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:
| 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 |
| 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 |
|
