DESC:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003

Complete

Specification
(See section 10 and rule 13)

Title: Development of High Potent Anti Snake Venom

The following specification particularly describes the nature of this invention and the manner in which it is to be performed.

Development of Highly potent Anti Snake Venom
Field of Invention
The present invention relates to a method to achieve high potent antibodies by modifying the conventional immunization of horses against snake venom by employing one or more adjuvant of nano range alone or in combination, optionally one or more pharmaceutically acceptable excipients and varying the venom concentration. The present invention also relates to a method to achieve high potent antibodies by modifying the conventional immunization of horses against snake venom by employing one or more adjuvant of nano range alone or in combination, optionally one or more pharmaceutically acceptable excipients and varying the venom concentration wherein the venom can be obtained from a single species or mixture of several species of snakes.
Background of the invention
Snake bite is a significant public health problem causing considerable morbidity and mortality worldwide, particularly in tropics. Snakebite is now recognized as a Neglected Tropical Disease by the World Health Organization (WHO). According to WHO estimates about 5 million people are bitten each year by poisonous snakes which results in 2.5 million envenomations, at least 100000 deaths, and 300000 amputations and other permanent disabilities. Majority of snakebite induced deaths occur in Asia and Sub-Saharan Africa. The mortality due to venomous snakebite in India is estimated 35000-50000 per annum, which is the highest in the world. The mortality due to venomous snakebite in India is continuous to be high due to various social, economic and cultural reasons.
Over 3000 species of snakes are known worldwide, of which around 600 are considered to be venomous.There are 2 important groups (families) of venomous snakes in south-east Asia - Elapidae have short permanently erect fangs. This family includes the cobras, king cobra, kraits, coral snakes, and the sea snakes. Viperidae have long fangs, which are normally folded up against the upper jaw, but when the snake strikes, are erected. There are 2 subgroups, the typical vipers (Viperinae) and the pit vipers (Crotalinae). Medically Important snakes of India include the so called “Big 4”, Russel’s viper (Daboia russelli), Cobra (Naja naja), Common Krait (Bungarus caeruleus) and Saw scaled viper (Echis carinatus) that occur throughout the country. The pit viper species - Malabar, green and the hump-nosed, sea snakes and others like the king cobra (Ophiophagus hannah), monocle cobra (Naja Kaouthia), Banded Krait (Bungarus fasciatus) and Echis sochureki are important causes in certain geographical areas. The principle effects of envenomation is on the nervous system, kidneys, heart, lungs, liver, blood coagulation system, vascular endothelium and local effects at the site of bite.
Snake envenomation is a serious health problem in many tropical and subtropicalregions in the world including India. The only accepted andspecific antidote to snake venom is the adminstration of snake antivenom (SAV). Thesnake antivenom is prepared by immunizing horses with venom to yield serum thatis rich in immunoglobulins capable of neutralizing snake venoms. A number ofapproaches have been adopted for the improvement in manufacturing protocol inorder to prepare the safe and effective antivenom. These primarily include a) use ofnewer, more effective and safe adjuvants b) use of purified fraction of venom asimmunogen and c) use of toxin subjected to immunomodulation making it less toxicbut more immunogenic. Antivenoms against snakes can be monospecific or polyspecific. The selection of monospecific antivenom for treatment is based on identification of the culprit snake and on the signs and symptoms present in patients after snakebite. When the identification of the envenoming species is not certain, it is better to use polyspecific antivenom that is effective against venoms of several species of snakes present in the area.
Conventional techniques produce snake antivenom using adjuvants like CFA, bentonite and aluminum salts resulting variable number of responding horses even 5-7% ofhorses do not give desired antibody levels in their sera. Again, larger quantity ofvenoms is required for immunization of horses, especially in the case ofimmunologically poor venom. Further, it was shown in different studies that bentonitewas the cause of the poor antibody response observed. Use of complete Freund’s adjuvant (CFA) is not preferred since it causes severe local reactions in horses.
So there was a constant need of an effective immunization process for the production of high potent horse antisera against medically Important snakes of India include the so called “Big 4”, Russel’s viper (Daboia russelli), Cobra (Naja naja), Common Krait (Bungarus caeruleus) and Saw scaled viper (Echis carinatus).
Surprisingly inventors of present invention developed a method to achieve high potent antibodies by modifying the conventional immunization of horses against snake venom by employing one or more non conventional adjuvant alone or in combination, optionally one or more pharmaceutically acceptable excipients and varying the venom concentration and achieved higher yield of antibodies thus nullifying all the problems related to prior art.

