FORM 2
THE PATENT ACT, 1970
COMPLETE SPECIFICATION
(SECTION 10)
TITLE : PROCESS FOR PREPARATION OF
BOVINE OESTROGEN CONJUGATES
ALKEM LABORATORIES LIMITED
510 Shah Nahar.Worlr,
Mumbai 400 013, Maharashtra,
India, An indian Company incorporated
under the Companies Act, 1956.
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES AND ASCERTAINS THE NATURE OF THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.


ORIGINAL
823/MUM/2003
18-08-2003

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FIELD OF INVENTION
This invention relates to bovine oestrogen conjugates.
It also relates to process for the preparation of bovine oestrogen conjugates. Further it relates to mettiod resulting in a product for the treatment of fracture and other bone related conditions using bovine oestrogen conjugates for accelerated and effective healing thereof.
PRIOR ART
Hormones known as oestrogens and vitamin D are reported to be responsible for absorption, transport and calcification of calcium in human body. Oestrogen conjugates are essentially conjugates of oestrone with sulfates, oestradiol sulfates, and, oestrioi sulfates. Oestrone confuages or sulfates are used in a variety of conditions associated with deficiency of oestrogens in humans including hormone replacement therapy (KRT) in menopausal syndrome, prurifis, sensile vaginitis, or hypogenitalism.
US Patent No. 2565115 of Bates et al refers to a method of obtaining conjugated oestrogen preparation from pregnant mares' urine. PCT Publication No WO 02/09732 A2 describes adsorber columns for concentrating and stabilizing conjugated oestrogens from pregnant mares' urine to avoid transportation of large volume of urine to a processing unit
It is difficult and expensive to look after pregnant mares. Therefore, preparation of oestrogen conjugates from pregnant mares' urine is expensive. Besides, oestrogen conjugates obtained from pregnant mares' urine comprise of 20-30% equilin and its metabolites. Therefore, purity level of oestrogen conjugates obtained from pregnant mares' urine is only 70-80%.

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Oestrogen conjugates are not reported to be used in the treatment of fracture, and, other bone related conditions, so far.
OBJECTS OF INVENTION
An object of the invention is to provide bovine oestrogen conjugates in high yield and purity.
Another object of the invention is to provide bovine oestrogen conjugates which is economical.
Another object of the invention is to provide a process for the preparation of bovine oestrogen conjugates in high yield and purity.
Another object of the invention is to provide a process for the preparation of bovine oestrogen conjugates which is economical.
Another object of the invention is to provide a process for the preparation of bovine oestrogen conjugates which is simple and easy and inexpensive to carry out
Another object of the invention is to provide a process resulting in a product for treatment of fracture and other bone related conditions by way of bovine oestrogen conjugates for accelerated, and, effective healing thereof.
DESCRIPTION OF INVENTION
Neither the US Patent nor the PCT publication discloses, suggests, motivates or points to preparation or isolation of oestrogen conjugates from bovine urine. Studies have shown, that, bovine urine is a rich source of oestrogen irrespective of their physiological states i.e. whether young,

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The invention is illustrated with the following experimental examples, which are not to be construed to limit the scope thereof
Example 1
Pooled cow urine (2 liters) was concentrated by spray drying. The concentrate was extracted 3 x 1 L each time. The acetone layer were pooled together, and, filtered through cotton fabric filter. The filtrate (extract) was dried by vaccum distillation of the solvent under reduced pressure to obtain a powder.
Yield of oestrogen conjugates: 3.5 g
Assay 90% oesfcjogens free from equilin and its metabolites.
Example 2
Pooled cow urine (3iiters) was concentrated by vacuum distillation under reduced pressure. The concentrate was extracted 4 times with acetone (1.5 liters). The acetone layers were pooled together and further processed as described in Example 1.
Yield of oestrogen conjugates: 3.8 g
Assay 92% ©estrogens free from equilin and its metabolites.
Example 3
Cow urine concentrate (120 g) obtained by spray drying of pooled cow urine (Two Liters) was made into a slurry with cow urine (500 ml) and extracted 4 times with acetone (1.5 liters). The acetone layers were pooled together and further processed as described in Example 1.
Yield of oestrogen conjugates: 4 g

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Assay 93% oestrogens free from equiiin and its metabolites.
Example 4
Pooled cow urine (1.5 liters) was filtered through cotton fabric filter and run through "Amberlite XAD - 7" column (polyacrylic ion exchange resin) manufactured by Rohm & Haas. The column was eluted with a mixture of (2 Liters) of acetone : benzene ( 70 : 30 v/v). The elute was vaccum distilled under reduced pressure. The concentrate was extracted with acetone and processed further as described in Example 1.
Yield of oestrogen conjugates: 3.5 g
Assay 96% oestrogens free from equiiin and its metabolites.
Example 5
The procedure of Example 4 was followed by elution of the colum with acetone (2 liters)
Yield of oestrogen conjugates: 3.5 g
Assay 96% oestrogens free from equiiin and its metabolites.

-7-WE CLAIM
1. Process for the preparation of bovine oestrogen conjugates comprising concentrating bovine urine, extracting the concentrate with an organic solvent of short carbon chain length (e.g. acetone, methyl ethyl ketone etc.) and drying the solvent extract.
2. Process as claimed in Claim 1, wherein the bovine urine is concentrated by spray drying.
3. Process as claimed in Claim 1, wherein the bovine urine is concentrated under reduced pressure of 650 to 700 m.m. of Hg.
4. Process as claimed in Claim 1, wherein the bovine urine is concentrated by adsorption on ion exchange resin column followed by elution with an organic solvent of short carbon chain length, such as acetone, methyl ethyl ketone and, evaporation of the solvent in the elute under reduced pressure.
5. Process as claimed in Claim 4, wherein the organic solvent used for elution of the column is acetone or mixture of acetone (60 to 80) and benzene(20 to 40).
6. Process as claimed in any one of the Claims 1 to 5, wherein the solvent used for extraction of the concentrate is acetone.
7. Process as claimed in any one of the Claims 1 to 6, wherein the solvent extract is dried by vacuum distillation i.e. under reduced pressure as in claim 3.
8. Process for the preparation of bovine oestrogen conjugates as herein described with reference to Examples 1-5.
For ALKEM LABORATORIES LTD.
(V. S Hegde)
Date : 18/08/2003 Constituted Attorney for the Applicant
To
The Controller of Patents,
The Patent Office MUMBAI.