FORM 2
THE PATENTS ACT, 1970 (39 of 1970}
& THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
[See section 10, Rule 13]
MAMMALIAN COLOSTRUM DERIVED
NANOPEPTIDES FOR
BROADSPECTRUM VIRAL AND RECURRENT INFECTIONS WITH A METHOD OF ISOLATION THEREOF;


PAWAN SAHARAN, AN INDIAN
NATIONAL, WHOSE ADDRESS IS A 2101-
04, MANSAROVAR NEELKANTH
HEIGHTS, POKHRAN ROAD NO.1, THANE (W) - 400 601, MAHARASHTRA, INDIA

THE FOLLOWING SPECIFICATION DESCRIBES THE INVENTION


FIELD OF THE INVENTION
The present invention relates to nanopeptides isolated from mammalian colostrum with antiviral and immunodulator activity.
BACKGROUND OF THE INVENTION
Colostrum is the pre-milk substance produced from the mother's breasts of all mammals during the first 24-48 hrs of lactation. Colostrum has been known as an immune booster since time immemorial. Colostrum triggers at least 50 processes in the newborn, including transferring all immune factors and the entire memory from mother's own immune system. Bovine colostrum is upto 40 times higher than human colostrums in immune factors including immunoglobulins, cytokines .interferon, lactoferrin and transfer factor. They are produced by T-lymphocytes and can transfer the ability to recognize a pathogen to naive cells.
Colostrum has antioxidant properties, natural anti-inflammatory properties and is a source of many vitamins, minerals, enzymes and amino acids. Colostrum rebuilds the immune system, helps the body ward off foreign invaders, accelerates healing of all body tissue, increases bone and lean muscle mass and replenishes an aging system.
"Colostrum stimulates the lymphoid tissue providing benefits in aged or immuno-deficient people"....Drs. Bocci, Bremen, Corradeschi, Luzzi and Paulesu; Journal Biology.
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"Researchers reported that colostrum stimulates maturation of B Lymphocytes (type of white blood cell) and primes them for production of antibodies, enhances growth and differentiation of white blood cells. Similar activity in cow and human colostrum can also activate Macrophages" ...Dr. M. Julius, McGil! University, Montreal: Science News.
"Bovine colostrum contains high levels of growth factors that promote normal cell growth and DNA synthesis" ...Drs. Oda, Shinnichi, et. al.;
Comparative Biochemical Physiology. "Drs. suggest that an important.role for growth factors is in promoting wound healing. Accelerated healing is possible for treatment with trauma and surgical wounds" ...Drs. Bhora, et. al.; Journal. Surg. Res.
US 20070212367 - This patent application discloses an immunologically active PRP isolated from mammalian colostral fluids for treatment of viral and non-viral diseases, a method and a system for processing mammalian colostral fluids and a pharmaceutical formulation. It is to be noted here that the invention is related to the PRPs.
W 098014473 - This patent application reJafes to the use of coJostrinin in the treatment of chronic disorders of the central nervous system and immune system. Colostrinin is the term given to PRPs. The nanopeptide as disclosed in this invention are isolated from PRPs. They are not present already in-isolated form.
A BRIEF DESCRIPTION OF THE INVENTION
According to an embodiment of the present invention relates to immunologically active nanopeptides isolated from mammalian colostrum. These series of
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nanopeptides include short chain proteins. These nanopeptides are in a segregated form. They are not binded by any other molecule. The motecular weight of these nanopeptides is in the range of 3115-10208 Daltons. The isolated nanopeptides have been designated as RADHA108 series. The use of these nanopeptides strengthens the immune system against diseases. There are no known side effects or drug interactions since it is isolated from the natural source and it can be taken safely by people of all ages.
The nanopeptide RADHA108 series, blocks the entry of viruses through GP120, 160, 180 and other receptor sites through which virus enters the immune and other cell types. Topographically, RADHA108 series nanopeptide docks on these, receptor sites mimicking the virus / antigen, closing the doors and windows used by foreign infectious agents.
The immunomodulatory action of RADHA108 series is to stimulate the maturation of immature thymocytes into either helper or suppressor T cells, depending on the need of the body. Helper T cells present antigens (such as viral protein) to B lymphocytes, which produce antibodies to that antigen. Helper T cells also help produce memory T cells, which retain the memory of an antigen in order to expedite the production of antibodies in the event the antigen is reencountered in the future. Suppressor T cells, on the other hand, deactivate other lymphocytes after an infection has been cleared to avoid damage to healthy tissues. RADHA108 series also promotes growth and differentiation of B cells in the response to an infection and the differentiation and maturation of macrophages and monocytes The activity of Natural Killer cells, cytotoxic cells of the innate immune system, was increased up to 5 times by RADHA108 series.
RADHA108 series modulates the cytokine system as well. It stimulates the production of a wide range of cytokines, including the pro-inflammatory cytokines tumor necrosis factor - alpha (TNF-a) and interferon gamma (INF - y) and anti¬inflammatory cytokines tnterleukin (IL6, IL10) immunomodulator.
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RADHA108 series are small nanopeptides and they are able to cross the blood brain barrier, and treat viral infections and neural disorders in brain. RADHA108 series helps in modulation of neural cells and thus helps in the treatment of Alzhiemer's and Parkinson's disease.
According to another embodiment of the present invention a pharmaceutical formulation comprises of RADHA108 series as an active pharmaceutical ingredient (API) and pharmaceutically acceptable carriers. Optionally, the formulation further comprises PRP as additional active ingredient. Also, other antiviral, bacterial and fungal drugs can be taken with RADHA108 series. The formulation is used for the treatment of wide range of diseases such as viral, bacterial, mycobacterial, fungal, parasitic, immune disorders including autoimmune disorders etc. RADHA108 series in combination with other active pharmaceutical ingredients act as a carrier
