Formation and Antitumor Activity of PNU-159682, A Major Metabolite of Nemorubicin in Human Liver Microsomes

Abstract

Purpose: Nemorubicin (3V-deamino-3V-[2 00 (S)-methoxy-4 00 -morpholinyl]doxorubicin; MMDX) is an investigational drug currently in phase II/III clinical testing in hepatocellular carcinoma. A bioactivation product of MMDX, 3V-deamino-3 00 ,4V-anhydro-[2 00 (S)-methoxy-3 00 (R)-oxy-4 00 -morpholinyl]doxorubicin (PNU-159682), has been recently identified in an incubate of the drug with NADPHsupplemented rat liver microsomes. The aims of this study were to obtain information about MMDX biotransformation to PNU-159682 in humans, and to explore the antitumor activity of PNU-159682.Experimental Design: Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P450s (CYP) were used to study MMDX biotransformation. We also examined the cytotoxicity and antitumor activity of PNU-159682 using a panel of in vitro-cultured human tumor cell lines and tumor-bearing mice, respectively.Results: HLMs converted MMDX to a major metabolite, whose retention time in liquid chromatography and ion fragmentation in tandem mass spectrometry were identical to those of synthetic PNU-159682. In a bank of HLMs from 10 donors, rates of PNU-159682 formation correlated significantly with three distinct CYP3A-mediated activities. Troleandomycin and ketoconazole, both inhibitors of CYP3A, markedly reduced PNU-159682 formation by HLMs; the reaction was also concentration-dependently inhibited by a monoclonal antibody to CYP3A4/5. Of the 10 cDNA-expressed CYPs examined, only CYP3A4 formed PNU-159682. In addition, PNU-159682 was remarkably more cytotoxic than MMDX and doxorubicin in vitro, and was effective in the two in vivo tumor models tested, i.e., disseminated murine L1210 leukemia and MX-1 human mammary carcinoma xenografts.Conclusions: CYP3A4, the major CYP in human liver, converts MMDX to a more cytotoxic metabolite, PNU-159682, which retains antitumor activity in vivo.

Highly Potent, Anthracycline-based Antibody–Drug Conjugates Generated by Enzymatic, Site-specific Conjugation

“…Nemorubicin, a morpholinyl analogue of doxorubicin, is significantly more potent than doxorubicin, also acts on multidrug-resistant cells and is not cardiotoxic at therapeutic doses (38,39). PNU-159682 is a liver metabolite of nemorubicin and is about three orders of magnitude more potent than its parent molecule on cultured human tumor cells (34).…”

“…PNU-159682 was described to be several orders of magnitude more potent than doxorubicin (34) and has recently shown promise as a payload for ADCs in the context of conventional chemical conjugation (32,33). Indeed, when conjugated to trastuzumab, a significantly increased cytotoxic activity was achieved in comparison with maytansine conjugates and even cells with moderate HER2 expression levels, that were not sensitive to Kadcyla, could efficiently be killed (Figs.…”

Doxazolidine Induction of Apoptosis by a Topoisomerase II Independent Mechanism

“…[37][38][39][40][41] An example is the bioactivation product of nemorubicin. 39,42 Nemorubicin, a non-alkylating anthracycline, is less than 3-fold more toxic than doxorubicin, but its bioactivation product, 1 (PNU-159682 39 ), an alkylating anthracycline, is over three orders of magnitude more toxic than doxorubicin (Scheme 3). 39 Second, increased potency of doxazolidine occurs in spite of a half-life with respect to hydrolysis to doxorubicin and formaldehyde of only 3 min.…”

“…39,42 Nemorubicin, a non-alkylating anthracycline, is less than 3-fold more toxic than doxorubicin, but its bioactivation product, 1 (PNU-159682 39 ), an alkylating anthracycline, is over three orders of magnitude more toxic than doxorubicin (Scheme 3). 39 Second, increased potency of doxazolidine occurs in spite of a half-life with respect to hydrolysis to doxorubicin and formaldehyde of only 3 min. 11 The rapid hydrolysis may lead one to believe that treatment with formaldehyde or co-treatment with doxorubicin and formaldehyde could produce the same effect as doxazolidine.…”

Enzymatic activity in turkey, duck, quail and chicken liver microsomes against four human cytochrome P450 prototype substrates and aflatoxin B1

“…[6][7][8][9] A widely used strategy to identify an enzyme involved in a metabolic pathway is to correlate the prototype enzymatic activity with the target substrate molecule activity. [10][11][12][13] In the same way, quantification of enzyme expression by immunoblot and correlation of the amount of protein expressed with the enzyme activity of interest has been used to characterize enzymatic activities. 14 The current knowledge about the role of poultry CYP enzymes in the metabolism of different xenobiotics and drugs is scarce.…”