BIOAVAILABILITY AND

BIO-EQUIVALENCE
 BIOAVAILABILITY
Bioavailability is the rate and extent (amount) of drug absorbed from the drug
product or dosage form after administration, which is absorbed into the
blood circulation or systemic circulation through biological membrane and
reaches at site of action.

.
 OBJECTIVES OF
BIOAVAILABILITY
Bioavailability data provides beneficial information relating to efficacy of the drug. The objectives
of the bioavailability study are:
To find out the extent of absorption from administered dose.
To find out the rate or time of absorption of drug.
To determine the amount of drug in the biological liquids or tissues.
To correlate relation between concentration of drug in the plasma and clinical response.
To compare bioavailability from different production batches.
To equate the bioavailability of same dosage form with different manufacturer.
To compare systemic bioavailability of different dosage form of same drug.
To design the dosage regimen for the patient.
Useful for bioequivalence studies.
Duration of activity can be determined.
To determine the selection of excipients for dosage regimen based on bioavailability studies.
 RELATIVE AND ABSOLUTE
BIOAVAILABILITY STUDIES
RELATIVE BIOAVAILABILITY
Bioavailability of marketed drug product/dosage form area under the curve as
recognized as standard accepted by Food Drug and Administration, the extent or
amount of drug from a test formulation area under the curve is compared to
reference drug product is called relative bioavailability.
The test formulation could be a novel/new dosage form, being introduced by the
manufacturer.
The comparative availability of two drugs given at the same dosage level as well as
same route of administration can be obtained as shown in the Fig. , using below
equation to estimate the relative bioavailability
E.g. Paracetamol Tablet 500 mg is available in the market, manufacturer is decided to market
Paracetamol capsules 500 mg, and relative bioavailability is determined by comparing area under the
curve of reference tablets dosage form is compared with test formulation (capsules) area under the
curve. Percent Relative bioavailability was calculated from the equation.
Relative bioavailability = [AUC]test/ [AUC]std × 100
When the doses are not equal, the equation becomes,
Relative bioavailability = [AUC]test/Dose test /[AUC]std/Dose std × 100
Consider, Paracetamol marketed tablet 500 mg is given orally and collected the blood
sample from at specified time intervals and analyse drug concentration by using
analytical technique, From the calculated data, the area integrated under plasma drug
concentration versus time curve is drawn and AUC is found to be 115 µg × h/ml and
from test formulation Capsule 500 mg plasma drug concentration is found to be 110
µg × h/ml.
The relative bioavailability of capsules formulation = (110/115) × 100 = 95.6%
So amount of drug absorbed from capsule dosage form is 95.6/100 × 500 = 478 mg.
 ABSOLUTE AVAILABILITY
The absolute bioavailability of drug is the systemic availability of drug from dosage form after extra vascular
administration such as oral, rectal, transdermal or subcutaneous administration compared with IV administration
of same drug. The absolute bioavailability of drug is usually measured by comparing AUC of extra vascular with
AUC of IV administration.
Absolute bioavailability (F) = [AUC]ev /[AUC]IV
Then the doses are not equal, the equation becomes,
Absolute bioavailability (F) = [AUC]ev/(Dose)ev
[AUC]IV/(Dose)IV
The fraction of drug, after injected through IV route considered as 100% bioavailability or F = 1.
Consider, 200 mg of tablets taken orally, the average values of AUC from 0-24 hours is 78 µg/ml and IV bolus
injection having 50 mg of drug, got the AUC from 0-24 hours is 39 µg /ml.
The Absolute bioavailability (F) = 78/200= 3.9/0.78 = 5 × 100 = 50%
39/50
 METHODS TO
ENHANCE THE
BIOAVAILABILITY
Co-Solvency: The solubility of poorly water-soluble drug can be improved by adding
water miscible solvent in which the drug has better solubility is known as co-solvents.
This is one of the most extensively used techniques because it is easy to prepare and
evaluate. Co-Solvents used in co-solvent mixtures are PEG 300, ethanol or propylene
glycol. E.g. Simvastatin drug solubility is increased by co-solvency technique.
pH Adjustment: Poorly water-soluble drug molecules can be protonated (base) or
deprotonated (acid) can possibly be dissolved in water by pH adjustment. E.g. Aspirin
Buffered Tablets
 Particle Size Reduction: The bioavailability is mainly depending on drug particle size.
By decreasing the particle size, increased surface area. Particle size reduction is done by
milling techniques using jet mill, colloid mills, and micronization and nanosuspension
techniques.
1.Micronization: In micronization the solubility of drug is related to drug particle size. By
decreasing the particle size, which increases the surface area, it enhances the dissolution
properties of the drug. E.g., Griseofulvin increased 50 times more solubility after
micronization.
.
 2.Nanosuspension: Nanosuspension is another method which is sub-micron colloidal dispersion of
drug particles, which are become stable by surfactants. E.g., Danazol nanosuspension.
3.Microemulsions: A microemulsion is a clear mixture of oil, hydrophilic surfactant and hydrophilic
solvent which dissolves a very poorly water- soluble drug. E.g. Felodipine microemulsion

