The Biowaiver Procedure: Its Application to Antituberculosis

Products in the WHO Prequalification Programme

STEFANIE STRAUCH,1 EKARAT JANTRATID,1,† MATTHIAS STAHL,2 LEMBIT RÄGO,2 JENNIFER B. DRESSMAN1
1
Institute of Pharmaceutical Technology, Goethe University, 60438 Frankfurt am Main, Germany
2
World Health Organization, 1211 Geneva 27, Switzerland

Received 1 July 2010; revised 26 August 2010; accepted 26 August 2010

Published online 6 October 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22349

ABSTRACT: In 2005, the World Health Organization (WHO) proposed that provided an active
pharmaceutical ingredient could meet certain criteria, bioequivalence could be evaluated with
a set of laboratory tests, obviating the need for expensive and time-consuming pharmacokinetic
studies in humans. The aim of this work was to determine whether this so-called “biowaiver”
procedure can be applied to antituberculosis products. Antituberculosis products from the WHO
Prequalification Programme, including three ethambutol, two isoniazid and one pyrazinamide
product, were investigated. In vitro dissolution data for these products were generated according
to the biowaiver method stipulated in the WHO Guidance, and the bioequivalence decision based
on these data was compared with that based on the corresponding in vivo pharmacokinetic data.
In no case was a “false positive” bioequivalence decision reached using the biowaiver procedure,
that is, all products deemed bioequivalent according to the biowaiver methods also proved to
be bioequivalent in the corresponding pharmacokinetic study. These findings open the way
to a simplified method of ensuring bioequivalence of antituberculosis drug products, thereby
improving access to high quality antituberculosis medicines for a greater number of patients.
© 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:822–830,
2011
Keywords: bioavailability; bioequivalence; biopharmaceutics classification system; dissolu-
tion; ethambutol; isoniazid; pharmacokinetics; pyrazinamide; tuberculosis

INTRODUCTION and Drug Administration (FDA) in the USA or the

European Medicines Agency (EMA) in Europe, which
Today around one-third of the world’s population
assess whether drug products can be approved and
(∼two billion people) is infected with Mycobacterium
also inspect them regularly once they are on the mar-
tuberculosis.1 Because tuberculosis (TB) is both con-
ket. Thus, it is probable that in many developing
tagious and spreads easily through the air, there are
countries, products with unproven therapeutic effi-
approximately nine million new TB cases per year
cacy are available. In order to help TB patients in
(based on 2006 Figures).1,2 The high incidence is fos-
these regions, (a) good quality, effective anti-TB prod-
tered by many factors, including insufficient med-
ucts must be available on the market and (b) every
ical care, lack of funds to pay for treatment, and
patient needs to have access to them through national
the growing numbers of immune-suppressed HIV pa-
health care systems.
tients. Therefore, TB is considered to be mainly a dis-
One major effort toward accomplishing this mission
ease of poverty.1 The majority of TB deaths appear
is the World Health Organization (WHO) Prequalifi-
in the developing countries.1 These countries often
cation Programme, which was established in 2001.
lack a well-established national or regional author-
The aim of this programme is to facilitate worldwide
ity for regulating medicines, such as the US Food
access to good quality medicines for the treatment of
Correspondence to: Jennifer B. Dressman (Telephone: + 49- the high-burden diseases, including TB, malaria, and
69-798-29-680; Fax: +49- 69-798-29-724; E-mail: dressman@em. HIV/AIDS, as well as to facilitate access to reproduc-
uni-frankfurt.de) tive health and influenza medications and zinc prepa-
† Ekarat Jantratid’s last address was Department of Pharmacy,
Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. rations for diarrhea.3 As part of this programme, the
Journal of Pharmaceutical Sciences, Vol. 100, 822–830 (2011) WHO evaluates the quality, safety, and efficacy of
© 2010 Wiley-Liss, Inc. and the American Pharmacists Association the medicinal products, based on both information

