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Bioavailability – The rate and extent (amount) of absorption of an unchanged

drug from its dosage form. (The systemic availability of an administered drug)

❖ Factors affecting bioavailability

1. Pharmaceutical factors related to the physicochemical properties of the drug

and characteristics of the dosage form.
2. Patient-related factors.
3. Route of administration.

Bioavailable Fraction (F) – The fraction of administered dose that enters the
systemic circulation.

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Objectives of Bioavailability Studies –

1. Primary stage of development of a suitable dosage form for a new drug

entity to obtain evidence of its therapeutic utility.
2. Determination of the influence of excipients, patient-related factors, and
possible interaction with other drugs on the efficiency of absorption.
3. Development of new formulations of the existing drugs.
4. Control of quality of a drug product to determine the influence of
processing factors, storage, and stability on drug absorption.
5. Comparison of availability of a drug substance from different dosage forms.

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Absolute Bioavailability (F) – The systemic availability of a drug administered
orally is determined in comparison to its intravenous administration.

Relative Bioavailability (Fr) – The systemic availability of a drug after oral

administration is compared with that of an oral standard of the same drug. (such as
an aqueous or non-aqueous solution or a suspension)
It is also called comparative bioavailability.

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 Measurement of Bioavailability

I. Pharmacokinetic Methods II. Pharmacodynamic Methods

1. Plasma level-time studies. 1. Acute pharmacological response.

2. Urinary excretion studies. 2. Therapeutic response.

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I. Pharmacokinetic Methods –
1. Plasma level time profile
✓ Assumption - Two dosage forms that exhibit superimposable plasma level
time profiles in a group of subjects should result in identical therapeutic
activity.
✓ With a single-dose study, serial blood samples for at least 2 to 3 biological
half-lives are to be taken.
✓ With i.v. dose, sampling should start within 5 minutes of drug
administration, and subsequent samples taken at 15-minute intervals.
✓ 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 to reach the steady state.

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Plasma level-time study parameters

1. Cmax: The peak plasma concentration which is

a function of both the rate and extent of
absorption. Cmax will increase with an increase
in the dose, as well as with an increase in the
absorption rate.
2. tmax: The peak time that gives an indication of
the rate of absorption. It decreases as the rate
of absorption increases. AUC
3. AUC: The area under the plasma level-time
curve that gives a measure of the extent of
absorption or the amount of drug that
reaches the systemic circulation. Determination of Cmax, tmax & AUC on single-dose study

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 ƮA
× ƮB

Determination of AUC and Css,max on multiple dosing up to steady-state

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2. Urinary Excretion Studies
✓ Assumption - Urinary excretion of an unchanged drug is directly proportional to
the plasma concentration of the drug.
✓ The study is particularly useful for –
1. Drugs extensively excreted unchanged in the urine. E.g., thiazide diuretics
2. Drugs that have urine as the site of action. E.g., urinary antiseptics like
nitrofurantoin
✓ This involves the collection of urine at regular intervals for 7 biological half-lives
and the determination of the amount of drug excreted in each interval and the
cumulative amount excreted.
✓ At each sample collection, total emptying of the bladder is necessary to avoid
errors

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Urinary excretion study parameters
1. (dXu/dt)max: The maximum urinary excretion
rate, obtained from the peak of plot
between the rate of excretion versus
midpoint time of the urine collection period.
Its value increases as the rate of and/or
extent of absorption increases.
2. (tu)max: The time for maximum excretion
rate, it is analogous to the tmax of plasma
level data. Its value decreases as the
absorption rate increases.
3. Xu: The cumulative amount of drug excreted
in the urine, is related to the AUC of plasma
level data and increases as the extent of
absorption increases.

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II. Pharmacodynamic Methods
1. Acute Pharmacological Response Method
✓ Acute pharmacological responses like changes in the ECG or EEG readings, pupil
diameter, etc. are measured.
✓ When pharmacokinetic methods are difficult, inaccurate, or non-reproducible,
this method is applied.
✓ Bioavailability can then be determined by the construction of a
pharmacological effect-time curve as well as dose-response graphs.
✓ Measurement of responses for at least 3 biological half-lives of the drug should
be done.
Disadvantages-
1. More variable and accurate correlation becomes difficult
2. The observed response may be due to the active metabolites.

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2. Therapeutic Response Method

✓ Observation of the clinical response to a drug formulation given to patients

suffering from a disease for which it is intended to be used.
✓ There are several drawbacks to this method-
1. Quantitation of observed response is too improper.
2. Patient would be able to receive only a single dose of the drug.
3. Study may be compromised due to drug-drug interaction during multiple
dosing.
4. Physiological status of the object may change.

