Biology Services

December 6th, 2006

2006 Copyright GVK Biosciences Private Limited

Biological Services

To provide support to Pharmaceutical and Biotech companies in discovery biology

Focus in providing support from hit-to-lead/ lead optimization and beyond Complements the existing strengths in Informatics & Med. Chem.

2006 Copyright GVK Biosciences Private Limited

Discovery Stages
Hit Generation Hit Validation Lead Optimization Candidate drug ID

Major activities
Primary/Functional assays Selectivity/Side effect profiling In vitro ADMET In vivo Efficacy/PK Exploratory Tox.

IND Clinical development

Safety Pharmacology Biochemical/Biomarker monitoring

2006 Copyright GVK Biosciences Private Limited

Biology Services at GVKBio: Current Status Biology Services

Pharmacology Phkinetics Tox & Safety Phcology

in vitro

in vivo

in vitro
- Absorption - Cyp inhib. - Micro. stab.

in vivo
- Exploratory tox. - acute - Mouse PK - repeated dose - rat, mouse - BBB -cytotoxicity - Rat PK

- Receptor assays - CNS - Cell based assays - Enzymes - Inflammation/ Pain - Metabolic dis.

- PhysChem

2006 Copyright GVK Biosciences Private Limited

Receptor Biology
G-protein coupled receptors: Validated assays
Adenosine (A1, A2A, A3), Adrenergic (a2A -a2C ; b1- b3, NET), Dopamine (D1,
D2L, D3, DAT), Histamine (H1, H2) Muscaranic (M1 - M5), Opioid (d, k, m), Serotonin (5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, 5-HT6, 5-HT7, 5HTT)
3H

Naloxone binding to Opioid m hrCHO-K1 cell membrane (Kd 3.2 nM; n = 3)

IC50 (n = 3) Ki (n + 3)

Naloxone
1.1e-9 0.8e-9

DAMGO
2.2e-9 1.7e-9

U69593
1.0e-6 0.8e-6

Validation of receptor binding assays for other GPCRs, ion channels and nuclear receptors are in progress
2006 Copyright GVK Biosciences Private Limited

Cell Based Functional Assays

GPCR + Ligand Exchange GDP to GTP at a subunit Changes in intracellular signaling mol. Alteration in gene transcription or proteins Functional response of the cell

Activation detected by using nonhydrolysable GTP analogues

PIP2/IP3 measurement cAMP or Ca2+ measurement

Reporter gene assays CREB, b-Gal, NFAT etc.

GVKs capability for cell-based assays IP3/PIP2 measurement for Gq coupled receptors cAMP measurement for Gs coupled receptor
2006 Copyright GVK Biosciences Private Limited

Enzyme assay
Proteases - Validated assays
Caspases (3, 8) Cathepsin (B,L) MMPs (1,2,3,7,9,12,13) Dipeptidyl Peptidase IV ACE TACE
Inhibition of MMPs by GM 6001 (Reference Compound)
125 100
% Inhibition

75 50 25 0 -25 -50 -12

MMP-1 MMP-2 MMP-3 MMP-7 MMP-12 MMP-13

IC50(nM) MMP1 1.16 MMP2 3.70 MMP3 3.65 MMP7 8.99 MMP12 1.19 MMP13 1.07
-9 -8 -7 -6

-11

-10

Kinases Standardization of following kinases is in progress (32P-ATP)

CDK1 PKA PKC (a, b, g, d) MAPK (MEK, ERK-1, p38-a) Src-tyrosine kinase

Concentration Log Drug [M]

CDK1/Cyclin B: Substrate - Peptide 80

60

Cdk1/CyclinB:Sub-Peptide

% Inhibition

40 20 0 -20 -7.5

IC50 (mM) Roscovitine - 2.7

-6.5

-5.5

-4.5

-3.5

Log Drug Conc(M)

The assays for other Kinases and proteases can also be considered for 2006 Copyright GVK Biosciences Private Limited development based on clients need

Whole Cell Based Assays

% inhibition of LPS

Cell Adhesion ICAM1 - LFA1 VCAM1 - VLA4 Whole Blood Assays COX-1 COX-2 Cell Proliferation PHA induced PBMNC proliferation Con A induced PBMNC proliferation Cytokine release Assays TNF-a IL-1b IL-6 Cytotoxicity Cell Viability

Inhibition of LPS induced IL-1

100

Release

% inhibition of LPS

SB203580
75 50
EC50 R 2.140e-007 0.9853

25 0 -9 -8 -7 -6 -5 -4

SB 20358 [Log]Conc (M)

100 75 50 25 0 -8

SKF-86002

EC50 R

1.614e-006 0.9788

-7

-6

-5

-4

-3

SKF-80002 [Log]Conc(M)
2006 Copyright GVK Biosciences Private Limited

In-vitro ADME
Physicochemical Properties
Solubility Lipophilicity
pKa LogP/LogD Oct./water partitioning

In-vitro Absorption

Plasma Protein binding

Stability Studies
In Biological Fluids
Plasma, serum, simulating fluids, buffer.