822 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011


submitted by the manufacturers and inspections of
the corresponding manufacturing and clinical sites.
Products that pass the evaluation are then published
in the WHO List of Prequalified Medicinal Products,
which is freely accessible via the WHO homepage
http://apps.who.int/prequal/.
In general, in order to obtain a marketing autho-
rization for a new multisource (generic) drug product,
the manufacturer has to demonstrate that its drug
product meets quality standards and is furthermore
bioequivalent to a well-established comparator prod-
uct (i.e., the innovator or an accepted reference prod-
uct). This is usually accomplished by the submission
of bioequivalence (BE) data generated in comparative
human pharmacokinetic (PK) studies to the respon-
sible authorities.4
In parallel with the prequalification of drug prod-
ucts, a new approach for BE testing, based on the
Biopharmaceutics Classification System (BCS), was
approved by the FDA in 2000.5 The BCS categorizes
active pharmaceutical ingredients (APIs) into four
classes according to their solubility and permeability
properties, both of which can be important limitations
to drug absorption after administration of solid oral
dosage forms.6 This approach is based on the hypoth-
esis that pharmaceutically equivalent drug products
demonstrating similar dissolution characteristics in
the gut lumen in vivo will have similar PK profiles
in vivo. Thus, the comparison of in vitro dissolution
profiles of a test product, for example, a new mul-
tisource product in the development phase with un-
known PK characteristics, and an approved reference
product, with known PK properties in vivo, could be
used to judge BE of the two products. Under these
circumstances, there would be no need to conduct
in vivo PK studies to demonstrate BE for the test
product. It is important to note that the dissolution
tests of test and reference products have to be carried
out according to explicitly prescribed specifications.
In 2005, the WHO adopted this so-called “biowaiver”
concept in its Guidance “Proposal to waive in vivo
BE requirements for WHO Model List of Essential
Medicines immediate-release, solid oral dosage forms
(WHO Technical Report Series No.937, Annex 8).”7
In order to minimize the patient risks associated
with an incorrect BE decision based on the biowaiver
approach, all API candidates are evaluated according
to the following aspects: BCS classification, indica-
tion, therapeutic index, interaction with food or excip-
ients as well as the risk of bioinequivalence and the
dissolution characteristics of the drug product. Fac-
tors that lead to low risks arising from an incorrect
biowaiver decision for a given API include (a) classi-
fication to BCS Class I (III), (b) a wide therapeutic
index, (c) few or no reported cases of bioinequivalence
in the public literature, and (d) “rapid” or “very rapid”
dissolution characteristics. By contrast, (a) classifi-
DOI 10.1002/jps
BIOWAIVER FOR ANTITUBERCULOTICS 823
cation to BCS Class IV, (b) a narrow therapeutic in-
dex, (c) “food effects” or interaction with excipients, (d)
published cases of bioinequivalence, and (e) slow and
incomplete dissolution result in higher risks to the
patient if an incorrect biowaiver decision is reached.
Thus, the WHO Guidance7 prescribes a risk–benefit
analysis for each API. Only in cases for which the
risks associated with an incorrect biowaiver decision
are outweighed by the potential benefits accrued from
the biowaiver approach, should the biowaiver proce-
dure be applied.7
The application of biowaiver-based approval for
new multisource products,5,7 as well as for drug
products already approved by the regulators when
the composition and/or manufacturing procedure is
changed,4,8 can result in large savings in resources
on the part of pharmaceutical companies. In turn, the
products can be distributed at lower prices, making
them more accessible to a larger percentage of the
population. This is a point that is especially crucial
for anti-TB drug products.
To justify the appropriateness of the biowaiver
strategy, it needs to be shown that products that
are declared to be bioequivalent using the biowaiver
methods are actually bioequivalent in vivo. This work
evaluated the appropriateness of biowaiver-based BE
for anti-TB products. For this purpose, the in vivo
PK data of the anti-TB products submitted to the
WHO Prequalification Programme were compared
with corresponding in vitro dissolution data. A selec-
tion of drug products from the WHO Prequalification
Programme containing ethambutol dihydrochloride
(ETB), isoniazid (INH), and pyrazinamide (PYR) were
investigated. First, the dissolution data of the multi-
source products were compared with the dissolution
profiles obtained from the recommended comparator
products. Second, PK data provided by the manufac-
turers to the WHO Prequalification Programme were
compared with the PK profiles of the recommended
comparator products using BE criteria. Third, the
concordance of the decisions based on the biowaiver
approach (in vitro comparative dissolution tests) and
