in vitro screening techniques to characterize ADMET properties

1. PRE CLINICAL IN-VITRO SCREENING FOR
EVALUATION OF DRUG’S ADMET
PROPERTIES: PROS AND CONS
By
T. Dilip Kumar
MS(pharm.) Pharmaceutics
Semester II
NIPER, Raebareli
I.D No 313/14

2. CONTENTS
 Introduction
 Partition coefficient
 Aqueous Solubility
 In Vitro Screening of Drug’s ADME-Tox Properties
Assay for evaluation of absorption.
Assay for Evaluation of Drug Distribution.
Assay for the evaluation of drug metabolism.
Assay for evaluation of drug excretion.
Assays for Evaluation of Drug Toxicity.
 Conclusion.
 References.
2

3. INTRODUCTION
 During the recent past, in vitro screening techniques to characterize ADME–Tox
profiles of compounds have been applied in early stages of the drug discovery
process to boost the success rate of discovery programmes and to progress better
candidates into drug development.
 ADME-Tox properties are vital determinants of the ultimate clinical success of a
drug.
 The ADME-Tox studies should be started as early as possible in the drug
discovery process to identify compounds that have good pharmacokinetic and
toxicological profiles enabling all the properties to be optimized simultaneously
3

4. PARTITION COEFFICIENT
 In Vitro Assay of Partition Coefficient:
 The assay of partition coefficient is based on miniaturised shake flask
method.
Aqueous phase- Dulbecco’s phosphate buffer solution pH 7.4.
Organic phase- n octanol.
 The compound is added to the octanol-buffer mixture and is allowed to
equilibrate between them. The amount of compound in the buffer phase is
determined by HPLC with photo diode array detector and the amount in the
organic phase is calculated by subtracting the amount in buffer from the total
amount of the compound which is determined from the calibration sample
4

5. • a) It can be performed with as little as 10nmol of
compound.
• b) The properties of octanol resemble to those of
lipid bilayer membranes and hence has been
suggested that distribution of chemicals in octanol
simulates their ability to diffuse across the
membrane passively to some extent.
Advantages
• a) The test compound need to contain
chromophore for photo diode array detection.
• b) Since the organic layer is not directly analysed
it doesn’t distinguish solubilisation in the organic
layer from precipitation.
Disadvantages
5

6. AQUEOUS SOLUBILITY
 In Vitro Assay of Aqueous Solubility:
Solubility by HPLC-UV:
In this method 200µM concentration of compound which is soluble in DMSO is
added to isotonic buffer of pH 7.4 and after 24hr of incubation at room temperature
the solubility of the compound is measured by chromatographic procedure with
photo diode array detection.
The advantage of this assay is that the compounds with low micro molar
concentration can be rapidly identified.
The disadvantage is the limit of quantification is often smaller than 1µM depending
on the absorbance of the compound.
6

7. IN VITRO SCREENING OF DRUG’S ADME-TOX
PROPERTIES
Absorption
-Artificial membrane.
-In Vitro cell culture
assay.
-Everted intestinal sac
Distribution
-Plasma protein
binding.
-Blood Brain Barrier
Metabolism
-Metabolic stability.
-Metabolite profiling.
-Drug- Drug
interaction assays.
Excretion
Toxicity
-Enzyme release.
-Neutral red uptake
-Macromolecular
synthesis
--MTT assay.
7

8. ASSAY FOR EVALUATION OF DRUG ABSORPTION
In-Vitro Cell Culture:
The absorption assay are performed by
placing the compounds to be studied in the
inner well (apical side) and monitoring the
amount of the test compound in the outer
well (basolateral side). Data are expressed
as apparent permeability coefficients
(Papp, cm sec−1), which are calculated by
using the following equation:
8
Figure 1: Caco 2 permeability assay

9. ASSAY FOR EVALUATION OF DRUG ABSORPTION
Everted intestinal sac: Intestinal segments are turned inside
out (everted), with both ends tied for intestinal absorption
studies.
The sacs are placed in a solution containing the drug to be
studied, and the amount that enters the lumen of the sac would
represent drugs that can be absorbed from the intestinal lumen
into the systemic circulation.
The advantage of using everted intestinal sac is that it might
provide data closer to human intestinal absorption than the
Caco-2 cell model but most challenging aspects of the use of
everted intestinal sacs is the maintenance of tissue viability,
9
Figure2:
Representation of
Everted Intestinal sac

