Metabolite in safety testing (MIST) for bioanalysis

Shefali R. Patel, Senior Scientist, Janssen Research & Devleopment (PA, USA)

Shefali is currently Senior Scientist in the Department of Drug Metabolism and Pharmacokinetics (DMPK) at Janssen Research & Development, where she is supervising the non-regulated bioanalysis group within East Coast Bioanalytical Department. In her current role, she is also partnering with CROs to oversee the regulated bioanalysis work for GLP and clinical studies. Shefali’s current interests includes microsampling techniques for sample collection as well as patient centric sample collection for home sampling. Additionally, she is applying microsampling techniques for rodent studies within J&J. She has 20 years of experience in Bioanalytical Sciences. Before joining Janssen in 2007, Shefali was a Research Chemist with Drug Metabolism Department for Merck Research Laboratories (NJ, USA). Prior to Merck she was employed as Scientist with Taylor Technology (CRO) in Princeton, NJ, USA.

What is MIST?

Once a drug is administered, it usually metabolizes in the body and forms metabolites. Depending on the number and abundance of metabolites formed, they may be associated with toxic effects. To ensure that the metabolites, which are formed do not cause any toxicological effects in humans, metabolite identification and profiling is needed. Most of the time, metabolites that are low in circulation do not cause any issues but metabolites with more than 10% of total drug-related material are of concern. The safety of those metabolites needs to be demonstrated in nonclinical studies (MIST testing). For MIST analysis human and nonclinical animal species exposure levels are compared and evaluated to understand which human metabolites are formed, in what abundance and are sufficiently covered in the safety testing studies. Metabolites such as migrating acyl glucuronides and pharmacologically active metabolites need quantitative monitoring but they do not fall under MIST coverage.

Guidance for MIST

FDA issued a guidance document in 2008 which provided recommendations on dealing with drug metabolites in safety testing for a small molecule. According to the guidance, metabolites comprising of more than or equal to 10% of the parent compound in the systemic circulation at a steady state needed to be evaluated for safety [1]. The plasma levels of these metabolites should be in the same range in at least one toxicology species. Additional safety studies might be required if that is not the case. In 2009, the International Conference on Harmonization [ICHM3(R2)] published guidance with a different threshold for relevant human metabolites (10% or greater of total drug-related material in circulation) [2]. In 2012, an ICHM3(R2) provided more information on the MIST guidance [3]. In 2016, the FDA issued a revision that superseded the MIST guidance from 2008. In 2020, the FDA issued revision 2 that superseded the guidance of the same name published in November 2016 [4]. The guidance has been revised to align with the ICH guidance for industry M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (January 2010). The recent FDA guidance discusses a general approach for assessing metabolite safety. The guidance document also acknowledges difficulties associated with synthesizing a specific metabolite as well as the inherent complexities that accompany its direct administration. The guidance also provides examples of when drug metabolites are formed at higher levels in humans and how the safety evaluation should be approached.

When to conduct MIST analysis

There are three key parameters for assessing MIST: metabolite detection, metabolite identification and metabolite quantitation. Quantification will need to cover metabolite abundance (if any metabolites are greater than 10% of total exposure and metabolite coverage in nonclinical safety species [5]. It is highly recommended that MIST evaluation be initiated as early as possible. If adequate exposure of human metabolites of concern in safety species is identified at a later stage it may cause significant delays in drug development. Metabolism may vary between humans and in animals hence metabolites may be generated differently in humans and animals. Some of the metabolites may form only in humans or at higher levels in humans than in animal species and vice versa. One misconception of MIST is that the percentage of a given metabolite in one of the toxicology nonclinical species should be higher than that in humans. That is not true. There can be a lower percentage of a metabolite in nonclinical species but if the dose is sufficiently high and if the absolute concentration that metabolite is higher in one of the nonclinical species than that in humans then it is sufficient for the MIST coverage for that metabolite. The MIST analysis is first performed in in vitro studies to evaluate metabolites in cross species (i.e. rat, dog, human, monkey, etc.). Metabolite profiles are then generated from in vivo studies. Data from metabolite profiles help to identify what metabolites are formed and how much. Samples from the animal species (toxicology studies) can be used to gather relevant information on circulating metabolites in nonclinical species. Once clinical studies are initiated, samples from multiple ascending doses are analyzed to gather information on circulating metabolites in human plasma at a steady state. In a nutshell, multiple metabolite analysis and testing at a different stage of drug discovery are needed to evaluate if any potential safety issues are arising from the metabolites. The required studies for MIST analysis are cross-species comparison, MIST exposure comparison including human Met ID (repeated dose studies) and human ADME Met ID (single dose, radiolabeled) [6]. Ideally, studies to assess risks due to metabolites should be completed before larger scale clinical trials.

