11

Bioavailability and Bi oequiva lence

as a vehic le fOl" the def:v ery a!

The ultimate goal of mos t oral dosa ge form s is to serve
s) of actio n. Bioa-vai labil rty IS a
drugs to the bloo d strea m for dist ribut ion to the site(
reach es the sys "~ c:m:t.llc0Xl
measurement of the rate and exten t (amo unt) of drug that
bioph arma ceuti cs g,ves subs lar.Li 3'
from a drug prod uct or a dosa ge form . T he study of
form ulatio n of a drug can rnar',t.erl!y
evidence that the meth od of manu factu re and th e final
ent types of bioa va3 a~ s!\xf 2s_
affect the bioav ailab ility of the drug . Ther e are.t wo differ
The first type invol ves an asse ssme nt of the bioav
ailab ility of a new drug forrntna:.xm
route s of admi nistra tion of the new
That is, phar maco kinet ic para mete rs follow ing different
um dosa ge regim en . Fina! ly the
drug are obtai ned and are utiliz ed in deve loping an optim
ation and its ~
new drug is form ulate d suita bly for an inten ded route of adm inistr
invol ves a com paris on of a test
is assessed . The seco nd type of bioav ailab ility study
that is prove d to have thera peut ic
formulation with that of a refer ence stand ard dosa ge form
as bioe quiv alen ce stud ies Laws
efficacy and safet y. T his type of stud ies are know n
Ther efore . laws allow martce1ing
rnandate that new drug prod ucts be safe and effec tive.
.. simil ar effica cy and safe ty to that
of the new drug prod uct if it shows bioeq uivalence . i.e
d to be bioeQ uival ent if the drug
of the ihno vato r prod uct . Two prod ucts are cons idere
un~ik ely to pr~~ ce ~rffer ~ m
~;~t r~tio n-tim e profi les are so simil ar that they are
ern 1s switchabthty, 1.e • a patie nt
c thera peut ic or ad vers e effec ts. T he m ajo r conc
bioe quivalent.
an exch ange one prod uct for the othe r if they are

387
388 Biopharmac eutics and Pharmacoki .
_ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ . : _ - - - - - - - - - -~ netics
11 .1 Definitions and Purpose of Bioavaialability Studies
Equivalence: Equivalence is a general term used to com pare products taking a spec·t·
c ~a ract eristic or fun ction or a defin ed set of con d ·t· . t . 1Ic
I ion s in o con sideration . In
b1opharmaceutics drug products are compared.
Chemical Name: Name used by the organic chemist to indicate the chemical structure
of the drug (e .g. 2-(acetyloxy)-benzoic acid) .
~ eneric Name : The established , non-proprietary or common name of the active drug
in a drug product (e.g. aspirin) .

Drug Product: The finished dosage form (e .g. a tablet or capsule) that contains the active
drug ingredient generally but not necessarily in association with inactive ingredients.
Brand Name: Trade name of a drug . The manufacturer or distributor of a drug product
assigns a name to distinguish his product from competitors' products (e .g. dispirin , aspro).
Chemical Equivalence: This term implies th at two or more drug products contain the
same labeled chemical substance as an active ingred ient in the same amou nt.
Pharmaceutical Equivalents: Drug prod ucts that contain the same active drug ingredient
(same salt, ester or chemical form) and are identical in strength, dosage form and the
route of ad ministration .
Therapeutic Equivalents: Therapeutic equivalents are drug prod ucts that contain the
same therapeutically active drug that should give the same therapeutic effect and have
an equal potential for adverse effects under conditions set forth in the labels of these
drug products. Therapeutic drug products may differ in certain characteristics , such as
color, scoring, flavor, packaging, preservatives , and expiration date. Therapeutic equivalent
drug products must be 1. safe and effective, 2. pharmaceuti cal equ ivalent, 3. bioequivalent,
4. adequately labeled , and 5. manufactured in com pliance with current good manufacturing
practices .
Bioequfvalence: Bioequivalence is defined in 21 CFR 320.1 as" the absence of a significant
difference in the rate and extent to wh ich the active ingredient or active mo iety is
pharmaceutical equivalents or pharmaceutical alternatives become available at the site of
drug action wh en administered at th e same molar dose under sim ilar conditions in an
appropriately designed study1 .

Absolute and Relative Bioavailability

Absolute Bioavailability of a drug in a drug product may be measured by comparing
the respect ive bioavaTTaomties after an oral and intravenous bo lus injection . Th is
measurement is valid as long as the volume of distribution (Vd) and elimination rate constant
(K) are independent of th e route of adm inistration .
The absolute bioavailability of a given drug using blood/plasma data may be calcu la~ed
by comparing the total areas under the plasma concentration -time curves obtained following
 I
q_
. ,.bili'I and 810&..:.. uw _:___ _ _ _ _ __ _ __ _ _ _ _ _ _ _ _ _ _3_8_9
_ _a_le_n_ce
, •1& ;,;
- ~f -

~ , ,,3ttat1on of equ, 1alent doses of the drug via an absorp1i on site a nd via the
-1; i:,.c,~ ::; route to the same subj ects on different occasion s .
,~, ✓

, ~ , ,:21 c,:a~ma coneentr a'bn-time curves obtained by administ ering equiva le nt doses
.,': 58 ms drug b-J thf: intraveno us route (bolus injection) and the extravas cular route
;' , ,, ~ ; €: sho-11n m Fig 11 1.
:"~
. _ [I- UCL,., / (Dose)e.,
.. . .. (11.1)
Ab3-0l1Jte b 102vallab1hty = (AUCl I (Dose),
I 1

-:,J;r uiative amounts of the uncha nged d rug or major metabolite excre ted in urine a re
• • 0 J~€:d to determin e the: absolute bioavaila bility.
1 ,

P<~J.,., I (Dose )e.,

Absolute b ioavailab ilw; = .. ... ( 11 .2 )
(X ~ J I I (Dose). 1

1/h~re, x; is the total amount of the uncha nged d rug o r m aj o r m etabolite e x c r e te d

· tr e urine in infinite: time.
The fraction of the dose absortJed (F ) is also used to denote abs o lute bioava ilability
-,1 a drug product. F value for drugs given by IV bo lus inj ection is equal to unity,
s ince
~l the drug e nters the systemic circulatio n. F value of drug products m ay range fro m zero
1
11here no drug reaches the syst emic circulatio n) to un ity (whe re t he tota l d rug reaches
!fie systemic circulatio n)
Relat ive Bioavaila bitity is defined as the ratio of b ioavailab ilitie s of a d rug p rodu ct
~rid a recogniz ed sta ndard . In general, a recog nized standa rd is e ithe r a competit o r 's
marketed d ru g product or a s t a nda rd dos age form a c c epta b l e Food and Drug by
;<Jministration or through administration via a d ifferer,t route. When the rec ognized sta ndard
·, a competito r's p roduct then it is termed a s "bioequiv alence". T h e b ioavaila bili1y of a
'';:t formulatio n is compared to the ava ilability of the drug in a selected dosage form us ing
:, r.ross-ove r study.

[AUC ]test I (Dose)test

Relative bioavaila bility = [AUCJstandard / (Do se)standard ... .. (11 .3)

Percent relative bioavailab ility is obta ined by multip lying this fraction by 100. Percent
~:lative b ioavailab ility c an be calculate d from the urinary d rug excretion d ata, provided
11t;h
anged drug is excreted in the urine.
0
[X u 11esl / (Dose J1es1
Percent rela tive bioavaila bility = ... .. ( 11.4)
[X ~ 1standard I ( Dose >standard
1

''l I
Nhere , [Xu' J, is the total amo unt of the unchange d drug or a major metabolite excreted
·he Urine in infinite time.
r
390 Blopharma ceutics and Pharmacok · .
- - - - - - - -:...:: 1net,c8
l he rnojor pnromotors roprosontlng tho rate and i:ixtent of drug absorption are ~
Discu
in dotoil l□tor In this choptor Fig . 11 .1). ssed
Cmn, : Tho ponk plosme drug co ncen trn tlon , Cmox Is used as a measurement for the
drug bloovolloblllty. C x has unils of mass/volume
1110
2 AU C is th o ores under the plasma concentrati on -time curve and is the measuremen t
of oxton t of bioavailabllity of th e drug
3 Tma~: IS th e time of peak plasma conce ntration (CmaJ
Cmax
AU C
Tmax
------
Fig. 11.1 Bioavailability Study Protocol.
11.2 Bioavailability Study ,
11.2.1 Bioavailability Studies fer New Drugs
In vivo bio ava ilabi lity studies are performed to r new drugs to establish essential
pharmacokinetic parameters including the rate of absorption, extent of absorption , rates
of excre tior, and metabolism , and eli mination ha lf--life after a single and multiple-dos e
administration . These essential pharmacokinetic parameters are useful in establ ishing
dosage regimens.
Bioavailabil ity studies are also conducted to determ ine th e influence of exipients,
manufacturing procedures , packaging materials and patient related factors on the biological
performance of a new drug formulation .
11 .2.2 Bioavailability Studies for Approved Drugs
Bioavailability studies for approved drugs are performed to develop a new dosage form
or to improve on existing dosage form . In approving a drug product for marketing , the
FDA must ensure that the drug product is sa i'e and effective for its labeled inqications
of use. In addition , the drug product must meet all applicable standa rds of identity, stren9 th •
quality and purity. Therefore, a drug product is first subjected to all applicable official
r alence
._-, 3 3 r,!!!,_tY and Bio equ i\
391
s b. .
~tof)f tes ts . Fin ally. the FDA req uire re
s to e ioav a,ta bility/phamaco kinetic studies and whe
:-,..· ~;a r)' bio equ iva len ce stu die nsu re the saf ety and· efficacy of the dru g pro duc t.
,•f~ - .
s. tes t dru g pro du t.
~ ~- \ ,3 e,c e stu die c 1sc om p d ·
-~ pro duc t) Tes t . are with a refe ren ce stan dar d (ge ner ally
· ".\ 3 ppr ove d dru g pro duc t 1s cal led b.1oequ1v · . .
• · ale nt 1f rt pro duc es a
• 1
iva len t to tha t of th f
. ~ilability equ e re ere nce sta nda rd .
~;.,\ ..
. .
,. certain situ atio ns 505 (b)(2) is also app1ied by innovato r.
res s! .
~?Cbon 505 (b)( 2): Thi s pro visi on exp NOA , on
elo ped by the app lica nt. y per mit s FDA to rely, for app rov al an
, ..: --ot dev
...
er r th
• oermits app rov al of app llca tion s oth ose for duplicate produc ts and per mit
s
. , , ce for suc h app rov als rt nan
I era ture or on an Age ncy find
ing of saf ety and /or
:- ~ •\ ene ss for an app rov ed odnrug product.
:~~- •
tion s include
Som e of the exa mp les of 505 (b)(2) app lica
ime n ,
h, route of administration , dosing reg
• Cha n~a in dos age form , strengt
t in a combination pro duc t,
• Substit utio n of an act ive ing red ien
ry stu die s
tho se considered limited con firm ato
• Ch ang e_in the form ulation beyond
app rop nat e to a 505 (j) app lication ,
.
app rove d indication for a listed dru g
• An app lica tio1 , for a not previou sly
• Rx/ OT C swi tch .

