was not statistically

significant.

The ratio (one 2 mg tablet : two 1 mg tablets (reference)) in AUCo-00 and Cmax and

the differences in T max T w Kel, and urinary recovery and the associated 90% confidence intervals are presented in the table below:

BU 9302 CAN Study

Pharmacokinetic parameters for bumetanide; mean ratio, mean difference and 90% confidence interval for one 2 mg tablet versus two 1 mg bumetanide tablets administered to healthy, fasting suUjeds (n=20)

90% confidence interval

Parameter Ratio1l Difference ErrorSD Lower Upper CL Ct

AU~l) 1.056 . 13.32.$ 95.8% 116.3% Cawc 1.101 - 25.92% 92.1% 131.6% TINX (Ius) . ~us 0.44 -0.489 0.187 T~ (hrs) - -0.01 0.15 -0.121 0.105 Kel (hrs1) - 0.02 0.09 -0.046 0.088 Urinary 24 hrs recovery (llg) - 24.39 150.68 -92.464 141.244 t) Data for test reference ie. one 2 mg tablet two 1 mg tablets Z) AUCo- = AU


1 INTRODUCTION AND RATIONALE

1.1 BUMET ANI DE

Bumetanide (Burinex~ is a potent loop diuretic which is reported to be 40 to

70 times more potent than furosemide on a milligram-for-milligram basis (1,2,3).

The dose response curve is linear at doses between 0.25 and 2.5 mg (1 ). Its general

therapeutic characteristics are similar to both furosemide and ethacrynic acid.

The absolute bioavailability of oral bumetanide reported in the literature ranges

from 59- 95%, with a median of about 80% (3,5,6). FoUowing oral administration,

diuresis occurs within 30 minutes and peaks at 60 to 180 minutes, with a duration

of action of approximately 6 hours (1). The usual oral dose of bumetanide is 0.5 to

2.0 mg daily. Higher doses are used in patients with renal failure.

1.1.1 Metabolism and Excretion

Published elimination half-lives for bumetanide range from 1 to 1.5 hours (6,7,8).

It undergoes hepatic and renal metabolism and elimination, with 80% of the drug

excreted within 48 hours (7,8). Most of bumetanide is renally excreted in the first

six hours after dosing (9). Fifty to sixty percent of the drug is excreted unchanged

in the urine (7). Metabolism of bumetanide occurs via the butyl side chain with the

3' alcohol being the major metabolite in the urine. In the bile and faeces, the major

metabolite is the 2' alcohol. All metabolites excreted either in the urine or bile are

conjugates, primarily as glucuronides (8).

1.1.2 Protein Binding

Bumetanide is 90-97% bound to albumin in plasma when evaluated using

Sephadex batch and ultrafiltration meUtods (7,10), with no apparent binding to

erythrocytes(7).

BU 9302 CAN Study

1.2 PRESENT STUDY: RATIONALE

For patients taking single oral doses of 2 mg bumetanide, the availability of a new

2 mg tablet in addition to the marketed 1 mg tablet will be convenient and may

~duce the risk of dosing error. Since the new 2 mg tablet of bumetanide may be

substituted in patients now taking single doses of two 1 mg tablets orally, it is

important that the relative bioavailability of the two formulation~ be known.


2 INVESTIGATIONAL PLAN

This section (Section 2) represents a synopsis of the Study Protocol. The protocol

is presented in Appendix ill.

2.1 STUDY OBJECTIVES

The objectives of the study were to compare the bioavailability of bum.etanide

following a single oral dose of 2 mg bumetanide given as either one 2 mg tablet or

as two 1 mg tablets (reference) of bumetanide in healthy subjects.

The assessment of bioequivalence of the two dosages is based on the

pharmacokinetic parameter area under serum concentration vs time curve, AUC.

2.2 STUDY DESIGN

This was a single-centre, randomised, cross-over, pharmacokinetic study

comparing the bioavailability of:

a) one 2 mg tablet of bumetanide, and

b) two 1 mg tablets of bumetanide,

both single oral, 2 mg doses of bumetanide administered following an overnight

fast.

Following each drug administration blood and urine samples were collected from

all subjects during the first 12 and 24 hours, respectively, for determination of

bumetanide concentration and calculation of pharmacokinetic parameters. AU Co-p

AUC()..a>t Cmax' Tmax and Tw for bumetanide were derived/calculated from the

serum concentration versus time curve (0-12 hrs post-dosing), and urinary

recovery was calculated from the data on urinary excretion, ie concentration of

bumetanide in urine fractions 0 - 24 hours post-dosing.

Individuals responsible for analysis of samples were blinded as to the identity or

orders of the study treatments.

BU 9302 CAN Study

2.2.1 Study phases:

The study was divided into three phases summarized in the diagram below:

Days:

Visit(s):

Phase 1:

Phase 2:

Phase 3:

-14 to -1 1 and 8 9 to 15

1 2and 3 4

Pre-study assessment (visit 1)

Physical examination and haematology and clinical biochemistry

assessments to assess the subject's eligibility for the study.

