CYP450 Time-Dependent Inhibition (TDI) Assay

The CYP450 time-dependent inhibition (TDI) assay sits at a nexus in the pipeline of drug development. Drug metabolism primarily occurs in the liver, relying on the cytochrome P450 (CYP450) enzymes, a superfamily of enzymes involved in catalysing the metabolic conversion of endogenous and exogenous compounds such as drugs, steroid hormones and fatty acids. More than 80 per cent of existing drugs are metabolised via one or more of these CYP enzymes, including CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4.

The inhibition effects drugs have on CYP450 enzymes can be classified into reversible and irreversible types. TDI is a type of irreversible inhibition where the inhibitor can form an irreversible covalent binding with the enzyme and cause permanent loss of function. This inhibition can trigger serious clinical drug-drug interactions (DDIs) and, after drug discontinuation, newly synthesized enzymes are required for recovery as the enzyme function doesn't immediately return.Hence, evaluation of the TDI risk of a drug at an early stage of the drug development is of paramount importance to ensure the safety and efficacy of the drug. Under the guidance of regulatory bodies like the EMA and FDA, TDI testing has become an integral part of the ADMET (absorption, distribution, metabolism, excretion, and toxicity) screening. This not only aids in assessing potential metabolic enzyme inhibition risks but also supports the prevention of clinical drug interactions.

Why Test Drugs for TDI?

• Evaluate metabolic enzyme inhibition risks: CYP450 Time-Dependent Inhibition can lead to reduced drug clearance, increased drug exposure, and subsequent toxic reactions. TDI testing can identify and mitigate this risk.
• Predict clinical drug interactions: TDI testing can foresee adverse interactions during a drug's metabolic process, ensuring drug safety and efficacy, and supporting normal drug development and utilization.
• Predict potential drug risks: Reactive metabolites binding covalently with P450 enzymes can trigger hapten formation, provoking autoimmune responses and causing severe health outcomes.
• Comply with market regulations: The TDI properties of drugs must be evaluated in new drug development under the mandates of major regulatory bodies including the FDA, EMA, and NMPA.

Creative Bioarray Offers Comprehensive CYP450 Time-Dependent Inhibition (TDI) Assay Services:

In the early drug screening phase, Creative Bioarray provides high-throughput, cost-effective TDI testings such as IC₅₀ shift. In later stages, comprehensive enzyme kinetic studies can be conducted to obtain parameters like K_I and K_inact for clinical implications.

IC₅₀ shift assay:

• Pre-incubation: Conduct pre-incubation at 37°C with or without NADPH, alongside a control group without inhibitor. A 30-minute pre-incubation time is typically used.
• Measurement phase: At each time point, add substrates to the pre-incubation system and commence enzyme activity measurements. Assess enzyme activity by determining substrate consumption via LC-MS/MS.
• Data processing: Calculate the IC₅₀ Shift fold by comparing IC₅₀ values with and without NADPH. A fold greater than a defined threshold (usually 1.5 or 2) indicates time-dependent inhibition.
  Matrix: Pooled human liver microsomes
  CYPs available: CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4
  Compound requirements: 5-10 mg of dry compound or 100 µL of 10 mM stock DMSO solution
  Deliverables: IC₅₀, Shifted IC₅₀, and report.

Determination of enzyme constants in vitro:

• Experimental setup: Select the relevant CYP450 enzyme subtype for the compound under study (typically using human liver microsomes) and an appropriate enzyme substrate, i.e., a CYP450 subtype-specific substrate.
• Pre-incubation: Mix the test compound with CYP450 enzymes and pre-incubate at a specified temperature over designated time periods, such as 0, 5, 10, 20, 30, and 60 minutes, to evaluate time-dependence.
• Measurement phase: Post-pre-incubation, immediately add the enzyme-specific substrate to start the reaction. Terminate the reaction at the predetermined time using a stop reagent. Subsequently, measure the concentrations of the CYP substrates by LC-MS/MS.
• Data processing: Plot curve graphs based on metabolic rates or metabolite generation amounts under varying time points and inhibitor concentrations to calculate K_inact (inactivation rate constant) and K_I (dissociation constant of the enzyme-inhibitor-substrate complex).
  K_obs = apparent inhibition rate constant, K_inact = theoretical maximum inhibition rate, K_I = inhibitor concentration at half maximum inhibition rate, [I] = inhibitor concentration.
• Result analysis: Evaluate the potential of time-dependent inhibition of the CYP450 enzyme by inhibitors based on obtained K_obs, K_I, and K_inact values, and assess TDI strength.
  Matrix: Pooled human liver microsomes
  CYPs available: CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4
  Compound requirements: 5-10 mg of dry compound or 100 µL of 10 mM stock DMSO solution
  Deliverables: K_obs, K_I, and K_inact, and report.

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Explore Other Options

• In Vitro Metabolism
• In Vitro Permeability and Transporters
• Drug-Drug Interaction
• High-throughput ADME Screening
• Physicochemical Characterization Assays

• CYP and UGT Reaction Phenotyping Assay
• Cytochrome P450 Induction Assay
• Cytochrome P450 Inhibition Assay
• UGT Induction
• UGT Inhibition

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