Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Cyclopentanohydrophenanthrene ring system doai
Reexamination Certificate
1998-04-21
2003-02-25
Badio, Barbara P. (Department: 1616)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Cyclopentanohydrophenanthrene ring system doai
C552S642000, C514S179000
Reexamination Certificate
active
06525039
ABSTRACT:
BACKGROUND OF THE INVENTION
The use of naturally occurring estrogenic compositions of substantial purity and low toxicity such as PREMARIN (conjugated equine estrogens) has become a preferred medical treatment for alleviating the symptoms of menopausal syndrome, osteoporosis/osteopenia in estrogen deficient women and in other hormone related disorders. The estrogenic components of the naturally occurring estrogenic compositions have been generally identified as sulfate esters of estrone, equilin, equilenin, 17-&bgr;-estradiol, dihydroequilenin and 17-&bgr;-dihydroequilenin (U.S. Pat. No. 2,834,712). The estrogenic compositions are usually buffered or stabilized with alkali metal salts of organic or inorganic acids at a substantially neutral pH of about 6.5 to 7.5. Urea has also been used as a stabilizer (U.S. Pat. No. 3,608,077). The incorporation of antioxidants to stabilize synthetic conjugated estrogens and the failure of pH control with tris(hydroxymethyl)aminomethane (TRIS) to prevent hydrolysis is discussed in U.S. Pat. No. 4,154,820.
One of the compounds described herein, 3&bgr;-hydroxy-5,7,9-estratriene-17-one 3-sulfate ester sodium salt is a minor component of PREMARIN (conjugated equine estrogens). The preparation of 3&bgr;-hydroxy-5,7,9-estratriene-17-one is been disclosed by D. Banedjee in Ind. Chim. Belge. Suppl. 2: 435 (1959); however, no utility is provided for this compound.
DESCRIPTION OF THE INVENTION
In accordance with this invention, there is provided 3&bgr;-hydroxy-5,7,9-estratriene-17-one or a pharmaceutically acceptable salt of its 3-sulfate ester. The structure of 3&bgr;-hydroxy-5,7,9-estratriene-17-one is shown below as compound I.
Pharmaceutically acceptable salts of 3&bgr;-hydroxy-5,7,9-estratriene-17-one 3-sulfate ester include, but are not limited to, the alkali metal salts, alkaline earth metal salts, ammonium salts, alkylammonium salts containing 1-6 carbon atoms or dialkylammonium salts containing 1-6 carbon atoms in each alkyl group, and trialkylammonium salts containing 1-6 carbon atoms in each alkyl group.
As 3&bgr;-hydroxy-5,7,9-estratriene-17-one 3-sulfate ester sodium salt is a minor component of PREMARIN (conjugated equine estrogens), this invention also provides 3&bgr;-hydroxy-5,7,9-estratriene-17-one 3-sulfate sodium salt in greater than one percent purity. This invention further provides a compound consisting essentially of a pharmaceutically acceptable salt of 3&bgr;-hydroxy-5,7,9-estratriene-17-one 3-sulfate ester; a compound consisting essentially of 3&bgr;-hydroxy-5,7,9-estratriene-17-one 3-sulfate ester sodium salt; and a compound consisting essentially of 3&bgr;-hydroxy-5,7,9-estratriene-17-one or a pharmaceutically acceptable salt of its 3-sulfate ester.
As used in accordance with this invention, treating covers treatment of an existing condition, ameliorating the condition, or providing palliation of the condition and inhibiting includes inhibiting or preventing the progress or development of the condition.
The compounds of this invention can be prepared from readily available starting materials. For example, the preparation of 3&bgr;-hydroxy-5,7,9-estratriene-17-one 3-sulfate ester sodium salt can be prepared from 3&bgr;-hydroxy-5,7,9-estratriene-17-one according to Scheme I. 3&bgr;-Hydroxy-5,7,9-estratriene-17-one can be prepared according to D. Banedjee in Ind. Chim. Belge. Suppl. 2: 435 (1959).
The compounds of this invention are estrogenic, as shown in the in vitro and in vivo standard pharmacological test procedures described below in which 3&bgr;-hydroxy-5,7,9-estratriene-17-one was evaluated as a representative compound of this invention.