Summary of the Invention
One of the embodiments of present invention discloses an improved method of immunization of horses to produce anti snake venom antibodies.
Another embodiment of present invention relates to an improved immunization process of horses by administering specific snake venom formulation of predefined concentration thus creation of antibodies take place.
Another embodiment of present invention relates to an improved immunization process of horses by administering specific snake venom formulation of predefined concentration wherein the formulation might contain venom, one or more adjuvant alone or in combination.
Another embodiment of present invention exemplifies different types of adjuvant of nano range that include, but not limited to, Bentonide, Freund's complete adjuvant, aluminium hydroxide, sodium alginate, calcium phosphate, Montanide and combinations thereof, preferably Montanide.
Yet another embodiment of present invention relates to varying concentration of snake venoms including, but not limited to, Russel’s viper (Daboia russelli), Cobra (Naja naja), Common Krait (Bungarus caeruleus), Saw scaled viper (Echis carinatus) and combinations thereof.
Another embodiment of present invention discloses composition of the immunogen introduced to the horse comprising of snake venom from a single species or mixture of several species of varying concentration, one or more non conventional adjuvant and optionally one or more pharmaceutically acceptable excipients.
Another embodiment discloses the venom concentration for
1. Russel’s viper (Daboia russelli) might be within 0.4 to 0.85 mg,
2. Cobra (Naja naja) might be within 0.5 to 0.8 mg,
3. Common Krait (Bungarus caeruleus) might be 0.3 to 1 mg and
4. Saw scaled viper (Echis carinatus) might be within 0.1 to 0.9 mg
of per mL of horse serum.
Another embodiment of present invention illustrates the pharmaceutically accepted excipients that are non functional additives such as preservatives or other pharmaceutically acceptable ingredients, more preferably cresol, used to preserve the formulation for a long period of time.

Detailed Description of the Invention
Antivenom (or antivenin or anti-venene) is a biological product used in the treatment of venomous bites orstings. Antivenom is created by injecting a small amount of the targeted venom into an animal such as a horse,sheep, goat, or rabbit, the subject animal will undergo an immune response to the venom, producing antibodiesagainst the venom's active molecule which can then be harvested from the animal's blood and used to treatenvenomation. Internationally, anti-venoms must conform to the standards of Pharmacopoeia andthe World Health Organization (WHO)
The present invention relates to a method to achieve high potent antibodies by modifying the conventional immunization of horses against snake venom by employing one or more adjuvant alone or in combination, optionally one or more pharmaceutically acceptable excipients and varying the venom concentration.
In this present invention the term “antibody” signifies a biological product that typically consists of venom neutralizing antibodies derived from a host animal, such as a horse or sheep. The host animal is hyperimmunized to one or more snake venoms, a process which creates an immunological response that produces large numbers of neutralizing antibodies against various components (toxins) of the venom. The antibodies are then collected from the host animal, and further processed into snake anti-venom for the treatment of envenomation.
The term “Immunization process” addresses a process which creates an immunological response that produces large numbers of neutralizing antibodies against various components (toxins) of the venom within an animal such as Horse, Sheep, Goat, Rabbit and like.
The term “Venom” or “Snake venom” signifies highly modified saliva containing zootoxins which facilitates the immobilization and digestion of prey. It is injected by unique fangs after a bite, and some species are also able to spit.
The precise role of the “adjuvant” has not been elucidated but it is thought that it acts in a controlled manner by decreasing the rate of release of venom and thus stimulating further the immunological response. The most commonly used adjuvants are Freund's, bentonite, aluminium hydroxide and sodium alginate, liposomes, montanide and combinations thereof. The present invention employed preferably Montanide.
The four venomous snake species responsible for causing the greatest number of medically significant human snake bite cases on the Indian Subcontinent (mostly in India) are sometimes collectively referred to as the “Big Four”. They are as follows:
1. Indian cobra, Naja naja
2. Common krait, Bungarus caeruleus
3. Russell's viper, Daboia russelii
4. Saw-scaled viper, Echis carinatus
One of the embodiments of present invention discloses the venom concentration as
5. Russel’s viper (Daboia russelli) might be within 0.4 to 0.85 mg,
6. Cobra (Naja naja) might be within 0.5 to 0.8 mg,
7. Common Krait (Bungarus caeruleus) might be 0.3 to 1 mg and
8. Saw scaled viper (Echis carinatus) might be within 0.1 to 0.9 mg
per mL of horse serum.
The unique embodiment of present invention discloses use of Montanide as an adjuvant in place of conventional adjuvant increases the yield of antibody in a cost effective way. The montanide adjuvant is a new generation of adjuvant that are ready to use, cost effective, safe and potent. They are biodegradable, compatible, non-toxic, noncarcinogenic, non-teratogenic and non-abortogenic.
The present invention successfully led to the development of novel immnunogenadjuvant configuration free from the adverse local reactions, while maintaining the therapeutic value of antibodies against venoms. This should lead to the improvement in the potency of the product and also in the overall safety of horses during the entire phase of immunization.

