Title of Invention

A PROCESS FOR PRODUCING PROTEINS HAVING PRONOUNCED CONTRACEPTIVE ACTIVITY

Abstract

The present invention relates to a process for preparing proteins having pronounced contraceptive activity which comprises in combination the following steps : (a) homogenization of mammalian brain in ice-cold buffer of pH varying between 7 and 8; (b) centrifugation of the tissue homogenate for 10-30 minutes; (c) high-speed centrifugation of the supernatant fluid from step (b) for around 40-60 minutes; (d) collection and storage of the supernatant fluid from step ( c ) called "cytosol" at low temperature; (e) chromatographic fractionation of "cytosol" using "sephadex-G-100" gel filtration pre-equilibrated with normal saline, discarding fractions collected in void volume; (f) rejection of first peak containing high molecular mass protein(s) and collection of second peak containing low molecular mass proteins and optionally followed by (g) conversion of the desired proteins into orally administrable dosage form in known manner. The proteins have no adverse side effects end after withdrawal of the compounds, the treated subjects regain normal cycle within 3-4 weeks time.

Full Text

The present invention relates to a process for preparing proteins saving pronounced contraceptive activity. More particularly this invention pertains to a process for preparing a protein fraction containing two major proteins of molecular masses 12 and 13 kDa, which for the first time has been isolated from cytosolic fraction of mammalian brain. These protein, including some minor proteins of comparable masses, exhibit remarkable contraceptive activity with virtually no adverse physiological side effects. Over the years, hormone-based contraceptives, commonly known as 'the pill', have been in use. These 'pills' usually contain steroids like estrogen and/or progesterone which on prolonged use have been shown to have various deleterious side effects, including an enhanced risk of cancer, blood clots in the legs, lungs, heart or brain and irregularity in the ovulation cycle even after the 'pill' is withdrawn with resulting difficulty in "conceive" even when desired. In recent years, importance of proteins of low molecular mass has been realised by many workers in this field. Recently in 2001, a protein called "CatSper" has been isolated which resembles a single, six-transmembrane-spanning repeat of the voltage-dependent Ca2+-channel four repeat structure. This protein is stated to belong to a principal part of the sperm-tail regulating the Ca2+-transport from environment to cell keeping sperm motile which plays an important role in fertilisation. The idea of providing a protein with relatively low molecular mass with desired physiological effect stemmed from isolation of an ATPase-modulator protein from rat brain cytosol in our Institute. One of these proteins was observed to inhibit Na+, K+-ATPase activity and another modulator protein was found to be effective for use as a tool for distinguishing the properties of Ca2+, Mg2+ and Ca2+-ATPases. Calcium is known to play an important role in sperm motility as well as in capacitation (fertilization), it is well known that Ca 2+-ATPase it, present both in male and female reproductive organs, hence it was surmised that the protein which affects and/or influence Ca -ATPase activities, could somehow have a role to play in regulating or controlling fertility. It was surprisingry found that modulators isolated from "Sephadex G-100" column fraction when administered orally to fertile female rats, acted as a potential contraceptive agent with virtually no adverse side effects. The principal object of the present invention is to provide a process for preparing protein having pronounced contraceptive activity. Another object of this invention is to provide a process for preparing proteins of low molecular masses of around 12 and 13 kDa. A further object of this invention is to provide a process for preparing low molecular mass proteins having contraceptive property but without any adverse physiological side effects. -2- A still farther object of this invention is to provide a process for the preparation of a contraceptive formulation in oral dosage form in admixture with physiologically acceptable excipient(s) or adjuvants. The above objectives are achieved by the present invention which provides a process for preparing proteins of molecular masses between 12 and 13 R Dasych as herein described having pronounced contraceptive activity which comprises in combination me following steps : ( a) homogenization of mammalian brain obtained from rodent, goat, sheep, pig and bovine sources, in ice-cold buffer of pH varying between 7 and 8, of composition such as herein described (b) centrifugation al 12,000 -15,000 g for 10-30 mins of the tissue homogenate; ( c) high-speed centrifugation of me supernatant fluid obtained from step (b) at not less than 100,000 g for around 43-60 mins; ( d ) collection and storage of supernatant from step ( c ) called "cytosol" and stored at -20°C for further processing; (e) chromatographic fractionation of "cytosol" using "Sephadex-G-100" gel filtration pre- equilibrated with normal saline, discarding fractions collected in void volume; ( f) rejection of first peak containing high molecular mass protein(s) from step (e ) and collection of second peak containing desiral fow molecular mass proteins and optionally followed by (g} conversion of the desired proteins into orally administrable dosage form in known manner. The buffer referred to above comprised of 25-50 mM Tris-HCl, 0.25 M Sucrose, 1 mM EDTA, 1 mM ECTA, 1 mM PMSF and 1 mM 2-mercaptoethanol / thioglycerol. The following features were observed as a result of biochemical characterization of second peak from "Sephadex-G-100" column : (i ) found to inhibit Na+ K+-ATPase; (ii) found