WO2005003173A1

WO2005003173A1 - Process of obtaining a freeze-dried complex from mammal gland secretion

Info

Publication number: WO2005003173A1
Application number: PCT/BR2003/000088
Authority: WO
Inventors: Luiz Fernando Mesquita
Original assignee: Luiz Fernando Mesquita
Priority date: 2003-07-07
Filing date: 2003-07-07
Publication date: 2005-01-13
Also published as: BR0318333A; AU2003236739A1

Abstract

Patent invention for 'Process of Obtaining a Freeze-Dried Complex from Secretion Extracted from mammal Gland, Use thereof in the treatment of Immunodeficiencies, and Method of Treatment Thereof'. The innovation proposed herein refers to a process of obtaining a freeze-dried complex of purified immunoglobulins from lacteal secretions of mammals, preferably from a family of bovines, caprines,equines, and ovines among others, which either mixed or unmixed and subjected to depuration, separation, purification, and heating steps, among others, the use thereof as an aid in treating immunodeficiencies, and method of treatment thereof.

Description

process of obtaining a freeze-dried complex from mammal gland secretion

Descriptive Report of the Patent Invention for, "Process of Obtaining a Freeze-Dried Complex from Secretion Extracted from Mammal Gland, Use Thereof in the Treatment of Immunodeficiencies, and Method of Treatment Thereof". Technical Field This invention refers to a process of obtaining a freeze-dried complex of purified immunoglobulins from lacteal secretions of mammals, the use thereof as an aid in treating immunodeficiencies, and the method of application thereof. Previous Techniques The use of proteins from animal tissues and/or extracts from mammal organs with pharmacological properties is known as described in international publication, WO 92/10197, which describes extracts from mammal organs, preferably she-goat liver, consisting of at least three different proteins and characterized by unique pharmacological and immunological properties . ZIMDUCK, as is named the innocuous compound formulated from the opaque liquid secreted by mammary glands of female mammals, preferably from a family of bovines. caprines, equines, and ovines among others, which either mixed or unmixed and subjected to depuration, separation, purification, and heating steps, among others, is indicated in fighting any type of major infection which could heavily depress the immunological system.

Overall Description of the Invention The process of obtaining the freeze-dried complex which is the object of this certificate follows the steps below: 1- extracting the lacteal secretion; 2- mixing the lacteal secretion extracted from female mammal glands, preferably three in number and in equal ratios, i.e. mixture ratio is generally one to one (1:1) depending on the amount of mammals at the time of composition and the time of lacteal extraction, considering that the chemical composition of a lacteal product varies from one week to the other in terms of nutrients and natural immunologies;

3- pasteurizing at temperatures not exceeding 65°C for up to 15 minutes; 4- separating the lacteal globulins by liquid chromatography coupled to phase transfer and conditioning of the freeze- dried protein material;

