Metabolite identification and stability

 

Our expertise in metabolite identification and stability

We provide our customers with world-class expertise in the various areas of small molecule drug metabolism, metabolite identification, stability, reactive metabolites charcaterisation and related drug interaction studies. Our in vitro metabolic studies can be conducted with liver microsomes, S9 fraction or intact hepatocytes. For cross-species comparison studies, microsomes, S9 fractions and cryoperserved hepatocytes are available in human, rat, mouse, dog, monkey, minipig and rabbit species.

Pilot studies

The NCE at one concentration (2 μM) is incubated with appropriate liver fraction in the presence of enzyme cofactors (NADPH, UDPGA, PAPS and/or GSH). Incubations without cofactors and without NCE are conducted as negative controls and to characterize the non-metabolism related disappearance of NCE. The samples are analyzed with UPLC/TOF-MS to monitor both metabolic stability and metabolite identification.

MetaboPilot

Quick and easy-to-order study for metabolite identification and stability, giving data on main biotransformations of NCE in human. All phase I and II metabolism pathways are enabled.

The tested compound is incubated with liver microsomes, S9 fraction or homogenate in the presence of necessary cofactors (NADPH, UDPGA, PAPS, GSH, depending on the enzyme source) as well as incubations without cofactors and without the test compound are conducted as negative controls and to differentiate the non-metabolism related disappearance of the test compound. The incubations are sampled at time points 0, 10 30 and 60 minutes. The samples are analysed with UPLC/TOF-MS to monitor both substrate depletion and metabolite formation.

metaboliteid_case_1
Suggested metabolic reactions and biotransformation sites for haloperidol in a MetaboPilot study. The four most abundant metabolites are marked with red font.

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CryoPilot

For metabolite identification, the primary hepatocytes, with all the major phase I and phase II metabolic transformation activity present, is considered a “golden standard” approach. CryoPilot study is conducted with pooled cryopreserved hepatocytes, being a simple study to give reliable information regarding metabolite identification and stability of NCE as well as tentative structures of formed metabolites at the same time. Hepatocytes contain most of the important drug metabolising enzymes and intact cellular environment and, therefore, they represent the most predictive in vitro model for predicting drug metabolism. The results is very comprehensive metabolite pattern, covering detection of expected and unexpected metabolites in a single UPLC/MS run -­ thus no need to predict metabolites without analyses.

The test compound is incubated with pooled cryopreserved hepatocytes, and samples are collected at time points 0, 30, 60, 120 and 240 minutes. The samples are analysed with UPLC/TOF-MS to monitor both substrate depletion and metabolite formation. The disappearance-data is used for for estimating in vitro clearance, T1/2 and liver intrinsic clearance. If logD/P, pKa and plasma protein binding information is available, also in vitro-in vivo extrapolation (including compound clearances and half- lives) will be given based on the results. The data acquired from 240 min samples are used for software-aided (MetaboLynx) and manually confirmed mining of metabolites, and the tentative structures of detected metabolites are identified based on accurate mass and in-source fragmentation ion data.

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Max studies

The NCE at higher concentration (10 μM) is incubated with appropriate liver fraction in the presence of enzyme cofactors. Also incubations without cofactors and without NCE are conducted as negative controls. The incubations are sampled at 2 time points. Metabolites are profiled and identified from the samples using UPLC/TOF-MS to monitor comprehensive metabolite identification.

MetaboMax

Incubation of the NCE with liver microsomes (with NADPH and UDPGA) or S9 fraction (with NADPH, UDPGA, PAPS and GSH) using time points 0 and 60 min.

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CryoMax

Incubation of the NCE with cryopreserved primary hepatocytes in the presence of NADPH, UDPGA, PAPS and GSH using time points 0 and 4h.

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met_id_tof
UPLC/TOF-MS ion chromatograms for the Study Compound and its metabolites.

MetaboCryo

The NCE at 3 different concentrations is incubated with cryopreserved primary hepatocytes in the presence of enzyme cofactors. Also incubations without cofactors and without NCE are conducted as negative controls and to differentiate the non-metabolism related disappearance of NCE. The incubations are sampled at 6 time points. The samples are analyzed with UPLC/TOF-MS to monitor both comprehensive metabolic stability and metabolite formation for metabolite identification.

