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

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.

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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 simultaneously3

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 sample4

• 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

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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.

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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

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:

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Figure 1: Caco 2 permeability assay

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,

intensive labour procedures and lot of time consumption.

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Figure2:

Representation of

Everted Intestinal sac

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.

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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.

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Figure 3: Screening assay of Metabolic

stability

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

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.

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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.

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Figure 4: Screening assay of Drug

Toxicity

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 lysosomes15

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.

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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.

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