CA2156162C - Cancer treatment - Google Patents

Abstract

The in vivo chemotherapeutic treatment of cancer cells in a living animal is improved by first administering to the animal, a compound which inhibits normal cell proliferation while promoting malignant cell proliferation, specifically a potent antagonist selective for intracellular histamine receptors, in an amount sufficient to inhibit the binding of intracellular histamine to the receptors in normal and malignant cells.
An enhanced toxic effect on the cancer cells from the chemotherapeutic agent is obtained while any adverse effect of the chemotherapeutic agent on normal cells, particularly bone marrow and gastro-intestinal cells, is inhibited. Certain fluoro derivatives useful in such procedure are novel compounds.

Description

TITLE OF INVENTION
CANCER TREATMENT
FIELD OF IPJVENTION
The present invention is concerned with the identification of compounds which increase the therapeutic index of chemotherapy drugs and which stimulate the growth of cancers, their use in the treatment of cancer and with certain novel compounds useful in such treatment.
BACKGROUND OF THE INVENTION
Over the last 50 years the treatment of a variety of human illnesses has vastly improved with the identification of active drugs and their introduction into clinical use. While perhaps not as dramatic as penicillin or insulin, various classes of agents, nonetheless, have improved the therapy and/or prognosis of common disorders, including (1) mental illness, especially schizophrenia (e.g. phenothiazines) and major depressive disease (e.g. tricyclic antidepressants and newer, non-tricyclic agents such as fluoxetine); (2) hayfever, asthma, urticaria and other acute allergic disorders (e.g. H1-antagonists); (3) peptic ulcer disease (e.g. H2-antagonists); (4) fungal diseases (imidazoles e.g. clotrimazole, ketoconazole); (5) breast cancer (e.g.
tamoxifen) ; and (6) hypertension, arrythmia and angina (Q-adrenergic antagonist). While these seemingly disparate classes of drugs have differing chemical structures, interactions, and indicated uses, in most cases the mechanisms by which they produce their effects are incompletely understood.
For example, although the phenothiazines are known to be antagonists of dopamine (D2) receptors, interactions at many other intracellular sites, including calmodulin, protein kinase C and calcium channels may be important to their activity. Similarly, while antidepressants are known to decrease the uptake of biogenic amine neurotransmitters into nerve endings 2 1 5 6 1 6 2 PCT/c"94/00087 (especially serotonin and norepinephrine) thereby increasing their concentration in synapses, a good correlation }-etween potency to inhibit the uptake of any specific amine and potency as antidepressant agents has not been shown.
As another example, while histamine antagonists appear to produce their antiallergic and antiacid effects through binding H1 and H. receptors, respectively, P450 microsomal enzymes, important in the metabolism of lipids and eicosanoids, have been identified as a major site of binding of the former, as well as of imidazoles. In addition, antidepressant drugs, such as doxepin, do not bind H2 receptors, yet are potent to inhibit acid secretion. As a final example, the antiestrogen tamoxifen is thought to inhibit breast cancer proliferation through binding estrogen receptors. Yet, it has been reported that tamoxifen is effective in 10k of breast cancers negative for estrogens receptors, suggesting additional mechanisms of action.
