4. DETAILED DESCRIPTION

FIELD QF INVENTION

The present invention relates to improving the reproduction efficiency in fish with a process by injecting a liquid peptide preparation that contains an analog of gonadotropin releasing hormone {GnRH)~ Buserelin and a brain neurotransmitter (dopamine) inhibitor- Pomperidone. The process induces spawning in fish to increase harvest in fish culture.

BACKGROUND

One of the most significant advancements in the field of aquaculture during recent times is the development of techniques to induce reproduction in fish. These techniques have allowed farmers to profitably breed and raise species that do not naturally reproduce in captivity, and to manipulate the timing of reproduction to suit production cycles.

Some species will not readily breed in captivity due to environmental or culture conditions that are different. from those found in nature, such as water temperature or substrate type. These conditions may cause stress or may not provide the cues needed to complete the reproductive process. Fish in captivity may not always reproduce at the most advantageous time, and alteration of the spawning cycle may be desirable. There are thousands of species of fish, with different requirements and with different survival conditions. This allows a farmer to:

1. obtain fish outside of the normal spawning season either to lengthen time for grow-out or to produce hybrids with other species;

2. improve efficiency by getting fish to spawn on a predetermined date; and

3. maximize survival by fertilizing and incubating eggs under hatchery conditions where successful techniques for altering the spawning cycle of fish have become a valuable tool.

The details of inducing spawning differ from species to species. They also differ according to the goals and means of the farmer

It must be emphasized that the common techniques do not apply to al) situations. Farmers should always thoroughly research the techniques that have been developed for their species of fish and select those that best fit the circumstances.

Many species offish will not readily reproduce under certain culture conditions. Others will, but not necessarily when the farmer desires. In these cases, induction of spawning can be of great value. Methods vary from species to species and situation to situation. However, at least two generalizations can be drawn. First, brooders are very vulnerable to rough handling. Care should always be used to avoid damaging these valuable animals. Second, a fish that does not have mature gametes will not produce viable eggs or sperm no matter how many times it is injected with hormones. Ripeness is the result of environmental factors working over a period of time, leading to maturation of the gonads and production of viable eggs.

Many procedures have been developed for inducing fish to undergo the last steps of spawning. Farmers should thoroughly research the procedures that have been developed for their species of fish through experimentation, and select those that best suit the circumstances. In addition, once the fish have spawned, there are many techniques involved in incubating and caring for the eggs, and caring for the hatched fry. These too must be thoroughly researched.

Fish must be handled with extreme gentleness. They are physically very vulnerable during spawning and may die if dropped or roughly handled.

When the brooders are kept in ponds or pens, all water quality parameters, such as temperature and oxygen, should be kept at optimal levels. A satisfactory amount of high quality, well balanced feed should be provided. If the fish are moved from one set of conditions to another, time must be allowed for acclimation. When handling the fish, gentle firmness should be the rule. Covering the fish with wet burlap or cloth, or covering their container to cut off light, will help to keep them calm. Tranquilizers may be used when fish are to be injected cr stripped.

There are two main strategies used to induce reproduction. The first is to provide an environment similar to that in which spawning occurs naturally. Catfish, for example, like to spawn in enclosed spaces such as hollow logs. A farmer can simulate this by putting milk cans in a pond. The presence of vegetation and an increase in temperature will usually work for goldfish. Changing the photoperiod in a hatchery can accelerate or delay maturation and ovulation in many salmon and trout species.

'The second strategy is to inject the fish with one or more naturally occurring reproductive hormones or their synthetic analogues. This is only effective in fish that are already in breeding condition and have mature eggs in which the germinal vesicle has migrated. Often the two strategies are used sequentially: the first to manipulate maturation, then the second to induce ovulation.

Numerous hormones have been used to induce reproduction. Two methods have emerged over the past few years that seem to offer the best chance for success at the least expense. They are injection of a GnRH analogue with dopamine antagonist, and injection of gonadotropin.

GnRH Analog with Dopamine Antagonist - Luteinizing Hormone Releasing Hormone (LHRH) is the name of a mammalian hormone that has been employed successfully to induce the reproductive hormonal cascade. In recent years, synthetic analogues of LHRH, referred to as LHRHa, have been developed that are far moire effective. Because they are purer and are not rapidly metabolized by fish/they remain active for longer periods.