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In Vitro Drug Dissolution Testing
✓ The rate of dissolution of a drug has an influence on its absorption
characteristics from the GIT.
✓ The best available tool today which can at least quantitatively assure the
bioavailability of a drug from its formulation is its in vitro dissolution test.

Factors to be considered-
➢ Dissolution apparatus- the design, size and shape of the container, nature &
speed of agitation, performance precision of apparatus, etc.
➢ Dissolution media- composition like simulated gastric fluid (0.1N HCl),
simulated intestinal fluid (phosphate buffer), volume, temperature,
maintenance of sink or non-sink condition, etc.
➢ Process parameters like a method of introduction of dosage form, sampling
techniques, changing the dissolution fluid, etc.

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Types of Dissolution Apparatus
1. Closed-compartment apparatus: 2. Open-compartment (continuous
➢ limited-volume apparatus flow-through) apparatus:
➢ operating under non-sink conditions ➢ brought in continuous contact with
fresh, flowing dissolution medium
(Sink condition maintained)

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Apparatus 1- (USP) Apparatus 2-
Rotating Basket Rotating Paddle
✓ cylindrical basket ✓ Paddle acting as a stirrer
made of 22 mesh ✓ distance of 2 cm from
✓ distance of 2 cm from the bottom
the bottom ✓ Tablets, capsules,
✓ capsules or tablets, suspensions, controlled-
suppositories, floating, release products
and delayed release ✓ Sinkers are
formulations. recommended for
capsules or floating
formulations

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Apparatus 3- Apparatus 4-
Reciprocating Cylinder Flow through cell
✓ Set of cylindrical flat- ✓ Consists of a reservoir for
bottomed glass vessels the dissolution medium and
equipped with a pump that forces the
reciprocating cylinders dissolution medium through
✓ Controlled release the cell holding the test
bead-type (pellet) sample (240 to 960 mL/h)
formulations. ✓ Formulations containing
poorly soluble drugs

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Apparatus 5- Apparatus 6-
Paddle over disc Cylinder Apparatus
✓ Consists of a sample ✓ Similar to Apparatus 1
holder or disc that ✓ Steel cylinder is used to
holds the product hold the sample instead
✓ The disc is placed at of a basket
the bottom of ✓ Sample is mounted on an
Apparatus 2 inert porous cellulosic
✓ Evaluation of material and adhered to
transdermal products the cylinder
✓ Evaluation of
transdermal products

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Apparatus 7-
Reciprocating disc
✓ Samples are placed on disc-
shaped holders which
reciprocate vertically
✓ The test is carried out at
320C and a reciprocating
frequency of 30 cycles/min.
✓ Evaluation of transdermal
products and non-
disintegrating controlled-
release formulations

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In Vitro—In Vivo Correlation (IVIVC)
✓ It is the predictive mathematical model that describes the relationship between
an in-vitro property (rate and extent of dissolution) of a dosage form and an in-
vivo response (plasma drug concentration or amount of drug absorbed).
Approaches –
1. By establishing a relationship, usually linear, between the in vitro dissolution
and the in vivo bioavailability parameters.
2. By using the data from previous bioavailability studies to modify the dissolution
methodology in order to arrive at meaningful in vitro-in vivo correlation.
Applications –
1. To ensure batch-to-batch consistency in the physiological performance.
2. In the development of a new dosage form with desired in vivo performance.
3. To assist in validating or setting dissolution specifications.

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 IVIVC developed
on the basis of

Plasma level Data Urinary Excretion Pharmacological

Data Response
➢ A linear relationship ➢ A linear relationship ➢ A linear relationship
between dissolution between dissolution between dissolution
parameters like parameters like parameters like
dissolution rate and dissolution rate and dissolution rate and
plasma level data like urinary excretion data acute
rate of absorption like rate of excretion pharmacological
(Cmax, Tmax) is of unchanged drug is response drug is
developed developed developed

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 IVIVC Levels
Level A Level B Level C Multiple level C
✓ Highly accurate ✓ Not a point-to-point ✓ Single point ✓ It is a correlation
✓ Point-to-point correlation correlation involving one or
relationship between in ✓ E.g., the mean in vitro ✓ It relates one several
vitro dissolution and the dissolution time is dissolution time pharmacokinetic
in vivo rate of absorption compared to the mean point to one parameters to the
✓ curves are residence time pharmacokinetic amount of drug
superimposable and have ✓ There are a number of in parameter dissolved at
the same mathematical vivo curves that will ✓ Useful only as a various time
equation produce similar mean guide in points
✓ Any change in residence time values formulation
manufacturing procedure ✓ So, one cannot rely upon development or
or modification in level B correlation to quality control
formula can be justified justify changes in
without the need for manufacturing or
additional human studies. modification in a formula
 Bioequivalence Studies
Equivalence: Compares drug products with respect to a specific characteristic to a
defined set of standards.

