WO2012007845A2 - A method of increasing the effect of an activated-potentiated form of an antibody - Google Patents

A method of increasing the effect of an activated-potentiated form of an antibody Download PDF

Info

Publication number
WO2012007845A2
WO2012007845A2 PCT/IB2011/002350 IB2011002350W WO2012007845A2 WO 2012007845 A2 WO2012007845 A2 WO 2012007845A2 IB 2011002350 W IB2011002350 W IB 2011002350W WO 2012007845 A2 WO2012007845 A2 WO 2012007845A2
Authority
WO
WIPO (PCT)
Prior art keywords
leu
antibody
pro
ala
activated
Prior art date
Application number
PCT/IB2011/002350
Other languages
French (fr)
Other versions
WO2012007845A3 (en
Inventor
Oleg Iliich Epshtein
Original Assignee
Oleg Iliich Epshtein
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from RU2010129291/15A external-priority patent/RU2525156C2/en
Priority claimed from RU2010129295/15A external-priority patent/RU2531049C2/en
Priority claimed from RU2010129298/15A external-priority patent/RU2526153C2/en
Priority claimed from RU2010129294/15A external-priority patent/RU2542414C2/en
Priority claimed from RU2010129292/15A external-priority patent/RU2523557C2/en
Priority claimed from RU2010129290/15A external-priority patent/RU2525155C2/en
Priority claimed from RU2010130353/15A external-priority patent/RU2542445C2/en
Priority claimed from RU2010130356/15A external-priority patent/RU2542453C2/en
Priority claimed from RU2010130355/15A external-priority patent/RU2530638C2/en
Priority claimed from RU2010130358/15A external-priority patent/RU2519695C2/en
Priority claimed from RU2010130348/15A external-priority patent/RU2531048C2/en
Priority claimed from RU2011110106/15A external-priority patent/RU2523451C2/en
Priority claimed from RU2011127052/15A external-priority patent/RU2503462C2/en
Priority claimed from RU2011127051/15A external-priority patent/RU2509572C2/en
Priority claimed from RU2011127058/15A external-priority patent/RU2536232C2/en
Priority claimed from RU2011127055/15A external-priority patent/RU2536230C2/en
Priority claimed from RU2011127053/15A external-priority patent/RU2565400C2/en
Priority claimed from RU2011127059/15A external-priority patent/RU2536234C2/en
Priority to BR112013000840A priority Critical patent/BR112013000840A2/en
Priority to NZ606767A priority patent/NZ606767A/en
Priority to SG2013002324A priority patent/SG187038A1/en
Priority to MYPI2013000107A priority patent/MY171206A/en
Priority to SE1350184A priority patent/SE1350184A1/en
Priority to UAA201300112A priority patent/UA112753C2/en
Priority to CN2011800443324A priority patent/CN103108657A/en
Priority to KR1020137003861A priority patent/KR20140014059A/en
Priority to EP11775838.3A priority patent/EP2593140A2/en
Priority to AU2011278038A priority patent/AU2011278038B2/en
Priority to EEP201300006A priority patent/EE201300006A/en
Priority to EA201300126A priority patent/EA030566B1/en
Priority to ES201390006A priority patent/ES2440393R1/en
Priority to JP2013519175A priority patent/JP2013538186A/en
Application filed by Oleg Iliich Epshtein filed Critical Oleg Iliich Epshtein
Priority to CA2804966A priority patent/CA2804966A1/en
Priority to DE112011102358T priority patent/DE112011102358T5/en
Priority to GB1302649.7A priority patent/GB2495884B/en
Publication of WO2012007845A2 publication Critical patent/WO2012007845A2/en
Publication of WO2012007845A3 publication Critical patent/WO2012007845A3/en
Priority to NO20130222A priority patent/NO20130222A1/en
Priority to DKPA201370079A priority patent/DK201370079A/en
Priority to FI20135143A priority patent/FI20135143L/en
Priority to FI20135152A priority patent/FI20135152L/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0004—Homeopathy; Vitalisation; Resonance; Dynamisation, e.g. esoteric applications; Oxygenation of blood
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/08—Drugs for disorders of the alimentary tract or the digestive system for nausea, cinetosis or vertigo; Antiemetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies

Definitions

  • the present invention relates to a method of increasing the effect of an activated-potentiated form of an antibody and a phamriaceutical formulation comprising an activated-potentiated form of an antibody to an endogenous biological molecule and an activated-potentiated form of an antibody to endothelial NO-synthase.
  • Nitric oxide is a gaseous molecule that has been shown to acts in the signaling of different biological processes.
  • Endothelium-derived NO is a key molecule in regulation of vascular tone and its association with vascular disease has long been recognized. NO inhibits many processes known to be involved in the formation of atherosclerotic plaque, including monocyte adhesion, platelet aggregation and vascular smooth muscle cell proliferation.
  • Another important role of endothelial NO is the protection of the vascular wall from the oxidative stress induced by its own metabolic products and by the oxidation products of lipids and lipoproteins. Endothelial dysfunction occurs at very early stages of atherosclerosis.
  • NO availability has been shown to modulate metabolism of lipoproteins. Negative correlation has been reported between plasma concentrations of NO metabolic products and plasma total and Low Density Lipoprotein [LDL] cholesterol levels while High Density Lipoprotein [HDL] improves vascular function in hypercholesterolaemic subjects.
  • LDL Low Density Lipoprotein
  • HDL High Density Lipoprotein
  • the loss of NO has considerable effect on the development of the disease. Diabetes mellitus is associated with increased rates of morbidity and mortality caused primarily by the accelerated development of atherosclerotic disease.
  • reports show that diabetics have impaired lung functions. It has been proposed that insulin resistance leads to airway inflammation. Habib et al., Nitric Oxide Measurement From Blood To Lungs, Is There A Link? Pak J Physiol 2007; 3(1 ).
  • Nitric oxide is synthesized by the endothelium from L-arginine by nitric oxide synthase (NO synthase). NO synthase occurs in different isoforms, including a constitutive form (cNOS) and an inducible form (iNOS). The constitutive form is present in normal endothelial cells, neurons and some other tissues.
  • the present invention provides a method of increasing the effect of an activated-potentiated form of an antibody to an endogenous biological molecule, said method comprising combining said endogenous biological molecule with an activated-potentiated form of an antibody to endothelial NO-synthase.
  • the method aspect of the invention includes administering said combination to a patient in need of treatment with said activated-potentiated form of an antibody.
  • said activated-potentiated form of an antibody to an endogenous biological molecule is an antibody to S-100 protein.
  • said activated- potentiated form of an antibody to an endogenous biological molecule is an antibody to prostate specific antigen.
  • said activated-potentiated form an antibody to an endogenous biological molecule is an antibody to insulin receptor.
  • said activated-potentiated form an antibody to an endogenous biological molecule is an antibody to antigiotensin receptor II.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a) an activated-potentiated form of an antibody to an endogenous biological molecule, and b) an activated-potentiated form of an antibody to NO synthase.
  • the pharmaceutical, composition pharmaceutically acceptable solid carrier.
  • the activated-potentiated form of an antibody to endothelial NO synthase contains a mixture of C12, C30, and C200 homeopathic dilutions impregnated onto the solid carrier.
  • the activated-potentiated form of an antibody to an endogenous biological molecule could be a monoclonal, monoclonal, or natural antibody.
  • the antibody to human insulin receptor is a polyclonal antibody.
  • the pharmaceutical composition comprises an activated- potentiated form of an antibody to an endogenous biological molecule prepared by successive centesimal dilutions coupled with shaking of every dilution.
  • the antibody to endothelial NO-synthase is monoclonal, polyclonal or natural antibody. It is particularly preferred that the antibody to endothelial NO-synthase is a polyclonal antibody.
  • the activated-potentiated form of an antibody to endothelial NO-synthase is prepared by successive centesimal dilutions coupled with shaking of every dilution.
  • Figure 1 Illustrates the effect of tested preparations on blood plasma glucose level of rats with streptozotocin-induced diabetes mellitus
  • Figure 2 Illustrates the effect of tested preparations on day 14 of injection on indicators of area under concentration-time curve (AUC) in the glucose tolerance test in rats with streptozotocin-induced diabetes mellitus.
  • AUC area under concentration-time curve
  • Figure 3 Illustrates the effect of tested preparations on blood plasma glucose level of rats with spontaneous non-insulin-dependent diabetes mellitus.
  • Figure 4 Illustrates the effect of tested preparations on day 28 of injection on indicators of area under concentration-time curve (AUC) in glucose tolerance test in rats with spontaneous non-insulin-dependent diabetes mellitus.
  • antibody as used herein shall mean an immunoglobulin that specifically binds to, and is thereby defined as complementary with, a particular spatial and polar organization of another molecule.
  • Antibodies as recited in the claims may include a complete immunoglobulin or fragment thereof, may be natural, polyclonal or monoclonal, and may include various classes and isotypes, such as IgA, IgD, IgE, lgG1 , lgG2a, lgG2b and lgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab')2, Fab', and the like.
  • the singular "antibody” includes plural “antibodies.”
  • activated-potentiated form or “potentiated form” respectively, with respect to antibodies recited herein is used to denote a product of homeopathic potentization of any initial solution of antibodies.
  • Homeopathic potentization denotes the use of methods of homeopathy to impart homeopathic potency to an initial solution of relevant substance.
  • 'homeopathic potentization may involve, for example, repeated consecutive dilutions combined with external treatment, particularly vertical (mechanical) shaking. In other words, an initial solution of antibody is subjected to consecutive repeated dilution and multiple vertical shaking of each obtained solution in accordance with homeopathic technology.
  • the preferred concentration of the initial solution of antibody in the solvent ranges from about 0.5 to about 5.0 mg/ml.
  • the preferred procedure for preparing each component, i.e. antibody solution is the use of the mixture of three aqueous or aqueous-alcohol dilutions of the primary matrix solution (mother tincture) of antibodies diluted 100 12 , 100 30 and 100 200 times, respectively, which is equivalent to centesimal homeopathic dilutions (C12, C30, and C200) or the use of the mixture of three aqueous or aqueous-alcohol dilutions of the primary matrix solution of antibodies diluted 100 12 , 100 30 and 100 50 times, respectively, which is equivalent to centesimal homeopathic dilutions (C12, C30 and C50).
  • an antibody is in the "activated-potentiated” or “potentiated” form when three factors are present.
  • the "activated-potentiated” form of the antibody is a product of a preparation process well accepted in the homeopathic art.
  • the "activated-potentiated” form of antibody must have biological activity determined by methods well accepted in modern pharmacology.
  • the biological activity exhibited by the "activated potentiated” form of the antibody cannot be explained by the presence of the molecular form of the antibody in the final product of the homeopathic process.
  • the activated potentiated form of antibodies may be prepared by subjecting an initial, isolated antibody in a molecular form to consecutive multiple dilutions coupled with an external impact, such as mechanical shaking.
  • the external treatment in the course of concentration reduction may also be accomplished, for example, by exposure to ultrasonic, electromagnetic, or other physical factors.
  • V. Schwabe "Homeopathic medicines", M., 1967, U.S. Patents Nos. 7,229,648 and 4,311 ,897 which are incorporated by reference in their entirety and for the purpose stated, describe such processes that are well-accepted methods of homeopathic potentiation in the homeopathic art. This procedure gives rise to a uniform decrease in molecular concentration of the initial molecular form of the antibody.
  • the required homeopathic potency can be determined by subjecting the intermediate dilutions to biological testing in the desired pharmacological model.
  • 'homeopathic potentization may involve, for example, repeated consecutive dilutions combined with external treatment, particularly vertical (mechanical) shaking.
  • an initial solution of antibody is subjected to consecutive repeated dilution and multiple vertical shaking of each obtained solution in accordance with homeopathic technology.
  • the preferred concentration of the initial solution of antibody in the solvent preferably, water or a water-ethyl alcohol mixture, ranges from about 0.5 to about 5.0 mg/ml.
  • the preferred procedure for preparing each component i.e.
  • antibody solution is the use of the mixture of three aqueous or aqueous-alcohol dilutions of the primary matrix solution (mother tincture) of antibodies diluted 100 12 , 100 30 and 100 200 times, respectively, which is equivalent to centesimal homeopathic dilutions C12, C30 and C200 or the mixture of three aqueous or aqueous-alcohol dilutions of the primary matrix solution (mother tincture) of antibodies diluted 100 12 , 100 30 and 100 50 times, respectively, which is equivalent to centesimal homeopathic dilutions C12, C30 and C50.
  • Examples of how to obtain the desired potency are also provided, for example, in U.S. Patent Nos. 7,229,648 and 4,311 ,897, which are incorporated by reference for the purpose stated.
  • the procedure applicable to the "activated-potentiated" form of the antibodies described herein is described in more detail below.
  • the claimed "activated-potentiated” form of antibody encompasses only solutions or solid preparations the biological activity of which cannot be explained by the presence of the molecular form of the antibody remaining from the initial, starting solution.
  • the "activated-potentiated” form of the antibody may contain traces of the initial molecular form of the antibody, one skilled in the art could not attribute the observed biological activity in the accepted pharmacological models to the remaining molecular form of the antibody with any degree of plausibility due to the extremely low concentrations of the molecular form of the antibody remaining after the consecutive dilutions.
  • the biological activity of the "activated-potentiated” form of the antibodies of the present invention is not attributable to the initial molecular form of the antibody.
  • Preferred is the "activated-potentiated” form of antibody in liquid or solid form in which the concentration of the molecular form of the antibody is below the limit of detection of the accepted analytical techniques, such as capillary electrophoresis and High Performance Liquid Chromatography.
  • Particularly preferred is the "activated- potentiated” form of antibody in liquid or solid form in which the concentration of the molecular form of the antibody is below the Avogadro number.
  • the "activated- potentiated" form of the antibodies contains molecular antibody, if any, at a concentration below the threshold dose for the molecular form of the antibody in the given biological model.
  • the combination pharmaceutical composition in accordance with this aspect of the invention may be in the liquid form or in solid form.
  • Each of the activated potentiated forms of the antibodies included in the pharmaceutical composition is prepared from an initial molecular form of the antibody via a process accepted in homeopathic art.
  • the starting antibodies may be monoclonal, or polyclonal antibodies prepared in accordance with known processes, for example, as described in Immunotechniques, G. Frimel, M., "Meditsyna", 1987, p. 9-33; "Hum. Antibodies. Monoclonal and recombinant antibodies, 30 years after" by Laffly E., Sodoyer R. - 2005 - Vol. 14. - N 1-2. P.33-55, both incorporated herein by reference.
  • Monoclonal antibodies may be obtained, e.g., by means of hybridoma technology.
  • the initial stage of the process includes immunization based on the principles already developed in the course of polyclonal antisera preparation. Further stages of work involve the production of hybrid cells generating clones of antibodies with identical specificity. Their separate isolation is performed using the same methods as in the case of polyclonal antisera preparation.
  • Polyclonal antibodies may be obtained via active immunization of animals.
  • suitable animals e.g. rabbits
  • the animals' immune system generates corresponding antibodies, which are collected from the animals in a known manner. This procedure enables preparation of a monospecific antibody-rich serum.
  • the serum containing antibodies may be purified, for example by using affine chromatography, fractionation by salt precipitation, or ion-exchange chromatography.
  • the resulting purified, antibody-enriched serum may be used as a starting material for the preparation of the activated-potentiated form of the antibodies.
  • the preferred concentration of the resulting initial solution of antibody in the solvent preferably water or a water-ethyl alcohol mixture, ranges from about 0.5 to about 5.0 mg/ml.
  • the preferred procedure for preparing each component of the combination drug according to the present invention is the use of the mixture of three aqueous-alcohol dilutions of the primary matrix solution of antibodies diluted 100 12 , 100 30 and 100 50 times, respectively, which is equivalent to centesimal homeopathic dilutions C12, C30, and C50 or diluted 100 12 , 100 30 and 100 200 times, respectively, which is equivalent to centesimal homeopathic dilutions C12, C30 and C200.
  • a solid carrier is treated with the desired dilution obtained via the homeopathic process.
  • the carrier mass is impregnated with each of the dilutions. Both orders of impregnation are suitable to prepare the desired combination dosage form.
  • the starting material for the preparation of the activated potentiated form that comprise the combination of the invention is polyclonal, animal-raised antibody to the corresponding antigen, namely, NO synthase and endogenous biological molecule.
  • the desired antigen may be injected as immunogen into a laboratory animal, preferably, rabbits.