Under natural conditions, there is a feedback mechanism in the fish that limits the release of gonadotropin. This mechanism uses a chemical called dopamine, which inhibits the action of LHRH. When dopamine is present in the fish, even LHRHa will have only limited success. A dopamine antagonist is often used to limit the effects of dopamine.

Gonadotropin - Two types of gonadotropin extracts have been used to induce ovulation in fish:

Human Chorionic Gonadotropin {HCG) and fish pituitary extract Pituitary extracts are made by removing the pituitary from a fish and extracting the hormones, which may then be injected into another fish. Carp, catfish, salmon, and other fish have been used for this. HCG offers three major advantages over the pituitary extract: 1) it is much less expensive, 2) it is more stable and thus has a longer shelf life, and 3) it comes in a purified form.

Two techniques are commonly used, sometimes in conjunction with one another.

• The first is manipulation of the culture environment to mimic some important quality in the fish's natural environment.

• The second is injection of hormones to stimulate spawning. The hormonas may be

natural hormones taken from fish or other animals, genetically engineered from bacteria, or synthetic analogues of naturally-occurring hormones.

STATEMENT OF INVENTION

The present invention is a breakthrough with the use of a combination of active ingredients of Buseretin and Domperidone into a special formula to get maximum benefits for enhanced reproduction in all species offish.

OBJECTS OF INVENTION

The present disclosure addresses the gaps in the existing procedures of improving the reproduction efficiency in fish and accordingly few objects of the present disclosure are listed hereunder -

• The main object of the present disclosure is to provide a reliable method of induced spawning and an aqueous solution so easy to administer with an injection comprising of active ingredients Buserelin IP and Domperidone BP to improve the reproduction efficiency in fish and to increase harvest in fish culture. It is also another object that the method stimulates the milt production for a longer period and for greater volume; moves fish forward In the spawning season; and spawns a population in a shorter period of time.

It is also another object to maximize reproductive potential in all species within a population.

It is also another object that the process induces maturation earlier than normal cycle {Species dependent) after administering the injection for fast results.

It is also another object that the process coordinates maturation during the normal spawning season. ,,.

It is still a further object that the process synchronizes and coordinates maturation in treated fish by significantly advancing maturation without affecting viability or fecundity.

It is stilt a further object that the process utilizes fish own endocrine system to safely induce maturation and coordinate spawning dates.

It is still a further object that the process induces maturation in advance of the spawning season even in difficult & complicated species of fish.

It is still a further object that the process also helps overcome problems of arrested maturation.

It is still a further object that the process comprises of potent-ovulating and sperm- forming agent optimizing reproduction in all species of fish.

It is still a further object that the process optimizes reproduction in many species of fish ensuring high predictability of ovulation with high egg count, fertility and viability in female fish.

It is still a further object that the process, wherein the carrier serves to increase milt production for long time in male fish.

It is still a further object that the process wherein the carrier induces advance maturation in all fish in a natural way without affecting gamete viability

It is still a further object that the process, wherein the carrier compresses and advances the spawning season and coordinates spawning dates It is still a further object that the process wherein the carrier comprises maximizing reproductive potential in all spawners within a population.

It is still a further object that the process spawns a population in a shorter period than normal.

It is still a further object that the process moves offspring through various stages as a tightly packed group according to spawning dates.

It is still a further object that the process conserves genetic material in a particular population, among populations and also in endangered species.

GENERAL TECHNICAL TERMINOLOGY

BASIC TERMINOLOGY

1. Basic Steps in Reproduction Process in Fish

Maturation- is the growth and development of the gametes (eggs in female fish and sperm in male fish] to a point where fertilization can occur

Ovulation- is the process of release of eggs from the ovary

Spawning- is the process of deposition of eggs and sperm so that they can unite.

2. Hormones

Hormones play a very critical role in the reproductive process in Fish. They are chemical messengers released into the blood by specific tissues, such as the hypothalamus, pituitary gland etc The hormones travel through the bloodstream to other tissues, which respond in a variety of ways.