Drug administration (visits 2 and 3)

The study included two drug administrations of a single oral2 mg

bumetanide dose: one 2 mg tablet or two 1 mg tablets administered

after an overnight fast. Subjects were stratified on the basis of

gender and randomised for order of treatment (see Section 2.5).

There was an interval of one week between these two study visits.

Following each dose of bumetanide 16 blood samples were taken

over 12 hours; and urine samples were collected in 6 fractions over

24 hours.

Follow-up assessment (visit 4)

Subjects had a follow-up assessment, including repeat haema-

tology and clinical biochemistry analyses performed within seven

days of the administration of the second dose of bumetanide.

2.2.2 Sample size calculation

Previous bioavailability studies have shown a 7-10% intrasubject coefficient of

variation for determination of bumetanide.

Assuming an intra-subject coefficient of variation of 15% for AUC, and a true

difference between formulations of less than 5%, 18 subjects will with 80%


2.3

2.3.1

2.3.1.1

2.3.1.2

2.3.1.3

2.3.1.4

2.3.1.5

2.3.2

2.3.2.1

probability result in a 90% confidence interval of the difference between formu-

lations in respect of bioavailability within= 20%, or equivalently a 90% confidence

interval of the ratio of AUC's within 80 to 125%, thus establishing bioequivalence

in accordance with both the EEC Guidelines on bioavailability and bioequivalence

and the equivalent Canadian HPB Guidelines.

A total of 20 subjects were entered into the study in order to ensure that the srudy

was balanced for the order of treatment within each gender.

CRITERIA FOR SELECTION OF STUDY SUBJECfS

Inclusion criteria

Male or female, between 18 and 55 years of age.

Within 15% of normal range for height and weight as published in the

Metropolitan Life Tables.

Judged healthy based on a medical history and physical examination.

Females of child-bearing ability (premenopausal and subjects not surgically

sterilized) were required to have a negative pregnancy test within seven days prior

to first treatment.

Signed informed consent was to be given by the subject prior to the

commencement of study procedures and following receipt of verbal and written

information about the study.

Although not an inclusion criterion per se, preference was to be given to non-

smokers.

Exclusion criteria

Known or suspected hypersensitivity to bumetanide.

BU 9302 CAN Study

2.3.2.2 History of significant gastrointestinal disorders.

2.3.2.3 Pre-treabnent haematology or clinical biochemistry results outside the reference

ranges ot abnormal renal function as determined by a calculated creatinine

clearance of less than 80 ml/ min (calculated from serum creatinine).

2.3.2.4

2.3.2.5

2.3.2.6

2.32.7

Use of any medication considered to be an enzyme inducer or inhibitor (eg.

phenytoin, cimetidine, etc.) or other medications which might interact with study

medication during the study period or during the 30 days prior to enrollment to the

study.

Donation of blood during the study period or during the 60 days prior to

enrollment in the study.

Participation in any other clinical trial during the study period or during the

60 days prior to enrollment in the study.

Known or suspected inability to comply with a study protocol or medication

schedule,

2.4 CRITERIA FOR EARLY WITHDRAWAL FROM THE STUDY

Subjects could be withdrawn for any of the following:

2.4.1 Voluntaty withdrawal: Subjects were free to withdraw from the study at any time

and for any reason.

2.4.2 Medical deterioration: An investigator was free to withdraw the subject from the

study fo:t medical reasons.

2.4.3 Adverse events: Any adverse event attributable to use of the study medication that

the investigator considered unacceptable.


2.4.4 Exclusion criteria: Any exclusion criterion becoming apparent during the course

of the study, including need for a medication listed in the exclusion criteria.

2.5 TREATMENT ASSIGNMENT METHOD

At visit 1, the subject was assigned a Case Record Form Book number. At visit 2,

subjects were assigned a randomisation code number in the order in which they

presented for the administration of their first dose of bumetanide .. The order of

treatment was randomi.sed in balanced blocks of ten subjects of the same sex to

allow the inclusion of an equal number of subjects of each gender. If a subject

dropped out of the study, he or she was replaced by the next subject (of the same

gender) to present for his/her first medication administration, and assigned the

same randomisation code number but with a letter added (eg. Sa replaced 5).

2.6 BLINDING OF THE STUDY

All study medication was provided in identical packaging. Each dose was packed

separately. The label on the medication bottle indicated only randomisation code

number and treatment number (first or second). Only the investigator actually

responsible for dispensing of doses had access to the randomisation schedule. The

same investigator was responsible for questioning the subject regarding adverse

events. It was not possible to blind subjects since the two dosages involved the

administration of different numbers of tablets.

Persons conducting the analysis of samples were blinded to the randomisation code.