Recently, there has been described the existence of unique intracellular histamine receptors, designated Hjc, in brain membranes and liver microsomes. The paradiphenyl-methane derivative, N,N-diethyl-2-[4-(phenylmethyl)-phenoxy]-ethanamine.HC1 (DPPE) is a potent antagonist of HIc. Surprisingly, the other classes of drugs mentioned above, including phenothiazines, Hl antagonists, serotonin (5HT1, and 5HT3) antagonists, triphenylethylene antiestrogens and 0-adrenergic antagonists also compete, with varying degrees of affinity, for both DPPE and Hic binding. While H2 antagonists and other imidazoles do not compete for DPPE
binding, they do compete for HIc, but with lower affinity than for compounds which bind both AEBS and HIc.
Through binding HIc, histamine functions as an intracellular messenger to mediate aggregation in blood platelets and is implicated in the proliferation of normal and malignant cells. A second messenger role for histamine at Hic also has been postulated in estrogen action and in brain function. Thus, it is possible that HIc binding may be common to the action of many classes of drugs, including phenothiazines, antidepressants, antiestrogens, histamine (Hl, H21 H3) antagonists, serotonin (5HT1, 5HT3) antagonists, 9-adrenergic antagonists and antifungal agents.
Recently, in published International patent application WO 92/11035, (U.S. Patent No. 5,798,339), there is described a novel method of treatment for cancer, combining DPPE or its analogues with chemotherapy drugs, such as doxorubicin (Adriamycin'). In animals and humans, this method of treatment results in the protection of normal stem cells, including bone marrow and mucosal epithelium, while enhancing the anticancer effects of chemotherapy on malignant cells. Although the mechanism of this differential action is not fully understood, in vitro studies indicate that DPPE inhibits normal cell proliferation, in the absence of toxicity, but stimulates malignant cell proliferation and cytotoxicity. Increased response to chemotherapy has been demonstrated in tumor-bearing animals treated concurrently with DPPE. In addition, DPPE also directly cytoprotects normal gut mucosa in vitro, an effect related to DPPE-induced increases in endogenous levels of the protective prostaglandin, PGI2 and reversed by indomethicin.
SUMMARY OF INVENTION
New data, provided herein, indicate that (1) DPPE
alone at low doses directly stimulates tumor cell growth in vivo and (2) increases the inflammatory response in skin elicited by the tumor-promoting phorbol ester, PMA
(phorbol myristate acetate). Several other classes of compounds, such as antidepressants, phenothiazines, triphenylethylenes, histamine (H1, H2, H3) antagonists, serotonin (SHT1, 5HT3) antagonists, Q-adrenergic antagonists and imidazole analogs, also have been WO 94/18961 Pt-17/CA94/00087 identified as producing the same results as those obtained for DPPE.
It now also has been fct=nd that tricyclic antidepressant drugs and the non-tricyclic agent, fluoxetine (Prozac'") , as well as Hl-antihistamines and 0-adrenergic antagonists, also compete for the binding of 3H-DPPE and 3H-histamine to HIc in rat liver microsomes or brain membranes and, likewise, promote tumor growth.
Accordingly, in one aspect of the present invention, there is provided a method for the treatment of cancer cells in an animal, which comprises:
(a) administering to the animal a compound which inhibits normal cell proliferation while promoting malignant cell proliferation in an amount sufficient to inhibit the binding of intracellular histamine in normal cells, and (b) subsequently administering to the animal at least one chemotherapeutic agent for the cancer cells in an amount toxic to the cancer cells. In this way, an enhanced toxic effect on the cancer cells is obtained from the at least one chemotherapeutic agent while adverse side effects of the at least one chemotherapeutic agent on normal cells, including bone marrow and gastro-intestinal cells.
The compounds used herein are not diphenylmethylene compounds of formula:

/ \ CH2 ~ / \ O - (CH2) n - N

3 0 XO yp Rs where X and Y are chlorine or bromine, o and p are 0 or 1, R1 and R2 are alkyl groups containing 1 to 3 carbon atoms or are joined together to form a heteroring with the nitrogen atom, and n is 1, 2 or 3.

4a It has been further found that certain fluoro analogs of DPPE exhibit an enhanced potency in inhibiting normal cell proliferation and in promoting malignant cell proliferation and such compounds are novel compounds.
Accordingly, in another aspect of the present invention, there is provided a compound having the formula:

Z O ( CH2 ) n _ N
-F Yp R2 wherein Y is fluorine, chlorine or bromine, Z is an alkylene group of 1 to 3 carbon atoms or a =C=O group, or the phenyl groups are joined to form a tricyclic ring, and p is 0 or 1, R, and R2 are each alkyl groups 5 containing 1 to 3 carbon atoms or are joined together to form a hetero ring with the nitrogen atom and n is 1, 2 or 3, as well as pharmaceutically-acceptable salts of such compounds.
Such compounds may be prepared by any convenient procedure depending on the identity of the variable groups. For example, for compounds where Z is a carbonyl group, the compound may be made by reacting a hydroxy substituted fluoro-benzophenone with a chloro-substituted amino-substitute alkyl group.
BRIEF DESCRIPTION OF DRAWINGS
Figures 1 to 10 are graphical representations of text data generated in certain experiments set forth in the Examples below.
GENERAL DESCRIPTION OF INVENTION
In the present invention, any compound which inhibits normal cell proliferation while promoting malignant cell proliferation is useful and is administered in an amount sufficient to inhibit the binding of intracellular histamine in normal cells. Such compounds generally exhibit a pKi of at least about 5, preferably at least about 5.5.
Specific compounds which are useful in the present invention are diphenyl compounds of the formula:

a Z 0 - (CH2) n - N (I) Xo Yp ~ 6 wherein X and Y are each fluorine, chlorine or bromine, Z is an alkylene group of 1 to 3 carbon atoms or a=C=0 group, o and p are 0 or 1, Rl and R2 are each alkyl groupL, containing 1 to 3 carbon atoms or are joined together tc form a hetero-ring with the nitrogen atoms and n is 1, 2 or 3. Pharmaceutically-acceptable salts of the diphenyl compounds may be employed.
Alternatively, the benzene rings may be joined to form a tricyclic ring, in accordance with the structure:
(II) !;::::IIIIiiiiIIIIIIIIIIi;:::IIIiIJ

In one preferred embodiment of the invention, the X group is f, Z is C=O, o is 1, and p is o. More preferably, such compounds have the formula:

O
F 0 ---(CH2)õ - N Rl (III) RZ

where n, R1 and R2 are as described above.
In one preferred embodiment, the group R, is a diethylamino group, although other alkylamino groups may be employed, such as dimethylamino, and, in another preferred embodiment, a morpholino group, although other heterocyclic ring groups may be employed, such as peperazino. o and p are usually 0 when Z is an alkylene group and n may be 2. In one particularly preferred embodiment of the compounds of formula I, Z is -CH2-, n is 2, o and p are each 0 and - N-'_ is a diethylamino group. This compound, namely N,N-diethyl-2-[4-(phenylmethyl)-phenoxy]ethanamine, in the form of its hydrochloride salt, is ab"+reviated herein as DPPE. In addition to a methyl group ]inking the benzene rings, other linking groups may be employed, such as =C=O. Other substitutions may be made on the benzene rings in addition to the halogen atoms, for example, an imidazole group. In a particularly preferred embodiment of the compounds of formula III, n is 2 and Rl is a diethylamino group. This compound, namely N,N=diethyl-2-[4-(4'-fluoro phenone)phenoxy] ethanamine, in the form of its hydrochloride salt, is abbreviated herein as DFPE. This compound exhibits a potency of two to four times that of DPPE in inhibiting normal cell proliferation and promoting malignant cell proliferation in HIc binding competition assays.
Other compounds which may be employed in this procedure include:
(a) tricyclic antidepressants, (e.g. amitriptyline, clomipramine, imipramine and like agents), (b) non-tricyclic antidepressants (e.g. fluoxetine and like agents), (c) phenothiazines (e.g. prochlorperazine, trifluoroperizine, chlorpromazine and like agents), (d) H1-antihistamines, e.g., loratadine, hydroxyzine, phenyltoloxamine, astemizole and the like, (e) Q-adrenergic agonists and antagonists (e.g., propanolol and the like), (f) serotonin (5HT, or 5HT3) antagonists, such as ondansertron (5HT3) and cyproheptadine (5HT1) , (g) imidazoles and imidazole-like compounds, including H2 antagonists, such as cimetidine and ranitidine, H3 antagonists, such as thioperamide and antifungal agents, such as ketoconazole, and (h) triphenylethylene derivatives, such as tamoxifen.
In general, the compounds which may be employed may have a chemical structure consisting of at least two phenyl rings, linked by a rigid third phenyl or non-phenyl ring, or by a non-rigid methyl, oxygen, or other moiety, with the phenyl ring structure being linked by an ether, sulfhydryl or other ring structure or group to a basic alkylamine or imidazole or amino-imidazole side chain, for example, the carboxyamide-amino-imidazole L651582.
Although this wide range of compounds may be employed to increase the therapeutic index of chemotherapy drugs, DPPE and its direct analogs may be a significantly better agent for combination with chemotherapy agents than the foregoing groups of compounds, since DPPE appears to be more potent and selective for HIc and does not interact with calmodulin, protein kinase C, or calcium channels and is only a weak antagonist at other common receptors, such as H1, 5HT and DZ.
For example, DPPE does not cause serious toxic effects in humans at clinically relevant doses to enhance chemotherapy (about 0.2-12 mg/kg, preferably less than about 10 mg/kg, with about 6 mg being an optimal dose), whereas, for example, at their relevant concentrations to antagonize HIc, the antidepressant group of drugs may cause cardiac arrythmias, H1 antagonists might cause marked sedation or even convulsions, and phenothiazines may cause dyskenesias.
EXAMPLES
Example I:
This Example illustrates the tumor promoting and pro-inflammatory response effects of DPPE alone.
Figure 1 shows the tumor-promoting effect DPPE (1 mg/kg or 4 mg/M2) given subcutaneously once daily x 3, to seven DBA/2 mice inoculated subcutaneously with 2 x 102 WO 94/18961 1561 6 2 ~ PCT/CA94/00087 L5178Y lymphoma cells 48 hours previously. A second group of 7 tumor cell-inoculated mice served as controls (saline injections, once daily x 3). By day 14, 7/7 DPPE
treated animals had palpable tumors as compared to 4/7 controls. At the end of 4 weeks, 6/7 controls had tumors with an aggregate surface area of 14.5 cm2 (mean = 2.1 .8 cm2/animal) , while 7/7 DPPE-treated animals had tumors with an aggregate surface area of 38.4 cm2 (mean = 5.5 t.7 cm2/animal). Thus, the tumor burden of DPPE-treated animals was approximately 2.5-fold greater than that of controls.
To investigate any effect of DPPE to increase PMA-induced inflammation in the same strain of mice (DBA/2), groups of 3 animals were shaved over the back and 48 hours later received a single topical application of 17 nM PMA in acetone. The PMA-treated mice then received either saline (control) or DPPE (4 or 32 mg/kg at time 0 and 24 hours). Three animals painted with acetone served as vehicle controls. Forty-eight hours later, the various groups were sacrificed by CO2 asphyxiation, the skin carefully excised, pinned to paper strips to prevent wrinkling, and immersed in formaldehyde. H and E-stained sections of skin were assessed for degree of inflammation.
It was observed that the animals who received DPPE
had a significantly greater inflammatory response to PMA
as compared to saline or acetone controls. The most intense inflammatory response was seen in animals receiving the high dose (32 mg/kg or 128 mg/M2) of DPPE, where increased mitotic activity in the epithelial layer was also noted as compared to the PMA and saline-treated groups. The results of the experiments reported in this Example clearly show that DPPE enhances the inflammatory response of the tumor promoter PMA. Indeed, since tumor promotion requires the presence of inflammatory response, and can be blocked by agents which inhibit inflammation by definition, DPPE functions as a co-promoter with PMA.