Complexation: Complexation with cyclodextrins has been used to increase the aqueous
solubility and drug stability. In the cyclodextrin structure, the ring is hydrophilic and lipophilic
core in which uniform sized organic molecules can form non-covalent inclusion complexes
result in improved chemical stability as well as aqueous solubility. E.g. Benzodiazepines,
Barbiturates.
 Supercritical Fluid (SCF) Process: Supercritical fluids are fluids in which temperature and
pressure are more than its critical temperature and also critical pressure allowing the properties
of both a liquid and a gas. At near critical temperature these are highly compressible letting
moderate changes in pressure to prominently alter the density and mass characteristics of fluid
that determine its solvent properties. Solubilized drug within the SCF (usually carbon dioxide),
they may be recrystallized.
Solid Dispersions: In this process, a poorly soluble drug is dispersed in a very soluble
solid hydrophilic matrix, which greatly enhances the dissolution of the drug. E.g.
Carbamazepine in polyethylene glycol 4000 solid dispersion, enhance the rate and extent
of dissolution of carbamazepine.
Hydrotropy: Hydrotropy is a solubilisation technique, where by addition of second
solute results in an increase in the aqueous solubility of other solute. E.g. Ibuprofen,
Riboflavin solubility can be increased by hydrotropy technique.
 Surfactants: Surfactants is used to improve the dissolution of poorly soluble drug has
been successfully adopted. Surfactants can decrease the surface tension and increase the
dissolution of lipophilic drugs in aqueous solution. Concentration of surfactants in the
range of 0.05-0.10% micelle formation will occur, drugs will entrap within the micelles.
This process is known as micellisation, which enhanced solubility of poorly soluble drugs.
E.g. Non-ionic surfactants include polysorbates, polyoxyethylated glycerides and fatty
acid esters of low molecular weight PEGs.
Salt forms: Salts have enhanced solubility and dissolution characteristics when compared with the
original drug. E.g. Atropine sulphate is more soluble than Atropine alone. Chlorpheniramine maleate
is more soluble than Chlorpheniramine alone.
Polymorphism: Drug can exist in many crystalline forms, the different forms are entitled as
polymorphs and the phenomena are called as polymorphism. The polymorphs are different from one
another with respect to physical properties such as solubility, hardness, density, melting point, and
compression characteristics. E.g.: Chloramphenicol palmitate exists in A, B and C forms. B form
shows better solubility, because it exists in metastable state. A and C forms are exist in unstable and
stable state.
Solid solutions: A binary mixture comprises solid solutes molecularly dispersed in a solid solvent.
Since, the two components are mixed in a homogenous one phase system. Solid solutions are also
called as molecular dispersion or mixed crystals. This is achieved by using fusion technique. E.g.
Succinic acid-Griseofulvin solid solution 6 to 7 times more soluble than Griseofulvin alone.
Eutectic Mixtures: Eutectic powder is defined as a mixture of powdered ingredients, which melt
when mixed with one another. Eutectic mixtures are the proportion of components which will give
the lowest melting point and liquefaction. This show more solubility than parent drug. E.g.
Paracetamol-Urea, Ibuprofen-Mannitol.
Hot Melt Extrusion (HME): Hot melt extrusion (HME) is the process in which heat
and pressure is applied to melt a polymer and drug, forced it through an orifice in a
continuous process. E.g. Valsartan solubility is improved by using hot melt extrusion
technique.
Spray Drying: Spray drying is the process of changing a mixture from its liquid form
to a powder. This is prepared by removing the moisture component from the liquid
solution by applying mechanical force. E.g. Heat sensitive drugs such as milk
products, liposomes.
Solvent evaporation: To dissolve both the drug and the carrier in a solvent and then
evaporate the solvent under vacuum to yield a solid solution. E.g. Meloxicam,
Naproxen, and Nimesulide using solvent evaporation technique.
Precipitation technique: In precipitation technique, the drug is dissolved in a suitable
solvent, which is then mixed to antisolvent to precipitate the crystals. E.g. Naproxen
crystals.
 Cryogenic techniques: Cryogenic techniques has been developed to improve the dissolution
rate of drugs by producing nanostructured amorphous particles with high degree of porosity at very
low temperature conditions. E.g. Repaglinide drug solubility increased by cryogenic technique.
 Another approach is to increase the permeation through biological membrane using the following
method.
1. Using Suitable Polymer: Biocompatible polymer chitosan has found a number of applications
in drug delivery including absorption of hydrophilic macromolecular drugs. When protonated at pH
6.5 is able to increase the paracellular permeability of peptide drugs through mucosal epithelia.
2. Self-Micro-emulsifying Drug Delivery Systems (SMEDDS): SMEDDS are isotropic
mixtures of oils, surfactants/co-surfactants and co-solvents. Once administered into the GI tract, they
are diluted with GI fluid and the gastric motility provides due to agitation oil-in-water (o/w) micro
emulsion (SMEDDS).
3. Spray Freeze Drying: Spray freeze drying is one of the techniques to enhance permeability
e.g. Spray freeze drying of oleonolic acid with PVP-40 as stabilizer and sodium caprate as
wetting agent and penetration enhancer produces stable, amorphous solid dispersion system with
better in vitro dissolution and more absorption in comparison with commercial OA tablets.
4. Nano Particulate System: Nanoparticles are produced by using suitable polymers. E.g;
PLGA can increase the drug across the BBB. Nanoparticles also increase the bio-distribution and
reduce the drug toxicity, so increases the therapeutic efficacy.
5.Lipid Technology: Straight chain fatty acids e.g. Lauric acid (C12) and long chain fatty
acids, such as oleic acid (C18) have been observed practically, to increase the permeability of a
series of hydrophilic drugs by widening the tight junction and/or altering the cytoskeleton of the
intestinal epithelial cells without cytotoxicity. Lipid based formulations can enhance the
permeation of BCS class III and IV Drugs. e.g. Liposomes are nanosized or microsized vesicles
containing a phospholipids bilayer that surrounds an aqueous core. The core may be the water
soluble drug and the outer membrane is hydrophobic layer, usually recognized from phagocytic
cells and is moved from the blood rapidly.
6. Ion Pairing: It involves the co-administration of hydrophilic or polar drug with a
suitable lipophillic counter ion, which improves the partition coefficient in the intestinal
tract. E.g. Atenolol is a ionisable drug which increases the oral bioavailbility by ion
pairing method.
7. Penetration Enhancers: Penetration enhancers are interacting with polar component
of lipid structure in the biological membrane, to facilitate the transport of drugs across
the through temporary rupture /injury of the biological membrane. E.g. Fatty acids such
as oleic acid and lanolin acid, certain bile salts, SLS etc.
 IN -VITRO DRUG
DISSOLUTION MODELS
 DISSOLUTION TESTING
 Dissolution and drug release tests are in-vitro tests that measure the rate and extent of dissolution or
release of the drug substance from a drug product, usually aq.medium under specified conditions.
 It is an important QC procedure for the drug product and linked to product performance in-vivo.

