Influence of Stereochemistry on the Bioactivation and Glucuronidation of 4-Ipomeanol

Abstract

A potential CYP4B1 suicide gene application in engineered T-cell treatment of blood cancers has revived interest in the use of 4-ipomeanol (IPO) in gene-directed enzyme prodrug therapy, in which disposition of the administered compound may be critical. IPO contains one chiral center at the carbon bearing a secondary alcohol group; it was of interest to determine the effect of stereochemistry on 1) CYP4B1-mediated bioactivation and 2) (UGT)-mediated glucuronidation. First, (R)-IPO and (S)-IPO were synthesized and used to assess cytotoxicity in HepG2 cells expressing rabbit CYP4B1 and re-engineered human CYP4B1, where the enantiomers were found to be equipotent. Next, a sensitive UPLC-MS/MS assay was developed to measure the IPO-glucuronide diastereomers and product stereoselectivity in human tissue microsomes. Human liver and kidney microsomes generated (R)- and (S)-IPO-glucuronide diastereomers in ratios of 57:43 and 79:21, respectively. In a panel of 13 recombinantly expressed UGTs, UGT1A9 and UGT2B7 were the major isoforms responsible for IPO glucuronidation. (R)-IPO-glucuronide diastereoselectivity was apparent with each recombinant UGT, except UGT2B15 and UGT2B17, which favored the formation of (S)-IPO-glucuronide. Incubations with IPO and the UGT1A9-specific chemical inhibitor niflumic acid significantly decreased glucuronidation in human kidney, but only marginally in human liver microsomes, consistent with known tissue expression patterns of UGTs. We conclude that IPO glucuronidation in human kidney is mediated by UGT1A9 and UGT2B7. In human liver, it is mediated primarily by UGT2B7 and, to a lesser extent, UGT1A9 and UGT2B15. Overall, the lack of pronounced stereoselectivity for IPO's bioactivation in CYP4B1-transfected HepG2 cells, or for hepatic glucuronidation, suggests the racemate is an appropriate choice for use in suicide gene therapies.

Fig. 1.

Cytotoxicity dose-response curves in HepG2 cells transfected with rabbit CYP4B1 (A) and human P+12 CYP4B1 (B) from treatment with (S)-, (R)-, racemic IPO, and control HepG2 cells treated with racemic IPO. Data shown are the mean ± S.D. from triplicate replicates.

Fig. 2.

Comparison of substrate depletion (A) and quantification of NAC/NAL adduct formation (B) in reconstituted CYP4B1 incubations after 30 minutes with 25 pmol of P450 and 100 μM IPO substrates. Data shown are the mean ± S.D. from triplicate replicates.

Fig. 3.

Chromatographic separation of (R,S)-IPO-glucuronide (A) and 4-methylumbelliferone glucuronide (B) by UPLC-MS/MS. Incubation of 1 µM (R,S)-IPO-glucuronide (C) and 1 µM 4-methylumbelliferone glucuronide (D) in the presence of H. pomatia (1000 U/mL) in potassium acetate, pH 5, for 24 hours at 37°C. XIC, Extracted Ion Chromatogram.

Fig. 4.

Representative chromatographic traces of (R)- and (S)-IPO-glucuronides. (A) Incubation of 100 µM IPO in Tris buffer (pH 7.5), 10 µg/mL alamethicin , 5 mM MgCl₂, and 5 mM UDPGA with 0.25 mg/mL human liver microsomes. Incubation of 100 µM (R)-IPO (B) and (S)-IPO (C) in human liver microsomes. (D) Incubation of 100 µM racemic IPO with human liver microsomes in the absence of UDPGA.

Fig. 5.

Diastereoselective glucuronidation of racemic IPO in human liver microsomes. Incubations were conducted with 100 µM IPO in potassium phosphate buffer (pH 6.0–8.0) and Tris buffer (pH 7.0–8.5) with 0.25 mg/mL microsomal protein, 10 µg/mL alamethicin, 5 mM MgCl₂, and 5 mM UDPGA for 60 minutes at 37°C. The Tris pH reflects the actual pH at 37°C. Results are reported as the specific activity mean ± S.D. of three determinations.

Fig. 6.

(R)- and (S)-IPO-glucuronide formation utilizing a panel of 13 recombinantly expressed UGT isoforms. Racemic IPO (100 µM) was incubated with rUGT (0.25 mg of total protein/mL), alamethicin (10 µg/mL), MgCl₂ (5 mM), and UDPGA (5 mM) for 60 minutes at 37°C. Results are reported as the specific activity mean ± S.D. of three determinations.