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Figure 1. In vitro metabolite (M) profiles of a drug candidate exposed to rat, dog and human hepatocytes. (All figures: Almac)

A drug entering the body undergoes a series of biotransformations via phase I and phase II metabolic pathways. During phase I, enzymatic processes in the liver and other tissues introduce reactive or polar (e.g. hydroxyl, thiol, amino, and carboxylic acid) groups, changing the compound’s chemical structure. These biotransformations make the drug more water-soluble and hence, easier to eliminate. The metabolites produced have a similar chemical structure to the parent drug and are more pharmacologically active at the therapeutic receptor sites.

In phase II, the drug is conjugated to produce a more water soluble and pharmacologically inactive metabolite. Phase II reactions can be catalyzed by transferase enzymes such as glutathione-S-transferases with the conjugated metabolite usually exiting the body through a detoxification mechanism. Moreover, the majority of phase II reactions are catalyzed by UDP glycosyltransferases. Other phase I/II biotransformations during the metabolism of the drug can take place and may involve a number of enzymes such as alcohol dehydrogenase, aldehyde dehydrogenase, ester and amide hydrolases, epoxide hydrolase, and flavine mono-oxygenases. Others include the sulfo-transferases, catechol-O-methyltransferase, and N-acetyltransferase.

The safety assessment of a drug candidate relies strongly on the metabolism data generated from animal studies. Unfortunately, preclinical animal safety studies produce a different metabolic profile than human studies. This is clearly illustrated by the analysis of in vitro metabolite profiles from a single drug candidate in the presence of a rat, dog, and human hepatocytes (Figure 1).

The findings of this study revealed that some of the in vitro metabolites formed in human hepatocytes were also present in both the rat and dog metabolic profiles. The most important difference was observed between rat and human hepatocytes which generated slightly different metabolite profiles.

One particularly striking disadvantage of the use of rodents is the production of a broad range of metabolites. A number of these metabolites are produced in small quantities in animals, but may be produced in significantly higher levels in human hepatocytes studies. This leaves the true metabolic fate of the drug uncertain and requires additional testing to evaluate the risk-reward of a particular metabolite. It is critical to have confidence in the right animal study in order for it to be possible to generate metabolites in significant amounts. These can be assessed against human metabolic profiles in developing a first-in-man study.

Metabolite in Safety Testing
The safety profile of drug candidates has raised new concerns about the toxicity of drug metabolites which are directly associated with the parent drug. Both the pharmaceutical industry and relevant regulatory agencies have taken a strong interest in this area since the publication of the first MIST (Metabolite in Safety Testing) paper in 2002. The next significant regulatory guidance was issued by the U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research in 2008. It was entitled “Safety Testing of Drug Metabolites” with the aim of clarifying the FDA position on when to identify and characterize metabolites. These specific guidelines only apply to small molecule, non-biological drug candidates. Anticancer agents, drug conjugates—other than acylglucuronides—and reactive intermediates are exempt from MIST guidelines.
MIST focuses on stable metabolites circulating in human plasma and recommends that in vivo metabolite evaluation in humans be performed as early as possible. The FDA guidance attaches an important emphasis on these circulating metabolites; in particular, all metabolites exceeding 10% of the parent drug, will require toxicological evaluation (Figure 2).


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Figure 2. MIST guidance decision flow diagram.

In regard to the ICH Topic M3 (R2) on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals guidelines, the evaluation of total exposure is when the metabolite has achieved 10% of parent drug systemic exposure at a steady state in order to initiate a toxicological investigation.

Complete data on human metabolites only becomes available after human mass balance studies (AME/MB) with carbon-14 radiolabeled material being used in most cases. This information is typically utilized in parallel with clinical Phase 2 studies. The requirement for safety drug metabolite studies can potentially delay the start of Phase 3 trials.

The MIST guidelines recommend that metabolites that are either absent in the animal species—or are present in humans at much lower levels—and whose area under curve (AUC) at steady-state is less than 10% that of the parent require no further investigation. In this regard, MIST requires efficient production systems to permit the production of human drug metabolites. In response to the fact that conventional chemical synthesis cannot always produce these metabolites, new approaches are being developed that typically employ the recombinant expression of human drug-metabolizing enzymes and whole-cell biotransformation processes.

Carbon-14 radiolabeling
The gold standard approach to quantify a metabolite is to synthesize a carbon-14-labeled version of the drug. By replacing a carbon-12 atom with radioactive carbon-14, researchers are left with a chemically identical analogue that enables the pathway of the drug to be traced in a biological system. Carbon-14 is preferred radioisotopes such as tritium because the exact position of the label can be selected based on the synthetic route employed for labeling. Carbon occurs in the skeleton of nearly all drug molecules, thereby allowing a chosen position for the radiolabeling site that is more likely to be metabolically stable.

