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MIST (Metabolites in Safety Testing)

In Vivo Services

In the preclinical development phase, toxicological studies conducted in preclinical species are used to demonstrate that the animals have safely been exposed to the drug at exposures in excess of those that patients will be exposed to under normal therapeutic conditions. 

A key component of human risk assessment for drug candidates is to include exposure to circulating metabolites of the drug in that risk assessment because drug toxicity could be mediated by these metabolites. 

Regulatory agencies (FDA and ICH) have provided recommendations on when and how to identify and characterize drug metabolites to ensure their toxicity has been adequately evaluated in preclinical species. Industry is encouraged to conduct such evaluations as early as possible during the drug development process. Metabolites identified only in human or metabolites present at higher levels in human than in any of the animal test species may be of concern. 

Industry Leading Comprehensive MIST Assessment 

Frontage has developed an approach that integrates a series of in vitro and in vivo studies that in combination demonstrate due diligence in meeting MIST regulatory expectations. This approach includes several inter-related experiments that are described below. 

Metabolite Response Factor Determination 

Relative Response Factor (RRF) is an analytical parameter used to control for other drug-related components in biological samples that contain the parent drug. RRF is used to correct for the difference in detector response of metabolites compared with parent drug. An LC/HRMS analysis is conducted using radiotracer (if available) or UV to generate peak areas for the metabolites and parent that are used to determine the RRF for the metabolites. 

Elimination of Matrix Interferences 

Often species-specific matrix interferences can result in differences in the ionization of metabolites and drug and can confound the relative comparisons required in determining metabolite safety coverage. This concern is addressed by conducting an UPLC-HRMS response linearity assessment of the drug and its metabolites across the human and animal species. 

Conducting Plasma AUC Pooling 

Hamilton RA et al.1 demonstrated that appropriate pooling of plasma across time points from several subjects or animals will generate a composite sample in which the relative abundance of each drug-related component is reflective of its AUC. The ability to analyze a single sample to measure all the drug-related components in plasma from each species enhances the reliability of these studies 

  1. Hamilton RA, Garnett WR, and Kline BJ, Determination of mean valproic acid serum level by assay of a single pooled sample, Clin Pharmacol. Ther., 29, (1981), 408-413. 

Identification of all Metabolites by HRMS 

The current best practice for identifying human metabolites in plasma samples dosed with non-labeled drug is to analyze the samples using high resolution mass spectrometry employing data processing approaches that will identify all metabolites present in the plasma samples.

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