Chemical Equivalence: Two or more drug products contain the same labeled
chemical substance as an active ingredient in the same amount.

Pharmaceutical Equivalence: Two or more drug products are identical in strength,

quality, purity, content uniformity, and disintegration and dissolution characteristics.

Bioequivalence: The drug substance in two or more identical dosage forms, reaches
the systemic circulation at the same relative rate and to the same relative extent i.e.
their plasma concentration-time profiles will be identical.

Therapeutic Equivalence: Two or more drug products elicit identical

pharmacological effects and can control the disease to the same extent.
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 Bioequivalence Experimental Study Design
1. Completely randomized designs
All treatments (factor levels) are randomly allocated among all experimental
subjects.
Advantages
1) The design is extremely easy to construct.
2) It can accommodate any number of treatments and subjects.
3) The design is easy to analyze even though the sample sizes might not be the
same for each treatment.
Disadvantages
1) Although the design can be used for any number of treatments, it is best
suited for situations in which there are relatively few treatments.
2) All subjects must be as homogeneous as possible. Any extraneous sources of
variability will tend to inflate the random error term.

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2. Randomized block designs
Subjects are sorted into homogeneous groups, called blocks and the treatments
are then assigned at random within the blocks.
Advantages
1) It can provide substantially more precise results than a completely
randomized design.
2) Different treatments need not have equal sample size.
Disadvantages
1) Missing observations within a block require a more complex analysis
2) The degrees of freedom of experimental error are not as large as with a
completely randomized design.

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3. Repeated measures, cross-over, and carry-over designs
✓ The same subjects are utilized for the study of different treatments.
✓ The administration of two or more treatments one after the other in a
specified or random order to the same group of patients is called a crossover
design or change-over design
✓ The carry-over effect may appear due to repeated treatments. To prevent
the same, one must always allow for a washout period (about 10 half-lives).
Advantages
1) Provide good precision due to utilization of the same subjects.
2) It is economic on subjects. Only a few subjects can be utilized.
Disadvantages
1) There may be an order effect connected with the position in the treatment
order.
2) There may be a carry-over effect connected with the preceding treatment or
treatments.

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4. Latin square designs
✓ Each subject receives each treatment during the course of the experiment.
✓ It is useful when three or more treatments are to be compared and carry-over
effects are balanced.

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Advantages –
1) It minimizes the inter-subject variability in plasma drug levels.
2) Minimizes the carry-over effects which could occur when a given dosage form
influences the bioavailability of a subsequently administered product
(intrasubject variability).
3) Minimizes the variations due to time effect.
4) Makes it possible to focus more on the formulation variables which is the key to
success for any bioequivalence study.
Disadvantages –
1) The randomization required is somewhat more complex than that for earlier
designs considered.
2) The study takes a long time since an appropriate washout period between two
administrations is essential.
3) When the number of formulations to be tested is more, the study becomes
more difficult, and subject dropout rates are also high.

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Bioequivalence Study Protocol

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METHODS FOR ENHANCEMENT OF BIOAVAILABILITY

Approaches

Pharmaceutical Pharmacokinetic Biological

➢ Modification of Modifying the chemical
formulation, structure by ➢ The Route of drug
manufacturing process ➢ Development of a administration may
or the physicochemical new chemical entity be changed
properties of the drug with desirable
without changing the features
chemical structure. ➢ Prodrug design

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Pharmaceutical Approaches –
A] Bioavailability enhancement by enhancing drug solubility or dissolution rate
1. Micronization
✓ Reducing the size of the solid drug particles to 1 to 10 microns commonly by
spray drying or by use of air attrition methods (fluid energy or jet mill).
✓ E.g., Griseofulvin
2. Nanonisation
✓ Drug powder is converted to nanocrystals of sizes 200 - 600 nm.
✓ E.g., Amphotericin B
✓ The main production technology currently in use is the dispersion of drug
nanocrystals in a liquid, typically water called nanosuspension.

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3. Supercritical Fluid Recrystallization –
✓ Particle size reduction via supercritical fluid (SCF) recrystallization processes.
✓ Supercritical fluids (e.g. carbon dioxide) are fluids whose temperature and
pressure are greater than their critical temperature (Tc) and critical pressure
(Tp), allowing them to assume the properties of both a liquid and a gas.
✓ At near-critical temperatures, SCFs are highly compressible, allowing
moderate changes in pressure to greatly alter the density and mass
transport characteristics of a fluid that largely determine its solvent power.
✓ Once the drug particles are solubilized within SCF, they may be recrystallized
at greatly reduced particle sizes.