5- determining the immunoglobulins by cellulose polyacrylamide electrophoresis, and molecular weight characterization. The compound thus obtained presents the following characteristics as oulined in the tests below: Test Results The samples of freeze-dried complex glycoproteins as characterized at USP have been defined by SDS - PAGE electrophoresis 6.25% and SDS-PAGE electrophoresis 12.5%. By using molecular weight gauges in SDS-PAGE electrophoresis 12.5% and previously treating the samples with β-2 mercaptoethanol, heated at 60 °C for 5 minutes, components with molecular weight of 66,000, 50,000, 29,000 23,500, 18,400, and 14,200 have been detected. Presence of albumin (66,000), heavy chain IgC (50,000), carbon anhydrase (29,000), light chain IgC (23,500), β-lactoglobulin, and α- lactoglobulin (14,200) has been confirmed a priori. In regard to the β-2 mercaptoethanol untreated samples, thus preventing breakage of disulfide bonds, presence of IgA (m.w. 160,000) and transferritin (m.w. 76,000) has been evinced. Presence of IgC, IgA, and transferritin has been confirmed by immunoelectrophoresis using specific antisera. Moreover, Ouchterlony i unodiffusion test confirms the aforementioned data, with IgC, IgA, and albumin standing out for their greater amount, while other proteins were found as trace. Technical reports issued by the Department of Pharmacology of University of Sao Paulo Biological Sciences Institute and the Technological Drug Development Center (USP CEDETEM) have confirmed the freeze-dried complex, object of this invention, to be free of acute, subacute, and chronic toxicity, thus suggesting that the product holds therapeutical characteristics required for treating immunodeficiencies, as shown next. Acute Toxicity Acute toxicity has been determined in male and female rats, mice, and rabbits divided into 8 groups of 8 animals. The intraperitoneally administered dosage ranged as follows: 137.85 - 179.20 - 232.96 - 302.86 - 393.71 - 551.82 - 665.36 - 864.97 - 1,124.26 - 1,461.80 - 1,900.35 - 2,470.45 mg/kg. Clinical signs or symptoms were not observed as regards the central nervous system (behavioral changes, sedation, convulsion, tremor, ataxia, catatonia, paralysis, etc.), the autonomic nervous system (miosis, mydriasis, salivation) , the cardiovascular system (bradycardia, arrhythmia) , the genitourinary system (vulval, mammarian, penile, circumanal inflammation) , skin and hair, mucous membranes and the eyes. Nonetheless, only at higher doses of 1,900.35 to 2,470.45 mg/kg, did loss of apetite, particularly in rabbits, sporadically appear on the first day of drug administration, however going back to normal on the second day. During the trial period mortality of animals was not found. Therefore, considering the amplitude (137.85 to 2,470.45 mg/kg) of the dosage used, it did not induce death among the animals, thus making determination of acute toxicity on the three species (mouse, rat, and rabbit) impossible. In spite of the maximum dose administered to the animals being equivalent to 148.23 g/60 kg, considering a 60 kg man, it is a dose scarcely applied neither therapeutically nor accidentally. Subacute Toxicity Subacute toxicity has been determined on male and female rats where the treatment group was given intraperitoneal doses of 393.71 and 1,124.46 mg/kg during 14 days. Males were observed to gain more weight than the females in both control and treatment groups. Moreover, neither sex gave signs of toxicity following drug administration. Analysis of variance did not detect significant differences in corporal weight gain between the treatment and respective control groups. Biochemical, hematological, and physiological tests carried out at the 15th day did not show any evidence of significant change in relation to controls. Chronic Toxicity Chronic toxicity has been determined on rats after 4 weeks of birth divided into two groups of males and females. These groups were subdivided into three subgroups. Intraperitoneal administration of two different concentrations of Zimduck was made to 6 subgroups of 40 animals each, during 32 weeks . Analysis of variance did not verify any difference between the treatment and control groups, either male or female. Change in signs and symptoms was not verified during that period, and neither the subgroups or the controls recorded death. Worth pointing out is that each subgroup started with 40 animals and finished with 20, since 40 animals from each group were sacrificed at the 4th, 8th, 16th, 24th, and 32nd week in order to run biochemical, hematological, and histological tests, these having not evinced any difference than the controls. The histological test did not show any inflammatory change in tissues surrounding the injection site. All acute, subacute, and chronic toxicity assays were conducted pursuant to World Health Organization requirements, Principles for Pre-Clinical Testing of Drug Safety, Tech. Rep. Ser. 341, 1996. The proposed dose for the product, once freeze- dried and carrying the required technical certificate of purity, should on the average be 2 g diluted in 2 mL distilled water by intramuscular means.

Claims

CLAIMS 1- "Process" consisting of extracting mammal gland secretion, characterized by steps of mixing the lacteal secretion, preferably three mammals in number and in equal ratios, i.e. mixture ratio is generally one to one (1:1), pasteurizing at temperatures not exceeding 65°C for up to 15 minutes, separating the lacteal globulins by liquid chromatography coupled to phase transfer, and determining the immunoglobulins by cellulose polyacrylamide electrophoresis. 2- "Freeze-Dried Complex from Secretion Extracted from Mammal Gland", according to the claim 1, characterized by presenting, when not treated with β-2 mercaptoethanol, IgG (m.w. 160,000) and transferritin (m.w. 76,000). 3- "Freeze-Dried Complex from Secretion Extracted from Mammal Gland", according to the claim 2, characterized by presenting, when treated with β-2 mercaptoethanol, albumin (m.w. 66,000), heavy chain IgG (m.w. 50,000), carbon anhydrase (m.w. 29,000), light chain IgG (m.w. 23,500), β- lactoglobulin (m.w. 18,400), and -lactoglobulin (m.w. 14,200). 4- "Use", as outlined in claims 1 and 2, characterized by being indicated as an aid in the treatment of immunodeficiencies. 5- "Method of Treatment" characterized by applying 2 g diluted in 2 mL distilled water by intramuscular (IM) means .