  • Polyclonal antibodies to NO synthase may be obtained using the whole molecule of bovine NO synthase of the following sequence:
  • Polyclonal antibodies to NO synthase may be obtained using the whole molecule of human NO synthase of the following sequence:
  • Val GLys er lie Thr Tyr Asp Thr Leu Ser Ala Gin Ala Gin Gin
  • Phe Arg lie Trp Asn Ser Gin Leu Val Arg Tyr Ala Gly Tyr Arg
  • Thr Glu Leu Cys lie Gin His Gly Trp Thr Pro Gly Asn Gly Arg
  • 601 605 610 615 lie Ser Cys Ser Asp Pro Leu Val Ser Ser Trp Arg Arg Lys Arg
  • fragment of NO synthase selected, for example, from the following sequences:
  • the exemplary procedure for preparation of the starting polyclonal antibodies to NO synthase may be described as follows. In 7-9 days before blood sampling, 1-3 intravenous injections of the desired antigen are made to the rabbits to increase the level of polyclonal antibodies in the rabbit blood stream. Upon immunization, blood samples are taken to test the antibody level. Typically, the maximum level of immune reaction of the soluble antigen is achieved within 40 to 60 days after the first injection of the antigen. Upon completion of the first immunization cycle, rabbits have a 30-day rehabilitation period, after which re-immunization is performed with another 1 -3 intravenous injections.
  • the immunized rabbits' blood is collected from rabbits and placed in a 50ml centrifuge tube.
  • Product clots formed on the tube sides are removed with a wooden spatula, and a rod is placed into the clot in the tube center.
  • the blood is then placed in a refrigerator for one night at the temperature of about 40°C.
  • the clot on the spatula is removed, and the remaining liquid is centrifuged for 10 min at 13,000 rotations per minute. Supernatant fluid is the target antiserum.
  • the obtained antiserum is typically yellow.
  • 10 ml of the antiserum of rabbits is diluted twofold with 0. 5 M NaCI, after which 6.26g Na 2 S0 4 is added, mixed and incubated for 12-16 hours at 4°C.
  • the sediment is removed by centrifugation, diluted in 10ml of phosphate buffer and dialyzed against the same buffer during one night at ambient temperature. After the sediment is removed, the solution is applied to a DEAE-cellulose column balanced by phosphate buffer.
  • the antibody fraction is determined by measuring the optical density of the eluate at 280 nm.
  • the isolated crude antibodies are purified using affine chromatography method by attaching the obtained antibodies to NO synthase located on the insoluble matrix of the chromatography media, with subsequent elution by concentrated aqueous salt solutions.
  • the resulting buffer solution is used as the initial solution for the homeopathic dilution process used to prepare the activated-potentiated form of the antibodies.
  • the preferred concentration of the initial matrix solution of the antigen-purified polyclonal rabbit antibodies to NO synthase is 0.5 to 5.0 mg/ml, preferably, 2.0 to 3.0 mg/ml.
  • polyclonal antibodies to endogenous biological molecule may also be obtained by a similar methodology to the methodology described for endothelial NO synthase antibodies using an adjuvant.
  • the resulting buffer solution is used as the initial solution for the homeopathic dilution process used to prepare the activated potentiated form of the antibodies.
  • the activated potentiated form of each component of the combination may be prepared from an initial solution by homeopathic potentization, preferably using the method of proportional concentration decrease by serial dilution of 1 part of each preceding solution (beginning with the initial solution) in 9 parts (for decimal dilution), or in 99 parts (for centesimal dilution), or in 999 parts (for millesimal dilution) of a neutral solvent, starting with a concentration of the initial solution of antibody in the solvent, preferably, water or a water-ethyl alcohol mixture, in the range from about 0.5 to about 5.0 mg/ml, coupled with external impact.
  • the external impact involves multiple vertical shaking (dynamization) of each dilution.
  • a 12-centesimal dilution (denoted C12) one part of the initial matrix solution of antibodies to NO synthase with the concentration of 3.0 mg/ml is diluted in 99 parts of neutral aqueous or aqueous-alcohol solvent (preferably, 15%-ethyl alcohol) and then vertically shaked many times (10 and more) to create the 1 st centesimal dilution (denoted as C1 ).
  • the 2nd centesimal dilution (C2) is prepared from the 1 st centesimal dilution C1. This procedure is repeated 1 1 times to prepare the 12th centesimal dilution C12.
  • the 12th centesimal dilution C12 represents a solution obtained by 12 serial dilutions of one part of the initial matrix solution of antibodies with the concentration of 3.0 mg/ml in 99 parts of a neutral solvent in different containers, which is equivalent to the centesimal homeopathic dilution C12. Similar procedures with the relevant dilution factor are performed to obtain the desired dilutions.
  • the intermediate dilutions may be tested in a desired biological model to check activity.
  • the preferred activated potentiated forms for antibodies comprising the combination of the invention is a C12, C30 and C200 dilutions for each activated-potentiated form.
  • each component of the composition e.g., C12, C30, C50, C200
  • the next-to-last dilution is obtained (e.g., until C1 1 , C29, and C199 respectively)
  • one part of each component is added in one container according to the mixture composition and mixed with the required quantity of the solvent (e.g. with 97 parts for centesimal dilution).
  • the active substance is possible to use as mixture of various homeopathic dilutions, e.g. decimal and/or centesimal (D20, C30, C100 or C12, C30, C50 or C12, C30, C200, etc.), the efficiency of which is determined experimentally by testing the dilution in a suitable biological model, for example, in models described in the examples herein.
  • various homeopathic dilutions e.g. decimal and/or centesimal (D20, C30, C100 or C12, C30, C50 or C12, C30, C200, etc.
  • the vertical shaking may be substituted for external exposure to ultrasound, electromagnetic field or any similar external impact procedure accepted in the homeopathic art.
  • the solid unit dosage form of the pharmaceutical composition of the invention may be prepared by using impregnating a solid, pharmaceutically acceptable carrier with the mixture of the activated potentiated form aqueous or aqueous-alcohol solutions of active components that are mixed, primarily in 1 :1 ratio and used in liquid dosage form.
  • the carrier may be impregnated consecutively with each requisite dilution.
  • the pharmaceutical composition in the solid unit dosage form is prepared from granules of the pharmaceutically acceptable carrier which was previously saturated with the aqueous or aqueous-alcoholic dilutions of the activated potentiated form of antibodies.
  • the solid dosage form may be in any form known in the pharmaceutical art, including a tablet, a capsule, a lozenge, and others.
  • inactive pharmaceutical ingredients one can use glucose, sucrose, maltose, amylum, isomaltose, isomalt and other mono- olygo- and polysaccharides used in manufacturing of pharmaceuticals as well as technological mixtures of the above mentioned inactive pharmaceutical ingredients with other pharmaceutically acceptable excipients, for example isomalt, crospovidone, sodium cyclamate, sodium saccharine, anhydrous citric acid etc), including lubricants, disintegrants, binders and coloring agents.
  • the preferred carriers are lactose and isomalt.
  • the pharmaceutical dosage form may further include standard pharmaceutical excipients, for example, microcrystalline cellulose, magnesium stearate and citric acid.
  • the example of preparation of the solid unit dosage form is set forth below.
  • 100-300 ⁇ granules of lactose are impregnated with aqueous or aqueous-alcoholic solutions of the activated potentiated form of antibodies to histamine, activated-potentiated form of antibodies to NO synthase and the activated potentiated form of antibodies to an endogenous biological molecule in the ratio of 1 kg of antibody solution to 5 or 10 kg of lactose (1 :5 to 1 :10).
  • the lactose granules are exposed to saturation irrigation in the fluidized boiling bed in a boiling bed plant (e.g.
  • aqueous-alcohol solution (3.0-6.0 mg/pill) of the combination of the activated- potentiated form of antibodies.
  • Each component of the combination used to impregnate the carrier is in the form of a mixture of centesimal homeopathic dilutions, preferably, C12, C30 and C200.
  • the activated-potentiated form of the antibodies described herein do not contain the molecular form of the antibody in an amount sufficient to have biological activity attributed to such molecular form.
  • the biological activity of the combination drug (combination, pharmaceutical composition) of the invention is amply demonstrated in the appended examples.
  • the combination of the invention is administered from once daily to four times daily, preferably twice daily, each administration including one or two combination unit dosage forms.
  • Sigma-1 ( ⁇ 1 ) receptor - an intracellular one which is localized in the cells of central nervous system, the cells of the most of peripheral tissues and immune component cells. Receptors exhibit a unique ability to be translocated which is caused by many psychotropic medications.
  • the dynamics of sigma-1 receptors is directly linked to various influences which are performed by preparations acting to the sigma-1 receptors. These effects include the regulation of activity channels, ecocytosis, signal transferring, remodeling of the plasma membrane (formation of rafts) and lipid transportation / metabolism. All this can contribute to the plasticity of neurons in a brain.
  • Sigma-1 receptors have a modulating effect on all the major neuromediator systems: noradrenergic, serotonergic, dopaminergic, cholinergic systems and NMDA- adjustable glutamate effects.
  • Sigma-1 receptor plays an important role in the pathophysiology of neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson), psychiatric and affective disorders and stroke; and it also takes part in the processes of learning and memory.
  • the ability of drugs to influence the efficiency of interaction of ligands with sigma-1 receptor indicates on the presence of neuroprotective, anti-ischemic, anxiolytic, antidepressant and anti astenic components in the spectrum of its pharmacological activity that allows the consideration of these drugs as effective preparations particularly for the treatment of cerebrovascular diseases.
  • Results are represented as percentage of specific binding inhibition in control (distilled water was used as control) (Table 1 ).
  • % of specific binding inhibition in control 100% - (specific binding during the test/ specific binding in control) * 100%).
  • the outcomes reflecting inhibition above 50% represent significant effects of the tested compounds; inhibition from 25% to 50% confirm mild to moderate effects; inhibition less than 25% is considered to be insignificant effect of the tested compound and is within background level.
  • AD Alzheimer's disease
  • scopolamine Injection of scopolamine into experimental animals (usually rats or mice) interrupts the ability to learn and leads to deterioration of memory.
  • the training session in the Morris water maze was conducted within 4 days of the scopolamine injection through 60 minutes after administration of tested drugs and 30 minutes after administration of scopolamine (4 sequential tests at interval of 60 seconds).
  • Morris' maze is a round reservoir (diameter - 150 cm, height - 45 cm) at 30 cm filled with water (26-28 ° C).
  • At 18 cm from the edge of the container there is hidden platform (diameter - 15 cm) buried on 1.5 cm below the water level. Cloudy water made by adding a non-toxic dye (e.g., milk powder) makes the platform invisible.
  • a non-toxic dye e.g., milk powder
  • the animal could not find the platform within 120 seconds, the animal was put on the platform and left for 60 seconds and the test was restarted.
  • the animals began to walk through the maze twice from each starting point. The tests were recorded on videotape and then analyzed for distance overcomes searching the platform in each trial and the latent period of searching for the platform.
  • the test was performed: the platform was removed from the maze and rats were given free float for 60 seconds. The time spent in the place where the platform used to be was recorded.
  • ULD ultra low doses
  • Acute cerebrovascular disease (brain stroke) ranks third among lethality causes in developed countries and one of the main causes of disability in humans (Gusev E.I., 2003; Janardhan V., Qureshi A.I., 2004).
  • the photo-induced thrombosis model meets almost all requirements to the experimental model of focal cerebral ischemia.
  • the method developed by Watson (Watson B. et ai, 1985) is based on the effect of light with wavelength 560 nm on photosensitive pigment Bengal rose introduced into the blood flow. Active oxygen forms are created and caused increase in adhesiveness of endothelium cells and platelets, and formation of clots closing vascular lumens.
  • the method of ischemic brain lesion induction by using photo-induced thrombosis is technically simple and to close to clinical forms of ischemic brain stroke.
  • a great advantage of this model is that it is non-invasive, i.e. does not require craniotomy and, therefore, more accurately reproduces clinical picture of cerebral thrombosis.
  • halogen lamp 24 V, 250 W
  • halogen lamp 24 V, 250 W
  • Sham-operated rats were subject to the same procedure except administration of bengal rose and exposure to halogen lamp light.
  • the intact group included 6 rats.
  • the animals were treated as follows. Group 1 - 2.5 ml/kg of the potentiated activated form of antibodies to a C-terminal fragment AT1 of human angiotensin II receptor (a mixture of aqueous dilutions C12, C30, C200) in combination with 2.5 ml/kg of distilled water, Group 2 - 2.5 ml/kg of the potentiated activated form of antibodies to endothelial NO-synthase (a mixture of aqueous dilutions C12, C30, C200) in combination with 2.5 ml/kg of distilled water, Group 3 - 5 ml/kg of the combination pharmaceutical composition (a mixture of aqueous dilutions C12, C30, C200 for each component), and Group 4 - 5 ml/kg of distilled water.
  • Group 1 - 2.5 ml/kg of the potentiated activated form of antibodies to a C-terminal fragment AT1 of human angiotensin II receptor
  • SBP Systolic blood pressure
  • the animals were given per orally, once a day and for 28 days, the following medications: Group 1 - 2.5 ml/kg of the activated potentiated form of antibodies to a C- terminal fragment AT1 of human angiotensin II receptor (a mixture of dilutions C12, C30, C200) in combination with 2.5 ml/kg of distilled water; Group 2 - 2.5 ml/kg of the activated potentiated form of antibodies to endothelial NO-synthase (a mixture of dilutions C12, C30, C200) in combination with 2.5 ml/kg of distilled water; Group 3 - 5 ml/kg of the combination pharmaceutical composition (a mixture of homeopathic aqueous dilutions C12, C30, C200 of each component); Group 4 - 5 ml/kg (10 ml/kg dose) of the comparison drug (losartan); and Group 5 - 5 ml/kg of distilled water.
  • SBP systolic blood pressure
  • the experimental notey investigated the effects of antibodies to the C-terminal fragment to the insulin receptor ⁇ -subunit affinity purified on antigen, in ultra-low dose, obtained by super dilution of the initial matrix solution 100 12 , 100 30 , 100 200 times (ULD anti-IR), antibodies to endothelial NO-synthase affinity purified on antigen, in ultra-low dose, obtained by hyper-dilution of the initial matrix solution 100 12 , 100 30 , 100 200 (ULD anti-ULD anti-eNOS), as well as the combination of ultra-low doses of antibodies to the C-terminal fragment to the insulin receptor ⁇ -subunit and ultra-low dose of antibodies to endothelial NO-synthase (ULD anti-IR + ULD anti-eNOS).
  • rats with blood plasma glucose level not less than 12 mmol/l were selected, divided into 7 groups (20 rats in each), which over 21 days were given distilled water (5 ml/kg/day, once daily intragastrically), insulin® (8 units/kg/day, subcutaneously), Rosiglitazone® (8 mg/kg/day, twice daily intragastrically), ULD anti-IR (2.5 ml/kg/day in a volume of 5 ml/kg/day, once daily intragastrically), ULD anti-IR + ULD anti-eNOS (5 ml/kg/day, once daily intragastrically), and also Rosiglitazone® and insulin® together or ULD anti-IR + ULD anti-eNOS and insulin®, according to regimes corresponding to each preparation (as described above).
  • Intact rats received distilled water in the same volume. On days 7, 14 and 21 of injection of preparations in rats, fasting blood plasma glucose level measured with enzymatic method (glucose oxidase method) with utilization of "glucose FKD" kits ( Russia).
  • Oral glucose tolerance test was performed on day 4 of the study (day 14 of administration of preparation) according to standard method (Du Vigneaud and Karr, 1925). The rats were starving at water for 18 hours. 60 min before the test they were last given test substances. Intact rats received distilled water in the same volume. Glucose was administered per os 50% w/w water glucose solution (1 g/kg of rat weight). Serum glucose of blood sample from tail vein was measured by using "Glucose FKD" kit (OOO "Pharamaceutical and clinical diagnostics, Russia, www.fkd.ru) at 0, 30, 60, 90, 120 min. Mean area under the curve (AUC) concentration of blood glucose over time was calculated.
  • Insulin injected at 1 / 2 of the effective dose (selected in the preliminary study) most effectively lowered glucose level in all observation periods (down to the level of the intact control). (Figure 1). It should be taken into account that short-acting insulin was used in the study and blood plasma glucose was measured 1 hour after its injection, which also influenced the effect of the 1 ⁇ 2 insulin dose on blood glucose level. Against this background it was not possible to fully determine what the effect of the combined use of insulin and rosiglitazone or insulin and complex ULD anti-IR + anti-eNOS is. Glucose tolerance disturbance (reduction in glucose utilization by the body) is one of the most important indicators in diagnostic and treatment of diabetes mellitus.
  • the experimental notey investigated the effects of antibodies to the C-terminal fragment to the insulin receptor ⁇ -subunit affinity purified on antigen, in ultra-low dose, obtained by super dilution of the initial matrix solution 100 12 , 100 30 , 100 200 times (ULD anti-IR), antibodies to endothelial NO-synthase affinity purified on antigen, in ultra-low dose, obtained by hyper-dilution of the initial matrix solution 100 12 , 100 30 , 100 200 (ULD anti-ULD anti-eNOS), as well as the combination of ultra-low doses of antibodies to the C-terminal fragment to the insulin receptor ⁇ -subunit and ultra-low dose of antibodies to endothelial NO-synthase (ULD anti-IR + ULD anti-eNOS).
  • Dizziness is the typical sign of lesion of the vestibular analyzer of various genesis including dysfunction of the vestibular nerve and cochlear system, circulatory embarrassment in vertebral basilar system, pathology of the central nervous system (CNS), etc.
