Title of Invention

THE PREPARATION OF TWO HIGHLY ACTIVE NOVEL FORMS OF GONADOTROPIN RELEASING HORMONES (GNRH).

Abstract

This invention relates to a process for the preparation of novel forms of gonadotropin releasing hormones (gnRH) comprising the steps of : extracting the hypothalami of Indian teleostean fish; in a manner such as herein described; subjecting the extract to the step of fractionation ; separating the active fraction by gel filtration followed by the step of elution; and purifying the same to obtain gonadotropin releasing hormones.

Full Text

FIELD OF INVENTION This invention relates to the preparation of two highly active novel forms of gonadotropin releasing hormones (GnRH) This invention further relates to two novel highly active forms of GnRH. PRIOR ART It is well known that brain gonadotropin releasing hormone (GnRH) plays a vital role in the reproduction of fish as in other vertebrates including mammals. GnRH is a decapeptide and is secreted from the hypothalamus. This neuropeptide binds to pituitary gonadotropin cell receptor and causes gonadotropin (GtH) release from the pituitary gland, GtH in turn acts on the gonad (testis and ovary), induces maturation of germ cells ultimately leading to spermiation and ovulation via the synthesis and secretion of gonadal sex stroids. In these events of reproduction, GnRH is the primary regulator, therefore the control of reproduction in different animals by using GnRH has been the practice of the day. Peptide structures of two forms of fish GnRH have so far been discovered, salmon GnRH and dogfish GnRH. Both contain 10 amino acids, amino terminal has a pyroglutamate residue, while carboxy terminal has an amidated glycine, only at 5 position there is a variation. A structural elucidation of mammalian GnRH and other two brain peptides are known in the art. Considerable research has been effected with mammalian GnRH including the development of antibody and GnRH-receptor gene expression. Sherwood et. al. in 1983 first discovered the structure of salmon GnRH. They purified salmon GnRH by using mammalian GnRH antibody. Hence, purification of salmon GnRH was based on immunological detection. Salmon GnRH did show biological activity in other fishes. A number of chemical analogs of mammalian and salmon GnRH have been prepared aiming mainly to (1) stabilizing the molecule against enzymatic degradation (2) increasing its binding to circulating proteins (3) enhancing the affinity of the molecules for receptors (Millar et. al., 1997). Salmon GnRH analog has been commercialized and available as Ovaprim. This also contains a dopamine agonist ie. Pimozide. Ovaprim is the only product available in the market for the induced breeding of fish. GnRHs from mammalian or piscine sources have been isolated or purified by detecting the molecule with immunological cross-reaction. There always remains a problem when a molecule is isolated by using a heterologous antibody. The molecule may be immunologically well recognised but biological activity of the same will be questionable. Since it is easy to isolate GnRH by immunological detection, everyone followed this method to purify GnRH. The need to purify murrel hypothalamic GnRH by developing a highly sensitive and consistent assay of GnRH biological activity remained. OBJECTS OF THE INVENTION An object of this inventin is to propose the preparation of two highly active notvel forms of gonadotropin releasing hormones. Yet another object of this invention is to develop a sensitive and dependable assay of GnRH, to obtain biological GnRH. Further objects and advantages of the invention will be more appararent from the ensuing. At the outset of the description which follows, it is to be understood that the ensuing description illustrates a particular form of this invention. However, such a particular form is only an exemplary embodiment, without intending to imply any limitation on the scope of the invention. BRIEF DESCRIPTION OF INVENTION According to this invention there is provided a method for the preparation of two highly active novel forms of gonadotropin releasing hormones (GnRH) comprising the steps of extracting the hypothalami of Indian teleostean fish such as murrel or carp, subjecting the same to the step of acetone fractionation, identifying the active fraction as the Acetone II fraction by radioimmunoassary, followed by gel filtration of the Acetone II fraction, eluting the fraction through anionic Mono Q and cationic Mono S columns, leading to GnRH I, GnRH II, followed by purification of each form through reverse phase chromatography to obtain pure forms of GnRH I and GnRH II. In accordance with this invention a sensitive and dependable assay of GnRH has been developed. The murrel pituitary gonadotropin is first purified and the antibody against it is raised which permits the development of a dependable, quick and sensitive RIA of murrel GtH. This is followed by the development of a murrel pituitary primary static cell culture system. In this pituitary cell culture system, test material during the purification steps is added and the amount