Methods of treatment

Abstract

A method of inducing inhibition or loss of cell proliferation in solid tumors utilizing a Vitamin D.sub.3 analog, as well as a method of inducing loss of cell proliferation in solid tumors utilizing trans retinoic acid and a Vitamin D.sub.3 analog.In another aspect, a method of inducing inhibition or loss of cell proliferation in solid tumors utilizing a Vitamin D.sub.3 metabolite, as well as a method of inducing loss of cell proliferation in solid tumors utilizing trans retinoic acid and a Vitamin D.sub.3 metabolite.

Description

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention relates to a method of inducing inhibition or loss of cell proliferation in solid tumors, particularly, breast tumors, which comprises administering to a host in need of such treatment an effective amount of a Vitamin D.sub.3 analog. In another aspect, the invention relates to a method of inducing inhibition or loss of cell proliferation in solid tumors, particularly, breast tumors which comprises administering to a host in need of such treatment an effective amount of trans retinoic acid and a Vitamin D.sub.3 analog. Preferably, the Vitamin D.sub.3 analog is selected from the group consisting of

1.alpha.,25-dihydroxy-26,27-hexafluorocholecalciferol; 1.alpha.,25-dihydroxy-22-ene-26,27-hexafluorocholecalciferol; 1.alpha.,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol; 1.alpha.,25-dihydroxy-23-yne-cholecalciferol; 1.alpha.,25-dihydroxy-16-ene-cholecalciferol; 1.alpha.,25-dihydroxy-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-1.alpha.-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-1.alpha.,25-dihydroxy-16-ene-23-yne-19-nor-cholecalciferol; 26,26,26,27,27,27-hexafluoro-25 -hydroxy-16-ene-23-yne-19-nor-cholecalciferol; and 26,26,26,27,27,27-hexafluoro-1.alpha.-fluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol.

In yet another aspect, the invention relates to a method of inducing inhibition or loss of cell proliferation in solid tumors, particularly, breast tumors, which comprises administering to a host in need of such treatment an effective amount of a Vitamin D.sub.3 metabolite, such as, 1.alpha.,25-dihydroxycholecalciferol (hereinafter also referred to as "D.sub.3 "), either alone or in combination with trans retinoic acid.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has now been found that a beneficial inhibition or loss of cell proliferation in solid tumors can be achieved utilizing a Vitamin D.sub.3 analog, especially one of those more particularly described hereinafter, alone or in combination with all trans retinoic acid (hereinafter also referred to as "RA").

Accordingly, the invention comprises a method of achieving an inhibition of or loss in cell proliferation in solid tumor in a host requiring such treatment by administering an effective amount of a Vitamin D.sub.3 analog selected from the group consisting of

1.alpha.,25-dihydroxy-26,27-hexafluorocholecalciferol (hereinafter also referred to as "HF"); 1.alpha.,25-dihydroxy-22-ene-26,27-hexafluorocholecalciferol (hereinafter also referred to as "22 HF"); 1.alpha.,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol (hereinafter also referred to as "16-23HF"); 1.alpha.,25-dihydroxy-23-yne-cholecalciferol (hereinafter also referred to as "23D"); 1.alpha.,25-dihydroxy-16-ene-cholecalciferol (hereinafter also referred to as "16D"); 1.alpha.,25-dihydroxy-16-ene-23 -yne-cholecalciferol (hereinafter also referred to as "16-23D"); 26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-1.alpha.-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol (hereinafter also referred to as "IF-16-23HF"); 26,26,26,27,27,27-hexafluoro-1.alpha.,25 -dihydroxy-16-ene-23-yne-19-nor-cholecalciferol (hereinafter also referred to as "19 nor"); 26,26,26,27,27,27-hexafluoro-25 -hydroxy-16-ene-23-yne-19-nor-cholecalciferol; and 26,26,26,27,27,27-hexafluoro-1.alpha.-fluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol.

The invention also comprises a method of inducing inhibition or loss of cell proliferation in solid tumors, particularly, breast tumors, which comprises administering to a host in need of such treatment an effective amount of a Vitamin D.sub.3 metabolite, preferably, 1.alpha.,25-dihydroxycholecalciferol, either alone or in combination with retinoic acid.

A preferred group of Vitamin D.sub.3 analogs, utilized in the methods of the invention, comprises the group:

1.alpha.,25-dihydroxy-26,27-hexafluorocholecalciferol; 1.alpha.,25-dihydroxy-22-ene-26,27-hexafluorocholecalciferol; 1.alpha.,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-1.alpha.-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-1.alpha.,25-dihydroxy-16-ene-23-yne-19-nor-cholecalciferol; 26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol; and 26,26,26,27,27,27-hexafluoro-1.alpha.-fluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol.