3. Hormonal Tissues in Fish

Below are the hormonal tissues involved in the hormonal process in fish:

Hypothalamus

A part of the brain that controls many internal body functions and the activity of the pituitary gland which produces gonadotropin releasing hormones.

Pituitary Gland An endocrine or hormone-producing gland found on the underside of the brain just behind the eyes. The Pituitary extract is an aqueous, alcoholic, or acetone extract of the pituitary gland used for artificial induction of spawning.

Gonad

An organ either the testes in male fish which produces sperm or ovary in female fish which produces eggs; Types of Hormones in Fish

A chemical formed in endocrine glands that affect the functions of specific tissues. Gonadotropin

A pituitary hormone that controls the production by the gonads (testes and ovary) of sperm and eggs.

Human Chorionic Gonadotropin (HCGl

A commercially available, semi-purified hormone that is used to induce ovulation and spermiation, i.e., egg and sperm production.

Luteinizing Hormone Releasing Hormone f LHRH)

Mammalian gonadotropin (hormone) that has been used to induce the reproductive cascade in fish.

Gonadotropin Releasing Hormone (GnRH)

A hormone produced by the hypothalamus travels from there to Pituitary G!and that stimulates the pituitary to release gonadotropin.

Synthetic Hormones

Synthetic Analogues of LHRH and GnRH referred to as LHRHa and GnRHa are developed which are proved far more effective as they are purer. And also they are not rapidly metabolized by fish, and thus they remain active for longer periods.

Dopamine

A chemical that inhibits the release of hormones from the pituitary and thereby blocks the pituitar/s response to injected LHRHa

Dopamine Antagonist fPA¾

A family of drugs that block action of dopamine PHARMACOLOGICAL DATA

Fish have evolved to reproduce under environmental conditions that are favourable to the survival of the young. Long before spawning, seasonal cues begin the process of maturation. In many fish, this can take up to a year. When the gametes have matured, an environmental stimulus may signal the arrival of optimal conditions for the fry, triggering ovulation and spawning. Examples of environmental stimuli are changes in photoperiod, temperature, rainfall, and food availability. A variety of sensory receptors detect these cues, including the eye, pineal gland (an organ in the dorsal part of the forebrain that is sensitive to light), olfactory organs, taste buds, and thermp-receptors.

The hypothalamus, located at the base of the brain, is sensitive to signals from sensory receptors and releases hormones in response to environmental cues. Principal among these hormones are gonadotropin releasing hormones (GnRH), which travel from the

hypothalamus to the pituitary gland. The pituitary is responsible for a wide variety of functions, including growth and reproduction. Certain cells of the pituitary receive GnRH and release gonadotropic hormones into the bloodstream. The gonadotropic hormones travel to the gonads, which synthesize steroids responsible for final maturation of the gametes.

A gonadotropin-releasiitg hormone analogue (GnRH analogue or analog) , also known as a luteinizing hormone releasing hormone agonist (LHRH agonist) or LHRH analogue is a synthetic peptide modeled after the human hypothalamic gonadotropin-releasing hormone (GnRH). A GnRH analogue is designed to interact with the GnRH receptor and modify the release of pituitary gonadotropins FSH and LH for therapeutic purposes. Shortly after the discovery of GnRH by Nobel laureates Guillemin and Schally researchers tried to modify the GnRH decapeptide with the intent to synthesize stimulating and blocking variants.

Two types of analogues need to be distinguished:

Agonists

A gonadotropfn-releasing hormone agonist (GnRH agonist) is an analogue that activates the GnRH receptor resulting in increased secretion of FSH and LH. Initially it was thought that agonists could be used as potent and prolonged stimulators of pituitary gonadotropin release, but it was soon recognized that agonists, after their initial stimulating action - termed a "flare" effect - eventually caused a paradoxical and sustained drop in gonadotropin secretion. This second effect was termed "downregulation" and can be observed after about 10 days. While this phase is reversible upon stopping the medication, it can be maintained when GnRH agonists use is continued for a long time. Antagonists

A gonadotropin-releasing hormone antagonist (GnRH antagonist) is an analogue that blocks the GnRri receptor resulting in an immediate drop in gonadotropin (FSH, LH) secretion. The GnRH antagonist is primarily used in 1VF treatments to block natural ovulation.

