BIOAVAILABILITY

AND
BIOEQUIVALENCE
Topics covered :
1. Historical aspects.
2. Definitions.
3. Objectives & significance of BA/BE
studies.
4. Factors affecting Bioavailability.
5. Measurement of Bioavailability.
6. Methods for enhancing Bioavailability.
7. Introduction to Bioequivalence.
8. Limitations of BA/BE studies.
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HISTORY:
Phenytoin toxicity in epileptic patients which
occurred in the year 1968 in Australia

The differences in the bioavailability observed

with different digoxin formulations in the year
1971.

3
 DEF : BIOAVAILABILITY

◦ “The term Bioavailability is defined as a rate

& extent (amount) of absorption of
unchanged drug from its dosage form.”
- Brahmankar & Jaiswal
“Bioavailability is a term used to indicate
the fractional extent to which a dose of
drug reaches its site of action or a
biological fluid from which the drug has
access to its site of action.”
- Goodman & Gillman
Factors affecting Bioavailability :
Patient related Routes of
Factors administration

BIOAVAIL
ABILITY
A ) Pharmaceutic factors :
1) Physicochemical properties of drug :
1. Drug solubility & dissolution rate.

2. Particle size & effective surface area.

3. Polymorphism & Amorphism.

 Amorphous > metastable > stable

4. Pseudopolymorphism (Hydrates / Solvates )

 Anhydrates > hydrates e.g. Theophylline, Ampicillin

 Organic solvates > non solvates e.g. fludrocortisone

5. Salt form of the drug.

 Weakly acidic drugs – strong basic salt e.g.barbiturates , sulfonamides.

 Weakly basic drugs – strong acid salt

6. Lipophilicity of the drug .

7. pKa of the drug & pH .

8. Drug stability.
2) Dosage form characteristics &
Pharmaceutic Ingredients :

1. Disintegration time (tab/cap)

2. Dissolution time.
3. Manufacturing variables.
4. Pharmaceutic ingredients ( excipients / adjuvants )
5. Nature & type of dosage form.
 Solutions> Emulsions> Suspensions> Cap> Tab> Enteric Coated Tab >
Sustained Release

6. Product age & storage conditions.

B ) Patient related factors :
1. Age
2. Gastric emptying time .
3. Intestinal transit time .
4. Gastrointestinal pH .(HCL > Acetic > citric )
5. Disease States .
6. Blood flow through the gastrointestinal tract .
7. Gastrointestinal contents :
a) Other drugs .
b) Food .
c) Fluids
d) Other normal g.i. contents
8. Presystemic metabolism (First – Pass effect ) by :
a) Luminal enzymes .
b) Gut wall enzymes .
c) Bacterial enzymes .
C ) Routes of administration :

Parenteral > Rectal > Oral > Topical

Route Bioavailability (%) Characteristics
Intravenous 100 (by definition) Most rapid onset
(IV)

Intramuscular 75 to ≤ 100 Large volumes often feasible;

(IM) may be painful

Subcutaneous 75 to ≤ 100 Smaller volumes than IM;

may be painful
(SC)

Oral (PO) 5 to < 100 Most convenient; first pass effects

may be significant

Rectal (PR) 30 to < 100 Less first-pass effects than oral

Inhalation 5 to < 100 Often very rapid onset

Transdermal 80 to ≤ 100 Usually very slow absorption;

used for lack of first-pass effects;
prolonged duration of action
 Introduction
 The dose available to patient – Bioavailable dose.

 The amount of drug that reaches the systemic circulation –

systemic availability.

 Bioavailable fraction (F), refers to the fraction of administered

dose that enters the systemic circulation.

Bioavailable dose
F =
Administered dose

It ranges from 0 to 1

Bioavailability normally expressed as %

Objectives of bioavailability studies

Primary stage of development of a suitable dosage form for new

drug entity

Determination of influence of excipients, patient related factors

and drug interaction on efficiency of absorption

Development of new formulations

Control of quality of drug product during early stages of

marketing

Comparision of availability of drug substance

Significance of Bioavailability
 Drugs having low therapeutic index, e.g. cardiac glycosides,

quinidine, phenytoin etc.and narrow margin of safety ( e.g.

antiarrythmics, antidiabetics, adrenal steroids, theophylline )

 Drugs whose peak levels are required for the effect of drugs,

e.g. phenytoin, phenobarbitone, primidone, sodium valporate,

anti-hypertensives,antidiabetics and antibiotics.