Metabolic stability studies

2006 Copyright GVK Biosciences Private Limited

Aqueous Solubility- Kinetic Solubility

Kinetic solubility

Test compound is dissolved in DMSO. The test compound of required concentration is added to the aqueous buffer at desired pH.
Equilibrate the MultiScreen plate for 90 minutes. The concentration of the soluble drug is analysed by LCMS-MS.

2006 Copyright GVK Biosciences Private Limited

Aqueous Solubility- Thermodynamic Solubility

Thermodynamic Solubility of the compound by Shaker Flask Method
Aqueous solubility-Thermodynamic Solubility

Test compound in solubility media and equilibrated with stirring for 24hrs and left unstirred for another 24hrs. The sample is filtered and filtrate concentration is analyzed by LCMS-MS The level of aqueous solubility is determined

Compound Name

Concentration (g/ml) Initial pH 2.3 0.32 Low pH 7.4 > 200 High 1.41 Low

Indomethacin

250

Ketoconazole

250

166.09 Moderate

2006 Copyright GVK Biosciences Private Limited

Absorption-MDCK/Caco2 Permeability study

MDCK - Unidirectional Permeability (96 well plates) Seed 45000cells/well/75ul media (apical well-AP) Add test compound (100uM) Collect basal and apical samples at different time point (15, 30, 60,120 min) Analyze Samples by LC-MS/MS Determine apparent permeability and percent recovery Ensure monolayer integrity using Lucifer yellow ( Fluorescence method) Bidirectional Permeability studies 24 well Caco-2 cell culture plates - Determine efflux ratio Caco-2 cell test compound is added 21 days after seeding the cells MDCK cells GVK results
Drugs applied Atenolol (low permeable drug) Propranolol (High permeable drug) Pindolol (medium permeable drug) Papp (nm/sec) GVK values Reported 8.7 + 2.25 18 + 9.2 889 + 54 101 + 13.4 1700 + 65 590 + 36
2006 Copyright GVK Biosciences Private Limited

PAMPA (Parallel artificial membrane permeability assay)

Non-cell based passive permeability assay Lecithin in dodecane artificial layer is used Test compound (dissolved in 5%DMSO or PBS) in donor plate and PBS in acceptor plate and incubated for 16 hours. UV absorption is measured from both donor and acceptor plates

Log of effective permeability (log Pe) is determined

2006 Copyright GVK Biosciences Private Limited

Protein Binding
Protein binding determined by Ultra-filtration Matrix: Human/Mouse/Rat/ specify Incubate plasma with the test compound for an hour Aliquot plasma into 10KD filter and spin at 3000g for 45 minutes Filtrate is injected into LCMS-MS to get the concentration. Percentage of free drug will be calculated % free drug =[ Concentration in the filtrate ] Concentration Retained in filter

2006 Copyright GVK Biosciences Private Limited

In-vitro Metabolism
In-vitro and Ex-vivo Metabolic studies Microtome, s9 fraction Various species like Rat/Human/Mouse

Metabolic stability at specific time Determination of Half life and Intrinsic clearance Metabolite Identification using in-vitro metabolic studies
Specific CYP inhibition screening Methods Activities of human CYP isozymes CYP1A2 CYP 2B6 CYP 2C9 CYP 2C19 CYP 2D6 CYP 3A4)

using selective substrates incubated with human microsomes.

2006 Copyright GVK Biosciences Private Limited

Microsomal Stability
Microsomal Stability
Assay Formats: Single time point or multiple time points.
90
% Compound Remaining after 30min

Microsomal Stability Study

Species: Human, Rat, mouse etc. (or any laboratory animal). Analytical Method: The analytical method is developed in LC-MS/MS, method is validated for recovery. General Procedure: The test compound is dissolved in suitable solvent (1M), incubated at 370C in incubation mixture. After each time point the samples is precipitated with Acetonitrile (depends on method). The samples are analyzed by LC-MS/MS

80 70 60 50 40 30 20 10 0
Imipramine Verapamil

Rat

Human

2006 Copyright GVK Biosciences Private Limited

CYP isozymes Inhibition Assay

Validated human CYP isozymes available with GVK Biosciences CYP1A2 CYP 2C9 CYP 2C19 CYP 2D6 CYP 3A4)
CYP2C9 Inhibition Assay
Enzyme Source
70

