Development and qualification of recombinant Göttingen Minipig CYP3A22 and CYP3A29

Abstract

In vitro drug metabolism assays are essential in early drug development to evaluate drug disposition. These assays often use hepatic subcellular fractions and recombinant enzymes originating from both human and nonclinical species. Within this domain, cytochrome P450 (CYP) 3A enzymes are considered key players for drug metabolism. Although recombinant CYP3A enzymes are available for several nonclinical species, isoform-specific enzymes from Göttingen Minipigs remain unavailable. Therefore, recombinant Göttingen Minipig CYP3A22 and CYP3A29 enzymes were developed in Escherichia coli. Their catalytic functionalities were examined using probe substrates (i.e., benzyl-oxy-methyl-resorufin and Luciferin-IPA), and clinically relevant human CYP3A4 substrates (i.e., midazolam and testosterone) along with known inhibitors (i.e., ketoconazole and troleandomycin). Gene sequence comparison to domestic pig revealed sequence variations for both r-CYP3A22 and r-CYP3A29. BOMR metabolism was detected in both constructs, but only r-CYP3A22 metabolized Luciferin-IPA. r-CYP3A22 exhibited a higher apparent affinity for midazolam, with a lower Km and comparable Vmax to r-CYP3A29, whereas r-CYP3A29 showed a greater maximal metabolic capacity toward testosterone, with a higher Vmax and similar Km as compared to r-CYP3A22. Ketoconazole exhibited potent reversible inhibitory effects on testosterone and midazolam metabolism in both enzymes. Troleandomycin strongly inhibited both constructs in a time-dependent manner; however, an IC50 shift was only measurable for r-CYP3A22. These results provide important insights into Göttingen Minipig CYP3A isoform-specific functionality which are relevant for species selection in future nonclinical studies.

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BibTeX RIS

BibTeX

@article{buyssens2026development,
  title = {{Development and qualification of recombinant Göttingen Minipig CYP3A22 and CYP3A29}},
  author = {Laura Buyssens and Alexandra Kiss and Miriam Ayuso and Zsolt Fekete and Emese Kis and Dávid Rakk and Chloé Bars and Chris Van Ginneken and Alain J. Labro and Steven Van Cruchten},
  journal = {Frontiers in Pharmacology},
  year = {2026},
  month = nov,
  doi = {10.3389/fphar.2026.1965447},
  url = {https://www.frontiersin.org/articles/10.3389/fphar.2026.1965447},
  note = {CC BY 4.0},
}

RIS

TY  - JOUR
TI  - Development and qualification of recombinant Göttingen Minipig CYP3A22 and CYP3A29
AU  - Laura Buyssens
AU  - Alexandra Kiss
AU  - Miriam Ayuso
AU  - Zsolt Fekete
AU  - Emese Kis
AU  - Dávid Rakk
AU  - Chloé Bars
AU  - Chris Van Ginneken
AU  - Alain J. Labro
AU  - Steven Van Cruchten
PY  - 2026
DA  - 2026/11/04
JO  - Frontiers in Pharmacology
DO  - 10.3389/fphar.2026.1965447
UR  - https://www.frontiersin.org/articles/10.3389/fphar.2026.1965447
AB  - In vitro drug metabolism assays are essential in early drug development to evaluate drug disposition. These assays often use hepatic subcellular fractions and recombinant enzymes originating from both human and nonclinical species. Within this domain, cytochrome P450 (CYP) 3A enzymes are considered key players for drug metabolism. Although recombinant CYP3A enzymes are available for several nonclinical species, isoform-specific enzymes from Göttingen Minipigs remain unavailable. Therefore, recombinant Göttingen Minipig CYP3A22 and CYP3A29 enzymes were developed in Escherichia coli. Their catalytic functionalities were examined using probe substrates (i.e., benzyl-oxy-methyl-resorufin and Luciferin-IPA), and clinically relevant human CYP3A4 substrates (i.e., midazolam and testosterone) along with known inhibitors (i.e., ketoconazole and troleandomycin). Gene sequence comparison to domestic pig revealed sequence variations for both r-CYP3A22 and r-CYP3A29. BOMR metabolism was detected in both constructs, but only r-CYP3A22 metabolized Luciferin-IPA. r-CYP3A22 exhibited a higher apparent affinity for midazolam, with a lower Km and comparable Vmax to r-CYP3A29, whereas r-CYP3A29 showed a greater maximal metabolic capacity toward testosterone, with a higher Vmax and similar Km as compared to r-CYP3A22. Ketoconazole exhibited potent reversible inhibitory effects on testosterone and midazolam metabolism in both enzymes. Troleandomycin strongly inhibited both constructs in a time-dependent manner; however, an IC50 shift was only measurable for r-CYP3A22. These results provide important insights into Göttingen Minipig CYP3A isoform-specific functionality which are relevant for species selection in future nonclinical studies.
SN  - 1663-9812
ER  -

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