of gonadotropin released into the medium is determined by GtH RIA. Hence, this GnRH biological activity assay is extremely dependable and will allow the isolation of a molecule which definitely contains cellular release of GtH property. Murrel GnRH I&II were isolated by this procedure and therefore their biological activity in laboratory cell culture system or in the field for induced breeding is unquestionable. The establishment of this biological assay, has made the isolation of mGnRH I possible which is an anionic GnRH and no anionic GnRH has yet been isolated from other sources. Murrel GnRH I does not give any cross reaction with anti-salmon GnRH antibody or anti-mammalian GnRH antibody. In accordance with this invention there is further provided two distinct distinct bioactive forms of GnRH from murrel brain. GnRH so far isolated from different vertebrates" brain has net cationic charge. Therefore in attempts to isolate GnRH, a cation exchanger column has always been used. The applicants tried to separate gel filtered Sephadex G-25 Peak II (using AC FII fraction in this chromatography), which has high GnRH activity, through a Mono Q column for Fast Performance Liquid Chromatography Mono Q is an anion exchanger. Very surprisingly an adsorbed material which is also a decapeptide and exhibits strong GnRH activity has been obtained. This is an unusual GnRH as it is adsorbed in an anionic exchanger column. Later, when Mono S (cationic exchanger) was available, the lyophilized material of Mono Q elute (unbound washed material through Mono Q) has been used and the second GnRH ie. cationic GnRH as the adsorbed material has been found). Since the Mono Q anionic GnRH was obtained earlier, it has been named murrel GnRH I and Mono S cataionic GnRH as GnRH II. Hence, although the discovery of an anionic form of GnRH was made possible with the above mentioned background, it is a considerable contribution in process technology as to one would use this particular step of separation and obtain an unique from of GnRH. The assay according to the invention is undertaken as follows: Pituitary cells from the murrel or carp are enzymatically dispersed (0.3% collagenase and 0.05% trypsin), washed and added into the NUNC microwell module (each has capacity of 400 ul volume) for short culture. 4 Each well contains 6x10 cells. Viability of cells is examined by trypan blue dye exclusion method. Test material containing GnRH activity is added to the pituitary cells and release of gonadotropic hormone (GtH) into the medium is estimated by GtH radioimmuno assay by raising anti- GtH antibody against murrel pituitary GtH. Based on this assay, murrel and carp GnRH have been purified. Purification steps are briefly described below. Murrel or carp hypothalami are extracted with 1N acetic acid, filtered through cheese cloth and subjected to acetone fractionation to obtain three different pellets, ACI, ACII and ACIII and only ACII exhibits GnRH activity ie. it releases substantial amount of GtH into the medium while ACI or ACIII does not show such activity. ACII fraction is then subjected to Sephadex G-25 gel filtration. A concurrent run of synthetic mammalian GnRH is given to identify the zone of decapeptide elution. Gel filtration also gives three different peaks, SG I, SGII and SGIII. It is SGII peak which clearly corresponds to the elution peak of synthetic mammalian GnRH indicating the zone of decapeptide elution and it is also SGII fraction which demonstrates GnRH activity. Pooled SGII fractions are lyophilized to reduce the volume and then subjected to FPLC Mono Q (anion exchanger) column chromatography. Adsorbed material is eluted by a continuous gradient of NaC1 . There is one unadsorbed peak (MQ I) and three adsorbed protein peaks (MQ II, MQ III and MQ IV). MQ II remarkably increases GtH release as compared to control while MQ III or MQ IV has no such activity. Surprisingly, Mono Q unadsorbed protein peak MQ I also indicates GnRH activity. Hence, this unadsorbed protein fractions are pooled lyophilized and passed through FPLC Mono S (cation exchanger) where it again shows one unadsorbed (MS I) and three adsorbed (SS III and MS IV) protein peaks and among these only MS II shows a highly significant GnRH activity. Hence, two forms of GnRH have been obtained, an anionic form ie. GnRH I (Mono Q) and a cationic form ie. GnRH II (Mono S). This is indeed very surprising as two almost equally potent bioactive forms of GnRHs has not yet been reported from any class of vertebrates. Both GnRH I and GnRH II have been further purified by reverse phase column chromatography (FPLC Pep-RPC) murrel GnRHs have been found to be more active even in carp, murrel GnRHs (I and II) have been used in field studies. During the process of purification, certain innovative steps have been adopted which very surprisingly led to two distinct forms of GnRHs (GnRH I and GnRH II) from the brain of murrel. Both are highly active and individually more than doubly active as compared to salmon GnRH or its analogues examined so far in different Indian fish pituitary cell culture system. These GnRH