FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10, Rule 13]
COMPOSITION FOR IMPROVING
ENDOMETRIAL THICKNESS DURING OVARIAN STIMULATION;
SANZYME LIMITED, A COMPANY
INCORPORATED UNDER THE
COMPANIES ACT, 1956, WHOSE ADDRESS IS A-2, SILVER BELLE, SRINIVAS BAGADKAR MARG, J.B. NAGAR, ANDHERI (EAST), MUMBAI - 400 059, MAHARASHTRA, INDIA.
THE FOLLOWING SPECIFICATION
PARTICULARLY DESCRIBES THE
INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.

Field of the Invention
The present invention relates to a pharmaceutical composition comprising combination of Follicle Stimulating Hormone (FSH) and Human Chorionic Gonadotropin (HCG) for improving endometrial thickness during ovarian stimulation.
Background of the Invention
During ovulation induction, stimulation with individual gonadotropins, such as FSH or combination of FSH and LH is at different time interval is a normal approach which has been in practice. For some patients, the dose of gonadotropin needed to provide adequate response is quite high and the use of low-dose regimens leads to very long duration treatment. Usage of 30 - 60 injections of FSH per cycle and around 5-15 injections of low dose hCG 200 IU, individually have been tried. However, with such frequent injections the treatment regimen becomes very lengthy, inconvenient for the patients and costly.
Attempts to improve the outcome of assisted reproduction treatment (ART) programmes in terms of improving patient friendliness, reducing the incidence of potential complications, such as cyst formation and development of ovarian hyper-stimulation syndrome (OHSS), increasing endometrial thickness for

improved implantation and cutting the global cost of ART have led to a growing interest in trying out various protocols over past decade. Although concentrated efforts were made to improve the outcome of ART cycles through various modifications of the stimulation protocol, the inter-cycle variations are many and as a result a standardized protocol for ovarian stimulation resulting in endometrial development has not been possible to date.
Clomiphene citrate has been used as the first line treatment for improving endometrial thickness in women seeking fertility treatment. However, it is observed not to provide the successful outcome. In some of the studies, Clomiphene citrate was given along with gonadotropin like HMG, however the endometrium was found to be thinner when Clomiphene citrate was combined with hMG (Gonen and Casper, 1990; Check et al., 1991; Saito et al., 1991). There are cases in which ovulation, proper follicular development and fertilization are achieved, and yet improper implantation of the embryo prevents pregnancy. In other cases, spontaneous abortion (miscarriage) occurs during the first trimester. Thus, it can be seen that even once follicular development, ovulation and fertilization have occurred there is no guarantee of a successful pregnancy and problems with implantation and early miscarriage are often encountered. In some patients, tendency to abort or failure to implant may eventually be overcome, but to do so requires repeated ART cycles, with consequent negative physiological and psychological effects on the patient. In other patients, these problems represent an essentially permanent stumbling block to pregnancy. These problems may be associated with thickness of the endometrium, which is

sensitive to hormone levels.
Whether, normal or "stimulated cycles" pre-ovulatory endometrial thickness, is predictive of prospective embryo (pregnancy) potential following In Vitro Fertilization/Embryo Transfer (IVF/ET). It has been shown that, optimum implantation potential requires the endometrium should be of sufficient thickness. Thus, increased endometrial thickness is associated with improved treatment outcome for patients undergoing ART treatment, particularly in vitro fertilization-embryo transfer.
For improving the outcome of assisted reproduction treatment (ART) it is highly desirable to provide regimen which can help improve endometrium thickness particularly in conjunction with ovarian stimulation and that such regimen is user friendly and cost effective.
Summary of the invention
It is an object of the invention to provide a composition which can help improve
endometrium thickness during ovarian stimulation.
It is another object of the invention that such composition provides user friendly and cost effective regimen.
It is further object of the invention to provide a composition which can help improve endometrium thickness during ovarian stimulation for improving the

outcome of assisted reproduction treatment.
It is still further object of the invention to provide a composition which can help improve endometrium thickness during ovarian stimulation in patients following a natural ovulatory cycle or in patients undergoing ovulation induction.
In a first aspect, the invention provides composition for improving endometrium thickness optionally and preferably during ovarian stimulation, in which the composition comprises combination of Follicle Stimulating Hormone (FSH) and Human Chorionic Gonadotropin (HCG).
In a second aspect, the invention provides a pharmaceutical formulation for improving endometrium thickness during ovarian stimulation, in which the composition comprises combination of highly purified hCG 50 IU to 1000 IU and highly purified FSH 5 IU to 50 IU per dosage.
In a third aspect, the invention provides a kit for improving endometrium thickness during ovarian stimulation, comprising daily doses of highly purified hCG 50 IU to 1000 IU and highly purified FSH 5 IU to 50 IU per dosage.
Description of the Invention
The present invention is directed towards providing a composition which can help improve endometrium thickness during ovarian stimulation and that such composition provides user friendly and cost effective regimen for improving the

