Method of using 4-(2-(P-((E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl) propenyl)phenoxy)ethyl)morpholine with cytostatics

The use of 4-[2-[p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-propeny l]phenoxy]ethyl]-morpholine, simultaneously, separately or sequentially in combination with a synergistically effective amount of cyclophosphamide in cancer prophylaxis or therapy for mammary tumors in mammals is described.

BRIEF SUMMARY OF THE INVENTION 
The invention is concerned with the use of 
4-[2-[p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthyl)propenyl] 
phenoxy]ethyl]-morpholine as an active substance for the manufacture of 
pharmaceutical preparations for supporting therapy with a cytostatic 
agent. In another aspect, the invention is concerned with a product 
containing 
4-[2-[p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propenyl 
]phenoxy]-ethyl]morpholine and a cytostatic agent as a combination 
preparation for the simultaneous, separate or sequential use in cancer 
prophylaxis or therapy particularly for cancers which are sensitive to 
such a combination. The invention is also concerned with a commercial pack 
containing as the pharmaceutically active substance 
4-[2-[p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propenyl 
]phenoxy]ethyl]morpholine together with instructions for its use in 
combination with a cytostatic agent for the simultaneous, separate or 
sequential use in cancer prophylaxis or therapy particularly for cancers 
which are sensitive to such a combination. The invention is also concerned 
with a method of cancer prophylaxis or therapy in mammals which comprises 
administering 4-[2-[p-[(E)-2 
-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propenyl]phenoxy]ethyl 
]morpholine simultaneously, separately or a sequentially in combination 
with a cytostatic agent particularly for cancers which are sensitive to 
such a combination. 
Therapy with cytostatic agents is frequently associated with undesirable 
effects which have their origin in the toxicity of the cytostatic agent. 
Examples of such undesirable effects are hair loss, weight loss or 
inappetence, bone marrow disorders, gastrointestinal disorders, myocardial 
changes, testicular changes, liver and kidney disorders. 
It has now been found that these undesirable effects during therapy with 
cytostatic agents can be reduced or avoided when the compound 
4-[2-[p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphthyl)propeny 
l]phenoxy]ethyl]morpholine (referred to hereinafter as "Compound Z") is 
administered simultaneously or sequentially.

DETAILED DESCRIPTION OF THE INVENTION 
Accordingly, the invention is concerned with the use of 
4-[2-[p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propenyl 
]phenoxy]ethyl]-morpholine as an active substance for the manufacture of 
pharmaceutical preparations for supporting therapy with a cytostatic 
agent. In another aspect, the invention is concerned with a product 
containing 
4-[2-[p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propenyl 
]-phenoxy]-ethyl]morpholine and a cytostatic agent as a combination 
preparation for the simultaneous, separate or sequential use in cancer 
prophylaxis or therapy particularly for cancers which are sensitive to 
such a combination. In another aspect, the invention is also concerned 
with a commercial pack containing as the pharmaceutically active substance 
4-[2-[p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthyl)propenyl] 
phenoxy]ethyl]morpholine together with instructions for its use in 
combination with a cytostatic agent for the simultaneous, separate or 
sequential use in cancer prophylaxis or therapy particularly for cancers 
which are sensitive to such a combination. In still another aspect, the 
invention is concerned with a method of cancer prophylaxis or therapy in 
mammals which comprises administering 4 
-[2-[p-(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propenyl]p 
henoxy]ethyl]morpholine simultaneously, separately or a sequentially in 
combination with a cytostatic agent particularly for cancers which are 
sensitive to such a combination. 
Compound Z, its manufacture and use as a medicament, for example, in the 
therapy of cancer, is described in European Patent Publication A-2 0 331 
983. 
Cytostatic agents whose undesirable effects can be reduced or avoided by 
simultaneous or sequential therapy with Compound Z are those of teh 
alkylating substance type such as, for example, cyclo-phosphamide; 
platinum derivatives; antimetabolites, for example, folic acid analogues 
such as methotrexate, or pyrimidine derivatives such as 5-fluororacil; 
anthracyclines such as doxorubicin; vinca alkaloids such as vinblastin or 
vincristin; as well as "Biological Response Modifiers" (BRM) such as 
interleukins, for example, IL-2 and inter-ferons, for example, IFN.alpha. 
and IFN.gamma., Colony Stimulating Factors, for example, G-CSF, GM-CSF and 
M-CSF, and retinoids, for example, temarotene. The reduction of the 
undesired effects is achieved by the fact that the substances described 
above can be administered in lower dosages in combination with Compound Z 
without reducing their antitumor activity. 