 NEED FOR DISSOLUTION TESTING:

 Evaluation of bioavailability.
 Batch to batch drug release uniformity.
 Development of more efficacious and therapeutically optical dosage forms.
 Ensures quality and stability of the product.
 Product development, quality control, research and application.
 APPARATUS-1(ROTATING BASKET)
 DESIGN:
 Vessel: -Made of borosilicate glass.

-Semi hemispherical bottom

-Capacity 1000ml
 Shaft : -Stainless steel 316

-Rotates smoothly without significance wobble(100 rpm)

 -Speed regulator
 Water bath:-Maintained at 37±0.5ºC
 USE: Tablets, capsules, delayed release suppositories, floating dosage
forms.
 METHOD(Rotating basket):
 Place the stated volume of the dissolution medium(±1 %) in the vessel and equilibrate
dissolution medium to 37±0.5°C.
 Place 1 tablet or capsule in the apparatus ,taking care to exclude air bubbles from the surface
of the dosage form unit and immediately operate the apparatus at the rate specified(100rpm).
 Withdraw a specimen from a zone midway between the surface of the dissolution medium and
the top of the rotating basket,not less than 1cm from the vessel wall at each times stated.
 Replace the aliquots withdrawn for analysis with equal volumes of fresh dissolution medium
at 37°C.
 Keep the vessel covered for the duration of the test and verify the temperature of the
mixture under test at suitable times.
 Perform the analysis as directed in individual monograph and repeat the test with additional
dosage form units.
 Advantages
 Full pH change during the test
 Can be easily automated which is important for
routine investigations.