Blood and urine samples were identified only by means of study subject number

and sample number.

BU 9302 CAN Study

2.7 BREAKING THE TREATMENT CODE

Due to the nature of the study, where both drug administrations consisted of doses

of the same active medication, sealed envelopes containing the treatment allocation

were not issued, but the entire randomisation code was made available to the

investigators in a single envelope.

2.8 STUDY MEDICATIONS

2.8.1 Test fm.mulation: bumetanide 2 mg tablet

Bumetanide 2 mg tablet, batch no. - expiry: October 1994, produced and

certified by Leo Pharmaceutical Products, Ballerup, Denmark. Each tablet contained

bumetanide 2 mg.

2.8.2 Reference formulation: bumetanide 1 mg tablet

Bumetanide 1 mg tablet, batch- expiry: June 1996, produced and certified by

Leo Pharmaceutical Products, Ballerop, Denmark. Each tablet contained bumet-

anide 1 mg.

2.9 STORAGE OF STUDY MEDICATION

Study tnedication was stored at room temperature, (ie. < 25C) in bottles protected .

against humidity.

2.10 ADMINISTRATION OF STUDY MEDICATION

Under the supervision of one of the investigators single oral bumetanide 2 mg doses

were taken in the morning following an overnight (at least 8 hours') fast, with

200 ml of water.


The detailed procedure for drug administration, fluid intake and meals prior to and

p~st dosing is described in Section 2.13.2.

2.11 DRUG ACCOUNT ABILITY

All study medications supplied by and returned to Leo Laboratories Canada Ltd.

were fully documented by the study monitor.

Study medication was administered to the subjects at the investigator site under the

investigators' supervision. The administration of these doses was accounted for by

the investigator.

2.12 CONCURRENT TREATMENT

Systemic conrurrent drug treatments, except for those medications listed in the

exclusion criteria, were to be continued without change throughout the study period

wherever possible. Any use of systemic concurrent medication and any changes to

such use were to be recorded in the Case Record Form.

BU 9302 CAN Study

2.13 STUDY PROCEDURES

The table summarizes the study procedures:

Day(s):

Infonned Consent

Medical History

Physical Examination

Blood sampling for haematology and clinical biochemistry(!)

Drug Administration

Recording of Adverse Events

Blood Sampling(2)

Urine Sampling(2J

14 to 1

"

1 8

..

l} includes a pre-study pregnancy test, in females of child-bearing ability. 2) for determination of bumetanide

2.13.1 Pre-study assessments (visit 1)

2.13.1.1 Medical history

9 to 15

,.

A medical history was taken at visit 1 to assess the subject's suitability for the study

with respect to the protocol eligibility criteria including smoking and drinking

habits. Any concurrent medication was recorded during this visit.

2.13.1.2 Physical examination

A physical examination was conducted during visit 1 to ensure that each subject

was in good health and to assess the subject's suitability for inclusion in the study.


2.13.1.3 Laborato:ry Assessments

The haematology and clinical biochemistry tests listed below were performed on all

subjects at visit 1 and within 7 days of last trial medication.

Haematology

B 1-haemoglobin B-erythrocyte count B-leukocyte count

Clinical Biochemistry

52-alkaline phosphatase S-alanine aminotransferase ( ALT) S-creatinine S-potassium S-sodium

Pregnancy Test

At visit 1 a urine choriongonadotropine test was performed on all female subjects

of child-bearing ability.

Haematology, clinical biochemistry assays and testing for pregnancy were

performed

Canada.

1 B =Blood

2 S = S~rum

BU 9302 CAN Study

2.13.2 Drug administration (visits 2 and 3)

Single oral doses of 2 mg bumetanide were taken with 200 ml of water at

approximately 8 a.m. on each study day (visits 2 and 3). The exact time of each

dose was recorded in the Case Record Form. All doses were administered under

the supervision of an investigator.

2.13.2.1 Fluid intake and meals

All subjects maintained an alcohol- and methylxanthine-free diet for at least 48

hours prior to drug administration. In the morning prior to dosing the subjects

took 200 ml of water or fruit juice. Subjects were not allowed to recline until at

least two hours after drug administration and physical activity was standardized

as much as possible to limit the effects on gastrointestinal blood flow and motility.

Subjects were not allowed to consume further liquid during the first hour post

dosing and not any meals until at least three hours after drug administration.

Subjects were encouraged to drink at least 200 ml of water or fruit juice each hour

thereafter. At 3 hours post-dosing subjects were served a standardized lunch.

2.13.2.2 Serum samples for determination of bumetanide

Venous blood samples (8 ml) were collected in siliconized tubes prior to the

ingestion of each dose of bumetanide (time 0) and at 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2,

2.5, 3, 4, 5, 6, 8, 10, and 12 hours after drug ingestion. The exact time of each

sample was recorded.

Samples were allowed to clot. Following centrifugation, serum was removed and

stored at -20 C until the time of liquid chromatographic assay.