According to another embodiment of the present invention a method of treatment of immune disorders including autoimmune disorders comprises of administration of pharmaceutically effective amount of formulation comprising of the nanopeptides RADHA108 series.
Wide range of diseases such as bacterial, mycobacterial, fungal, parasitic, viral can be effectively treated. The diseases and conditions which can be treated by the formulation comprising of RADHA108 series are, but not limited to AIDS (HIV), tuberculosis, Hepatitis A, B & C,allergies, alzheimer, benign prostatic hyperplasia, cancer, hypertension, lupus (discoid and systemic), thrush, autism, perthe disease, premenstrual syndrome and endometriosis, prion disease (kuru and creutzfeld-Jakob syndrome), psoriasis, Sjogren's syndrome, spinal muscular atrophy, thrombocytopenia (idiopathic and autoimmune), burns, infections, insect bites, diaper rash, herpetic lesions, perthes disease, pharangitis (bacterial), porphyria, Raynaud's phenomenon, sarcoidosis, celiac disease, chronic Pancytopenia, Crohn's disease, diabetes (type II), fibromyalgia rheumatica, mononucleosis, multiple sclerosis, Perth's disease, rheumatoid arthritis*osteo
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arthritis, spinal muscular atrophy (Werding-Hoffman's disease), Brown Recluse spider bite, burns {1st, 2nd, 3rd degree), corneal regeneration, diarrhea, endometriosis (inflammatory aspect), Guillain Barres syndrome, hemolytic anemia, idiopathic thrombocytopenia purpura, kuro (Prion disease), myasthenia Gravis, lupus, tuberculosis, HIV, Hepatitis A, B & C, rabies, Herpes Simplex I&II, Acute & Chronic Viral Infections, Viral Respiratory Infections, Dengue Fever, Human Papilloma Virus, Pharangitis (Viral), SARS, Rabies, Rota viral Diarrhea, Asthma, Tuberculosis, Alzheimer, Hepatic Cellular Carcinoma, Hypertension, Lupus (discoid and systemic), Oral Thrush, Autism, Premenstrual Syndrome and Endometriosis, Rheumatoid & Osteo Arthritis, Spinal Muscular Atrophy.
According to another embodiment of the present invention the dose of the formulation comprising of the nanopeptides RADHA108 series is a dose of 3 ml in a spray form inside the mouth on buccal mucosa (4 times a day) circulated for one minute before swallowing. Taking 12 glasses of pure water daily with yoga / exercise as part of treatment regime activates the nanopeptides .
According to another embodiment of the present invention the formulation can be applied topically or as a skin/transdermal patch. The other route by which RADHA 108 series can be applied is through the nasal, intravenous route.
According to another embodiment of the present invention a process for the isolation of RADHA108 series nanopeptides comprises of collecting the colostrum after delivery from mammal, cooling the colostrum to 4 deg C within 30 min to preserve the biological activity of RADHA108 series. Freezing the colostrum to -20 deg C till future processing, for production, the colustrum is gradually thawed at room temperature over night to bring the temperature to 5 deg C, pumping the colostrum to the cream separator for removal of fats at 1440 RPM at 45 deg C, increasing the temperature to 72 deg C for 15 sec for pasteurization, cooling at 48 deg C and receiving the cofostrums into cheese vat, adding enzyme Rennet to the cheese vat for 60 min, cutting of the curd and
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separation of colostrum whey, cooling the whey to 25 deg C, separating cream from the whey in a cream separator, pasteurizing the whey, passing the whey to a series of filters upto molecular exclusion nano ultrafiltration for separation to NMWL "lOKda to obtain nanopeptide series, gradually adding the preservatives to the nanopeptides RADHA108 series drop by drop over a period of one hour.
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The patients who were administered RADHA108 series were not on any other drugs especially, anti-retroviral and antibiotics therapies at the time of administration of RADHA108 series or its formulation thereof. RADHA108 series is effective in a stand-alone or monotherapy.
WHO approved end points for assessing the therapeutic usage of Radha 108 series were scientifically analysed through the Indian Government sponsored and monitored study on 2 sets of patients at LTMMC and LTMG municipal tertiary care hospital in Sion, Mumbai with end point analysis for weight gain and clinical symptoms carried out the Sion Hospital and viralogical (viral load) and immunological {absolute CD4 count analysis) being, done at two separate internationally recognized center (Institute of Immunoheamatology, Indian Council Of Medical Research Institute in Mumbai and Metropolis Central Laboratory having approval from USA pathological association and accreditation from NABL, department of science and technology Government of India.
The statistical analysis of both these sets of studies were undertaken at the premier medical research institute in India; All India Institute of Medical Sciences New Delhi.
A. Initially, 50 participants were evaluated in the clinic once a week through the first 4 weeks of therapy, and then every 2 weeks for the remaining 8 weeks of the study. They were evaluated about clinical and physical symptoms via symptom assessment form, physical examination, side effect of treatment and compliance to the drug on follow up visit. Blood tests to measure CD4 cell count, HIV viral load, heamoglobin, white blood cell count, liver function test and renal function test were done at baseline and at end of study.
CD4 and CD8 cell count were analyzed via Flow Cytometry and Viral Load via Polymerase Chain Reaction at the beginning and at the end of 12 weeks. .
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Primary efficacy end points of study was the effect of this therapy on the markers of HIV disease including clinical symptoms and physical findings, weight gain/ loss along with the general well being of the patients and HIV viral load and CD4 cell count, which showed consistently positive results in both the trials.
Weight Gain: Consistent weight gain was observed in all patients on follow-up visits, which was one of the primary end point (Table 1). The average weight gain after 12 weeks of treatment was 4.73 kg per patient, which is statistically highly significant (p<0.001).
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Table 1: changes in mean weight after treatment

