Biopharmaceutical system , methods of permeability , generic biologics, generic substitution

Siddhapura Pratik ( M.Pharm Pharmaceutics)

CONTENT
1. BIOPHARMACEUTICS CLASSIFICATION SYSTEM
2. METHODS OF PERMEABILITY: IN-VITRO, IN-SITU AND IN-VIVO
METHODS
3. GENERIC BIOLOGICS (BIOSIMILAR DRUG PRODUCTS)
4. CLINICAL SIGNIFICANCE OF BIOEQUIVALENCE STUDIES
5. SPECIAL CONCERNS IN BIOAVAILABILITY AND BIOEQUIVALENCE
STUDIES
6. GENERIC SUBSTITUTIONSiddhapura Pratik ( M.Pharm Pharmaceutics)

1. Biopharmaceutics classification system
1.1 Introduction
1.2 Factor affecting on biopharmaceutical classification system
1.3 Biopharmaceutical classification system [Classes]
1.4 Application of biopharmaceutical classification system
1.5 Class boundaries
1.6 Biowaiver
1.7 Criteria of biowaiver
1.8 ConclusionSiddhapura Pratik ( M.Pharm Pharmaceutics)

 The Biopharmaceutical Classification system was first developed in
1995, by Amidon et & his colleagues.
1.1 Introduction
 Definition:
 “The biopharmaceutical classification system is a scientific
framework for classifying a drug substance based on its aqueous
solubility & intestinal permeability & dissolution rate”.
 To saved time fast screening is required so drug substances are
classified on basis of solubility and permeability. This classification is
called as Biopharmaceutical classification system.Siddhapura Pratik ( M.Pharm Pharmaceutics)

1.2 Factor affecting on biopharmaceutical
classification system
The biopharmaceutical classification system has been developed to provide A
scientific approach to allow for to prediction in vivo pharmacokinetics of oral
immediate release (IR) drug product by classifying drug compound based on
their.
1. Solubility
2. Permeability
3. DissolutionSiddhapura Pratik ( M.Pharm Pharmaceutics)

SOLUBILITY
The maximum amount of solute dissolved in a given solvent under standard
condition of temperature, pressure and pH.
Solubility is the ability of the drug to be solution after dissolution.
The higher single unit dose is completely soluble in 250 ml at pH 1-6.8
(37 ̊C).

PERMEABILITY
Permeability of the drug to pass the biological membrane which is the
lipophilic.
Permeability is indirectly based on the extent of absorption of a drug
substance.
Drug substance is considered to be highly permeable , when the extent of
absorption in human determined to be 90% or more of administered drug or
compare to in vivo reference dose.Siddhapura Pratik ( M.Pharm Pharmaceutics)

DISSOLUTION
It is process in which solid substance solubility in given solvent i.e mass
transfer from solid surface to liquid phase.
Using USP apparatus I at 100 rpm or USP apparatus II at 50 rpm.
Dissolution media [900 ml].
1. 0.1 N HCl or simulated gastric fluid (pH 1.2) without enzyme.
2. pH 4.5 buffer & pH 6.8 buffer.
3. Simulated intestinal fluid without enzyme.

1.3 Biopharmaceutical classification
system [Classes]

CLASS I
Ideal for oral route administration.
Drug absorbed rapidly.
Drug dissolved rapidly.
Rapid therapeutic action.
Bioavailability problem not expected for immediate release drug product.
e.g. Metoprolol, propranolol, diltiazem.

CLASS II
Oral route for administration.
Drug absorb rapidly.
Drug dissolve slowly.
Bioavailability is controlled by dosage form and rate of release of the drug
substance.
E.g. nifedipine, naproxen.

CLASS III
Oral route of administration.
Drug absorbance is limited.
Drug dissolve rapidly.
Bioavailability is incomplete if drug is not release or dissolve in absorption
window.
E.g. cimetidine, metformin , insulin.

CLASS IV
Poorly absorbed orally administration.
Both solubility & permeability limitation.
Low dissolution rate.
Slow or low therapeutic action.
An alternate route of administration may be needed.
E.g. taxol, chlorthiazole, cefexime trihydrate.