standard PK analysis of in vivo data was evaluated.
MATERIALS
The chemicals used in this study were all of analytical
grade and purchased commercially. ETB (≥99% pure,
lot # 077K1295), INH (≥99% pure, lot # 013K0813),
and PYR (≥99% pure, lot # 062K1334) reference sub-
stances were obtained from Sigma–Aldrich Chemie
GmbH, Steinheim, Germany.
Aside from ETB-91-400A tablets, which were re-
ceived directly as a gift from the manufacturer, and
INH-ref-100, which was purchased from Phoenix
Pharmahandel, Hanau, Germany, all products tested
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011
824 STRAUCH ET AL.
Table 1. Excipients Present in the Antituberculosis Drug
Products Tested
Number of Productsa
Tested Containing the
Excipient Named Excipient
Magnesium stearate
Corn starch/Maize starch
Colloidal silicon dioxide/Colloidal
anhydrous silica
Talc
Dicalcium phosphate/Calcium
hydrogen phosphate
Microcrystalline cellulose
Sodium starch glycolate
Croscovidone/Copovidone
Disodium edetate
Hypromellose
Macrogol/Macrogol 6000
Povidone
Ethyl cellulose
Propylene glycol
Titanium dioxide
a For ETB-380–400A and PYR-ref-500 the excipient composition was
not provided by the manufacturer.
in this study were obtained from the WHO. The ex-
cipients used in the various drug products are shown
in Table 1.
METHODS
In Vitro Dissolution Study
All dissolution tests were carried out according to
the WHO7 biowaiver specifications using the USP
Apparatus 2 (paddle assembly) and three standard
dissolution media [Simulated Gastric Fluid without
pepsin at pH 1.2 (SGFsp ), acetate buffer at pH 4.5,
and Simulated Intestinal Fluid without pancreatin at
pH 6.8 (SIFsp ), which was slightly modified by replac-
ing potassium dihydrogen phosphate with equimo-
lar amounts of sodium dihydrogen phosphate]. The
medium volume used was 900 mL per vessel with a
medium temperature of 37 ± 0.5◦ C, and the rotational
speed was set at 75 rpm. The samples were taken af-
ter 5, 10, 15, 20, 30, and 45 min. Sampling was per-
formed manually using 5 mL glass syringes connected
with stainless steel sampling devices and cylindrical
polyethylene filter sticks at the end of each sampling
device. The volume withdrawn from the vessels at
each sampling time point was approximately 5 mL,
and the sample volume was replaced at each sampling
point. After withdrawal, the samples were filtered
through a 0.45 :m PTFE filter, suitably diluted and
then analyzed by ultraviolet (UV) spectrophotometry
(INH, PYR) or high-performance liquid chromatog-
raphy (HPLC) method (ETB). All experiments were
conducted in six replicates.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011
Quantitative Analysis Via UV Spectrophotometry
A U-3000 Spectrophotometer (Hitachi Ltd., Tokyo,
Japan) was used for the quantification of INH and
PYR. The UV spectra of the two APIs in SGFsp , ac-
etate buffer, pH 4.5, and SIFsp were recorded, and the
7
maximum absorbance was determined at 263 nm for
6
5 INH and 268 nm for PYR. To analyze the dissolution
samples, a calibration curve prepared using the dis-
5 solution medium was freshly constructed at the corre-
4 sponding wavelengths over a concentration range of
2–15 :g/mL for INH and 4–24 :g/mL for PYR. Sam-
3
3 ples from the dissolution tests were suitably diluted to
2 API concentrations within the calibration range and
2 analyzed for INH and PYR, respectively.
2
2 Quantitative Analysis Via HPLC
2
1 The quantification of ETB was performed by an HPLC
1 method, slightly modified from a WHO publication.9
1 A Lichrosorb
R
(Merck KGaA, Darmstadt, Germany)
RP-Select B (125 × 4 mm, particle size: 5 :m) col-
umn was used for ETB analysis by adjusting the col-
umn temperature to 40◦ C. The injection volume was
20 :L, the flow rate was 2.2 mL/min, and the run
time was set to 6 min. The mobile phase consisted of
94% copper acetate buffer, 0.2% triethylamine, and
5.8% methanol. The detection wavelength was set at
270 nm. Calibration standards in each buffer were
prepared by diluting a stock solution consisting of
pure ETB standard substance dissolved in the re-
spective buffer to obtain concentrations of ETB in the
range 60–950 :g/mL. The peak areas of the calibra-
tion standards were measured in duplicate at 270 nm,
and calibration curves were generated by plotting the
ETB concentrations against the corresponding peak
areas.
Dissolution Testing Required for Application
of the Biowaiver
For drug product approval based on the biowaiver
procedure, strict dissolution requirements have to be
met. These criteria depend on the BCS Class of the
API as defined in regulatory guidances, such as those
of the FDA,5 the WHO,7 and the EMA.10 The BCS
classifications of ETB, INH, and PYR have been re-
ported previously:
• ETB is classified variously as BCS Class I/III11
(borderline) or III,7,12,13
• INH as BCS Class I12 , I/III7,14 (borderline) or
III,13 and
• PYR as BCS Class I12 , I/III7 (borderline) or
III.13,15
According to these literature data and in order to
guarantee patient safety, all three APIs were treated
conservatively in this study as BCS Class III (highly
DOI 10.1002/jps
 BIOWAIVER FOR ANTITUBERCULOTICS 825