outcome of assisted reproduction treatment.
An association of various cycle characteristics and treatment outcome has been evaluated since the introduction of assisted reproduction technologies. One such parameter, which has been evaluated by several groups, is that of endometrial thickness (Check et al., 1991; 1993; Dickey et al., 1992; Noyes et al., 1995; Rinaldi et al., 1996; Yuval et al., 1999; De Geyter et al., 2000; Bassil, 2001; Schield et al., 2001). Adequate proliferative and secretory changes are necessary for successful implantation to occur.
Endometrial thickness is regarded as a reflection of the degree of endometrial proliferation in the absence of intrauterine pathology, and is measured in the midsagittal plane during transvaginal ultrasound scan.
Adequate endometrial development is required for pregnancy to occur, the endometrium has been found to be significantly thicker in cycles that resulted in pregnancy. Pregnancy rates were found to be higher when the endometrium reached at least 10 mm thickness.
Studies conducted world over suggest that adequate endometrial development is one of the factors that play a significant role in IVF outcome. Therefore, for clinicians providing IVF for infertile couples as much as follicle growth, to ensure sufficient endometrial development is achieved is a challenge.

The inventors have unexpectedly discovered that administration of the composition comprising combination of highly purified hCG and highly purified FSH at low doses during the ovarian stimulation can improve endometrium thickness and that it can have positive effect on outcome of assisted reproduction treatment. Conventionally offered treatments such as with that of clomiphene citrate either alone or in combination with hormones or other agent as well as treatment with estrogen supplement have not found to be satisfactory in providing the successful regimen for increasing the endometrial thickness during ovarian stimulation so as to improve the final outcome of assisted reproduction treatment. So far combination of Follicle Stimulating Hormone (FSH) and Human Chorionic Gonadotropin (HCG) has not been attempted for improving the endometrial thickness during ovarian stimulation.
The composition of the present invention gives rise to surprising advantages in terms of increasing the endometrial thickness during ovarian stimulation which can help to improve the final outcome of assisted reproduction treatment with the help of combination of Follicle Stimulating Hormone (FSH) and Human Chorionic Gonadotropin (HCG) during ovarian stimulation. Further, the low dose requirement of both the agents, particularly FSH also can help in lowering the cost and improving the safety of ART cycles through a reduction of the total dose of FSH administered during ovarian stimulation.
The present invention accordingly provides a compositions comprising combination of Follicle Stimulating Hormone (FSH) and Human Chorionic

Gonadotropin (HCG) for improving endometrium thickness during ovarian stimulation. The composition comprises the combination of FSH and HCG in optimum therapeutic concentrations which may provide synergistic activity thereby reducing the dose of both the agents. Thus, such composition provides user friendly and cost effective regimen for improving the outcome of assisted reproduction treatment.
In one embodiment, the present invention provides a composition for improving endometrium thickness during ovarian stimulation comprising combination of highly purified hCG 50 IU to 1000 IU and highly purified FSH 5 IU to 50 IU per dosage.
Highly purified FSH and hCG to be used in the composition of the present invention may be obtained from natural sources, e.g. isolated from urine, pituitary or placenta and further purified so as to have low endotoxin levels. They may also be obtained using recombinant DNA technology. Biologically-active analogues thereof include peptidic analogues, non-peptidic analogues and chimeras. It is preferred that human FSH and hCG are used in the present invention. The highly purified hCG to be used in the composition of present invention has reduced endotoxin level of less than 0.03 EU/IU, preferably less than 0.01EU/IU. Such highly purified hCG is contemplated to be without structural damage or loss of potency.
In one preferred embodiment, the present invention provides a composition for

In one specific embodiment, the invention also provides, pharmaceutical formulations comprising composition of the present invention for improving endometrium thickness during ovarian stimulation comprising combination of 5 IU to 50 IU of highly purified FSH and 50 IU to 1000 IU of highly purified hCG having reduced endotoxin level of less than 0.03 EU/IU, preferably less than 0.01EU/IU per dosage.
The pharmaceutical formulations of the present invention may be formulated in dosage form suitable for oral or any other form of administration.
The pharmaceutical formulations of the invention may be formulated for administration by any convenient route, often in association with a pharmaceutically and/or veterinarily acceptable carrier. It is preferred that the pharmaceutical formulations are formulated for parenteral administration.
It is preferred that the hCH and FSH to be administered subcutaneously in In vitro fertilisation (IVF) / Intracytoplasmic Sperm injection (ICSCI) procedures.
The pharmaceutical formulations for parenteral administration will usually be sterile.
Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents are also

