Lorne J. Brandes

Evidence that the antiestrogen binding site is a histamine or histamine-like receptor

N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine X HCl (DPPE), a compound selective for the antiestrogen binding site, is structurally similar to the aminoethyl ether group of antihistamines. Our studies now reveal that H1-, but not H2-antagonists, also compete for this site in the order: DPPE = hydroxyzine = perchlorperazine greater than phenyltoloxamine greater than pyrilamine greater than diphenhydramine. The affinity of these compounds for the antiestrogen binding site correlates with their in vitro cytotoxicity against MCF-7 and EVSA-T human breast cancer cells. Tamoxifen, DPPE and hydroxyzine also bind to H1 receptors present in digitonin-solubilized rat liver microsomes, but with less affinity than pyrilamine, which is selective for this site; the ratio of H1 to antiestrogen binding sites in this preparation is 4:1. The data suggest that the antiestrogen binding site may be, in whole or in part, a receptor for histamine different from H1 and H2.

H3 receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450.

1 Thioperamide (TP), an imidazole and a highly potent, specific antagonist of the histamine H3 receptor, inhibited the secretion of cortisol from bovine isolated adrenocortical cells (IC50 0.20 μm) and, in the rat (5 mg kg−1) prevented both basal and stress‐induced secretion of corticosterone. 2 In adrenocortical microsomes, low affinity binding of [3H]‐histamine (KD 27.7 μm) was potently inhibited by TP (Ki 0.33 μm). 3 In adrenocortical microsomal membranes, both histamine and TP yielded type II difference absorption spectra, characteristic of the interaction between imidazole and cytochrome P450 enzymes. Dissociation constants for binding to P450, calculated from spectral data, were 15.9 μm and 1.5 mm for histamine, and 0.3 μm and 3.7 μm for TP. 4 In view of previously reported evidence for an intracellular mediator role of histamine in platelets, the present findings suggest a physiological role for histamine in the modulation of adrenal P450 monooxygenases that generate adrenocortical steroids. 5 The results suggest that direct adrenocortical inhibition by thioperamide at a non‐H3 intracellular site must be taken into account in studies designed to elucidate functional roles of H3 receptors.

Evidence that tamoxifen is a histamine antagonist

Recently we reported that both the triphenylethylene antiestrogen tamoxifen, and the novel compound N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine. HCl (DPPE), which is selective for the antiestrogen binding site, may be histamine antagonists and have suggested that the antiestrogen binding site may be a growth-promoting histamine receptor different from H1 and H2 (?H3). We now show that along with established H1-antagonists, tamoxifen and DPPE specifically block the histamine-induced (H1) contraction of canine tracheal smooth muscle in the order: pyrilamine = hydroxyzine greater than tamoxifen = 4-hydroxytamoxifen greater than DPPE. The H1-antagonist hydroxyzine, which competes about equally with DPPE for the antiestrogen binding site, is up to 10(3) times stronger than DPPE in blocking histamine-induced muscle contraction. This shows that H1 antagonism is distinct from binding to the antiestrogen binding site and suggests that if the latter is a histamine receptor, it is not H1; presumably tamoxifen and DPPE compete for this novel site in addition to, and with greater affinity than, H1.

A diphenylmethane derivative specific for the antiestrogen binding site found in rat liver microsomes

A new para-diphenylmethyl derivative, N,N-diethyl-2-[(4-phenylmethyl)-phenoxy]-ethanamine X HCl (N,N-DPPE) has been synthesized which binds with high affinity to the anti-estrogen binding site found in male rat liver microsomes. However, no evidence of significant interaction with the estrogen receptor can be observed at or below 10 microM in rat uterine cytosols; 10 nM N,N-DPPE fails to significantly induce progesterone receptor in MCF-7 cells. Tamoxifen also binds to anti-estrogen binding site but, unlike N,N-DPPE, binds significantly to estrogen receptor at much lower concentrations and induces MCF-7 progesterone receptor. This property of high affinity for anti-estrogen binding site but not for estrogen receptor may make N,N-DPPE an important probe for the study of anti-estrogen binding site and its biological relevance.