Furthermore, it has been found that the simultaneous administration of 
Compound Z and interleukin or doxorubicin inhibits the tumor growth of 
tumors which are sensitive to such a combination to a greater than an 
additive inhibitory effect. In combination with cyclophosphamide (CY), the 
toxicity of CY was largely offset. 
The undesirable effects during tumor treatment with Compound Z and other 
cytostatic is documentated by the following test results: 
A. Treatment of Chemically-Induced Mammary Tumors in Rats With 
Cyclophosphamide and Compound Z 
Female rats aged 50 days were given a single oral dosage of 12 mg of 
7,12-dimethylbenz(a)anthracene in 0.5 ml of arachis oil. After about 8 
weeks, the rats which had developed mammary tumors were divided into 
groups of 10. One group was given only feed without the addition of test 
substance; one group was given 75 mg/kg/day (7 times weekly) of Compound Z 
as a feed additive; one group was given 10 mg/kg/day of cyclophosphamide 
intra-peritoneally (5 times weekly); one group was given Compound Z and 
cyclophosphamide in the indicated dosages. The treatment extended over 10 
weeks. Thereafter, the number of tumors, tumor volumes and mortality were 
determined. The results are set forth in Table 1 and FIGS. 1-3. 
TABLE 1 
__________________________________________________________________________ 
Tumor incidence 
Average number 
Average tumour 
Number of 
Body weight 
Group Compound(s) 
in the group [%] 
of tumors/rat 
burden [cm.sup.3 ] 
surviving rats 
[g] 
__________________________________________________________________________ 
Start of test 
1 Control 
100 2.8 .+-. 0.5 
2.8 10 251 .+-. 5 
2 Z 100 3.1 .+-. 0.5 
4.0 10 256 .+-. 4 
3 Cy 100 2.4 .+-. 0.4 
2.6 10 266 .+-. 6 
4 Cy + Z 100 2.7 .+-. 0.4 
3.2 10 252 .+-. 5 
After 6 weeks 
1 Control 
100 6.0 .+-. 0.8 
100.2 10 267 .+-. 10 
2 Z 100 4.0 .+-. 0.6 
9.6 9 246 .+-. 6 
3 Cy 100 5.7 .+-. 0.5 
10.3 10 249 .+-. 5 
4 Cy + Z 80 1.7 .+-. 0.4 
1.5 10 243 .+-. 5 
After 
10 weeks 
1 Control 
100 6.6 .+-. 0.8 
119.5 9 285 .+-. 8 
2 Z 100 3.4 .+-. 0.6 
10.9 9 255 .+-. 6 
3 Cy NU NU NU 0 NU 
4 Cy + Z 80 2.8 .+-. 0.8 
3.6 10 253 .+-. 5 
__________________________________________________________________________ 
NU = not used 
The data obtained show the tumor-inhibiting effect of the two test 
compounds, which appears to be additive in the case of combined 
administration. It surprisingly emerged that all experimental animals 
which had been given cyclophosphamide and Compound Z survived the entire 
test duration, while the unaccompanied administration of cyclophosphamide 
led to the premature death of all experimental animals. 
FIG. 1 shows the tumor incidence expressed as the percentage of the 
tumor-bearing rats of a group. 
FIG. 2 shows the average tumor burden per animal, which is the product of 
the average number of tumors and the average tumor volume per rat. 
FIG. 3 shows the survival rate of the test animals. All animals which were 
treated with the combination of Z and cyclophosphamide survived to the end 
of the test, whereas in the cyclophosphamide-treated group all animals 
died prematurely with symptoms of anaemia, weakness and weight loss. These 
data shows that Compound Z clearly reduces the toxicity of 
cyclophosphamide. 
B. Treatment of Chemically-Induced Mammary Tumors in Rats with 
Interleukin-2 and Compound Z. 
