Identification of the Biotransformation Pathways of a Potential Oral Male Contraceptive, 11β-Methyl-19-Nortestosterone (11β-MNT) and Its Prodrugs: An In Vitro Study Highlights the Contribution of Polymorphic Intestinal UGT2B17

Abstract

11β-Methyl-19-nortestosterone dodecylcarbonate (11β-MNTDC) is a prodrug of 11β-MNT and is being considered as a promising male oral contraceptive candidate in clinical development. However, the oral administration of 11β-MNTDC exhibits an ~200-fold lower serum concentration of 11β-MNT compared to 11β-MNTDC, resulting in the poor bioavailability of 11β-MNT. To elucidate the role of the first-pass metabolism of 11β-MNT in its poor bioavailability, we determined the biotransformation products of 11β-MNT and its prodrugs in human in vitro models. 11β-MNT and its two prodrugs 11β-MNTDC and 11β-MNT undecanoate (11β-MNTU) were incubated in cryopreserved human hepatocytes (HHs) and subjected to liquid chromatography-high resolution tandem mass spectrometry analysis, which identified ten 11β-MNT biotransformation products with dehydrogenated and glucuronidation (11β-MNTG) metabolites being the major metabolites. However, 11β-MNTG formation is highly variable and prevalent in human intestinal S9 fractions. A reaction phenotyping study of 11β-MNT using thirteen recombinant UDP-glucuronosyltransferase (UGT) enzymes confirmed the major role of UGT2B17 in 11β-MNTG formation. This was further supported by a strong correlation (R² > 0.78) between 11β-MNTG and UGT2B17 abundance in human intestinal microsomes, human liver microsomes, and HH systems. These results suggest that 11β-MNT and its prodrugs are rapidly metabolized to 11β-MNTG by the highly polymorphic intestinal UGT2B17, which may explain the poor and variable bioavailability of the drug.

Keywords: 11β-MNT; 11β-MNTDC; 11β-Methyl-19-nortestosterone; LC-MS; MetID; UGT2B17; biotransformation; metabolite identification; oral male contraceptive.

Figure 1

11β-Methyl-19-nortestosterone dodecylcarbonate (11β-MNTDC) and 11β-Methyl-19-nortestosterone undecanoate (11β-MNTU) are prodrugs metabolized by esterases into their active metabolite, 11β-Methyl-19-nortestosterone (11β-MNT) (A). Structural similarities amongst testosterone, dimethandrolone (DMA), and 11β-MNT (B). Red spheres in (B) indicate oxygen atom.

Figure 2

Volcano plot showing fold-differences and p-values of m/z features detected in a 2 h incubation of human hepatocytes with 11β-MNT as compared to the untreated (control) samples (A). P represents the parent active metabolite (11β-MNT); M1 and M3–M7, six identified metabolites; M2, four potential structural isomers. The black dotted line indicates −log10 (p-value, i.e., 1.33). Relative MS intensity of 11β-MNT metabolites in human hepatocyte incubation (B).

Figure 3

Box and Whisker plot representing 11β-MNT and its major metabolites, M4, i.e., 11β-MNTG (A) and M7 (B) showing signal in the treatment (50 µM of 11β-MNT) compared to the untreated group in human hepatocytes.

Figure 4

The levels of dehydrogenated 11β-MNT and 11β-MNTG formed in human hepatocyte incubations of 50 µM 11β-MNT (A) and 10 µM 11β-MNTU (B), and 11β-MNTDC (C). HH1–4: (Average of all four hepatocytes with SD). HH1 and HH2 represent UGT2B17 high expressors, whereas HH3 and HH4 are the null expressors of UGT2B17.

Figure 5

Metabolite/11β-MNT ratio of dehydrogenated 11β-MNT and 11β-MNTG in the pooled human intestinal (A) and liver (B) S9 fraction incubations with 50 µM of 11β-MNT, 11β-MNTDC, and 11β-MNTU.

Figure 6

Normalized 11β-MNTG levels (with internal standard) detected in the incubation of individual recombinant UGT isoforms. SD represents triplicate analysis.

Figure 7

Correlation of UGT2B17 abundance and relative 11β-MNTG levels in HIMs (A), HLMs (B), and human hepatocytes (C).