to modulate Ca2+-ATPases, i.e. it inhibited Ca2+-ATPase and stimulated Mg2+- dependent Ca2+-ATPase; (iii ) found to inhibit protein kinase C and A Biochemical characterization of major proteins isolated from the second peak when separated through anion exchange column ("FPLC-Mono Q") surprisingly revealed that one of them (13 kDa) specifically inhibited Na+ K+-ATPases, and the other (12 kDa) was found to be capable of modulating Ca2+-ATPases, in other words, it inhibited Ca2+ -ATPase and stimulated Mg2+-dependent Ca2+-ATPase. These observations justify the aforesaid proteins to be termed as "modulators of Ion transporting-ATPases". From the foregoing, it will be reasonable to presume that either or both these proteins or conjugate / multiple cascades including some minor proteins are involved in contraceptive activity and / or relevant function(s) in females by prolonging estrus and / or metestrus phase. The proteins are acidic (negatively charged) in nature with isoelectric point around 6.0. The proteins could retain their biochemical activity at 60°C for at least 30 mins and was found to be -3- stable for around one month in normal saline solution at 10-12°C. Repeated freeze-thaw can damage the contraceptive action of the proteins. No tryptophan residue was found to be present in the proteins isolated by us. Since the low molecular mass proteins surprisingly restrict sperm motility in vitro, it may be useful for an LU.D. A further unique possibility is that these proteins can also be administered to male partners, without restricting to females alone. The daily dosage of the drug is surprisingly small, often in picogram quantities and administrate in aqueous suspension form which is not only easy to apply but is also highly cost-effective and within common persons ' reach. It has been observed that at mis concentration, the estrus cycle lasts for around 1.5-2 days which has been found to be optimum in preventing ovulation. Lower concentration was found to be ineffective while higher concentration or a prolonged treatment (> 25 cycles) leads to irreversible effect. In this specification certain abbreviations have been used for the sake of convenience, such as for instance, - EGTA - Ethylene glycol tetraacetic acid, EDTA - Ethylene diamine tetraaoetic acid, PMSF - Phenyl methyl sulphonyl fluoride, SOS - Sodium dodecyl sulphate., PAGE - Polyacrylamide gel eleetrophoresis, and so on. SGOT - Serum Glutamate Oxaioacetate Transaminase SGPT - Serum Glutamate Pyruvate Transaminase Isolation of protein fraction showing pronounced contraceptive activity has been carried out by following a sequence of steps narrated hereinbefore. Some of the important parameters of the individual steps are given below : (h) Homogenization of mammalian brain obtained from rodent, goat, sheep, pig and bovine sources, is effected from freshly sacrificed animals or frozen at 60°C, and is carried out in ice cold buffer wherein the ratio of brain and buffer is optimally maintained at 1 ; 2-10 wt: vol. Ratio. (i ) The fraction from the second peak obtained by chromatographic fractionation on being subjected to a continuous gradient gel (10-20% SDS PAGE) separates into the desired proteins of low molecular masses 12 and 13 kDa showing pronounced contraceptive activity, and ( j ) Biochemical characterization of second peak from Sephadex G-100 column was also found to inhibit protein kinase C and protein kinase A. The contraceptive proteins are studied closely and at great length and the observations on effects thereof were noted in tabular forms. -4- Experimental Data - TABLE 1; Protein concentration study to monitor estrus phage Serial No. No. of virgin female taken for the experiment Av. Body weight of rat (gms) Protein / rat / day (ppn) Duration of treatment (cycle) Duration of citrus phage per cycle day) I. 5 150 110 10-15 1 2. 5 150 220 10-15 1 3. 5 150 330 10-15 1 4. 5 150 440 10-15 1.5 5. 5 150 660 10-15 2.0 6. 5 150 880 10-15 3.0 7. 5 150 1320 10-15 3.5 It is well known that fertility may be suppressed or inhibited in the females' by preventing ovulation due to prolong estrus i.e. protein in question increased oestrogen level. Normally, complete cycle of a rat continues for 4-5 days of which estrus stage is only for 12-24 hrs. We, therefore, have used an effective dose 4.4-6.6 pgm/gm/day where estrus continues for 1.5-2 days. Metestrous normally continues for 21 hrs, but treated rat shows 36 - 48 hrs ( 1.5 - 2 days ) of metestrous period whereas diestrous period found to be short lived. TABLE 2: Effect of protein to monitor pregnancy No. of animals Protein concentration / gm body wti day (pgm) Duration of treatment (cycle) No. of animal conceived 10 3.3 2-10 10 12 4.4 25 0 10 5.8 18 0 8 6.6 15 0 -5 - Animal Experiment - Isolation of modulator proteins: Mammalian brain cytosol was loaded on a saline equilibrated sephadex-G-100 column. Use modulator proteins were eluted as second peak and found to be low molecular mass-protein on sodium dodecyl polyacrylamide gel electrophoresis. The second peak was collected and the O.D. value was adjusted to 0.2 at 280 nm by diluting with saline solution whenever required From this solution, 1-1.5µl (2.2-3.3 ng protein) was diluted to 500µl with saline. An aliquot of lµl (4.4 - 6.6 pgm) /gm body wt of rat/day was administered orally. Choice of animals : To check normal cycle of female rats, initially several matured males and females were purchased from local supplier and put on regular laboratory diet for about 15 days before mating experiments were started Rats were mated and litters produced in our laboratory animal house . They were grown up under balanced diet When they became matured virgin, the vagina] smear was tested regularly under microscope. Vaginal fluid was drawn with 50µl of normal saline through a micro pipette and kept on an oil free glass