  • Dizziness as a manifestation of kinetosis accompanied with other vestibular- vegetative disorders which include three types of reactions: the vestibular-motor (nystagmus and the reaction of deviation), vestibular-sensory (in addition to dizziness, nystagmus is (or reaction of post rotation), defensive movements) and vegetative (nausea, vomiting, sweating, palpitation, heat feeling, pulse and blood pressure fluctuations).
  • Group I was given ULD anti-S100+anti-eNOS
  • Group 2 was given ULD anti-S100
  • Group 3 was given anti-eNOS.
  • CCEAC Coriolis
  • the safety criteria were character, evidence and terms of emergence of probable adverse events (AE) in the treatment period connected with medication intake; influence of studied drugs for indexes which characterize the function of central nervous system (CNS) (reaction on moving object (RMO)), the time of simple motor reaction (TSMR); the dynamics of physical and functional factors (heart rate (HR), systolic and diastolic blood pressure (SBP, DBP), Stange's test; exercise tolerance (index of Harvard step-test). Safety was assessed after single dose administration and after 7-day course administration of the combination ULD anti-S-100 and ULD anti-eNOS.
  • CNS central nervous system
  • TSMR time of simple motor reaction
  • HR heart rate
  • SBP systolic and diastolic blood pressure
  • Stange's test exercise tolerance (index of Harvard step-test).
  • Safety was assessed after single dose administration and after 7-day course administration of the combination ULD anti-S-100 and ULD anti-eNOS.
  • the study was parallel and conducted in the first half of a day with participation of, as a rule, 4 persons in a day, one person for drug or placebo.
  • the next three weeks were washout period, at the end of which the new drug or placebo was prescribed to subjects of each group; the cycle of study was being repeated (Visit 1 , the course intake of a drug; Visit 2).
  • Visit 1 the course intake of a drug
  • Visit 2 the course intake of a drug
  • each subject took part in four cycles of study. That is, each subject participated in each group with a three-week washout period between each cycle. This allowed the researcher to level the influence of individual peculiarities of a test person on the treatment effect.
  • the monocomponent preparation ULD anti-S100 had anti motion sickness action as better indexes of tolerance of CCEAC test, recovery time of nystagmus and recovery than in the placebo group evidenced (Table 8, Visits 1 and 2), but the efficacy of ULD anti-S100 was inferior to composition of ULD anti-S100 + anti-eNOS.
  • the monocomponent preparation ULD anti-eNOS did not show anti motion sickness effect since the results of CCEAC tests and subsequent recovery period had no significant difference from the placebo group (Table 8, Visits 1 and 2).
  • Visit 1 one-day intake
  • Visit 2 course intake
  • the safety analysis included data from all the subjects who participated in the study. During the observation period a well tolerance of studied preparations was noticed. No adverse events associated with drug administration identified. All the subjects of studied groups completed treatment in the terms established by the study protocol; there was not persons early dropped out.
  • Step-test index 50.0 ⁇ 2.03 — 50.1 ⁇ 1.99
  • the study using an experimental motion sickness demonstrated the effectiveness of the combination composition ULD anti-S100 + anti-eNOS and monocomponent preparation ULD-S100.
  • the studied drugs increase the stability of the subjects to the kinetic effect after simulation of the clinical and physiological effects of motion sickness contributing to more mild clinical process of motion sickness and earlier recovery of the subjects after cessation of treatment.
  • the anti motion sickness effect of the combination composition increases the efficiency of individual components.
  • the effectiveness of the combination composition ULD anti-S100 + anti-eNOS in the control of the vestibular- autonomic and sensory reactions of a body in experimental motion sickness increases at course intake.
  • ULD anti-eNOS in the form of monopreparation does not have a protective effect against motion sickness but when combined with ULD anti-S100 significantly enhances the anti motion sickness effect of the last one which manifests itself as at one-day so at short course intake of the drug.
  • the best ability to adjust the transient processes that is to influence to the reactivity of the parasympathetic and sympathetic parts of ANS as well as adaptive capabilities of ANS in a state of motion sickness (to increase the tolerance to sudden changes in a body position) was observed in the composition ULD anti-S100 + anti-eNOS which is an important component of anti motion sickness properties of the drug.
  • Composition ULD anti-S100 + anti-eNOS and monocomponent preparation ULD anti-S100 when using them as anti motion sickness preparation including when performing an operator functions are safe and do not adversely impact on the physical and psycho- physiological parameters.
  • Combination composition ULD anti-S100 + anti-eNOS and ULD anti-S100 can be recommended for the prophylaxis and relief of kinesia in motion disease (including sea, air and car sicknesses) to persons with low and moderate degree of stability.
  • the combination composition has high safety and no adverse effects on the quality of professional activity.
  • control group patients received 300 mg tablets impregnated with pharmaceutical composition containing water-alcohol solutions (6 mg/tablet) of activated-potentiated forms of polyclonal affinity purified rabbit brain-specific proteins antibodies S-100 (anti-S100) in ultra low doses (ULD) obtained by super dilution of initial solution (with concentration of 2.5 mg/ml) in 100 12 , 100 30 , 100 200 times.
  • water-alcohol solutions (6 mg/tablet) of activated-potentiated forms of polyclonal affinity purified rabbit brain-specific proteins antibodies S-100 (anti-S100) in ultra low doses (ULD) obtained by super dilution of initial solution (with concentration of 2.5 mg/ml) in 100 12 , 100 30 , 100 200 times.
  • Psychoorganic syndrome is characterized by the following triad of signs: weakness of memory, loop of intelligence, incontinence of affect (Walther Buel triad).
  • MINI international neuropsychiatric mini-interview
  • immunomodulators including plant and homeopathic preparations.
  • Patients are the staff of the center which directly related to the conducted study and/or are family members of the research center staff's which directly associated with the ongoing study.
  • the "family members” are a husband (wife), parents, children, brothers (sisters).
  • Treatment phase lasted from Visit 1 to Visit 4 for 84 ⁇ 5 days on average.
  • Visit 4 (Day 84 ⁇ 5) was the first endpoint of the study followed by a follow-up observation.
  • follow-up phase continued from Visit 4 to Visit 5 (Day 168 ⁇ 5 on average).
  • control group patients received 300 mg tablets impregnated with pharmaceutical composition containing water-alcohol solutions (3 mg/tablet) of activated-potentiated forms of polyclonal affinity purified rabbit brain-specific proteins antibodies S-100 (anti-S100) in ultra low doses (ULD) obtained by super dilution of initial solution (with concentration of 2.5 mg/ml) in 100 12 , 100 30 , 100 200 times, of equivalent mixture of centesimal homeopathic dilutions C12, C30, C200
  • Alzheimer's disease is characterized by dementia (acquired dementia, stable impairment of cognitive activity with certain loss of previously acquired knowledge and practical skills, difficulties or impossibility to gain new knowledge).
  • MINI international neuropsychiatric mini-interview
  • immunomodulators including plant and homeopathic preparations.
  • Patients are the staff of the center which directly related to the conducted study and/or are family members of the research center staff's which directly associated with the ongoing study.
  • the "family members” are a husband (wife), parents, children, brothers (sisters).
  • the patients were randomized into two study groups: a group of patients receiving ULD anti- Si 00 (3 patients, women - 100%, men - 0%, mean age - 59.0 ⁇ 3.6 years old), a group of patients receiving ULD anti-S100 + anti-eNOS (3 patients, women - 66.66 % men - 33.33 %, mean age - 59.0 ⁇ 4.36 years old).
  • Treatment phase lasted from Visit 1 to Visit 4 for 84 ⁇ 5 days on average.
  • Visit 4 (Day 84 ⁇ 5) was the first endpoint of the study followed by a follow-up observation.
  • follow-up phase continued from Visit 4 to Visit 5 (Day 168 ⁇ 5 on average).
  • Group 1 - the active drug group was given 300 mg tablets impregnated with an aqueous-alcohol solutions (6 mg/tab) of activated-potentiated form of polyclonal rabbit antibodies to brain specific S-100 protein (anti-S-100), and to endothelial NO-synthase (anti-eNOS) in ultra low dose (ULD anti-S-100 + ULD anti-eNOS), purified on antigen, obtained by super dilution of initial solution (with concentration of 2.5 mg/ml) in 100 12 , 100 30 , 100 200 time, equivalent to mixture of centesimal homeopathic dilutions C12, C30, C200;
  • Group 2 the comparison group was given 300 mg tablets impregnated with an aqueous-alcohol solution (3 mg/tab) of activated-potentiated forms of polyclonal rabbit antibodies to brain-specific S-100 protein purified on antigen in ultra low dose (ULD anti- S100) obtained by super dilution of initial solution in 100 12 , 100 30 , 100 50 times, of equivalent mixture homeopathic dilutions C12, C30, C50.
  • ULD anti- S100 ultra low dose
  • Group 3 - the control group (placebo) was given of 300 mg tablets having excipients (lactose monohydrate - 267 mg, microcrystal cellulose - 30 mg, magnesium stearate - 3 mg).
  • composition ULD anti-S100 + anti-eNOS or ULD anti-S100 influenced on both clusters of symptoms of ADHD which was confirmed by dynamics of assessments by two sections of the scale with ADHDRS-IV-Home Version. Moreover, the treatment with the composition ULD anti-S100 + anti-eNOS was significantly higher than the effectiveness of therapy with monopreparation ULD anti-S100 in the degree of influence on the intensity of implications and attention deficit and hyperactivity/impulsivity.
  • PWV pulse wave velocity
  • Table 20 shows the dynamics in the rates of pulse wave velocity in the carotid- femoral (CF) (elastic type) and carotid-radial (CR) (muscle type) segments of arteries.
  • CF carotid- femoral
  • CR carotid-radial
  • (#) denotes difference from the group receiving ULDs of Abs to C-terminal fragment AT1 receptor angiotensin II with verifiable difference in p value of ⁇ 0.05.
  • ULD denotes ultra-low doses.
  • Group 3 After 6 months of treatment, only group 3 showed a proven effect of the claimed pharmaceutical composition on the stiffness of muscular type arteries.
  • Group 1 which received ULD of antibodies to a C-terminal fragment of angiotensin II AT1 -receptor, and group 3 which received the combination pharmaceutical composition of the invention showed a proven increase in the stiffness of elastic type arteries.
  • 80 patients CHF of ll-IV functional class (FC), left ventricular ejection fraction (LVEF) less than 40%
  • FC left ventricular ejection fraction
  • LVEF left ventricular ejection fraction
  • the background therapy was not discontinued (bisoprolol ⁇ -blocker, ACE inhibitor enalapril, aspirin (unless contraindicated); administration of diuretics, nitrates, digoxin was also admitted).
  • the groups were comparable in the initial study parameters: in age and sex, and severity (class of CHF and LVEF) and duration of the disease. Before and after treatment, the patients were evaluated for the life quality (Minnesota and Kansas questionnaires), morphological parameters of the heart, and tolerance to physical exercise.
  • Table 3 shows the results of the study in the form of dynamics in the basic parameters of the treatment efficacy.
  • the patients in group 1 treated with ULD of antibodies to a C-terminal fragment of angiotensin II AT1 -receptor showed a significant improvement of the life quality, improvement of the left ventricular systolic function, and an increased tolerance to physical exercise.
  • Group 2 showed a proven decrease in the anxiety and depression levels and in the life quality, which were evaluated using the Kansas questionnaire.
  • the combination of activated (potentiated) forms of antibodies to a C-terminal fragment of angiotensin II AT1 -receptor and to endothelial nitric oxide synthase (NO- synthase) in the pharmaceutical composition of the invention (combination drug) provides an unexpected synergistic therapeutic effect implying an enhanced influence on vascular remodeling and endothelium dysfunction that is critical for the CHF process and progression, as also on the improvement of the patients' life quality, on morphological parameters of the heart and tolerance to physical exercise, which is confirmed by clinical trials.
  • $, $$ - difference from the group receiving ULDs of Abs to endothelial NO-synthase is verifiable at p values of 0.05 and 0.01, respectively.
  • HADS denotes HADS total score
  • FC CHF denotes patients with chronic heart failure, functional class
  • FF LV denotes fraction of functioning of left vertical.
  • Benign prostatic hyperplasia is one of the most frequently occurring disorders in males (Bruskewitz R.C., 2003; Rosen R., 2003): on the one hand, epidemiological studies, carried out in Russia, point to a gradual increase in frequency of BPH from 11.3% in 40-49 year olds to 81.4% in 80 year olds (Gorilovskiy, L.M., 1999); on the other hand, demographic studies conducted by WHO confirm a significant increase in the population over 60 years old, surpassing any other age group growth.
  • the main symptoms of benign prostatic hyperplasia are lower urinary tract symptoms, which can cause significant discomfort and decrease quality of life (Bruskewitz R.C., 2003; Lepor H., 2004; O'Leary M.P., 2005).
  • the disease can be accompanied by complications, such as acute urinary retention, urinary tract infection, erythruria, kidney failure (Stepanov, V.N., 1999; Jacobsen S.J., 1997; Lepor H., 2004).
  • BPH is also associated with development of erectile dysfunction in patients (Bruskewitz R.C., 2003; Daly MP, 2005).
  • a necessary inclusion criterion was absence of intake of the following medications in the medical records: finasteride, dutasteride, or other experimental drug 6 months prior to inclusion in the study, a1 -adrenoreceptor blockers and herbal medications 4 weeks prior to the inclusion into the study, any inhibitors of phosphodiesterase type 5 and other erectile dysfunction treatments 4 weeks prior to the inclusion into the study.
  • the study did not include patients undergone invasive methods of treatment of BPH, including transurethral prostatic resection, thermotherapy, transurethral needle ablation, stent angioplasty and other; with malignant oncological disease, acute urination delay, bladder stones, urethral stricture, Marion's disease, genitourinary system infections in the phase of active inflammation and others.
  • ULD anti-PSA + ULD anti-eNOS is also more effective compared to ULD anti-PSA in improving erectile function in patients.
  • the total IIEF (International Index of Erectile Dysfunction) score increased by 19% in patients (in ULD anti-PSA group by 10.5%), an average increase of IIEF score in ULD anti-PSA + ULD anti-eNOS group was 8% vs 4.5% in a ULD anti-PSA group.
  • the pharmaceutical compositions showed excellent safety profile, no adverse effects related to the administered medications were observed in the course of study.
  • ULD anti-PSA + ULD anti-eNOS showed better efficacy compared to that of ULD anti-PSA in treating urination problems caused by benign prostatic hyperplasia.
  • a greater positive effect of ULD anti-PSA + ULD anti-eNOS on erectile function of patients compared to ULD anti-PSA was revealed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Epidemiology (AREA)
  • Endocrinology (AREA)
  • Hematology (AREA)
  • Neurosurgery (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Diabetes (AREA)
  • Psychiatry (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Hospice & Palliative Care (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Otolaryngology (AREA)
  • Reproductive Health (AREA)
  • Gynecology & Obstetrics (AREA)

Abstract

The preset invention provides a method of increasing the effect of an activated-potentiated form of an antibody to an endogenous biological molecule by combining said endogenous biological molecule with an activated-potentiated form of an antibody to endothelial NO-synthase. The present invention also provides a pharmaceutical composition comprising a) an activated-potentiated form of an antibody to an endogenous biological molecule, and b) an activated-potentiated form of an antibody to NO synthase.

Description

A METHOD OF INCREASING THE EFFECT OF AN ACTIVATED- POTENTIATED FORM OF AN ANTIBODY
FIELD
The present invention relates to a method of increasing the effect of an activated-potentiated form of an antibody and a phamriaceutical formulation comprising an activated-potentiated form of an antibody to an endogenous biological molecule and an activated-potentiated form of an antibody to endothelial NO-synthase.
BACKGROUND
Nitric oxide (NO) is a gaseous molecule that has been shown to acts in the signaling of different biological processes. Endothelium-derived NO is a key molecule in regulation of vascular tone and its association with vascular disease has long been recognized. NO inhibits many processes known to be involved in the formation of atherosclerotic plaque, including monocyte adhesion, platelet aggregation and vascular smooth muscle cell proliferation. Another important role of endothelial NO is the protection of the vascular wall from the oxidative stress induced by its own metabolic products and by the oxidation products of lipids and lipoproteins. Endothelial dysfunction occurs at very early stages of atherosclerosis. It is therefore possible that deficiency in local NO availability could be a final common pathway that accelerates atherogenesis in humans. In addition to its role in the vascular endothelium, NO availability has been shown to modulate metabolism of lipoproteins. Negative correlation has been reported between plasma concentrations of NO metabolic products and plasma total and Low Density Lipoprotein [LDL] cholesterol levels while High Density Lipoprotein [HDL] improves vascular function in hypercholesterolaemic subjects. The loss of NO has considerable effect on the development of the disease. Diabetes mellitus is associated with increased rates of morbidity and mortality caused primarily by the accelerated development of atherosclerotic disease. Moreover, reports show that diabetics have impaired lung functions. It has been proposed that insulin resistance leads to airway inflammation. Habib et al., Nitric Oxide Measurement From Blood To Lungs, Is There A Link? Pak J Physiol 2007; 3(1 ).