Female rats with mammary tumors induced as described under A were given 75 
mg/kg/day of Compound Z orally as a feed additive; or 1.mu./kg/day of 
interleukin-2 i.p., 5 times weekly; or both test compounds. The duration 
of the treatment was 10 weeks. The dosage of IL-2 is an exceptionally low 
dosage which is very well tolerated. The results are given in Table 2 and 
FIGS. 4 and 5: 
TABLE 2 
__________________________________________________________________________ 
Tumor incidence 
Average number 
Average tumor 
Surviving rats 
Body weight 
Group Compound(s) 
in the group [%] 
of tumors/rat 
burden [cm.sup.3 ] 
[%] [g] 
__________________________________________________________________________ 
Start of test 
1 Control 
100 3.0 .+-. 2.0 
3.6 .+-. 2.1 
100 287 .+-. 26 
2 Z 100 2.7 .+-. 0.6 
6.0 .+-. 5.0 
100 275 .+-. 4 
3 IL-2 100 2.8 .+-. 0.7 
3.4 .+-. 1.2 
100 285 .+-. 10 
4 IL-2 + Z 
100 2.5 .+-. 0.6 
4.0 .+-. 2.7 
100 293 .+-. 9 
After 6 weeks 
1 Control 
100 4.0 .+-. 2.0 
17.7 .+-. 11.5 
100 305 .+-. 30 
2 Z 100 3.7 .+-. 1.0 
14.2 .+-. 6.5 
100 264 .+-. 6 
3 IL-2 100 3.0 .+-. 0.5 
25.4 .+-. 13.4 
100 296 .+-. 11 
4 IL-2 + Z 
83 1.5 .+-. 0.6 
3.0 .+-. 2.3 
100 265 .+-. 13 
After -10 weeks 
1 Control 
100 5.5 .+-. 3.5 
18.8 .+-. 12.4 
100 320 .+-. 25 
2 Z 100 3.3 .+-. 0.5 
18.2 .+-. 12.1 
100 261 .+-. 11 
3 IL-2 100 3.5 .+-. 0.6 
33.9 .+-. 17.9 
100 314 .+-. 13 
4 IL-2 + Z 
50 1.0 .+-. 0.6 
2.6 .+-. 2.4 
100 254 .+-. 9 
__________________________________________________________________________ 
FIG. 4 shows that tumor incidence in the animals treated with Z or IL-2 
remained unchanged compared with the control group. On the other hand, the 
combined treatment with Z and IL-2 led to the complete disappearance of 
the tumors in three out of six test animals. 
FIG. 5 shows the average number of tumors in the case of treatment with Z 
or IL-2, in the case of treatment with Z and IL-2 and without treatment 
(control group). 
At the commencement of treatment the average tumor count was 3.4 per 
animal. This count doubled in the control group in the course of 10 weeks. 
The treatment with Z alone led to a reduction by 48% in the 5th week and 
thereafter to a slight increase by about 10%. The treatment with IL-2 
alone had no effect on the number of tumors. In the combined treatment, 
the number of tumors had decreased by 67.5% after the 10th week. 
C. Treatment of Chemically-Induced Mammary Tumors in Rats with Doxorubicin 
and Compound Z. 
Female rats with mammary tumors induced as described under A were treated 
for 12 weeks with 75 mg/kg/day of Compound Z as a feed additive; or with 
0.2 mg/kg of doxorubicin i.p. or with both test compounds. The treatment 
was effected 5 times weekly. The dosage of doxorubicin is an exceptionally 
low dosage with very good tolerance. The results are given in Table 3 and 
FIGS. 6-8. 