have been found to be remarkably more active than carp GnRHs. In field experiments for the induced breeding of Indian carps, biopotency of murrel GnRH I or GnRH II astonishingly exceeds the potency of salmon GnRH. This is the first time when two highly biologically active GnRH molecules are available from the brain of the same fish. Till date, although more than one immunoreactive forms have been identified in fish brain but isolation of more than one biologically active forms has not been reported. By using fractionation steps of acetic acid extracted from the brain of Indian freshwater murrel, a major amount of the extraneous materials could be removed. This is such an effective step, that, if followed rigorously, acetone fractionated material of murrel GnRH (50 ug/Kg fish) provides 100% success in induced breeding of carps. Although this is a very simple and very inexpensive process to isolate active GnRH material, definitely highly effective procedure to obtain biologically active GnRH. The murrel GnRH I&II isolated by the process of our invention have been found to be three times more active than the commercially available Ovaprim. Another interesting feature is the summation effect of murrel GnRH I&II since they occupy two distinct receptors, specific for each type of GnRH in the pituitary cell membrane. By adding calcium and pimozide with murrel GnRH I and plus II, field trials for induced pimozide with murrel GnRH I plus II, field trials for induced breeding of different carps was undertaken. Ovaprim was also used concurrently to compare the effect. Our material (combined GnRH I plus II) was far more active as compaed to Ovaprim both in terms of dose and also in terms of reductin of spawning time. Ovaprim is an imported material and is the only product in the global market for induced breeding of fish. Since murrel GnRH I&II are much more active than Ovaprim it would be a highly competitive product in the international market. WE CLAIM 1. A process for the isolation and purifification of novel forms of gonadotropin releasing hormones (BnRH) comprising the steps of: extracting the hypothalami of Indian teleostean fish in a manner such as herein described: subjecting the extract to the step of fractionation: separating the active fraction by gel filtration followed by the step of elution and purifying the same to obtain gonadotropin releasing hormones. 2. A process as claimed in claim 1 wherein said gonadotropin releasing hormones (BnRH) are Mono Q anionic GnRH (BnRH I) and Mono 8 cationic BnRH (BnRH II). 3. A process as claimed in claim 1 wherein the hypothalami of Indian teleostean fish, such as murrel or carp are extracted. 4. A process as claimed in claim 1 wherein for the step of fractionation,acetone is used. 3. A process as claimed in claim 1 wherein the active fraction is identified as the Acetone II fraction. 6. A process as claimed in claim 1 wherein the active fraction is identified by an assay comprising the steps of enzymatically dispersing pituitary cells from the fish brain followed by washing and adding into the NUNC microwell module for short cul- ture, examining the viability of the test cells, followed by adding test material containing BnRH into the medium, estimating the release of BtH released into the medium by BtH radioimmuno- assay. 7. A process as claimed in claim 6 wherein viability of the cells is examined by tryptan blue dye exclusion method. 8. A process as claimed in claim 6 wherein in the step of BtH radioimmunoassay, anti-BtH antibody is raised against murrel pituitary BtH. 9. A process as claimed in claim 1 wherein for the step of gel filtration, a Sephadex B25 column is used. 10. A process as claimed in claim 1 wherein the active fraction is eluted through FPLC Mono Q anionic and FPLC Mono 8 cationic columns. 11. A process as claimed in claim 1 wherein the step of elution leads to BnRH I and GnRH II from the anionic MONo Q and cationic Mono S columns respectively. 12. A process as claimed in claim 1 wherein GnRH 1 and BnRH II are purified by reverse phase chromatography using a FPLC pep— RRC column. 13. A process for the isolation and purification of novel forms of gonadotropin releasing hormones (BnRH) substantially as herein described and illustrated. This invention relates to a process for the preparation of novel forms of gonadotropin releasing hormones (GnRH) comprising the steps of: extracting the hypothalami of Indian teleostean fish; in a manner such as herein described; subjecting the extract to the step of fractionation; separating the active fraction by gel filtration followed by the step of elution; and purifying the same to obtain gonadotropin releasing hormones.

Documents:

438-cal-1998-granted-abstract.pdf

438-cal-1998-granted-claims.pdf

438-cal-1998-granted-correspondence.pdf

438-cal-1998-granted-description (complete).pdf

438-cal-1998-granted-form 1.pdf

438-cal-1998-granted-form 3.pdf

438-cal-1998-granted-form 5.pdf

438-cal-1998-granted-letter patent.pdf

438-cal-1998-granted-pa.pdf

438-cal-1998-granted-reply to examination report.pdf

438-cal-1998-granted-specification.pdf