Metabolite Biosynthesis and Structural Characterization

Obtaining reference standards of disproportionate and/or unique metabolites in human and evaluating their potential pharmacological activity and toxicity (MIST）play an important role in the successful regulatory filings of drugs. The metabolite biosynthesis by in vitro incubation or in vivo dosing in animals can efficiently synthesize metabolites and support the accurate structures identification of the metabolites.

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Overview
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Workflow
Case Study
Regulatory Guidance
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FAQs
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Overview

It is very challenging to obtain the precise structures of the specific or disproportionate metabolites quickly and cost-effectively. The biosynthesis platform of WuXi AppTec DMPK Service can biosynthesize target metabolites by in vitro or in vivo approach based on study result of MetID, and combination with preparative liquid chromatograph for separation, high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) for structural characterization. The targeted metabolites can be biosynthesized efficiently and their precise structures can be characterized. This technique has the following advantages: 1. synthesize rapidly and accurately, no need for multiple matching with the target metabolites in in vivo or in vitro samples; 2. alternative plan when chemical synthesis is difficult; 3. the process is relatively simple and less time-consuming.

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Assays

Metabolite biosynthesis and structural characterization via in vitro incubation system
- Confirmation of the target metabolite and optimization of the in vitro incubation system
- Scale up production of the target metabolite through in vitro system
- Isolation and purification of the target metabolite
- structural characterization via NMR
Metabolite biosynthesis and structural characterization via in vivo administration system
- Confirmation of the target metabolite and optimization of the in vivo system
- Scale up production of the target metabolite through in vivo system
- Isolation and purification of the target metabolite
- Structural characterization via NMR

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Workflow

The workflow of metabolite biosynthesis

The metabolite biosynthesis process mainly includes optimization and scale-up of the experiment system (screening of animal species, matrix types, and experiment conditions), isolation and purification of the target metabolites, and structural identification by HRMS and NMR.

Case Study

The case for the biosynthesis of the oxidative metabolite of carbazeran

The metabolite was observed to be abundant (~60% relative abundance) in human hepatocytes (Figure 1A). After comparing the metabolite profiles of carbazeran in liver microsomes, S9, and hepatocytes, it was hypothesized that the oxidation metabolite was a product of the AO enzyme. Considering the accessibility of the bio-matrix and cost, the monkey cytosol enriched in the AO enzyme was chosen for further optimization of the incubation conditions (Figure 1B). Eventually, more than 95% of carbazeran was metabolized to the oxidative metabolite. After isolation and purification by preparative LC, this metabolite was characterized as 7-oxo-carbazeran by NMR (Figure 2&3).

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- LC-UV Chromatograms of Carbazeran in human hepatocytes after incubation 2 h (A) and in monkey cytosol after incubations 4 h (B)
  
  Figure 1
- Carbazeran 1 H NMR Spectrum
  
  Figure 2
- 7-oxo-carbazeran 1 H NMR Spectrum
  
  Figure 3

Regulatory Guidance

Safety Testing of Drug Metabolites Guidance for Industry

M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals

Featured Strengths

Comprehensive platform
Targeted synthesis
Short turnaround time
Customized delivery
10+ years’ experience
50+ projects completed

FAQs

How long is the TAT（turnaround time）of a biosynthesis project?

The TAT of biosynthesis projects is generally 4-6 weeks, including process optimization, scaling-up production, separation and purification, and structural characterization of the target metabolites by NMR.
What are the deliverables of metabolite biosynthesis?

There are three deliverables based on different experimental requirements, 1) Precise metabolite structure based on HRMS and NMR, 2) Metabolite standards (powder or stock solution)，3) Precise metabolite structure based on HRMS and NMR + metabolite standards (powder or stock solution).
What are some challenges associated with the structural characterization of metabolites?

The precise structure characterization of the metabolites primarily relies on high-quality HRMS data and NMR data, with the metabolite standard being a prerequisite for obtaining these HRMS and NMR data. Due to the complexity of biological samples (in vitro incubated system or animal excrements), the low concentration of metabolites, and unknown structures, obtaining sufficient metabolites and isolating and purifying them to achieve mg-level high-purity standards is very challenging. Based on the metabolite biosynthesis platform provided by WuXi AppTec DMPK, we are capable of efficiently synthesizing metabolite standards and achieving precise characterization of the structure of metabolites.

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Metabolite Biosynthesis and Structural Characterization

Overview

Assays