Nitric oxide is synthesized by the endothelium from L-arginine by nitric oxide synthase (NO synthase). NO synthase occurs in different isoforms, including a constitutive form (cNOS) and an inducible form (iNOS). The constitutive form is present in normal endothelial cells, neurons and some other tissues.
The therapeutic effect of an extremely diluted form (or ultra-low form) of antibodies potentized by homeopathic technology (activated potentiated form) has been discovered by Dr. Oleg I. Epshtein. U.S. Patent No. 7,700,096 discloses a homeopathically potentized form of antibodies to endothelial NO-synthase. The homeopathically potentized form of antibodies to endothelial NO-synthase is marketed in the Russian Federation and other countries under the name Impaza®.
There is a continuing need for a method of increasing the effect of an activated- potentiated form of an antibody.
SUMMARY
In accordance with one aspect, the present invention provides a method of increasing the effect of an activated-potentiated form of an antibody to an endogenous biological molecule, said method comprising combining said endogenous biological molecule with an activated-potentiated form of an antibody to endothelial NO-synthase. Preferably, the method aspect of the invention includes administering said combination to a patient in need of treatment with said activated-potentiated form of an antibody.
In one variant, said activated-potentiated form of an antibody to an endogenous biological molecule is an antibody to S-100 protein. In another variant, said activated- potentiated form of an antibody to an endogenous biological molecule is an antibody to prostate specific antigen. In another variant, said activated-potentiated form an antibody to an endogenous biological molecule is an antibody to insulin receptor. In another variant, said activated-potentiated form an antibody to an endogenous biological molecule is an antibody to antigiotensin receptor II. In accordance with another aspect, the invention provides a pharmaceutical composition comprising a) an activated-potentiated form of an antibody to an endogenous biological molecule, and b) an activated-potentiated form of an antibody to NO synthase. Preferably, the pharmaceutical, composition pharmaceutically acceptable solid carrier. Preferably, the activated-potentiated form of an antibody to endothelial NO synthase contains a mixture of C12, C30, and C200 homeopathic dilutions impregnated onto the solid carrier. The activated-potentiated form of an antibody to an endogenous biological molecule could be a monoclonal, monoclonal, or natural antibody. Preferably, the antibody to human insulin receptor is a polyclonal antibody.
It is contemplated that the pharmaceutical composition comprises an activated- potentiated form of an antibody to an endogenous biological molecule prepared by successive centesimal dilutions coupled with shaking of every dilution. It is also contemplated that the antibody to endothelial NO-synthase is monoclonal, polyclonal or natural antibody. It is particularly preferred that the antibody to endothelial NO-synthase is a polyclonal antibody. It is contemplated that the activated-potentiated form of an antibody to endothelial NO-synthase is prepared by successive centesimal dilutions coupled with shaking of every dilution.
DESCRIPTION OF THE FIGURES
Figure 1 - Illustrates the effect of tested preparations on blood plasma glucose level of rats with streptozotocin-induced diabetes mellitus
Figure 2 - Illustrates the effect of tested preparations on day 14 of injection on indicators of area under concentration-time curve (AUC) in the glucose tolerance test in rats with streptozotocin-induced diabetes mellitus.
Figure 3 - Illustrates the effect of tested preparations on blood plasma glucose level of rats with spontaneous non-insulin-dependent diabetes mellitus.
Figure 4 - Illustrates the effect of tested preparations on day 28 of injection on indicators of area under concentration-time curve (AUC) in glucose tolerance test in rats with spontaneous non-insulin-dependent diabetes mellitus. DETAILED DESCRIPTION
The invention is defined with reference to the appended claims. With respect to the claims, the glossary that follows provides the relevant definitions.
The term "antibody" as used herein shall mean an immunoglobulin that specifically binds to, and is thereby defined as complementary with, a particular spatial and polar organization of another molecule. Antibodies as recited in the claims may include a complete immunoglobulin or fragment thereof, may be natural, polyclonal or monoclonal, and may include various classes and isotypes, such as IgA, IgD, IgE, lgG1 , lgG2a, lgG2b and lgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab')2, Fab', and the like. The singular "antibody" includes plural "antibodies."
The term "activated-potentiated form" or "potentiated form" respectively, with respect to antibodies recited herein is used to denote a product of homeopathic potentization of any initial solution of antibodies. "Homeopathic potentization" denotes the use of methods of homeopathy to impart homeopathic potency to an initial solution of relevant substance. Although not so limited, 'homeopathic potentization" may involve, for example, repeated consecutive dilutions combined with external treatment, particularly vertical (mechanical) shaking. In other words, an initial solution of antibody is subjected to consecutive repeated dilution and multiple vertical shaking of each obtained solution in accordance with homeopathic technology. The preferred concentration of the initial solution of antibody in the solvent, preferably water or a water- ethyl alcohol mixture, ranges from about 0.5 to about 5.0 mg/ml. The preferred procedure for preparing each component, i.e. antibody solution, is the use of the mixture of three aqueous or aqueous-alcohol dilutions of the primary matrix solution (mother tincture) of antibodies diluted 10012, 10030 and 100200 times, respectively, which is equivalent to centesimal homeopathic dilutions (C12, C30, and C200) or the use of the mixture of three aqueous or aqueous-alcohol dilutions of the primary matrix solution of antibodies diluted 10012, 10030 and 10050 times, respectively, which is equivalent to centesimal homeopathic dilutions (C12, C30 and C50). Examples of homeopathic potentization are described in U.S. Patent. Nos. 7,572,441 and 7,582,294, which are incorporated herein by reference in their entirety and for the purpose stated. While the term "activated-potentiated form" is used in the claims, the term "ultra-low doses" is used in the examples. The term "ultra-low doses" became a term of art in the field of art created by study and use of homeopathically diluted and potentized form of substance. The term "ultra-low dose" or "ultra-low doses" is meant as fully supportive and primarily synonymous with the term 'activated-potentiated" form used in the claims.
In other words, an antibody is in the "activated-potentiated" or "potentiated" form when three factors are present. First, the "activated-potentiated" form of the antibody is a product of a preparation process well accepted in the homeopathic art. Second, the "activated-potentiated" form of antibody must have biological activity determined by methods well accepted in modern pharmacology. And third, the biological activity exhibited by the "activated potentiated" form of the antibody cannot be explained by the presence of the molecular form of the antibody in the final product of the homeopathic process.
For example, the activated potentiated form of antibodies may be prepared by subjecting an initial, isolated antibody in a molecular form to consecutive multiple dilutions coupled with an external impact, such as mechanical shaking. The external treatment in the course of concentration reduction may also be accomplished, for example, by exposure to ultrasonic, electromagnetic, or other physical factors. V. Schwabe "Homeopathic medicines", M., 1967, U.S. Patents Nos. 7,229,648 and 4,311 ,897, which are incorporated by reference in their entirety and for the purpose stated, describe such processes that are well-accepted methods of homeopathic potentiation in the homeopathic art. This procedure gives rise to a uniform decrease in molecular concentration of the initial molecular form of the antibody. This procedure is repeated until the desired homeopathic potency is obtained. For the individual antibody, the required homeopathic potency can be determined by subjecting the intermediate dilutions to biological testing in the desired pharmacological model. Although not so limited, 'homeopathic potentization" may involve, for example, repeated consecutive dilutions combined with external treatment, particularly vertical (mechanical) shaking. In other words, an initial solution of antibody is subjected to consecutive repeated dilution and multiple vertical shaking of each obtained solution in accordance with homeopathic technology. The preferred concentration of the initial solution of antibody in the solvent, preferably, water or a water-ethyl alcohol mixture, ranges from about 0.5 to about 5.0 mg/ml. The preferred procedure for preparing each component, i.e. antibody solution, is the use of the mixture of three aqueous or aqueous-alcohol dilutions of the primary matrix solution (mother tincture) of antibodies diluted 10012, 10030 and 100200 times, respectively, which is equivalent to centesimal homeopathic dilutions C12, C30 and C200 or the mixture of three aqueous or aqueous-alcohol dilutions of the primary matrix solution (mother tincture) of antibodies diluted 10012, 10030 and 10050 times, respectively, which is equivalent to centesimal homeopathic dilutions C12, C30 and C50. Examples of how to obtain the desired potency are also provided, for example, in U.S. Patent Nos. 7,229,648 and 4,311 ,897, which are incorporated by reference for the purpose stated. The procedure applicable to the "activated-potentiated" form of the antibodies described herein is described in more detail below.
There has been a considerable amount of controversy regarding homeopathic treatment of human subjects. While the present invention relies on accepted homeopathic processes to obtain the "activated-potentiated" form of antibodies, it does not rely solely on homeopathy in human subjects for evidence of activity. It has been surprisingly discovered by the inventor of the present application and amply demonstrated in the accepted pharmacological models that the solvent ultimately obtained from consecutive multiple dilution of a starting molecular form of an antibody has definitive activity unrelated to the presence of the traces of the molecular form of the antibody in the target dilution. The "activated-potentiated" form of the antibody provided herein are tested for biological activity in well accepted pharmacological models of activity, either in appropriate in vitro experiments, or in vivo in suitable animal models. The experiments provided further below provide evidence of biological activity in such models. Human clinical studies also provide evidence that the activity observed in the animal model is well translated to human therapy. Human studies have also provided evidence of availability of the "activated potentiated" forms described herein to treat specified human diseases or disorders well accepted as pathological conditions in the medical science.
Also, the claimed "activated-potentiated" form of antibody encompasses only solutions or solid preparations the biological activity of which cannot be explained by the presence of the molecular form of the antibody remaining from the initial, starting solution. In other words, while it is contemplated that the "activated-potentiated" form of the antibody may contain traces of the initial molecular form of the antibody, one skilled in the art could not attribute the observed biological activity in the accepted pharmacological models to the remaining molecular form of the antibody with any degree of plausibility due to the extremely low concentrations of the molecular form of the antibody remaining after the consecutive dilutions. While the invention is not limited by any specific theory, the biological activity of the "activated-potentiated" form of the antibodies of the present invention is not attributable to the initial molecular form of the antibody. Preferred is the "activated-potentiated" form of antibody in liquid or solid form in which the concentration of the molecular form of the antibody is below the limit of detection of the accepted analytical techniques, such as capillary electrophoresis and High Performance Liquid Chromatography. Particularly preferred is the "activated- potentiated" form of antibody in liquid or solid form in which the concentration of the molecular form of the antibody is below the Avogadro number. In the pharmacology of molecular forms of therapeutic substances, it is common practice to create a dose- response curve in which the level of pharmacological response is plotted against the concentration of the active drug administered to the subject or tested in vitro. The minimal level of the drug which produces any detectable response is known as a threshold dose. It is specifically contemplated and preferred that the "activated- potentiated" form of the antibodies contains molecular antibody, if any, at a concentration below the threshold dose for the molecular form of the antibody in the given biological model.
The combination pharmaceutical composition in accordance with this aspect of the invention may be in the liquid form or in solid form. Each of the activated potentiated forms of the antibodies included in the pharmaceutical composition is prepared from an initial molecular form of the antibody via a process accepted in homeopathic art. The starting antibodies may be monoclonal, or polyclonal antibodies prepared in accordance with known processes, for example, as described in Immunotechniques, G. Frimel, M., "Meditsyna", 1987, p. 9-33; "Hum. Antibodies. Monoclonal and recombinant antibodies, 30 years after" by Laffly E., Sodoyer R. - 2005 - Vol. 14. - N 1-2. P.33-55, both incorporated herein by reference.
Monoclonal antibodies may be obtained, e.g., by means of hybridoma technology. The initial stage of the process includes immunization based on the principles already developed in the course of polyclonal antisera preparation. Further stages of work involve the production of hybrid cells generating clones of antibodies with identical specificity. Their separate isolation is performed using the same methods as in the case of polyclonal antisera preparation.
Polyclonal antibodies may be obtained via active immunization of animals. For this purpose, for example, suitable animals (e.g. rabbits) receive a series of injections of the appropriate antigen, for example, NO synthase. The animals' immune system generates corresponding antibodies, which are collected from the animals in a known manner. This procedure enables preparation of a monospecific antibody-rich serum.
If desired, the serum containing antibodies may be purified, for example by using affine chromatography, fractionation by salt precipitation, or ion-exchange chromatography. The resulting purified, antibody-enriched serum may be used as a starting material for the preparation of the activated-potentiated form of the antibodies. The preferred concentration of the resulting initial solution of antibody in the solvent, preferably water or a water-ethyl alcohol mixture, ranges from about 0.5 to about 5.0 mg/ml.
The preferred procedure for preparing each component of the combination drug according to the present invention is the use of the mixture of three aqueous-alcohol dilutions of the primary matrix solution of antibodies diluted 10012, 10030 and 10050 times, respectively, which is equivalent to centesimal homeopathic dilutions C12, C30, and C50 or diluted 10012, 10030 and 100200 times, respectively, which is equivalent to centesimal homeopathic dilutions C12, C30 and C200. To prepare a solid dosage form, a solid carrier is treated with the desired dilution obtained via the homeopathic process. To obtain a solid unit dosage form of the combination of the invention, the carrier mass is impregnated with each of the dilutions. Both orders of impregnation are suitable to prepare the desired combination dosage form.