slide which was tested under microscope and stained with haematoxyline and eosine, if necessary. Mating experiments to check the fertility of virgin male and female: A group of normal female rats were subjected for mating with proper weight containing matured males. Males were marked properly and every week they were interchanged between different cages. Male, female ratio was 1:3 per cage. Sperm positive females were identified by testing vaginal fluid under microscope. Design of experiments to check the reversible contraceptive effect of the proteins: When it was sure about the normal cycle of female rats, examining from their regular proestrus, estnu, metestrus and diestrus phases respectively, they were chosen for experiments. A group of virgin was subjected to protein treatment without mating (kept in absence of male). Everyday vaginal fluid was tested under microscope to examine different stages of the cycle. They were treated with the protein, upto 10-15 cycles followed by mating with different matured males without protein (refitment Females were found to conceive on discontinuation of the protein (thereby establishing that the effect is reversible). Mating experiments after protein treatment: A group of fertile males and females were subjected for mating experiment in a 1:3 ratio under protein treatment Regularly their vaginal fluid were checked to examine the different phase of cycle and sperm positivity, if any. In the same way mating experiments were continued upto 2-25 cycles with 40 fertile female rats. A group was taken with BSA as a control. Side effects - No side effect, on the other hand it prevents white discharge, reduce the irregular bleeding during menopause, reduce skin-dryness and loss of appetite and can be used as womanizing agent for virgin al so in addition to the primary effect in acting as a contraceptive among females. -6- Dose - age relation - Four to six monts old animals (body weight 140 - 160 gm.) were used for the study with a concentration range of the protein 4.4 to 6.6 pgm / gm body weight / day. It has been observed that at this concentration the estrous cycle last for about 1.5 - 2 days which was found to be optimum in preventing ovulation. Lower concentration was found to be ineffective while higher concentration or a protracted treatment (>25 cycles) lead to irreversible effect Table 3 : Toxicity data System SGOT (IT / ml serum) SGPT (U / ml serum) Alkaline Phoiphataie (U/mlserum) Sugar (PP) * (mg/dl) Control Treated Control Treated Control Treated Control Treated Expt. 1 Expt. 2 88 90 98 96 35 36 40 40 465 466 473 477 94 98 96 92 * from blood at 30 °C (12 : 12 hr light: dark) fed with balanced diet SGOT, SGPT and alkaline phosphatase data indicate liver function is normal during treatment further animal experiments were conducted to determine two different aspects of the use of proteins prepared by the process of the present invention, namely, (a) effect of application of subject products on premature rats, and (b) effect of withdrawal of protein on animals. A further study ( c ) was carried out to find out effects on variation in the mode of administration of proteins, e.g. from oral to subcutaneous, i.m. or i.v. routes. The results the findings are given below: a. Effect of application of proteins on premature rats : Premature female rats (before start of estrus cycle) treated with an optimum dose of the protein(s) of the present invention for 3-4 weeks through oral route, showed (i) no change in body weight, (ii) no loss of appetite, (in) no other visible change like loss of hair and (iv) irritation vis-a-vis the control. In this experiment one set of rats serving as control was treated with normal saline. After withdrawal of protein(s), the treated rats were kept isolated for a couple of weeks to allow them to attain maturity and consequent appearance of estrus. When allowed to mate with mature fertile males, the female rats conceived and gave birth to normal siblings. -7- b. Effect of withdrawal of proteins (i) Conception - After withdrawal of the proteins preceded by administration of normal dose, the female animals conceived immediately on / after the next cycle. If the protein dose was higher than optimum, occasionally the animals were found to conceive after 3-4 cycles commencing from withdrawal. (ii) Sibling condition - Siblings were normal and healthy. (iii) Weight of siblings - Weights were within normal ranges. All studies were made under appropriate control. ( c ) Effect of administration of proteins by Injection mode instead of oral route : Two sets of female rats (3 each) were selected for this experiment One set was subjected to protein treatment by intra muscular (i.m.) injection. Another set (control) was given normal saline via i.m. route. In the course of treatment they were mated with fertile male rats routinely at their estrus phase. None of the protein-treated rats were found to conceive through they were sperm positive in every cycle of their estrus phase. The protein-treated rats were found to have glossy coat of hair and looked cheerful during the course of treatment On the other hand, control group of animals conceived and gave birth to normal siblings. The histopathology study of protein treated ( both for short and long term ) ovary shows immature / atretic graffian follicles which have not undergone ovulation i.e. the protein I in question prevents Ovulation and there is no adverse effects on other organs. After withdrawal of proteins, the first set of animals was found to attain normal cycle within 3-4 weeks' time. The study established that in'Injection mode, effects of protein persist for a little while longer than oral administration. The histopathology study of protein treated ( both for short and long term ) ovary shows Immature / atretic graffian follicles which have not undergone ovulation