New evidence that the antiestrogen binding site may be a novel growth-promoting histamine receptor (?H3) which mediates the antiestrogenic and antiproliferative effects of tamoxifen

Using as a probe [3H]-DPPE (N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine HCl), a novel compound selective for the antiestrogen binding site (AEBS), new evidence is presented that this site could be a growth-promoting histamine receptor of a type not previously described (?H3). In the rat uterus, DPPE alone at a concentration of 4 mg/kg acts as an estrogen antagonist, unlike TAM alone which is a partial estrogen agonist. In the presence of exogenous estradiol, both TAM and DPPE are partial antagonists. This suggests that the antiestrogenic effects of tamoxifen are mediated through AEBS/?H3 while the estrogenic effects are mediated through ER.

A diphenylmethane derivative selective for the anti-estrogen binding site may help define its biological role.

By employing as a probe the new compound, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine X HC1 (N,N-DPPE), which preferentially binds the anti-estrogen binding site, it is demonstrated that this site appears to contribute to the growth inhibitory action of tamoxifen on MCF-7 human breast cancer cells, even at lower concentrations of this anti-estrogen (1 X 10(-7) M to 1 X 10(-6) M) at which the major effect is clearly mediated via estrogen receptor. The combination of N,N-DPPE and tamoxifen is additive and this effect is not abolished by 17 beta-estradiol. This suggests that the anti-estrogen binding site is not simply a passive reservoir for binding tamoxifen, but may itself mediate the cytotoxic effects of specific ligands.

Results of a clinical trial in humans with refractory cancer of the intracellular histamine antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine-HCl, in combination with various single antineoplastic agents.

PURPOSEnWe assessed N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine-HCl (DPPE) potentiation of chemotherapy in vitro and performed a pharmacokinetic study and phase I/II trial of DPPE, combined with various single agents, in patients with advanced refractory cancer.nnnPATIENTS AND METHODSnIn vitro chemopotentiation by DPPE was assessed in drug-sensitive and -resistant (multidrug resistant-positive [MDR+]) human tumor cells using a colony survival assay. The effect of DPPE and verapamil on the intracellular concentration of daunorubicin in MDR+ cells was compared. For the clinical study, subjects with progressive malignancy received a weekly infusion of a maximally tolerated dose of DPPE (240 mg/m2) over 80 or 440 minutes, in conjunction with a single chemotherapy drug to which, in most cases, the patients tumor was previously resistant. Concentrations of DPPE in blood and urine were determined by high-performance liquid chromatography (HPLC).nnnRESULTSnIn vitro, micromolar concentrations of DPPE potentiated (fivefold to 10-fold) chemotherapy cytotoxicity to both drug-sensitive and -resistant cells, but did not inhibit the p-glycoprotein pump; in vivo, serum levels of DPPE were 3 to 5 mumol/L at the end of 80 minutes and 1 to 2 mumol/L after 440 minutes of infusion. Of 48 patients monitored for a minimum of four DPPE/chemotherapy treatment cycles, 16 (33%) progressed, 12 (25%) stabilized, 12 (25%) improved, and eight (17%) responded (one complete and seven partial remissions). Four of 11 subjects who did not respond to the 80-minute infusion regimen improved with the 440-minute infusion; one had a partial remission of melanoma. In more than 600 patient-treatments, bone marrow toxicity was negligible (mean absolute neutrophil count [ANC] > 2.0 x 10(9)/L). Acute CNS symptoms associated with DPPE infusions were of relatively short duration (1 to 4 hours); delayed toxicity attributable to DPPE consisted of mild nausea and/or fatigue (1 to 2 days).nnnCONCLUSIONnAlthough preliminary, the results suggest that more structured trials should be performed to determine whether DPPE may increase the therapeutic index of certain chemotherapy drugs.

N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine in combination with cyclophosphamide: an active, low-toxicity regimen for metastatic hormonally unresponsive prostate cancer.