11.3 Bioavailability Stu dy Protocol

req ui res the
ess me nt of b ioa vai lab ility of sev era l dru g pro duc ts mo st ofte n
ne ass e . T he
ent of dru g and /or me tab olit e lev els in eith er the blo od or the urin
Tea sur em adm inis trat io n,
form depends on the dos e and route of
~.oavaila bilit y of a dru g from a dos age lity stu dy is to
of adm inis trat ion , sub jec ts and dos age form . The aim of bioa vailabi
:.me . The refo re,
the dos age form infl uen ce on the biological performance of the d rug
.:nd out nce s
of sufficient sensitivity to det ect diff ere
~,e bioa vail abi lity stu dy pro toc ol use d sho uld be
tha t are attribu table only to dos age
form var iab ility
:\ the rate and e xte nt ot abs orp tion ele me nts of the
sho uld voi d v·~r :.Jbilitie s due to oth er fac tors . Tab le 11 .1 lists the
and 2
':{)a\';;,i ::b1lity stu dv µrc toc ol.
oun tere d
of the drug, analytical difficulties are enc
Sr, •;et ,me s, due '.J inh ere nt properties s, so oth er
1
pre clu de the me asu rem ent of dru g or me tabolite levels in body fluid
'lat will e
e
use d to ass ess bioa vail abil ity. The drug may be labe~ed with a rad ioa ctiv
s techniq ues are per form ed
may be measured , or studies ma y be
,. lag, a pha rma col ogi cal or clinical respon se
al on anim als
 392 Biop harm aceu tics and Pharmaco
-~~~- - - - - - - - - - - - - - - - - - - - - - - -- - -...:.:kinetics
.
Table 11.1 ----:::
Bioavailab ility Stud y Protocol.
~
A Study objective
8 . Study design
1. Experimen tal desig n
2. W ash out period
3. Drug products:
(a) Test prod uct(s) and (b) Recog nized sta ndard
4. Route of administra tion
5. Dosa ge regimen
6. Frequency and duration of samp ling
7. Randomiza tion of drug admin istration
8. Single-versus multi ple-d ose study desig n
9. Subj ects
(a) Healt hy subjects versus patients
(b) Subje ct selection
(i) medi cal history,
(ii) Phys ical exam ination ,
(iii) Labo ratory tests
(c) Study conditions
10. Analysis of biolog ical fl uids
C. Meth ods of Asse ssm ent of Bioav ailabl ility
1. Plasm a data
2. Urine data
3. Acute pharmacolo gical effect
.4. Clinical respo nse
D. Analysis and Prese ntation of Data
1. Statistical treatment of data-Analysis of varia
nce (A NOVA )
2. Format of data

11.3.1 Study Obj ective

The objective of the bioavailab ility study decid es the
study protocol. A study..design meant
for estimating essential pharmaco kinetic paramete rs
diffe rs significantly from 3 bioequivalence
study mea nt for com paring the test fo rmulation
with refer ence to a standard .
11.3.2 Study Design
Various factors have to be cons idere d in cond uctin
g a bioavailability study since the rate
and extent of absorption of a drug into the syste mic
circulation , its distributio n and elimination
are influenced by a variety of facto rs . Subj ect facto
rs such as age, sex, disease state,
. gene I h I
food ha bits, · ·
ra ea th cond 1t1on, body weig ht of subje cts, expe · n· time
rimental desig
of adm inistration , time of sampling, analytical meth
od used and comp artment model used
· · .
in estim ating Pharmaco k.met·1c parameters/bioavailability cont
ribute to the observ ed blood
 ,oavai/abiHty and Bioequivalence 3! 3

·t ·1s necessary to consider all these impor:an: facwni

. Therefore • 1
~ n time
;eentra . profile
~oli tudY design .
"a s
tion of study design is based on
eelee
V • Number of formulations to be compared
• characteristics of the drug and its dispos ition
, study object ives
• Availab ility of subjects
, Inter- nad intra-subject variabilities
, Duration of the study o rthe number of periods allowed
• Cost of add ing a subject relative to that of adding one period
• Dropout rates
ubject
For examp le: If the intra-subject variability is the same as or larger tna"' the in:er-s
variability, the inference on the differe nce in average bioava ilabd'ty \',~
be t.ie scr..:e
regardless of which design is used .

Actually, a crossover design in this situation is a poor choice . becaJS e b'od.. --;
resuft'S u-:
.r.ter.al or.
the loss of some degrees of freedom and will actually lead to a wider cor..~cence
the difference between formulations.
In the following sections various factors are discussed keeping t.ia b.oeQ L"'\~~ce s ~
1

also in mind. However, they are valid for simple bioavailabihtv shJd,es a so.

P,a ~ I Desig n
s or.d to tl\'OtO
The aim of experimental designs is to minimize the e, pf•rinn""l,) \'3r~
1 \"OhJntecrs
a biaf 1~ a parallel design, two formulations are administered to t\'t"U gro"';,s of
The nta:ot
To avoid a bias, formul ations may be administered rondon,~, to the votunt~rs.
ted lt has
disadvantage of this design is that the inter-subject variation is not ~'\fl9 correc
QTaetet than
been proved beyond doubt that most of the times mtec•su bj~t vJn3ti\ )tl is
th e va riation between any formul ations. Therefore , n cross-oVEW" des~n IS protcrreo rn
n
bioavailability/bioeq uivalence tria ls to avoid influence of o mter•subfect ,·cnatlO

Parallel design may be considered as on nlternohve to a CfOSSO\Cr d~n tf

1th ,ntrasub;«"t \'arim:, 1 ty
• The intersubject variabi lity is rolatively small cornpored \'
\:.' ,..tmpi ~
• The drug is potentially toxic or has a very long ehnlinJtton hatt hro Fo,
tamoxifen , triptorelin , torem1fene, goser1l111 acetate . nsv1rapme
r 394
- - -- - - -- - - - - - - - - - Biopharmaceutics and Pharma
----- - Cok,n~~
• The population of interest consisits of very ill - tl"S
patients, and ---- - .:._
• The cost fo r increasing the number of su bjec
ts is much less than that of ,
additional treatment period .
ado,n']
Othe r examples include bioequivalence study
of topical antifungals like keto
clotrimazole . /
Cro ss-o ver Des ign , ;i~c
Usually, a substan~ ter- sub ject variability exist
conazc,1e

s in the drug levels achieved from

given dose of medicatio n in a pane l of subjects
. The cross-over design minimizes the
of inter-subject va riabi lity in the study by using
e:rri
each subject as his or her own con t:
Generall y, two types of cross-over designs are
used in bioavailability trails . They are, _
Latin Squ are cross-ov er design and 2. Balanced
Incomplete Block Design (BIBO ). 1
Latin Squ are Cross -Ov er Des ign
A standard approach for cond ucting a comparative
bioavailabilir.i study is to use a randomized ,
balanced , cross-over desi gn called a Latin squa
re or complete cross-over design. In this
design, 1. each subject receives just once each
formulation , and 2. each formulation is
administ ered just once in each study period . The
basic elements of this desig n are shown
in Table 11.2 , whe re the first desi gn is a two-way
cross-over desig n. The three- and four-
way cros s-over designs shown in the table
11 .2 repr ese' lt one of the several poss ible
combina tions .
In a two-way cross-over stud y, 12 su bjects are
used to study the bioequiva lencie s of
two form ulations, treat ment A and treatme nt
8 . During the first study period, subjects 1
to 6 receive treatmen t A, wh ile subj ects 7 to 12
rece ive treatment B. A second study period
is initiated after the was hout perio d, during whic
h a complete elimination of the drug and
its major metabolit es takes plac e. In the second
stud y period, subjects 1 to 6 now receive
treatmen t B and su bjects 7 to 12 receive trea
tme nt A. The refore , each subject acts as
his own cont rol. This design has seve ral adva
ntages, liste d under,
1. It minimizes the effect of inter-subject vmia
t'i!ity in the study by using each subject
as his or her own control.
2 . It minimizes the ~ rry-over ~ wh ich
cou ld occu r when a given dosage form
influ ences the bioa vaiTaffilityof a subseque ntly
adm inistered dosage form since each
form ulati on is preceded and succeed ed by othe
r fo rmulation s or dosage forms .
3. It minimizes the time effect on bioavailabilty
since each dosage form is adm ·inis
· tered
in each stu dy period .
4 . It requ ires less number of subjects to get
meaning ful results.
 ,,nrl /j/()P( 111lv11li> l1PO l9S
,tfl/11/J/Y
,,,, Tnhlo 11 .2 I rl llil ~.q11nrn I )fltlllJlll'l

rwo-WRY crotu1ovor I
0 1011p Nn U11/1/1wl11 Ill Clft1II/> I1,111/11u,11/ lo, I 111fl,,d N•1
I II
I
1 1,'1,:1,'1 ,ll,'1 /\ II
? /.(\,0, 10,11 ,1:.1 II /\

Throo-Wny CroHOVOr
01011p No
S11/1/oolt1 In n,, 1111 i I 1,11Jlf1 ,m ii fr,, 11,11/c,(J M,
II 111

1 1,i,3.4 ,fi,0 A C II

I
~ / ,0,0,10, 'I 1,1~ II (\ C
13,14 ,'l ll,10,1 / , 10 C I\ A
:l
Four-Woy Croooovor
G,ou,, No. 811/)/00ID In GrOU/1 f 1011 /rnoi-,1 for f.Jm//Jfl No
I II Ill IV

1. 1.~.3,4,,0,0 A lJ C D
2 7,0,0,10,1 1,12 u 0 A C
3. 13,1'1-,16,16,17,10 C A D u
18,20,21,22,23,2'1 0 C [3 A

The major disadvantages of the Latin-square claolgn ( rabis 11 .2) aro tho followin
g
1. It requires longer time to comple te the study since o wt1shout ponod c>usts
between
two study periods. The higher the blologlcal half-llfo of druo, tho lonoor will be tho
time required for completing the study.
2. The time to complete the trial depends on the numbor -formulo tions ovoluJtcd In \ht
study. It takes a longer time to complete tho study as tho numbo r o f formulJ t1ons
increases.
3· Increased number of study periods leads to high subjo1.: t dropou ts c1nd tho study
becomes difficult.
4
· ~edical ethics does not allow too many trials on o aubjoct continuously ror
o toneof
time.
These disadvantages can be overcome by u0e of n bolonced lncompluto hloci-. de~ 1gn
8ilan
Ab Ced Incom plete Block Doelgn
¥1~hal~nced ~ncomplete Block De-sign (BIBO) ollmln~!es mony of tho dlfficu)Uo ,,.ountorcd
0
Latin square design (l able 11 3 & 11 .4 ). f he sollont footur<h of tll, · ,e$1Qn .uo
~ Each subject receives not morn than two forrnulatlons
Each formulation Is administered the sume number of times
3·. Each
Pair of formulations occurs tow,thor In tllo snmo nurnbo r of sub1oct"
, ◄

396 Biopharmaceuti cs and Pharmacok· .

- - - - - - -..:. : ___ inet,cs
Table 11 .3 st,ows BI BO for four formulations A , 8 , C and D. In this design , as d: ; - : :
. hf I t· . ussed
above , each subject recei~es two formul ations, .eac orm.u a ,on 1s ad ~in,stered
· ·
six tirne
and each pair of formulation s occurs togeth er rn two subJects (the pairs are AB, AC , AD8
BC , BO and CD) . ,
Table 11.3 Balanced Incomplete Block Design (BI BO) for Four Formulations.