 Disadvantages
 Basket screen is clogged with
gummy particles.
 Hydrodynamic „dead zone“ under the basket
 Degassing is particularly important
 Mesh gets corroded by HCl solution
 APPARATUS-2 (PADDLE
 DESIGN:

 Vessel: -Same as basket apparatus

 Shaft: -The blade passes through the shaft so that the bottom of the blade
fuses with bottom of the shaft.
 Stirring elements: -Made of Teflon
For laboratory purpose
-Stainless steel 316
 Water-bath: -Maintains at 37±0.5°C

 Sinkers : -Platinum wire used to prevent

tablet/capsule from floating
METHOD
 Itconsists of a special coated paddle formed from a blade and a shaft that
minimizes turbulence due to stirring.
 The coated material is inert.
 The paddle is attached vertically to a variable -speed motor that rotates at a
controlled speed.
 The tablet or capsule is placed into a round-bottom dissolution flask and the
apparatus is housed in a constant temperature water bath maintained at 37°C.
 Most common operating speeds are 50rpm for solid oral dosage forms and 25 rpm
for suspensions.
 A sinker ,such as few turns of platinum wire may be used to prevent a capsule or
tablet from floating.
 Used for film coated tablets that stick to the vessel walls or to help to position
tablet/capsule under the paddle.
 Advantages
 Robust

 Easy to use

 pH change possible

 Can be easily automated which is important for routine investigations

 Disadvantages

 Hydrodynamics are complex, they vary with site of the dosage form in the vessel

(sticking, floating) and therefore may significantly affect drug dissolution

 Sinkers for floating dosage forms
 APPARATUS-3(RECIPROCATING
CYLINDER)
 DESIGN:
 Vessel: -Set of cylindrical flat bottom glass vessels
-Set of reciprocating cylinders

-stainless steel fittings(type 316) and screens

made of non-absorbing or non-reactive materials.

 Agitation type: -Reciprocating-5-35 rpm

 Volume of dissolution medium:-200-250ml
 Water bath:- Maintain at 37±0.5°C
 USE: Tablets, beads, controlled and extended
release formulations
METHOD(Reciprocating cylinder):
 Place the stated volume of dissolution medium in each vessel of the apparatus,
assemble the apparatus, equilibrate the dissolution medium to 37±0.5 and remove
the thermometer
 Place one dosage form unit in each of the cylinders taking care to exclude the
air bubbles from the surface of each dosage unit and immediately operate the
apparatus as specified in the monograph.
 During the upward and downward stroke, the reciprocating cylinder moves
through a total distance of 9.9 to 10.1cm.
 Within the time interval specified raise the cylinders and withdraw a portion of the
solution under test from a zone midway between the surface of the dissolution
medium and bottom of each vessel.
 Advantages
 Easy to change the pH
 Hydrodynamics can be directly influenced by varying the dip rate

 Disadvantages
 Small volume (max. 250 ml)
 Limited data
 APPARATUS-4 (FLOW THROUGH CELL
 DESIGN:
 Reservoir : -For dissolution medium
 Pump : -Forces dissolution medium through cell

-Holding a sample
-Flow rate 10-100ml/min
-Laminar flow is maintained

 Water bath:- Maintain at 37±0.5°C

 USE:
 Low solubility drugs ,micro particulates ,implants, suppositories controlled release
formulations
METHOD(Flow through cell):
 The flow through cell is transparent & inert mounted
vertically with filters.
 Standard cell diameters are 12 & 22.6 mm.
 The bottom cone usually filled with glass beads of 1mm
diameter.
 Tablet holder used for positioning special dosage form e.g.
inlay tablets.
 Place the glass beads into the cell as specified in the
monograph.
 Place one dosage unit on top of the beads or on a wire carrier.
 Assemble the filter head and fix the parts together by means
of a suitable clamping device.
 Introduce by the pump of the dissolution medium warmed
to 37±0.5 through the bottom of the cell to obtain the
flow rate specified and measured with an accuracy of 5%.
 Collect the eluate by fractions at each of the times stated.
Advantages
easy to change media pH
pH-profile possible
Sink conditions maintained