PURPOSEnThe intracellular histamine antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine. HCl (DPPE), potentiates chemotherapy cytotoxicity to malignant cells but protects normal tissue, including bone marrow, gut, and hair. We assessed the response to and clinical toxicity of DPPE/cyclophosphamide therapy in 20 patients with advanced hormonally unresponsive prostate cancer, 19 of whom were symptomatic.nnnPATIENTS AND METHODSnSubjects received a maximally tolerated dose of DPPE (6 mg/kg) intravenously (IV) over 80 minutes. Cyclophosphamide (600 to 800 mg/m2; maximum dose, 1,500 mg) was administered over the last 20 minutes of DPPE infusion. Treatments (usually outpatient) were given once weekly for 4 weeks, followed by a 1-week delay, and then 2 of every 3 weeks as long as the patient was deemed to benefit.nnnRESULTSnFive of seven patients (71%) with measurable soft tissue disease had a partial remission (PR). Three of 16 (19%) with assessable bone disease responded (one complete remission [CR] and two PRs). Nine of 18 (50%) with an elevated serum level of prostate-specific antigen (PSA) had more than a 50% (mean +/- SD, 78% +/- 14%) decrease. Eleven of 13 (85%) with bone pain had partial or complete resolution of this symptom; the PSA level and bone scan improved in six and two of these subjects, respectively. Acute treatment toxicity consisted of nausea/vomiting (six of 20) and ataxia (20 of 20), which correlated with peak serum levels of DPPE. Delayed effects (24 to 48 hours) consisted mainly of tiredness and mild nausea; one patient developed hemorrhagic cystitis. Bone marrow and hair follicle toxicity was negligible in 14 and 15 patients, respectively.nnnCONCLUSIONnDPPE/cyclophosphamide appears to be an active regimen for metastatic prostate cancer, with the added benefit of relatively low toxicity.

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)phe- noxy]ethanamineu2009·u2009HCl (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 thin-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 (Ks=4.1u2009±u20090.4u2009μM), CYP3A4 (Ks= 31u2009±15u2009μM) and CYP1A1 (Ks=40u2009±u20099u2009μM), but not with CYP2B6. (2) In correspondence with the binding studies, DPPE was metabolized by CYP2D6, CYP3A4 and CYP1A1; no metabolism occurred with CYP2B6. (3) HA evoked “type II” (heme iron binding) absorbance-difference spectra with all four isozymes, with Ks values in the range 80–600u2009μM. DPPE inhibited HA (600u2009μM) binding to CYP2D6 (IC50=4u2009μM, 95% CI=1.8–8.9u2009μM) and CYP1A1 (IC50=135u2009μM: 95% CI=100–177u2009μM), but stimulated HA (500 and 1000u2009μM) binding to CYP3A4 (EC50=155u2009μM, 95% CI=104–231u2009μM). 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=3u2009μM, 95% CI=0.5–25.5u2009μM), and an additional 70% inhibition occurred at the second site (IC50=350u2009μM, 95% CI=215–570u2009μM). 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 Ks 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.

Intracellular histamine and liver regeneration: high affinity binding of histamine to chromatin, low affinity binding to matrix, and depletion of a nuclear storage pool following partial hepatectomy.

We have demonstrated in rat hepatocytes that 3H-histamine binds specifically to novel low (microM) and high (nM) affinity sites, designated HIC to denote their intracellular location. Low affinity HIC sites are associated with microsomes, while both low and high affinity HIC sites are associated with the nucleus. A growth-regulatory action of intracellular histamine at HIC, independent of the rise in cytosolic calcium, has been demonstrated in mitogen-stimulated lymphocytes. We now report that the high affinity HIC sites in liver cell nuclei are associated exclusively with chromatin, while only low affinity sites are found in the residual material containing the nuclear matrix. Moreover, hepatocyte nuclei contain histamine (approximately 1 ng/mg protein), unaffected by incubation for up to 18 hours with the histidine decarboxylase inhibitor, alpha-FMH, suggesting a slow rate of turnover typical of a storage pool. A decrease in nuclear histamine parallels a rise in DNA synthesis in the first 24 hours after partial hepatectomy. Our findings support a role for a nuclear pool of pre-formed histamine in the mediation of liver regeneration.