- Subject -- Treatment
-
for Period No.
I II
-- -
1 A B
--
2 B A
3 A C
4 C A
5 A D
6 D A
7 B C
8 C B
9 B D
10 D B
11 G; D
12 D C

.I
Table 11 .4 Randomization Scheme Used in a Bioavailability Study.

Group No. Subjects in Group Treatment for Period No.

I II Ill
1. 1,2,3,4,5,6 A C B
2. 7,8 ,9,10,11,1 2 B A C
3. 13,14,15,16,17,18 C B A

Repl.icated Cross over design

Replicated crossover designs are critical when an individual BE approach is used to allow
estimation of within-subject variances for the T and R measures and the subject-by-
fo rmulation interaction variance component
Following is the four-period, two-sequence, two-formu lation design recommended for
replicated BE studies

Period
1 2 3 4
Sequence 1
--
2
T
R
R
y
T R

--- R T

C
~ a nd B ioequivalence 3 97
~ ig n , the s ame lots of the T a nd R formula tions should be used fOf the reµficcrt
ed
eftfl~_.~ :on. Each penod should be separa ted t,y an adequa te washou t period.
,i t : , u

-" r repncate<I crossov er designs are possib le . For exampl e, a three-p eriod
design
CJ~ t>elOW, could be used .
.: sn
;--

P eriod
1 2 3
Sequenc e 1 T R T
2 R T R

/-. greate r number of s ubje cts w ould be e ocourag ed fo_r the three-p eri~ design
compar ed
·:; the recomm ended four-p e riod desig n t o achieve the same statistic al po,,ve
r to CXlndud e

other Design s
::or the h ig hly v a.-iaoJe-..Gru gs(HVD ) and hl._g_h!y variable d rug product s (HVDP )
having within
subject variabili ty of m o re tha n 30% , FDA h as recomm ended . " referenc e scaled
averag e
bioequiva lence approac h" where the b ioequiva lence limits are no1 determi ned by
the samp(e
size. Rather, they are scaled bas ed on the w ithin-sub ject variabili ty o f the ref erence
product ..
For drugs with an expe cted w ithin-subj ect variabili ty of 30% or greater. a B
E study with
three-per iod , refere n ce- replicate d, crossov er design with sequen ces o f TRR
. RTR. and
RRT is propose d . Specific ally, s u bjects receive a single dose of the test product
once and
reference product twice on separat e occasio ns with random assignm ent to the t
hree possi:bt e
sequence s of product administra tion . This partial replicate design a llows for the
estimat ion
of withinsu bject variabili ty for the referenc e product . Treatme nts should be
separat ed by a
washout period of adequa te duration such t h at the drug of interest can no lon ger
be detecte d
'" plasma. that would be accept able is 24. The three-pe riod desig n was selecte
d ovei- a
four-perio d design because of efficienc y. The o nly advanta ge of the fo ur period
design is
!hat it a llows the calc u lation of the variability of the test product . The test produ
18 ct variabil ity
not u sed in the propose d statistic al metho, V '
11 ,,
.3.3 Washo u t Period
111
a L atin Square cross-o ver study design each subject receives each formul
a tion and
: en in BIBO each subject receives two formula tion ~ a\differ e nt occas!o ns_. The
t ~ e int~rvaJ
eli ~ ee~ the two treatme nts is called "washou t pen od . Wash_o ut penod ,s requir
ed _for _tne
"lh~ 1nat1on of the adminis tered dose of a d rug so as t o avo id t~e cartyo v e~- A gu,dehn
e,
10
Ch ha_s come into use for a crossov er d esign for most drugs 1s that a penod o_f ~t le~st
Of half- hves should be a llowed b e tween t reatmen t s . T h is should ensure an ehmmat
99 ton
firs -9 % of t he adminis te red dose and a maximu m carryov e r of le ss th a n 0
. 1 % from the
t tre atment . Washo ut period is a func tio n of the half-life and the d ose of the d r ug
 398 Biopl10r111 oce ut ics and Pharmacok·
riot1c1
1
administer ed . The numb er of washout period s in o s tudy dopa
nds upon tho type or er -.....
desig n used and the numb er of formulations to bo ovolu oted
. In the case of dlgito xl~ssover
has a half-l ife of 6 to 9 days. the total stu dy perio d excoe
. . ds one year If four forrn~I W~lch
have to be evalu ated using .
the Latin Sq uare desig n. 8 eca use a vary large numbat10118
drugs have been found to have a half-l ife betwe e n ·1 and
10 hours, a washout Per. of
of 1 vree k w as usua lly found suita ble in most of the repor
ted studie s . eriod
It shoul d be noted that the meta bolite s of the drug shoul d
also be elimin ated irorn the
body befor e the comm ence ment of next treatm ent. U nfortun ately,
the exact metabolic sche
of all drugs is not know n . W hethe r all the meta bolite s of
the drug are eliminated or ~~
is unknovvn . However, as most of the meta bolite s are more
water-solub le than the parent
drug and have a short er resi denc e time in th e body th
an th e parent drug molecule, It
is as sumed that their elimination occur s well before the elimin
ation of th e paren t compounds.
11.3.4 Drug Prod ucts
Tes t P rodu cts : Te s t p rodu ct ( s ) may be new drug
form ul a t ions de velop ed by a
pharm aceut i cal techn ologis t or new dosa ge form s of an existi
ng drug. A test product may
be com pared to a refere nce stand ard recog n ized by the
Food and Drug Adm inistration
for getting approval for mark eting the drug produ ct. Test produ
cts are generally evaluated
for follow ing reaso ns .
1. To se lect best dosa ge form of a new drug or existi ng
dru g amon g diffe rent dosage
fo rm s (e.g . Ta blet , caps ule , emu lsion , an d susp ensio n).
2 . To se lect the best formu l atio n of a new d ru g o r ex ist ing
drug among different
formu lati ons th at have show n equal perfo rma nce in in vitro
tests.
3 . To compare biolog ical perfo rman ce of a test produ ct to that
of a recog nized standard
(i. e . bioeq uiva lence studi es).

Refe renc e Stan d a rd

A chem ical or gene ric prod uct has to be com pared
w ith some stand ard dosage form to
verify its in-vivo perfo rm ance . In gene ral , the Food a nd Drug
Admi nistra tion (FDA ) accepts
any innovator's drug produ ct as a refere nce stand ard . The innov
ator is the on e who origina lly
receiv ed appro val from the FDA to market the produ ct in the
country. Some times, several
manu factu rers may hold appro val for certa in drugs . T heref
o re , any one of the permitted
drug produ cts can be used as a refere nce sta nda rd .
In many of these instan.
ces, the FDA wou ld reque st that on ly one of these products
be used as a refere nce produ ct in order to obtai n a more
easily comp arable data.
.
G enera lly highe st dose is the refere nce listed drug . There are some excep f10 ns in case
where the safety of the volun teer is a conce rn .
.
For exam ple , atomo xeti ne HCI (60 mg ), glime piride (1 mg), . ) terazo cin
rispe ndone ( 1 mg '
(2 mg), Lamo trigine (2 x 25 mg) , olanz epine (5 mg ).
 ' ·i·ty and Bioequivalence 399
/abl I
,,;(JfJV~
~ -f Adm inis trat ion
O
es ora lly adm inis.tere d dosa r srudie s.
0 ute •
. ge orms are subjected fOf bco ava Uab ility
~ f the t,rn · nsd erm .a .
:er , dos age form s adm inis tere d by oth er routes such as buc caf . ,,.a
~ost rape ...',:
ular sho uld a lso be eva luat ed for their biological perform anc e The •,..a
How ;asc 2
rate and extent of abs orp tion of the dru
a of the se dos ag e form s dep end s on the
,~tr_
~til1tY se dos ag e form s.
the
d . . t d h variation rn thei-- p,erforma'"'ce
fof111
nera l, ora lly a minis ere dosage form s sho w a muc
In ge ·
r-su
b. t
Jee an
d .
intra -sub
·
Ject variations. Alf the dos age f0<ms adm ·n <:.te~ed
se of ,nte · vailabiHty ass ess men t
bec8 U extr ava scu Iar rou t e d O req uire . a b1oa
bY an
_3_5 Sin gle - Ve rsu s Mu ltip le- Dose
Study Design
11 -do se stud ies are bet :e ... for ihe
ther a sing le dos e s tud ies are bett er or mul tiple
a s,n gfa
b. I·1 b·I·t f prod uct? It is always drff1CU't to giv e
Whe essrnent of the Ioav a a 11Y o a drug
hly so as to u:id erst a"l( ! L'-le
ass wer. How eve r, .it is pos sible to disc uss the issu e thor oug
d .d . h ·~ t~e dos age
ans
epts invo lved in ec1 mg t e dos age regimen for a bioa vailabilrty stu dy
conc . . ·es a ·e u5.. _a y
qu1valence purp ose s. sing le-d ose stud
forms are to be ev alua ted only for b1oe ,~ es car . be
bioavaila bility of mos t tablets and ca;:
sufficient. Th is is bec aus e the relative ose eve 's
usually, this is pred ictiv e of mtr !i:>, 'e-d
determin ed on a sing le-d ose bas is, and , as
s me ant fo r a sing le dos e adm inis trati on for a the rap eut ic ben e""i'! sue
Dosage form .ag e
the reli ef of hea dac he nee d only sing le-d ose stud ies. How eve ·. cer .a --, cos
analgesics for --es
d to ach ieve spe cial rele ase prof iles of drugs may requ ire mu' ~:J'e- dos e st.Jd
forms des igne JSC J a"'
mpl e, tim e-re l eas e pro duc ts, ente ric-c oate d pre par atio ns . som e . ~rar
For exa :i e-e ose
. Eve n the dru gs that und ergo the first-pass met abolism do need a r J ~
injections c1e ,: a---::1 o~
y. Use of an imp rop er stud y des ign lead s to the coll ect io n o~ lns uff·
stud e o,.. :\'.. :ce-
dos age regi men s suc h as load.'lQ dos
inapprop riate data . Fur the r, if spe cial ess a1y
-dos e stud y design ma y a:SO be nec
a-da y dos ing are to be use d , a mul tiple
ts
11 .3.6 Ad m inis tra tion of Dr ug Pro duc .;:a !~ Af:e •
subjects sho uld be bas ed on ran dom
Administ ratio n of dru g prod ucts to the the s.:. oje cts
the adm inis trat ion of the dru g prod ucts , blood sam ples are with dra ,vn from
a sam ple from e ach sub ject and l'le
· t~ta J
poin ts. It take s som e time to take
~t fixed time 20 m....'lutes
diffe renc e betw een first sub ject and the last sub ject may ran ge frt>m 10 to
lime pt:n g sch edu le
and technicians in the stud y. tf the sam
?ePending upo n the num ber of subjects nce s can
seq uen tial man ner , stgn ific ant diff ere
not follo wed rigo rou sly in the sam e
15
ted sam nr .......
of the drug in the bod y and the sta
~onceivably exis t in the actu al duration · t ' d.ff Id ...-. •~
a sub sfa nt:a j
tirne g·Iven for eac h sub ject . This 10 to 20 mIn u es I eren ce \WO rep res ent
nts
dru g con cen trat ions obs erve d in the bloo d. If und er thes e con dtlt ons trea tme
change in the to the ftrs t
uen tial man ner (suc h as trea tme nt A
:re adm inis tere d to the sub jects in a seq
7 to 12, and trea tme nt C to volu nte
ers 13 to 18
voluntee rs, trea tme nt 8 to volunteers sam plm g wm
een the time of adm inis trat ion and
:~a~hown in tab le 11 .4 ), the erro r betw seq uen ttat
trea tme nt gro up. Thi s is bec aus e of
ua11y inc rea se from trea tme nt group to
 Biopharmaceutics and Pharmacok·1 .
400 net1cs
· · t t· f drug products to different treatme nts. To avo id th is type ~ ff
a d min is ra ,on o . d fd . . ect
randomized administration of drug produ_cts is used .. The or ~r o osrn g rs no~ sequential
but a part of each treatment is given fi rst, a part rn the middle and a part rn the last.