Example 1:
The present invention was undertaken to evaluate various types of Montanide emulsion and nonemulsion adjuvants for snake antivenom preparation in Ponies.
Here three different types of adjuvants of the montanide group were used:
1. Aqueous based nanoparticles IMS 3012,
2. W/OAV emulsion ISA 206 and
3. W/OAV emulsion ISA 35.
4. An Incomplete Freund's adjuvant (IFA, water-in -oil W/0) emulsionwas used as a control adjuvant through the present study.
Composition
1. Snake venoms (Cobra venom (CV), Krait Venom (KV), Russell’s viper venom (RV) and Saw scaled viper venom (SSV).
2. Sterile syringes (2 ml, 5 ml and 10ml)
3. Freund’s incomplete adjuvant and Montanide adjuvants
4. Sterile Normal Saline.
PROCEDURE
Preparation of inoculums
Equal volume of adjuvant was added with venom solution and made up total require volume with Normal saline. The adjuvant Freund’s incomplete adjuvant, Montanide ISA206 and Montanide ISA35 were added to the antigen solution drop by drop with constant emulsification.
Area for Immunization
The poney to be immunized shall be brought into the trevice and immunization shall be done.
The schedule of Immunization
1. The schedule of immunization with Freund’s incomplete adjuvant (FIA)as per table given below.
Days Venom dose (mg)
Volume of inoculum (mL) Route of administration Adjuvant Injections
CV KV RV EV
0 day 0.10 0.01 0.10 0.01 1.0 s/c FIA F1
15thday 0.10 0.02 0.10 0.02 1.0 s/c FIA F2
23rdday 0.20 0.03 0.20 0.03 1.0 s/c FIA I injection
35thday 0.20 0.04 0.20 0.04 1.0 s/c FIA II injection
43rdday 0.25 0.05 0.25 0.05 1.0 s/c FIA III Injection
51stday 0.30 0.07 0.30 0.06 1.0 s/c FIA IV Injection
59thday 0.40 0.09 0.30 0.07 1.0 s/c FIA V Injection
67thday 0.50 0.10 0.40 0.08 1.0 s/c FIA VI Injection
75thday 0.50 0.20 0.45 0.09 1.0 s/c FIA VII Injection
83rdday 0.60 0.30 0.50 0.10 1.0 s/c FIA VIII Injection
91thday 0.70 0.40 0.60 0.10 1.0 s/c FIA IX
Injection

Blood sample à Check titres on 99th day
The titers were checked with individual venom and accordingly immunization was continued with increased doses if required.
2.) The schedule of immunization with Montanide IMS 3012 adjuvant as per table given below.
Days Venom dose (mg)
Volume of inoculum (mL) Route of administration Adjuvant Injections
CV KV RV EV
0 day 0.10 0.01 0.10 0.01 2.0 s/c Montanide
IMS 3012 F1
15thday 0.10 0.02 0.10 0.02 2.0 s/c Montanide
IMS 3012 F2
23rdday 0.20 0.03 0.20 0.03 2.0 s/c Montanide
IMS 3012 I injection
35thday 0.20 0.04 0.20 0.04 2.0 s/c Montanide
IMS 3012 II injection
43rdday 0.25 0.05 0.25 0.05 2.0 s/c Montanide
IMS 3012 III Injection
51stday 0.30 0.07 0.30 0.06 2.0 s/c Montanide
IMS 3012 IV Injection
59thday 0.40 0.09 0.30 0.07 2.0 s/c Montanide
IMS 3012 V Injection
67thday 0.50 0.10 0.40 0.08 2.0 s/c Montanide
IMS 3012 VI Injection
75thday 0.50 0.20 0.45 0.09 2.0 s/c Montanide
IMS 3012 VII Injection
83rdday 0.60 0.30 0.50 0.10 2.0 s/c Montanide
IMS 3012 VIII Injection
91thday 0.70 0.40 0.60 0.10 2.0 s/c Montanide
IMS 3012 IX
Injection

Blood sample à Check titres on 99th day
The titers were checked with individual venom and accordingly immunization was continued with increased doses if required.
3. The schedule of immunization with Montanide ISA206 adjuvant as per table given below