All GnRH analogues are contraindicated in pregnancy (pregnancy category X).

PHARMACODYNAMICS & PHARMACOKINETICS

GnRH

GnRH Is synthesized in the hypothalamus and controls the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary. FSH. and LH stimulate the gonads (ovaries in females, testes in mates), leading to the production of androgens in males.

GnRH is released in a pulsatile manner. This intermittent release is crucial for the proper synthesis and release of FSH and LH

Pulsatile (physiologic) release of GnRH results in increased gonadotropin release. Continuous administration of GnRH results in suppression of gonadotropin release via down-regulation of GnRH receptors, a process called desensitization.

GnRH agonists can be administered via pulsatile or continuous fashion, depending on the desired effect (stimulation or suppression of gonadotropins respectively).

GnRH antagonists simply block the GnRH receptor, resulting in reduced release of FSH and LH.

GnRH agonist administration has been the treatment of choice for central precocious puberty. Continuous administration of the agonist, instead of permanently stimulating gonadotropin secretion, deeply suppresses LH and FSH levels and induces a marked inhibition of gonadal activity and regression of clinical symptoms. This inhibitory effect is due both to specific kinetic parameters relative to natural GnRH, and to marked alterations of the biosynthetic pathways of gonadotropin subunits. The half disappearance time of infused agonists is 3-10 fold that of natural GnRH. This means that the residence time of GnRH agonists is significantly longer than that of GnRH. The resistance of agonist to enzymatic degradation, mainly due to the substitution of a hydrophobic D-amino acid for glycine 6, is one of the factors involved in the increased availability of GnRH super agonists. The paradoxical effects of GnRH super agonists are still incompletely understood. In children long-term treated with depot formulations of triptorelin or leuprorelin, alpha- subunit secretion is markedly increased, and remains sensitive to exogenous GnRH, which demonstrates that the gonadotrophs are not totally desensitized. Despite the sustained stimulation of a-subunit secretion, no deleterious side effects, either during therapy or during post-therapy follow-up, have been reported in children treated with GnRH agonists. It should be noted that a!pha-subunit responsiveness to exogenous GnRH decreases progressively after several years of treatment, although it is never completely abolished. On the other hand, LH beta-subunit secretion is suppressed as evidenced by radioimmunoassay of LH beta-subunit in serum chromatographic fractions from children treated with triptorelin. This differential pattern of secretion parallels that of mRNA levels in rat pituitary after in vivo exposure to triptorelin. Both pharmacodynamic and pharmacokinetic data can help diagnose the situations of resistance or escape

Domperidone

Domperidone is a dopamine antagonist with anti-emetic properties domperidone does not readily cross the blood-brain barrier. In domperidone users, especially in adults, extrapyramidal side effects are very rare, but domperidone promotes the release of prolactin from the pituitary. Its anti-emetic effect may be due to a combination of peripheral (gastro kinetic) effects and antagonism of dopamine receptors in the chemoreceptor trigger zone, which lies outside the blood-brain barrier in the area postrema. Animal studies, together with the low concentrations found in the brain, Indicate a predominantly peripheral effect of domperidone on dopamine receptors.

Absorption

In fasting subjects, domperidone is rapidly absorbed after oral administration with peak plasma concentrations at 30 to 60 minutes. The low absolute bioavailability of oral domperidone (approximately 15%) is due to an extensive first-pass metabolism in the gut wail and liver. Although domperidone's bioavailability is enhanced in normal subjects when taken after a meal, patients with gastro-intestinal complaints should take domperidone 15- 30 minutes before a meal. Reduced gastric acidity impairs the absorption of domperidone. Oral bioavailability is decreased by prior concomitant administration of cimetidine and sodium bicarbonate. The time of peak absorption is slightly delayed and the AUC somewhat increased when the oral drug is taken after a meal. Distribution

Oral domperidone does not appear to accumulate or induce its own metabolism; a peak plasma level after 90 minutes of 21 ng/ml after two weeks oral administration of 30 mg per day was almost the same as that of 18 ng/ml after the first dose. Domperidone is 91-93% bound to pjasma proteins. Distribution studies with radio labelled drug in animals have shown wide tissue distribution, but low brain concentration. Small amounts of drug cross the placenta in rats.