Multiple-dose study
Multiple-dose study may be required to determine the bioavailability of a drug product in
the following circumstances.
• There is a difference in the rate of absorption but not in the extent of absorption.
• There is excessive variability in bioava ilability from subject to subject.
• The conce ntration of th e active dru g in gred_ient or t he~a peu tic moiety, or its
metabo lite(s) , in th e blood re sultin g from a sin gle dose ts too low for accurate
determination by analytical method.
• The drug product is an extended release dosage form.
Examples for the drugs for which multiple-dose study is performed are rivastigmine
tartarate( oral capsules), quatiapine fu merate (tablets oral).

11 .3. 7 Sampling
The biological sample to be used in the study has to be decided before the commencement
of a bioavailability study. If the bioavailability of a given dosage form is to be evaluated
by a blood level study, some estimate of the area under the serum concentration versus
time curve, peak serum concentration, and time of peak serum concentration must be
obtained from the study. Therefore, the frequ ency of sampling and the duration of sampling
are very important since these factors can markedly influence the "apparent" results obtained
in a given study. While the blood sampling schedule required to evaluate bioavailablity
will vary with the drug , it is possible to list a few factors that will ensure a satisfactory
determination of the blood-level profile. The sampling scheme should be frequent enougi1
to define the absorption phase, the peak, and the elimination phase during a drug's time
course in the body.
In order to estimate the rate of absorption it is necessary to have enough data points
in the absorption phase and hence, the frequency of sampling is more in th is phase. Even
though the frequency of collection of samples is monitored, it may not be possible to get
the time of peak serum concentration directly from the data and is generally estimated
from the data. Since the relative amount of the drug absorbed is determ ined from ~he
AUC parameter, there must be sufficient sam pling points to allow for proper evaluation
of_t~e ~rea under the blood-level curve. The absorption rate, volume of distribution, and
eh_mmat,on rate, all influence the apparent drug concentration one obtains in a given sa~ple.
It rs necessary to see that all these factors influence each dosage form equally. To estimate
th e AUC from_the data, sampling has to be ca rried out till the concentration of the drug
reaches the_lt~ea~ elimination phase. For first-order process , the time necessary fo~~
complete e_llmination wo~ld be infinity. However, for all practical purposes it is a p~n~o
of 10 half-hves for any given drug. A rule of thumb sampling in a blood-level study 15uld
sample for t~ree to five half-lives of the drug. If the half-life is not known, sampling sho
proceed until 1/1O or 1/20 of the peak levels are reached.
 . blllfY end Bloequlvalence
.08votl8- - - -
~ case of urina ry excretion studies the .
401
1n the tudies are used whe n It is eith ' same principles apply. Gen erally, urina ry
~cretion :rna, or seru m or whe n ethical cone:ldnot ~oss lble to mea
sure a given drug in the
!,ood, pla riod of time (e.g. subj ects such as ertttons do_
not allow the colle ction of sam ples
ver 3 pe tudie s are · 1 it involves non • ~a tentS, children). The advantages of urina ry
o tion s . . -inva sive method of s
~ere ·n the urine 1·
is often greate th
~f the drUQ t and 3. the amo unt of ther an that tn
. amp mg, 2 . the conc entrat',on
bl~od /s~rum allow ing easy estim ation
of the drUQbl od -leve l stud y th drug excreted tn unne is obtained direc tly.
ase of a O . t ' e amo unt of the drug in the body is estim ated In the
charrn acok ineti c para me e_rs . On th e _other hand , urina usin g
ry excr etion meth od has seve ral
~isadvantage~ as well. 1 · _urina ry excre.t,o~ studies are
not useful in estimating the abso rptio n
rate of a rapid ly ~bso rbmg drug . This is beca use
it is diffic ult to obtain man y sam ples
during the absorptt~n phase to define the abso rption
rate accu rately. Subjects feel diffic ulty
. emptying the urina ry bladder frequ ently and it leads
to a carryover effec t on the next
;arnple. 2. In so~ e cases, !he ':'etabolites of the drug
are also concentrated in the sam ple
that interferes w 1th th e e stimat ion of the unchange
d drug in the urine sam ple .
However, in orde r to obta in useful data , it is necessary
to plan the frequ ency and dura tion
of sampling care fully. If possible, urine samples shou
ld be collected for 1 O half- lives of
the drug to ensu re a 99 .9% of elim ination of the drug
in urine . If the eliminati on half- life
isnot known , a plot of the cumulative amount of drug
excreted over time will reac h a plate au
at some point in time cons isten t with a comp lete
elimination of the drug .
11.3.8 Sele ctio n of Subject s
Healthy Sub jects Vers us Patients: Bioavailability
stud ies are desi gned to find out the
dosage form biologica l performance. Hence, it is nece
ssary to minim ize all possible varia tions
if it is not possible to elim inate them . Use of healthy
volunteers avoid s much of va riatio ns
that are possible with patients. However, it is true to
say that the condition s shou ld mim ic
as much as poss ible the actu al conditions of usag
e for patients. The variables asso ciated
with most dise ase state s make it impossible to desig
n a mea ning ful bioequiv alence test .
Some of the spec ific problems associated with testin
g in patie nts are give n belo w:
1. It is diffic ult to obtain many patients in a given
place .
2. The seve rity of a disease varies from one patie
nt to anot her.
3. Ethical cons idera tions do not allow withdrawal of
many bloo d sam ples from the patie nts
for a long er time .
4. It is not ethic al to adm inister a dosage form to a patie
nt who se ther apeu tic effic acy
is unkn own .
5. Since treat men t of a disease involves use of several
drugs simu ltaneousl y, the effec t
of these drug s on the bioavailabi_lity ?.f the drug to be
tested shou ld be know n befo re
the inter preta tion of the bioava1lab 1hty test results
.
Therefore , normal subjects are preferred in the bioav
ailability stud ies over patie n~s.
Examples for the drugs for which patie nts are used
in bioa vaila bility stud ies inclu de
Anticancer drug s like azacitidin e, nabilone, temozolom
ide, cape citab ine. leup rolid e
acetate .
 402 8 ioph orm ace utic s and Pharma .
- - cok1ni.t·
Solcctlon of Subjocts ~
If it is ac-coptod tlint 1,onlt1,y vollmloorn
slloulcl bo use d in bioavailability stud
quo~t,on onC' 1ncci, is. whot is moo nt ies, th
ll y "l1oolthy" volunteers? Hea lthy mea
11 .,vin q on ovl'I nll good slnto o'f phy slcn l lion Ith. It Is asc ns a e next
ortained by vital signs s~~tm
tPmpClraturo . pulso. 1ospi tt1lion , blood
pre ssuro , and labo ratory .tests on bloo
WBC count. 11omoolob1n. blood sugo1, d (RBc cou~~
olc.), urin e (pH , albumin , sugar etc.
hv<'r func tion tost 5 such as scrum ) and also r ·
alkaline pho sph atase and serum glut
transammaso Dependi ng upon l11e ami c-oxaloa ~Y
dru g products use d .in the study, ce cet1c
bo incl ude d or excluded . rtain tests rnay

Age , sax and body weight also infl~

en~e the blood l~vel profile of a drug
general 21 -year old, male subjec ts product.
weighing 150-lb ar~ idea l _to act as
study It 1s difficult to ootain a sufficie volunteers in th10
nt number of subJects with these spe
hence acceptable norma l ranges are cifications an~
20-50 yea rs of age and 120 to 200 lb
Males are preferred over females of bod y weight
bec ause menstrual cycle, pregnancy
menopause stages tha t occur in fem , lactation and
~ les ma~ affe~t t~~ bloo d leve_l profiles
Howeve r, females are also included of the drug.
rn the b1oava1lab1hty study taking abo
consideration. ve points into
Medical history of the subjects has to rev
iewed critically by a panel of experts . For
a person who has undergone gastrec exam ple,
tomy shows normal values for the gen
tests used, but he may not absorb cert era l screening
ain drugs like normal volunteers becaus
in the gastric pH . e of difference
The selected subjects should be dist
ributed randomly to differen t groups
achieve a uniform distribution of the in orde r to
ava ilable volunteers with respect to
body weight and to avoid bias. age , sex , and