within the scope of the invention. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. The formulations can be administered through a prefilled syringe, an auto-injector or a multidose auto-injector.
In one of the embodiment, the present invention provides a pharmaceutical formulation comprising composition of the present invention for improving endometrium thickness during ovarian stimulation comprising combination of 5 IU to 50 IU of highly purified FSH and 50 IU to 1000 IU of highly purified hCG, and pharmaceutically acceptable suitable excipients such as Lactose, Sucrose etc. which may aid in the stabilization of the lyophilized product. Such formulation is filled into glass ampoules.
In another embodiment, the present invention provides pharmaceutical formulation comprising the composition for improving endometrium thickness during ovarian stimulation comprising combination of 5 IU to 50 IU of highly purified FSH and 50 IU to 1000 IU of highly purified hCG, formulated as a unit dosage in the form of a solid ready for dissolution to form a sterile injectable solution for intramuscular or for subcutaneous use. The solid usually results from lyophilisation. Typical excipients and carriers include sucrose, lactose, sodium chloride, buffering agents like sodium phosphate monobasic and sodium phosphate dibasic. The solution may be prepared by diluting with water for

injection immediately prior to use.
Oral and other enteral pharmaceutical formulations need not be sterile and may be presented In unit- or multi-dose form. Oral pharmaceutical formulations may be in the form of solids, such as powders, granules, tablets, capsules (for example hard or soft gelatin capsules) or lozenges, or liquids, such as syrups or elixirs. Fillers and/or carriers may be present as appropriate, and those skilled in the art of pharmaceutical formulation will be able to provide such additional or alternative excipients as may be necessary or desirable; flavouring agents are one example. Any pharmaceutical formulation intended for oral administration may be formulated for enteric resistance, so as to assist delivery to the small intestine by avoiding or mitigating any digestion of the compound(s) as may occur in the stomach or the proximal part of the small intestine. Tablets or capsules may be enteric coated, for example by conventional procedures. Liquid pharmaceutical formulations may be effectively rendered enteric resistant by including or being co-administered with a suitable agent such as medium-chain triglycerides.
Enteral pharmaceutical formulations other than oral pharmaceutical formulations include rectal compositions, which may be in the form of a suppository. Suppositories will generally include a suppository base, such as cocoa butter. Again, particular formulations containing the active ingredient(s) may routinely be prepared by those skilled in the art of pharmaceutical formulation.
In one more embodiment, the present invention provides a kit for improving endometrium thickness during ovarian stimulation, comprising daily doses of

highly purified hCG 50 IU to 1000 IU and highly purified FSH 5 IU to 50 IU.
The daily dose of the composition comprising combination of 5 IU to 50 IU of highly purified FSH and 50 IU to 1000 IU of highly purified hCG or pharmaceutical formulation comprising the same may be administered once a day from day 2 to about day 11 or 12 of the stimulatory cycle.
Preferred features of each aspect of the invention are as for each other aspect, mutatis mutandis.
The composition of the present invention comprising of a stable combination of physiological gonadotropin and the analog of another physiological gonadotropin can be targeted to given to women in older age groups or those having poor ovarian reserves where large amount of gonadotropins are required. In conclusion, a newer treatment modality using controlled dosage forms of highly purified HCG and highly purified FSH can be used for female patients seeking infertility treatment, using much lower doses of HCG in place of LH due to its longer half-life and also FSH in unconventional doses rather than 325 - 475 IU. The proposed invention of hCG in the range of about 50 IU to about 500 IU and FSH in the range of about 10 IU to about 100 IU is a valuable regimen for use in patients, where OHSS and intolerance to higher doses of FSH are a problem in clinical settings in Assisted Reproductive Therapy (ART) procedures of Intrauterine Insemination (IUI), In vitro fertilisation (IVF), Intracytoplasmic Sperm injection (ICSCI), Zygote Intrafallopian Transfer (ZIFT), Gamete Intrafallopian Transfer (GIFT) and other procedures of future.

The composition of the present invention comprising combination of hCG and FSH in a unit formulation may act synergistically and reduce the total gonadotropin required to be administered. The composition may also reduce the total number of injections to be taken by the subject. Thus, the composition is advantageous with respect to providing ease of administration, reduced number of administrations and cost effective.
While the present invention is described as above in general and with the help of various embodiments, it is apparent that various modifications may be made to the above or embodiments can be altered to provide other embodiments which utilize the processes of this invention. Such embodiments are construed to be within the scope of the present invention.
The invention will now be described further in the following non-limiting examples.
Example 1
Adult mature female Wistar rats weighing 180 - 220 gms were chosen for the study with composition comprising a combination of highly purified HCG and FSH for improvement in endometrial thickness [ET] during ovarian stimulation and also improvement in follicular counts.

