The document discusses various in vitro, in vivo, and in situ methods for determining drug absorption. In vitro methods include physicochemical techniques like measuring partition coefficients as well as more complex methods like using everted intestinal sacs or cell culture models. In vivo methods directly measure drug levels in blood/urine over time or indirectly via pharmacological response. In situ techniques simulate in vivo conditions by perfusing drug solutions through an intestinal segment while keeping the blood supply intact. The document provides detailed descriptions of techniques like the everted sac method, Caco-2 cell cultures, and Doluisio intestinal perfusion method.

1. Seminar
on
Methods of determining
absorption
Tenzin Jangchuk
1st M.Pharm
Dept.- Pharmaceutics

2. Absorption
Absorption is defined as the process of movement
of unchanged drug from the site of administration
to the systemic circulation.

3. Different methods of determining drug
absorption are:
A.In vitro method
B.In Vivo method
C.In situ method

4. 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 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.

5. 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 techniques:
a). Everted small intestinal sac technique
b) Everted sac modification
c) Circulation techniques
d) Everted intestinal ring or slice techniques
7. Diffusion cell method
8. Cell culture techniques

6. 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.

7.  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 this pH.
The log D measured at intestinal pH will give a much better
idea about extent of drug permeability across GI membrane
than log P.

8. 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.

9.  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)

10. 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 culture methods.
Disadvantage
 They are based on approximation and oversimplification of the actual in vivo
conditions.

11.  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

12. 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.

13. 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.

14. Fig.1: Brush border membrane vesicles Fig.2: Structure of microvilli

15.  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.

16. 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.

17. 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.

18. 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

19. Fig. Everted sac technique

20. 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.

21. 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.

22.  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.

23.  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.

24. 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.

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

26. 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.

27.  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.

28. 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.

29. Fig. Diffusion cell for studying drug uptake from GIT

30. 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.

31. 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.

32. Fig. CaCo-2 cell monolayer to study drug absorption

33.  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.

34.  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.

35. 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 method

36. 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
absorption.

37.  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.

38. 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.

39. IN SITU METHOD
 It simulates 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.

40. I- Absorption of drug from small intestine
Absorption of drug from small intestine is described in two methods:
i. Perfusion technique:
 Doluisio method
 Single pass perfusion method
ii. Intestinal loop techniques

41. 1. Perfusion technique
a) Doluisio method
 In this technique, adult male rats are fasted for about 16-24 hours before the
experiments.
 A midline abdominal incision is made and the intestinal segment to be
perfused is identified.
 A L shaped glass inlet cannula is secured into the segment and the outlet
cannula is placed approx. 15-50cm from the inlet cannula. These are secured
with sutures and the intestine is replaced in the abdominal cavity.
 Dissected rats are connected by means of tubing to syringes of capacity of 10-
30ml.
 After washing the intestinal segment with normal saline , the syringe is filled
with a solution of drug which is delivered to the intestinal segment and is
then collected in the second syringe and analysed for the drug content.

42. Note: an identical syringe is affixed to the ileal cannula. {second syringe}
 The effective permeability (P) of the drug can be calculated by equation-
Cout = exp -p(2πrl)
Cin Q
Where, r=radius of the gut lumen
l= length of the gut lumen
Q= perfusate flow rate(ml/min)
p= permeability of the compound (cm/min)
Cout and Cin = concentration of the drug leaving the perfused segment
and conc. of drug entering the segment.

43. Fig. Doluisio method for drug uptake study through
rat intestine.

44. b. Single pass perfusion
 In this technique, the drug solution passes through the
intestinal segment just once.
 This technique is superior to Doluisio method in that
precise adjustment of hydrodynamic conditions that can
influence blood circulation and puts stress on intestinal
wall can be controlled.

45. Fig. Single pass perfusion technique for studying drug
uptake.

46. 2. Intestinal loop technique
 Here, single or multiple intestinal loops are used for studying
the absorption.
 Adult male rats are fasted before the experiment under the
anaesthesia and abdominal midline incision is made and the
small intestine is exposed.
 A proximal ligature is placed around the intestine from the
pylorus and a distal ligature is secured at a distance of 4’’ distal
to the proximal ligature.

47. The drug solution is introduced into the lumen of the loop by
means of a syringe which is secured by the proximal ligature.
After injection, the needle is removed, the proximal ligature is
tightened and the loop is replaced into the abdominal cavity
and the incision is closed.
After a specific time, the animal is sacrificed, the intestinal loop
is rapidly excised and homogenized and the amount of
unabsorbed is determined.

48. Advantage
 Simple and reproducible
Disadvantage
 Only one sample can be obtained from the experimental
animal.

49. II- Absorption from the stomach
 Fasted adult male rats are anaesthetized, stomach is exposed
and the cardiac end is ligated.
 An incision is made in the pylorus in which the cannula is
introduced and ligated.
 The lumen is washed several times with saline and
subsequently with 0.1N HCl solution containing 0.15M NaCl.
 The drug solution of known concentration is introduced into
the stomach and after 1 hour, the solution is removed from
the gastric pouch and assayed for drug content.

50.  The gastric pouch may also be homogenized and analysed for
drug.
 In order to obtain number of samples as a function of time, the
following modifications is done:
 Drug solution is introduced into the gastric lumen via the cardiac
cannula.
 A polyethylene tubing connected to a 2ml syringe equipped with a three
way stopcock is attached to the duodenal L shaped glass cannula.
 At specific intervals, the stomach contents are sampled by withdrawing
the solution into the syringe.

51. Advantage:
 has the capability of absorbing the most acidic drug form and
the very weakly basic drugs.
E.g. salicylic acid, aspirin, thiopental, and antipyrine, which are
un-dissociated in the acidic gastric contents were readily
absorbed.
Disadvantage:
 In situ techniques equate absorption with the loss of drug from
the GI lumen.
 A drug is accumulated or metabolized in the gut wall and one will
get an overestimate of the amount of drug absorbed.

52. Reference:
o Biopharmaceutics and pharmacokinetics
- Dr. Shobha Rani R.
o Biopharmaceutics and pharmacokinetics
- DM Brahmankar and Sunil B. Jaiswal
- J.S Kulkarni, A.P. Pawar, V.P. Shedbalkar

53. Important questions:
1. In vitro - in vivo correlation
2. Explain in vitro methods for determination of drug
absorption.