In a preferred embodiment, the starting material for the preparation of the activated potentiated form that comprise the combination of the invention is polyclonal, animal-raised antibody to the corresponding antigen, namely, NO synthase and endogenous biological molecule. To obtain the activated-potentiated form of polyclonal antibodies to NO synthase, the desired antigen may be injected as immunogen into a laboratory animal, preferably, rabbits. Polyclonal antibodies to NO synthase may be obtained using the whole molecule of bovine NO synthase of the following sequence:
SEQ ID NO: 1
Met Gly Asn Leu Lys Ser Val Gly Gin Glu Pro Gly Pro Pro Cys
1 5 10 15
Gly Leu Gly Leu Gly Leu Gly Leu Gly Leu Cys Gly Lys Gin Gly
16 20 25 30
Pro Ala Ser Pro Ala Pro Glu Pro Ser Arg Ala Pro Ala Pro Ala
31 35 40 45
Thr Pro His Ala Pro Asp His Ser Pro Ala Pro Asn Ser Pro Thr
46 50 55 60
Leu Thr Arg Pro Pro Glu Gly Pro Lys Phe Pro Arg Val Lys Asn
61 65 70 75
Trp Glu Leu GLys er He Thr Tyr Asp Thr Leu Cys Ala Gin Ser
76 80 85 90
Gin Gin Asp Gly Pro Cys Thr Pro Arg Cys Cys Leu GLys er Leu
91 95 100 105
Val Leu Pro Arg Lys Leu Gin Thr Arg Pro Ser Pro Gly Pro Pro
106 110 115 120
Pro Ala Glu Gin Leu Leu Ser Gin Ala Arg Asp Phe He Asn Gin
121 125 130 135
Tyr Tyr Ser Ser He Lys Arg Ser GLys er Gin Ala His Glu Glu
136 140 145 150
Arg Leu Gin Glu Val Glu Ala Glu Val Ala Ser Thr Gly Thr Tyr
151 155 160 165
His Leu Arg Glu Ser Glu Leu Val Phe Gly Ala Lys Gin Ala Trp
166 170 175 180 Arg Asn Ala Pro Arg Cys Val Gly Arg He Gin Trp Gly Lys Leu 181 185 190 1 95
Gin Val Phe Asp Ala Arg Asp Cys Ser Ser Ala Gin Glu Met Phe 196 200 205 210
Thr Tyr lie Cys Asn His He Lys Tyr Ala Thr Asn Arg Gly Asn 211 215 220 225
Leu Arg Ser Ala He Thr Val Phe Pro Gin Arg Ala Pro Gly Arg 226 230 235 240
Gly Asp Phe Arg He Trp Asn Ser Gin Leu Val Arg Tyr Ala Gly 241 245 250 255
Tyr Arg Gin Gin Asp GLy ; er Val Arg Gly Asp Pro Ala Asn Val 256 260 265 270
Glu He Thr Glu Leu Cys He Gin His Gly Trp Thr Pro Gly Asn 271 275 280 285
Gly Arg Phe Asp Val Leu Pro Leu Leu Leu Gin Ala Pro Asp Glu 286 290 295 300
Ala Pro Glu Leu Phe Val Leu Pro Pro Glu Leu Val Leu Glu Val 301 305 310 315
Pro Leu Glu His Pro Thr Leu Glu Trp Phe Ala Ala Leu Gly Leu 316 320 325 330
Arg Trp Tyr Ala Leu Pro Ala Val Ser Asn Met Leu Leu Glu He 331 335 340 345
Gly Gly Leu Glu Phe Ser Ala Ala Pro Phe Ser Gly Trp Tyr Met 346 350 355 360
Ser Thr Glu He Gly Thr Arg Asn Leu Cys Asp Pro His Arg Tyr 361 365 370 375
Asn He Leu Glu Asp Val Ala Val Cys Met Asp Leu Asp Thr Arg 376 380 385 390
Thr Thr Ser Ser Leu Trp Lys Asp Lys Ala Ala Val Glu He Asn 391 395 400 405
Leu Ala Val Leu His Ser Phe Gin Leu Ala Lys Val Thr He Val 406 410 415 420
Asp His His Ala Ala Thr Val Ser Phe Met Lys His Leu Asp Asn 421 425 430 435
Glu Gin Lys Ala Arg Gly Gly Cys Pro Ala Asp Trp Ala Trp He 436 440 445 450
Val Pro Pro He Ser GLy ; er Leu Thr Pro Val Phe His Gin Glu 451 455 4 60 4 65
Met Val Asn Tyr He Leu Ser Pro Ala Phe Arg Tyr Gin Pro Asp 466 470 475 480
Pro Trp Lys GLy Ser Ala Thr Lys Gly Ala Gly He Thr Arg Lys 481 485 4 90 495
Lys Thr Phe Lys Glu Val Ala Asn Ala Val Lys He Ser Ala Ser 496 500 505 510
Leu Met Gly Thr Leu Met Ala Lys Arg Val Lys Ala Thr He Leu 511 515 510 525 Tyr Ala Ser Glu Thr Gly Arg Ala Gin Ser Tyr Ala Gin Gin Leu 526 530 535 540
Gly Arg Leu Phe Arg Lys Ala Phe Asp Pro Arg Val Leu Cys Met 541 545 550 555
Asp Glu Tyr Asp Val Val Ser Leu Glu His Glu Ala Leu Val Leu 556 560 565 570
Val Val Thr Ser Thr Phe Gly Asn Gly Asp Pro Pro Glu Asn Gly 571 575 580 585
Glu Ser Phe Ala Ala Ala Leu Met Glu Met Ser Gly Pro Tyr Asn 586 590 595 600
Ser Ser Pro Arg Pro Glu Gin His Lys Ser Tyr Lys He Arg Phe 601 605 610 615
Asn Ser Val Ser Cys Ser Asp Pro Leu Val Ser Ser Trp Arg Arg 616 620 625 630
Lys Arg Lys Glu Ser Ser Asn Thr Asp Ser Ala Gly Ala Leu Gly 631 635 640 645
Thr Leu Arg Phe Cys Val Phe Gly Leu GLy Ser Arg Ala Tyr Pro 646 650 655 660
His Phe Cys Ala Phe Ala Arg Ala Val Asp Thr Arg Leu Glu Glu 661 665 670 675
Leu Gly Gly Glu Arg Leu Leu Gin Leu Gly Gin Gly Asp Glu Leu 676 680 685 690
Cys Gly Gin Glu Glu Ala Phe Arg Gly Trp Ala Lys Ala Ala Phe 691 695 700 705
Gin Ala Ser Cys Glu Thr Phe Cys Val Gly Glu Glu Ala Lys Ala 706 710 715 720
Ala Ala Gin Asp He Phe Ser Pro Lys Arg Ser Trp Lys Arg Gin 721 725 730 735
Arg Tyr Arg Leu Ser Thr Gin Ala Glu Gly Leu Gin Leu Leu Pro 736 740 745 750
Gly Leu lie His Val His Arg Arg Lys Met Phe Gin Ala Thr Val 751 755 760 765
Leu Ser Val Glu Asn Leu Gin Ser Ser Lys Ser Thr Arg Ala Thr 766 770 775 780
He Leu Val Arg Leu Asp Thr Ala Gly Gin Glu Gly Leu Gin Tyr 781 785 790 795
Gin Pro Gly Asp His He Gly He Cys Pro Pro Asn Arg Pro Gly 796 800 805 810
Leu Val Glu Ala Leu Leu Ser Arg Val Glu Asp Pro Pro Pro Pro 811 815 820 825
Thr Glu Ser Val Ala Val Glu Gin Leu Glu Lys GLy er Pro Gly 826 830 835 840
Gly Pro Pro Pro Ser Trp Val Arg Asp Pro Arg Leu Pro Pro Cys 841 845 850 855
Thr Leu Arg Gin Ala Leu Thr Phe Phe Leu Asp He Thr Ser Pro 856 860 865 870 Pro Ser Pro Arg Leu Leu Arg Leu Leu Ser Thr Leu Ala Glu Glu 871 875 880 885
Pro Ser Glu Gin Gin Glu Leu Glu Thr Leu Ser Gin Asp Pro Arg 886 890 895 900
Arg Tyr Glu Glu Trp Lys Trp Phe Arg Cys Pro Thr Leu Leu Glu 901 905 910 915
Val Leu Glu Gin Phe Pro Ser Val Ala Leu Pro Ala Pro Leu Leu 916 920 925 930
Leu Thr Gin Leu Pro Leu Leu Gin Pro Arg Tyr Tyr Ser Val Ser 931 935 940 945
Ser Ala Pro Asn Ala His Pro Gly Glu Val His Leu Thr Val Ala 94 6 950 955 960
Val Leu Ala Tyr Arg Thr Gin Asp Gly Leu Gly Pro Leu His Tyr 961 965 970 975
Gly Val Cys Ser Thr Trp Leu Ser Gin Leu Lys Thr Gly Asp Pro 976 980 985 990
Val Pro Cys Phe lie Arg Gly Ala Pro Ser Phe Arg Leu Pro Pro 991 995 1000 1005
Asp Pro Tyr Val Pro Cys lie Leu Val Gly Pro Gly Thr Gly lie 1006 1010 1015 1020 Ala Pro Phe Arg Gly Phe Trp Gin Glu Arg Leu His Asp lie Glu 1021 1025 1030 1035 Ser Lys Gly Leu Gin Pro Ala Pro Met Thr Leu Val Phe Gly Cys 1036 1040 1045 1050 Arg Cys Ser Gin Leu Asp His Leu Tyr Arg Asp Glu Val Gin Asp 1051 1055 1060 1065 Ala Gin Glu Arg Gly Val Phe Gly Arg Val Leu Thr Ala Phe Ser 1066 1070 1075 1080 Arg Glu Pro Asp Ser Pro Lys Thr Tyr Val Gin Asp lie Leu Arg 1081 1085 1090 1095 Thr Glu Leu Ala Ala Glu Val His Arg Val Leu Cys Leu Glu Arg 1096 1100 1105 1110 Gly His Met Phe Val Cys Gly Asp Val Thr Met Ala Thr Ser Val 1111 1115 1120 1125 Leu Gin Thr Val Gin Arg lie Leu Ala Thr Glu Gly Asp Met Glu 1126 1130 1135 1140 Leu Asp Glu Ala Gly Asp Val lie Gly Val Leu Arg Asp Gin Gin 1141 1145 1150 1155 Arg Tyr His Glu Asp lie Phe Gly Leu Thr Leu Arg Thr Gin Glu 1156 1160 1165 1170 Val Thr Ser Arg lie Arg Thr Gin Ser Phe Ser Leu Gin Glu Arg 1171 1175 1180 1185 His Leu Arg Gly Ala Val Pro Trp Ala Phe Asp Pro Pro Gly Pro 1186 1190 1195 1200 Asp Thr Pro Gly Pro
1201 1205 Polyclonal antibodies to NO synthase may be obtained using the whole molecule of human NO synthase of the following sequence:
SEQ ID NO:2
Met Gly Asn Leu Lys Ser Val Ala Gin Glu Pro Gly Pro Pro Cys
1 5 10 15 Gly Leu Gly Leu Gly Leu Gly Leu Gly Leu Cys Gly Lys Gin Gly
16 20 25 30 Pro Ala Thr Pro Ala Pro Glu Pro Ser Arg Ala Pro Ala Ser Leu
31 35 40 45
Leu Pro Pro Ala Pro Glu His Ser Pro Pro Ser Ser Pro Leu Thr
46 50 55 60
Gin Pro Pro Glu Gly Pro Lys Phe Pro Arg Val Lys Asn Trp Glu
61 65 70 75
Val GLys er lie Thr Tyr Asp Thr Leu Ser Ala Gin Ala Gin Gin
76 80 85 90
Asp Gly Pro Cys Thr Pro Arg Arg Cys Leu GLys er Leu Val Phe
91 95 100 105
Pro Arg Lys Leu Gin Gly Arg Pro Ser Pro Gly Pro Pro Ala Pro
106 110 115 120
Glu Gin Leu Leu Ser Gin Ala Arg Asp Phe lie Asn Gin Tyr Tyr
121 125 130 135
Ser Ser lie Lys Arg Ser GLys er Gin Ala His Glu Gin Arg Leu
136 140 145 150
Gin Glu Val Glu Ala Glu Val Ala Ala Thr Gly Thr Tyr Gin Leu 151 155 160 165
Arg Glu Ser Glu Leu Val Phe Gly Ala Lys Gin Ala Trp Arg Asn
166 170 175 180
Ala Pro Arg Cys Val Gly Arg lie Gin Trp Gly Lys Leu Gin Val
181 185 190 195
Phe Asp Ala Arg Asp Cys Arg Ser Ala Gin Glu Met Phe Thr Tyr
196 200 205 210 lie Cys Asn His lie Lys Tyr Ala Thr Asn Arg Gly Asn Leu Arg
211 215 220 225
Ser Ala lie Thr Val Phe Pro Gin Arg Cys Pro Gly Arg Gly Asp
226 230 235 240
Phe Arg lie Trp Asn Ser Gin Leu Val Arg Tyr Ala Gly Tyr Arg
241 245 250 255
Gin Gin Asp GLy Ser Val Arg Gly Asp Pro Ala Asn Val Glu lie
256 260 265 270
Thr Glu Leu Cys lie Gin His Gly Trp Thr Pro Gly Asn Gly Arg
271 275 280 285
Phe Asp Val Leu Pro Leu Leu Leu Gin Ala Pro Asp Glu Pro Pro
286 290 295 300
Glu Leu Phe Leu Leu Pro Pro Glu Leu Val Leu Glu Val Pro Leu
301 305 310 315
Glu His Pro Thr Leu Glu Trp Phe Ala Ala Leu Gly Leu Arg Trp
316 320 325 330
Tyr Ala Leu Pro Ala Val Ser Asn Met Leu Leu Glu lie Gly Gly
331 335 340 345 Leu Glu Phe Pro Ala Ala Pro Phe Ser Gly Trp Tyr Met Ser Thr
34 6 350 355 ' 360
Glu lie Gly Thr Arg Asn Leu Cys Asp Pro His Arg Tyr Asn lie
361 365 370 375
Leu Glu Asp Val Ala Val Cys Met Asp Leu Asp Thr Arg Thr Thr
376 380 385 390
Ser Ser Leu Trp Lys Asp Lys Ala Ala Val Glu lie Asn Val Ala
391 395 400 405
Val Leu His Ser Tyr Gin Leu Ala Lys Val Thr lie Val Asp His
406 410 415 420
His Ala Ala Thr Ala Ser Phe Met Lys His Leu Glu Asn Glu Gin
421 425 430 435
Lys Ala Arg Gly Gly Cys Pro Ala Asp Trp Ala Trp lie Val Pro
436 440 445 450
Pro lie Ser GLys er Leu Thr Pro Val Phe His Gin Glu Met Val
451 455 4 60 4 65
Asn Tyr Phe Leu Ser Pro Ala Phe Arg Tyr Gin Pro Asp Pro Trp
466 470 475 480
Lys Gly Ser Ala Ala Lys Gly Thr Gly lie Thr Arg Lys Lys Thr
481 485 4 90 4 95
Phe Lys Glu Val Ala Asn Ala Val Lys lie Ser Ala Ser Leu Met
496 500 505 510
Gly Thr Val Met Ala Lys Arg Val Lys Ala Thr lie Leu Tyr Gly
511 515 510 525
Ser Glu Thr Gly Arg Ala Gin Ser Tyr Ala Gin Gin Leu Gly Arg 526 530 535 540
Leu Phe Arg Lys Ala Phe Asp Pro Arg Val Leu Cys Met Asp Glu
541 545 550 555
Tyr Asp Val Val Ser Leu Glu His Glu Thr Leu Val Leu Val Val
556 560 565 570
Thr Ser Thr Phe Gly Asn Gly Asp Pro Pro Glu Asn Gly Glu Ser
571 575 580 585
Phe Ala Ala Ala Leu Met Glu Met Ser Gly Pro Tyr Asn Ser Ser
586 590 595 600
Pro Arg Pro Glu Gin His Lys Ser Tyr. Lys lie Arg Phe Asn Ser
601 605 610 615 lie Ser Cys Ser Asp Pro Leu Val Ser Ser Trp Arg Arg Lys Arg
616 620 625 630
Lys Glu Ser Ser Asn Thr Asp Ser Ala Gly Ala Leu Gly Thr Leu
631 635 640 645
Arg Phe Cys Val Phe Gly Leu GLys er Arg Ala Tyr Pro His Phe
646 650 655 660
Cys Ala Phe Ala Arg Ala Val Asp Thr Arg Leu Glu Glu Leu Gly
661 665 670 675
Gly Glu Arg Leu Leu Gin Leu Gly Gin Gly Asp Glu Leu Cys Gly
676 680 685 690
Gin Glu Glu Ala Phe Arg Gly Trp Ala Gin Ala Ala Phe Gin Ala
691 695 700 705
Ala Cys Glu Thr Phe Cys Val Gly Glu Asp Ala Lys Ala Ala Ala
706 710 715 720 Arg Asp lie Phe Ser Pro Lys Arg Ser Trp Lys Arg Gin Arg Tyr 721 725 730 735
Arg Leu Ser Ala Gin Ala Glu Gly Leu Gin Leu Leu Pro Gly Leu 736 740 745 750
He His Val His Arg Arg Lys Met Phe Gin Ala Thr He Arg Ser 751 755 760 765
Val Glu Asn Leu Gin Ser Ser Lys Ser Thr Arg Ala Thr He Leu 766 770 775 780
Val Arg Leu Asp Thr Gly Gly Gin Glu Gly Leu Gin Tyr Gin Pro 781 785 790 795
Gly Asp His He Gly Val Cys Pro Pro Asn Arg Pro Gly Leu Val 796 800 805 810
Glu Ala Leu Leu Ser Arg Val Glu Asp Pro Pro Ala Pro Thr Glu 811 815 820 825
Pro Val Ala Val Glu Gin Leu Glu Lys Gly Ser Pro Gly Gly Pro 826 830 835 840
Pro Pro Gly Trp Val Arg Asp Pro Arg Leu Pro Pro Cys Thr Leu 841 845 850 855
Arg Gin Ala Leu Thr Phe Phe Leu Asp He Thr Ser Pro Pro Ser 856 860 865 870
Pro Gin Leu Leu Arg Leu Leu Ser Thr Leu Ala Glu Glu Pro Arg 871 875 880 885
Glu Gin Gin Glu Leu Glu Ala Leu Ser Gin Asp Pro Arg Arg Tyr 886 890 895 900
Glu Glu Trp Lys Trp Phe Arg Cys Pro Thr Leu Leu Glu Val Leu 901 905 910 915
Glu Gin Phe Pro Ser Val Ala Leu Pro Ala Pro Leu Leu Leu Thr
916 920 925 930
Gin Leu Pro Leu Leu Gin Pro Arg Tyr Tyr Ser Val Ser Ser Ala
931 935 940 945
Pro Ser Thr His Pro Gly Glu lie His Leu Thr Val Ala Val Leu
94 6 950 955 960
Ala Tyr Arg Thr Gin Asp Gly Leu Gly Pro Leu His Tyr Gly Val
961 965 970 975
Cys Ser Thr Trp Leu Ser Gin Leu Lys Pro Gly Asp Pro Val Pro
976 980 985 990
Cys Phe lie Arg Gly Ala Pro Ser Phe Arg Leu Pro Pro Asp Pro
991 995 1000 1005
Ser Leu Pro Cys lie Leu Val Gly Pro Gly Thr Gly lie Ala Pro
1006 1010 1015 1020
Phe Arg Gly Phe Trp Gin Glu Arg Leu His Asp lie Glu Ser Lys
1021 1025 1030 1035
Gly Leu Gin Pro Thr Pro Met Thr Leu Val Phe Gly Cys Arg Cys
1036 1040 1045 1050
Ser Gin Leu Asp His Leu Tyr Arg Asp Glu Val Gin Asn Ala Gin
1051 1055 1060 1065
Gin Arg Gly Val Phe Gly Arg Val Leu Thr Ala Phe Ser Arg Glu
1066 1070 1075 1080
Pro Asp Asn Pro Lys Thr Tyr Val Gin Asp lie Leu Arg Thr Glu
1081 1085 1090 1095 Leu Ala Ala Glu Val His Arg Val Leu Cys Leu Glu Arg Gly His 1096 1100 1105 1110
Met Phe Val Cys Gly Asp Val Thr Met Ala Thr Asn Val Leu Gin 1111 1115 1120 1125
Thr Val Gin Arg lie Leu Ala Thr Glu Gly Asp Met Glu Leu Asp 1126 1130 1135 1140
Glu Ala Gly Asp Val lie Gly Val Leu Arg Asp Gin Gin Arg Tyr 1141 1145 1150 1155
His Glu Asp lie Phe Gly Leu Thr Leu Arg Thr Gin Glu Val Thr 1156 1160 1165 1170
Ser Arg lie Arg Thr Gin Ser Phe Ser Leu Gin Glu Arg Gin Leu 1171 1175 1180 1185
Arg Gly Ala Val Pro Trp Ala Phe Asp Pro Pro Gly Ser Asp Thr 1186 1190 1195 1200
Asn Ser Pro
1201 1203
To obtain polyclonal antibodies to NO synthase, it is also possible to fragment of NO synthase, selected, for example, from the following sequences:
SEQ ID NO: 3
Pro Trp Ala Phe
1192 1195
SEQ ID NO: 4 Gly Ala Val Pro
1189 1192
SEQ ID NO: 5
Arg 1185
His Leu Arg Gly Ala Val Pro Trp Ala Phe Asp Pro Pro Gly Pro 1186 11 90 11 95 1200
Asp Thr Pro Gly Pro
1201 1205
SEQ ID NO: 6
Ala Phe Asp Pro Pro Gly Pro 11 94 1195 1200
Asp Thr Pro Gly Pro
1201 1205
SEQ ID NO: 7
His Leu Arg Gly Ala Val Pro Trp Ala Phe Asp
1186 1 190 11951196
SEQ ID NO: 8
His Leu Arg Gly Ala Val Pro Trp Ala Phe Asp Pro Pro Gly Pro 1186 1190 11 95 1200
Asp Thr Pro Gly Pro
1201 1205
The exemplary procedure for preparation of the starting polyclonal antibodies to NO synthase may be described as follows. In 7-9 days before blood sampling, 1-3 intravenous injections of the desired antigen are made to the rabbits to increase the level of polyclonal antibodies in the rabbit blood stream. Upon immunization, blood samples are taken to test the antibody level. Typically, the maximum level of immune reaction of the soluble antigen is achieved within 40 to 60 days after the first injection of the antigen. Upon completion of the first immunization cycle, rabbits have a 30-day rehabilitation period, after which re-immunization is performed with another 1 -3 intravenous injections.