Duration in weeks Mean weight (N = 50)
basal 50.48 ±10.97
2 50.77 ±11.26
3 51.38 ±10.94
4 52.33 ±10.73
6 53.89 + 11.17
8 54.59+10.89
10 55.44 + 11.07
12 55.21 ± 9.42
Clinical parameters: All the clinical symptoms disappeared during the 12 weeks of treatment. These reductions are statistically significant (p<0.05).
Fatigue / malaise: 88 % of the total study cases had symptoms of fatigue at basal. After treatment at the end of 2nd week proportion of symptoms of fatigue had a significant fall from basal. After 6th week onwards only one or two patients had fatigue (Figure 1).
Figure 1: percentage of cases with fatigue / malaise after the treatment
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Diarrhoea: -18 % of the total study cases had diarrhea at basal and after treatment from 5th week onwards all the patients had relief from diarrhea (Figure 2).
RI7JUNZ00
ii

Figure 2 : Percentage of cases with diarrhea after the treatment

■th
Fever: Fever was reported by 24 % of total study cases at basal and from 7 week onwards not a single patient had fever with significant fall started 4th week onwards.

Cough ; 28 % of the total study cases had a symptom of cough at basal which had significant fall from 3rd week onwards .and all improved 10th week onward (Figure 3).
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Figure 3 : percentage of cases with fever / cough after therapy.


Skin rash and Herpes Zoster: 14 % and 12 % of total study cases had skin rash and herpes zoster at basal respectively and from 4th week onwards not a single patient had suffered from skin rash.
Similarly, nausea, vomiting and disturbed sleep were improved significantly during therapy with RADHA108 series.
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Absolute CD4 cell count and HIV viral load: CD4 cell count was available for 48 patients with pre and post treatment values. There was increase in CD4 count on the average by 51 (median CD4 cell counts from 312 to 363). This is of borderline statistical significance (p = 0.06). 30 out of 48 (62.5 %) patients showed rise in CD4 count and 18 patients (37.5 %) showed decrease in CD4 cell count. CD4 cell count range with number of patients at baseline and at the end of study is shown in figure 4.
Figure 4 : No. of patients at baseline and at end of treatment with CD4 cell count range.