soluble, poorly permeable) compounds. For products

Age (yrs.)
containing BCS Class III APIs, the WHO7 biowaiver

18–55

18–45

18–35
18–50

Adult

Adult
dissolution criterion is that both the multisource and
comparator products must be “very rapidly dissolv-
ing,” i.e. ≥85% release within 15 min in all three me-

Gender

Female
Male/

Male

Male

Male

Male

Male
dia. This strict criterion ensures a maximal contact
time between the dissolved drug and the intestinal
mucosa, thus facilitating absorption and ensuring the

Asian Indian
best possible BA of the compound.16 If a drug product

South Asian
Population
Caucasian
fails to meet the WHO7 dissolution criteria, BE must

a
be demonstrated with PK studies in humans.
Evaluation of the Dissolution Profiles

Period (days)
The dissolution profiles in the three standard dissolu-

Wash-out
tion media of all the tested products were constructed

4–7

7
7
from the average of the percentage of cumulative
drug dissolved from six vessels at each sampling time
point.

400 (Single dose)

300 (Single dose)

500 (Single dose)

300 (Single dose)
400 (single dose)
PK Data Interpretation

400 × 2 (Single
Dose (mg)
According to the WHO Guideline,4 two pharmaceu-

dose)
tical products are bioequivalent if they are pharma-
ceutically equivalent or pharmaceutical alternatives,
and their BAs, in terms of peak and total exposure
[area under the curve (AUC)] after administration of
the same molar dose under the same conditions, are Prandial

Fasted

Fasted

Fasted

Fasted

Fasted

Fasted
similar to such a degree that their effects can be ex- State
pected to be essentially the same.
Therefore, for a multisource product to be bioe-
quivalent to an accepted comparator,17 it must be
Two-treatment, two-period,