To obtain antiserum containing the desired antibodies, the immunized rabbits' blood is collected from rabbits and placed in a 50ml centrifuge tube. Product clots formed on the tube sides are removed with a wooden spatula, and a rod is placed into the clot in the tube center. The blood is then placed in a refrigerator for one night at the temperature of about 40°C. On the following day, the clot on the spatula is removed, and the remaining liquid is centrifuged for 10 min at 13,000 rotations per minute. Supernatant fluid is the target antiserum. The obtained antiserum is typically yellow. 20% of NaN3 (weight concentration) is added in the antiserum to a final concentration of 0.02% and stored before use in frozen state at the temperature of -20°C or without NaN3 at the temperature of -70°C. To separate the target antibodies to gamma interferon from the antiserum, the following solid phase absorption sequence is suitable:
10 ml of the antiserum of rabbits is diluted twofold with 0. 5 M NaCI, after which 6.26g Na2S04 is added, mixed and incubated for 12-16 hours at 4°C. The sediment is removed by centrifugation, diluted in 10ml of phosphate buffer and dialyzed against the same buffer during one night at ambient temperature. After the sediment is removed, the solution is applied to a DEAE-cellulose column balanced by phosphate buffer. The antibody fraction is determined by measuring the optical density of the eluate at 280 nm.
The isolated crude antibodies are purified using affine chromatography method by attaching the obtained antibodies to NO synthase located on the insoluble matrix of the chromatography media, with subsequent elution by concentrated aqueous salt solutions.
The resulting buffer solution is used as the initial solution for the homeopathic dilution process used to prepare the activated-potentiated form of the antibodies. The preferred concentration of the initial matrix solution of the antigen-purified polyclonal rabbit antibodies to NO synthase is 0.5 to 5.0 mg/ml, preferably, 2.0 to 3.0 mg/ml.
The polyclonal antibodies to endogenous biological molecule may also be obtained by a similar methodology to the methodology described for endothelial NO synthase antibodies using an adjuvant. The resulting buffer solution is used as the initial solution for the homeopathic dilution process used to prepare the activated potentiated form of the antibodies.
The activated potentiated form of each component of the combination may be prepared from an initial solution by homeopathic potentization, preferably using the method of proportional concentration decrease by serial dilution of 1 part of each preceding solution (beginning with the initial solution) in 9 parts (for decimal dilution), or in 99 parts (for centesimal dilution), or in 999 parts (for millesimal dilution) of a neutral solvent, starting with a concentration of the initial solution of antibody in the solvent, preferably, water or a water-ethyl alcohol mixture, in the range from about 0.5 to about 5.0 mg/ml, coupled with external impact. Preferably, the external impact involves multiple vertical shaking (dynamization) of each dilution. Preferably, separate containers are used for each subsequent dilution up to the required potency level, or the dilution factor. This method is well-accepted in the homeopathic art. See, e.g. V. Schwabe "Homeopathic medicines", M., 1967, p. 14-29, incorporated herein by reference for the purpose stated.
For example, to prepare a 12-centesimal dilution (denoted C12), one part of the initial matrix solution of antibodies to NO synthase with the concentration of 3.0 mg/ml is diluted in 99 parts of neutral aqueous or aqueous-alcohol solvent (preferably, 15%-ethyl alcohol) and then vertically shaked many times (10 and more) to create the 1 st centesimal dilution (denoted as C1 ). The 2nd centesimal dilution (C2) is prepared from the 1 st centesimal dilution C1. This procedure is repeated 1 1 times to prepare the 12th centesimal dilution C12. Thus, the 12th centesimal dilution C12 represents a solution obtained by 12 serial dilutions of one part of the initial matrix solution of antibodies with the concentration of 3.0 mg/ml in 99 parts of a neutral solvent in different containers, which is equivalent to the centesimal homeopathic dilution C12. Similar procedures with the relevant dilution factor are performed to obtain the desired dilutions.The intermediate dilutions may be tested in a desired biological model to check activity. The preferred activated potentiated forms for antibodies comprising the combination of the invention is a C12, C30 and C200 dilutions for each activated-potentiated form. When using the mixture of various homeopathic dilutions (primarily centesimal) of the active substance as biologically active liquid component, each component of the composition (e.g., C12, C30, C50, C200) is prepared separately according to the above-described procedure until the next-to-last dilution is obtained (e.g., until C1 1 , C29, and C199 respectively), and then one part of each component is added in one container according to the mixture composition and mixed with the required quantity of the solvent (e.g. with 97 parts for centesimal dilution).
It is possible to use the active substance as mixture of various homeopathic dilutions, e.g. decimal and/or centesimal (D20, C30, C100 or C12, C30, C50 or C12, C30, C200, etc.), the efficiency of which is determined experimentally by testing the dilution in a suitable biological model, for example, in models described in the examples herein.
In the course of potentiation and concentration decrease, the vertical shaking may be substituted for external exposure to ultrasound, electromagnetic field or any similar external impact procedure accepted in the homeopathic art.
The solid unit dosage form of the pharmaceutical composition of the invention may be prepared by using impregnating a solid, pharmaceutically acceptable carrier with the mixture of the activated potentiated form aqueous or aqueous-alcohol solutions of active components that are mixed, primarily in 1 :1 ratio and used in liquid dosage form. Alternatively, the carrier may be impregnated consecutively with each requisite dilution.
Preferably, the pharmaceutical composition in the solid unit dosage form is prepared from granules of the pharmaceutically acceptable carrier which was previously saturated with the aqueous or aqueous-alcoholic dilutions of the activated potentiated form of antibodies. The solid dosage form may be in any form known in the pharmaceutical art, including a tablet, a capsule, a lozenge, and others. As an inactive pharmaceutical ingredients one can use glucose, sucrose, maltose, amylum, isomaltose, isomalt and other mono- olygo- and polysaccharides used in manufacturing of pharmaceuticals as well as technological mixtures of the above mentioned inactive pharmaceutical ingredients with other pharmaceutically acceptable excipients, for example isomalt, crospovidone, sodium cyclamate, sodium saccharine, anhydrous citric acid etc), including lubricants, disintegrants, binders and coloring agents. The preferred carriers are lactose and isomalt. The pharmaceutical dosage form may further include standard pharmaceutical excipients, for example, microcrystalline cellulose, magnesium stearate and citric acid.
The example of preparation of the solid unit dosage form is set forth below. To prepare the solid oral form, 100-300 μιη granules of lactose are impregnated with aqueous or aqueous-alcoholic solutions of the activated potentiated form of antibodies to histamine, activated-potentiated form of antibodies to NO synthase and the activated potentiated form of antibodies to an endogenous biological molecule in the ratio of 1 kg of antibody solution to 5 or 10 kg of lactose (1 :5 to 1 :10). To effect impregnation, the lactose granules are exposed to saturation irrigation in the fluidized boiling bed in a boiling bed plant (e.g. "Huttlin Pilotlab" by Hiittlin GmbH) with subsequent drying via heated air flow at a temperature below 40°C. The estimated quantity of the dried granules (10 to 34 weight parts) saturated with the activated potentiated form of antibodies is placed in the mixer, and mixed with 25 to 45 weight parts of "non- saturated" pure lactose (used for the purposes of cost reduction and simplification and acceleration of the technological process without decreasing the treatment efficiency), together with 0.1 to 1 weight parts of magnesium stearate, and 3 to 10 weight parts of microcrystalline cellulose. The obtained tablet mass is uniformly mixed, and tableted by direct dry pressing (e.g., in a Korsch - XL 400 tablet press) to form 150 to 500 mg round pills, preferably, 300 mg. After tableting, 300 mg pills are obtained that are saturated with aqueous-alcohol solution (3.0-6.0 mg/pill) of the combination of the activated- potentiated form of antibodies. Each component of the combination used to impregnate the carrier is in the form of a mixture of centesimal homeopathic dilutions, preferably, C12, C30 and C200.
While the invention is not limited to any specific theory, it is believed that the activated-potentiated form of the antibodies described herein do not contain the molecular form of the antibody in an amount sufficient to have biological activity attributed to such molecular form. The biological activity of the combination drug (combination, pharmaceutical composition) of the invention is amply demonstrated in the appended examples. Preferably, for the purpose of treatment, the combination of the invention is administered from once daily to four times daily, preferably twice daily, each administration including one or two combination unit dosage forms.
The invention is further illustrated with reference to the appended non-limiting examples.
EXAMPLES
Example 1.
Study of effect of a complex preparation containing ultralow doses (ULD) of activated - potentiated forms of polyclonal affinity purified rabbit antibodies to brain- specific protein S-100 (anti-S100) and endothelial NO-synthase (anti-eNOS), obtained by super-dilution of initial matrix solution (concentration: 2.5 mg/ml) (10012, 10030, 100200 times), equivalent to a blend of centesimal homeopathic dilutions C 2, C30, C200 (ratio: 1 :1 ) (ULD of anti-S100+anti-eNOS), as well as its components - activated - potentiated form of polyclonal affinity purified rabbit antibodies to ultralow doses (ULD) of brain- specific protein S-100 (anti-S100), purified on antigen, obtained by super-dilution of initial matrix solution (10012, 10030, 100200 times, equivalent to a blend of centesimal homeopathic dilution C12, C30, C200, and activated - potentiated form of polyclonal rabbit antibodies to ultralow dose of endothelial NO-synthase (ULD of anti-eNOS), obtained by super-dilution of initial matrix solution (10012, 10030, 100200 times), equivalent to a blend of centesimal homeopathic dilution C12, C30, C200 in vitro on binding of standard ligand [3H]pentazocine to human recombinant σ1 receptor was evaluated using radioligand method. Potentiated distilled water (blend of homeopathic dilutions C12+C30+C200) was used as test preparations control.
Sigma-1 (σ1 ) receptor - an intracellular one which is localized in the cells of central nervous system, the cells of the most of peripheral tissues and immune component cells. Receptors exhibit a unique ability to be translocated which is caused by many psychotropic medications. The dynamics of sigma-1 receptors is directly linked to various influences which are performed by preparations acting to the sigma-1 receptors. These effects include the regulation of activity channels, ecocytosis, signal transferring, remodeling of the plasma membrane (formation of rafts) and lipid transportation / metabolism. All this can contribute to the plasticity of neurons in a brain. There is evidence that the sigma-1 receptors have a modulating effect on all the major neuromediator systems: noradrenergic, serotonergic, dopaminergic, cholinergic systems and NMDA- adjustable glutamate effects. Sigma-1 receptor plays an important role in the pathophysiology of neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson), psychiatric and affective disorders and stroke; and italso takes part in the processes of learning and memory. In this regard, the ability of drugs to influence the efficiency of interaction of ligands with sigma-1 receptor indicates on the presence of neuroprotective, anti-ischemic, anxiolytic, antidepressant and anti astenic components in the spectrum of its pharmacological activity that allows the consideration of these drugs as effective preparations particularly for the treatment of cerebrovascular diseases.
During the test (to measure total binding) 20 μΙ of complex preparation of ULD of anti-S100+anti-eNOS or 10 μΙ of ULD of AB to S100 or 10 μΙ of ULD of AB to NOS were transferred in the incubation medium. Thus, the quantity of ULD of anti-S100+anti- eNOS, transferred into the test basin when testing the complex preparation was identical to that of ULD of AB to S100 and ULD of AB to NOS tested as monopreparations, which allow comparing the efficiency of the preparation to its separate components. 20 μΙ and 10 μΙ of potentiated water were transferred in the incubation medium.
Further, 160 μΙ (about 200μg of protein) of Jurkat cell line membranes homogenate (human leukemic T-lymphocyte line), and finally, 20 μΙ of tritium-labeled radioligand [3H]pentazocine ( 5 nm) were transferred.
In order to measure non-specific binding, 20 μΙ of non-labeled ligand- haloperidol (10 μΜ) were transferred in the incubation medium instead of the preparations or potentiated water.
Radioactivity was measured using a scintillometer (Topcount, Packard) and scintillation blend (Microscint 0, Packard) following the incubation within 120 minutes at 22°C in 50 mM Tris-HCI buffer (pH = 7,4) and filtration using fiberglass filters (GF/B, Packard). Specific binding (during the test or control) was calculated as a difference between total (during the test or control) and non-specific binding.
Results are represented as percentage of specific binding inhibition in control (distilled water was used as control) (Table 1 ).
Table 1.
Figure imgf000028_0001
Effect of the preparations and potentiated water on binding of standard ligand
[3H]pentazocine to human recombinant σ 1 receptor
Note: % of specific binding in control = (specific binding during the test/ specific binding in control)* 100%;
% of specific binding inhibition in control = 100% - (specific binding during the test/ specific binding in control) * 100%). The outcomes reflecting inhibition above 50% represent significant effects of the tested compounds; inhibition from 25% to 50% confirm mild to moderate effects; inhibition less than 25% is considered to be insignificant effect of the tested compound and is within background level.
Therefore, the conditions of this test model showed that the complex preparation of ULD of anti-S100+anti-eNOS is more efficient than its separate components (ULD of anti-S100 and ULD of anti-eNOS) in inhibiting the binding of standard radioligand [3H]pentazocine to human recombinant σ1 receptor; ULD of anti-S100, transferred into the test basin, namely 10 μΙ, inhibit the binding of standard radioligand [3H]pentazocine to human recombinant σ1 receptor, but the effect intensity is inferior to that of the complex preparation of ULD of anti-S100+anti-eNOS; ULD of anti-eNOS, transferred into the test basin, namely 10 μΙ, had no effect on the binding of standard radioligand [3H]pentazocine to human recombinant σ1 receptor; potentiated water, transferred into the test basin, namely 10 μΙ or 20 μΙ, had no effect on the binding of standard radioligand [3H]pentazocine to human recombinant σ1 receptor.
Example 2.
Alzheimer's disease (AD) is a neurodegenerative disease that is characterized by lowering of cognitive functions, memory deterioration, confused consciousness, and emotional changes. Although the main cause of this pathology is nowadays considered the accumulation of beta amyloid which leads to the formation of beta-amyloid plaques and neurofibrillary tangles in brain tissues; AD is also accompanied by a deficiency of cholinergic system. This is the basis of a most common way of modeling of AD in animals with the help of antagonist of cholinergic system of scopolamine. Injection of scopolamine into experimental animals (usually rats or mice) interrupts the ability to learn and leads to deterioration of memory.
Various methods were used to assess cognitive functions of rats and mice, including Morris water maze. The essence of this test is that the animals are released into a container with cloudy water from different points are forced to look for a hidden fixed platform. The advantage of this method is that it allows the researcher to monitor the process of animal training (the formation of ideas about the spatial alignment of the platform no matter where the animal was placed in the water) so as to assess the memory strength (for this the test is conducted when the platform is removed).