Disadvantages
Deaeration necessary

labor intensiveof media
high volumes
 APPARATUS-5(PADDLE-OVER-DISK)
 DESIGN:
 Vessel
 Shaft
 Stirring elements- rotating speed 25-50 rpm

 Sample holder:-disk assembly that hold a product in such a way

that release surface is parallel with paddle

-Paddle is directly attached over disk assembly

-Samples are drawn between surface off the medium

and top of the paddle blade
 Volume:900ml
 Temperature:32°C
METHOD(Paddle over disk)
 This method is used for testing the release of drugs from transdermal
products.
 The apparatus consists of a sample holder or disc assembly that holds the
product.
 The entire preparation is placed in a dissolution flask filled with specified medium
maintained at 32ºC.
 The paddle is placed directly over the disc assembly.
 The disk assembly holds the system flat and is positioned such that release surface is
placed parallel with the bottom of the paddle blade.
 Vessel is covered to minimize evaporation during test.
 Samples are drawn midway between the surface of dissolution medium and the top
of the paddle blade at specified times.
 USE: Transdermal patches, ointments, floaters , emulsions.
 Modification: Disk design and volume
 Advantages:
 Easy to handle
 Sink conditions are maintained.
 Membrane effect is minimum.

i.e. drug is placed on a disc at the bottom.

 Disadvantages:
 Disk assembly restricts the patch size
 APPARATUS-6(ROTATING CYLINDER)
 DESIGN:
 Vessel:- In place of basket, cylinder is used.
 Shaft :-Stainless steel 316

 Sample :- Mounted to cuprophan (inner porous cellulosic material)

an entire system adheres to cylinder.
- Dosage unit is placed in cylinder and release from side
out.
 Water-bath: maintained at 32±0.5°C
 USE:
 Transdermal patches cannot be cut into small size.
 Solid dosage forms, pH profile , small volumes
 METHOD( Rotating cylinder):

 Use the assembly from apparatus 1 except to replace the basket and shaft with a stainless steel cylinder
stirring element.
 The temperature is maintained at 32±0.5°C.
 The dosage unit is placed on the cylinder with side out .
 The dosage unit is placed to the exterior of the cylinder such that long axis of the system fits around the
circumference of the cylinder and removes trapped air bubbles.
 Place the cylinder in the apparatus and immediately rotate at the rate specified in the individual
monograph.
 Samples are drawn midway between the surface of the dissolution medium and the top of the rotating
cylinder for analysis.
 Advantages: -Equipment (apparatus 1)available
with the manufacturers can be used with
modification as apparatus 6.
 Disadvantages:-Large volume of medium is
required.
-Drug gets diluted & causes
difficulties in analysis
-Difficult to clean the
cylinder.
 APPARATUS-7(RECIPROCATING-DISK)
 DESIGN:
 Vessel:-Flat bottomed cylindrical vessel

-Volume of dissolution medium

 Shaft :
 Sample : -Placed on disk shaped holders
 Agitation :-Reciprocation

-Reciprocating frequency 30 cycle/sec

 Water-bath:-Maintain at 32±0.5°C
 USE:
 Transdermal patches
 UNOFFICIAL METHODS
1.ROTATING/STATIC DISK METHOD
 Developed by late Eino nelson and described by Levy and Sahli.
 In this method ,the drug is compressed in a non-disintegrating disc without
excipients.
 The disc is mounted in a holder so that only one face of the disc is exposed to the
dissolution medium.
 The holder and disc are immersed in medium and held in a fixed position as in
static disc method and rotated at a given speed in rotating disc method.
 Samples are collected at predetermined times.
 Surface area of the drug through which dissolution occurs is kept
dissolution rate.
2.BEAKER METHOD:
 Reported by Levy and Hayes(1960).

 Dissolution medium, 250ml of 0.1N HCl at 37°C placed in a 400ml beaker.

 Agitation by three blade polyethylene stirrer,5cm diameter and rotates at 60
rpm.
 Stirrer immersed to a depth of 2.7 cm in medium and in the center.
 Tablets are placed in a beaker and test was carried out.
 Samples are removed and assayed for the content.