Two-treatment, two-period,

Two-treatment, two-period,

Two-treatment, two-period,

Two-treatment, two-period,

Two-treatment, two-period,
two-sequence, cross-over

two-sequence, cross-over

two-sequence, cross-over

two-sequence, cross-over

two-sequence, cross-over

two-sequence, cross-over
demonstrated that the 90% confidence interval (CI)
around the geometric mean ratios (GMRs) for the
Study Design

Cmax , AUC0-t, and AUC0-∞ are within the 80–125%

BE limits of the comparator.4,18 These traditional BE
limits are based on the requirement that BAs of the
test and the reference products should not differ more
than 20% to ensure the same clinical efficacy.19 In cer-
ETB, ethambutol dihydrochloride; INH, isoniazid; PYR, pyrazinamide.
tain cases, if Cmax is of less importance for the clinical
efficacy and safety compared with AUC, the accep-
Participants

tance criteria for Cmax (not for AUC) can be widened

Number of

a Ethnic background is not described in the study report.

to, for example, 75%–133%.4 The range used must

18

24

24

24
24

24

be defined prospectively and should be justified, tak-

ing into account safety and efficacy considerations.4
Further, for “highly variable drugs,” that is, drugs
Pharmacokinetic Study Design

with within-subject variability of ≥30% for Cmax and/

or AUC, the wider acceptance limits of 75%–133% or
INH-ref-100 (3
ETB-ref-400

PYR-ref-500
Comparator

even 70%–142% might be appropriate.20

tablets)

The PK data needed for this study were obtained

from BE studies submitted to the WHO Prequalifica-
tion Programme by the manufacturers of the multi-
source products.3 All BE studies were run according
to standard conditions: randomized, two-treatment,
ETB-380-400A

ETB-91-400A

ETB-91-400B

PYR-91-500A
INH-91-300A

INH-91-300B

two-period, two-sequence crossover. Study details can

be found in Table 2. The GMRs of Cmax , AUC0-t, and
Table 2.

Product
Generic

AUC0-∞ were calculated from the individual data

of 18 (ETB-380–400A) and 24 subjects (all other
tested products), respectively. Subsequently, the 90%

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011
826 STRAUCH ET AL.

CIs of all tested multisource products were com-
puted according to the step-by-step approach from
Balthasar.21 Because all included BE studies were
analyzed retrospectively, ethical approval was not re-
quired for this study.
Comparison of In Vitro and In Vivo Data-Based
BE Decisions
The results of all in vitro ETB, INH, and PYR disso-
lution studies were compared with those of the cor-
responding in vivo PK studies. In this way, the cor-
respondence between the BE decisions based on the
biowaiver procedure and the in vivo PK data could be
evaluated.
RESULTS
Dissolution Tests
Three ETB, two INH, and one PYR multisource prod-
ucts as well as the recommended comparators were
subjected to the biowaiver dissolution tests. Results
are shown in Figures 1–3.
ETB-91-400B released more than 85% of the la-
beled amount of the API in 15 min and would there-
fore meet the WHO7 criteria of “very rapidly dis-
solving.” By contrast, the other two ETB multisource
products, ETB-380-400A and ETB-91-400A, as well
as the comparator ETB-ref-400 failed to comply with
WHO7 requirements for either very rapid or rapid
dissolution.
For INH and PYR, all tested products (INH-91-
300A, INH-91-300B, PYR-91-500A, and the compara-
tors INH-ref-100 and PYR-ref-500) demonstrated
very rapid dissolution characteristics according to the
WHO7 Guideline.
PK Data
The upper and lower limits of the 90% CIs for Cmax ,
AUC0-t, and AUC0-∞ for all tested multisource prod-
ucts are demonstrated in Table 3.
For ETB-91-400A, ETB-91-400B, INH-91-300B,
and PYR-91-500A, all calculated 90% CIs fell within
the 80–125% BE range of the corresponding compara-
tor. By contrast, the 90% CIs of ETB-380-400A for
Cmax (0.9667–1.2965) and for AUC0-∞ (0.9827–1.2564)
failed to fall within the 80–125% BE range of the com-
parator. Furthermore, for INH-91-300A, the 90% CI
results for Cmax (1.0474–1.3293) exceed 125%, but for
AUC0-t and AUC0-∞ , they lie within the BE limits of
80%–125%. However, for INH products, the use of ex-
panded BE limits of 75%–133% has been accepted by
the WHO Prequalification Programme.3
DISCUSSION
Evaluation of the Dissolution Data
The recommended comparators suggested by
the WHO17 for ETB multisource products are
Myambutol
R
400 ( Riemser Arzneimittel AG, Greif-
swald - Insel Riems, Germany; lot # 810850, exp. date
11/2013) and Myambutol
R
400 (Teofarma S.r.l., Valle
Salimbene (PV), Italy; lot # 0504B, exp. date 10/2010).
Because neither Myambutol
R
400 (Teofarma) nor

