Metabolites in Safety Testing Guidance

A disconnect is sometimes seen between pharmacokinetic (PK) data and the in vivo pharmacodynamic (PD) behavior of a drug, suggesting that metabolites might be partly responsible for observed efficacy and/or toxicity. In this situation, the exact structure of the metabolite can be obtained and synthesized for subsequent testing in in vitro potency assays. However, it is not common to test metabolites in in vivo toxicology studies because the toxicology species is assumed to be exposed to the metabolite when the parent compound is initially dosed. Nevertheless, it is possible that the exposure to metabolites in humans is higher than that in preclinical toxicology studies. A detailed flow chart has been proposed (see Fig. 6.1) to ensure that sufficient metabolite information is obtained in preclin-ical settings. This flow chart is based on the 2008 FDA (US Food and Drug Administration Guidance for Industry 2008) and 2009 ICH (ICH Harmonized Tripartite Guideline 2009) guidance and is referred to as the Metabolites in Safety Testing (MIST) guidance.

If a metabolite contributes to <10% of the parent systemic exposure (FDA guidance) or the total drug-related exposure (ICH guidance) at steady state, no further testing is needed. Of course a big difference exists between these two criteria, especially when the parent drug contributes to a relatively small percentage of the total drug-related exposure. In most cases, the ICH guidance is the more practical of the two, but it can be applied correctly only if the total drug-related exposure is known, and this requires human mass balance study using radiolabeled material.

If a metabolite contributes to >10% of the systemic exposure, preclinical testing may be required if exposure to the metabolite at the maximum tolerated dose exceeds that observed in humans at the highest clinical dose. The definitive assessment requires pre-clinical and clinical studies with radiolabeled material so that the absolute exposure of the metabolites can be quantified. However, radiolabeled material is frequently not available until later in the development stage.

The following experiment (Walker et al. 2009) can assess the liability of a metabolite in the absence of radiolabeled material:

1. Mix 50% plasma at the maximum tolerated dose from one of the preclinical toxicology species with 50% blank human plasma.

2. Mix 50% human plasma at the highest clinical dose with 50% blank plasma from one of the preclinical toxicology species.

3. Extract the drug and metabolite from the plasma samples.

4. Analyze the samples back-to-back, profiling the metabolites with LC-MS/MS.

Since the matrix is the same in both samples, the absolute abundance of the MS signal for each metabolite can be compared. The shortcomings of this approach are that (1) unanticipated metabolites may be missed and (2) determining if a metabolite contributes to >10% of the parent systemic exposure or total drug-related exposure may be difficult because the parent compound and metabolite may have different MS ionization responses.

Figure 6.1. FDA proposed Metabolites in Safety Testing (MIST) flow chart for assessing sufficient metabolite coverage in human plasma compared to preclinical species.

Monitoring UV response can circumvent the latter disadvantage, but this technique is prone to endogenous interference. Quantitative NMR is another option for monitoring metabolites in the absence of radiolabeled material (Vishwanathan et al. 2009).

AUC pooling is a method of pooling samples from multiple time points to get a single sample with concentrations of the parent drug and its metabolites that represent the total area under the curve (AUC) of the concentration-time profile (Hop et al. 1998).

Cpooi = concentration of pooled sample. tm — last timepoint.

The volumes of the individual aliquots from each timepoint used to create the pooled sample can be determined by:

v0 : v1 : v2 ...Vj ...vm — kAt0 : kAtj : kAt2 ... kAtj... kAtm v — volume of aliquot taken from each timepoint. k — proportionality constant.

At0 — t1 - t0, At1 — t2 - t0, At2 — t3 - t1 ... Atj

Example: If PK samples were collected for the following time points and k — 10, then the following volumes will need to be pooled.

Timepoint (in h)



























AUC pooling can reduce the number of samples for bioana-lysis from PK studies. This methodology may be even more valuable for metabolism studies to determine the relative amount of the parent drug and its metabolites that an animal is exposed to.

The following caveats have to be taken into consideration when interpreting the MIST guidance:

• Studies should preferably be done at steady state because the half-lives of metabolites may be different from that of the parent compound.

• Pharmacological and toxicological behavior is likely to be dependent on free plasma exposure, and, therefore, differences in plasma protein binding between the parent compound and metabolites should be considered.

• The guidance does not require both preclinical toxicology species to form the metabolite at adequate exposures.

• "Some metabolites are not of toxicological concern (e.g., most glutathione conjugates) and do not warrant testing." (ICH Harmonised Tripartite Guideline 2009).

• Even if the preclinical exposure of a metabolite is less than that encountered in the clinic, preclinical safety studies with this metabolite may not be realistic or practical (e.g., glucuronides).

• "The nonclinical characterization of metabolites with an identified cause for concern (e.g., a unique human metabolite) should be considered on a case-by-case basis." (ICH Harmonized Tripartite Guideline 2009).

• "This guidance does not apply to some cancer therapies where a risk-benefit assessment is considered." (US Food and Drug Administration Guidance for Industry 2008).

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