Quantification methods for MIST quantification

Quantification methods that can be applied to quantitate MIST samples are the radiometric method, mixed matrix method, NMR method and conventional bioanalytical methods. In radiometric methods, radiolabeled compounds are synthesized. The human radiolabel ADME study remains the cornerstone of addressing MIST guidance [5]. In the mixed matrix method, metabolite coverage in nonclinical species is determined by mixing equal volumes of pooled human plasma with the blank plasma of animal species and vice versa. The sample is then analyzed using a full scan high-resolution mass spectrometer [7]. This approach does not require synthetic standards or radiolabeled compounds and provides comparable results to conventional bioanalytical assays. The NMR method can be applied to quantitate metabolites in clinical studies where authentic standards might not be available. NMR methods have not gained more acceptance due to relatively lower sensitivity than LC−MS as NMR instruments are not readily available in the lab as an LC−MS instrument. The conventional quantitation approach requires the synthesis of authentic standards as well as quantification methods to meet health authority guidelines. With conventional quantitation methods, a tiered approach for sample analysis can be applied depending on the stage of the drug development.

Bioanalytical considerations for MIST

A major challenge for bioanalysis of metabolites is the availability of authentic standards to quantitate reliable and accurate results in biological samples. Metabolite standards are not available commercially so they will need to be synthesized in proper quantities to support bioanalytical methods. If the analysis of metabolites is needed for GLP studies, then a proper certificate of analysis along with expiration dates will be needed as per the FDA guidelines. Stable isotopic labeled internal standards would be ideal for metabolite quantification but they are difficult to synthesize and expensive to generate. Another analytical consideration is appropriate sample volume especially for rodent studies where the sample volume is limited, and multiple analytes need to be measured. Enough sample volume for regular analysis along with reanalysis as well as incurred sample analysis will be needed. Hence analytical methods with multiple analytes in one assay should be ideal for supporting MIST nonclinical studies. While developing analytical assays stability experiments should be given proper emphasis since some of the metabolites might be unstable and may influence the analytical performance. Long term stability experiments should be designed so they are enough to cover all the MIST sample storage in the freezer. If MIST study analysis is supported via a qualified assay, emphasis on method quality should be given. Appropriate experiments to evaluate matrix effect, ion channel cross talk and carryover should be carried out. Proper analog internal standard selection where no stable isotopic labeled internal standard is available should be performed to compensate variations on instrument performance.

Tiered approach for MIST samples with conventional bioanalytical methods

Depending on the study type as well as the stage of drug development, quantification of metabolites should be performed via a tiered approach – screening, qualified or validated assays [8].

Screening methods are applied where relative quantification is satisfactory to detect and identify the metabolites. Screening methods apply to discovery, in vitro and non-GLP studies. Reference standards may not be available for supporting the screening methods. Data generated from screening methods may provide information on the abundance of metabolites and help guide if qualified methods will be required for the nonclinical and first-in-human studies.

Qualified methods are applied where absolute concentration data is needed with proper documentation for decision making. Reference standards should be available for the qualification and appropriate experiments with pre-set acceptance criteria should be performed. Qualified assays are usually performed to support non-GLP, GLP studies as well as first-in-human studies. Qualified assays should have a minimum of accuracy and precision run as well as stability experiments to support studies.

Validated methods are applied when metabolites are found to be active metabolites or are 10% or greater of total drug-related material in circulation. Metabolite methods should be validated as per regulatory guidance [9,10]. Reference standards with a proper certificate of analysis and expiration date should be synthesized and provided for the method. In special circumstances when an appropriate reference standard is not available/difficult to synthesize then qualified methods can be applied for absolute quantitation.

Conclusions

MIST analysis is conducted at various stages of drug development and covers multiple studies starting from discovery to clinical studies. The purpose of each study is different to better understand metabolite formation in multiple species. Evaluating metabolites early in drug development is important in understanding impact on the efficacy and safety. Recent guidance by FDA has clearly defined the need and expectations for the MIST analysis.