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metabolites_and_stab
Stability of the main metabolites (M1, M4, M5, M7 and M8) at two time points (0, 60 min) +/- cofactors (NADPH and UDPGA) in mouse in vitro system.

MetaboRad

The 14C labeled NCE at one concentration (about 2-5 μCi/ml) is incubated with liver microsomes or cryoperserved primary hepatocytes in the presence of enzyme cofactors.The incubations are sampled at 2 time points (0 and 60 min for microsomes; 0 and 240 min for hepatocytes). Similar incubations are conducted with non-labelled NCE. The samples from 14C labeled NCE are analyzed with HPLC with online radioactivity detector (HPLC/RAD) and the samples from non- labeled NCE are analyzed with UPLC/TOF-MS.

MetStab

Straightforward study to characterize metabolic stability of NCEs and to calculate enzyme kinetic values for intrinsic clearance and in vivo prediction. Used for hit molecule characterisation, lead molecule selection and to aid lead optimisation.
The test compound is incubated at 3 different concentrations with pooled liver microsomes or cryopreserved primary hepatocytes in the presence of appropriate enzyme cofactors. Samples are withdrawn at 4 time points (0, 10, 20 and 40 min) and analyzed with UPLC/TOF-MS or UPLC/MS-MS for comprehensive metabolic stability. The disappearance rate is used to calculate in vitro clearance and to predict in vivo hepatic clearance using physiological assumptions.

metabolic_stability
Metabolite stability for a test compound with two concentrations, +/- cofactors at 0 + 3 times points.

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metabolic_stability_2
Metabolite stability for a test compound in two time points, cross species comparison.

Reactive metabolite characterization

Reactive drug metabolites are known to be one of the factors behind unexpected drug-induced toxicity and therefore their identification early in the drug discovery process is of big importance.
>>Follow this to link more on our unique experience with reactive metabolites characterisation. >>Also see our comprehensive MIST (metabolites in safety testing) service offering from this link.

We help you to understand more on the metabolism studies and help you to choose the right analysis for each project and molecule. Contact us from this link.

Background of in vitro metabolism studies

Drug molecules face a variety of oxidizing (Cytochrome P450s, MAO, FMO, ADH) and conjugating (UGT, SULT, GST, MT) enzymes in the body. These enzymes catalyse reactions that render drugs into more hydrophilic metabolites that can be excreted from the body. The pace and capacity of metabolism dictate the duration of action of drugs in the body. Sometimes the metabolites possess pharmacologic activity or even toxicity. Therefore, the knowledge of metabolic stability and metabolite profile is essential already in the early phase drug discovery. In addition, identification of the metabolite structures is needed to find sites in the molecules undergoing metabolism (so called “soft-spots”) and thus disappearance of the parent compound. This information can be used to alter the relevant chemical structure of the compound and thus to improve unfavorable parent compound stability.

In vitro metabolism studies are typically conducted as studies comparing the metabolism in several species. The differences in species specific metabolism is then to be used in selection of the proper model organism for in vivo pharmacokinetic and toxicity studies. >>more on our MIST – metabolism in safety studies expertise and cross species metabolism differences.

Assay systems for in vitro metabolism studies

In vitro metabolite identification and stability studies can be conducted with liver microsomes, liver homogenates, S9 fraction or intact hepatocytes. Microsomes contain mainly drug metabolizing phase I enzymes and UGTs. In addition to microsomal enzymes, S9 fraction contains soluble cytosolic proteins thus covering most of the phase I and phase II metabolic enzymes. Cryopreserved primary hepatocytes contain most of the important phase I and phase II metabolic enzymes and an intact cellular environment, therefore representing the most predictive in vitro model for predicting in vivo drug metabolism.

Cryopreserved primary hepatocytes are considered to be the most accurate model for metabolite identification and stability. Cryopreserved hepatocytes are available commercially from multiple vendors, coming from different species, as pooled or form a single individual and other stratification criteria (like sex, age groups etc.). Whilst fresh hepatocytes are also available, they are less used for metabolite identification/stability studies, but rather for example to CYP enzyme transcript/protein induction studies.