Preferred Vitamin D.sub.3 analogs utilized in the methods of the invention are 1.alpha.,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol; 1.alpha.,25-dihydroxy-26,27-hexafluorocholecalciferol; and 1.alpha.,25-dihydroxy-16-ene-23-yne-cholecalciferol.

A particularly preferred Vitamin D.sub.3 analog, utilized in the methods of the invention, is 1.alpha.,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol.

Processes for preparing 1.alpha.,25-dihydroxy-26,27-hexafluoro-cholecalciferol are set forth in U.S. Pat. No. 4,358,406, issued Nov. 9, 1982 which is hereby incorporated by reference.

Processes for preparing 1.alpha.,25-dihydroxy-22-ene-26,27-hexafluoro-cholecalciferol are set forth in U.S. Pat. No. 4,613,594, issued Sep. 23, 1986 which is hereby incorporated by reference. Processes for preparing 1.alpha.,25-dihydroxy-23-yne-cholecalciferol are set forth in U.S. Pat. No. 4,804,502, issued Feb. 14, 1989 which is hereby incorporated by reference. Processes for preparing 1.alpha.,25-dihydroxy-16-ene-cholecalciferol and 1.alpha.,25-dihydroxy-16-ene-23-yne-cholecalciferol are set forth in U.S. Pat. No. 5,087,619, issued Feb. 11, 1992 which is hereby incorporated by reference. Processes for preparing all rans retinoic acid are set forth in U.S. Pat. No. 3,746,730, issued Jul. 17, 1973 which is hereby incorporated by reference. Processes for preparing

1.alpha.,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-1.alpha.-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-1.alpha.,25-dihydroxy-16-ene-23-yne-19-nor-cholecalciferol; 26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol; and 26,26,26,27,27,27-hexafluoro-1.alpha.-fluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol are set forth in U.S. patent application Ser. No. 971,788, filed Nov. 5, 1992, which is hereby incorporated by reference.

The methods of the invention can be utilized for the treatment of solid tumors, in particular, breast tumors, such as, those comprising MDA-MB231 cells which are devoid of estrogen receptors, T-47D cells which possess estrogen receptors and the like. The inhibition or loss in cell proliferation in solid tumors can be demonstrated utilizing the procedures described herein below.

A colony assay can be used to evaluate inhibition or loss of solid tumor cell proliferation on tumor cells with the Vitamin D.sub.3 analogs or metabolites referred to herein, particularly, 1.alpha.,25-dihydroxycholecalciferol; 1.alpha.,25-dihydroxy-26,27-hexafluorocholecalciferol; 1.alpha.,25-dihydroxy-22-ene-26,27-hexafluorocholecalciferol; 1.alpha.,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol; 1.alpha.,25-dihydroxy-16-ene-cholecalciferol; and 1.alpha.,25-dihydroxy-16-ene-23-yne-cholecalciferol.

METHOD

Cells of the cell line utilized are trypsinized, counted and plated 250 cells/5 ml minimal essential medium (MEM) containing 10% heat inactivated fetal calf serum. The test compound is added 48 hours after plating for an exposure time of 120 hours. Thereafter, the medium is aspirated and replaced with drug-free medium, and 14-16 days later the medium is aspirated. The colonies formed are fixed and stained with 0.5% methylene blue in 50% methanol. Colonies which contain greater than 500 cells are counted. The percent loss in colony formation is determined as follows: ##EQU1##

The results are provided in Tables I-IV.

TABLE I______________________________________EFFECT OF VITAMIN D ANALOGS ON COLONY FORMATIONBY HUMAN BREAST CARCINOMA CELLS (T-47-D) Concentration Exposure time % loss inDrug (.mu.M) (hr) colony formation.sup.a______________________________________D.sub.3 0.05 120 29*D.sub.3 0.1 120 94*16 D.sub.3 0.01 120 30.5 .+-. 6.416-23 D.sub.3 0.05 120 82.0 .+-. 11.323D.sub.3 0.1 120 72.0 .+-. 29.7HF 0.01 120 83.5 .+-. 12.022HF 0.01 120 51.0 .+-. 45.216-23-D.sub.3 HF 0.01 120 100 .+-. 0______________________________________ .sup.a mean .+-. S.D. n = 2 *only one test was run at this concentration, accordingly no standard deviation is provided. TABLE II______________________________________EFFECT OF VITAMIN D ANALOGS ON COLONY FORMATIONBY HUMAN BREAST CARCINOMA CELLS (MDA-MB231) Concentration Exposure time % loss inDrug (.mu.M) (hr) colony formation.sup.a______________________________________D.sub.3 0.1 120 25.0 .+-. 8.716 D.sub.3 0.01 120 42.0 .+-. 20.516-23 D.sub.3 0.05 120 30.0 .+-. 16.123D.sub.3 0.1 120 33.3 .+-. 14.9HF 0.01 120 23.7 .+-. 19.822HF 0.01 120 29.0 .+-. 15.916-23-D.sub.3 HF 0.01 120 26.7 .+-. 17.3______________________________________ .sup.a mean .+-. S.E. n = 3