Example II:
This Example shows the HZ, binding and tumor promoting effects of certain corr.pounds and the antiproliferative effect of DPPE and certain compounds.
5 Figure 2 shows the potency of two tricyclic agents, namely amitriptyline and doxepin, to compete for 3H-DPPE
binding in liver microsomes. The Kd value for DPPE is 65 nM while the Ki for doxepin is 5 M and for amitriptyline is 10 M. Doxepin and fluoxetine also compete for 3H-10 histamine binding to HIc in brain membranes (Kf = 10 M;
Fig. 3).
Figures 4A and 4B demontrate the tumor-promoting effects of the tricyclic agent, amitriptyline, and the non-tricyclic agent, fluoxetine, in C3H mice injected subcutaneously into the gluteal region with 1 x 105 C-3 fibrosarcoma cells. The doses employed were equivalent to therapeutic human doses (80 mg/M2 for amitriptyline and 20-40 mg/M2 for fluoxetine). The experiments were blinded so that the individual measuring the first appearance of palpable tumor was unaware of the treatment group (saline control vs antidepressant drug; n=10 in each group).
It may be seen from this data that, in both experiments, the control animals did not develop tumors until day 6, whereas in the fluoxetine-treated animals, tumors appeared on days 3, 4 and 5 post-injection and, in the amitriptyline-treated animals, tumors appeared on days 4 and 5 post-injection. Thus, in both experiments, 4/10 of antidepressant-treated animals, but no controls had tumors by day 5 (8/20 vs 0/20 controls, both experiments combined).
Conversely, Figure 5 shows that, like DPPE, both amitriptyline and fluoxetine inhibit, in the absence of cytotoxicity, the proliferation of concanavalin A-stimulated normal lymphocytes (ICso = 10 to 20 M) . Thus, although weaker than DPPE, these agents inhibit the proliferation of normal stem cells while increasing the proliferation of tumor cells.

WO 94/18961 215c1c2 PCT/CA94/00087 Figure 6A shows that propanolol (a 0-adrenergic antagonist) inhibits histamine binding to Hjc in microsomes and Fig-re 6B shows that propanolol inhibits normal lymphocyte mitogenesis. In a C-3 fibrosarcoma murine model, propanolol significantly increased tumor weight on Day 23, as seen in Figure 7. Similarly loratidine (a tricyclic non-sedating Hl-antihistamine) potently promoted tumor growth, as seen in Figure 7, and also inhibited concanavalin A-stimulated mitogenesis (Figure 8). Astemizole (a non-sedating Hl-antihistamine) similarly is potent to inhibit histamine binding and concanavalin A-stimulated mitogenesis (data not shown) and, in two separate experiments, to potently stimulate the growth of C-3 fibrosarcoma, as shown in Figure 9.
The compounds for whicY} binding and proliferation data are provided in this Example, therefore, mimic the profiles of DPPE to inhibit normal cell proliferation but to promote malignant cell proliferation (Example I). On the basis of his profile, these agents, at the proper dose level, may be predicted to increase the therapeutic index of chemotherapy drugs in the procedure of W092/11035.
Example III
This Example illustrates the chemical synthesis of N,N-diethyl-2-(4-(4'-fluorophenone)phenoxy] ethanamine.
Diethylaminoethyl chloride.HC1 (2 grams) was dissolved in 50 ml H20 made basic with potassium hydroxide, extracted four times with 25 ml toluene to form the base and dried overnight in the presence of NaZSO4. Five grams of 4-fluoro-4'-hydroxy-benzophenone was added to a heated mixture of 50 ml of distilled toluene containing sodium hydride (600 mg). The DEAE
base (step 1) was added drop-wise to the benzophenone/toluene and the mixture was refluxed for twenty hours. The mixture was cooled to room temperature and then washed three times with approximately 150 ml of toluene. The toluene wash was taken to dryness. The resulting precipitate was taken up in ethanol and was recrystallized using etheral.HC1. The crystallization was repeated a second time.
Thin layer chromatography of the resulting crystals showed a single product with a melting point of 128'C, and a molecular weight of 351.5. The IR spectrum of this compound shows a C = 0 stretch. The structure of DPPE
was confirmed by mass spectroscopy and NMR as follows:

0 CHzCH3 F C O OCHz CHzPT . HCl CHzCH3 The morpholino-analogue also was prepared using the above-described procedure, but substituting 4-(2-chloroethyl)morpholine.HC1 for DEAE.HC1.
ExamBle IV
This Example illustrates the binding characteristics and antiproliferative properties of DPPE.
DFPE competes for ['H] DPPE binding in rat liver microsomes with a K; value of approximately 70 nM. The K; value for DFPE approximates the Kd value for DPPE in the same assay. DFPE competes for [3H] histamine binding in rat cortical membranes with a Ki value of 0.3 x 10"6 M.
This compares to a K; value for DPPE in the same assay of 0.9 x 10'6 M; thus DFPE is approximately three times more potent than DPPE in inhibiting histamine binding at a non-Hl, non-HZ site (HIc) in brain membranes (Brandes, L.J. et al, Cancer Research, 47:4025-4031, 1987).
DFPE antagonizes phorbol myristate acetate (PMA)-induced platelet aggregation with an IC50 = 20 M; this compares to an ICso value for DPPE in the same assay of 80 M. Thus, DFPE is approximately four times more potent than DPPE in antagonizing PMA-induced platelet aggregation.
The ability of DFPE and DPPE to inhibit/kill the growth of MCF-7 human breast cancer cells after seven days incubation at 37'C in vitro is shown in Figure 10.

The IC50 value for DFPE is 3.0 x 10"6 M. This compares with an IC50 value for DPPE of 6.5 x 10'6 in the same assay.
Thus, DFPE possesses novel antihistaminic properties, antagonizes the effects of phorbol myristate acetate on platelet aggregation, and is antiproliferative cyclotoxic to MCF-7 human breast cancer cells, all with a potency approximately three to four times greater than that of DPPE.
Since DPPE has been demonstrated to be antiestrogenic in vivo, to augment the effects of tamoxifen in the rat uterus in vivo, a similar spectrum of in vivo activity is expected for DFPE, but with an overall potency two to four fold greater than that observed for DPPE. In addition DFPE may be used in place of DPPE in the cancer treatment method described herein to improve the therapeutic index of conventional chemotherapy drugs.
SUMMARY OF DISCLOSURE
In summary of this disclosure, the present invention provides identification of compounds and classes of compounds which stimulate cancer growth and which enable the therapeutic index of chemotherapy agents to be improved. Novel compounds also are described.
.25 Modifications are possible within the scope of this invention.

Claims (9)

14~

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. The use of a compound which inhibits normal cell proliferation while promoting malignant cell proliferation for the manufacture of a medicament useful in the treatment of cancer cells in an animal wherein said compound is a diphenyl compound of the formula:

wherein Y are each fluorine, chlorine or bromine, Z is an alkylene radical of 1 to 3 carbons or a =C=O group, or the phenyl groups are joined to form a tricyclic ring, p is 0 or 1, R1 and R2 are each alkyl groups containing 1 to 3 carbon atoms or are joined together to form a hetero-ring with the nitrogen atom and n is 1, 2 or 3, or a pharmaceutically-acceptable salt thereof.

2. The use of a compound which inhibits normal cell proliferation while promoting malignant cell proliferation for the manufacture of a medicament useful in the treatment of cancer cells in an animal wherein said compound is one having the formula:
where R1 and R2 are each alkyl groups containing 1 to 3 carbon atoms or are joined together to form a hetero-ring with the nitrogen atom and n is 1, 2 or 3, or a pharmaceutically-acceptable salt thereof.

3. The use of claim 1 or 2 wherein the group is a diethylamino group, a dimethylamino group, a morpholino group, or a piperazino group.

4. The use of claim 3 wherein is a diethylamino group and n is 2.

5. The use of claim 4 wherein said compound is in the form of its hydrochloride salt.

6. The use of any one of claims 1 to 5 wherein said normal cells include bone marrow and gastrointestinal cells.

7. A compound of the formula:

wherein n is 2 and is a diethylamino group.

8. The compound of claim 7 in the form of its hydrochloride salt.

9. The use of a compound as claimed in claim 7 or 8 for the manufacture of a medicament useful in the treatment of cancer cells in an animal.