ADME Services 


PHARMout offers ADME (Absorption, Distribution, Metabolism, Excretion) services to provide fast and improved decision making for our clients to support drug discovery programs. Our cost-efficient standardized and customized assays per client's requirement for ADME parameters will help strengthen your ability to meet your drug discovery and pre-clinical goals. Our ADME capabilities include:


 Metabolite Identification and Profiling

    • In Vitro Metabolism: Liver Microsomes, Hepatocytes, S9, and Recombinant Enzymes
    • In Vivo Metabolism: plasma, blood, urine and feces
    • Small Molecules and Peptides
    • UPLC with LC/Orbitrap Mass Spectrometer
    • Structural Elucidation

 

 ADME Screening

    • Metabolic, Plasma, Blood, Intestinal Fluid and Buffer Stability
    • Plasma Protein Binding
    • Permeability and Transporters: Caco-2 and MDCK
    • Cyp450 Phenotype
    • CYP450 Inhibition and Induction

 

Microsomal Stability

    • Test Compound Concentration: 3uM (different concentrations available)
    • Microsome Concentration: 0.5 mg/mL
    • Time-points: 0, 15, 30, 45, 60 minutes
    • Final DMSO Concentration: < 1.0%
    • Compound Requirement: 20 uL of 10 mM solution in DMSO
    • Cofactor: NADPH
    • Control: Blank, minus Cofactor (60 min. only)
    • Positive Control: Verapamil, Diazepam
    • Analysis: LC/MS/MS
    • Data Deliveries: Half-life (t1/2), Intrinsic Clearance (CLint)

 

Plasma Protein Binding

    • Assay: Equilibrium Dialysis at 37 °C, 4 hours or TBD
    • Test Article Concentration: 5 uM (different concentration available)
    • Replicates: 2
    • Compound Requirements: 20 uL of 10 mM solution in DMSO
    • Control: Verapamil
    • Analysis: LC/MS/MS (both plasma and buffer standards preparation)
    • Data Deliveries: Fraction unbound in 100% plasma recovery

 

 CACO-2 Permeability

    • Test Article Concentration: 10 uM in HBSS-MES Buffer
    • Replicates: 2
    • Incubation Period: 37 °C, 60 minutes
    • Assay Buffer: HBSS-HEPES (pH 6.5/7.4 or pH 7.4/7.4, Apical/ Basolateral)
    • Compound Requirements: 20 uL of 10 mM solution in DMSO
    • Control: Atenolol, Propranolol and Talinolol
    • Analysis: LC/MS/MS Quantitation
    • Data Deliveries: Papp, Eflux Ratio

 

CACO-2 Substrate/ Inhibitor

    • Test Article Concentration: 10 uM in HBSS-HEPES Buffer Single Point, or 5 Points IC50
    • Replicates: 3
    • Incubation Period: 37 °C, 60 minutes
    • Assay Buffer: HBSS-HEPES (pH 7.4/7.4, Apical/ Basolateral)
    • Compound Requirements: 50 uL of 10 mM solution in DMSO
    • Control Inhibitor: 50 uM Verapamil
    • Control Substrate: 5 uM Loperamide
    • Analysis: LC/MS/MS Quantitation
    • Data Deliveries: Papp, Eflux Ratio in presence and absence of inhibitor, IC50

 

Cytochrome P450 Inhibition

    • CYP Isoforms Available: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4
    • Replicates: 2 or 4
    • Pre-Incubation Time: 30 minutes
    • Test Article Concentration: 25 uM (single point); 0, 0.1, 0.25, 1.0, 2.5, 10 and 25 uM
    • Compound Requirements: 100 uL of 50 mM solution in DMSO
    • Substrate and Controls: Isoform   Substrate
                                                 CYP1A2   Phenancetin
                                                 CYP2B6   Bupropion
                                                 CYP2C8   Paclitaxel
                                                 CYP2C9  Diclofenac
                                               CYP2C19   S-Mephenytoin
                                                 CYP2D6   Dextromethorphan
                                                 CYP3A4   Midazolam
                                                 CYP3A4   Testosterone 
    • Analysis: LC/MS/MS for Specific Metabolites
    • Data Deliveries: Mean% inhibition following pre-incubation IC50, Standard Error of IC50

 

Cytochrome P450 Induction 

    • Test System: Cryopreserved Human Hepatocytes
    • Replicates: 3
    • Exposure Period: 72 hours (Media changed every 24 hours)
    • Test Article Concentration: 0.1, 1.0 and 10 uM (or Custom)
    • Compound Requirements: 30 uL of 10 mM solution in DMSO
    • CYP Isoforms: CYP1A2, CYP2B6 and CYP3A4
    • Negative Control: Vehicle 0.1% DMSO
    • Positive Controls: Omeprazole (CYP1A2), Phenobarbital (CYP2B6) and Rifampin (CYP3A4)
    • Probe Substrate: Phenacetin (CYP1A2), Bupropion (CYP2B6) and Testosterone (CYP3A4)
    • Analysis: LC/MS/MS for Specific Metabolites: Acetamenophen (CYP1A2), Hydroxybupropion (CYP2B6) and 6β-Hydroxytestosterone
    • Data Deliveries: Assay method, basal and induction activity, fold inductive above vehicle control, % positive control induction

 

 



Copyright © 2018 Pharmout Laboratories, Inc. All rights reserved.