Estrogen Receptor Binding
An initial evaluation examined the competitive binding properties of 3&bgr;-hydroxy-5,7,9-estratriene-17-one to the human estrogen receptor (hER-&agr;) prepared as a soluble cell extract (cytosol). In this standard pharmacological test procedure, 3&bgr;-hydroxy-5,7,9-estratriene-17-one demonstrated no specific binding activity. However, when estrogen receptor binding was analyzed using a whole cell test procedure, specific binding was clearly demonstrated. This test procedure indicated an IC
50
of 7.9×10
−9
M for 3&bgr;-hydroxy-5,7,9-estratriene-17-one. This would be compared with a K
i
for estrone, equilin and equilinen of 51, 67 and 375 nM, respectively.
In Vitro Cotransfection Test Procedure
In this standard pharmacological test procedure, hER-&agr; over-expressed in Chinese hamster ovary (CHO) cells infected with adeno-2x-ERE-tk-luciferase, an estrogen responsive reporter gene construct, cells were exposed to varying concentrations (10
−12
-10
−5
M) of 3&bgr;-hydroxy-5,7,9-estratfiene-17-one for 24 hours.
Cells were also exposed to 17&bgr;-stradiol at 10
−9
M. Following the 24-hour treatment, cells were lysed and cell extracts assayed for luciferase activity. The results provided that 3&bgr;-hydroxy-5,7,9-estratriene-17-one had an EC
50
of approximately 50 nM. Using a similar test procedure, previous data indicate a 5.6 nM EC
50
for estrone.
In Vivo Uterotropic Activity
Immature rats were treated with 100 &mgr;g 3&bgr;-hydroxy-5,7,9-estratriene-17-one for three days (S.C.) as well as additional groups (n=6) of rats treated with 0.5 &mgr;g ethinyl estradiol and vehicle as positive and negative controls, respectively. Rats treated with 3&bgr;-hydroxy-5,7,9-estratriene-17-one had a uterine weight of 54.63±5.2 mg, whereas control rats had uterine weight of 29.78±1.84 mg demonstrating that 3&bgr;-hydroxy-5,7,9estratriene-17-one was estrogenic. Rats treated with ethinyl estradiol (0.5 &mgr;g) had a uterine weight of 92.68±7.6 mg.
The results of these standard pharmaco logical test procedures demonstrate that the compounds of this invention are estrogenic.
A representative compound of this invention (3&bgr;-hydroxy-5,7,9-estratriene-17-one) was evaluated in a standard pharmacological test procedure which measured the cardioprotective effects of the compound tested.
Briefly, Female rats, weighing 180-200 g, were pretreated with DES (0.25 mg/kg, i.p.) 18 hr prior to the study. Uterine horns and aortae were removed from pentobarbital-overdosed animals, cleaned of adherences and cut into 1 cm and 2-3 mm segments, respectivey. The preparat ions were mounted in orgy baths containing Jalon solution at 30° C. and Krebs solution at 37° C., respectively, and aerated with 95% O
2
and 5% CO
2
. The contractions were monitored with force-displacement transducers on a Grass polygraph recorder. DMSO produced no effects on the tissues in the present study and thus, was used as a vehicle for dissolving the test compounds.
Both uterine and aortic smooth muscles were contracted with 60 mM KCl. When potassium-induced contraction reached steady-state, cumulatve-concentration response curves were determined for test drug or vehicle. Increasing concentrations of the compound to be evaluated were added when the effect of the previous concentration reached steady state (not longer than 20 min). The EC
50
was de fined as the concentration of test compound which produced 50% relaxation.
EC
50
values of 3.9×10
−5
and 4.0×10
−5
were obtained in uterine muscle and thoracic aorta, respectively, for 3&bgr;-hydroxy-5,7,9-estratriene-17-one. These results demonstrate that the compounds of this invention provide cardioprotective protection, particularly in the treatment of ischemic disease and hypertension.
Based on the results obtained in the standard pharmacological test procedures, the compounds of this invention are useful in providing estrogen replacement therapy following ovariectomy or menopause, and in relieving symptoms related to estrogen deficiency, including vasomotor symptoms, such as hot flushes, and other menopausal related conditions, such as vaginal atrophy, vaginitis, and atrophic changes of the lower urinary tract which may cause increased urinary frequency, incontinence, and dysuria. The compounds of this invention are useful in preventing bone loss and in the inhibition or treatment
Kagan Michael Z.
Raveendranath Panolil
Shah Syed M.
Badio Barbara P.
Milowsky Arnold S.
Wyeth
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