TABLE 3 
__________________________________________________________________________ 
Tumor incidence 
Average number 
Average tumor 
Number of 
Body weight 
Group Compound(s) 
in the group [%] 
of tumors/rat 
burden [cm.sup.3 ] 
surviving rats 
[g] 
__________________________________________________________________________ 
Start of test 
1 Control 
100 5.2 .+-. 0.8 
17.7 .+-. 6.4 
7 290 .+-. 10 
2 Z 100 5.7 .+-. 0.8 
17.9 .+-. 6.4 
7 306 .+-. 9 
3 Dox 100 3.8 .+-. 0.7 
11.8 .+-. 7.0 
7 301 .+-. 16 
4 Dox + Z 
100 4.4 .+-. 6.0 
15.0 .+-. 6.0 
7 292 .+-. 15 
After 6 weeks 
1 Control 
100 6.5 .+-. 1.0 
96.2 .+-. 31.4 
7 299 .+-. 14 
2 Z 100 4.1 .+-. 0.9 
14.1 .+-. 5.4 
7 273 .+-. 6 
3 Dox 100 4.2 .+-. 0.9 
91.1 .+-. 54.4 
7 301 .+-. 9 
4 Dox + Z 
71 2.5 .+-. 1.0 
6.6 .+-. 5.7 
7 254 .+-. 9 
After 
12 weeks 
1 Control 
100 6.4 .+-. 0.8 
233.4 .+-. 105.3 
100 318 .+-. 12 
2 Z 100 4.4 .+-. 0.9 
36.5 .+-. 22.2 
100 275 .+-. 9 
3 Dox 100 4.5 .+-. 0.7 
197.8 .+-. 132.1 
100 338 .+-. 28 
4 Dox + Z 
71 1.1 .+-. 0.4 
1.2 .+-. 1.1 
100 251 .+-. 9 
__________________________________________________________________________ 
FIG. 6 shows tumor incidence expressed as the percentage of tumor-bearing 
animals in each group. The individual treatment with Z or doxorubicin 
showed no change here vis-a-vis the untreated control group. 
FIG. 7 shows that while the average number of tumors was reduced only 
slightly by treatment with the individual preparations, the combined 
administration showed a synergistic effect. A synergistic effect was also 
observed in the case of the parameter "average tumor burden per rat" (FIG. 
8), which is the product of the average number of tumors and the average 
tumor volume. 
D. Antiproliferative Activity of Compound Z in Combination with Interferon 
on Human Tumor Cells in Vitro 
Human mammary cell lines BT-20 (oestrogen receptor-negative) and ZR 75-1 
(oestrogen receptor-positive) were used for this test. The cells were 
cultivated in RPMI 1640, which contained 10% FCS, and plated out on tissue 
culture plates. One day after the plating out, the cultures were treated 
with Compound Z and interferon individually and in combination for 14 
days. Culture medium and test compounds were renewed every 2-3 days. Cell 
counts were undertaken at the beginning of the test at each change of the 
medium and at the end of the test. The interferons used were human hybrid 
rIFN-.alpha.AD, human interferon rIFN.alpha..sub.2 (active ingredient of 
`Roferon`) and rIFN.gamma.. 
The results are presented in FIGS. 9-12. The results obtained with 
IFN.alpha.A/D and IFN.alpha..sub.2 were the same, therefore only one curve 
is given for interferon. 
FIG. 9 shows that the concentration of 10 U/ml, which is almost inactive in 
the case of sole administration, clearly enhances the activity of Compound 
Z. 
FIG. 10 shows that an almost complete inhibition of the cell proliferation 
was achieved with higher, but still non-toxic, dosages of the combination. 
FIG. 11 shows that similar results were achieved with rIFN.gamma. as with 
IFN.alpha.A/D. 
Also in the case of cell line ZR 75-1 the treatment with the combination of 
Compound Z and interferons led to a greater inhibition of the cell 
proliferation than the treatment with the individual components (FIG. 12 
and 13). 
Treatment of Human Bronchial Squamous Cell Carcinoma (Riedacher LXFE) in 
the Athymic Mouse 
About 1 mm.sup.3 of tumor tissue was transplanted subcutaneously in the 
athymic mouse for this test. The treatment (50 mg/kg/day of arotinoid 
p.o.; 50,000 or 100,000 U/ml of interferon .alpha.A/D i.p., 5 times weekly 
for 4 weeks) was begun as soon as the transplants had grown to a diameter 
of about 0.5 cm. The tumor growth was determined weekly by measuring the 
size and small diameter of the tumors. Adriamycin (1 mg/kg/day i.p.) was 
used as the positive control. 
The results are presented in FIG. 14. From this, it will be evident that 
the lower interferon dosage, which on its own had no influence on the 
tumor growth, clearly reduced the tumor growth in combination with 
compound Z. 
In the use in accordance with the invention, Compound Z can be employed in 
the dosages and formulations which are known from European Patent 
Publication A2-0 331 983. The dosing in an individual case must be fitted 
to the requirements of the patient and to the other cytostatic or BRM used 
in parallel, which lies in the purview of the specialist knowledge.