The document outlines GVK Bio's in-vitro ADME screening services that evaluate metabolism, distribution, and toxicity of drug candidates through various solubility assays. It details methodologies for determining solubility, protein binding, permeability, and metabolic stability across different species using advanced techniques such as LC-MS/MS and Caco-2 assays. The importance of these assays is emphasized in predicting oral bioavailability and pharmacokinetic properties essential for drug development.

1. CMYK
ADME SERVICES
Our In-vitro ADME screening service offers a portfolio of Solubility
assays for investigating: metabolism, distribution and toxicity.
permeability, solubility & physicochemical properties, Solubility is one of the most important physicochemical
GVK BIO delivers consistent, accurate compound data properties. We determine equilibrium solubility by dried DMSO
with cost-efficiency. method. We can also determine solubility in aqueous buffer (pH
1-9), organic solvents (DMSO, Ethanol, etc), formulations and
In-vitro ADME Capabilities excipients by pION/Multiscreen/HPLC/UV/MS/MSMS methods.
Physicochemical studies
• Log D/Log P
Protein Binding
• Solubility(Kinetic/Equilibrium)
In-vitro binding studies with plasma have proven to be a valuable
• Chemical stability tool for predicting In-vivo protein binding. We determine the
protein binding by both ultra filtration and rapid equilibrium
• Biological matrix stability (serum/ plasma/
dialysis. Using RED device we determine protein binding in
microsomes/ blood /hepatocytes/tissue homogenates)
microsomes. Plasma and tissue homogenate across various
Absorption/Distribution Assays species (Rat/mice/human &dog).
• Caco2 and PAMPA permeability assay
• Pgp substrate / inhibitor assay
Caco2 Permeability Assay
• Protein binding
Caco2 cells are the most frequently
• Blood/Plasma Partitioning Ratio used In-vitro models to assess
intestinal permeability. Permeability
Metabolism/Excretion across Caco2 cell monolayer is used
• Half life/clearance determination using to predict human permeability of drug
microsomes/Hepatocytes /S9 fractions/Cyp across species candidates, to perform in-depth
(Human/Rat/Mouse/Dog/ monkey) mechanistic and absorption studies,
to study the effects of transporters on
• CYP Inhibition (CYP1A2, CYP2C9, CYP2C19, permeability. We can determine
CaCo-2 Cells
CYP2D6, CYP3A4) apparent permeability (Papp) / efflux Semi-permeable
• Pathway determination (Phase I and Phase II) ratio / Unidirectional / Bidirectional membrane
by LCMS.
• Metabolite identification using Microsomes / Hepatocytes
and
• Characterization of potential metabolites using microsomes
and Hepatocytes across species (Human/Rat/Mouse/Dog/ PAMPA
monkey) (Parallel artificial membrane permeability assay)
Log D/Log P The Parallel Artificial Membrane Permeability Assay (PAMPA)
assay is used as an in-vitro model of passive, transcellular
Log D is determined by the shake-flask method, by dissolving
some of the solute in a volume of octanol and water/buffer and permeability. As well as for the prediction of oral absorption and
measure the concentration of the solute in each solvent. Log D is brain penetration. Effective permeability (log Pe) may be
determined by HPLC-UV with confirmation by mass. measured by pION/Multiscreen/LCMS.
CMYK

2. CMYK
ADME SERVICES
Microsomal Microsomal Stability
CYP2D6 inhibition by Furafyline CYP2D6 inhibition by Qunidine
Stability A
B
C
1 2 3 4 5 6 7 8 9 10 1112
D
96 - Well Plate
Metabolic stability plays E
F
G
H
110 EC50 3.092e-006 80 EC50 1.015e-008
an important role in the 90 R2 0.9968 70 R2 0.9900
Chemicals
success of drug 70 60
candidates. First pass 50
% Inhibition
% Inhibition
50
40
metabolism is one of Add Hepatocytes 30
30
the major causes of or Microsomes 10
20
0
poor oral bioavailability 10
-10
and short half life and -8 -7 -6 -5 -4
0
-10 -9 -8 -7 -6 -5
the study influences
Incubate
both oral bioavailability Log drug Con [M] Log drug concentration [M]
and half life. The half life
/ clearance / % Extract
metabolized can be CYP2D6 inhibition by Sulphaphenazole CYP3A4 - Ketoconazole
determined in
microsomes / LCMS Analysis
S9 fractions / 35 EC50 6.372e-007
110
EC50 1.884e-007
100
hepatocytes. 30
2
R 0.9431 90
R2 0.9979
80
25
% Inhibition 70
20 60
Hepatocyte metabolic stability
% Inhibition
50
15 40
10 30
Per orally administered drug may undergo first-pass metabolism 5
20
10
which influences the pharmacokinetic properties such as the 0 0
-9 -8 -7 -6 -5 -4 -10
clearance, the half-life or the bioavailability. Cryopreserved -10 -9 -8 -7 -6
hepatocytes are used to calculate half life/clearance/% parent Log drug Concentrations [M] Log drug concentration [M]
compound remaining.
Human hepatocytes metabolic stability Metabolite identification using
Light Sight Software
% Parent compound remaining
Metabolic stability and the identification of formed metabolites has
100
become an important tool. In-vitro metabolite identification &
90
80 characterization of the test compounds is determined using Q
70 trap with light sight software.
60
50
40
30 Bioanalysis
20
10
LC-MS/MS
0 ?
3200 QTRAP
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API3200
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7-E
no
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API4000
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HPLC
Compound details ? RRLC with PDA
Agilent 1200
?
Shimadzu Prominence with UV
? RRLC with HTS PAL
Agilent 1200
CYP Inhibition Others
CYP inhibition occurs either as reversible inhibition, quasi- ? (Molecular Devices)
Spectramax
irreversible inhibition or irreversible inhibition. CYP inhibition is a
fluorescent based/LCMS assay. Specific isoforms of CYP ?
BMG Polarstar
(CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are ? Handler (Tomtec)
Qudra4 Liquid
used to determine the inhibition (IC50).
GVK Biosciences Private Limited
28A, IDA, Nacharam, Hyderabad 500 076, India. T 91 40 66281823
F 91 40 6628 1505 E-mail: bdbio@gvkbio.com Website: www.gvkbio.com
CMYK