Enhanced cancer growth in mice administered daily human-equivalent doses of some H1-antihistamines: predictive in vitro correlates.

BACKGROUND: Present studies of drug-induced tumor growth promotion have evolved from earlier investigations into the mechanism of action of N,N-diethyl-2-[4-(phenylmethyl)phenoxy[ethanamine.HCl, a tamoxifen derivative which potently inhibits lymphocyte mitogenesis in vitro and stimulates tumor growth in vivo. It is thought that potency to bind to intracellular histamine receptors (HIC), some of which are on cytochromes P450, may correlate with tumor growth-promoting activity. PURPOSE: We assessed the effectiveness of five in vitro assays in predicting in vivo tumor growth stimulation by the H1-antihistamines loratadine, astemizole, cetirizine, hydroxyzine, and doxylamine. METHODS: Potency of each agent was ranked 1-5 in each of the following in vitro assays: 1) inhibition of [3H]histamine binding to microsomal HIC, 2) inhibition of histamine binding to microsomal P450, 3) inhibition of the P450-catalyzed demethylation of aminopyrine, 4) inhibition of lymphocyte mitogenesis, and 5) stimulation of tumor colony formation. An overall rank score was assigned to each drug and correlated with tumor growth stimulation in vivo. Two laboratories conducted in vivo studies in a blinded fashion. Female C57BL and C3H mice were given a subcutaneous injection on day 1 of syngeneic B16F10 melanoma cells (5 x 10(5)) or C-3 fibrosarcoma cells (1 x 10(5)), respectively. Mice were randomly assigned to treatment groups, then received a single, daily intraperitoneal injection of an estimated human-equivalent dose (or range of doses) of antihistamine or vehicle control for 18-21 days before being killed. Tumors were surgically removed and wet weights compared statistically among groups. RESULTS: The cumulative potency of each drug in affecting tumor growth or growth mechanisms in the five in vitro assays ranked as follows: Loratidine and astemizole ranked highest and were equally potent, followed in decreasing order by hydroxyzine, doxylamine, and cetirizine. A significant correlation (r = .97; P < .02) was observed between the rank order of potency of the antihistamines in all five in vitro assays and the rank order to enhance tumor growth in vivo: Loratidine and astemizole significantly (P < .001) promoted the growth of both melanoma and fibrosarcoma, hydroxyzine significantly (P < .001) promoted the growth of melanoma, while doxylamine and cetirizine did not promote the growth of either tumor. CONCLUSION: Data demonstrate that the in vitro assays predicted the propensity of each H1-antihistamine to stimulate cancer growth in vivo. IMPLICATION: These in vitro tests may prove valuable to screen potential tumor growth promoters.

Stimulation of malignant growth in rodents by antidepressant drugs at clinically relevant doses.

Tricyclic antidepressants, such as amitriptyline (Elavil), and the nontricyclic agent, fluoxetine (Prozac), bind to growth-regulatory intracellular histamine receptors, associated with anti-estrogen binding sites in microsomes and nuclei. The prototype anti-estrogen binding site/intracellular histamine receptor ligand, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl, inhibits normal cell proliferation in vitro but stimulates tumor growth in vivo. Because of their structural similarity to N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl, we carried out studies to determine whether amitriptyline and fluoxetine stimulate tumor growth and/or development in rodents at concentrations relevant to the treatment of human depression (equivalent human dose range, approximately 100-150 mg/day for amitriptyline and approximately 20-80 mg/day for fluoxetine). All experiments were performed blinded. In studies of growth stimulation of transplantable syngeneic tumors, groups of mice were inoculated s.c. with C-3 fibrosarcoma cells or given i.v. or s.c. injections of B16f10 melanoma cells, followed 24 h later by daily i.p. injections of saline, amitriptyline, or fluoxetine. Tumor latency (fibrosarcoma), aggregate tumor weight (s.c. injected melanoma), or time to death from pulmonary metastasis (i.v. injected melanoma) was determined; drug-induced stimulation of DNA synthesis in C-3 fibrosarcoma cells in vitro was correlated with tumor growth acceleration in vivo. In a mammary carcinogenesis model, the effects of chronic saline, amitriptyline, or fluoxetine administration on the rate and frequency of development of mammary tumors in rats fed dimethylbenzanthracene (DMBA) were compared. Eight of 20 amitriptyline- or fluoxetine-treated mice developed fibrosarcoma tumors by day 5, as compared to none of 20 saline controls (P less than 0.002). Similarly, 20 of 21 DMBA-treated rats receiving the antidepressant drugs developed 33 mammary tumors by week 15 as compared to 5 tumors in 4 of 7 DMBA-treated rats receiving saline (P less than 0.001). For both models, tumor latency decreased 30-40% and, in the DMBA model, tumor frequency increased greater than 2-fold in the antidepressant-treated rats as compared to controls. Stimulation of fibrosarcoma growth in vivo correlated with a corresponding bell-shaped drug-induced increase in DNA synthesis in vitro. While the median time to death from pulmonary metastases did not differ among groups given i.v. injections of melanoma cells, a significant (P less than 0.01) stimulation of growth of s.c. injected melanoma was observed in mice receiving the antidepressants.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

The antiproliferative potency of histamine antagonists correlates with inhibition of binding of [3H]-histamine to novel intracellular receptors (HIC) in microsomal and nuclear fractions of rat liver.