10. ASSAY FOR EVALUATION OF DRUG DISTRIBUTION
 Plasma Protein Binding Assay: The extent of binding of drug to plasma influences
the way in which it distributes into tissues in the body.
 Equilibrium dialysis is used to determine the extent of binding of a compound to
plasma proteins. A semi-permeable membrane separates a protein-containing
compartment from a protein-free compartment. The system is allowed to equilibrate
at 37°C. The test compound present in each compartment is quantified by LC-
MS/MS.
 The extent of binding is reported as a fraction unbound (fu) value which is calculated
by the equation:
 PC=Test compound concentration in protein-containing compartment.
PF = Test compound concentration in protein-free compartment.
10

11. ASSAY FOR THE EVALUATION OF DRUG METABOLISM
Metabolic Stability:
The 96 wellplates are preloaded with test
compounds in an isotonic buffer (e.g. Krebs-
Henseleit buffer) and then the liver microsomes are
added to the wells to initiate the study.
The test compounds are extracted from the assay
and are then centrifuged to filter the reaction
mixture through the porous membrane into a new
96-well recipient plate, where the samples are
analyzed by LC–MS.
11
Figure 3: Screening assay of Metabolic
stability

12. ASSAY FOR THE EVALUATION OF DRUG METABOLISM
The use of microsomes allows evaluation of
the readiness of drugs to undergo oxidative
metabolism.
For some drugs undergoing phase II
metabolism or metabolism involving non-
microsomal enzymes the use of microsomes
could create a bias towards phase I oxidation,
which might not be the key pathway in vivo,
and could lead to erroneous conclusions on
metabolic stability. 12
Advantages
Disadvantage
s

13. ASSAY FOR EVALUATION OF DRUG EXCRETION
 Excretion is probably the least studied, renal, faecal and biliary excretions are
generally studied using whole animals, with no appropriate in vitro surrogates.
 Recently, a technology has been developed using hepatocytes cultured in a
sandwich
format to allow the development of bile cannaliculi in vitro. This technology has
been applied to evaluate hepatobiliary excretion as well as transporter mediated
drug–drug interactions.
13

14. ASSAY FOR EVALUATION OF DRUG TOXICITY
Enzyme release Assay:
The viable cells are treated with test compound in 96 well
plate at either single or multiple concentration.
After the treatment period, the cells are lysed and the ATP
content per well is quantified based on chemiluminescence
using a luciferin-luciferase assay.
This study can be performed with all cell types, including
cryopreserved human hepatocyte.
14
Figure 4: Screening assay of Drug
Toxicity

15. ASSAY FOR EVALUATION OF DRUG TOXICITY
Neutral Red Uptake:
 Neutral red is a dye that is preferentially absorbed into the lysosomes of viable cells.
 In this assay, after compound treatment, the cells are incubated with neutral red to
allow uptake. The cells are then destained and the red color quantified at 540 nm
using a microplate reader.
 Advantage: This assay can also be used with any cell type.
 Disadvantage: This assay measures viability as lysosomal activity, and so results
might be inaccurate if the chemicals tested have differential effects on lysosomes
15

16. CONCLUSION
 ADME/Tox properties are important parameters for the selection of drug candidates
for development. . Drug candidate selection involving both pharmacological
properties and ADME/Tox screening should lead to an enhanced probability of
clinical success.
 One goal of ADME/Tox screening should be the development of a database
correlating chemical structures and biological endpoints
 Future directions should include high content assays (assays yielding extensive
information, such as expression genomics and proteomics) as well as the
development of extensive databases correlating chemical structure and ADME/Tox
drug properties.
16

17. REFERENCES
 Albert P, Li. (2005) ‘Preclinical in vitro screening assays for drug-like properties,’
Drug Discovery Today: Screening Technologies, 2(2), pp. 179-185.
 Albert P, Li. (2001) ‘Screening for human ADME/Tox drug properties in drug
discovery’, Drug Discovery Today, 6(7), pp. 357-366.
 Ania de la, N. Rolando, R. (2008) ‘Current methodology for the assessment of
ADME-Tox properties on drug candidate molecules’, Biotecnología Aplicada, 25(2),
pp. 98-110.
 Hongshi, Y. and Adedayo, A. (2003) ‘ADME–Tox in drug discovery:integration of
experimental and computational technologies’, Drug Discovery Today, 8(18), pp.
852-861.
17

18. 18