Metabolism

Domperidone undergoes rapid and extensive hepatic metabolism by hydroxyiation and N- dealkylation in vitro metabolism experiments with diagnostic inhibitors revealed that CYP3A4 is a major form of cytochrome P-450 involved in the Nrdealkylation of domperidone whereas CYP3M, CYP1A2 AND CYP2E1 are involved in domperidone aromatic hydroxyiation.

Excretion

Urinary and faecal excretions amount to 31 and 66% of the oral dose respectively, the proportion of the drug excreted unchanged is small (10% of faecal excretion and approximately 1% of urinary excretion). The plasma half-life after a single oral dose is 7-9 hours in healthy subjects but is prolonged in patients with severe renal insufficiency.

TECHNICAL DETAILS & DESCRIPTION SUMMARY OF PRODUCT CHARACTERISTICS

Injectable Solution (Buserelin 20 ug/ml + Domperidone 10 mg/ml) For intraperitoneal or intramuscular Injection in fish.

Injectable solutionis a liquid peptide preparation that contains an analog of gonadotropin releasing hormone (GnRH) and a brain neurotransmitter (dopamine) inhibitor. The GnRH in the injection elicits the release of stored gonadotropins from the pituitary. The dopamine inhibitor (Domperidone) serves to remove other inhibition of GnRH release. Release of stored pituitary gonadotropins may aid spawning by stimulating ovulation and spermiation in sexually mature fish. Name of the Veterinary Medicinal Product

Buserelin & Domperidone Injection (For Veterinary use only) - Pack Size 10 ML.

Qualitative and Quantitative Composition

injection {Buserelin & Domperidone Injection} prepared in Propylene Glycol .

stable product. The composition is as follows:

The invention consists of a sterile injection and each ml of injectable solution contains 20 micrograms of Buserelin and 10 milligrams of Domperidone.

Buserelin is a longer acting synthetic peptide analog of the naturally occurring gonadotropin- releasing hormone (GnRH/LHRH). Substitution of glycine In position 6 by D-serine and that of glyclnamlde tn position 10 by ethyl amtde, leads to a nanopeptide with a significantly enhanced LHRH effect. The effects of Buserelin on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release are approximately 20 to 170 times higher than those of natural LHRH.

Chemical Sequence: pGlu-His-Trp-Ser-Tyr-0-Ser(tBu)-Leu-Arg-Pro-NHEt

Chemical Formula: C60-H86-N16-O13

Pharmaceutical Form

Solution for Injection, clear, colorless liquid

CLINICAL PARTICULARS

Benefits To induce spawning in Tilapia, _, Carps, Catfish and PANGASIUS

The Injection can be used in all species of fish. The forms of GnRH that naturally occur in the brains of fishes differ throughout the more than 25,000 fish species. However, the Buserelin form of GnRH is present in most of these fish and thus the GnRH in the injection is the peptide of choice. Among the other types of GnRH found in the other fishes, there is such a conservation of form and function that the injection works successfully. The Injection has been used successfully in all major types of fish including:

• Ornamentals

• Salmonids

• Cyprinids (goldfish, carp)

• Catfish

• Pangasius

• Perch (sea bream, tilapia). Other benefits indude:

• Stimulates milt production for a longer period and for greater volume,

• Moves fish forward in the spawning season,

• Maximizes reproductive potential in all spawners within a population,

• Conserves genetic material in a population, between populations or in endangered stocks.

• Coordinates maturation during the normal spawning season:

• Spawns a population in a shorter period of time,

• Maximizes hatchery labor and infrastructure by concentrating efforts,

• Moves offspring through fife stages as a tightly- packed group according to spawning date,

• Reduces overlap of hatchery duties due to protracted development of hatchery fish.

• May re-start stalled maturation,

• Using the Injection, post-transport may reduce the incidence of transport shock.

Contraindication

Do not use in animals with known hypersensitivity to the active ingredient

Special Warning for each target species

No data available

Special precautions for use None

Special precautions to be taken by the person administering the veterinary medicinal product to animals

Avoid eye and skin contact with the solution for injection. In case of accidental eye contact, rinse thoroughly with water. Should skin contact with the product occur, wash the exposed area immediately with soap and water.

