Outsourcing Preclinical Drug Safety Studies
By Kyle Kramer, XenoTech LLC
Cytochrome P450 Inhibition
P450 Enzymes Involved in Drug Metabolism
Cytochrome P450 Induction
Metabolism Studies
NMEs As Inducers Of P450 Enzymes
Experimental Design and Study Specifics
XenoTech's Services
Benefits of our Services
In vitro drug metabolism studies play a critical role in the drug development process. United States and European regulatory authorities now encourage the use of appropriate in vitro methods to examine drug metabolism and drug interactions (inhibition and induction). Studies of this type are critical for preclinical drug safety assessment for prediction of inter-individual pharmacokinetic variability and potential drug-drug interactions.
New drugs should be evaluated early in drug development for the potential to inhibit cytochrome P450 enzymes. Human liver microsomes are a stable, reliable, and the most accepted in vitro system for measuring the potential of drugs and new molecular entities (NME's) potential to inhibit cytochrome P450 enzymes. XenoTech's scientists have performed experiments of this type for nearly a decade. We have the capacity to examine NMEs for their potential to inhibit any and all of the human hepatic cytochrome P450 enzymes. Our protocols are custom written for each client, and we can determine an IC50 or Ki value under GLP or non--GLP conditions. In addition, we routinely screen all chemicals for their potential to cause metabolism--dependent (mechanism-based) inhibition of P450 enzymes. Our Study Directors can extrapolate in vitro data to assess the clinical potential for cytochrome P450 inhibition in viva.
Identifying the P450 Enzymes Involved in Drug Metabolism (Reaction Phenotyping)
Establishing whether a drug is a cytochrome P450 substrate and, if so, which P450 enzyme(s) is/are involved in its biotransformation can help predict pharmacokinetic variability and adverse drug reactions. The research tools used for reaction phenotyping include:
- correlation analysis of the sample-to-sample variation in the metabolism of a drug by a bank of human liver microsomes
- inhibition of the reaction of interest by selective chemical inhibitors or specific inhibitory antibodies
- confirmation of the results obtained from (i) and (ii) by the use of cDNA-expressed enzymes.
The importance of reaction phenotyping was recently highlighted in the guidelines issued by the FDA for studying drug metabolism. For example, if a drug is metabolized by a P450 that is either polymorphicaly expressed or varies enormously from one individual to another, then this drug will exhibit wide variation in its pharmacokinetic behavior (oral bioavailability and/or clearance).
Cytochrome P450 Induction
Many cytochrome P450 enzymes in human liver are inducible by drugs and other chemicals. Cytochrome P450 induction increases the metabolic clearance of drugs, which results in lower blood levels, which can compromise the drug's therapeutic goal. The generally accepted research tools for studying induction of P450 enzymes include:
- in vivo treatment of laboratory animals followed by ex vivo analysis of liver microsomal samples
- induction of P450 enzymes in cultured rat or human hepatocytes.
XenoTech has years of experience in examining drug candidates as P450 inducers in both of these systems.
Cytochrome P450 metabolism can vary widely from one species to another. In fact, P450 metabolism in rodents or other laboratory animals may have little or no relevance to humans. However, species differences in drug metabolism can be assessed by studying the in vitro metabolism of a drug by liver microsomes or hepatocytes from a variety of species and strains of laboratory animals. This can help identify the most relevant animal model for pharmacokinetic and toxicity studies. It should be noted that species differences in the toxicity of drugs can often be explained by species differences in metabolism.
Rationale For Examining NMEs As Inducers Of P450 Enzymes
It is prudent to evaluate the potential of a new molecular entity (NME) to induce cytochrome P450 enzymes, as recommended by the FDA. Clinically important consequences of enzyme induction include:
- enhanced biotransformation of contraceptive steroids, cyclosporin and warfarin by inducers of CYP3A and CYP2C enzymes
- enhanced activation of acetaminophen to its hepatotoxic metabolite, N-acetylbenzoquinoneimine, by CYP2E1, and possibly by CYP3A enzyme inducers
- increased elimination of HIV reverse transcriptase inhibitors by CYP3A inducers.
Experimental Design and Study Specifics
In vivo treatment with ex vivo analysis: For analysis of in vivo induction of cytochrome P450 enzymes, XenoTech or the sponsor can treat laboratory animals with the test article. However, it is often cost effective to use portions of liver removed from animals used in short-term toxicology studies. XenoTech conducts ex vivo analysis of P450 enzyme induction by examining both enzyme activities and protein levels (as detected by western immunobloting). Studies are designed to identify if the drug is similar to the following prototypical inducers of P450: b-naphthoflavone (a CYP1A inducer), phenobarbital (a CYP2B inducer), isoniazid (an inducer of CYP2E1), pregnenolone-16-a-carbonitrile (a CYP3A inducer), clofibric acid (a CYP4A inducer), or a mixed inducer such as Aroclor 1254.
In vitro induction: Hepatocytes isolated from human or rat liver are cultured in a sandwich of extracellular matrix which allows establishment of near-normal morphology and the restoration of hepatocellular function, including secretion of albumin and formation of bile cannuliculi. After a suitable incubation period, primary cultures of hepatocytes are treated with various concentrations of test article for two to three days. Positive and negative controls are included in all experiments. Following treatment, cells are harvested and microsomes are prepared to access P450 induction as described above.
In addition to the types of studies listed above, XenoTech offers a host of other services related to Phase-II metabolism of drugs, covalent binding of reactive intermediates, development of HPLC methods, and large-scale metabolite generation. We also review reports, offer consultation, and teach introductory and advanced continuing education courses. All of our laboratory services are offered as either GLP or non-GLP, but all of our services are protocol driven, with the experimental design based upon information provided by our sponsors.
To date, XenoTech's scientists have provided services like those described above to over forty organizations in the pharmaceutical or chemical industry over the last 10 years. In addition, XenoTech's scientists have consulted on metabolism-related issues to over fifty organizations worldwide, including the US Food and Drug Administration.
The team of Xenotech's senior scientific staff, led by Andrew Parkinson, has lectured on in vitro techniques at numerous pharmaceutical companies, scientific meetings, and the FDA's Center for Drug Evaluation and Research. In addition, we have written reviews and published over 100 publications on various aspects of drug metabolism. XenoTech consistently anticipates the needs of the pharmaceutical industry and regulatory agencies. It is for this reason that Dr. Parkinson's research group has provided in vitro metabolism contract research services since 1986, more than a decade before FDA released its guidelines in 1997. You may not have heard of us, but the scientific group at XenoTech was instrumental in regulatory approval for some of the top selling drugs in the world. We have conducted studies for over forty pharmaceutical/chemical companies, including five of the ten largest pharmaceutical companies in the world.
XenoTech offers a host of services designed specifically to help you meet regulatory needs, and we have the experience to design in vitro studies and interpret their clinical significance.
- Save time and money by selecting the most appropriate human model
- Avoid idiosyncratic drug responses in clinical trials
- Avoid selecting lead candidates that are substrates for polymorphic cytochrome P450 enzymes
- Evaluate the potential of new drugs to induce or suppress cytochrome P450 enzymes in a clinically relevant model
- Identify potential drug-drug interactions that may affect labeling requirements
- Predict variability in pharmacokinetic behavior
For more information: Kyle Kramer, manager, Marketing and Sales, XenoTech LLC, 3800 Cambridge St., Kansas City KS 66103. Tel: 913-588-7530. Fax: 913-558-7572.