Moreover, carbon-14-labeled compounds generally exhibit greater radiochemical stability than their tritium-labeled counterparts, as a result of the higher specific activity of tritium-labeled material. This has the effect of increasing the risk of significant autoradiolysis, (radiochemical decomposition), during storage or usage of the radiolabeled compound. Carbon-14 is also detectable at very low levels using scintillation counting, making it useful for studies in which doses that run close to the pharmacological threshold are common.

Carbon-14 radiolabeled metabolites
Preclinical in vitro metabolism studies form the starting point for carbon-14-radiolabeled drugs using hepatocytes from humans and animals. This study can be implemented using standard protocols to test for metabolites—which are radiolabeled at concentrations of 10 μmol/L—when incubated with the hepatocytes cells for a period of several hours.

This approach is necessary to evaluate the absolute concentrations of circulating metabolites. Additional safety evaluation may be required for an appropriate risk assessment for human metabolites that are structurally distinct from the parent drug but are present in circulation at high concentrations (e.g. greater than 1µM), in particular if human exposure is greater than that observed in animals. The central premise of these studies is to see if metabolites present in human in vitro systems are also found at comparable levels in animals. This enables metabolites (MIST) safety testing to be carried out correctly before entering human AME/MB studies.

Bio-analytical methods
There are an array of analytical techniques for analyzing the isolation of carbon-14-radiolabeled metabolites from preclinical plasma pooling methods. These can include a combination of high-performance liquid chromatography/ultraviolet/mass spectrometry (HPLC/UV/MS) by the use of a radio-detection configuration to obtain reliable AUC values of metabolites present in the plasma of preclinical safety studies. When guided by real-time peak analysis, HPLC can be used to collect radioactive fractions. Additionally, quantification of major metabolites in human plasma from single- and escalating-repeat-dose studies can be carried out using nuclear magnetic resonance spectroscopy (NMR). The labeling of drugs with carbon-14 has the added benefit of facilitating metabolite detection, easing purification, and enabling the radiochemical concentration of the metabolite in question.

As a consequence, the data generated will help to provide metabolite profiles, whilst providing clues to the chemical structure of the metabolite. As a result, this makes it more valuable for use in vivo metabolism studies where drug doses close to the pharmacological threshold are normally utilized.

Using carbon-14 for AME studies
Carbon-14 labeling has emerged as an even more useful tool because of advances in accelerated mass spectrometry (AMS). AMS is a costly, but highly sensitive technique for detecting carbon-14 and other radiolabels. The sensitivity of AMS allows human microdosing to be carried out with significantly lower levels of radioactivity (50 nCi) and sub-therapeutic doses. Small quantities of carbon-14-labeled API—typically about 10 kBq per study—are used to analyse the ratio of carbon-14 to carbon-12 and provide information on drug metabolism and pharmacokinetics (DMPK). In comparison, human mass balance studies require roughly 4 MBq of carbon-14 labeled investigational medicinal product per individual.

The benefit of these low-dose AMS studies lies in their ability to be performed at the earliest stage in the clinical development of the drug candidate. These studies provide information on the metabolism profile and rapidly highlight any difficulties, aiding Phase 2 studies through the selection of the correct preclinical study. These measures are also beneficial to managing project time lines and clinical costs in later stage development.

Taking a new approach
The MIST guidelines and ICH Topic M3 (R2) documents provide a reliable approach to assessing the safety of drug metabolites in humans. MIST places strong emphasis on obtaining preclinical human data on the pharmacokinetics of an investigational drug as early as possible. The guidelines can be met through the use of in vitro studies, implementing limited animal studies, and finishing with human AME studies using carbon-14-labeled drugs. The radiolabeling approach can help exclude potential reactive metabolites and serve as a useful tool for determining which circulating metabolites may need a MIST investigation. The presence of low levels of parent compound—as a result of extensive metabolism—highlights the significant challenges in assessing all metabolites present in greater than 10% of the parent drug in accordance with the MIST recommendations. Furthermore, the recent revision of the ICH M3 guidance is applicable to studies for the selection of circulating human metabolites requiring separate safety assessment criteria.

About the Author
Dr. Kitson is an investigator at Almac and has more than 10 years’ experience in the synthesis of carbon-14 radiolabeled compounds. He is also the editor-in-chief of Current Radiopharmaceuticals and a scientific committee member of the International Isotope Society (UK Group). He is a recipient of the 2006 Wiley Journal of Labeled Compounds and Radiopharmaceuticals award for radiochemistry.