When administering the product, care should be taken to avoid accidental self-injection by ensuring that animals are suitably restrained, and the application needle is shielded until the moment of injection. Because of the potential for effects on reproductive function, women of child-bearing age should handle the product with caution. Pregnant women should not administer the product In case of accidental self-injection, seek medical advice immediately and show the package insert or label to. the physician.

Adverse reactions (frequency and seriousness)

None

Use during pregnancy, lactation or lay

No data available

Interaction with other medicinal products

None known

Amount to be administered and administration route

For intraperitoneal or intramuscular use as a spawning aid in most of the varieties of Fish Dosage

As Directed by Veterinary Doctor

OR

Female fish~0.5 ml/kg

Male fish 0.1-0.2 ml/kg

Overdose (symptoms, emergency procedure, antidotes)

No specific overdose reactions known

Withdrawal period No withdrawal period is reported

PHARMACEUTICAL PARTICULARS

List of exciplents

Propylene Glycol

Incompatibilities

In the absence of compatibility studies, this veterinary medicinal product must not be mixed with other veterinary medicinal products.

Shelf-life

Shelf-life of the veterinary medicinal product as packaged for sale: 2 years

Special precautions far storage

Do not store above 25 °C

Do not freeze.

Nature and composition of immediate packaging

Solution packed in sterile vial (glass type-l amber with a Bromo butyl rubber plug and an aluminium crimp cap) containing 10 ml solution for injection, Such single vial labelled, placed in a mono carton, such 10 carton shrinked, and such 56 shrinked packed in a shipper.

Special precautions for the disposal of unused veterinary medicinal products or waste materials derived from the use of such products

Any unused veterinary medicinal product or waste materials derived from such veterinary medicinal products should be disposed of in accordance with local requirements.

Chemical, Pharmaceutical and Biological Documentation

control of the starting materials

Ingredients

Buserelin & Domperidone along with other exciplents

PRODUCTION FLOW CHART

Buserelin & Domperidone

Buserelin (GnRH) present in all cultured fishes, and some studies show better binding to pituitary receptors and better release of gonadotropin than is achieved with mammalian GnRH. However, there is no practical superiority of [D-ArgA ³ , Pro9 NEt] GnRH (the most commonly available one) over commonly available analogues of mammalian GnRH such as [D-Ala6, Pro9 NEt] LHRH. Fish culturists should certainly take the lead from published reports of the use of GnRH in their species, but when cost - GnRH is more expensive than mammalian - and easy availability are factors (as they usually are), analogues of mammalian GnRH are perfectly adequate. The fact that GnRH is only the "on" part of an "on-off" control system for gonadotropin release means that the analogue one uses may be less important than whether it is accompanied by a dopamine inhibitor. Domperidone is a medication developed by Janssen Pharmaceutica that is a peripheral, specific blocker of dopamine receptors. It is administered orally, rectally, or. intravenously. Domperidone is given In order to relieve nausea and vomiting; to increase the transit of food through the stomach (as a prokinetic agent through increase in gastrointestinal peristalsis); and to increase lactation (breast milk production) by release of prolactin. It is also used fn the scientific study of the way dopamine (an important neurotransmitter) acts in the body.

b) Stability

Stability study at room temp of the injection at

Objective '

Experimental determination of the physical and chemical stability of active drugs i.e., GnRH & Domperidone maintained ' throughout the period of validity to 25c +-2c and relative humidity of 60c +-5%

Design of the Assay

Properties to Determine

By the characteristics of specialty of the injection, during the stability test they wilt be controlled,

Physical characteristic (color, appearance, transparency and ph value)

Chemical content of the active drugs.

Analysis Periodicity

The physical and chemical test are made with a quarterly regularity during the first semester year and semester during the second. A sample of each batch is analyzed by triplicate for the physical-chemical test

Sample to Assay

The stability test takes place on three pilot batches of the specialty. The packages are identified with an individually labelled.