i.e. the protein is question depresses Oogenesis and prevents Ovulation and there is no adverse effects on other organs. To sum up, it may be Inferred from the Examples and experiments aforesaid, the proteins prepared by the process of this invention are, inter alia, :- (i) anti-ovulatory, (ii) non-spermicidal, (iii) non-antizygotic and (iv) non-abortive. -8- Advantages : i. Being different from conventional contraceptive drugs which are seroidal in nature, the side effects are virtually absent Also the proteins have been found to rectify white discharge during application. ii. During treatment some secondary effects beneficial for users appeared as emergent characteristics : due to prolonged estrus and / or metestrus phase, more epithelial cells secrete from vaginal fluid resulting in improved sexual receptivity and mating tendency. iii. Higher number of epithelial cells either block the sperm motility, or sperms get aggregated over island of epithelium celts, as was evident from microscopic studies on sperm positive females. iv. The effective dosage is in picogram quantities, thereby rendering this new drug easy to administer and highly cost-effective. v. The proteins of the present invention have no toxic or other deleterious side effects on living bodies. vi. When administered within specified time limit after onset of pregnancy, the compound is capable of bringing about termiaation if applied in high doses. While the invention has been described in detail and with reference to the specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without deviating or departing from the scope of the invention. Thus the disclosure contained herein includes within its ambit the obvious equivalents and substitutes as well. Having described the invention in detail with reference to the examples and experimental data, it will now be defined by means of claim appended hereinafter. -9- We claim: 1. A process for preparing proteins of moleanlar masses between 12 and 13 k Da such as herein described having pronounced contraceptive activity which comprises in combination the folIowing steps : (a) homogonization of mammalian brain obtained from rodent, goat, sheep, pig and bovine sources, in ice coldbuffer of pH varying between 7 and (b) centrifugation of the tissue homogenate for 10-30 minutes: (c) high-speed centrifugation of the supernatant fluid from step (b) for around 40-60 minutes: (d) collection and storage ol the supernatant fluid from step ( c ) called "cylosoI" at low temperature: (e) chromatographic fractionation of' 'cytosol" using "sephadex-G-100" gel filtration pre-equilibrated with normal saline, discarding fractions collected in void volume: (f) rejection of first peak containing high molecular mass protein(s) and collection of second peak containing desired low molecular mass proteins and optionally followed by (g) Conversion of the desired proteins into orally administrable dosage form in known manner. 2. A process as claimed in Claim I, wherein centrifugation of tissue homogenate in step (b) is carried out at 12,000-15000 g. 3. A process claimed in Claim 1, wherein high-speed centrifugation in step (c) is carried out at not less than 100,000 g. 4. A process as claimed in Claims 1-3. wherein storage of "cytosol" from step ( c ) is done at a low temperature of around -20°C. 5. A process as claimed in Claim 1, wherein the buffer medium used lor homogenization of mammalian brain has the following composition :- 25-50 mM Tris-HCI, 0.25 M Sucrose. 1 mM EDTA. 1 mM EGTA, 1 mM PMSF and 1 mF mercaptoethanol or thioglycerol. 6. A process as claimed in Claims 1 and 5. wherein homogenization of mammalian brain is effective from freshly sacrificed animals or frozen at around -60°C. -10- 7. A process as claimed in Claims 1, 5 and 6, wherein homogenization is carried out in ice-cold buffer and the ratio of brain and buffer is optimally maintained at 1:2 - 10 wt:vol. ratio. 8. A process as claimed in Claim I, wherein the fraction from the second peak obtained by chromatographic fractionation as in steps (e) and (f) is subjected to a continuous gradient gel (10-20% SOS PAGE) whereby it separates into the desired proteins of masses 12 and 13 kOa showing pronounced contraceptive activity. forming a stable suspension in water or normal saline at 10°-12°C. 9. A process as claimed in Claims 1 and 8, wherein the proteins isolated from the said second peak of Sephadex G-100 column inhibit protein kinase C and protein kinasc A, are acidic in nature, having an isoelectric point of around 6. 10. A process preparing proteins of molecular masses between 12 and 13 k Da having pronounced contraceptive activity, substantially as hereinbefore described with particular reference to the examples and experiments given hereinbefore. -11- The present invention relates to a process for preparing proteins having pronounced contraceptive activity which comprises in combination the following steps : (a) homogenization of mammalian brain in ice-cold buffer of pH varying between 7 and 8; (b) centrifugation of the tissue homogenate for 10-30 minutes; (c) high-speed centrifugation of the supernatant fluid from step (b) for around 40-60 minutes; (d) collection and storage of the supernatant fluid from step ( c ) called "cytosol" at low temperature; (e) chromatographic fractionation of "cytosol" using "sephadex-G-100" gel filtration pre-equilibrated with normal saline, discarding fractions collected in void volume; (f) rejection of first peak containing high molecular mass protein(s) and collection of second peak containing low molecular mass proteins and optionally followed by (g) conversion of the desired proteins into orally administrable dosage form in known manner. The proteins have no adverse side effects end after withdrawal of the compounds, the treated subjects regain normal cycle within 3-4 weeks time.