Days Venom dose (mg)
Volume of inoculum (mL) Route of administration Adjuvant Injections
CV KV RV EV
0 day 0.10 0.01 0.10 0.01 1.0 s/c Montanide
ISA 206 F1
15thday 0.10 0.02 0.10 0.02 1.0 s/c Montanide
ISA 206 F2
23rdday 0.20 0.03 0.20 0.03 1.0 s/c Montanide
ISA 206 I injection
35thday 0.20 0.04 0.20 0.04 1.0 s/c Montanide
ISA 206 II injection
43rdday 0.25 0.05 0.25 0.05 1.0 s/c Montanide
ISA 206 III Injection
51stday 0.30 0.07 0.30 0.06 1.0 s/c Montanide
ISA 206 IV Injection
59thday 0.40 0.09 0.30 0.07 1.0 s/c Montanide
ISA 206 V Injection
67thday 0.50 0.10 0.40 0.08 1.0 s/c Montanide
ISA 206 VI Injection
75thday 0.50 0.20 0.45 0.09 1.0 s/c Montanide
ISA 206 VII Injection
83rdday 0.60 0.30 0.50 0.10 1.0 s/c Montanide
ISA 206 VIII Injection
91thday 0.70 0.40 0.60 0.10 1.0 s/c Montanide
ISA 206 IX
Injection

Blood sample à Check titres on 99th day
The titers were checked with individual venom and accordingly immunization was continued with increased doses if required.
4. The schedule of immunization with Montanide ISA35 adjuvant as per table given below

Days Venom dose (mg)
Volume of inoculum (mL) Route of administration Adjuvant Injections
CV KV RV EV
0 day 0.10 0.01 0.10 0.01 1.0 s/c Montanide
ISA 35 F1
15thday 0.10 0.02 0.10 0.02 1.0 s/c Montanide
ISA 35 F2
23rdday 0.20 0.03 0.20 0.03 1.0 s/c Montanide
ISA 35 I injection
35thday 0.20 0.04 0.20 0.04 1.0 s/c Montanide
ISA 35 II injection
43rdday 0.25 0.05 0.25 0.05 1.0 s/c Montanide
ISA 35 III Injection
51stday 0.30 0.07 0.30 0.06 1.0 s/c Montanide
ISA 35 IV Injection
59thday 0.40 0.09 0.30 0.07 1.0 s/c Montanide
ISA 35 V Injection
67thday 0.50 0.10 0.40 0.08 1.0 s/c Montanide
ISA 35 VI Injection
75thday 0.50 0.20 0.45 0.09 1.0 s/c Montanide
ISA 35 VII Injection
83rdday 0.60 0.30 0.50 0.10 1.0 s/c Montanide
ISA 35 VIII Injection
91thday 0.70 0.40 0.60 0.10 1.0 s/c Montanide
ISA 35 IX
Injection

Blood sample à Check titres on 99th day

The titers were checked with individual venom and accordingly immunization was continued with increased doses if required.
Table 1: Comparison of antibody yield within existing technology and present invention
Existing Technology Present invention
18 vial per litre of horse plasma 50 vial per litre of horse plasma

The invention having been disclosed in connection with the foregoing embodiments, additional variations will now be apparent to persons skilled in the art. Various modifications and variations to the above described development of anti-snake venom of high potent can be made without departing from the scope of the invention.
From the foregoing it will be understood that the embodiments of the present invention described above are well suited to provide the advantages set forth, and since many possible embodiments may be made of the various features of this invention and as the process herein described may be varied in various parts, all without departing from the scope of the invention, it is to be understood that all matter hereinbefore set forth or shown in the accompanying process is to be interpreted as illustrative and that in certain instances some of the features may be used without a corresponding use of other features, all without departing from the scope of the invention.
The following specific and non-limiting steps for functioning need to be construed as merely illustrative, and do not limit the present disclosure in any way whatsoever.
Date: November 19, 2018
,CLAIMS:We Claim,
1. A highly potent anti snake venom, wherein the process of preparation employs an improved method of immunization of horses to produce anti snake venom antibodies by application of one or more adjuvants of nano range.
2. A highly potent anti snake venom according to claim 1, wherein the said adjuvant of nano range that include, but not limited to, Bentonide, Freund's complete adjuvant, aluminium hydroxide, sodium alginate, calcium phosphate, Montanide and combinations thereof.
3. A highly potent anti snake venom according to claim 1 and 2, wherein the said adjuvant of nano range is preferably Montanide.
4. The process for preparing highly potent anti snake venom, wherein the process comprises of injecting of immunogen to horses wherein the immunogen comprises of snake venom from a single species or mixture of several species of varying concentration, one or more adjuvants in nano range, more preferably Montanide and optionally one or more pharmaceutically acceptable excipients.
5. The process for preparing highly potent anti snake venom according to claim 4, the venom concentrations for
1. Russel’s viper (Daboia russelli) might be within 0.4 to 0.85 mg,
2. Cobra (Naja naja) might be within 0.5 to 0.8 mg,
3. Common Krait (Bungarus caeruleus) might be 0.3 to 1 mg and
4. Saw scaled viper (Echis carinatus) might be within 0.1 to 0.9 mg per mL of horse serum.

6. The process for preparing highly potent anti snake venom according to claim 5, the pharmaceutically acceptable excipients consists of preservatives, more preferably cresol, used to preserve the formulation for a long period of time.