Storage Conditions

The batches subjected to stability test are kept in conditioned stability chamber to an average temp, of 25c +-2 c and one relative humidity of 60 +- 5 % in a closed chamber and safe from the light Temperature and Humidity is register in the data sheet attached to the stability chamber- Assay Method

The assay method used for the obtaining data for the calculation of the stability are described and validated, (by HPLC) Result

tn order to facilitate the study and interpretation of the result they will be grouped in tables of chemical results throughout the time that lasts the test At the end of the period of validity, the injection does not have to present "significant changes" the content of active drugs must be minimum 95% of the initial value. And the physics! and chemical properties do not have to present significant variation with respect to the initial value, always being included within the limits.

Conclusions

Once concluded all the studies of stability passed two years from the date of manufacture, we can confirm that

i. The physical does not vary significantly from the determinate on time zero ii. ' The chemical properties do not vary significantly from the determinate on time zero iii. The identification of the ingredients is correct throughout all the study of stability. iv. The quantitative determination of the ingredient stayswithin the established limits, throughout the period of stability.

v. Significant changes in the product do not exit during the period of the stability.

This allows to assure that the Injection has a period of expiry of two years counted as of the date of manufacture.

PHARMACEUTICAL-CLINICAL OOCCUMENTATION

Pharmacological Report

Pharmacodynamics, Action and Clinical

Description

The injectable solution is a liquid peptide preparation that contains an analog of gonadotropin releasing hormone (GnRH) and a brain neurotransmitter (dopamine) inhibitor. The GnRH in the injection elicits the release of stored gonadotropins from the pituitary. The dopamine inhibitor (Domperidone) serves to remove other inhibition of GnRH release. Release of stored pituitary gonadotropins may aid spawning by stimulating ovulation and spermiation in sexually mature fish.

Indications

For intraperitoneal or intramuscular use as a spawning aid in fish. Warning

i. Care should be taken to avoid accidental contact or self-injection. In the event of accidental self-injection, seek medica! advice immediately.

ii. Use in a well-ventilated area. Wear gloves, goggles and suitable protective clothing. iii. Not for use in humans.

iy. Keep put of the reach of children.

Inhalation

May be harmful If breathing becomes difficult, move to fresh air, and contact a physician. Ingestion

May be harmful. If the person is conscious, wash out mouth with copious amounts of water. Contact a physician. ^'

Eye Contact

May be harmful. In case of contact, flush with copious amounts of water for at least 15 minutes. Assure adequate flushing by separating eyelids with fingers. Contact a physician.

Skin Contact

May be harmful. In case of contact, flush with copious amounts of water for at least 15 minutes. Remove contaminated clothing and wash before re-using. Contact a physician.

Other Health Information

The toxicological properties of GnRH have not been thoroughly investigated. The actions are similar to luteinizing hormone releasing hormone (LHRH, Gonadotropin releasing hormone, GnRH) in humans. LHRH is the key mediator in the liberation of the pituitary gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH). LHRH may modify reproductive ability by influencing plasma gonadotropin levels and concomitantly gonadal steroid levels.

Directions for Use

The injection is packaged ready for use in liquid form. Precise amounts for individual injections should be withdrawn directly from the bottle into a syringe. Due to the viscosity of the product, use of syringes with Luer Lock tips or permanently attached needles is strongly recommended. Use of syringes with slip tips is not recommended since needles could inadvertently eject under pressure. Cleanliness

Syringes/needles should be sterile prior to use.

Loading

Withdraw only enough solution from the bottle of the Injectable Solution as will be required for the weight of the fish. With the needle pointed upward and directed away from your face, squeeze the syringe gently to expel any trapped air.

Injection

Hold the fish firmly and insert the needle Into the abdominal cavity/belly between the pelvic fin and the vent or into the muscle on either side of the, dorsal fin. For intra-abdominal injection in smaller species, It may be helpful to secure the fish on a soft wet surface such as a sponge, and to place the fish on its side and inject into the rear abdominal cavity to avoid contact with internal organs. Inject the solution carefully. After injection, gently place the fish into a container of clean aerated water. . .

Sedation for Handling ftRecovery

If necessary, sedate fish prior to injecting the solution.