1.4 APPLICATION OF BIOPHARMACEUTICAL
CLASSIFICATION SYSTEM
To predict in vivo performance of drug product using solubility and
permeability measurements.
Aid in earliest stages of drug discovery research.
To use in biowaiver considerations.
For research scientist to decide upon which drug delivery technology to
follow or develop.
Also for the regulation of bioequivalence of the drug product during scale
up and post approval.

1.5 CLASS BOUNDARIES
Highly soluble
The highest dose strength is soluble in ≤250 ml water over a pH range of 1 to 7.5.
The volume estimate – a glassful ( 8 ounce)
Highly permeable
When the extent of absorption in humans is determined to be ≥ 90% of an
administered dose.
Rapidly dissolving
When ≥ 85% of the labelled amount of drug substance dissolves within 30
minutes using USP apparatus I to II in a volume of ≤ 900ml buffer solutions.Siddhapura Pratik ( M.Pharm Pharmaceutics)

1.6 BIOWAIVER
“in vitro instead of in vivo bioequivalence testing”
Definition:
It is an exemption from conducting human bioequivalence studies
when the active ingredients meet certain solubility and
permeability criteria in vitro and when the dissolution profile of
the dosage from meets the requirements for an IR dosage form.

1.7 CRITERIA OF BIOWAIVER
Rapid and similar dissolution
High solubility
High permeability
Wide therapeutic window
Excipient used in dosage form are same as those present in approved drug
product

1.8 CONCLUSION
Biopharmaceutical classification system aims to provide regulatory tools
for replacing certain bio-equivalence studies by a accurate in vivo
dissolution tests.
The in vivo pharmacokinetics of drug depends largely on the solutility and
permeability.
Many laboratories are engaged to find better means to estimates in vivo
behaviour of the drug after oral administration by using simple in vitro
dissolution tests.

Methods of permeability
In Vitro In Situ In Vivo
2.Methods of permeability

IN VITRO METHOD
In vitro method are carried out outside of the body and are used to
determine the permeability of drug using live animal tissues.
In vitro models have been introduced to assess to the major factors
involved in the absorption process and predict the rate and extent of drug
absorption.
Here, the intestine of lower experimental animals such as rats , guinea pigs,
rabbits are taken for the study.

THE DIFFERENT IN VITRO METHODS ARE:
Physicochemical methods
1. Partition coefficient
2. Artificial membranes
3. Chromatographic retention indices
4. Brush border membrane vesicles (BBMV)
5. Isolated intestinal cells
6. Tissue technique:
Everted small intestinal sac technique
Everted sac modification
Circulation techniques
Everted intestinal ring or slice techniques
7. diffusion cell method
8. Cell culture techniques.Siddhapura Pratik ( M.Pharm Pharmaceutics)

1. PARTITION COEFFICIENT

Partition coefficient between an oil and water phase , log p, is one of the
easiest property of a drug molecule that can be determined.
It provides a measure of the lipophilicity of a molecule and can be used to
predict to what extent it will cross the biological membrane.
Eg. Octanol is selected as an oil phase as it has similar properties to biological
membranes.
It’s important to note that log p does not take the degree of ionization into
consideration and hence log D is used.

Log D is the distribution coefficient where aqueous phase is at a particular
pH and thus it takes into account the ionization of the molecule at ths pH.
The log D measured at intestinal pH will givea much better idea about
extent of drug permeability across GI membrane than log P.

2. ARTIFICAL MEMBRANES
 Artificial membranes are very useful in studying passive membrane
permeability as they are reproducible and are suitable for high throughput
screening.
 In this method, PAMPA model is used.
Parallel artificial membrane permeability assay (PAMPA)
 PAMPA is a method which determines the permeability of substances from a
donor compartment, through a lipid infused artificial membrane into an acceptor
compartment.
 The artificial membrane is like a phospholipid membranes supported by
filter material.
 It is prepared by pipetting a solution of lipids in an inert organic solvent on
a supporting filter material which is placed on 96- well microtitre plate.