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4. Use of Surfactants –
✓ Surfactants enhance the dissolution rate primarily by promoting wetting
and penetration of dissolution fluid into the solid drug particles.
✓ They are generally used in concentrations below their critical micelle
concentration (CMC) values since above CMC, the drug entrapped in the
micelle structure fails to partition in the dissolution fluid.
✓ Nonionic surfactants like polysorbates are widely used.
✓ E.g., steroids like spironolactone

5. Use of Salt Forms

✓ Salts have improved solubility and dissolution characteristics in comparison to
the original drug.
✓ Alkali metal salts of acidic drugs like penicillins and strong acid salts of basic
drugs like atropine are more water-soluble than the parent drug.

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6. Solid Dispersion –
✓ The dispersion of one or more active
ingredients in a hydrophilic inert carrier
matrix at a molecular level.
✓ It is prepared by the melt (fusion)
method and solvent evaporation
technique.
✓ The main mechanism behind the
enhancement of solubility by solid
dispersion is the drug is precipitated in
an amorphous form.

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7. Molecular encapsulation by Cyclodextrins
✓ Cyclodextrins are having the ability to form
molecular inclusion complexes with
hydrophobic drugs having poor aqueous
solubility.
✓ The hydrophobic cavity of the cyclodextrin can
accommodate the lipophilic drug and the outer
surface is hydrophilic which greatly enhances
Functional and structural features of a cyclodextrin
the solubility and dissolution rate of the molecule showing an encapsulated drug.
compound.
✓ E.g., thiazide diuretics, barbiturates,
benzodiazepines and a number of NSAIDs

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B] Bioavailability enhancement by enhancing drug permeability through
membrane
1. Lipid Technologies
(a) Lipid solutions & suspensions – Lipophilic drugs like steroids can be
dissolved in oils like triacylglycerols
(b) Coarse emulsions, microemulsions, SEDDS, SMEDDS – These systems are
formed using an oily vehicle (or a mixture of a hydrophilic phase and a
lipophilic phase) a surfactant with a high HLB and if required, a co-
surfactant
(c) Solid lipid nanoparticles - The liquid lipid is replaced by a solid lipid leading
to the formation of solid lipid nanoparticles
Homogenization of melted lipids at elevated temperature, and
Homogenization of a suspension of solid lipids at or below room temperature.
(a) Liposomes - lipid bilayers surrounding an aqueous space. Liposoluble drugs
can be embedded in the fatty regions, while hydrophilic substances are held
in the aqueous internal spaces of these globular vesicles.
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2. Ion Pairing –
✓ Co-administration of a hydrophilic or polar drug with a suitable lipophilic
counterion
✓ Improves the partitioning of the resultant ion-pair (relatively more lipophilic)
into the intestinal membrane.
✓ E.g., Atenolol
3. Penetration Enhancers –
✓ Compounds that facilitate the transport of drugs across the biomembrane are
called as penetration/permeation enhancers or promoters.
✓ Used mainly in cases of hydrophilic drugs having difficulty penetrating the
lipid structure of the biomembrane.
✓ Acts in the interaction of its lipid part with the polar component of membrane
phospholipids.

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C] Bioavailability enhancement by enhancement of drug stability
1. Enteric coating –
✓ Utilize polymeric coatings that are insoluble in the gastric media and
therefore, prevent or retard drug release in the stomach.
✓ Such systems release the drug in the alkaline environment of the intestine.
✓ E.g., Bioavailability of drugs that are unstable are gastric media like
omeprazole, erythromycin, penicillin V can be improved
2. Complexation –
✓ Complexation of drugs by agents like cyclodextrins, caffeine, sodium
salicylate, sodium benzoate can be used to increase the stability of drugs in
the GI environment.

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3. Use of metabolism inhibitors –
✓ Co-administration of a drug with low bioavailability and its metabolism
inhibitor, which can selectively inhibit any of the metabolic processes, would
result in increased fractional absorption and hence a higher bioavailability.
✓ E.g., co-administration of ketoconazole and grapefruit juice with cyclosporin,
which contains the inhibitory components, can significantly decrease the pre-
systemic metabolism

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D] Bioavailability enhancement by gastrointestinal retention
✓ Gastro-retentive drug delivery systems (GRDDS) are designed on the basis of
delayed gastric emptying and CR principles and are intended to restrain and
localize the drug delivery device in the stomach or within the upper parts of
the small intestine
✓ Excipients that are bio-adhesive or that swell on hydration when
incorporated in an oral dosage form, can promote gastro-retention and
absorption by –
1. Increased contact with epithelial surfaces
2. Prolonging residence time in the stomach
3. Delaying intestinal transit.

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