The synergistic compositions and methods of the invention can be utilized for the treatment of solid tumors, in particular, breast tumors. The inhibition or loss of solid tumor cell proliferation of the method of the invention comprising administering a combination of trans retinoic acid and Vitamin D.sub.3 analogs or metabolites may be demonstrated utilizing the procedure described above. In order to determine if the interactions between the drugs was synergistic the data on colony formation were analyzed according to F. Valeriote and H. Lin, Synergistic Interaction of Anticancer Agents: A cellular perspective, Cancer Chemother. Rep. 59, 895 (1975). The results on percent loss in colony formation are presented in Tables III and IV below.

TABLE III______________________________________EFFECT OF ALL-TRANS RETINOIC ACID (RA) AND 16-23D.sub.3ON COLONY FORMATION BY HUMAN BREASTCARCINOMA CELLS (MDA-MB-231) Concentration Exposure time % loss inDrug (.mu.M) (hr) colony formation.sup.a______________________________________RA 1.0 120 1.7 .+-. 0.916-23-D.sub.3 0.05 120 30.0 .+-. 16.1RA + 16-23-D.sub.3 1.0 + 0.05 120 55.7 .+-. 10.6______________________________________ .sup.a mean .+-. S.E. n = 3 TABLE IV______________________________________EFFECT OF ALL-TRANS RETINOIC ACID (RA) AND 16-23D.sub.3 HFON COLONY FORMATION BY HUMAN BREASTCARCINOMA CELLS (MDA-MB-231) Concentration Exposure time % loss inDrug (.mu.M) (hr) colony formation.sup.a______________________________________RA 1.0 120 1.7 .+-. 0.916-23-D.sub.3 HF 0.01 120 26.7 .+-. 17.3RA + 16-23-D.sub.3 HF 1.0 + 0.01 120 64.3 .+-. 18.7______________________________________ .sup.a mean .+-. S.E. n = 3

METHOD

Tetrazolium based MTT assay

T47D cells are seeded at densities of 4.times.10.sup.3 (cells/well) and 8-10.sup.3 (cells/well) (day zero). Twenty four hours after (day one) seeding the test compound is added to each well. On the second through seventh days, 50 .mu.l 3(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide is added to each well. The plates are incubated at 37.degree. C. for 2.5 hours. Then, the plates are centrifuged for 5 minutes at 800.times.g, supernantant is aspirated from the wells and 50 .mu.l 100% ethanol is added to each well. The plates are then shaken for 15 minutes to solubilize formazan. A blue color developes in replicating cells. The optical density is measured in anautomatic plate reader at dual wavelength of 570/660 nm and compared with the optical density of a control. The IC.sub.50 value reported is the concentration of compound tested at which 50% of cell proliferation is inhibited as determined by the method of Reed and Muench, as described in Reed, L. J., and H. Muench, A Simple Method of Estimating 50% endpoints, Am. J. Hyg. 27:493-497 (1938).

The results are as set forth in Table V below.

TABLE V______________________________________IC.sub.50Test Compound 4 .times. 10.sup.3 cells/well 8 .times. 10.sup.3 cells/well______________________________________16-23HF 1.3 .mu.M 1.61 .mu.M1F-16-23HF 14.2 nM 22.3 nM19 nor 9.0 nM 15.2 nMD.sub.3 3.7 nM 8.9 nM______________________________________

The percent loss in colony formation is an index of the irreversible loss of the proliferative potential of the tumor cells and is directly proportional to the tumor kill potency of the Vitamin D.sub.3 analog or metabolite, alone or in combination.