 Procedure
 test compound is added to the donor compartment in buffer solution
of pH 7.4 and permeation takes place through artificial membrane into
the acceptor compartment.
 An aliquot is collected and the concentration of drug permeated is
measured by suitable assay technique.
Fig. Parallel Artificial Membrane Permeation assay(PAMPA)Siddhapura Pratik ( M.Pharm Pharmaceutics)

USES
 mainly used to determine the intestinal permeability
 Prediction of passive transport
 Trans-cellular permeability of drugs
Advantage
 Used for screening large no. of compounds.
 Provides information on solubility, lipophilicity and ionization status of a drug.
 It is much lesser labor intensive than cell culturemethods.
Disadvantage
 They are based on approximation and oversimplification of the actual in vivo
conditions.Siddhapura Pratik ( M.Pharm Pharmaceutics)

 A modification of this system is immobilized liposome
chromatography(ILC) and on ILC, many compounds with same log P
have been shown to demonstrate variable membrane partitioning
based on their logs.
Liposomes , lipid bilayers produced from mixture of lipids are also
useful in investigating passive diffusion of drugs through lipid
membranes and also used to study passive absorption of many
monocarboxylic acids.
 A modified version of this is the filter-immobilized artificial membrane
(filter-IAM) permeability assay.
 This coupled with an instrument PSR4p (Permeability solubility
retention, pION, USA) can be used for high throughput permeability
screens

3. CHROMATOGRAPHIC RETENTION INDICES
Immobilized artificial membranes(IAM) chromatography along with
physicochemical parameters is used for evaluation of passive intestinal
absorption.
IAM packings are prepared by covalently immobilizing monolayers of
membrane phospholipids to silica particles.
Micellar liquid chromatography(MLC) is also used for the prediction of
passive drug absorption and in this system retention of drug mainly
depends on hydrophobic, electronic and steric interactions.
In general, chromatographic techniques are easy in operation and
have high analytical sensitivity.

4.BRUSH BORDER MEMBRANE VESICLES
(BBMV)
 A brush border is the name for the microvilli covered surface of
simple cuboidal epithelium and simple columnar epithelial cells,
found in the small intestine.
Both animal and human tissue can be used for this.
Procedure:
- intestinal tissues are treated with calcium chloride precipitation
method using centrifugation.
- the pellets obtained after centrifugation is resuspended in buffer
which results in the formation of vesicles.

Fig.1: Brush border membrane vesicles Fig.2: Structure of microvilliSiddhapura Pratik ( M.Pharm Pharmaceutics)

 Vesicles are mixed with drug in buffer solution and filtered after a
period of time
The amount of drug taken up by the vesicles gives an account of drug
absorption.
Advantage
 useful for mechanic studies of drug absorption process.

5. USING ISOLATED INTESTINAL CELLS
Here, the small intestine is perfused with enzyme solutions that
release the cells and the cells are treated with chelating agents or
enzymes.
 The freshly isolated cells are suspended in buffer solution.
 At the time of experiment, the cells are separated, resuspended in
buffer containing the drug under O2/CO2 and shaken well.
After a specific period of time, the cells are separated by filtration,
extracted and drug absorbed is determined.

6.TISSUE TECHNIQUES
a). Everted small intestinal sac technique:
this method involves isolating a small segment of the intestine of a
laboratory animal such as rat, inverting the intestine and filling the
sac with a small volume of drug free buffer solution.
Both the segments are tied off and the sac is immersed in an
ERLENMEYER FLASK containing a large volume of buffer solution
that contains the drug.
The flask and its contents are then oxygenated and the whole
preparation is maintained at 37°C and shaken mildly.
At predetermined time intervals, the sac is removed and the
concentration of drug in the serosal fluid is determined/assayed for
drug content.Siddhapura Pratik ( M.Pharm Pharmaceutics)

ADVANTAGE
-THE EPITHELIAL CELLS OF THE MUCOSAL SURFACE ARE
EXPOSED DIRECTLY TO THE OXYGENATED MUCOSAL FLUID.
-Prolongs the viability and integrity of the preparation after
removal from the animal.
-Convenience and accuracy with respect to drug analysis.
Disadvantage
- Difficulty in obtaining more than one sample per intestinal
segment

Fig. Everted sac technique

B)- EVERTED SAC MODIFICATION
 In this method, the test animal is fasted for a period of 20-24 hr and water is
allowed.
 The animal is killed and the entire small intestine is everted. Segments, 5-15 cm in
length are cut from a specific region of the intestine.
 The distal end of the segment is tied and the proximal end is attached to the
cannula. The segment is suspended in a mucosal solution which contains the drug.
 A drug free buffer is then placed in the serosal compartment.