The effectiveness in rats with Scopolamine amnesia of the combination pharmaceutical composition of the present invention containing activated-potentiated forms of polyclonal affinity purified on antigen of rabbit brain-specific proteins S-100 (anti-S100) and to endothelial NO-syntheses (anti-eNOS) in ultra low doses (ULD) obtained by super dilution of storage stock solution (with concentration of 2.5 mg/ml) in 10012, 10030, 100200 times, equivalent to centesimal homeopathic dilutions C12, C30, C200 (ULD anti-S100 + anti-eNOS) is studied.
In a study of the effectiveness of the drug ULD anti-S100 + anti-eNOS in rats with scopolamine amnesia (a model of Alzheimer's disease) 48 male rats of the Rj: Wistar (Han) line (weight 180-280g) were used. During 4 days the rats were subdermally injecting with normal saline (n = 12, intact) or scopolamine in doze of 0.5 mg / kg (n = 36) (scopolamine-induced amnesia). Rats with scopolamine-induced amnesia were divided into three groups and administered with distilled water (7.5 ml / kg, n = 12, control group 1 ), or ULD anti-S100 (7,5 ml / kg, n = 12, group 2) or ULD anti-S100 + anti-eNOS (7,5 ml / kg, n = 12, group 3) intragastrically for 9 days (4 days prior to the injection of scopolamine, 4 days against the background of scopolamine and 1 day after the last scopolamine injection).
The training session in the Morris water maze was conducted within 4 days of the scopolamine injection through 60 minutes after administration of tested drugs and 30 minutes after administration of scopolamine (4 sequential tests at interval of 60 seconds). Morris' maze is a round reservoir (diameter - 150 cm, height - 45 cm) at 30 cm filled with water (26-28 ° C). At 18 cm from the edge of the container there is hidden platform (diameter - 15 cm) buried on 1.5 cm below the water level. Cloudy water made by adding a non-toxic dye (e.g., milk powder) makes the platform invisible. For each test the animal was placed in a maze in one of the initial points that are equidistant from the hidden platform and the animal was allowed to find the platform. If the animal could not find the platform within 120 seconds, the animal was put on the platform and left for 60 seconds and the test was restarted. During the four tests in random order the animals began to walk through the maze twice from each starting point. The tests were recorded on videotape and then analyzed for distance overcomes searching the platform in each trial and the latent period of searching for the platform. On day 5 the test was performed: the platform was removed from the maze and rats were given free float for 60 seconds. The time spent in the place where the platform used to be was recorded.
The administration of scopolamine significantly worsened the ability of animals to learn. In the control group the time spent by animals searching for platforms and the distance that animals swam searching for the platform, significantly increased (Table 2, 3). The test shows that the memory of animals in the control group worsened: the animals in this group spent less time in the place where the platform used to be located than intact animals (Table 4). The administration of ULD anti-S100 didn't lead to improvement of the studied parameters (Tables 2, 3, 4). The administration of ULD anti- Si 00 + anti-eNOS led to some improvement in learning which resulted in a shortening of the latent time of the platform search time (Table 2) and covered distance (Table 3) within 4 days of training and an improvement of memory as reflected in increase of the time spent in a place where the platform used to be located (Table 4).
Table 2.
Latent period of the platform search,
Figure imgf000031_0001
Control, n=12 100.1 ±6.8*** 92.4±9.3*** 81.4±10.7*** 77.7±9.4***
ULD anti-S100, n=12 106.8±7.0 99.3±7.8 95.6±9.0 80.4±11.1
ULD anti-S100 + anti- 94.4±7.2 90.7±8.2 78.3±8.6 60.1±10.2 eNOS, n=12
* - difference from intact is significant, p<0.05
Table 3.
Distance overcome to search the platform, cm
Figure imgf000032_0001
- difference from intact is significant, p<0.05
Table 4.
Time spent in a place where the platform used to be located, sec.
Figure imgf000032_0002
Control, n=12 18.4+.2.8*** 18.8±1.9*** 18.811.7***
ULD anti-S100, n=12 13.3±2.1 21.5±2.6 17.611.3
ULD anti-S100 + anti-eNOS,
19.1±4.8 23.8±2.2 21.2+2.5 n=12
*** - difference from intact is significant, p<0.05
Thus, in model of Alzheimer's disease, the administratoin of the complex ULD anti-S100 + anti-eNOS was more effective in comparison with administration of ULD anti-S100 and vehicle.
Example 3.
The preclinical research studied the ultra low doses (ULD) of activated - potentiated forms of polyclonal affinity purified rabbit antibodies to brain-specific protein S-100 (anti-S100) purified on antigen, and endothelial NO-synthase (anti-eNOS), obtained by super-dilution of initial matrix solution (concentration: 2,5 mg/ml) (10012, 10030, 100200 times), equivalent to a mixture of centesimal homeopathic dilutions C12, C30, C200 (ratio: 1 :1 ) (ULD anti-S100+anti-eNOS) in treating ischemic stroke caused by prefrontal cerebrocortical photothrombosis in rats.
Acute cerebrovascular disease (brain stroke) ranks third among lethality causes in developed countries and one of the main causes of disability in humans (Gusev E.I., 2003; Janardhan V., Qureshi A.I., 2004).
The photo-induced thrombosis model meets almost all requirements to the experimental model of focal cerebral ischemia. The method developed by Watson (Watson B. et ai, 1985) is based on the effect of light with wavelength 560 nm on photosensitive pigment Bengal rose introduced into the blood flow. Active oxygen forms are created and caused increase in adhesiveness of endothelium cells and platelets, and formation of clots closing vascular lumens. The method of ischemic brain lesion induction by using photo-induced thrombosis is technically simple and to close to clinical forms of ischemic brain stroke. A great advantage of this model is that it is non-invasive, i.e. does not require craniotomy and, therefore, more accurately reproduces clinical picture of cerebral thrombosis.
Thirty seven male Wistar rats (weight: 150-180 g; age: 2-3 months) were included in the study of the activity of ULD anti-S100+anti-eNOS in rats with ischemic stroke caused by prefrontal cerebrocortical photothrombosis. Bilateral focal ischemic injury in prefrontal cerebral cortex in rats was induced using the photochemical thrombosis method by Watson (Watson B. D. et ai, 1985) as modified by I.V. Viktorov {Romanova G.A. et al, 1998). Bengal rose (3% solution) was injected in the jugular vein of anesthetized rats (n=37) (anesthesia: chloral hydrate 300 mg/kg, intraperitoneally). Using a fiber optic bundle (3 cm in diameter) the light beam from halogen lamp (24 V, 250 W) was delivered to the skull surface above the frontal cortex of the left and right cerebral hemispheres to induce photothrombosis. Sham-operated rats (n=6) were subject to the same procedure except administration of bengal rose and exposure to halogen lamp light. The intact group included 6 rats.
Five days before and 9 days after stoke induction the following preparations were administered to rats with photothrombosis: distilled water (control-photothrombosis, 5 ml/kg daily, n=12), ULD anti-S100 (5 ml/kg daily, n=7) or ULD anti-S 00+anti-eNOS (5 ml/kg daily, n=6). On Day 8 after the operation (or sham operation) conditioned passive avoidance reflex (CPAR) test was performed to assess learning capability and memory in rats. Rats were placed in a unit consisting of illuminated site and connected dark chamber, where animals were exposed to electric foot-shock of 0,45 mA due to which usually preferred dark chamber became dangerous. Development of conditioned passive avoidance reflex was tested on the next day. At that, rats were placed in the illuminated chamber. Latent period of the first entry in the dark chamber was recorded. If a rat avoided the dark chamber for a long time, a conclusion was made that it remembered the danger (electric shock). The longer the latent period of entry in the dark chamber, the better the memory. Volume of the stroke lesion was morphologically assessed in a proportion of rats of experimental groups on Day 9.
In control rats photothrombosis caused formation of a large stroke area and, therefore, leaded to memory impairment: CPAR reproduction worsened by 9.6% compared to intact rats and by 22.9% compared to sham-operated (Table 5). Administration of ULD anti-S100 reduced the stroke volume by 42.2% and improved memory by 14.0% compared to control-photothrombosis group. Administration of ULD anti-S100+anti-eNOS was more effective: the stroke volume reduced by 44.0% , and conditioned reflex reproduction - by 33.4% compared to control-photothrombosis group.
Therefore, administration of the complex preparation of ULD anti-S100 + anti- eNOS was more efficient than monocomponent preparation of ULD anti-S100.
Table 5.
Figure imgf000035_0001
Example 4.
Study of the combination of "activated" potentiated forms of antibodies to a C- terminal fragment of the angiotensin II AT1 -receptor, in a mixture of homeopathic dilutions of C12, C30, C200, with the activated potentiated form of antibodies to endothelial NO-synthase, in a mixture of homeopathic dilutions of C12, C30, C200, in SHR rats in a model of hypertension.
The combination of the "activated" potentiated form of antibodies to a C-terminal fragment of the angiotensin II AT1-receptor, in a mixture of homeopathic dilutions of C12, C30, C200, and the activated potentiated form of antibodies to endothelial NO- synthase in a mixture of homeopathic dilutions of C12, C30, C200, was studied, in solution form, in the SHR rat hypertension model. Investigations were conducted on 40 SHR line male rats from (weight 350+50 g, age 4.5 - 5 months) with hypertension, which were divided into 4 groups of 10 animals each.
For 28 days, the animals were treated as follows. Group 1 - 2.5 ml/kg of the potentiated activated form of antibodies to a C-terminal fragment AT1 of human angiotensin II receptor (a mixture of aqueous dilutions C12, C30, C200) in combination with 2.5 ml/kg of distilled water, Group 2 - 2.5 ml/kg of the potentiated activated form of antibodies to endothelial NO-synthase (a mixture of aqueous dilutions C12, C30, C200) in combination with 2.5 ml/kg of distilled water, Group 3 - 5 ml/kg of the combination pharmaceutical composition (a mixture of aqueous dilutions C12, C30, C200 for each component), and Group 4 - 5 ml/kg of distilled water.
Systolic blood pressure (SBP) of awake rats was measured with the aid of an indirect method in a tail artery (using a cuff) once a week and 9 hours after the last administration of medicines.
All tested compositions demonstrated hypotensive effect (p<0.05): by 28th day, systolic blood pressure (SBD) decreased in comparison with the initial level in Group 1 by - 20.6%; in Group 2 by 14.4%; in Group 3 by 27.6%. In the control Group 4, SBD changes were 1.6% in comparison with the initial values. The results demonstrate a clear synergistic hypotensive effect of the combination pharmaceutical composition.
Example 5.
Study of the combination of the activated potentiated forms of antibodies to a C- terminal fragment of angiotensin II AT1 -receptor, in a mixture of homeopathic dilutions of C12, C30, C200, with the activated potentiated form of antibodies to endothelial NO- synthase, in a mixture of homeopathic dilutions of C12, C30, C200, in NISAG rats in a model of hypertension.
The combination of the activated potentiated form of antibodies to a C-terminal fragment of angiotensin II AT1 -receptor, in a mixture of homeopathic dilutions of C12, C30, C200, and the activated potentiated form of antibodies to endothelial NO-synthase in a mixture of homeopathic dilutions of C12, C30, C200, was studied, in solution form, in the NISAG rat hypertension model. Investigations were conducted on 50 NISAG line male rats (weight 300 g, age 4 months) with hereditary stipulated stress-sensitive arterial hypertension, which were divided into 5 groups by 10 animals each.
The animals were given per orally, once a day and for 28 days, the following medications: Group 1 - 2.5 ml/kg of the activated potentiated form of antibodies to a C- terminal fragment AT1 of human angiotensin II receptor (a mixture of dilutions C12, C30, C200) in combination with 2.5 ml/kg of distilled water; Group 2 - 2.5 ml/kg of the activated potentiated form of antibodies to endothelial NO-synthase (a mixture of dilutions C12, C30, C200) in combination with 2.5 ml/kg of distilled water; Group 3 - 5 ml/kg of the combination pharmaceutical composition (a mixture of homeopathic aqueous dilutions C12, C30, C200 of each component); Group 4 - 5 ml/kg (10 ml/kg dose) of the comparison drug (losartan); and Group 5 - 5 ml/kg of distilled water.
Two times a week, 2 to 6 hours after administration of VSD antibodies and losartan, systolic blood pressure (SBP) was measured by an indirect method in a tail artery (using a cuff). The Table 6 shows the dynamics of changes in systolic blood pressure in NISAG line rats, measured by indirect method.
Table 6.
Indicator Initial SBP in SBP after 28 Δ in % of the mmHg days of comparison initial level medicine with the initial
administration level, in
in mmHg mmHg
VSD antibodies to C- 176 150 -26 -14.7% terminal fragment AT1 of human angiotensin II
receptor
VSD antibodies to 175 164.5 -10.5 -6% endothelial NO-synthase
Combination medicine on 179.5 140 -39.5 -22% the basis of VSD
antibodies to C-tailed
fragment AT1 of
angiotensin II receptor
and to endothelial NO- synthase
Losartan 173.5 140.5 -33 -19%
Control (distilled water) 181 178 -3 -1.6%
Example 6.
The experimental studiy investigated the effects of antibodies to the C-terminal fragment to the insulin receptor β-subunit affinity purified on antigen, in ultra-low dose, obtained by super dilution of the initial matrix solution 10012, 10030, 100200 times (ULD anti-IR), antibodies to endothelial NO-synthase affinity purified on antigen, in ultra-low dose, obtained by hyper-dilution of the initial matrix solution 10012, 10030, 100200 (ULD anti-ULD anti-eNOS), as well as the combination of ultra-low doses of antibodies to the C-terminal fragment to the insulin receptor β-subunit and ultra-low dose of antibodies to endothelial NO-synthase (ULD anti-IR + ULD anti-eNOS).
In the study, 150 male Wistar were used (weight at beginning of study 250-300 g, age 3.5-4 months). 10 rats were intact. The rest were intravenously injected with streptozotocin at the dose of 50 mg/kg (experimental model of diabetes mellitus). 72 hours after injection of streptozotocin, rats with blood plasma glucose level not less than 12 mmol/l were selected, divided into 7 groups (20 rats in each), which over 21 days were given distilled water (5 ml/kg/day, once daily intragastrically), insulin® (8 units/kg/day, subcutaneously), Rosiglitazone® (8 mg/kg/day, twice daily intragastrically), ULD anti-IR (2.5 ml/kg/day in a volume of 5 ml/kg/day, once daily intragastrically), ULD anti-IR + ULD anti-eNOS (5 ml/kg/day, once daily intragastrically), and also Rosiglitazone® and insulin® together or ULD anti-IR + ULD anti-eNOS and insulin®, according to regimes corresponding to each preparation (as described above). Intact rats received distilled water in the same volume. On days 7, 14 and 21 of injection of preparations in rats, fasting blood plasma glucose level measured with enzymatic method (glucose oxidase method) with utilization of "glucose FKD" kits (Russia).
Oral glucose tolerance test (OGTT) was performed on day 4 of the study (day 14 of administration of preparation) according to standard method (Du Vigneaud and Karr, 1925). The rats were starving at water for 18 hours. 60 min before the test they were last given test substances. Intact rats received distilled water in the same volume. Glucose was administered per os 50% w/w water glucose solution (1 g/kg of rat weight). Serum glucose of blood sample from tail vein was measured by using "Glucose FKD" kit (OOO "Pharamaceutical and clinical diagnostics, Russia, www.fkd.ru) at 0, 30, 60, 90, 120 min. Mean area under the curve (AUC) concentration of blood glucose over time was calculated.
Injection of streptozotocin led to a substantial increase in blood plasma glucose of rats in comparison with intact animals (18 mmol/l versus 3.5 mmol/l, p<0.05). In the ULD anti-IR group, on day 7, 14 and 21 of injection of preparation, glucose level was lower than in the control group by 22-28% on average; however, differences did not reach a statistically significant level. The combination of ULD anti-IR and anti-eNOS was more efficacious; the decrease in glucose level on days 14 and 21 of the experiment were 47% and 42%, respectively (p<0.05 versus control). The reference preparation, Rosiglitazone, also lowered glucose level by day 14 and 21 of the experiment; at that, the effect reached statistical significance on day 14 of the experiment only (36%, p<0.05 versus control).
Insulin, injected at 1/2 of the effective dose (selected in the preliminary study) most effectively lowered glucose level in all observation periods (down to the level of the intact control). (Figure 1). It should be taken into account that short-acting insulin was used in the study and blood plasma glucose was measured 1 hour after its injection, which also influenced the effect of the ½ insulin dose on blood glucose level. Against this background it was not possible to fully determine what the effect of the combined use of insulin and rosiglitazone or insulin and complex ULD anti-IR + anti-eNOS is. Glucose tolerance disturbance (reduction in glucose utilization by the body) is one of the most important indicators in diagnostic and treatment of diabetes mellitus. In intact animals, in the oral glucose tolerance test (day 14 of injection of preparations), complex preparation ULD anti-IR + ULD anti-eNOS and insulin most effectively increased glucose tolerance when administered alone. Rosiglitazone also reduced the area under concentration over time curve (increased glucose tolerance); however, its efficacy was not statistically significant versus the control group (Figure 2).