The individual components of the method comprising administering trans retinoic acid and a compound selected from the group consisting of

1.alpha.,25-dihydroxy-26,27-hexafluorocholecalciferol; 1.alpha.,25-dihydroxy-22-ene-26,27-hexafluorocholecalciferol; 1.alpha.,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol; 1.alpha.,25-dihydroxy-23-yne-cholecalciferol; 1.alpha.,25-dihydroxy-16-ene-cholecalciferol; 1.alpha.,25-dihydroxy-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-1.alpha.-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol; 26,26,26,27,27,27-hexafluoro-1.alpha.,25-dihydroxy-16-ene-23-yne-19-nor-cholecalciferol; 26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol; and 26,26,26,27,27,27-hexafluoro-1.alpha.-fluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.

The individual components of the method of comprising administering trans retinoic acid and 1.alpha.,25-dihydroxycholecalciferol may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.

A composition of the invention may be administered by any of the modes by which the components may be administered, for example, orally, intravenously or the like. The dosage regiment may be regulated according to the potency of individual compounds employed, the mode of administration, and the needs of the host mammal depending on factors such as the degree and the severity of the disease state and age and general condition of the host mammal being treated.

In the method of the invention, trans retinoic acid and/or a Vitamin D.sub.3 analog or metabolite can be administered in unit dosage forms such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, suppositories, transdermal compositions, aerosols and the like. Such dosage forms are prepared according to standard techniques in the art.

The dosages or therapeutically effective amounts utilized in the methods of the invention may be varied depending upon the requirements of the host, the severity of the condition being treated and the particular compound being employed in the method. Determination of dose amount for a particular administration is within the skill of the art. Generally, treatment is initiated at lower dosages and increased as needed by small increments until the optimum effect is reached. Exemplary dosages utilized in the methods of the invention are in the range of 0.00025-0.01 mg Vitamin D.sub.3 analog or metabolite per day, with appropriate monitoring of blood calcium levels and adjustment of the dosages as needed. For convenience, the total daily dosage may be administered in divided doses.

Exemplary dosages of trans retinoic acid can be in the range of from about 10 to about 20 mg per day, preferably 14 mg per day. Generally the trans retinoic acid is present in a ratio of from about 100 to about 1000 parts to one part of the Vitamin D.sub.3 analog or metabolite.

Oral dosage forms comprising compounds of formula I of the invention may be incorporated in capsules, tablets and the like with one or more pharmaceutically acceptable carrier materials.

Illustrative of the pharmaceutically acceptable carrier materials utilized in the pharmaceutical dosage forms are binders, such as gum tragacanth, acacia, corn starch or gelatin; an excipient, such as, dicalcium phosphate; disintegrating agents, such as, corn starch, potato starch, algenic acid and the like; lubricants, such as, magnesium stearate; sweetening agents, such as, sucrose, lactose or saccharin; flavoring agents, such as, peppermint, oil of wintergreen or cherry. Various other materials may be present as coating or to otherwise modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, a dye and a flavoring such as cherry or orange flavor.

Exemplary of formulations which may be utilized in the methods of the invention are:

EXAMPLE 1

______________________________________Capsule FormulationItem Ingredients mg/capsule______________________________________1 1.alpha.,25-dihydroxy-26,27- 0.005 0.010 hexafluorocholecalciferol2 Butylated Hydroxyanisole 0.016 0.0163 Butylated Hydroxytoluene 0.016 0.0164 Glycerin 16.000 16.0005 PEG 400 143.963 143.958 TOTAL 160.000 160.000______________________________________ Manufacturing Procedure Note

Perform all manufacturing steps under a nitrogen atmosphere and protect from light.

1. Warm the mixture of items 4 and 5 to 55.degree. C. 2. Dissolve items 2 and 3 in the solution from Step 1. 3. Dissolve item 1 in the solution from Step 2.

EXAMPLE

______________________________________Capsule FormulationItem Ingredients mg/capsule______________________________________1 1.alpha.,25-dihydroxy-22-ene- 0.00025 26,27-hexafluorocholecalciferol2 Polyethylene glycol 400 200.003 Butylated Hydroxyanisole 0.1004 Ascorbyl palmitate 1.00______________________________________ Manufacturing Procedure Note

Perform all manufacturing steps under a nitrogen atmosphere and protect from light.

1. Dissolve items 1, 3 and 4 in item 2 and encapsulate.

EXAMPLE

______________________________________Capsule FormulationItem Ingredients mg/capsule______________________________________1 1.alpha.,25-dihydroxy-26,27- 0.005 0.010 hexafluoro-16-ene-23-yne cholecalciferol2 Butylated Hydroxyanisole 0.016 0.0163 Butylated Hydroxytoluene 0.016 0.0164 Neobee M-5 159.963 159.958 TOTAL 160.000 160.000______________________________________ Manufacturing Procedure Note

Perform all manufacturing steps under a nitrogen atmosphere and protect from light.