R
Myambutol 400 (Riemser) was able to meet the
dissolution criterion for “very rapidly dissolving,”
biowaiver-based approvals cannot be currently ap-
plied to new multisource products containing ETB,
so BE decisions for ETB products must currently be
made based on PK data. Of the multisource products,
ETB-91-400B proved to be “very rapidly dissolving,”
whereas ETB-380-400A and ETB-91-400A failed to
meet this criterion (Fig. 1). Contingent on identi-
fication of a “very rapidly dissolving” comparator,
ETB-91-400B could be approved with the biowaiver
approach, whereas BE of the other two products
would still have to be accessed in a PK study.
With respect to the corresponding biowaiver
monographs,14,15 all tested INH and PYR multisource
products would be eligible candidates for an approval
based on the biowaiver procedure. In this study, all
INH and PYR test products (INH-91-300A, INH-91-
300B, PYR-91-500A, and the comparators INH-ref-
100 and PYR-ref-500) released more than 85% of the
labeled amount of the API within 15 min in all three
standard buffers at pH 1.2, 4.5, and 6.8 (Figs. 2 and
3). Therefore, the biowaiver procedure would lead to

Table 3. 90% Confidence Intervals of All Multisource Products Tested

90% CIs

Test Product Comparator Cmax AUC0-t AUC0-∞ Bioequivalence

ETB-380-400A ETB-ref-400 0.9667–1.2965 0.9869–1.2012 0.9827–1.2564 No
ETB-91–400A 0.9172–1.2294 0.9181–1.0513 0.9295–1.0515 Yes
ETB-91-400B 0.8593–1.0819 0.9965–1.1791 0.9827–1.1403 Yes
INH-91-300A INH-ref-100 (3 tablets) 1.0474–1.3293a 1.0419–1.1299 1.0416–1.1272 Yes
INH-91-300B 0.9543–1.1306 0.9718–1.0637 0.9841–1.0604 Yes
PYR-91-500A PYR-ref-500 0.9554–1.0698 0.9596–1.1055 0.9349–1.1491 Yes

ETB, ethambutol dihydrochloride; INH, isoniazid; PYR, pyrazinamide.

a
Since Cmax values are more variable than, for example, the AUC-ratios the WHO Prequalification Programme accepts the use of
the expanded BE limits4 of 0.75–1.33 for INH-91–300A.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011 DOI 10.1002/jps
 BIOWAIVER FOR ANTITUBERCULOTICS 827

Figure 1. Comparison of the mean plasma ETB concentration–time profiles (left column)
following a single oral dose and the corresponding dissolution profiles (right column). Top: ETB-
380–400A vs. ETB-ref-400 (comparator); Middle: 2 tablets ETB-91–400A vs. 2 tablets ETB-ref-
400 (comparator); and Bottom: ETB-91–400B versus ETB-ref-400 (comparator). The PK data
represent mean ± SEM of plasma ETB concentration. The dissolution data represent mean
± SD of the percentage of ETB dissolved at each sampling time point: •, ◦, simulated gastric
fluid without pepsin (SGFsp );
, , acetate buffer; , , simulated intestinal fluid without
pancreatin (SIFsp ). Filled symbols indicate test product profiles, open symbols with dotted lines
indicate comparator profiles. The horizontal and vertical dotted lines in the dissolution profiles
intersect 85% dissolved and 15 min, representing the WHO biowaiver criterion of “very rapidly
dissolving.”