Human recombinant (insect Sf9 cell) 7-ethoxy-3-cyanocoumarin (CEC) CEC + NADPH 7-hydroxy-3cyanocoumarin + NADP+ Fluorescence Crespi et al., Anal Biochem. (1997)

Substrate Reaction

% Inhibition

45 20 -5 -30 -2 -1

Method Ref. compound Sulfaphenazole

0 1

IC50 0.0365M
2

References

Concentration M
2006 Copyright GVK Biosciences Private Limited

In-vivo Pharmacokinetics
Single Dose Pharmacokinetics Study (Rats,Mice, Guinea pigs, Rabbits) by various routes in cannulated animals Dose Proportionality Studies Absolute Bioavailability Multiple Dose Pharmacokinetics & Toxicokinetics Tissue Distribution studies In-vivo Blood Brain Barrier Studies PK/PD relationship studies

Pharmacokinetic Analysis of Non-clinical Studies (WinNonlin)

Multiple Dose Administration Various Route of Administration
Oral, IV, IP, SC, IM, ID, IT, Ocular, Dermal

Blood collection in Small animals and Rabbits Surgical Techniques ( Cannulation of jugular, carotid and femoral)

2006 Copyright GVK Biosciences Private Limited

Pharmacokinetic Study in rat

Single Dose Intraperitoneal Pharmacokinetic Study of Compound XXX in male Sprague Dawley rats at a dose of 3 mg/kg.
Intraperitoneal PK of com pound XXX in fed m ale Sprage Daw ley rats at a dose of 3 m g/kg

Parameter
200.00
Mean Concentration ( ng/ml)

Mean data ( n=4)

0.08

Mean +/- SEM 150.00

TMax (hr) C Max ( ng/ml) kel (1/hr)

186.04 0.29 2.39 260.07

100.00

50.00

t1/2 (hr) AUC ( 0-t ) (hr * ng/ml)

2.00 4.00 6.00 8.00 10.00

0.00 0.00

Tim e ( hr)

AUC( 0- ) (hr *ng/ml)

284.06

2006 Copyright GVK Biosciences Private Limited

Bio-analytical Expertise
Method Development in Biological Matrix
Blood Plasma/serum CSF Urine Faeces Tissues (Liver, spleen, brain, lung etc)

Analysis by LCMS-MS Validation using Bio-analytical FDA guidelines

Recovery Linearity Accuracy and Precision Method Transfer

Bioanalysis of Drug and Metabolites

In-vitro metabolic studies PK/TK studies

2006 Copyright GVK Biosciences Private Limited

Animal Models
Analgesic Activity

Writhing

Phenylbenzoquinone induced

Metabolic Disorders

Acetic Acid Induced

STZ/Alloxan induced diabetic model OGTT/IVGTT in mice and rats Fructose induced Lipidaemia model Diet induced diabetic model Type II diabetes model Triton induced lipidaemia model Cholesterol fed Model

Formalin induced Hot Plate Tail flick Tail suspension

Carrageenan induced pain

Hyperalgesia Model

Inflammation

Carrageenan induced inflammation Ear Swelling test in mice Arthritis Model

Adjuvant

LPS induced sepsis model Inflammatory Bowel Disease Model

2006 Copyright GVK Biosciences Private Limited

Exploratory Toxicology studies in Rodents

Repeated dose study

SD rats, Wistar rats, Swiss mice

Observations

Body weights, clinical signs & Mortality Pathology

Haematology : Clinical Chemistry Necropsy/ Histopathology examination Organ weight and preservation of tissues/ organs Toxicokinetics / MoE

Target Specific monitoring as per sponsors recommendations

2006 Copyright GVK Biosciences Private Limited

Infrastructure

Recently built well equipped laboratories

Standard counters Multimode readers Dispensers

AERB (Atomic Energy Regulatory Board) approved radioactive facility P-II cell culture facility GLP compliant animal house for in vivo studies IT interface for online data generation, management & report submission

2006 Copyright GVK Biosciences Private Limited

We are willing to work as per your need

Integrated research services - Complements Informatics & Med. Chem. Capabilities and accelerate lead optimization Capability to co-develop assays/animal models in co-operation with the client Clients specific standards, ligands/membrane preparations Semi automated data management across all platforms Client specific dedicated templates for data transfer

2006 Copyright GVK Biosciences Private Limited

Thank You!

2006 Copyright GVK Biosciences Private Limited