Documents:

00501-cal-2002-abstract.pdf

00501-cal-2002-claims.pdf

00501-cal-2002-correspondence.pdf

00501-cal-2002-description(complete).pdf

00501-cal-2002-description(provisional).pdf

00501-cal-2002-form-1.pdf

00501-cal-2002-form-18.pdf

00501-cal-2002-form-2-1.1.pdf

00501-cal-2002-form-2.pdf

00501-cal-2002-form-3.pdf

00501-cal-2002-form-5.pdf

00501-cal-2002-p.a.pdf

501-CAL-2002-CORRESPONDENCE.pdf

501-CAL-2002-FORM 27.pdf

501-cal-2002-granted-abstract.pdf

501-cal-2002-granted-claims.pdf

501-cal-2002-granted-correspondence.pdf

501-cal-2002-granted-description (complete).pdf

501-cal-2002-granted-examination report.pdf

501-cal-2002-granted-form 1.pdf

501-cal-2002-granted-form 18.pdf

501-cal-2002-granted-form 2.pdf

501-cal-2002-granted-form 3.pdf

501-cal-2002-granted-form 5.pdf

501-cal-2002-granted-letter patent.pdf

501-cal-2002-granted-pa.pdf

501-cal-2002-granted-reply to examination report.pdf

501-cal-2002-granted-specification.pdf