Hits to Leads ADMET assays

These are examples of the type of ADMET assays that CXR can deliver to identify potential liabilities of compound early in discovery. Please contact us to discuss your requirements and we will tailor these studies to your particular needs.

Initial Studies

Metabolic stability
Measure compound metabolism in rat & human microsomes, LC/MS/MS to measure disappearance of parent compound.
HRN microsomes can be used to determine FMO metabolism

Determination of oral absorption & bioavailability in vivo
Can be performed in mice, rats and/or HRN mice
cassette or individual compound dosing n=3, 8 time points over 6 hours, compound blood concentration measured by MS/MS and the half-life, AUC and clearance calculated.
Use of HRN mice improves bioavailability & provides insights into the role of metabolism in compound disposition

Early demonstration of in vivo efficacy
First in vivo studies using HRN increases probability of demonstrating initial efficacy

Preliminary toxicity
Incubate compound with rat and human hepatocytes, measure LDH leakage and [ATP] depletion to estimate TD50

Metabolism Studies

Human microsome studies – reaction phenotyping
Determine which cytochrome P450’s are involved in metabolising each compound
Incubate with a panel of pre-characterised human microsomes, n=12, and determine loss of parent with MS/MS.
Perform Spearman Rank correlation of disappearance of compound with known CYP activity to identify those CYPs responsible for metabolising the compound

Analysis using recombinant human P450 enzymes
Spearman Rank results confirmed and extended using recombinant human P450 isoforms.
Unequivocal identification of P450s involved.

CYP P450 inhibition using human microsomes
Pool of pre-characterised human microsomes
Incubate microsomes with test compounds and cocktail of P450 substrates (n=5)
Use MS to determine appearance of substrate metabolites and determine which P450’s are inhibited by the test compound to determine IC50 values.

Metabolic Kinetics

Across species comparison of in vitro metabolism
Hepatocytes from all species of interest (eg humans, dog, monkey and rat) are incubated with test compound and the loss of parent measured using MS/MS. The Km and Vmax for each species is calculated and the intrinsic clearance of each compound is calculated in each species. 

Detailed analysis of metabolic processing across species
Hepatocytes from each species of interest (eg human, dog and rat) are incubated with radio-labelled compound and both Phase 1 and Phase 2 metabolites determined, HPLC radiolabel detection and LC/MS. 

Detailed determination of PK and bioavailability in rats
Detailed PK study with two routes of administration (iv and oral ) in groups of 3 rats with blood samples at 12 time-points taken over 24 hours. Samples are analysed using MS/MS and full PK parameters calculated

Pre-development systemic toxicity study 

 This study will identify potential target organ toxicity and estimate the toxic potency of the compounds and /or metabolites prior to entering full GLP safety studies to prevent any unexpected results

Groups of 6 rats (3 male and 3 female), 3 dose levels, oral dose, daily for 7 days,
body weight, clinical observations taken daily. Killed day 8, weighed, post mortem. 
Histopathology on brain, thyroid, kidney, liver, spleen, testes/ovaries, uterus, adrenals and heart. 
Clinical chemistry
Haematology
Liver microsomes are analysed for CYP P450 content and activities.
Satellite groups for transcriptional profiling
HRN used to determine toxicokinetics or parent vs metabolite toxicity

Please contact us to discuss your specific requirements and we will design a study package to address your needs.

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