Steroidal erythropoietic agents and therapeutic compositions and methods

~69~37 This inventlon relates to certain novel non-pyrogenlc steroidal erythropoietic agents, and in particular to therapeutic compositions containing the known l9-noretiocholanolone.

These compositions are useful in the stimulation of erythropoiesis.

Erythropoiesis is ~he process of formation of red blood cells.

The ~erm anemia implies an abnormally low number of circulating red cells or a decreased concentration of hemoglobln in the blood.

The appearance of anemia reflects either marrow failure or excassi~e red cell loss, or both.

Marrow failure, i.e., reduced erythropoiesls, may occur ~-as a result of a nutritional deficiency~ toxic exposure, tumor invasion, or other and sometimes unknown causes. ;~ For the treatment of anemias of bone marrow failure ~hypoplastic and aplastic anemias), it has been proposed to use substances which might stimulate the marrow, such as androgens or corticosterioids.

Campbell et al United States patent 3,383,282 discloses various 3,5-androstadiene-3i17diol derivatives as possessing erythropoietic activity.

Schmidl m et al United States paten~s 3,519,659 and 3,519,660 disclose various prednisolone deTivatives h~ving antileukemia activity. ~ It is known that erythropoietic activity is exhibited by ~metabolltes of certain androgenic, anabolic9 or progestational steroids.

Thus, Levere et al.

Proceedings o~ a Symposium held in conjunction with the American Society of Hematology, December 4, 1971, Chapter III, discloses that etiocholanololle, a human metaboiite of testosterone, possesses erythropoietic activity.

Jepson, ibid.j Chapter II, discloses that nandrolone ~l9-nortestosterone; 17-~-hydroxy-19-nor-4-androsten-3-one), an anabolic steroidJ possesses ery~hropoietic activity similar to testosterone.

This substance, however, has the~drawback of exhibi~ing androgenic side-effects.

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It is known in ~he form of its decanoate, described in DeWitt et al United , ~ ~ ', .

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~;943~7 States patent 2,998,423.

Etiocholanolone possesses the substantial drawback of being a pyrogen in man.

ln accorda~ce with the present invention, it has been found that a certain estrane derivative which contains the 5~-H coniguration exhibits erythropoietic activity, while at the same time it is nonpyroge~ic and exhibits little or no androgenic sid~ efects. ;

The compound found to be non-pyrogenic and active in stimulating erythropoiesis is 19-noretiocholanolone ~3~-hydroxy-5~-estrane-17-one).

l9-Noretiocholanolone is a known compound and is disclosed by Engel et al, J.

Biol, Chem. 231, 1 9 159 ~1958).

This compound is also disclosed in an article by Counsell in Tetrahedron, Vol. 15, 202-211 (1961).

19-norethiocholanolone may also be synthesized by hydrogenating nandrolone 17-acetate to the corresponding 5~-3 keto-17~-acetate by the method described in J.

Org.

Chem. 31. 2394 ~1966), then hydrogenating the 3-keto group to form the 3~-hydroxy group using lithium-aluminum tri-tert.- ;

butoxyhydride, protecting the 3d-hydroxy group and hydrolyzing the 17~acetate, oxidising the 17~-hydroxy group to 17-keto with CrO3-pyridine, and finally remo~ing the 3~-protPoting group.

This protecting group is another group, such as the methoxymethyl ether, trimethylsilyl ether or tetrahydropyranyl et~er group, or an ester group such as the pivalate or benzoate.

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~C~6~3~ The ~ollowlng exampleq illustrate the therapeutlc compositions - ~of the inventlon and their applicat~on, but are not to be regarded as llmitlng: :

EXAMPLE 1 Erythropoietin Bioassay l9-noretiocholanolone was administered to mice as a single subcutaneous injection of 2.5 mg. in a 2-propanediol vehicle, at various intervals following induced hypoxia.

The f~rst injection was made on the third day post-hypoxia; on the i~th post-hypoxic day, 0.5~v Ci59FeC13 was injected intra~enously; the percent 59Fe incorporation into the red cells was detern~ned on da~ 7 post-hypoxia.

The l9~noretiocholQnolone stimulated radioiron incorporation significantly, the figure for % RBC-59Fe incorporation being 5.82 ~ 1.21 (p< 0.05~.

EXAMPLE 2 Ra~ Marrow Bioassay l9-noretiocholanolone was added in 1~ 1 of 2-propanediol to rat bone marrow, (S-D ~ ~,fJ100 - 150 g.3 at the initiation~of the cultures;

about 72 hou~s later 0.5 Ci59Fe, bound to transferrin,~was added to the . "' '" '' ':

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'". ':', ., ' 437 cultures; radioheme was extracted 6 hours later and qu~ntitated. The data suggest that 19-noretiocholanolone is active in this system:

Concentration (M) 3xlO 7 3xlO 3x15 9 3xlO 10 54 46 172 180~47.9 ~ , .4C-Labeled Hemo~lobin Human marrow cultures were treated with 19-noretiocholanolone for three days; 3~ Ci of 14C-valine was added for the last 24 of culture.

Hemoglobin was isolated simultaneously from cells cultured with either 2propanediol or the steroid (3 x 10 M~.

The specific activity ( 4C-cpm/ A540) of each was calculated and the ratio determined.

The data show that the steroid was stimulatory:

4C-hemoglobinl/ l9-noretiocholanolone 1-34 1/ ratio of the specific activity of a steroid-treated culture to a 2 propanediol-treated culture.

E~AMPLE A uman Marrow Culture6 ~ i Radioiron lncorporation into heme was determined in~the same manner as in the rat marrow cultures of Example 2.

The 19-noretiocholanolone was evaluated at a concentration of 3xlO M except in the marrow obtained from a patient with no demonstrable~disease;;where a conoentration of 5xlO lOM was used.

The test data are as follows:

~ Fe-Heme Incorporation : : ~ ;

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NoSystemicMycosis Hemolytic Rhabdomyo DemonstrableLupus ~ Fungoides Anemia SarCQma Dlseasethrematosus~ 144 ~ I48 ~ ~ 93 105 ~1/ The vehicle is considered as 100%.

-The foregoi~g data indicate~that l9-noretiocholanolone ~ ~ 4 ~ . ' ~.' '~" ::

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1S~6~37 stimulates erythropoesis both in vivo and in vitro.

The foregoing compounds are adapted for the administration thereof to humans and other warm-blooded animals in amounts effective to stimulate erythropoesis~ such amounts being generally in the range from about 5 to about 500 mg. per unit dosage. The usual method of administration is parenterally~ for which purpose the compound may be prepared in a form suitable for injection as a solution or suspension in m ml. ampoules. The following is an example of such a preparation~ EXAMPLE 5 10 ~ The dosage form can be prepared by admixing 500 g. of 19 B i noretiocholanolone .~ 3~ k~ into 2 liters of sterilesesame oil containing about 500 mlO of benzyl alcohol as a preservative, and heating the resulting mixture to about 80 C. to obtain a solution.

The solution is allowed to return to room temperature and the volume is increased to 10 liters by addition of sesame oil. The solution is filtered through a bacteriological membrane fil~er and is packaged into dosage forms, e.g.~ vials of 2 or 5 ml. or ampoules of 1 ml. The strength of the steroid solution is about 50 mg./cc.

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