 Drugs that are absorbed by an active transport,e.g. amino acid

analogues. Purine analogues etc.

 Drugs which are disintegrated in the alimentary canal and
liver,e.g.chlorpromazine or those which under go first pass
metabolism.
 Formulations that give sustained release of drug.
 Any new formulation has to be tested for its bioavailability
profile.
 Drugs with steep dose response relationship i.e. drugs
obeying zero order kinetics / mixed order elimination kinetics
( e.g. warfarin , phenytoin, digoxin, aspirin at high doses,
phenylbutazone)
Considerations in bioavailability studies

Bioavailability – absolute vs. relative

Single dose vs. multiple dose

Human volunteer – healthy subject vs. patients

Absolute Bioavailability ( F )
Def :
“When the systemic availability of a drug
administered orally is determined in
comparison to its intravenous administration ,is
called as Absolute Bioavailability”

Dose (iv) x AUC (oral)

% Absorption = ------------------------------- X
100
Dose (oral) x AUC (iv)
Relative Bioavailability (Fr)
When systemic availability of drug after oral administration is compared with that of an oral standard of same drug

(such as an aqueous or non aqueous solution or suspension) it is referred as relative bioavailability

It is used to characterize absorption of drug from its formulation

Relative Bioavailability == [AUC]test /(Dose)std

[AUC]std/(Dose)test
Single dose vs. multiple dose

Multiple
Single dose
dose
Difficult
Very common
to control
& easy

More
Less exposure
exposuretotodrug
drug&&less
tedious
tedious

Time
Difficult
consuming
to predict steady state
Time to reach steady state

Concentration due to
repeated doses

Concentration due to a single dose

Single dose vs. multiple dose
 Multiple dose study has several advantages
like

1. More accurately reflects the manner in which

the drug should be used.

2. Drugs levels are higher due to cumulative

effect which makes its determination possible
even by less sensitive analytical methods.
Single dose vs. multiple dose

3. Better evaluation of the performance of

controlled release formulation is possible.

4. Small intersubject variability is observed

which allows use of fewer subjects.

5. Nonlinearity in pharmacokinetics, if present,

can be easily detected.
Human volunteer – healthy subject vs. patients

 Ideally, the bioavailability study should be

carried out in patients for whom the drug is
intended to be used.

 Advantages :
1. Patient is benefited from the study.

2. Reflects better therapeutic efficacy of drug.

Human volunteer – healthy subject vs. patients

3. Drug absorption pattern in disease state can be

evaluated.

4. Avoids the ethical quandary of administering

drug to healthy subjects.
Human volunteer – healthy subject vs. patients

There are some drawbacks of using patients as

volunteers.

◦ Stringent conditions such as fasting state required is

difficult to be followed by the patients.

Studies are therefore performed in young (20-40

yrs.), healthy males adult volunteers.
Human volunteer – healthy subject vs. patients

Female volunteers are used only when drugs

such as oral contraceptives are to be tested.

No. of subject- extent of intersubject variability

–minimum required to obtain reliable data.

They must be informed about the importance of

 Study
 conditions to be followed
 Possible hazards if any.
Human volunteer – healthy subject vs. patients

Medical examination should be performed.

Drug washout period for minimum of ten

biological half lives must be allowed for
between two studies in same subject.
Measurement of Bioavailability

Pharmacokinetic
(Indirect )

1.Plasma level time studies

2.Urinary excretion studies

 Plasma level time studies
 This is the method of choice when compared to urine data

 Method is based on the assumption that two dosage forms

that exhibit super impossible plasma level time profiles in a
group of subjects should result in identical therapeutic
activity
 single dose study
Collection of serial blood samples for a period of
2 to 3 biological half-lives

their analysis for drug concentration and

making a plot of concentration versus time

plasma level – time profile

Plasma concentration-time profile
 Parameters considered
Cmax: (Peak plasma concentration)
Maximum concentration of the drug obtained after the
administration of single dose of the drug
Expressed in terms of μg/ml or ng/ml

tmax: (time of peak conc.)

Time required to achieve peak concentration of the drug after
administration
Gives indication of the rate of absorption
Expressed in terms of hours or minutes

AUC (area under curve) is the measurement of the extent

of the drug bioavailability
The extent of bioavailability can be
determined by following equations :

[AUC]oral Div
F=
[AUC]iv Doral

[AUC]test Dstd
Fr=
[AUC]std Dtest

D-----dose administered
 With multiple dose study, the method involves drug
administration for at least 5 biological half-lives with a dosing
interval equal to or greater than the biological half-life(i.e.
administration of at least 5 doses) to reach the steady state.
 A blood sample should be taken at the end of previous dosing
interval and 8 to 10 samples after administration of next dose.
 The extent of bioavailability is given as:

[AUC]test Dstd τtest

Fr= where, τ---time interval
[AUC]std Dtest τstd
Bioavailability can also be determined from
the peak plasma concentration at steady state
Css, max according to the following
equation :
(Cssmax) test D std Ʈ test
Fr=
(Cssmax)std Dtest Ʈstd
 Urinary excretion studies :
Urinary excretion α plasma concentration of drug

 Mainly used in drugs extensively excreted unchanged in urine.

 E.g. Thiazide diuretics

Sulfonamides

Urinary antiseptics : nitrofurantoin ,

Hexamine.
 METHOD FOR URINARY EXCRETION STUDIES

 It involves collection of urine at regular intervals for a time span

equal to 7 biological half lives

 Then analysis for unchanged drug in the collected sample

 Then determined the amount of drug excreted in each interval

and cumulative amount excreted
a. (dXu / dt)max : Maximum urinary excretion rate

b. (tu ) max :Time for maximum urinary excretion rate

c. Xu∞ :Cumulative amount of drug excreted in the urine.

 Three Important Parameters in urine
excretion data for single dose study
1) (dxu/dt)max :(Maximum urinary excretion rate)
◦ Its value increases as rate and/or extent of absorption increases
◦ Obtained from peak of plot between rate of excretion versus midpoint
time of urine collection period
2) (tu) max:
◦ Time for maximum excretion rate
◦ Its value decreases as absorption rate increases
◦ Analogues of tmax of plasma level data
3) Xu:Cumulative amount of drug excreted in urine
◦ Related to AUC of plasma level data
◦ It increases as the extent of absorption increases
The extent bioavailability is calculated from
equtions
 F= ( Xu∞) oral Div
( Xu∞) iv D oral

 Fr= ( Xu∞) test Dstd

( Xu∞) std D test

 with multi dose steady state the equation for bioavailability

 fr = ( Xu,ss) test D std Ʈ test

( Xu,ss) std D test Ʈstd
 Where ( Xu,ss) is the amount of drug excreted unchanged
 ADVATAGES OF URINARY EXCRETION STUDIES

 These method is useful when there is a lack of sufficiently sensitive

analytic tech to measure concentration of drugs in plasma with
accuracy
 Convenince of collecting urine samples.
 Direct measurement of absolute and relative bioavailablity is possible
without the neccesity of fitting the data to a mathematic model
 When coupled with plasma level-time data,it can be used to estimate
renal clearance of unchanged drug, by

CLR=total amount of drug excreted unchanged / AUC

 PHARMACODYNAMIC METHOD
It is also called as direct method

Acute pharmacological response

Therapeutic response
ACUTE PHARMACOLOGICAL RESPONSE

 Acute pharmacological effect such as change in ECG or EEG

readings, pupil diameter etc is related to the time course of a given
drug
 Bioavailability can be determined:
◦ By construction of pharmacologic effect - time curve

◦ By dose-response graphs

Disadvantages :
More variable

Active metabolite interferes with the result.

 THERAPEUTIC RESPONSE
This method is based on observing the clinical response
to a drug formulation given to a patient suffering from
disease for which it is intended to be used
Disadvantages :
Improper quantification of observed response.
E.g.: for anti-inflammatory drugs, reduction in inflammation is
determined
InVitro Drug Dissolution rate and
bioavailabity

• Disintegration studies

• Dissolution studies
Disintegration test :
The early attempts to establish an indicator of drug bioavailability
focused on disintegration as the most pertinent in-vitro parameter.

The first official disintegration test appeared in the United States

Pharmacopeia (USP) in 1950

A solid dosage form must disintegrate before significant

dissolution & absorption can occur

Meeting the disintegration test requirement only ensures that the

dosage form (tablet) will break up into sufficiently small particles
in a specified length of time..
Limitations

It does not ensure that the rate of solution of the drug is
adequate to produce suitable blood levels of the active ingredient

While the test for tablet disintegration is very useful for quality
control purposes in manufacturing, it is a poor index of
bioavailability.
In vitro drug dissolution Testing models

Factors to be considered:
1. Factors relating to dissolution apparatus
2. Factors relating to dissolution fluid
3. Process parameters
DISSOLUTION MEDIUM EXAMPLE
Water Ampicillin caps., butabarbital
sodium tabs.
Buffers Azithromycin caps.,
paracetamol tabs.
HCL solution Cemetidine tabs.
Simulated gastric fluid Astemizole tabs., piroxicam
caps.
Simulated intestinal fluid Valproic caps., Glipizide tabs.

Surfactant solution Clofibrate caps, danazol caps

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Ideal features of dissolution apparatus
 Simple in design
 Fabrication dimensions are specified and reproducible
 Easy introduction of dosage
 Controlled, uniform, non-turbulent liquid agitation
 Minimum mechanical abrasion
 Maintain sink conditions
 Eliminates evaporation of dissolution medium
 Ease to draw samples
 Facilitates good laboratory equipment
 Sensitive to reveal process changes
 Permits evaluation of disintegrating, non-disintegrating,
dense, floating tablets or capsules etc.
 2 principal types of Dissolution Apparatus

Closed compartment Open compartment

apparatus apparatus

3rd type :- DIALYSIS SYSTEMS:- Very poorly aq. Soluble

drugs
1.Closed Compartment Apparatus:
It is a basically limited volume apparatus operating under non-sink
condition
The dissolution fluid is restrained to the size of the container

2.Open Compartment Apparatus:

In this the dosage form is contained in a column which is brought
in continuous contact with fresh, flowing dissolution medium (sink
condition)
OFFICIAL DOSSOLUTION MONOGRAPHS

I.P. USP B.P. E.P.

Type I paddle basket apparatus basket apparatus paddle apparatus

apparatus
Type II basket paddle apparatus paddle apparatus basket apparatus
apparatus
Type III Reciprocating flow through cell flow through cell
cylinder apparatus apparatus
Type IV flow through cell
apparatus
Type V Paddle over disk
Type VI cylinder

Type VII reciprocating holder

According to USP 30 dissolution apparatus used are:

USPAPP DESCRIPTION ROT.SPEED

.

I BASKET 50-120 rpm

II PADDLE 25-50 rpm

III RECIPROCATING 6-35 rpm

CYLINDER
IV FLOW-THROUGH CELL N/A

V PADDLE OVER 25-50 rpm

DISK

VI CYLINDER N/A

VII RECIPROCATING 30 rpm

HOLDER / DISK
USP Apparatus I- Basket Apparatus
•Useful for Drug Product:
Capsules, Beads, Delayed release /
Enteric Coated dosage forms,
Floating dosage forms

•Standard volume: 900/1000 ml

1, 2, 4 liter vessels

•Agitation
– Rotating stirrer
– Usual speed: 50 to 100 rpm
•Advantages:
1) more than 200 monographs.
2) Full pH change during the test
3) Can be easily automated which is important for routine investigation.

•Disadvantages:
1) Disintegration-dissolution interaction
2) Hydrodynamic Dead zone under the basket.
3) Degassing is particularly important
4) Limited volume-sink condition for poorly soluble drugs.
USP Apparatus-II - Paddle Apparatus.

•The dosage unit is allowed to sink

to the bottom of the vessel before
rotation of the blade is started.
•A small, loose piece of no reactive
material such as not more than a few
turns of wire helix may be attached
to dosage units that would otherwise
float.
• Useful
for :
– Tablets
– Capsules
•Standard volume: 900/1000 ml
•Agitation
– Rotating stirrer
– Usual speed: 25 to 75 rpm

Advantages:
•Easy to use
•Robust
•Can be easily adapted to apparatus 5
•long experience
•Can be easily automated which is important for routine investigations.
Disadvantages:
•media change is often difficult
•Hydrodynamics are complex, they vary with site of the dosage form in
the vessel (sticking, floating) and therefore may significantly affect drug
dissolution.
USP Apparatus III – Reciprocating cylinder

The assembly consists of a set of

cylindrical, flat-bottomed glass vessels;
a set of glass reciprocating cylinders;
stainless steel fittings (type 316 or
equivalent) to fit the tops and bottoms
of the reciprocating cylinders; and a
motor and drive assembly to
reciprocate the cylinders vertically
inside the vessels.

Useful for: Tablets, Beads, controlled

release formulations

Standard volume: 200-250 ml/station

Advantages:
1) Easy to change the pH-profiles
2) Hydrodynamics can be directly influenced by varying the dip rate.

Disadvantages:
1) small volume (max. 250 ml)
2) Little experience
3) Limited data
USP Apparatus IV – Flow through cell
•The assembly consists of a reservoir and a pump for the Dissolution
Medium; a flow-through cell; a water bath that maintains the Dissolution
Medium at 37 ± 0.5oC.
•The pump forces the Dissolution Medium upwards through the flow-
through cell.
•Place the glass beads into the cell specified in the monograph.
•Place 1 dosage unit on top of the beads or, if specified in the
monograph, on a wire carrier.
•Assemble the filter head, and fix the parts together by means of a
suitable clamping device.
•Introduce by the pump the Dissolution Medium warmed to 37 ± 0.5oC
through the bottom of the cell to obtain the flow rate specified in the
individual monograph.
•Collect the elute by fractions at each of the times stated.
Useful for:
•Low solubility drugs
•Micro particulates
•Implants & Suppositories
•Controlled release formulations
Variations: (A) Open system & (B) Closed system

Advantages:
•Easy to change media pH
•PH-profile possible
•Sink conditions
•Disadvantages:
•De-aeration necessary
•High volumes of media
•Labor intensive
USP Apparatus V – Paddle over disk
•Use the paddle and vessel assembly from Apparatus 2 with the addition
of a stainless steel disk assembly designed for holding the transdermal
system at the bottom of the vessel.
•The temperature is maintained at 32°C ± 0.5°C
• The disk assembly holds the system flat and is positioned such that the
release surface is parallel with the bottom of the paddle blade
•Borosilicate Glass
•17 mesh is standard (others available)
•Accommodates patches of up to 90mm
USP Apparatus VI – cylinder
•Use the vessel assembly from Apparatus 1 except to replace the basket
and shaft with a stainless steel cylinder stirring element and to maintain
the temperature at 32 ± 0.5oC during the test.

•The dosage unit is placed on the cylinder at the beginning of each test
•Place the cylinder in the apparatus, and immediately rotate at the rate
specified in the individual monograph.
USP Apparatus VII – reciprocating holder
•The assembly consists of a set of volumetrically calibrated solution
containers made of glass or other suitable inert material a motor and
drive assembly to reciprocate the system vertically and a set of suitable
sample holders.
•The solution containers are partially immersed in a suitable water bath
of any convenient size that permits maintaining the temperature
Dissolution acceptance criteria
Q is defined as percentage of drug content
dissolved in a given time period.
Objectives of dissolution profile
comparison
Development of bioequivalent drug products.
Demonstrating equivalence after change in
formulation of drug product.
Biowaiver of drug product of lower dose
strength in proportion to higher dose strength
product containing same active ingredient and
excipients.
2. OPEN FLOW-THROUGH COMPARTMENT SYSTEM

 The dosage form is contained in a small vertical

glass column with built in filter through which a
continuous flow of the dissolution medium is
circulated upward at a specific rate from an
outside reservoir using a peristaltic or centrifugal
pump.
 Dissolution fluid is collected in a separate
reservoir.
 E.g. lipid filled soft Gelatin capsule

78
79
 Useful for
• low solubility drugs
• microparticulates
• implants
• suppositories
• controlled release
formulations

 Variations
• open system
• closed system

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 Advantages
 No stirring and drug particles are exposed
to homogeneous, laminar flow that can be
precisely controlled. All the problems of
wobbling, shaft eccentricity, vibration,
stirrer position don’t exist.
 There is no physical abrasion of solids.
 Perfect sink conditions can be maintained.
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 Disadvantages
 Tendency of the filter to clog because of the
unidirectional flow.
 Differenttypes of pumps, such as peristaltic
and centrifugal, have been shown to give
different dissolution results.
 Temperature control is also much more
difficult to achieve in column type flow
through system than in the conventional
stirred vessel type.
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3. DIALYSIS SYSTEM
 Here, dialysis membrane used as a selective
barrier between fresh solvent compartment
and the cell compartment containing dosage
form.
 Itcan be used in case of very poorly soluble
rugs and dosage form such as ointments,
creams and suspensions.

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84
Reference
 D.M.Brahmankar, Biopharmaceutics and
pharmacokinetics- A Treatise; Vallabh
Prakashan, page no. 315–332.
 Leon Shargel, Applied Biopharmaceutics
& Pharmacokinetics; 4th edition, page
no. :-247-260
Thank
You !