3.FLASK STIRRER METHOD

 Developed by Poole(1969).
 It includes RBF and a stirring element similar to that of beaker method.
 RBF used to avoid the formation of moulds of particles in different positions on the flat
bottom of a beaker.
4.PERISTALSIS METHOD:
 To stimulate hydrodynamic condition of GIT tract in an in-vitro dissolution device.
 It consists of rigid plastic cylindrical tubing fitted with septum and rubber stopper at both ends.
 Dissolution chamber consists of a space between septum and lower stopper.
 Dissolution medium is pumped with peristaltic action through the dosage form.

5.ROTATING BOTTLE METHOD:

 It consists of rotating rack to hold sample drug products in bottle and they are capped tightly &
rotated in 37°C temperature bath.
 Sample are decanted through a 40 mesh screen and residue are assayed.
6.DIALYSIS METHOD:
 Cell consist of 32mm inflated membrane.
 Plugged at the lower end by tight fitting cylindrical perspex box.
 Upper end of the tube held by thin perspex ring inserted into the tube and secured by an elastic
band.
 The cell suspended , from the arm of the tablet disintegration apparatus and containing the
dosage form in 150ml of distilled water at 37°C.
 The cell is raised or lowered 30times a min, into 150ml of distilled water at same temperature.
 Agitation by slight flexing and stretching of the dialysis membrane as it enters and leaves the bath.
 Rotated at 60rpm.
7.DIFFUSION CELL
 Static or flow through diffusion cells are used to characterize in-
vitro drug release and drug permeation kinetics from a topical drug
product eg: Ointment, cream or transdermal drug product.
 The Franz diffusion cell is static diffusion system used to
characterize drug permeation through skin model.
 The skin is mounted on the Franz diffusion cell and the drug
product is placed on the skin surface.
 The drug permeates across the skin into a receptor
fluid compartment that may be sampled at various
times.
 This system is used for selection of appropriate
formulation that has optimum drug delivery.
IN VITRO—IN VIVO CORRELATION (IVIVC)
A simple in vitro dissolution test on the drug product will be insufficient to predict its
therapeutic efficacy.
 Convincing correlation between in vitro dissolution behaviour of a drug and its in vivo
bioavailability must be experimentally demonstrated to guarantee reproducibility of
biological response.
 In vitro-in vivo correlation is defined as the predictive mathematical model that
describes the relationship between an in-vitro property (such as the rate and extent of
dissolution) of a dosage form and an in-vivo response (such as the plasma drug concentration
or amount of drug absorbed).
The main objective of developing and evaluating an IVIVC is to enable the dissolution test
to serve as a surrogate (alternate) for in vivo bioavailability studies in human beings.
THE APPLICATIONS OF
DEVELOPING IVIVC
 To ensure batch-to-batch consistency in the physiological performance of a drug product
by use of such in vitro values.
 To serve as a tool in the development of a new dosage form with desired in vivo
performance.
To assist in validating or setting dissolution specifications (i.e. the dissolution
specifications are based on the performance of product in vivo)

There are two basic approaches by which a correlation between dissolution testing
and bioavailability can be developed:
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.
Some of the often used quantitative linear in vitro-in vivo correlations are –
1. Correlations Based on the Plasma Level Data: Here linear relationships between
dissolution parameters such as percent drug dissolved, rate of dissolution, rate constant for
dissolution, etc. and parameters obtained from plasma level data such as percent drug
absorbed, rate of absorption, Cmax, tmax, Ka, etc. are developed; for example, percent drug
dissolved versus percent drug absorbed plots.
2. Correlation Based on the Urinary Excretion Data: Here, dissolution parameters are
correlated to the amount of drug excreted unchanged in the urine, cumulative amount of drug
excreted as a function of time, etc.
3. Correlation Based on the Pharmacological Response: An acute pharmacological effect
such as LD50 in animals is related to any of the dissolution parameters.
Statistical moments theory can also be used to determine the relationship such as mean
dissolution time (in vitro) versus mean residence time (in vivo).
 IN VITRO-IN VIVO
CORRELATION LEVELS
Level A – The highest category of correlation, it represents a point-to-point relationship
between in vitro dissolution and the in vivo rate of absorption (or in vivo dissolution) i.e.
the in vitro dissolution and in vivo absorption rate curves are superimposable and the
mathematical description for both curves is the same.
Advantages of level A correlation are as follows –
1. A point-to-point correlation is developed.
2. The in vitro dissolution curve serves as a surrogate for in vivo performance. Any
change in manufacturing procedure or modification in formula can be justified without
the need for additional human studies.
3. The in vivo dissolution serves as in vivo indicating quality control procedure for
predicting dosage form‘s performance.
Level B –
Utilises the principles of statistical moment analysis. The mean in vitro dissolution time
is compared to either the mean residence time or the mean in vivo dissolution time.
 However, such a correlation is not a point-to-point correlation since there are a number
of in vivo curves that will produce similar mean residence time values.
 It is for this reason that one cannot rely upon level B correlation to justify changes in
manufacturing or modification in formula.
So the in vitro data cannot be used for quality control standards.
Level C – It is a single point correlation. It relates one dissolution time
point (e.g. t50%, etc.) to one pharmacokinetic parameter such as AUC,
tmax or Cmax. This level is generally useful only as a guide in
formulation development or quality control with its obvious limitations.
Multiple Level C – It is correlation involving one or several
pharmacokinetic parameters to the amount of drug dissolved at various
time points.
 BIOEQUIVALENCE STUDIES
Bioequivalence: It is a relative term which denotes that 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 without
significant statistical differences. .
The factors include:
(a) Use of different formulation materials (E.g. Lubricants, diluents, disintegrants etc.)
(b) Use of similar materials from different vendors or suppliers.
(c) Validity of process variables.
(d) Method of manufacture differs.
(e) Equipment's used in the manufacturing process.
 PHARMACEUTICAL
EQUIVALENTS
FDA defines the term pharmaceutical equivalents as follows: Drug products are said to be
pharmaceutical equivalents if they contain the same active ingredients and are
indistinguishable in their strength or concentration, dosage form and route of
administration. These products may or may not contain same excipients. e.g. Two tablet
formulation ingredients as given in Table.
The Tablets A and Tablet B contains same Active ingredients, but different excipients are
identical in their route of administration,
Pharmaceutical Alternatives: Pharmaceutical alternatives are those drug products that
have the same therapeutic moiety, but variety of salts, esters or complexes of that moiety or
are different dosage forms or strength. E.g. The two formulations of drug products are given
in Table 4.3.

Here, A and B products contain same active ingredients, but not pharmaceutical equivalents
because the dosage form is different, one dosage form is tablet and another one is
suspension, these are pharmaceutical alternatives.
Bioequivalence Study Design: A study design is used to estimate the essential
pharmacokinetic parameters differ significantly from bioequivalence study meant for
comparing the test formulation with reference product.
The simplest and most common experimental design in comparison with
bioavailability of two drug products is cross over design study.
Crossover Design Study: In this method, the effect of intersubject variability in the
study with each subject as its own control.
Usually, two types of cross over design are used for the bioavailability studies.
1. Latin Square Cross over design
2. Balanced Incomplete block design
 LATIN SQUARE CROSS OVER
DESIGN
In this, every subject receives just once every formulation and every formulation is administered
only one time in every study.
Design is two ways, three ways or four ways cross over design.
In two ways cross over study, during selection of the subjects age, gender, weight, disease states are
taken into consideration.
 Here, 12 subjects are used for the study of bioequivalence of two formulations, treatment A and
treatment B.
During first study, subjects 1- 6 receive treatment A, while subjects 7-12 receive treatment B.
A second study is started after the washout period; here we need to wait for complete removal of the
drug from the body.
In the second study period, subjects 1 - 6 now receives treatment B and subjects 7 - 12 receives
treatment A as show in the Table
Disadvantages of this study: It requires lengthier time to complete the study, for the
reason that wash out period exist among two study periods due to more biological half life
of drug and increased number of study period indications to high subjects dropouts and
the study is tiresome.
Washout period: The time interval in the middle of two treatments is called washout
period. Complete or 99% elimination of administered dose of a drug based on the half life
of the drug. At least 6 to 7 biological half-lives should be permitted between the
treatments.
BALANCED INCOMPLETE
BLOCK DESIGN
If more than three formulations Latin square design is not advisable.
Balanced incomplete block design eradicates many of the difficulties encountered with
Latin square design.
If each volunteers predictable to receive at least two formulations, then such studies can
be done by using this design. E.g. Below Table shows four formulations A, B, C and D.
In this design each subjects receives two formulations, and each formulation is
administered six times.
PARALLEL GROUP DESIGN
In this design, subjects are separated into two groups’ odd number and even number
subjects, receives different formulation as shown in the below table.
 No washout period is necessary.
This design is more useful for drugs having long half
REPLICATED CROSSOVER
STUDY DESIGN
Intra subject variability is observed for highly variable drugs.
Here not necessarily to expose a large number of healthy subjects.
Replicated crossover designs are useful for the determination of individual
bioequivalence to estimate the intra subject variability for both the test and reference drug
products.
A four period, two series, two formulation design is shown in the Table
 MEASUREMENT OF
BIOAVAILABILITY OR METHODS OF
ASSESMENT OF BIOAVAILABILITY
INDIRECT METHODS OR
PHARMACOKINETIC
METHODS
From human plasma data:
(a) The peak plasma drug concentration (Cmax)
(b) The time of ultimate plasma concentration (tmax)
(c) Area under the curve (AUC)
THE PEAK PLASMA DRUG
CONCENTRATION (CMAX)
The peak plasma concentrations obtained after extra vascular administration of drug
product, collect the blood samples at predetermined time intervals from the human body and
measure the drug concentration at each time intervals.
Concentration of drug in each time, blood sample is determined using suitable assay
technique (e.g. HPLC method), these concentrations are expressed in µg/ml or ng/ml is
plotted using graph paper with respect to time.
 The point of concentration of drug maximum in the plasma is known as peak or maximum
drug concentration (Cmax). From the drug level curve, we can judge the drug concentration
in plasma, within therapeutic level or toxic levels as shown in the Fig.
THE TIME OF ULTIMATE PLASMA
CONCENTRATION (T MAX)
The time taken to reach ultimate peak plasma concentration after oral
administration is known as the time of peak plasma concentration (tmax).
It is expressed in hours or minutes. On set of action and onset of time are estimated
by based on the rate of absorption.
 AREA UNDER THE CURVE
(AUC)
 The total integrated area under the plasma drug concentration and time profile.
It is useful to estimate the total amount or extent of drug enters into the systemic
circulation.
It is estimated in µg.h/ml or ng.h/ml.
 It is one of the significant parameters to consider for estimating the
bioavailability.
 URINARY EXCRETION DATA
The urinary excretion of unchanged drug is directly comparable to the unchanged plasma
drug concentration.
Based on drug excreted in the urine bioavailability can be determined.
Methods to estimate the unchanged drug from urine: Collection of urine at specified intervals
for a time span which is equivalent to 7 biological half-lives, unchanged drug present in each
interval is collected and analyzed the cumulative drug content is excreted.
Graphical representation of urinary excretion rate versus time at midpoint of urine collections
as shown in the Fig.
THE MAXIMUM URINARY
EXCRETION DATA (DXU/DT)
Maximum urinary rate increases, when rate and amount of drug absorption
increases in the blood, the rate of excretion of the unchanged drug in urine
generally follows first order process.
Plot is obtained from urinary excretion rate versus time at midpoint of urine
collection as shown in the Fig.
The maximum point peak is (dXu/dt)max, which is used to estimate the
bioavailability of the products.
THE TIME FOR MAXIMUM
EXCRETION (T U)MAX
It is the time at which maximum excretion rate occurs.
It decreases .as absorption rate decreases. It is analogues
to tmax Plasma level data.
THE CUMULATIVE EXTENT OF DRUG EXCRETED IN
THE URINE (X∞ U)
The cumulative extent of drug excreted in the urine corresponds to (X∞u) is directly
correlated to the amount/extent of drug absorbed.
 The excretion of drug into urine continues till the drug level in blood falls to zero.
Using this value bioavailability can be estimated.
The amount of bioavailability is calculated from equation:
PHARMACODYNAMICS OR
DIRECT METHODS
Pharmacodynamics methods are utilized to assess the
bioavailability when, it is difficult to assess the bioavailability by
pharmacokinetic method or lack of sensitive analytical method.
The two pharmacodynamics methods are used for the
determination of bioavailability.
1. Acute pharmacological response
2. Clinical response
ACUTE PHARMACOLOGICAL
RESPONSE
 Easily measurable pharmacological response methods are EEG, heart rate,
ECG, BP, pupil diameter etc.
Bioavailability can be determined by plotting Pharmacological effects versus
time curve along with dose response graph.
The disadvantage of this technique is tends to be more inconstant, the
accurate linear relationship between observed pharmacological response
versus drug level is difficult to assess.
CLINICAL RESPONSE
 It
is directly the therapeutics effects of dosage form after
administration are observed from the clinical response.
Practically, it is difficult to predict the bioavailability, as a result
of differences both in pharmacokinetics and pharmacodynamics
behaviour of the drug among individuals.
Number of factors affecting pharmacodynamics drug behavior
can includes drug tolerance, age, drug addiction, drug interactions
and unknown pathophysiological factors.