a positive BE decision for all tested INH and PYR
multisource products.
Evaluation of the PK Data
The PK parameters of ETB-380-400A, ETB-91-400A,
and ETB-91-400B were determined in three separate
BE studies,1 all of which compared the multisource
products with ETB-ref-400 (Fig. 1). The study de-
signs (Table 2) in all three cases were nearly iden-
tical, with differences limited to the following: (a) in
1 In-house data from the WHO Prequalification Programme.

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828 STRAUCH ET AL.

Figure 2. Comparison of the mean plasma INH concentration-time profiles (left column)
following a single oral dose and the corresponding dissolution profiles (right column). Top: INH-
91–300A versus 3 tablets INH-ref-100 (comparator); and Bottom: INH-91–300B vs. 3 tablets
INH-ref-100 (comparator). The PK data represent mean ± SEM of plasma INH concentration.
The dissolution data represent mean ± SD of the percentage of INH dissolved at each sam-
pling time point: •, ◦, simulated gastric fluid without pepsin (SGFsp );
, , acetate buffer; ,
, simulated intestinal fluid without pancreatin (SIFsp ). Filled symbols indicate test product
profiles, open symbols indicate comparator profiles. The horizontal and vertical dotted lines in
the dissolution profiles intersect 85% dissolved and 15 min, representing the WHO biowaiver
criterion of “very rapidly dissolving.”

Figure 3. Comparison of the mean plasma PYR concentration-time profiles (left column)
following a single oral dose and the corresponding dissolution profiles (right column): PYR-
91–500A vs. PYR-ref-500 (comparator). The PK data represent mean ± SEM of plasma PYR
concentration. The dissolution data represent mean ± SD of the percentage of PYR dissolved at
each sampling time point: •, ◦, Simulated Gastric Fluid without pepsin (SGFsp );
, , acetate
buffer; , , simulated intestinal fluid without pancreatin (SIFsp ). Filled symbols indicate test
product profiles, open symbols indicate comparator profiles. The horizontal and vertical dotted
lines in the dissolution profiles intersect 85% dissolved and 15 min, representing the WHO
biowaiver criterion of “very rapidly dissolving.”