Fig. Everted sac modification
 For determining the rate of drug transfer, the entire volume of
serosal solution is removed from the sac at each time interval
with the help of a syringe and replaced with fresh buffer
solution.

 The amount of drug that permeates the intestinal mucosa is
plotted against time to describe the absorption profile of drug at
any specific pH.
Advantage
 A number of different solutions may be tested with a single
segment of the intestine.
 Simple and reproducible.
 It distinguishes between active and passive diffusion.
 It determines the region of small intestine where absorption is
optimal, in the case of active transport.

 Used to study the effect of pH, surface active agents,
complexation and enzymatic hydrolysis.
DISADVANTAGE
- The intestinal preparation is removed from the animal as
well as from its normal blood supply. Under these conditions,
the permeability characteristics of the membrane are
significantly altered.
- The rate of transport of drug as determined from the
everted sac technique, may be slower than in the intact
animal.

C). CIRCULATION TECHNIQUES
 In this method, small intestine may or may not be everted.
 This involves isolating either the entire small intestine of small lab
animal or a segment and circulating oxygenated buffer containing
the drug through the lumen.
 Drug free buffer is circulated on the serosal side of the intestinal
membrane and oxygenated.
 Absorption rate from the lumen to the outer solution are
determined by sampling both the fluid circulating through the
lumen and outside.

ADVANTAGE
• This method is applicable to kinetic studies of the factors
affecting drug absorption.
• Both surfaces are oxygenated.
• Eversion is not necessary.

D). EVERTED INTESTINAL RING OR SLICE
TECHNIQUE
 In this technique, the entire small intestine is isolated from the fasted experimental
animal and washed with saline solution and dried by blotting with filter paper.
 The segment is tied at one end and by placing on glass rod it is carefully everted
and cut into small rings.
 The everted intestinal rings are then incubated in drug containing buffer
maintained at 37c with constant oxygenation.
 Under optimal conditions, rings remain viable for up to 2 hours and the transport
of drug is stopped by rinsing the rings with ice cold buffer and drying them.

 At selected time interval, the tissue slices are assayed for drug content
and expressed as mol/gm/time.
Advantage
• Simple and reproducible.
• Kinetic studies can be performed.
• Each animal can act as its own control as many rings can be prepared
from each segment of the intestine isolated.
• Mechanism of drug absorption can be studied by changing the
experimental conditions.
Disadvantage
• Extreme care is needed to maintain to viability of the tissue
throughout the experiment.
• tissue needs to be disrupted completely for the determination of drug
contents, which complicates the assay procedure.
• polarity of the absorption cannot be assayed.

7. DIFFUSION CELL METHOD
In this method, small segments of small intestine are mounted
between two glass chambers filled with buffer at 37 C.
Diffusion cell consist of two compartments :-
i. Donor compartment - which contains the drug solution and the
lower end of which contains the synthetic or natural GI membrane
that interfaces with the receptor compartment.
ii. Receptor compartment - which contain the buffer solution.

Fig. Diffusion cell for studying drug uptake from GIT

8. CELL CUTURE TECHNIQUES
Cell culture is the complex process by which cells are grown under
controlled conditions, generally outside their natural environment.
In this technique, differentiated cells of the intestine, originating
from CaCo2 cells ( cells of carcinoma of colon) are placed on
synthetic polycarbonate membrane previously treated with an
appropriate material such as collagen which on incubation aids
reproduction of cells while not retarding drug permeation
characteristics.
 These models are based on the assumption that passage of drugs
across the intestinal epithelium is the main barrier for drugs to
reach the circulation.

Human intestinal cell lines are generally divided into four different groups:
 Type I : These cells differentiate spontaneously under normal culture
conditions and hence are polarized (i.e. apical and basolateral surface),
form domes, have tight junctions and brush border (eg.Caco-2 cells).
 Type II : These cells differentiate into enterocytes- type cells only under
specific culture conditions e.g. HT29 in presence of glucose, HT29 clone can
differentiate into mucus cells.
 Type III : These cells form domes but do not express any biochemical or
morphological markers of differentiated cells.
e.g. T84, SW116 and Col115 cell lines.
 Type IV : These cells do not differentiate.
e.g. HCA7 and SE480 cell lines.

Fig. CaCo-2 cell monolayer to study drug absorption

 CaCo-2 is the most widely used cell line and CaCo-2 are a human
colon carcinoma cell line.
 The CaCo2(colon cancer cells) cell line is a continuous line of
heterogeneous human epithelial colorectal adenocarcinoma cells.
 Solution of drug is placed on this layer of cultured cells and the
system is placed on this layer of cultured cells and the system is
placed(R.C) in a bath of buffer solution.
The drug that reaches the latter compartment is sampled and
analysed periodically.

 CaCo2 cells express tight junctions, microvilli and a number
of enzymes and transporters that are characteristic of
enterocytes.
 CaCo2 monolayer is widely used across the pharmaceutical
industry as an in vitro model of the human small intestine
mucosa to predict the absorption of orally administered
drugs.
 The correlation between the in vitro apparent permeability
across CaCo2 monolayers and the in vivo fraction absorbed
is well established.
 Cell culture models have been employed in the screening of
the intestinal permeability of libraries of new drug entities
that have been generated through combinatorial chemistry
and high throughput pharmacological screening.

IN VIVO METHODS
In vitro and in situ techniques gives us an idea about absorption , but in
vivo method gives us an idea about some important factor that influence
absorption such as gastric emptying. Intestinal motility and the effects of
drugs on the GIT can be determined.
The in vivo method can be classified into:
1. Direct method
2. Indirect methodSiddhapura Pratik ( M.Pharm Pharmaceutics)

DIRECT METHOD
The drug levels in blood or urine is determined as a function of time. For
this , a suitable sensitive reproducible analytical procedure should be
developed to determine the drug in the biological fluid.
In this method, blank urine or blood sample is taken from the test animal
before the experiment.
The test dosage form is administered to the animal and at appropriate
intervals of time the blood or urine sample are collected and assayed for the
drug content.
From the data ,we can determine the rate and extent of drug absortption

DIRECT METHOD
In this method, the experimental animal chosen should bear some
resemble to man.
It is reported that pigs most closely resemble to man but are not used due
to the handling problems.
The other animal that can be used are dogs, rabbits and rats.

INDIRECT METHOD
When the measurement of drug concentration in blood or urine is difficult
or not possible , but a sensitive method is available to test the activity, then
absorption studies can be done by this indirect method.
In this method, pharmacological response of the drug is related to the
amount of drug in the body.
The response is determined after the administration of a test dosage form,
LD 50 appears to be dependent on the rate of the absorption of drug.

IN SITU METHOD
It stimulated the in vivo conditions for drug absorption and are based on
perfusion of a segment of GIT by drug solution and determination of
amount of drug diffused through it.
In situ refers to those method in which the animal’s blood supply remains
intact in which the rate of absorption determined from these methods may
be more realistic than those determined from in vitro techniques.
These models are powerful tools to study the mechanistic aspects of these
important process.
Acts as a bridge between in vivo and in vitro methods.

In situ method
Absorption of drug from
small intestine
Absorption of drug from
stomach
Perfusion technique
Intestinal loop
techniques
Doluisio method
Single pass perfusion
method

DOLUISIO METHOD

SINGLE PASS PERFUSION METHOD

GENERIC BIOLOGICS
(BIOSIMILAR DRUG PRODUCTS)
Traditional drugs are called small-molecule agents because the active
ingredient is usually a single, discrete chemical entity.
Biologic drugs are complex products that are derived from biologic sources
(human, animal, microorganisms, or yeast). Biologics include viruses, genes,
blood and body tissues, antibodies, toxins or antitoxins, vaccines, and
related products used for treating disease. Until now, it has not been
possible to develop generic versions of these products because of their
complex manufacturing requirements and the difficulty in defining their
exact composition.

The U.S. Food and Drug Administration (FDA) requires manufacturers to
submit a proposed product for approval as a biosimilar drug or an
interchangeable drug, rather than as a generic equivalent. Biosimilars are
very similar to the original brand name biologic drug they are compared to.
Only minor differences in clinically inactive components are allowable in
biosimilar products. Interchangeable drugs, additionally, are expected to
provide the same benefits and risks as the original brand name biologic.
Zarxio is a biosimilar drug to filgrastim (a drug used in cancer patients to
help maintain their white blood cell counts). It is the first drug approved by
the FDA as a biosimilar, not as an interchangeable, product. This means that
Zarxio may be prescribed by a doctor, but it cannot be substituted
(interchanged) for filgrastim by a pharmacist without the approval of the
prescribing doctor.
GENERIC BIOLOGICS

Ongoing scientific developments may allow the creation of generic biologic
products in the next several years. The advantage of generic biologic drugs
for manufacturers, pharmacies, and consumers is that they could be freely
interchanged and compete against one another for inclusion on a hospital
or health plan drug list. Having different brands of very similar biologic
products, as with epoetin, a hormone to increase red blood cell count, does
not offer all the benefits of generically equivalent products.
GENERIC BIOLOGICS

CLINICAL SIGNIFICANCE OF
.BIOEQUIVALENCE STUDIES
Clinical interpretation is important in evaluating the results of a bioequivalence
study.
Differences of less than 20% in AUC & Cmax between drug products are unlikely
to be clinically significant in patients.
A small ,statistically significant difference if the study well controlled & the
number of subjects is sufficiently large.
Above MEC & do not reach the MTC.
Elderly or patients.
Normal healthy volunteers.
Minimize product to product variability by different manufactures & lot to lot
variability with a single manufacture.Siddhapura Pratik ( M.Pharm Pharmaceutics)

SPECIAL CONCERNS IN BIOAVAILABILITY
AND BIOEQUIVALENCE STUDIES
For certain drugs and dosage forms , systemic bioavailability and
bioequivalence are different to ascertain for e.g. Cyclosporine, verapamil
are considered to be highly variable.
The number of subjects required to demonstrate bioequivalence for these
drug products may be excessive , requiring more than 60 subjects.
The intrasubject variability may be due to the drug itself or to the drug
formulation or to both.
The FDA has held public forums to determine whether the current
bioequivalence guidelines need to be changed for these highly variable
drugs.

GENERIC SUBSTITUTION
Generic substitution is the term applied to the substitution of a
prescribed branded drug by a different form of the same active substance.
The generic is usually unbranded
Brand name : zovirax® ( API is acyclovir)
Generic name : acyclovir
However, therapeutic classes such as hormones and drugs with narrow
therapeutic index (anticonvulsants, anticoagulants ,etc.) may not be
appropriate for generic substitution because this drug cause serious
toxicity or therapeutic failure when minor change occur in dose size
during manufacturing process.

Branded Medicine(patented drug)

Branded Generic Medicine
(Standard company)

Generic Medicine

REFERENCE
Brahmankar, D. M and Jaiswal, Sunil. B (2009). Biopharmaceutics and
Pharmacokinetics – A Treatise. 2 nd edition. Vallabh Prakashan, Page no. 27-
29 and 332 - 335.
Dr. Tipnis H.P. and Dr. Bajaj Amrita, Principles and applications of
Biopharmaceutics and Pharmacokinetics, 1 st edition 2002, reprint 2005,
career publication, Page no. 332-340.
 Leon shargel, Susanna wu-pong, Andrew B.C.Yu , Applied Biopharmaceutics
& Pharmacokinetics, 5 th edition 2005, published by the Mc Graw hills
companies, page no. 431-436 & 482-484.

 Biopharmaceutics and pharmacokinetics - Dr. Shobha Rani R.
 Biopharmaceutics and pharmacokinetics - J.S Kulkarni, A.P. Pawar, V.P.
Shedbalkar
Web site :
https://www.msdmanuals.com/en-kr/home/drugs/brand-name-and-
generic-drugs/generic-biologic-drugs
https://www.mabxience.com/products/biosimilar/generics-biologics-
biosimilars-whos-who/
https://www.slideshare.net/
REFERENCE

Biopharmaceutical system , methods of permeability , generic biologics, generic substitution

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