1. Warm 10% of item 4 to 55.degree. C. 2. Dissolve items 2 and 3 in the solution from Step 1. 3. Dissolve item 1in the solution from Step 2. 4. Add the remaining item 4 to the solution from Step 3 and mix well.

EXAMPLE

______________________________________Capsule FormulationItem Ingredients mg/capsule______________________________________1 1.alpha.,25-dihydroxy-22-ene-23 0.005 0.010 yne-cholecalciferol2 Butylated Hydroxyanisole 0.016 0.0163 Butylated Hydroxytoluene 0.016 0.0164 Neobee M-5 159.963 159.958 TOTAL 160.000 160.000______________________________________ Manufacturing Procedure Note

Perform all manufacturing steps under a nitrogen atmosphere and protect from light.

1. Warm 10% of item 4 to 55.degree. C. 2. Dissolve items 2 and 3 in the solution from Step 1. 3. Dissolve item 1 in the solution from Step 2. 4. Add the remaining item 4 to the solution from Step 3 and mix well.

EXAMPLE

______________________________________Capsule FormulationItem Ingredients mg/capsule______________________________________1 Trans Retinoic Acid 10.002 1.alpha.,25-dihydroxy-16-ene-23 0.01 yne-cholecalciferol3 Purified Beewax 7.854 Hydrogenated Soyhean Oil 7.855 Hydrogenated Vegetable Oil 31.406 Butylated Hydroxyanisole 0.117 Soybean Oil 107.288 Disodium Edetate 0.50 TOTAL 165.00______________________________________ Manufacturing Procedure Note

Perform all manufacturing steps under a nitrogen atmosphere and protect from light.

1. Melt items 3, 4 and 5 by heating at 70.degree. C., and mix well. 2. Dissolve item 6 in the mixture from Step 1. 3. Cool the mixture from Step 2 to 35.degree.-40.degree. C. 4. Add item 8 to the mixture from Step 3 and mix well. 5. Add item 7 to the mixture from Step 4, mix well and cool to room temperature. 6. Add items 1 and 2 to the mixture from Step 5 and disperse well until a uniform suspension is obtained.

EXAMPLE

______________________________________Capsule FormationItem Ingredients mg/capsule______________________________________1 Trans Retinoic Acid 10.0002 1.alpha.,25-dihydroxy-26,27- 0.00025 hexafluoro-16-ene-23-yne cholecalciferol3 Butylated Hydroxyanisole 0.0164 Butylated Hydroxytoluene 0.0165 Glycerin 16.0006 PEG400 133.86775 TOTAL 160.000______________________________________ Manufacturing Procedure Note

Perform all manufacturing steps under a nitrogen atmosphere and protect from light.

1. Warm the mixture of items 5 and 6 to 55.degree. C. 2. Dissolve items 3 and 4 in the solution from Step 1. 3. Add items 1 and 2 to the solution from Step 2 and mix well.

EXAMPLE

______________________________________Capsule FormulationItem Ingredients mg/capsule______________________________________1 Trans Retinoic Acid 10.002 Purified Beewax 7.853 Hydrogenated Soybean Oil 7.854 Hydrogenated Vegetable Oil 31.405 Butylated Hydroxyanisole 0.116 Soybean Oil 107.247 Disodium Edetate 0.50 TOTAL 165.00______________________________________ Manufacturing Procedure Note

Perform all manufacturing steps under a nitrogen atmosphere and protect from light.

1. Melt items 2, 3 and 4 by heating at 70.degree. C., and mix well. 2. Dissolve item 5 in the mixture from Step 1. 3. Cool the mixture from Step 2 to 35.degree.-40.degree. C. 4. Add item 7 to the mixture from Step 3 and mix well. 5. Add item 6 to the mixture from Step 4, mix well and cool to room temperature. 6. Add item I to the mixture from Step 5 and dispense well until a uniform suspension is obtained.

Claims

1. A method of inducing inhibition of cell proliferation in a solid breast tumor comprising administering to a host in need of said treatment from about 10 mg to about 20 mg of trans retinoic acid and from about 0.00025 mg to about 0.10 mg of a Vitamin D.sub.3 analog selected from the group consisting of 1.alpha.,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol, 1.alpha.,25-dihydroxy-26,27-hexafluorocholecalciferol and 1.alpha.,25-dihydroxy-16-ene-23-yne-cholecalciferol.

2. A method in accordance with claim 1 wherein the Vitamin D.sub.3 analog is 1.alpha.,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol.