Science Pool

Cytochrome P450 (CYP) induction plays an important role in the pharmacokinetics of a drug and can have consequences for drug efficacy through the reduction of plasma half-life, or drug toxicity if elevated levels of toxic metabolites are formed. These effects are commonly observed when one drug has an effect on a co-administered medication – a term known as drug-drug interactions or DDI.

Transcriptional gene activation, mediated by nuclear receptors such as the aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), is the most common mechanism of CYP induction. These receptors correlate directly to the expression of CYP1A2 (AhR), CYP2B6 (CAR), and CYP3A4 plus the CYP2C enzymes (PXR). Therefore, receptor activation can be used as an early indicator of potential changes in CYP enzyme expression.

A less common mechanism for CYP induction is through mRNA or enzyme stabilisation. In this case, certain drugs don’t necessarily stimulate CYP enzyme expression, but rather, slow down CYP protein degradation.

In drug discovery, cell-based transactivation assays can be used for identifying CYP induction potential. In this assay, stably or transiently transfected cell lines containing the nuclear receptor to be evaluated and reporter gene vectors are used. Activation of the response elements following receptor heterodimerisation serves as a suitable proxy for CYP induction. Results are typically reported as E_max and EC₅₀, or a concentration-dependent fold activation relative to vehicle control.

For more advanced drug development, such as IND-enabling studies and NDA submission, CYP induction is typically evaluated as part of a more extensive DDI package. At this stage, cryopreserved human hepatocytes are the preferred model with at least three donors assessed to account for inter-individual variability in response. The hepatocytes are typically incubated with the test compound over 48 to 72 hours, and CYP enzyme induction is evaluated by measuring mRNA levels and/or measuring the catalytic activity of an isoform-specific probe substrate. Because mRNA detection isn’t subjected to the masking effects of time dependent inhibition, regulatory authorities such as the US FDA and EMA recommend this method. However, if protein stabilisation is expected, catalytic activity analysis should also be conducted. Once again, results are typically reported as an E_max and EC₅₀, or concentration-dependent fold increase in response relative to the control. In addition, measurement of test compound over several time points on the last day of incubation is encouraged.

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The current Sars-CoV-2 pandemic has shown what a powerful threat pathogens can be to human civilisation. However, not only viruses threaten the human population. Every year, at least 700,000 people worldwide die of drug-resistant diseases, including 230,000 people who die from multidrug-resistant tuberculosis. An increasing number of diseases, including respiratory tract infections, sexually transmitted infections and urinary tract infections, has become untreatable, while lifesaving medical procedures are becoming much riskier. Last year, the U.S. CDC listed 18 antibiotic-resistant bacteria and fungi that are a threat to humans. Novel anti-infectives are urgently required to address this unmet medical need.

Evotec is dedicated to the fight against resistant pathogens and the development of novel anti-infectives. In 2018, the Company acquired Sanofi´s infectious disease unit and thereby laid the foundation for accelerating an comprehensive R&D portfolio to combat infectious diseases. Moreover, the Company entered into a five-year partnership with the Bill & Melinda Gates Foundation in June 2019 to discover new treatment regimens that better address tuberculosis (“TB”), a severe global health burden and one of the leading lethal infectious diseases worldwide. Also in 2019, Evotec and Lygature announced their cooperation in a new initiative for the development of novel antibacterial agents against Gram-negative bacteria called “GNA NOW”.

In August 2020, Evotec entered into a new partnership with Resolute Therapeutics to combat infectious diseases and antimicrobial resistance. 

Evotec has established a leading-edge platform enabling the discovery and development of new therapies and therapeutic approaches to treat and prevent serious and life-threatening infections. The Company´s anti-infectives platform includes

• EvostrAIn™ – An extensive range of geographically diverse human pathogenic bacteria and fungi including isolates that are susceptible and resistant to current antimicrobial drugs.
• In vitro and in vivo microbiology encompassing Gram positive and Gram negative pathogens (including anaerobes) in a wide range of animal models with a broad range of endpoints.
• Translational in vitro and in vivo PK/PD and mathematical modelling with emphasis on in vitro Hollow Fibre Systems to mimic defined drug exposure profiles.
• In vitro and in vivo virology, focusing on respiratory viruses such as RSV, HRV, influenza virus and human coronavirus (enabling COVID-19 work).
• In vitro and in vivo mycology: human pathogens including Candida spp. Aspergillus spp. and parasitology

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PK/PD modelling is an important aspect of dose prediction of antibiotics for both preclinical and clinical development and is a requirement of both the European Medicines Agency (EMA) and US Food and Drug Administration (FDA). This technique facilitates efficient dose-response study designs and assists in identifying optimal dosing regimens to ensure clinical efficacy and to suppress drug resistance.

Cyprotex is increasingly supporting its parent company, Evotec, in the modelling and simulation area. For example, mathematical modelling techniques have been applied in the rational design of antibiotic efficacy studies to reduce the number of animals required to determine the magnitude of efficacy and pharmacodynamic driver and promote acceptance of such data by regulatory authorities.

Our research poster, titled Pharmacokinetic/Pharmacodynamic Modelling of Antibiotic Efficacy, was presented at the Alderley Park 3R’s Poster Event on 17th October 2018. The event was jointly hosted by AstraZeneca, Cancer Research UK and Agenda Life Sciences. The poster was awarded 1st prize in the event for its contribution to the 3R’s concept of Replacement, Reduction and Refinement.

The contemporary definitions of the 3Rs are:
• Replacement: accelerating the development and use of models and tools, based on the latest science and technologies, to address important scientific questions without the use of animals.
• Reduction: appropriately designed and analysed animal experiments that are robust and reproducible, and truly add to the knowledge base.
• Refinement: advancing animal welfare by exploiting the latest in vivo technologies and by improving the understanding of the impact of welfare on scientific outcomes.

In silico modelling and simulations can be used to improve study designs leading to a significant reduction in the number of animals required to achieve experimental objectives.

You can read more in our poster.

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