Previously, we identified in rat liver microsomes, low (microM) affinity histamine receptors (HIC), associated with antiestrogen binding sites (AEBS). N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE), a potent AEBS ligand, is a specific HIC antagonist. Through binding HIC, newly-formed intracellular histamine mediates, and DPPE inhibits, human platelet aggregation. We now provide evidence that histamine, mobilized from cytoplasmic stores, is a mediator of the mitogenic response to concanavalin A in mouse spleen cells. DNA synthesis and intracellular histamine levels are decreased over time by the histidine decarboxylase inhibitor, alpha-fluoromethylhistidine. For DPPE, H1 and H2 antagonists, rank order of potency to inhibit [3H]-histamine binding to HIC in rat liver microsomes correlates with antiproliferative potency. DPPE also competes for [3H]-histamine binding at low and high affinity sites in rat liver nuclei (IC50 approximately 2 microM). Thus, histamine may mediate growth through two intracellular subtypes of HIC.

Does intracellular histamine mediate mast cell histamine release?

Previously, we demonstrated that through binding a novel intracellular receptor of microM affinity (HIC), histamine mediates, and the HIC antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine. HCl (DPPE) inhibits, platelet aggregation and serotonin granule secretion; the latter response is dependent upon the same processes that mediate histamine release from mast cell granules. We now show that, as for platelet serotonin release, DPPE blocks concanavalin A-stimulated mast cell histamine release with a potency (IC50 = 30 microM) greater than the H1-antagonist, pyrilamine (IC50 = 150 microM) or the H2-antagonist cimetidine (IC50 = 5 mM), correlating with rank order of potency to inhibit 3H-histamine binding in rat brain membranes and liver microsomes. We postulate that histamine release from mast cells is mediated at HIC by second messenger intracellular histamine. However, unlike platelets, mast cells do not appear to rely on newly synthesized histamine. Rather, as for calcium, histamine may be mobilized from bound stores to mediate histamine secretion.

Correlation of the antiproliferative action of diphenylmethane-derivative antiestrogen binding site ligands with antagonism of histamine binding but not of protein kinase C-mediated phosphorylation.

The nonestrogen receptor-mediated antiproliferative action of antiestrogen binding site (AEBS) ligands, including triphenylethylene antiestrogens and phenothiazines, has been linked to their ability to inhibit protein kinase C (PKC). Recent studies indicate that some diphenylmethane derivatives inhibit growth, are potent AEBS ligands, and antagonize histamine binding at an AEBS-related histamine site different from H1 and H2. Three novel diphenylmethane derivatives, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine.HCI (DPPE), 4-decanoyl-DPPE (dec-DPPE), and 4-benzylphenyl decanoate (BPD) were studied in an attempt to determine whether PKC or histamine interactions best correlate with their antiproliferative effects. Platelet aggregation and the phosphorylation of a platelet Mr 47,000 protein (p47) induced by phorbol-12-myristate-13-acetate (PMA) represent two processes mediated by PKC. DPPE inhibits PMA-induced aggregation [50% inhibitory concentration (IC50) = 31.2 +/- 2.4 (SEM) x 10(-6) M] but does not significantly inhibit either PMA-induced phosphorylation of Mr 47,000 protein (IC50 greater than 500 x 10(-6) M), or binding of [3H]phorbol dibutyrate to platelets. dec-DPPE is a more potent inhibitor of PMA-induced platelet aggregation (IC50 = 18.8 +/- 0.7 x 10(-6) M), a weak inhibitor of Mr 47,000 phosphorylation (IC50 = 80-200 x 10(-6) M), but is without effect on [3H]phorbol dibutyrate binding. BPD, which lacks the alkylaminoethoxy side chain necessary for binding to the AEBS/DPPE site, is devoid of anti-PMA effects. These results are compared to the inhibition of [3H]histamine binding in rat cortex membranes (Ki value for DPPE = 0.83 +/- 0.62 x 10(-6) M; Ki value for dec-DPPE = 6.6 +/- 3.5 x 10(-6) M; BPD is inactive) and growth inhibition of MCF-7 cells (IC50 value for DPPE = 4.5 x 10(-6) M; IC50 value for dec-DPPE = 1.5 x 10(-5) M; BPD is ineffective at all concentrations tested). Thus, while dec-DPPE is a more potent inhibitor of PKC-mediated phosphorylation, DPPE is a more potent inhibitor of histamine binding and is correspondingly more antiproliferative than dec-DPPE. The results support a relationship between antagonism of histamine binding and growth inhibition but argue against an association between the antiproliferative effects of DPPE and dec-DPPE and inhibition of PKC. The findings for DPPE suggest that platelet response to PMA, antagonized by diphenylmethane-type AEBS-ligands, may be mediated, at least in part, by mechanisms other than activation of protein kinase C-dependent phosphorylation.