2.13.2.3 Urine samples for determination of bumetanide

Urine was collected prior to each dose (pre-dose void), and with the following

intervals after the administration of bumetanide doses: 0 to 2, 2 to 4, 4 to 8, 8 to 12,

and 12 to 24 hours. The volume of urine collected for each interval was recorded,


as was the pH of the urine. An aliquot of each sample was stored at -20C until

the time of liquid chromatographic assay.

2.13.2.4 Bumetanide Assays

Assays for bumetanide in both serum and urine were performed by-

The Netherlands, using a validated high performance liquid

chromatography (for details see Appendix III).

Serum and urine samples having been stored at -20C were shipped frozen from

Canada to Holland, in two lots via overnight courier. Only samples for patients

who completed the study according to the protocol were shipped for analysis.

2.13.2.5 Recording of Adverse Events

At visits 2 and 3, when subjects received bumetanide doses, the investigator was to

record any adverse event spontaneously reported by the subject, or observed by the

investigator. In addition, at twelve hours after the ingestion of each dose (at the

time of catheter removal), the investigator asked the subject, "Since taking the

medication, have you had any problems?" If the answer was "NO," then no further

questions were asked If the answer was "YES," or if any adverse event had been

spontaneously reported by the subject or observed by the investigator, then the

investigator recorded the nature, time of onset, time of resolution and severity of

the adverse event and judged whether there was a causal relationship of the event

to the study medication.

Any serious or unexpected adverse event was to be reported to the sponsor within

24 hours.

2.13.3 Follow-up Assessments (visit 4)

Within 7 days of last drug adminishation a follow-up assessment including repeat

haematology and clinical biochemistry as described in section 2.13.1.3 wa8

perfonned. Adverse events were recorded as described above except that the

BU 9302 CAN Study

duration of the adverse event was to be recorded as the number of days that the

subject had experienced the adverse event. The outcome of the event was to be

recorded, and all adverse events which were ongoing at visit 4 were to be

followed-up to determine the outcome.

Safety analysis took into consideration any reports of adverse events and any

significant changes in laboratory parameters from the baseline to the follow-up

laboratory assessment.

2.14 PARAMETERS FOR ANALYSIS

The principle parameters to be analysed were the serum and urine concentrations

of bumetanide. Bumetanide serum concentration versus time curves were to be

plotted for each subject following each single dose of bumetanide to characterize the

important bioavailability parameters of the drug (AUc;,_p AUC(}..a>f

AUCo.T/ AUC().cot Cmax, Tmax and T~). Further details regarding the analyses

are provided in Appendix I.

2.15 COMPliANCE WITH ETHICAL RESPONSIBnmES

The study was approved by the Canadian Health Protection Branch prior to the

commencement of subject recruitment.

All investigators signed a statement to confirm:

a) that the study would be conducted to conform with the Declaration of Helsinki II as adopted by the 18th World Medical Assembly, 1964, and subsequent amendments, Tokyo, 1975, Venice, 1983, and Hong Kong, 1989.

b) that the protocol would be approved by the Research Ethics Board, prior to enrollment of the first study

subject.

c) that the subjects' 'Written, informed consent to participate in the study would be obtained prior to enrollment in the study.


All subjects rf!ceived verbal and written information. This information emphasized

that participation in the study was voluntary and that the subject was free to

withdraw from the study at any time.

Subjects enrolled in the study were covered by the liability insurance of Leo

Pharmaceutical Products.

BU 9302 CAN Study


3 RESULTS

Individual subject data are listed in Appendix ll. Data are listed in order of

randomisation code number. The reference to Case Record Form Book number for

individual subjects is presented in Appendix D, Table 11.1.

3.1 STUDY PERIOD

3.2

3.2.1

3.2.1.1

The first subject attended visit 1 (pre-study assessment) on June 14, 1993.

The first and last bumetanide dosages were administered on June 22, 1993 and

August 7, 1993, respectively.

The last subject attended the last study visit (follow-up assessment) on August 9,

1993.

The dtuation of the study was, therefore, 2 months.

Individual data on visit dates are included in Appendix ll, Table ll.l.

STUDY POPULATION

Disposition of Study Subjects

Recruitment of Subjects

A total of 22 subjects were recruited for the study.

No subjects withdrew from the study prior to randomisation and first

administration of bumetanide (ie visit 2).

BU 9302 CAN Study

Two subjects (randomisation code numbers and . ) were withdrawn from the

study during visit 2 (visit with first dosing) due to blood sampling problems

resulting from poor veins. Both - subjects had received one dose of

medication at time of withdrawal. The subjects were replaced and subsequently,

two additional - subjects were enrolled in the study (for further details see

Appendix IL Table ll.lS).

