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This document has been doY..111oaded from www.leo-pharma.com subject to the terms of use state on the website. It conta1ns data and results :regarding approved and non-approved uses, formulations or treatment regimens, and it is provided for transparency and informational purposes only. The content does not reflect the complete results from all studies related to a product. As a document of scientific natur,e it is not to be seen as a recommendation or advice regarding 1he use of any products and you must always consult the specific prescribing information approved for the product prior to any prescription or use.
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
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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
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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.
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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
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Page6
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
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BU 9302 CAN Study Page7
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
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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
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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
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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
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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.
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BU 9302 CAN Study Page 13
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.
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Page 14 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:
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BU 9302 CAN Study Page 15
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
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Page 16 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
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BU 9302 CAN Study Page 17
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:
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Page 18 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
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BU 9302 CAN Study Page 19
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).
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Page 20 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.
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BU 9302 CAN Study Page21
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.
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Page 22 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%
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BU 9302 CAN Study Page23
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.
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Page 24 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.
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BU 9302 CAN Study Page25
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.
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Page 26 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.
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BU 9302 CAN Study Page27
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.
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Page 28 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.
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BU 9302 CAN Study Page29
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
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Page 30 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,
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BU 9302 CAN Study Page31
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
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Page 32 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.
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BU 9302 CAN Study Page 33
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.
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Page34 BU 9302 CAN Study
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BU 9302 CAN Study Page35
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).
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Page36 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.
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BU 9302 CAN Study Page37
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.
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Page 38 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.
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BU 9302 CAN Study Page39
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.
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Page 40 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
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BU 9302 CAN Study Page41
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.
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Page 42 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
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BU 9302 CAN Study Page 43
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
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Page 44 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%
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BU 9302 CAN Study Page45
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
Headache 2 11.00 moderate unlikely 20.25
Nausea 2 10.00 mild possible 19.00
Dizziness 2 15.30 mild possible 16.15
Headache 3 15.00 mild unlikely 19.30
Headache 3 15.00 moderate unlikely 18.00
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
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Page46 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
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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.
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BU 9302 CAN Study Page49
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
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Page 50 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.
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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.
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7 REFERENCES
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