Home
 
Email Newsletter icon, E-mail Newsletter icon, Email List icon, E-mail List icon Sign up for our Email Newsletter
 
Apredica

ADME Tox Service Summary:

ADME Tox Service Details:

In Vivo Bioanalysis: Research Grade Level (non-GLP)

Bioanalytical services for pharmacokinetic in vivo studies, including:

  • Pilot screening studies - rapid PK for candidate selection
  • Pre-formulation development
  • Single compound and series of compounds on cassette dosing
  • Bioanalytical method development for parent and metabolites
  • PK parameter calculations

Tissue Extraction Method Development

Tissue extraction:

  • Plasma
  • Red blood cells
  • Brain
  • Liver
  • Kidney
  • Lung
  • Heart
  • Skeletal muscle
  • Urine

In Vitro ADME Screen Level I:

Solubility Screen

source Test matrix is 75 mM PBS buffer, pH 2-8 (1% DMSO)
substrate Test article
method Analyses by HPLC/UV or turbidimetry
results Data are expressed in µM

Test Article Stability

source Test matrix options include 1N HCl, 1N NaOH, 30% H2O2 and buffer 60C
substrate Test article
time points 0 and 60 min.
method Analyses by HPLC/UV and LC/MS/MS
results % stable relative to T=0 minutes. Optional identification of degradation products.

Metabolism Screen 1:
Metabolic Stability: Interspecies Comparison

source Test matrix options include microsomes or S9 and/or plasma (efficacy test species + human)
substrate Test article
time points 0 and 60 min, or 0, 15, 30, 45, 60 min.
method Analyses by either (+)- or (-)-ESI LC/MS
standards Assay dependent (verapamil, propranolol, warfarin, labetalol, phenacetin)
results Data are expressed as % remaining parent or t1/2

In Vitro ADME Screen Level II:

CaCO-2 Permeability

source Test matrix is 21-day CaCO-2 cells seeded in 24-well assay plates
substrate Test article
time points 0 and 120 min.
method Analyses by either (+)- or (-)-ESI LC/MS
standards Atenolol, propranolol, sulfasalazine. TEER determination and Lucifer Yellow passage for leakage tests.
results Data are expressed as Papp

Protein Binding

source Test matrix options include plasma or purified proteins
substrate Test article
time points 4 hrs.
method 96-well Equilibrium Dialysis or ultrafiltration. Analyses by either HPLC or (+)- or (-)-ESI LC/MS
standards Warfarin, acetylesalicylic acid, propranolol
results Data are expressed as % bound

Metabolism Screen 2:
rCYP450 Inhibition (Screen)

source Test matrices are microsomes containing individually expressed human P450. rCYP450: 2C9, 2C19, 2D6, 3A4, 1A2, 2B6, 2A6, 2C8, 2E1, and 3A5
substrate Assay dependent
time points Assay dependent
method Fluorescence detection
standards Assay dependent
results % inhibition relative to inhibitor-free data

In Vitro ADME Screen Level III:

Metabolism Screen III:
rCYP450 Inhibition (IC50)

source Test matrices are microsomes containing individually expressed human P450. rCYP450: 1A2, 2B6, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5
substrate Assay dependent
time points Assay dependent, inhibitor pre-incubation optional
method Fluorescence or LC/MS/MS detection
standards Assay dependent
results IC50 value in µM

Cell-based Toxicity

source HepG2, NIH 3T3, HaCaT cells
time points Acute: 2 hrs. Chronic: 24 - 48 hrs
method Cell necrosis marker
standards Taxol, acetylsalisylic acid
results EC50 value in µM

In Vitro ADME Screen Level IV:

CYP Identification and Metabolite Profile

source Test matrix is pooled human microsomes for CYP450 activity and efficacy species, plasma and tissues
substrate Test article
time points 0 and 120 min.
method Analyses by either (+)- or (-)-ESI LC/MS/MS
standards Warfarin, propranolol and testosterone
results Data are expressed by ion masses, signal abundances and retention times. Optional structural elucidation includes product ion scan of selected masses.

rCYP450 Inhibition (pHLM, LC/MS/MS)

source Test matrix is pooled human microsomes
substrate Assay dependent (e.g., Phenacetin (1A2), tolbutamide (2C9), S-mephenytoin (2C19), bufuralol (2D6) and midazolam (3A4/5))
time points 30 min.
method Analyses by either (+)- or (-)-ESI LC/MS/MS
standards Isoform-selective inhibitors
results IC50 or Ki