3.3 BASELINE CHARACfERISTICS OF SUBJECT

The baseline characteristics for all subjects entered in the study are presented in this

section. The following section on pharmacokinetic results only include the subjects

who completed the study according to the protocol

3.3.1 Sex distribution and age

A total of 22 subjects, 10 males and 12 females, were included in the analysis of

bioavailability. The mean age was 30 years (range 20-42). The individual subject

demographic data are presented in Appendix IT, Table IT.2.

3.3.2 Medical history

A medical history taken at visit 1 confinned the eligibility of all subjects to

participate in the study. No subject had a history of significant gastrointestinal

disease, nor were there any significant concurrent illnesses. Individual medical

histories are recorded in Appendix IT, Tables ll.3 and ll.4.

Several subjects in the study were taking other medications. These included

antihistamines or decongestants for seasonal allergies, analgesics and oral

contraceptives. One subject (randomisation code number .) was taking

beclomethasone and salbutamol by inhaler for asthma. One subject (randomisation

code number .) was receiving levothyroxine supplementation. Two subjects

(randomisation code numbers al> were taking vitamin or mineral supplements.


The medications taken by subjects are listed in Appendix II, Table II.5. All subjects

were non-smokers, except for randomisation code number 1, smoking less than 5 dgarettes per day, and number . who was an ex-smoker (for at least 14 days

prior to the study). All subjects had their alcohol consumption recorded as less

than 5 drinks per week except for randomisation code number. having 5-10

drinks per week (1 drink= 6 oz wine, 2 oz beer, or 1~ oz liquor). Individual data

on tobacco and alcohol consumption are given in Appendix II, Table II.6.

3.3.3 Physical examination

All subjects were considered to be healthy according to the physical examination.

The mean values for the subjects height, weight, and vital signs at enrollment, ie.

heart rate, respiratory rate, blood pressure, and temperature are given in Table L

Table 1: Height, weight and vital signs follCJWing physical examination of subjects (n=22)

at time of enrollment

Mean so Range Height (em) male n = 10 176.20 7.04 164-188

female n = 12 159.83 5.41 149-168 Weight (kg) male n = 10 78.64 8.51 61.1-87.5

female n = 12 60.28 8.44 48.5-80.0 Heart rate (beats/min) n = 22 70.4 6.1 60-80

Respiratory rate/min n = 22 13.3 1.8 12-18

Diastolic blood pressure (mmHg) n = 22 76.4 5.8 62-88

Systolic blood pressure (mmHg) n = 22 115.5 13.2 98-144 Temperature, oral (0 Q n = 22 36.4 0.2 35.9-36.9

Individual subject data concerning height, weight and vital signs are presented in

Appendix ll, Table ll.3.

BU 9302 CAN Study

3.3.4 Baseline Haematology and Oinical Biochemistry

The mean values for laboratory assessments made prior to randomisation of subjects

in the study are summarized in Section 3.6.2, with comparison to follow-up values.

At enrollment no subject had laboratory values outside the reference range, and all

female subjects of childbearing potential had a negative pregnancy test. Creatinine

clearance (ml/ min) was calculated from serum creatinine values. according to the

formular for males:

(140- age1>) x body weigh~)

serum creati.nine3> x 50 x60

for males. Females- males X 0.85 e>years, 2>kg, J)~ol/1).

Individual data are presented in Appendix II, Tables ll.l2, 11.13, and ll.14.

3.4 PROTOCOL DEVIATIONS

Violation of protocol eligibility criteria occurred in one subject, a -

(randomisation code no. 1>, who was included in the study despite having a creatinine clearance calculated from the serum creatinine value of 69 ml/ minute,

which was below the 80 ml/ minute minimum indicated in the protocol's eligibility

criteria. This subject had a serum creatinine within the reference range.

3.5 PHARMACOKINETIC RESULTS

The serum concentrations, urine excretion and pharmacokinetic parameters for

bumetanide presented below are calculated from the individual data obtained from

the 20 subjects who received both dosages in the cross-over study. The two

subjects who were withdrawn at the first dosing day due to problems with blood

sampling have not had any sample assayed and are only accounted for regarding

safety assessments.


Each of the two bumetanide doses (2 mg) was taken in the morning following an

overnight fast, under supervision of the investigator. Subjects maintained an alcohol

and methylxanthin-free diet for 48 hours prior to each study day and fluid intake

was restricted during study days (for individual data see Appendix II, Table II.7).

3.5.1 Serum concentrations of bumetanide after administration of 2 mg doses of

bumetanide

Actual sampling times were recorded during the drug administration and sampling

phases of this study. In some instances, these sampling times varied slightly from

the intended time according to the study protocol Actual sampling times, rather

than proposed times, were used for the calculation of all pharmacokinetic

parameters.

Serum concentrations of bumetanide were determined by HPLC assay. The figures

I and II below show the arithmetic and semi-log plots of the mean serum

concentration versus time curves, and Table II lists the mean concentrations of

bumetanide after the adminstration of the two dosages of bumetanide to fasting

subjects. Bumetanide was detected in all first post-dosing samples (at 0.25 hrs) and

was still detectable in some samples at 8-10 hours post dosing. The peak mean

concentration was 96.04 and 77.36 ..-.gil following one 2 mg tablet and two 1 mg

tablets, respectively, and occurred at 0.75 hours.

BU 9302 CAN Study

Fig 1: Setum concentTation of bumetanide versus time following a 2 mg single dose in healthy, fasting subjects (n=20), mean values

J.l.g/1

100

90

80 70 eo 50 40 30 20 10

l- One 2 ~ 1abl8t I --lWo 1 mg l8blala

o~~~~~~~~=T~ 0 1 2 3 4 5 6

Hours 8 10 12

Fig II: Serum concentration of bumetanide versus time following a 2 mg single dose in healthy fasting subjects (n=20), mean values, log scale

1- One 2 mg tablet 1 ~lWO 1 mg tabt8ts

1 . .. .. ...

0.1

0.01

0.001

0 1 2 3 4 5 6 8 10 12

Hours


Table II: Serum concentrations of bumetanide folluwing a single 2 mg dose in healthy, fasting subjects (n=20), mean values

Serum concentration of bumetanide (pgll)

Time11 of Treatment Treatment:

Sampling (hrs) one 2 mg tablet, n = 20 two 1 mg tablets, n =20

Mean so Range Mean so Range

0 o.oo2> 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.25 2620 21.76 0.00 78.80 20.56 30.60 0.00 134.00

0.5 78.10 49.08 5.60 171.00 63.31 44.52 1.86 164.00

0.75 96.04 45.24 11.20 176.00 77.36 40.18 4.44 157.00

1.0 89.49 38.68 13.70 163.00 76.08 34.77 5.72 124.00

1.25 79.10 30.65 12.70 137.00 69.04 28.92 6.21 110.00

1.5 68.56 25.46 15.80 126.00 66.52 22.62 6.37 106.00

2 57.09 20.10 30.80 %.80 55.04 23.11 12.20 108.00

2.5 42.82 21.48 13.10 87.00 41.832) 16.09 18.50 75.10

3 3121 14.48 12.50 63.30 32.562) 14.73 15.40 64.30

4 14.98 8.45 5.06 40.50 16.28 9.47 6.02 37.30

5 8.14 4.52 3.06 22.00 9.48 6.14 2.34 24.90

6 4.04 2.57 0.00 10.40 4.82 3.23 0.00 12.50

8 1.01 1.37 0.00 4.35 0.92 1.53 0.00 4.49

10 0.00 0.00 0.00 0.00 0.19 0.59 0.00 2.17

12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

1) Hours post dosing 2) n " J9

Individual data on serum concentrations of bumetanide and actual collection times

are presented in Appendix ll, Table ll.S. Individual serum concentrations of

bumetanide versus time curves in arithmetic plot are shown in Appendix I.

BU 9302 CAN Study

3.5.2 Pharmacokinetic parameters for bumetanide

3.5.2.1

Table ill lists the mean pharmacokinetic parameters for bumetanide observed or

derived from the serum concentration versus time curves for the one 2 mg and the

two 1 mg dosing groups of 20 subjects. cmax and r max are the mean of the actually observed values. T ~ is calculated from the observed values using linear regression.

AUC~T was calculated using the trapezoidal method. Since all subjects had serum

concentrations "0" before or at the last sampling point (12 hrs), no calculation of

residual area under serum concentration versus time curve was relevant. The AUC

calculated from time 0 to last sampling time point with measurable concentration

therefore is the AUCo-, i.e. AUCo- = AUCo.T

Table ill: Pharmacokinetic parameters for bumetanide foUowing a 2 mg single dose in

healthy, fasting subjects (n=20), metm tJlllues

Tratment

l'hml.acokindic Treatment: one 2 mg tablet. n = 20 Treatment: two 1 mg tablets, n = 2D param.m-

Mean SD Range Mean SD Range


3.5.2.2 Mean pharmacokinetic parameters for two 1 mg tablets administered to fasting

subjects n = 20) The mean AUC0_00 for bumetanide administered as two 1 mg tablets to fasting

volunteers was determined to be 210.27 hrs x 11gll (range 135.07- 319.84 hrs x

fA.g/1), and the mean

BU 9302 CAN Study

Tabl~ IV: Urinary recovery of bumetanide following a 2 mg single dose in healthy fasting subjects (n=20), mean values

Bumetanide amount in urine (J&g)

Samplin~) Treatment: one 2 mg tablet. D = 20 Trutment two 1 mg tablets, n = 20 Interval (hrs)

Mean SD Range Mean SD Range

0- 2 531.()6 201.17 162.90 960.48 496.72 2.29.22 48.06 865.78

2- 4 248.06 88.97 38.85 368.72 .2.52.34 10'7.87 126.90 490.56

4- 8 115.00 65.91 1.17 288.80 116.74 53.09 26.80 243.00

8- 12 23.70 19.58 1.61 72.48 24.98 16.96 7.85 58.96

12-24 7.90 5.96 0.00 21.28 10.55 10.99 0.00 43.87

0- 24 925.72 196.51 597.31 1222.88 901.33 235.65 458.44 1231.49

1) Hours post dosing

IndiVidual data are pre sented tn Al pp endix u, Table 11.10.

3.5.4 Comparative bioavailability of bumetanid~ 2 mg and 1 mg (reference) tablets

Table V compares the various pharmacokinetic parameters for bumetanide 1 mg

and 2 mg tablets, showing the ratio of the parameter for one 2 mg tablet, and two

1 mg tablets and the associated 90% confidence intervals.

Table V: Pharmacokinetic parameters far bumetanide; mean ratio, mean difference and 90% confidence interval for one 2 mg tablet versus two 1 mg bumetJJnide tablets administered to healt , tin su ects (n=20)

90% confidence interval

Paramei2r Ratio1l Difference Enor Lower Upper so CL CL

AUCo.,..1> 1.056 13.32% 95.8% 116.3%


The ratio of AU Co- of 2 mg vs reference is 1.056, and the 90% confidence interval

is 95.8% to 1163%. Thus, bioequivalence was established as the 90% confidence

interval was within 80-125% limit according to EEC Guidelines on bioavailability

and bioequivalence and the equivalent Canadian HPB Guidelines.

Potency correction: Calculations of the ratios and 90% confidence intervals for

AUCo- and Cmax were also performed on data corrected for measured content

(according to information in Analysis Certificate, Appendix IV). The results of these

calculations showed the following ratios and (90% confidence limits): AUCo.ao 1.045

(94.8- 115.1 %), Cawc 1.09 (91.1- 130.3%).

3.6 SAFETY OF STIJDY DRUGS

3.6.1 Adverse Events

Five subjects reported a total of 10 adverse events. The most frequently reported

adverse event was headache, reported by three subjects. Details on severity and

duration of the individual adverse events are given in the Table VI and in

Appendix ll, Table ll.ll.

Table VI: Adverse events reported

Subject Time of Relationship to nmeof

Adverse event ) Code no. Visit no. onset Severity study drug resolution

Occasional cough 3 14.00 mild unlikeJy 20.00

Headache 3 11.00 moderate unlikely 19.00


Nausea 2 10.00 mild possible 19.00

Dizziness 2 15.30 mild possible 16.15

Headache 3 15.00 mild unlikely 19.30


Ughtheadedness 2 9.00 mild possible 14.00

Weakness in legs 2 9.00 mild unlikely 22.00

Ughtheadedness 3 9.00 mild unlikely 14.00

) all reported by subject

BU 9302 CAN Study

No subject withdrew from the study due to adverse events. No serious or

unexpected adverse events were experienced and no persistant adverse event was

reported.

3.6.2 laboratory abnormalities

No clinically significant deviations from baseline values were seen for individual

subjects at the follow-up assessment for haematology and clinical biochemistry

parameters. The comparison between the pre-treatment and the end of study

assessments is presented in Tables VII and VITI. Individual laboratory results for

the pretreatment and end-of-study haematology and clinical biochemistry tests are

presented in Appendix ll, Tables ll.12a-b and ll.13.

Table VII: Haematology parameters, before and after two doses of 2 mg bumetanide to healthy subjects

Ref.

HAEMATOLOGY TEST Mean SD Range Range Unit

B-haemoglobin:

Pre-treatntent n = 22 143.23 10.66 121 164 M=135-180 g/ 1 End of study n = 20 139.20 13.13 115 169 F=llS-165

B-erythrocytes:

Pre-treatment n = 22 4.68 0.41 4.00 5.42 M=4.5-6.5 101211

End of study n = 20 4.57 0.50 3.67 5.50 F=4.0-5.5

B-leukocytes:

Pre-treatment n=22 6.42 1.18 4.50 8.70 4.0-11.0 to9/ 1

End of study n =20 6.57 1.25 4.30 9.40

BU9302 CAN Study Page47

Table VII: Clinical biochemistry parameters, before and after two doses of2 mg bumetanide to healthy subjects

Ref. CLlNlCAL BIOCHEMISTRY Mean so Range Range Unit S.Alkaline phosphatase: Pre-treabnent n =22 59.55 14.98 37 97

End of study n :::20 60.25 15.96 35 109 35-110 U/1

S.Alanine aminotransferase: Pre-treabnent n :::22 19.64 6.84 8 34 5-40 U/ 1

End of study n = 20 17.75 8.42 5 35

S-Creati.nine: Pre-treabnent n=22 82.18 13.82 62 118 M=65-125

End of study n = 20 85.80 15.37 62 112 F=S0-110 f.'IDOl/1 S.Sodium: Pre-treabnent n = 22 14059 2.32 137 147 End of study n =20 139.40 1.67 136 143

135-147 mmol/1

S.Potassium: Pre-treabnent n = 22 4.27 0.31 3 4 End of study n =20 4.23 0.35 3 4

3.5-5.2 mmol/1

4 COMPLIANCE WITH GOOD CLINICAL PRACI'ICE

The present study was carried out in accordance with the Canadian Drugs

Directorate Guideline: Conduct of Clinical Investigations, covering the assurance

that data generated from the study is credible and that the right, integrity and

confidentiality of subjects are protected. Prior to start of the study the protocol was

approved by the Canadian Health Protection Branch and by the Research Ethics

Board, . Furthermore, the investigator signed a

statement confirming that the study was to be conducted to conform with the

Declaration of Helsinki II.


5 DISCUSSION

This study was conducted to compare the bioavailability of bumetanide given as

either one 2 mg tablet or as two 1 mg tablets in healthy volunteers.

This was a single-centre, randomised, single-blind, cross-over, pharmacokinetic

study. According to the protocol 20 subjects, 10 males and 10 females, should

complete the study including i) pre-study eligibility assessments, ii) two single dose

drug administrations with a one week interval, iii) follow-up assessment. Two

- were withdrawn the day following the first drug administration due to

problems with blood sampling, they did not contribute to evaluation of

bioavailability, and were substituted by two additional-. Thus, a total of 22

subjects passed the eligibility assessments and 20 subjects completed the entire

study period. All20 contributed with the scheduled number of 16 blood samples

during the first 12 hours after each dosing as well as with wine samples, 6

collections during 24 hours post dosing, except for one subject who missed the

baseline blood sample before first dosing and another subject who missed two

blood samplings at 2.5 and 3 hours after the first dose. The interval between the 2

doses of bumetanide was scheduled to be one week but ranged from 7 to 14 days.

Overall the subjects' compliance with the study protocol was good.

Subjects were randomised for the order of treatment. Both of the- subjects

who dropped out of this study were replaced with substitutes who were assigned

the same randomisation code numbers but with a letter added (eg. 3a replaced 3).

Only the investigator actually responsible for dispensing of doses had access to the

. randomisation schedule. It was not possible to blind subjects since the two dosages

involved the administration of different numbers of tablets. Persons conducting the

analysis of samples were blinded. At the end of the study all study medication was

accounted for.

The assays for bumetanide in serum and urine were performed at

The Netherlands, where a validated HPLC method was available.

Samples were stored at -20 C and shipped frozen, in two lots by overnight courier.

Only samples for patients who completed the study according to the protocol were

BU 9302 CAN Study

shipped for analysis. Both shipments of samples arrived at

., The Netherlands, in good condition within 18 hours of the departure from

Canada.

From the results for bumetanide concentration in serum and urine and the

calculations of mean pharmacokinetic parameters it can be concluded that the two

doses are bioequivalent. The pharmacokinetic parameters agreed well with

previously reported data for bumetanide although the recovery in urine in this

study was in the lower end of the .range.

One- subject had a creatinine clearance (69 ml/min) calculated from serum

creatinine below the limit for inclusion (80 ml/ min). This subject had a serum

creatinine within the reference range. The investigator felt, however, that including

this subject would jeopardize neither. health nor the results of the study. The

subject passed through the study without incident.

A total of 5 subjects reported 10 adverse events. Headache was the most frequently

reported (3 subjects). All adverse events resolved within day of onset. No patient

withdrew from the study due to adverse events. No serious or unexpected adverse

events were experienced by subjects in this study.


6 CONCLUSIONS

The present study in 20 healthy volunteers demonstrated that a newly developed

2 mg tablet formulation of bumetanide and two 1 mg tablets of bumetanide

(reference) administered in the fasting state are bioequivalent, according to the

criteria in the guidelines established by the EEC and the Canadian Health Protection

Branch. The study demonstrated that AUC following administration of the 2 mg

tablet was not statistically significantly different from that of two 1 mg tablets. The

ratio of AUCo- for the two dosages (testreference) is 1.056 and the 90%

confidence interval is 95.8-116.3%, thus being within the 80-125% limit for

bioequivalence.

BU 9302 CAN Study


7 REFERENCES

1. Flamenbaum W, Friedman R Pharmacology, therapeutic efficacy, and adverse effects ofbumetanide, a new "loop" diuretic. Pharmacotherapy 1982;2:213-22.

2. Ramsay LE, Mcinnes GT, Hettiarachi Jet al. Bumetanide and furosemide: a comparison of dose response cwves in healthy men. Br J Clin Pharmacol1978;5:243-7.

3. Brater DC Oinical pharmacology of loop diuretics. Drugs 1991;41(suppl 3):14-22.

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BU 9302 CAN Study

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