Histamine and growth: interaction of antiestrogen binding site ligands with a novel histamine site that may be associated with calcium channels.

N,N-Diethyl-2-[(4-phenylmethyl)-phenoxy]ethanamine hydrochloride (DPPE) is a novel paradiphenylmethane derivative with antiproliferative and antiestrogenic properties. Like tamoxifen (TAM), DPPE binds to the microsomal antiestrogen binding site with high affinity (Kd approximately 50 nM), but, conversely, not to estrogen receptor or calmodulin. We now demonstrate that DPPE competes for [3H]histamine binding in rat cerebral cortex with an affinity (Ki = 4.5 +/- 2.6 X 10(-6) M) significantly greater than that of the H1 antagonist pyrilamine (Ki = 7.2 +/- 2.2 X 10(-5) M), despite the previous demonstration that pyrilamine is up to 1000 times more potent than DPPE in antagonizing histamine-induced contraction in canine tracheal smooth muscle. DPPE demonstrates antiproliferative activity against MCF-7 cells at concentrations between 1 X 10(-7) and 1 X 10(-5) M; the IC50 value of DPPE for growth inhibition at 7 days in this assay is 5 X 10(-6) M, a value equivalent to its Ki value for histamine binding. DPPE also competes for [3H]verapamil binding in membranes from whole rat brain with an affinity equal to that for verapamil (Kd = 4.0 +/- 1.8 X 10(-7) M); however, verapamil competes for [3H]DPPE binding in brain membranes and rat liver microsomes with an affinity markedly lower (Ki approximately 1 X 10(-4) M) than that of DPPE, suggesting allosteric interactions between the verapamil and DPPE sites. Unlike DPPE, verapamil is not antiproliferative in vitro against MCF-7 cells at concentrations up 1 X 10(-5) M, but, like DPPE, is cytotoxic at concentrations of 1 X 10(-4) M. In immature oophorectomized rats, verapamil or DPPE alone is antiuterotropic; however, verapamil shows no antagonism of exogenous estradiol on uterine growth, as opposed to DPPE which is a partial antagonist. Thus, the antiproliferative and antiestrogenic properties of DPPE either are not associated with calcium channel antagonism, or result from a qualitatively different effect on channels than verapamil. The in vitro antiproliferative effect of DPPE (7.5 X 10(-6) M) on MCF-7 cells at 72 h is significantly reversed by 10 mM L-histidine (70.2 +/- 12.6% reversal) and L-methionine (92.4 +/- 11.1% reversal), but not by L-ornithine, L-arginine, L-phenylalanine, or exogenous histamine. At lower concentrations of TAM (0.75 X 10(-6) M), where growth inhibition is estrogen-reversible, L-ornithine, but not L-histidine or L-methionine, causes significant reversal of growth inhibition (66.8 +/- 13.3%; p less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)

The antiproliferative properties of tamoxifen and phenothiazines may be mediated by a unique histamine receptor (?H3) distinct from the calmodulin-binding site.

N,N-diethyl-2-[(4-phenylmethyl)-phenoxy]-ethanamine HCl (DPPE), a novel histamine antagonist (?H3), which selectively binds with high affinity to the antiestrogen-binding site (AEBS/?H3), inhibits the activity of calmodulin-dependent myosin light chain kinase (MLCK) only at concentrations greater than 1 mM, as opposed to tamoxifen (TAM), which has an IC50 = 4 microM in the same assay. This suggests that the antiestrogen-binding site is distinct from the site on calmodulin which binds TAM and phenothiazines. However, at an in vitro concentration of 1 X 10(-6) M, the antiproliferative effects of DPPE and several phenothiazines, which also compete for binding to AEBS/?H3, are about equal; this suggests that affinity for AEBS/?H3 rather than that for the calmodulin-binding site may correlate with clinically relevant antigrowth effects of these compounds.

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.

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.