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011 DOI 10.1002/jps

the ETB-380-400A study, 18 healthy subjects were
enrolled, as opposed to 24 subjects in both of the
other cases, (b) the study of ETB-380-400A included
both female and male healthy volunteers, whereas
the other two studies only involved male healthy sub-
jects, and (c) two tablets (800 mg) were administered
as a single dose in the study of ETB-91-400A com-
pared with one tablet (400 mg) used in the studies
investigating ETB-380-400A and ETB-91-400B, re-
spectively. For ETB-91-400A and ETB-91-400B, all
calculated 90% CIs (Table 3) fell within the 80–125%
BE range of the comparator. Based on this in vivo
evaluation, both products are bioequivalent to ETB-
ref-400. By contrast, the 90% CIs of ETB-380-400A for
Cmax (0.9667–1.2965) and for AUC0-∞ (0.9827–1.2564)
failed to fall within the 80%–125% BE range of the
comparator. Thus, ETB-380-400A cannot be consid-
ered bioequivalent with ETB-ref-400.
The in vivo PK studies1 of INH-91-300A and INH-
91-300B were conducted with nearly identical study
designs (Table 2). The calculated upper and lower 90%
CI limits (Table 3) of INH-91-300B fall completely
within the usual BE range of 80%–125%. For INH-91-
300A, the 90% CIs for AUC0-t and AUC0-∞ lie within
the limits of 80%–125%, but for Cmax (1.0474–1.3293)
results slightly exceed 125%. However, for INH prod-
ucts, the WHO Prequalification Programme3 has ac-
cepted the use of expanded BE limits of 75–133%. On
this basis, both multisource products tested fulfill the
PK requirements for BE to INH-ref-100 (3 tablets).
Because all 90% CIs (Table 3) of the PYR multi-
source products lie inside the 80%–125% BE range of
PYR-ref-500, PYR-91-500A is judged to be bioequiva-
lent with the comparator.
Comparison of In Vitro and In Vivo Data
ETB-91-400A and ETB-91-400B both proved to be
bioequivalent to ETB-ref-400 in the corresponding
PK studies, even though dissolution was considerably
faster than that of the comparator ETB-ref-400. The
product ETB-380-400A was unable to meet either dis-
solution or PK criterion for BE. As the excipient com-
position for ETB-380-400A was not provided by the
manufacturer (Table 1), it is not possible to draw any
conclusions about possible excipient effects on either
in vitro or in vivo performance.
The results suggest that there is a low risk of a
false-positive2 BE decision based on the biowaiver
procedure for ETB products. On the contrary, meet-
ing the biowaiver dissolution criteria appears to be
a more difficult task than demonstrating BE in PK
studies.
2 A false-positive BE decision means that products that are in fact
not bioequivalent in vivo are declared to be bioequivalent by the
biowaiver dissolution procedure.
DOI 10.1002/jps
BIOWAIVER FOR ANTITUBERCULOTICS 829
For all tested INH and PYR multisource prod-
ucts, the “positive” biowaiver decisions are consis-
tent with the results of the corresponding in vivo PK
studies (Figs. 2 and 3). Hence, for both these APIs
the biowaiver procedure can serve as a surrogate for
in vivo PK results.
CONCLUSION
ETB, INH, and PYR are all conservatively classified
as BCS Class III, and therefore products with these
APIs must be “very rapidly dissolving” to qualify for
a biowaiver-based approval. All INH and PYR drug
products tested proved to be “very rapidly dissolv-
ing,” and therefore eligible for an approval based on
the biowaiver procedure. The in vivo PK data of the
INH and PYR multisource products showed that each
tested product is bioequivalent to its comparator, indi-
cating that the biowaiver procedure may be a suitable
alternative to in vivo BE studies for these APIs.
Because the comparator ETB-ref-400 was not able
to meet the “very rapidly dissolving” criterion, appli-
cation of the biowaiver procedure to ETB products is
not currently possible. As a result, in vivo PK studies
would be required to test BE of ETB products with a
comparator. It is recommended that a more suitable
comparator, that is, one with “very rapid dissolution”
be identified, if feasible.
In no case was there a “false-positive” BE decision
reached when using the biowaiver procedure, i.e. the
biowaiver procedure did predict BE for a product that
was subsequently shown to be not bioequivalent in
the corresponding PK study. These results suggest
that the biowaiver procedure can be applied to ap-
proval of single-API preparations containing either
ETB, INH, or PYR. On the one hand, the biowaiver
procedure simplifies and reduces the costs of demon-
strating BE, and on the other hand, it appears not
to place the patient at any additional risk. According
to Polli,22 the in vitro dissolution testing should in
fact be viewed as the preferred method to judge BE:
it reduces costs, assesses product performance more
directly, and offers benefits in terms of ethical con-
siderations. The findings of this work thus open the
way to a simplified method of ensuring BE of anti-TB
products.
ACKNOWLEDGMENTS
One of our co-authors, Dr. Ekarat Jantratid, passed
away after submission of the manuscript. The authors
wish to thank Bill and Melinda Gates Foundation
(BMGF) and UNITAID for their financial support to
WHO Prequalification Programme, which made this
study possible.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011
830 STRAUCH ET AL.
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