11 .3.9 Study Conditions

The selected subjects should be ma
intained on a uniform diet and none
have taken af'1'/ drug at least one wee of them should
k prior to the study. Before the com
the study it is necessary to define the men cem ent of
study conditions such as the fasting
the adminis trat ion , time period after period before
drug product administrat ion during
continued, standard diet to be given whi ch fasting is
after fasting, fluid intake and volume
etc . The quality and quantity of food to be allowed.
intake drastically affects the bioavail
drug s. In general , bioa vailability trails ability of some
are conducted on subjected that has
fasted overnigh t.
11.3.10 Analysis of Biological Samp
les
Ideally, the biological samples collected
as per the sam pling proced ure have
immediately after the study. But mo st to be analyzed
of the time s, the samples are stored
before they are subjected to a chemic for several da~s
al analysis. The storage of biologic
an impo_ rtant aspect i~ a bioavailabil ity study, al samples 15
since , during storage the sample may nd
a chemical degradation , adsorption u ei~:i
on to the walls of the contain er, etc
method used for the estimation of . The ana lyt •
the acti ve ingredie nt responsible for th
efficacy must be selective and sensitiv the era~ eut,c
e Nonspecific analytical metho ds mea
of the unchanged drug and metabo suring a mixture
lit~s are less des irable even in well-co
ntrolled cross-
~ d 8 ;0 equivale nce
·1itY an
403
l;1sb' oruQS that_undergo the first-pass effect exhibit different unchanged dru~
~ t\JdieS· tiO depending on the rate of absorption . If the analytical method used is
l ~lite r~t,el1 the results of the study may not reveal the difference in therapeutic effi~cy
~t~"c'between dru~ products. MoSt of the manufacturers use the latest analytical
I' ~ists the analysts of the samples. Only one analytical method should be used
,,t :iaue fo:is of all the sample~ of a stud~. There may be more than one selective and
~~~e anal~thod s for ~he analysis of the 91~en chemical moiety. Any one of them may
\'I ilNe r,, the analysis bu~the results obtained with one analytical method in one study
t~\ed for compared with th0 se obtained in a other study with a different analytical
i~
·~1d not be rnmary, the ana lyt·,ca I method used must be specific to the active chemica · l
~ t11od· In suhould exhibit high sensitivity.
..e ands
tdtetf i::stimation of Drugs and/or its Metabolites in Biological Fluids
11 ~ . . b
\I, 3· . uivalence testing is ase d on the premise
.
that equivalent blood levels produce
b1oeQ . I ·t . . .
-,rJ, ient pharmacolog1ca _respon~es, ~ 1s ob~1ous that one must be certain that equ~alen1
~uwa ts are measured mthe b1olog1cal fluids after administration of chemical equwatenl
~ p0ne\he development of highly sensitive and specific instrumental techniques, coupled
-Aucts. . xt t·
:I"" advances in select1v~ e rac 1~n a~d s_ epar~tion ~rocedures, has enabled a res_~ ue
,~~sis of drugs and their ~etabohtes m b1olog1cal fluids with a high degree of prec1slon
: accuracy. In most ~nalyt1cal problems , the final ~uantitative analytical step is the lea_st
@'icult part of the ent1~e pr~cedu~e. In the a~alys1s of blood or urine, the most easily
ib[ainable and useful b1olog1cal flu ids, the maJor problem is to extract quantitatively and
tien separate the intact drug from its major metabolites or even to separate a mixture
~ two or more drugs from their metabolites.

Ideally, the pharmacologically active component should be measured in the b;o~g-~I

sample. The analytical method used must be specific to the active component and should
~ good sensitivity. Since usually there are several potential methods of analysis that
iieusable, the type of procedure employed should be mentioned and also, a concise
~atement as to whether the unchanged drug, metabolite , or some other combination of
ocoducts measured should be included with the bioavailab ility data.
1
1 \fMethods of Assessment of Bioavailability
~ere are several methods of assessing bioavailability in humans and the selection of
1i rneth0d
, ~eth depends on the purpose of the study, nature of the dosage form, and the a1a\ ticat
I1ietn~~ of drug measurement. The methods available are classified as pharmacok.inelic
\ s and Pharmacodynamic methods.
I Th
•,i drue assumption in using pharmacokinetic methods for the assessment of bioavailability
' uid 9 Products is that th ere exist a linear relation between the drug level in the a biological
1

and th
~elho erapeutic re sponse . Therefore , these methods are also known as mdtrect
~'i10/ s· Because the free or therapeutlcally active drug can be accurately measured in
I 'Cai fluids, plasma and urine data give the most objective information on bioavailabihty.
I
404 f31ophorm acou ti co and Pharrna,, ~
0 tr,,;l1
,~
11.4.1 Indirect Methods or Pharmacoklnotlc Met
hod s
The parameters th at are useful in determining
1ho bioo vailabillty of a druo frrm1
product based on indirect meth ods aro:
;, <fr,,,,
1 . Plasma Data
(a) The tim e of pea k pl as ma conce ntration
(tmm,)
(b) The peak plas ma conc entra tion (Crnox)
(c) The area unde r th e plas ma conc entration
-tim e curv e (AUC)
2. Urine Data
(a) Th e rate of drug excretion in th e urin e (dXu
/ dt)
(b) The cum ulative amount of drug excreted
in th e urin e (X: )
(c) The time for maximum urinary excretion
(t: )
11 .4.1 .1 Plasma Data
This is the most widely used and accepted meth
od for the assessment of bioavailab ility
of drug products. For some drugs like sirolimus
, indapam ide, tacro limus, whole blood and
for som e drug s like tript orel in , amin osal icyli
c acid , seru m is used for assessing
bioavailab ility.
The basic assumption in this method is that drug
products that are bioequivalent produce
superimposable plasma level -time curves. By defin
ition , bioavailability is a meas ure of the
rate and extent of absorption of a drug from a
drug product. The parameters t and c
are the measures of the rate of absorptio n of the
drug wh ile the parameter AUC i/a meas:
of the extent of absorption .

(a) The time of peak plasma concentration

(tmax ): The time required to reach the
maximum drug concentration in plasma after
drug administration is known as the
time of peak plasma concentration (tmax) (Fig.
11 .2) . It indicates the time at which
the rRte of absorption is maximum follow ing drug
adm inistration . At tmax• the rate of
drug absorption exactly equals the rate of drug elimi
nation . Therefore , when comparing
drug products, tmax can be used as an approxim
ate indicator of the drug absorption
rate . Sma ll tmax value indic ate that less time
is requ ired to reac h peak plasm a
concentration . In other words, small t means a more rapid absorption . Unrts for
max
tmax are units of time (e.g. hours, minutes)
.
Mathematiacally, tmax is a function of both abso
rption and elimination rate cons tants.
It is highly dependent on the sampling scheme,
and it is therefore difficult to use
it to detect differences between two products,
especially when tmax values are less
than 2 hrs.
·
In contrast, for slow-reeleasin · · ed
g dosage forms, plasma concentrations are main
tain
at a plateau for long time and tmax does not
reflect the rate of bioavailability.
 ~,/flt,ilitY and Bioequ .
•1111'/l. ,.,..
i,..~· ,,
,vatenCe
·/ st8tistical comparisio
n1Y undertaken fa ns of t
rapid re"''i)(
0 rt1e clinical rele/ ance. ease for cthe .tnno
50
aecause Cmax reflects ornPoun~ator and
and Cmax. extent of absor . s andtor generic
When Products 405
bl f he peak plasma con Plion , th tm,, is kno!re usually
the maximu m plas centrat1· ere is st n to have
· · t • rn a d on (C rang
( · rati on of th e dr rug con "'" )·· Th
gdrT1'· n1s correlarion bet
suff1c1entiY absorbed ug (Fig centrar e Peak ween AUC
in plaS ma is with in th to elicit a. r11.2). C ton Obtai: ~:sma concent .

w~~~~
ug/ml or ng/ml. esponse"'"
erapeutic Ievels als . anfollowing
. Itprovides mdic . an ration represents
extravas
aecause Cmax reflect or reachingot~ndicates that the dr~~'~:
and Cmax s extent of absorptio x,c levels. C"'" er,s the drug level
expressed

It should be recogn· n, there is t as

absorption a nd is i~zfled th at Cma, .IS only
uenced . s rang correlation betw een AUC

eased on its c linic by extent of an indirect me

I applicability [it
concentration) in the abo absorption. asure of the rate of drug
a .strong dy andt may reIate
. II case can be mad refltoects . the h.ighest d .
c 1n1ca y appropriate one to
e r hat (alth ough cpatients . risk for tox,c,ty
r_u~ exposure (e.g.
assessing rat ma, ts a poor meas and/or efficacy]
e of absorption9 . ure, it is the most
I

v>
(I)
...J
00
2
Cl
CQ

~ C
0:

Time (hrs)

Fig, 11 .2 Plasma drug level versus time curves following oral administration.

(c) The area under the plasma concentration-time curve (AUC): The area under
the plasma concentration-time curve, AU C, is a measure of the extent of drug absorption
from a dosage form or the fraction of the dose that reaches the systemic circulation.

The [AUC]; is the sum of [AUG]~ and [Auer , where 't' is th• Jast time point of
. [AUG]' is calculated bYthe Trapezoidal rule and the [AUC]\'

asma sam ple collection. o11 6 .6)

Pl obtained fro m the Equation · ·
is • (11 .5)
.... .... ..(11
[AUC]~ "" [AUG]\ + [AUG],

[Auer C G'/K
r

406 Biopharmaceutics and Pharmacok· .

1net1cs
Where . C* is the concentration of the drug in the last plasma sample an~
overall elimination rate constant. AUC is independent of the route of administ tf!le
and processes of drug elimination as long as the elimination process do as not ch~a '0 n
The units for AUC are concentration-time/volume (e.g. µg-hr/ml). nge,