Dose

A general dose of injection is 0.5 ml per kilogram of bodyweight. This dose may vary among finfish species and physiological state of individual animals. Environment and temperature also play a significant role in the reproductive process and may affect dose and timing. For most species, only a single dose of the injection is required; but treatment offish with a single dose of the injection is only effective in fish that are within or near their natural spawning season. For other species, a split dose may be needed. For split dosing, a loading dose of 10% of the total dose should be injected, followed by injection of the remaining 90% of the total dose at least 6 hours later. In warm water species, ovulation may occur in as little as 4 hours post treatment, so fish should be monitored accordingly. Signs of ovulation/spermiation may include a noticeable swelling and "softening ⁰ of the abdomen; presence of eggs in the water; the ability to easily express eggs from the female and milt from males; and when sexes are held together in the same tank, onset of spawning behaviour (e.g., males "chasing" females). However, it is important to note that the interval between treatment and the onset of ovulation is variable between species and

temperature, and treated fish should be disturbed as little as possible to reduce stress during final maturation. Males may respond in shorter periods of time than females of the same species. Dose Calculation

The dose is calculated based on the weight of the fish.

Use of a needle guard to limit the penetration of the needle into the fish may be helpful, and use of a tuberculin or similar syringe is also recommended for accurate dosing in small fish and to minimize trauma at the injection site. Intramuscular injection may result in bleeding at the injection site. Swelling, ulceration, whiteness, redness, or hypo pigmentation - may also be observed at the injection site.

Storage &Handling

Store at room temperature and protect from heat & light

Disposal

Contact your State Environmental Control Agency, or the Hazardous Waste Representative at the nearest EPA Regional Office for guidance pertaining to disposal of unused product.

APPLICATION - TESTS - RESULTS

The process is tested in all major species of fish. The forms of GnRH that naturally occur in the brains of fishes differ throughout the more than 25,000 fish species. However, the Buserelin form of GnRH is present in most of these fish and thus the Buseretin in the present process is the peptide of choice. Among the other types of GnRH found in other fishes, there is such a conservation of form and function that the present process works successfully.

Not all fish species can be bred efficiently by stimulating reproduction with GnRH agonists. Hence the development of combination products composed of GnRH agonists as BUSERELIN and dopamine receptor antagonists as Domperidone meant a breakthrough for fish farming as they allowed reliable induction and synchronization of ovulation and spawning. The synthetic stimulators yield spawn of much higher quality than the pituitary extracts used in controlled fish reproduction.

Under natural conditions, there is a feedback mechanism in the fish that limits the release of gonadotroph^. This mechanism uses a chemical called Dopamine, which inhibits the action of GnRH. When a dopamine is present in the fish, even GnRH will have only limited success. A dopamine antagonist is often used to limit the effects of dopamine, when GnRH and dopamine antagonist are used in conjunction (with the injection), reproductive success dramatically increases. Numerous hormones have been used to induce reproduction. Two methods have emerged over the past few years that seem to offer the best chance for success at the least expense. They are injection of GnRH analog with dopamine antagonist and injection of

Gonadotrophin.

Salmon GnRHa and Domperidone combination is widely used in Europe and Northern America mainfy for Salmon and Trout culture. Over, ten years of research of developing the sGnRHa and Domperidone formulation, it is concluded that Buserelin and Domperidone combination works much better than sGnRHa and Domperidone. Salmon and trout are not the kind of species which can live well in ASIAN waters and that is the reason Asian countries do not even culture these species. Carp, Cat Fish, Tilapia and other kind of fish species which are cultured in Asian countries are giving excellent results with the injection of Buserelin and domperidone formulation, when compared to sGnRHa and domperidone formulation.

In most te!eost fish, including the Thai carp (Barbonymusgonronotus), a surge of gonadotropin secreted by the pituitary initiates the final maturation of gonads and ovulation, and triggers the onset of spawning. The hypothalamus regulates this surge through the interaction of two other hormones, gonadotropin-releasing hormone (GnRH) and dopamine (an agent that inhibits the release of GtH).

Fish reared artificially, will spawn if dosed with the injection. In Thailand, this proved a very effective and reliable way of inducing several freshwater fish species to spawn. Also found that Buserelin (BUS), a mammalian luteinizing hormone, is the most effective of the several forms of GnRHa.