Inbred female 3-4 month old Wistar rats, weighing 180 - 220 gms grams were chosen for the study. Ovarian stimulation protocol as described in literature were used for the study to evaluate the efficacy of the composition comprising a combination of highly purified HCG and FSH.
Female rats, with regular estrus patterns were chosen after microscopic examination of vaginal smears. The phases studied were Di-estrus, Pro-estrus and Estrus. The animals were allowed to rest and acclimatize to the day-night cycles of the laboratory.
The Study
1. Six groups of 36 rats [n =32] were chosen for the study
2. Rats were fed normally with no dietary restrictions and any form of pre-treatment
3. Blood was collected from the tail vein for analysis of the hormones FSH,LH and Prolactin
4. The injections were administered 48 hrs prior the next meta-estrus cycle a nd continued till the estrous phase
5. The dosages of 100 IU highly purified HCG, 25 IU highly purified FSH were scaled down and adjusted as per the rats body-weight prior to administration
6. The Treatment Groups:

(a) Control Group [n=16]
(b) HP-HCG - IU + HP-FSH 75 IU [n=16]

The control group had no treatment at all. The injections were administered with 1 ml disposable syringes causing minimal trauma so as to not stress the animal. 7. Hormonal Estimations
Blood was collected from the tail vein analysis of FSH, LH were carried out at the beginning of the study and immediately after sacrifice using the cervical dislocation using ELISA
8. Post sacrifice, ovaries were removed surgically and were damped with tissue paper and weighed, The ovaries were then fntroc/uced into glass vials containing formaldehyde diluted with normal saline.
9. Endometrial thickness was measured using the modified method using a USG and the endometrial thickness [[μm]
10. The ovaries were examined for follicular counts.
Table 1: Endometrial Thickness Measurement:

Groups Endometrial Thickness [μ m]
Control Pre-treatment 640.39 ±13.80

Post-treatment 665.27 ±18.96
Treatment Groups Pre-treatment 637.59 ±19.80

Post-treatment 778.34 ± 22.75
The above data showed encouraging results suggesting that the dosage range of

the study could be of immense value to non responders using modified dosage regimens of highly purified HCG and FSH. Table 2:

Groups FSH [ng/mL] LH [ng/mL] Total
Follicular
Count Viable
Follicle Counts
Control Pre- 450.03 ± 45.9 295. 29 ± 48.75 43.001 8.30 19.231 2.50

Post- 496.23 ±
42.01 343. 32 ± 26.75

Treatment Groups Pre- 550.161 49.75 392. 65 ± 33.75 63.00 ± 5.30 26.231 2.50

Post- 700.42 ± 68.16 642. 54 1 42.75

We Claim:
1. A composition for improving endometrium thickness during ovarian stimulation comprising combination of highly purified hCG 50 IU to 1000 IU and highly purified FSH 5 IU to 50 IU per dosage.
2. A composition for improving endometrium thickness during ovarian stimulation comprising combination of 5 IU to 50 IU of highly purified FSH and 50 IU to 1000 IU of highly purified hCG having reduced endotoxin level of less than 0.03 EU/IU, preferably less than 0.01EU/IU per dosage.
3. The composition as claimed in claim 1 or claim 2, optionally comprising of pharmaceutically acceptable diluents, carrier or excipient.
4. The composition as claimed in claim 1 or claim 2, optionally comprising other active drugs selected from but not limiting to gonadotropin releasing hormone, gonadotropin releasing hormone agonists, gonadotropin releasing hormone antagonists, preparations with luetinizing hormone activity, progesterone preparations and aromatase inhibitors.
5. A pharmaceutical formulation comprising the composition of claim 1 or 2 and pharmaceutically acceptable diluents, carrier or excipient.
6. The pharmaceutical formulations as claimed in claim 5, wherein the formulation is formulated for once a day administration from day 2 to about day 11 or 12 of the stimulatory cycle.
7. The pharmaceutical formulations as claimed in claim 5, wherein the formulation is formulated as a unit dosage in the form of a solid ready for dissolution to form a sterile injectable solution for intramuscular or for

subcutaneous use.
8. The pharmaceutical formulations as claimed in claim 5, wherein the formulation is formulated as a solution for injection, comprising any of the excipients and buffers.
9. A kit for improving endometrium thickness during ovarian stimulation, comprising daily doses of highly purified hCG 50 IU to 1000 IU and highly purified FSH 5 IU to 50 IU per dosage.
10. The kit as claimed in claim 9 wherein, highly purified hCG has reduced endotoxin level of less than 0.03 EU/IU, preferably less than 0.01EU/IU per dosage.