N,N-diethyl-2-[4(phenylmethyl)phenoxy] ethanamine (DPPE), a chemopotentiating and cytoprotective agent in clinical trials:: interaction with histamine at cytochrome P450 3A4 and other isozymes that metabolize antineoplastic drugs

Purpose: N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine . HCl (DPPE), an intracellular histamine (HA) antagonist with chemopotentiating and cytoprotective properties, is currently in phase 2 and 3 clinical trials in breast and prostate cancer. DPPE modulates growth at in vitro concentrations that antagonize HA binding to cytochromes P450 in rat liver microsomes. HA inhibits P450 metabolism of some drugs. Recent in vitro studies in human colon cancer cells have linked DPPE enhancement of paclitaxel, doxorubicin and vinblastine cytotoxicity to inhibition of the P-glycoprotein (P-gp) pump. Many substrates of P-gp are also substrates of CYP3A4, a P450 isozyme that metabolizes a variety of antineoplastic agents and is highly expressed in some malignant tissues. Therefore, we assessed whether (a) DPPE and HA interact at CYP3A4 and other P450 human isozymes, and (b) DPPE inhibits the catalytic activity of CYP3A4. Methods: Using spectral analysis, we measured DPPE and HA binding to insect microsomes that express human P450 isozymes 1A1, 2B6, 2D6 or 3A4. Employing this-layer chromatography, we assessed the metabolism of DPPE by each isozyme and DPPE inhibition of testosterone metabolism by CYP3A4 and by rat liver microsomes. Results: (1) DPPE evoked "type I" (substrate site binding) absorbance-difference spectra with CYP2D6 (K-s = 4.1 +/- 0.4 mu M). CYP3A4 (K-s = 31 +/- 15 mu M) and CYP1A1 (K-s = 40 +/- 9 mu M), but not with CYP2B6. (2) In correspondence with the binding studies, DPPE was metabolized by CYP2D6, CYP3A4 and CYP1A1: 170 metabolism occurred with CYP2B6. (3) HA evoked "type II" (heme iron binding) absorbance-difference spectra with all four isozymes, with K-s values in the range 80-600 mu M. DPPE inhibited HA (600 mu M) binding to CYP2D6 (IC50 = 4 mu M. 95% CI = 1.8-8.9 mu M) and CYP1A1 (IC50 = 135 CIM: 95% CI = 100 177 mu M). but stimulated HA (500 and 1000 mu M) binding to CYP3A4 (EC50 = 115 mu M, 95% CI =104-231 mu M). DPPE did not affect HA binding to CYP2B6. (4) DPPE inhibited the metabolism of testosterone by CYP3A4. The concentration/effect curve was biphasic: DPPE inhibited metabolism by 30% at the first site (IC50 = 3 mu M, 95% CI = 0.5-25.5 mu M). and an additional 70% inhibition occurred at the second site (IC50 = 350 mu M 95% CI = 215-570 mu M). A similar result was observed with rat liver microsomes. Conclusion: DPPE is a substrate for CYP3A4, CYP2D6 and CYP1A1, but not CYP2B6. DPPE inhibits testosterone metabolism by interacting at two sites on CYP3A4, the first correlating with its K-s value to bind the substrate site and the second. with its EC50 value to enhance HA binding to the heme iron. We postulate that (1) the inhibitory effect of DPPE on CYP3A4 activity is mediated directly at the substrate site and indirectly by its enhancement of the binding of HA to the heme moiety, (2) in tumor cells that express high constitutive levels of CYP3A4, potentiation of chemotherapy cytotoxicity by DPPE results, in part, from inhibition of CYP3A4-mediated metabolism and P-gp-mediated efflux of antineoplastic drugs, (3) in normal cells that express low constitutive levels of the isozyme, cytoprotection by DPPE results, in part, from induction of CYP3A4 and P-gp, resulting in an increase both in metabolism and efflux of antineoplastic drugs.