In Vitro ADME Assays and Services

• Aqueous Solubility Assays
  • Kinetic solubility
  • Thermodynamic solubility
• Log D_7.4

• Human liver enzymes or recombinant enzymes
• Major CYP450 isozymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4 and 2E1
• Reversible, metabolic and time-dependent inhibition (IC50 shift)
• Initial screening assessments (% inhibition) or definitive determinations (IC50, Ki)
• K_inact/K_I determinations
• Determine formation of MIC and/or covalent binding
• Activity of CYP450 enzymes measured via LC-MS/MS analysis
• Drug-drug interaction experiments with known co-medications

• Human liver microsomes or recombinant enzymes
• CYP450-selective chemical inhibitors to determine which enzyme(s) are involved in metabolism
• Metabolite-monitoring option

• Multiple human CYP450 enzymes: 1A2, 2B6, 2C9, 2C19 and 3A4
• Enzyme catalytic activity or mRNA levels using human cryopreserved hepatocytes
• Catalytic activity of CYP450 isoforms measured via LC-MS/MS analysis

• Carboxyl esterases (CES): Reaction Phenotyping
• FMO assays: Reaction Phenotyping
• MAO-A/B assays: Inhibition and Reaction Phenotyping
• UGT assays: Inhibition, Induction and Reaction Phenotyping

• Plasma, serum, whole blood, microsomes, tissue homogenates or purified proteins (albumin, AGP)
• Human, rodent and nonrodent species
• Initial screening assessments or definitive binding determinations
• Ex vivo samples (plasma, tissue homogenates)

• Separate free compound from melanin-bound by centrifugation
• Synthetic melanin
• Also available from sepia (cuttlefish)
• Initial screening assessments (% bound) or definitive binding determinations (kd, Bmax)

• Liver microsomes, S9
• Non-hepatic microsomes available
• Human, rodent and nonrodent species
• Range of formats: % remaining or intrinsic clearance (T_1/2, CL_int)
• Metabolite-monitoring option

• Pooled cryopreserved hepatocytes
• Fresh primary hepatocytes available
• Human, rodent and nonrodent species
• Range of formats: % remaining or intrinsic clearance (T_1/2, CL_int)
• Metabolic monitoring option

• Microsomes, S9, hepatocytes, ex vivo samples, plant or sediment material
• Human, rodent and nonrodent species
• Initial screening assessments (profiling) or more definitive determinations (ID) using accurate mass

• Ex vivo matrices (plasma, serum, blood, tissue, homogenates, CSF), aqueous buffers, formulations
• Range of formats: % remaining pr T_1/2 determinations

• Fresh whole blood
• Human, rodent and nonrodent species
• Blood/plasma ratio, Kp

• Caco-2 (human epithelial colorectal adenocarcinoma cell line), MDCK (wild type, canine), MDCK-MDRI (transfected with human Pgp) and MDCK-BCRP (transfected with human breast cancer resistance protein)
  • Cell monolayers available every week
• Uni-directional (A-B flux) or bi-directional (efflux)
• Measurement of analyte levels on apical and basolateral sides of cell monolayer using LC-MS/MS bioanalysis

• Stably transfected cells (HEK293) overexpressing the transporter of interest: MATE1, MATE2, OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2 and more

• Substrate and inhibition assay formats
• Initial screening assessments or definitive determinations

• hERG Inhibition using the Charles River ChanTest^® hERG-HEK stably transfected cell line on the Sophion Qube automated electrophysiology platform

• Cell Proliferation and Cytotoxicity in HepG2 cells

• Ames MPF, Ames II, mini Ames
• Bacterial Mutation Test (Ames, OECD 471)
• Mammalian Cell Gene Mutation Test (HPRT Gene, OECD 476)
• Mammalian Chromosome Aberration Test (OECD 473)
• Mammalian Cell Gene Mutation Test (TK Gene, OECD 490)
• Mammalian Cell Micronucleus Assay (OECD 487)
• Fluorescence In Situ Hybridization (FISH)

As compound potency improves during hit-to-lead and lead optimization, in vitro ADME and DMPK assays provide necessary data to establish insight into the key physicochemical properties and structural motifs, ensuring the efficacy and safety of compounds.

Yes, while standard protocols help to maximize throughput and reduce lead times, Charles River can assist in customizing a comprehensive strategy that provides the targeted candidate ADME profile.

Kinetic solubility interprets the performance of compounds within in vitro ADME assays and biology screens while also assessing the effect the structural modifications have upon solubility within chemical series. Experimental logP/logD assessments are also available using miniaturized shake-flask methodology to reinforce in silico predictions.

Charles River uses validated metabolic stability screens to assess a compound’s vulnerability to metabolic instability whether through CYP450-mediated or other types of oxidations, conjugations or other biotransformations. High resolution mass spectrometry is used to identify putative metabolites, elucidate their structures, and characterize the metabolic fate of compounds.