The mean HIV log viral load has statistically significantly dropped from 4.63 to 4.18 after 12 weeks of treatment (p = 0.03). Similar trend is also seen in the pre treatment and post treatment median viral loads (from 92458 vs. 25332, p<0.001).
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B. Another study was carried out with 51 HIV positive patients by the administration of the formulation comprising RADHA108 series for twelve weeks. The results obtained are as follows:
Weight gain: Weight monitored on every visit showed significant gain in ail 51 HIV patients with mean weight gain of 4.68 ±1.9 kg after 12 weeks (Figure 5)
Figure 5: mean weight in kg during RADHA108 series therapy

Clinical parameters: All the clinical symptoms had disappeared during the 12 weeks of treatment (most of them had disappeared within 3 weeks). These reductions are statistically significant (p < 0.05).
Fatigue / malaise: 100 % of the total study cases had symptoms of fatigue at basal. After treatment at the end of 2nd week proportion of symptoms of fatigue had a significant fall from basal. After 6th week onwards all patients had relief from fatigue (Figure 6).
Figure 6 : No. of patients with fatigue / malaise after therapy
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Fever and diarrhea: 100 % of the total study cases had fever and diarrhea at basal and after treatment from 3rd week onwards. All the patients had relief from diarrhea and fever (Figure 7).
Figure 7 : No. of patients with fever / diarrhea with RADHA108 series therapy.

Skin rash and Herpes Zoster: All the patients had HIV associated skin rash and herpes zoster at basal and became asymptomatic after 3rd week onwards with treatment.(Figure 8)
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Figure 8 : No. of patients having HIV associated skin rash and herpes zoster
after therapy

Similar improvement seen in nausea, vomiting, cough and disturbed sleep and all patients became asymptomatic from 3rd week onwards.
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Absolute CD4 cell count: CD4 cell count was available for ali 51 patients with pre and post treatment values. There was increase in CD4 count on the average by 27 (median CD4 cell counts from 276 to 305). This value is statistically significant (p = 0.042). C04 cell count range with number of patients at baseline and at the end of study is shown in figure 9
Figure 9 : No. of patients and CD4 cell count range

HIV viral load: After RADHA108 series 12-week treatment, at the end of study mean viral load showed a significant fall (p < 0.001) from the baseline.
Side effects: All patients tolerated RADHA108 series well with no side effects.
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C. The output of the statistical analyses of the study carried out on 50 HIV positive patients is as follows:

Sr. No. Parameter Before After 12 Weeks P-value
I. Physical (N=47)
I. Weight 50.48(10.97) 55.21 (9.42) <0.001
II. Clinical Symptoms (N=50)
1. Fatigue 44 (88%) 0 (0%) <0.Q01
2. Diarrhoea 9(18.0%) 0 (0%) 0.004
3. Nausea 8(16%) 0 (0%) 0.008
4. Vomiting 7(14%) 0 (0%) 0.02
5. Fever 12(24%) 0 (0%) <0.001
6. Cough 14(28%) 0 (0%) <0.001
7. TB 6(12%)'" 0 (0%) 0.03
8. Disturbed Sleep 13(26%) 0 (0%) <0.001
9. Rash On Skin 7(14%) 0 (0%) 0.02
10. Herpes Zoster 6(12%) 0 (0%) 0.03
III. Virologica!
1. Log of HIV-I,
RNA n=34
Median H1V-I
RNA
25,h Percentile
75( Percentile 5.11 (0.090) 206057 62884 508038 4.103(1.32) 25280 1665 87511 <0.001 <0.001
IV. Immunological
1. CD4 count
(N=48)
Median
25th Percentile
75th Percentile 312.5
275.5 430 363.5
294.2 435 0.06
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These results show that informational nano-peptides are effective and safe drug in increasing weight and general well being of patients, decrease in viral load and increase in CD4 cell count. Thus, informational nanopeptides as active pharmaceutical ingredients hold good promise for the treatment of HIV patients across all age groups.
The nanopeptides are active in the treatment of various diseases and conditions as explicated above. They in no way should be restricted to the treatment of onty HIV. Also, the name of series of nanopeptides (i.e.RADHA108 series) is a terminology used by the Applicant for the ease to identify the series. Any other name .of the series woll not deviate or curb it from being nanopeptides isolated from colostrum.
Dated this 27th day of June, 2008
FOR PAW AN SAHARAN

(SAIMA SAGHIR ANSARI) KRISHNA &SAURASTRI
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