For many drugs, AU C is directly proportional to the administered dose . Nonline .
between the administered dose and AUC , in general, indicates the existence ~~ty
saturable kinetics of absorption and/or elimination . When the AUC is not direc/
proportion al to the dose, bioava ilability of the drug is difficult to evaluate . Y
In the ca se of multiple dose studies, AUC for one dosing interval at the steady-stat
level is a measure of the extent of drug absorption from the dosage form . The followin e
equations are used to estimate th e fraction of the dose absorbed , F (i .e., thg
bioavailability of the drug product). e

F = _[A_U_C_J-=-oracc.r. _x_
[D_o_s_el_r,
[AUC].v X [Dose lora1 .... .(11. 7)

If doses are equal, then

[AUC]oral
F = [AUC]iV ... ..(11 .8)

Wh ere F = Absolute bioava ilability

[AUC]text X [Dose] standard

Fr= .... .(11 .9)
[AUC] standard X [Doseltest

If doses are equa l, then

Fr = ..... (11 .10)
Wh ere, Fr = Relative bioava ilability.

Truncated AUC .
The common measures used in bioequivalence study are AUC and Cmax.
Estimation of AUC requires frequent blood samples. For long half-life drugs, sampling
for long periods of time may become cumbersome. To resolve this issue some investigators
have su ggested the use of truncated AU C in bioequ ivalence stud ies for long half-life
drugs.

For example , pi mozide , sirolimus , entacavir, sa lefivacin succinate , atavaquone ,

apriprazole, anastrazole.

Partial AUC

Here AUC is tru ncated at t max for the innovator compound .

~ 407
. ·ty and Bioequ iva lence
af/ab'''
exposu r~
e;oBv allY admi nistere d im11~edi~ te r~leas_e products, measur es of peak and total
efficac y tna ts C.:,
r:or or ufficie nt. Howev er, in s1~uat1 ons in which clinical sa fety and/or
of drug at"jl) ' r.,' ,,.J
aY be s Kin etic-pha rm acody nam,c stu dies indicate that bettet control
re via use of partJa J
,,.,harrria~:rnic circ~ lation m ay b e w arran ted, assessm ent of early exposu
P10 sY5 be indicate d .
1~ cs n1aY
p,.!J rnple zo lp id em ext1end ed re lease table ts.
f or exa
Data
2 Urine · t e
11 .4-1· . to estr~a te r
. excretio n of the u nchang ed drug versus ti me data can be used th8
eral observ ation t
unna~lab ility of a d rug product . This method is ba sed on the gen f thP
. ava1 t· f proporti ona l to the concen tration o -
bl0 ra te of urinary excre ion o a drug is directly
the . the blood. In other words , the same fra ction of absorb
ed drug a lways reache s
ted a s the ratio of th e
drug ~ne uncha nged . Therefo re , the bioavai labi lity can be calcula
1
th e unmount of the unchan ged drug recover ed in urine foll ow ing the admini stration of test
1 lan,trty
~o~~ ;andard formu lations . Exampl e for which urine data is used to asse ss b 1oava
is alendronate .
s ignifica nt q .Ja .,~::= es
In order to obtain valid estimat es, the drug must be excrete d in
s . Th is method is u~ful
in urine and urine sample s must be collecte d for at least 7 half-l ive
tor all the drugs that are e>ecreted in urine unchan ged in sign
ifica nt qua ntities . Unna- y
va ila b ility since the drug
metabolite excretio n data is not used for the estimat ion of b ioa
a nd th e rates of
can undergo metabo l ism at differen t sites includin g the gu t a nd liver
metabolism may va ry becaus e of various factors .
(a) The averag e rate of drug excreti on in urine (.6.Xu / ~t)
: The r a te of excr etion
s . s ince , •t depen ds
of the unchan ged drug in uri ne general ly follows first order proces
on the blood concen tration of the drug . Therefo re , a plot of ~
J ...\t versus tun e al
drug conce nt~alio n
the mid point of urine co llection (t') is a mirror image of the plasma
, the maxim um rate
versus time curve (compa re Figures 11 .2 and 11 .3 ). In Fig . 11 .3
s ponds to the time
of drug excretio n would be at point B, (~ Xu/ ~t)max . and it corre
te the b ioavail abi.~ty
at which C max occurs . Hence, (~ Xu / ~ t)max is used to estima
of drug produc ts .
rine in infinit e tim e (Xu) :
(b) The cumula tive amoun t of drug excrete d in the u
B
.....0
_
c::
.S:
....
~
-
xu ....._
Ct) O
~'~-
~ OIJ
·c: 2
;:) "ti
4-
0
CIJ (.'
A
""
0:::
Time (hrs)
Fig. 11.3 Rate of urinary excretion of drug versus time plot.
408 Blopharmaceutics and Pharmac k'
~ nett,·c~
The cumulative amount of the drug excreted in th e urine ~=-- is;_ directly related~ .J ! __ ••
amount of the drug absorbed . The drug excretion into urine continues till th to the
level in blood falls to zero . Therefore, urine samples must be collected for a ~o~rug
time (at least for 7 half-lives ) in order to get a better estimate of X'. A gr ger
u aph of
cumulative amount of the unchanged drug excreted into the urine versus time is sh
in Fig . 11 .4. The cumulative amount of th e drug excreted up to point C corresp own
Onds
to X~. This va lue is used for the estima tion of bioava ilability.
Absolute bioavailability,
bO
2,,..._
-0 bO
<+-- E
-0-._,
C ~
·-!::c::
::,
0 C
E ~
ell · -
~ c::
B
.2'.: .s
]::, ~
u
E
::I
><
~
u
Time (hrs)
Fig. 11.4 Cumulative amount of unchanged drug excreted·in urine versus time following oral
administration.
0
[X u_
F = ____; 1oral
_ X [Dose 11v
_ __
.. .. .( 11.11 )
rx: 11v X [Dose loral
Re lative bioavailability,
Fr=
rx: ltest X [Dose lstandard
..... (11 .12)
rx: lstandard X [Dose l test
When doses admin istered are eq ual,
Absolute bioavailability,
F = rx: loral
..... ( 11 .13)
[X~] v 1
and Relative bioavallabillty,
[X~ltest
Fr - -- - - - - ... .. (1 1.14)
- [X~; 1standard
~
. biliW and Bioequivalence
~~ . ~ - . ~. //
A09
8;08 8 tirne fo r maximum urinary excretio , . .
c) fl1 "T. • • n (tu) · Th r: t,m9 :Jt pr,,,n1 C ,r, Fs~ / ~.
( thing but tu. It 1s the time required f
is no or the abtorptron and a corr. f1J,,} ~ ,,:tfr:1 .. :~' ✓,,-
drug fo llowing the admini strat·
of the . . ion of a drug product t' f(} a u:;.c;fu' :;~ --tV " ~ •~ ,,
· equivalence studies comparin '1
in b10 t d 9 several drug products oio,;e it tJ 0 rsie::;,~ J '~;
1
th the ra e an extent of dr b • •
of bO ug a sorp t,on from a drug produ<.,i .
steady st ate: _For udie~ at st eady state , the follovting characteri~U~ -:ti ,,..J't1 ~
st
calculated during one do~mg interval or cycle, C ,at steady state : AUC< 0-T J (;; -- .,
and peak-tro ug h fluctua ti on , PTF = (Cmax _ c m,n)/Cav, where Cav = 1-.. UC (0-Trf,
denotes th e average steady state concentration .
For confirmative b_ioequiva lence assessment. Primary characteristics for the ,~.bar d
extent of absorption shoul d be stipulated prospectively in the study protocof11
11.4.2 Direct M ethods or Pharmacodynamic Methods
The pharmacokinetic methods are based on the assumption that the drug concentrat,on
in blood or the _d rug excretion in the urine are related to the observed therapeutic effect.
Pharmacodynamic methods are used when the assessment of b ioavailabilit'J D":(
pharmacokinetic methods is not possible due to non-availability of a sensitive a'"'a ·,t-·ca
method for the measurement of the drug or the analytical method lacks sufficient accurac/
and/or reproducibility. The two pha rmcodynamic methods used fo r the estim a+j o n of
bioavailability are based on the measurement of:
1. Acute pharmacological effect
2. Clinical response
11.4.2.1 Measurement of Acute Pharmacological Effect
In order to estimate the bioavailability of a drug product accurately by this m ethod. the
following criteria should be met.
1. An established dose-related response curve .
2. An easily measurable pharmacological response such as heart rate . ECG. b lood
pressure, pupil diameter, etc.
Experimentally, the pharmacological effect is measured at different time intervals following
t~e administration of a drug product. A plot of observed pharmacological effect versus
tirne is made in order to get a smooth curve. The area under this curve is a measure
of the performance of the drug product and is used for the estimation of the bioava tlabifr+u
ih I . I " 7·
e frequency of measurement of the pharm aco og,ca effect and the total duration of
the study affect the results . The study should be conducted for at least three times th
h~tf-life of the drug and a measurement of the pharm acological response should be .d e
With . . ma e
h sufficient frequency to permit a reasonable estimate of the total area under the curve.
•rie . . . t . .
Ieve1
main drawback of this method is that an accura e 1mear relationsh ip between the d
· d 'ff. rug
and observed pharmacological response ,s 1 1cult to obtain .
 410 Biopharm aceutics and Pharmacokinet·ICS
11.4.2.2 Clinica l Res ponse
Theoretically, this method seems to be the best among the met_h ods disc~ssed so far
erved differences in th erapeuti~
But, practically, it is not. This is beca use of the fact th at obs_
response fo llowing differe nt fo rmu lati ons ca n not be attrib ute d o~ly to th e formulat ion.
th
Differences in the cli nical response may be due to differences both in e pharmacok inetic
and pharmacodynamic behavior of the drug among individ ~~l s. The drug may be available
to the systemic circu lation from a drug product at a su_ff1 c1ent rate and extent, b~t rnay
not elicit a cl inical response in an individual becau se his receptors are less sensitive to
the drug compared to others. This is because of diffe rences in pha rm ac~dynamics of the
drug in a particular patient. Various factors affecting a pharmacodyna m,~ dr~g behavior
may include age, drug tolera nce , drug interactions and unknown pathoph~s1olog1cal fa ctors.
In addition, quantification of clinica l response is too inaccurate to be useful 1n the assessment
of bioava ilability of dru g prod ucts. Examples mesalam ine, ketoconazole , clotrimazole.
/n vitro studies: In some cases in vitro studies are recomm ended for bioequivalence studies.
Binding studies: in case of sevelamer carbona te and seve lamer HCL tablets two in vitro
teste are suggested by FDA.
(i) in vitro eq uilibrium binding which is considered as a pivotal bioequivalen ce stu dy and
(ii) in vitro kin etic binding which is used to support the pivotal eq uilibrium bi nding study.
The bioequivalence is based on Langmuir binding consatant k2 from the eq uilibrium
binding stu dy.
Similarly in vitro stu dies are also recommende d for calci um acetate ( in vitro phosphate
binding), colesevelam hydrochloride and cholestyram ine p/
11 .5 Statistical Analysis of the Data
The pu rpose of a bioavailabil ity test is to find out whether the test formu lation gives a
blood-level profile identical (i. e., superimposable) to that observed for a reference sta ndard
product or not. Due to th e limitation in sa mpling techniq ues the resultant blood-level curve
is infl uenced by both the number and duration of blood sa mples obta ined in any study.
Unfortunately, due to biolog ical and experimental variations , some differences always exist
and it is necessary to ascertain whether these differences are simply cha nce occurrences
or a~e du~ to actual differe nces in treatment adm in istered to the subjects. By conevention,
all b1oequ1valence data are expressed as a ratio of th e average response (AUC and Cma)
for Test/Referen ce. The statistical crite ria for acceptance of a generic prod uct (test) are
based on 90% confidence interva ls (C ls) and not based upon diffe rences in average values
for AUG and Cmax· The 90% cis for the test product must fa ll within 80% to 120% of the
Reference pro~uct based_o~ no~ log transform ed data. The use of log-transform ed data
tends to nor~allze the sta t1 st1cal distri bution of the data . Therefore , based on log-transformed
da ta, the 90 1/o Cls for the tets product must fa ll with in 80% to 125% of the Reference product.
The_U.S. Food a nd ~ rug Administratio n (FDA) currently uses bioeq uivalence(BE) limits
for fasting state be studies tha t are based on 90% confidence interva l for ratio of difference
of the test and reference produ cts c
. . max
and AU C fall'tng WI'th'In 800,10 t0 1250,10. The FDA has
aIso propose d that BE I1m1ts be used similarly fo r AUC dC ments for fed state
BE studies. an max measure
r
·iability and Bioequi va lence
0;o a~
1/ rn·e cases , regulato ry agencie s ha 411
- - . - - - - - - - - - - - - -- ~~
?
1ri 5 allY higher variabili ty of C com s consider ed a wider BE limn for c because of
tYP'c may pared to AUC (C d ""'"'
tl1e 2003 FDA guidanc e requi res that th
fh; . ana a regulato ry) .
be based on the exposur e measu e evaluatio n of ABE (average bioequiv
atence )
5noul three measu res of system ic expo:~~e AUC(O-t) , ~UC(0-" ), and C,nay. There fore
. for
~se f the average s , and 90% confide . ' geometr ic average , aritheme tic average s ,
rat10 0 the test a nd reference formul ~~e in~ervals should be provided . In add ition. AB~
oetwe;; confiden ce inte rvals for the r t· a ions is conclude d at the 0.05 significa
nce level ,f
th ego ,o temic exposur e are co m a ios of the popular
t . . ion average s for t h h
e t ree res
measu
of ~he (:o%
lill'llts O
5 I
and 125% fo r the n!~n e 1Y w1th in 80% ~nd 125% . In addition , the tradition
al
arrow therape utic ra nge drugs remaine d unchang ed
for the th ree .;xpos~ re measure s of narrow therapeu tic range drugs . Furtherm ore , the
2003 FDA g_ui ance oes not allow the round ing off of the lower and upper limits of the 90%
confidence intervals . In ot~er wo rd s , to conclude ABE at the 5% significa nce lev el, the towe r
lirnit should be at lea st SO 1/o an dthe upper limit should be no more than 125%
.
Sta_tis~ical meth_ods ar~ used to evaluate the data in order to identify th e different sources
of variation and , if possible , to measure th e contribut ion of each identified v ariable and
isolate the specific observa tion of primary interest. The analysis of variance
(ANOVA ). a
statistical proced ure used for a crossove r design, is used w idely in bioavaila bility testing
and is th e procedu re that will be encounte red most frequent ly by the health scientist . In
order to understa nd the applicat ion of AN OVA in bioavaila bility testing , the fo llowing
example
is used.
11.5.1 Analys is o f Var ian ce (AN OVA)
Purpose of Bioava ilability S tudy : To fi nd out w hether the test p roduct
(t ablet ) is
bioequiva lent to the stand ard product or not.
Study Design : Latin Sq uare cross-ov er design and rand omized dru g admini
stration .
Wash out Period: O ne w ee k (half-life of dru g is 2 hours).
Recogniz ed standard : Ma rketed Tablets
Frequency and Duratio n of Samplin g: Blood sa mples were collected at 0.0
, 0.25 , 0.5 ,1 .0.
2.0, 3.0, 5.0 , 7 .0 , 10 .0 , 12 .0 , 16 .0 , 20 .0 and 24 .0 hou rs .
Single or Multiple Dose Study Design : Single dose study design .
Subjects: Twelve health y ad ult male voluntee rs between the ages of 20 to 35 (mean 25
Yea~s) and weighing betw een 62 to 90 kg (mean 72 kg ) were select~d for the study. The
SUbJects are ra ndom ly assigned to 2 groups of 6 eac~ and two formulat io ns are
adminis te red
randomly accordin g to a 2 x 2 Latin Square design .
Study Conditio ns: A ll subjects fasted overnigh t _P~ior to rec~iving th e test tablet or standard
tablet the fo llowing morning . The tablet is adm1~1~t ere~ w ith 200 ml of w ater. Fasting of
subjects is continu ed fo r 6 hours after the a?mm1st _
rat1on of tablets . Blo od sam ples a re
taken just prior to the dose and at specified time points as pe r schedul e into heparini zed
tubes. The plasma fraction is separated from th e blood and analyzed for the
d rug co nte nt
by HPLC .
....__
 .............
~ I

.1
412 Biopl1a rmaceutics and Pharmacokin t'
- - e ics
-------.:::
Asse ,sment of Bioavailabllity
th
Plasm a data were used to estimate th e area .under _th e curv_e by ~ Trapezoidal rule up
to the last time point and th e remaining AU C 1s obtained ~y integ ration me th0 d. The total
AU C or each treatm ent in each volun teer is prese nted in Table 11 .5.
- Table 11.5 The AUC 's Obtained in th e Bioavailability Study
---
2nd week Standard
- -- ----
Ri
-----
(Ri)
2
Subject 1st week Test
76 83 159 25281 -- I
1
-
2 59 54 113 12769
81 177 31329 -
3 96
4 68 61 129 16641
5 86 79 165 27225
6 57 68 125 15625
Standard Test
7 98 84 179 32041
8 64 50 114 12996
9 66 61 127 16129
10 57 48 105 11025
11 70 74 144 20736
12 88 91 179 32041
Oi = 882 Oi = 834 = 253838
I
Average AUG Test = 70.83 and Standa rd = 72.1 7; Ft= s850 and Fs = 866 and x = 171 6
Diff1 rences in the estimated values of A UC for test and standard arise because of the
order o : administration , subjects, form ulations and error. The total sum of squares observed
is due o all the sources of variation just mentioned . T he error sum of squares is the total
sum o squares minus the su m of squares due to order, subjects , and form ulations.
Ord ~r sum of squares 11
,,
L Oi 2 - C .F
= ns ... .. (1 1.15)
Wh re Oi =
order and C.F = correction facto r and n = num ber of determinations in
each o ·der (subjects) s
C .F = (LX)2 = (sum of all va lues)2
= (1715>2 = 122694
N total no. of determinations 24
1:0i is the summation of 1st week and 2nd week values. Hence,
II
Ord r sum of squares I
2
= (882) + (834) 2
~ "" " " .t -~
 .,·iy and Bioequivalence 413
1
l vB'·1stJ '
s ubject sum of squ ares
eel"I 2
eetv' tR i C .F -
- --- n,
..... ( 11 .1 6 )
·Ri2 :::: sum of squ ares of values in each row and n = numbe r of trea tments
~ i I
1/J~ e
e., 2)· subjects sum of squ ares
(' ween
2 2
eet ( 159
= ,:,___+ _113
__ + ..._
+ 179 2 )
__.!.., - 122694
2
= 2 53838 _ 122694 = 422 5
2
n Formu lations su m of squares
8etwee
rFi 2
= - - C .F ... . . (11 .17)
ns
Where 1:Fi = sum o! al_l v~l~es of each formulation i.e., sum of the test and sum of t he
d AUC va lues in md1v1d uals . In th is the case sum of the test (SFt = 850) and
~~ dar '
!he sum of th e standard (S Fs = 866) and n 5 = 12.
Between Form ulations sum of squares
(850 2 + 866 2 )
12 - 122694 = 10.67
Total sum of squares
= :Exi2 - C. F . ... . (11 .1 8 )
Where I.xi2 = sum of squ ares of individ ual AUC values in the table 11 . 5 .
Total su m of squares
= (76 2 + 83 2 + 59 2 + ... .... + 91 2 ) - 122694 = 4 7 08
Now, erro r sum of sq uares ca n be calculated .
Erro r su m of sq uares
= Total SS - Order SS - Su bject SS - Formulation SS .. . .. (1 1 .19)
= 47 08-96 - 4225-10 .67 = 376 .33
0
Now, we can constru ct an ANOVA table using the above values and rea lizing that d eg rees
~freedom , DF = N- 1. Mean sum of squares (MS) is obtained by dividing the su m of squares
h
Wi! DF. F rati os are c alc ulated by divid ing the MS by e rro r MS .
Therefore , Sum of squares (SS)
MS - ... .. (1 1 .20 )
- Degrees of Freedom (OF)
MS of pa rameter
Ms of Pa rameter F rat io = Error MS ... .. (11 .2 1)
~ 414 Biopharmaceutics and Pharmacokinetics·
The F, ,,o means . we have to see F value under f 1=1 and f2=1 0 . For most oft
bioavailability studies the level of significance accepted is 95% confidence limits (p>o.o~e
Therefore, we have to look for a tabled value under f1 =1 and f2= 10 at 95% confiden ).
level to obtain the F 1 10 value (F 1 10 value is 4.96 at p>0. 05) . The calc ulated va lu
· , . e IS
:e
less than table value . Therefore , the F test for formulations and order of administratio
are not significant, suggesting that the values of total absorption from both the formulation:
are similar.
ANOVA
Source OF 55 MS F ratio
Subjects 11 4225 384 .1 F11 .10 = 10.2
Formulations 1 10.67 10 .67 F1.10 = 0 .28
Order 1 96 96 • F1,10 = 2 .55
Error 10 376.33 37.63 -
Total 23 4708
Conclusion: Th e test tablet is bioeq uivalent to the standard tablet used in the study.
For the example shown in Table 11.5, 95 % confidence limits on the difference between
the form ulations for "area under the curve" can be constructed as per the procedure given
below.
Find th e "t" value from t-table for DF=10 at p>0.05 (t with DF=10 at p>0.05 is 2.23).
The 95% confidence limits
= (averag e of sta ndard - average of test)
±t EMS J (1/ ni+1/ nj ) ..... (11.22)
Where ,
t = 't' va lue from t-table for given degrees of freedom at p>0.05
EMS= error mea n sq uare
ni = numbe r of subj ects in ith group
ni = number of subjects in jth group
The 95% confidence limits
= (72 .17-70 .83) ±. 2.23 37.63 (1/12 +1 / 12)
= 1.34 ±. 5.58
= (-4.24 to 6.92)
The true difference between the AUCs for th e formulations lies between -4 .24 and 6 -92
with a probability of 95% .
 .,
~ oil~

. ·ty and Bioequivale nce

·iabt/1
•
415
~, NOVA t o r Multi ple Compariso ns
11,5,z. Aes a bioavail ability stu dy in~olve s more than two formulation s. In such cases . the
~~nY t1f11
51 n bY th e above metho? will be either the fo rmulations are same or different If
•0nc1LJ ~usion drawn out of a bi~avai lability study involving four formulations (labeled
as
t~e cone d D) is that th ey ~re different , th en, whether all th e formulations are different
~.e.C 1
~: another or a parti cul ar formulati on is di fferen t fro m the other formulations is
1ron :wn - In ot~er w?~d s, out of th e four fo rmulati ons, whi ch are bioequivalen t. which are
unkf1 n not be ident1f1ed by the F value only. For th is purpose, it is necessary to use
not ca_ Newman- Keu ls Test.
18
sts 11ke . ..
order to un ~er~t~ nd t_h~ significance _ of these statistical tests , let us consider one
lri A bioava1lab1 llty trail 1s conducted using 4 x 4 Latin Square design for the assessmen t
eta~~~~ivalence of fo ur fo rmu lations_labeled as A, B , c and B. The AUC s of plasma versus
olb data are used to assess th e b1oeq u1valence . The data is presented in Table 11 .6 .
t1i rn: desired to know w~ether_th~ -forrnul_aiton s A, B, C , and o are bioequivale~ t or not.
1111 15 found that there 1s a s1gnif1cant differen ce in means of the treatments . it may be
1 ' .
. d to find wh ich pairs , among all possible pairs of means are different. Analysis of
~~ '
. nee table can be constructed as per the proced ure described for repeated measures
vard,athe F ratio can be used to fi nd out whether there is a siginiftcant difference between
~ .
h means of treatmen ts or not. Table 11.7 shows the procedure to be followed in constructing
:h: ANOVA table fo r calculati ng the F ratio. The fo llowing equations may be used for
calculating the sum of sq uares of various sources of variation .
Table 11.6 AUC's of Formulations Used in the Bioavailability Study
Formulation
I
Subjects A B C 0 I
1 42 35.6 39.6 35.3 I
- ,---
I
2 41 34.0 40.2 33.9 I
- - I
3 39 33.2 38.9 34 .3 I
4 36 34 .9 37.0 33.8 --r- I
Total 158 137 .7 155.7 137 .3 _ . l _
-
C .F = (588.7) 2/16 = 21660.48
Total sum of squares (S .S)
= (42 .0 2 + 35 .6 2 + ... + 33.8 2) - C .F
= 21786 .25 - 21660.48 = 125 .77
Treatment sum of squares (Between formul ations) (T. S .S)
= (158 .0 2 + 137 .7 2 + ... + 137. 3 2)/4 - C .F
= 2 1754.77 - 21660 .48 = 94 .29
~
~
416 Biopharmace utics and Pharm acoki .
netics
Between subjects sum of sq uares (S.. S.S)
-------..:::
= (152 .52 + 149.12 +... + 141 .72)/4 -C.F
= 21676 .78 - 21660 .48 = 16.23
Within Subject sum of squares or Error sum of squares or a residual sum of squar
(E.S.S) = S.S - S.S.S-T.S .S = 125.77 - 16.23 - 94 .29 = 15 _1
9 es
Now, the ANOVA table can be constructed using the formul ae given in the Table
11
From the ANOVA Ta ble 11.8, it is evident that the calculat~d F value (18 .60) is grea/·
than the critical value of 3.86 (F3,9 at p >0.05) . Therfore, it can be concluded that t;'
means of the treatments differ significantly. Now, we are interested to find wh ich pa· 8
. . d'ff t F
am ong all possible pairs of means, are I eren . or th·Is purpose Newman-Keuls Tirs '
· · f est
1s quite use uI.
Table 11.7 Formulae Used for the Con struction of ANOVA Table for Repeated Measure
$,
~
Sources of variation
Sum of
Degrees of Freedom
Mean Square -
Squares F
Total sum of Squares S.S nk- 1 (4 x 4 -1 = 15) -
Between Treatm ent T.S.S K - 1 (4 - 1 = 3) s21 -_-
T. S.S
sum of Squares - sf ,sf
k -1
Between Subjects sum S.S.S n - 1 (4 -1 = 3) s2 = S.S.S S~ IS~
of Squares . 5
n- 1
Within Subjects or E.S.S. (n - 1)(k - 1)= 9
Error Sum of Squares
s2= E.S.S
P (n -1 ) (k - 1)
Table 11.8 Analysis of Variance of the Data (ANOVA).
Sources of variation Sum of Squares Degrees of Freedom Mean Square F
Total sum of Squares 125.77 15
Between Treatment 94.23 3 31 .4 1 18.60
sum of Squares
Between Subjects sum 16.30 3 3.213
5.43
of Squares
Within Subjects or 15.19 9 1.69
Error Sum of Squ ares
In Newman-Keuls test, for k treatments (formulation s) the number of pairs is k(k-1)/2·
Th e k treatm ent means are fi rst arran ged in order of increasing magnitude.
According ly, in th is ca se, 34 .33 (D), 34.43 (8), 38.93 (C) , 39 .50 (A)
Consider the pair means of A versu s D; iur these a standrad error (SE) is computed
using the respective samples sizes n and nd and the val ue of
0
s~ (mean square of error
or within subjects), see Table 11 .8 .
 417
. bilifY and Bio equival enc e :._-
8 t1a
.., ~~ ~ ~- - - - - -----------------_
B~ ~ or (SE )
~ dara ~" ~
= [( Si;/2)(1 /na + 1/nd )J1'2
= [(1 .69 /2 ) ( 1/4+1 /4 ))1 '2
.. ... (11 .23 )
= 0 .65
den tize d ran ge " den oted by q :
Also nee ded is the va lue of the "stu
Mea n of A - Mea n of D .. .. . (11 .24 )
Q=
SE
39.50- 34.33
q = = 7 .953
0 .65
g tes ted .
the valu e , w , equ al to the num ber of mea ns in the ran ge of the pair bein
And = 3 etc .,) . The
; for .the pair A , B , the valu e w = k-1
(for the pair A,0 , the valu e w = ~ = 4 e of q_a .~.w
calculated valu e of q ~om equ ~tio n 11
.24 Is then com par ed with the criti cal valu
), the -wit hin
the 'q' tabl e , usin g a des ired con fide nce valu e a (usu ally 0 .05 or 0 .01
rrom If the calc ula ted
the valu e of w pre viou sly def ined .
samplen deg ree s of free dom , v, and ula ted
pair of mea ns diffe r sign ifica ntly at the a leve l. For exa mp le , the calc
tabl e valu e of q = 5 .91 at a = 0 .05
q > qµ vw • then the , for
q for A:D pair is 7 .953 whi ch is larg er than the
w = 3. A sim ilar test is mad e on eac h pai r of the me ans
.
3 degrees of free dom (v) and
n ·Ia rge st"
r of com par ison of mea ns is "larg est" aga inst "sm alle st" (k vs 1 ) , the
The orde ins t " sm alle st9
, etc ; the n "se con d-la rge st" aga
ag ains t "se con d-s ma lles t" (k vs 2) Tab le 11 .9 sho ws
(k-1 vs 1), and "se con d-la rge st" aga
inst "sec ond -sm alle st" (k-1 vs 2) .
the abo ve data .
the results of New man - Keu ls test on
Conclusions:
bioe quiv alen t.
1. Form ulat ions A & C and B&D are
ntly from
atio ns A&C sho we a high er bioa vai labi lity and the y diff er sign ifica
2. For mul
B&D.

Test on the Dat a Pre sen ted in tab le

11 .5 .
Table 11.9 Res ults of New man -Ke uls

SE w q P<0 .05
Com pari son Diff eren ce of Mea ns
= 5 .18 0 .65 4 7 .9 68 YES
A vers us D 39.5 0- 34.3 3
39.5 0 - 34.3 3 = 5 .08 0 .65 3 7 .814 YES
A vers us B
0 .65 2 0 .885 NO
A vers us C 39.5 0 - 38.9 3 = 0 .58
0 .62 3 7 .083 YES
C vers us D 38.9 3 - 34 33 = 4 .60
0 .65 2 6 .929 YES
38.9 3 - 34.4 3"" 4 .50
- C vers us B
34.4 3 - 34.3 4 - 0 .10 0 .65 2 0 .154 NO
- B vers us D
Deg rees of free dom - 9
, Biopharmaceutics and Pharrnacok· .
◄

418 - - - - - - - - --...:.:- inet,cs

~
Likely Questions
1 Defin e the following
(a) Generic name
(b) Brand name ,
(c) Pharmaceu tical equivalen ts,
(d) Therapeutic eq uivalent.
2. How do you estimate the absolute bioa vai lability and relative bioavailability ?
3. What are th e two types bioa vailability studies ?
4 . Give the table showing bioavailability protocol.
5. What are th e advantages and disadvantages of a parallel design over a cross-over
design ?
6. What are the merits and demerits of a Latin-Square design ?
7. What are the salient features of a Ba la nced Incomplete Block Design (BIBO) ?
8. Give a four-way cross-over design for fo ur products A , B, C and D.
9. Why a wa shout period has to be given in a bioavailability study ?
1o. What are the difficulties encountered in using patients as subjects in bioavailability
study ?
11 . Write a noted on th e analysis of biolog ical samples.
12. Wh at are the different methods availa ble fo r the assessment of bioavailability ?
13. What are the important pharmacokinetic parameters calculated from the plasma data
for the assessment of bioavaila bility ? W hat is their significa nce ?
14 . How do you assess the bioava ilability of a prod uct using the cumulative amount of
a drug excreted in urine in infi nite time (X: ) data ?
15. Write about the pharmacodynamic methods used for the assessment of bioavailab1lity

References
1. 21 Code of Federal Regulations Part 320 , Section 320.27 .. Guidelines on Design of
Multiple-dose In Vivo Bioavailability Study. Food and Drug Administration, Department
Of Health and Human Services, April 2009.
http://www.accessdata .fda.gov/scri pts/cdrh/cfdoc s/cfcfr/C FRSearch.cfm?fr-320.27
2. Bi~equivalence Approaches for Highly Variable Drugs and Drug Products, By Sam H.
Ha1dar, Barbara Davit, Mei-Ling Chen, Dale Conner, laiMing Lee, Qian H. Li.Robert
Lionberger, Fairouz Makhlouf, Dowrat Patel, Donald J. Schuirmann, and Lawrence
X. Yu , Pharmaceutical Research, Vol. 25 , No. 1, January 2008.
3. Bioequivalence studies in Drug Development methods and applications, by Dieter
Hauschke, Volker Steinijsns and Iris Pigeot, Page NO: 7.