Example 7.
The experimental studiy investigated the effects of antibodies to the C-terminal fragment to the insulin receptor β-subunit affinity purified on antigen, in ultra-low dose, obtained by super dilution of the initial matrix solution 10012, 10030, 100200 times (ULD anti-IR), antibodies to endothelial NO-synthase affinity purified on antigen, in ultra-low dose, obtained by hyper-dilution of the initial matrix solution 10012, 10030, 100200 (ULD anti-ULD anti-eNOS), as well as the combination of ultra-low doses of antibodies to the C-terminal fragment to the insulin receptor β-subunit and ultra-low dose of antibodies to endothelial NO-synthase (ULD anti-IR + ULD anti-eNOS).
In the study, 36 male Goto-Kakizaki rats were used (weight at beginning of study 250-280 g, age 10-12 weeks). Rats of this line are characterized by spontaneous development of non-insulin-dependent diabetes. The animals were divided into 3 groups (12 rats in each) and received either distilled water (5 ml/kg, once daily intragastrically), or ULD anti-IR (2.5 ml/kg once daily intragastrically), or ULD anti-IR + ULD anti-eNOS (5 ml/kg, once daily intragastrically) for 28 days. Blood plasma glucose level was measured with the help of a glucose analyzer (Beckman, Fullerton, California, USA) before beginning injection of preparations and on day 4, 8, 12, 16, 20, 24, 28 of injection of preparations. On day 28, a glucose tolerance test was carried out (glucose p.o., 1 g/kg).
Injection of ULD anti-IR led to a significant (p<0.05) drop in blood plasma glucose level of rats; however, the use of complex ULD anti-IR + ULD anti-eNOS was more efficacious (p<0.001 versus control) (Figure 3). The results were confirmed by glucose tolerance test data carried out on day 28 of injection of preparations (Figure 4). Injection of ULD anti-IR led to an increase in glucose tolerance (statistically insignificant drop by 44% AUC versus control). At the same time, the reduction in this parameter (AUC) caused by injection of complex ULD anti-IR + ULD anti-eNOS was 62% and it was statistically significant versus control (p<0.05).
Example 8.
The following preparation were used: 300 mg tablets impregnated with aqueous alcoholic solution (3 mg / tab.) activated - potentiated form of polyclonal rabbit brain- specific proteins antibodies S-100, purified on an antigen, in ultra low dose (ULD anti- Si 00) received by super dilution of initial solution (with concentration of 2.5 mg / ml) in 10012, 10030, 100200 times, of equivalent mixture of centesimal homeopathic dilutions C12, C30, C200; 300 mg tablets impregnated with pharmaceutical composition contained aqueous-alcohol solutions of (6 mg/tab) activated - potentiated forms of polyclonal affinity purified rabbit antibodies to brain-specific protein S-100 (anti S-100) and to eNOS (anti-eNOS) in ultra low dose (ULD), received by super dilution of initial solution (with concentration of 2.5 mg/ml) in 10012, 10030, 100200 times, of equivalent mixture of centesimal homeopathic dilutions C12, C30, C200; (ULD anti-S100+anti- eNOS); 300 mg tablets impregnated with aqueous-alcohol solution (3 mg/tab.) of activated - potentiated form of polyclonal rabbit eNOS antibodies purified on antigen in ultra low dose (ULD anti-eNOS), received by super dilution of initial solution (with concentration of 2.5 mg/ml) in 10012, 10030, 100200 times, of equivalent mixture of centesimal homeopathic dilutions C12, C30, C200; and as placebo 300 mg tablets containing excipients: lactose (lactose monohydrate) - 267 mg, microcrystal cellulose - 30 mg, magnesium stearate - 3 mg.
The effectiveness of the studied drugs in the treatment of dizziness (vertigo) and other symptoms of motion sickness was evaluated on kinetosis model or motion diseases/motion sicknesses which occurs by various vestibular vegetative disorders. Dizziness is the typical sign of lesion of the vestibular analyzer of various genesis including dysfunction of the vestibular nerve and cochlear system, circulatory embarrassment in vertebral basilar system, pathology of the central nervous system (CNS), etc. Dizziness as a manifestation of kinetosis accompanied with other vestibular- vegetative disorders which include three types of reactions: the vestibular-motor (nystagmus and the reaction of deviation), vestibular-sensory (in addition to dizziness, nystagmus is (or reaction of post rotation), defensive movements) and vegetative (nausea, vomiting, sweating, palpitation, heat feeling, pulse and blood pressure fluctuations).
Double blind placebo controlled comparative study were conducted in parallel groups consisting of 15 somatically healthy subjects - males and females aged from 15 to 60 years (mean age 33.3±0.75 years) with low (n=5; 33%) or mean (n=10; 67%) degree of motion sickness resistance in order to test anti motion sickness properties of various compositions. Group I was given ULD anti-S100+anti-eNOS, Group 2 was given ULD anti-S100 and Group 3 was given anti-eNOS.
To simulate the condition of motion sickness and evaluate the effectiveness of studied drugs the most appropriate and recognized kinetosis models - test with a continuous cumulative effect of accelerations by Coriolis (CCEAC) was used. Initial tolerance of CCEAC test in all study subjects was not more than 5 minutes. Vestibular- vegetative disorders provoked by kinetic effect (CCEAC) were registered with usage of complex of diagnostic methods including subject's examination, quantitative evaluation of disorders of vestibular-vegetative sensitivity (Halle scale), analysis of heart rate variability (HRV), and self-esteem of functional condition (WBAM - well-being, activity, and mood). As the criteria of efficiency of conducted therapy the dynamics of tolerance and extent of recovery period at kinetic influence were assessed as well as alteration of indexes' evidence of sensory-motor reactions (nystagmus), HRV indexes (with usage of Biocom Wellness Scan system, developed by AWS, LLC in accordance with International Standard of European Cardiologists Association and North American Electrophysiology Association) and WBAM data. The safety criteria were character, evidence and terms of emergence of probable adverse events (AE) in the treatment period connected with medication intake; influence of studied drugs for indexes which characterize the function of central nervous system (CNS) (reaction on moving object (RMO)), the time of simple motor reaction (TSMR); the dynamics of physical and functional factors (heart rate (HR), systolic and diastolic blood pressure (SBP, DBP), Stange's test; exercise tolerance (index of Harvard step-test). Safety was assessed after single dose administration and after 7-day course administration of the combination ULD anti-S-100 and ULD anti-eNOS.
All the subjects during 1 month before being involved into the study had not taken any drugs. After screening the subjects were randomized into 4 groups (Groupl - ULD anti-S100+anti-eNOS, Group 2 - ULD anti-S100, Group 3 - ULD anti-eNOS, and Group 4 - placebo).
On the first day of the study (Visit 1 ) the initial functional and psycho-physiological state of the subjects was registered, the subjects were then given 5 tablets of the respective ULD antibodies, followed by administration of the CCEAC test. The duration of the test was registered; vegetative-vestibular disorders and AEs related to motion sickness were detected with the help of a complex diagnostic examination. In the next 2-6 days the subject were given 1 tablet three times a day of the prescribed drug. At the 7th day (Visit 2) the subjects were given the same dosage as on the first day (Visit 1 ). The complex of diagnostic studies was conducted before and after the CCEAC test. The study was organized in such way as study crew would work only with one subject. The study was parallel and conducted in the first half of a day with participation of, as a rule, 4 persons in a day, one person for drug or placebo. The next three weeks were washout period, at the end of which the new drug or placebo was prescribed to subjects of each group; the cycle of study was being repeated (Visit 1 , the course intake of a drug; Visit 2). Thus, during the study each subject took part in four cycles of study. That is, each subject participated in each group with a three-week washout period between each cycle. This allowed the researcher to level the influence of individual peculiarities of a test person on the treatment effect. The analysis of drug efficiency was conducted on the data of all the test subjects who has completed the full course of studied drug intake according to study protocol (n=15). The evidence factors of symptoms of motion sickness (vertigo, nausea, inactivity, skin pallor, sweatiness, etc.) after kinetic influence (CCEAC) against the background of single-day intake of studied drugs evidenced that all the study subjects have gained roughly the same state of motion sickness as far as the evidence of assessed symptoms of vegetative dysfunction on Halle's scale by physician-researcher was not differed significantly in all groups (Table 7, Visit 1 ). However, while the kinetic affect which cause similar symptoms of motion sickness was different in four groups and was dependent on the drug which was taken by the subjects of the study (Table 8, Visit 1 ). One-day intake of ULD anti-S100 + anti-eNOS preparation led to most clearly anti motion sickness effect which manifested itself not only in significantly more time of tolerance of CCEAC test (104.10 ± 13.14 sec. vs. 68.50 ± 6.57 sec. - in the group of ULD anti-S100; 75.00 ± 6.79 sec. - in the group of ULD anti-eNOS and 61 .30 ± 3.15 sec. - in the placebo group) but also in the least time of nystagmus (9.90 ± 1 .20 sec. vs. 13.50 ± 1 .51 ; 16.10 ± 1.68 and 13.30 ± 1.12 sec, respectively) and in maximal rapid recovery (96.90 ± 13.54 sec. vs. 194.20 ± 18.45; 202.50 ± 21 .72 and 241 .70 ± 38.41 sec, respectively).
Roughly similar indexes were registered at Visit 2 after receiving a course of drugs. To achieve the similar symptoms of motion sickness (Table 7, Visit 2) the longest time of kinetic impact was applied to the subjects who has been receiving the composition of ULD anti-S100 + anti-eNOS (Table 8, Visit 2) for 7 days. The most pronounced anti motion sickness effect of the composition of ULD anti-S100 + anti- eNOS was expressed in significantly less time of nystagmus (9,50 ± 1 ,38 sec, p <0.01 ) and duration of the recovery period (1 17.90 ± 15.65 sec; p <0.01 ). The monocomponent preparation ULD anti-S100 had anti motion sickness action as better indexes of tolerance of CCEAC test, recovery time of nystagmus and recovery than in the placebo group evidenced (Table 8, Visits 1 and 2), but the efficacy of ULD anti-S100 was inferior to composition of ULD anti-S100 + anti-eNOS. The monocomponent preparation ULD anti-eNOS did not show anti motion sickness effect since the results of CCEAC tests and subsequent recovery period had no significant difference from the placebo group (Table 8, Visits 1 and 2). Comparative analysis of indexes of CCEAC test in the groups of ULD anti-S100 + anti-eNOS and ULD anti-S100 in one-day intake of the drugs has shown that the addition of ULD anti-eNOS increased the tolerance of the kinetic effect on the 52%, reduced the nystagmus time on 27% and contributed to the reduction the recovery period after the end of the kinetic effect on 50% including the duration of dizziness - on 49%. However, the greatest contribution of the component of ULD anti- eNOS introduced the effectiveness of combined preparation (compositions of ULD anti- Si 00 + anti-eNOS) in course intake of a drug which was expressed in excess of 30% of the result achieved in the group of ULD anti-S100 by factors of tolerance of kinetic effect and nystagmus duration (in each of the parameters). In addition, the growth of the effect on Visit 2 by indexes of tolerance of CCEAC test and duration of the nystagmus in relation to data of Visit 1 when taking the composition ULD anti-S100 + anti-eNOS in comparison to monocomponent preparation ULD anti-S100 was expressed in a greater degree as confirmed by alteration of these indexes on 30% and 4% (versus 21 % and 0% in the ULD anti-S100 group). In assessing the effectiveness of anti motion sickness properties of drugs the special attention was paid to the possible impact of drugs on the stability of autonomic nervous system (ANS) in particular, shifting of the balance between its sympathetic and parasympathetic divisions. For this purpose, at each visit HRV parameters were analyzed at the rest condition and when performing the functional tests (breathing and orthostatic tests).
Table 7
Indexes of Halle's scale depending on applied preparation after the
performance of CCEAC test
Figure imgf000045_0001
ULD anti-S100 13.30±0.65 12.30±0.46
ULD anti-eNOS 13.10±0.78 12.00±0.55
Placebo 13.40±0.77 13.30±0.45
Table 8
The dynamics of indexes of CCEAC test depending on applied preparation
Figure imgf000046_0001
Notes: for determination of significant difference between groups the Kruskal-Wallis test was used. If the
test showed a significant difference of p <0.05 for comparison between groups against each other
the Mann-Whitney test was used.
* the significant difference in comparison with placebo, p<0,05;
** the significant difference in comparison with placebo, p<0,01 ;
*** the significant difference in comparison with placebo, p<0,001.
the significant difference in comparison with ULD anti-S100 + anti-eNOS, p<0,05;
the significant difference in comparison with ULD anti-S100 + anti-eNOS, ρΟ,ΟΊ ;
the significant difference in comparison with ULD anti-S100 + anti-eNOS, p<0,001.
The analysis of HRV at the rest condition (in sitting position) before and after the CCEAC test (Table 9) detected that in subjects receiving study drugs had a tendency to an increased rate of SDNN indicating an increase in heart rate variability due to parasympathetic influence on heart rhythm. In response to a kinetic effect in all treatment groups the value of RMS-SD increased which characterizes the activity of the parasympathetic component of autonomic regulation. In the groups receiving the composition ULD anti-S100 + anti-eNOS and ULD anti-S100 showed an increase in HF which also indicated a shift in autonomic balance toward parasympathetic link. Thus, after conducting CCEAC tests in all groups there was an increase of parasympathetic effects on heart rate.
Table 9
The HRV parameters of the study participants at rest
before and after the kinetic action Visit 1 (one-day intake) Visit 2 (course intake)
Parameter After the After the After the drug After the drug intake CCEAC test intake CCEAC test
ULD anti-S100 + anti-eNOS group (M±SD)
SDNN, msec. 57.7±5.51 68.2±7.42 59.4±5.03 65.6±4.66
RMSSD, msec. 43.1±6.77 51.4±9.22 47.0±6.21 47.6±5.33
TP, msec.2 979.0±186.06 1678.3±397.1 1067.2±167.24 1381.01166.30
1#
LF, msec. 2 437.5±709.6 709.6±178.72 391.9±75.61 588.5±87.48
HF, msec 171.5±51.08 228.4±76.79 206.5±58.32 218.5±43.96
LF/HF, c.u. 4.2±0.82 4.9±0.83 3.3±0.83 4.2±0.91
ULD anti-S100 group (M±SD)
SDNN, msec. 60.9±4.62 70.9±5.90 59.1 ±4.80 68.8±4.87
RMSSD, msec. 44.3±5.39 50.6±6.56 42.4±4.63 47.8±5.57
TP, msec. 2 832.2±124.93* 1342.8±217.0 841.4±149.93 1288.0±163.52
9 #
LF, msec. 2 315.2±52.38* 550.9±72.44# 313.6±66.71 540.7±87.57#
HF, msec. 2 151.4±41.19 247.0±69.53# 138.3±38.42 187.1 ±39.80
LF/HF, c.u. 3.0±0.54 4.0±0.72 2.8±0.53 4.0±0.52
ULD anti-eNOS group (M±SD)
SDNN, msec. 67.4±7.73 78.6±6.14 65.8±8.68 69.0±5.23 RMSSD, msec. 53.0±8.86 58.4±7.68 59.6±12.45 52.2±5.30
TP, msec. 1307.8±324.24 1841.1 ±359.7 1232.3±292.51 1275.4±172.47
9#
LF, msec.2 576.5±167.07 849.9±194.2# 527.2±167.07 562.1 ±89.38
HF, msec. 313.3±139.90 285.3±65.92 218.9±74.78 216.3±63.72
LF/HF, c.u. 3.6±0.87 3.9±0.82 3.7±1.14 3.8±0.58
Placebo group (M±S D)
SDNN, msec. 64.6±6.10 75.7±6.42 61.1 ±6.72 70.8±6.79
RMSSD, msec. 50.9±7.74 53.1±6.62 44.6±6.63 44.3±5.31
TP, msec.2 1062.2±150.02 1917.8±318.9 898.8±169.62 1418.5±227.59
6# #
LF, msec. * 440.6±77.30 832.4±181.15 334.8±75.94 611.4±113.64#
HF, msec. 253.9±59.95 266.7±61.94 166.0±48.14 174.1 ±44.96
LF/HF, c.u. 3.4±0.72 5.0±1.33 3.4±0.93 4.8±0.83
Note: * the significant difference in comparison with the placebo, p≤0,05);
# the significant difference in comparison with baseline parameters, p<0,05.
The analysis of HRV in transition states showed that one-day intake of composition ULD anti-S100 + anti-eNOS increased the reaction time (13.9 ± 1.14; p≤ 0.05) and the stabilization time (24.2 ± 1.28; p≤ 0.05) in comparison with the ULD anti- Si 00 and placebo. The same factors exceeded the value of the placebo group and after the kinetic effect which demonstrated the positive effect of the combined drug on the reactivity of the ANS (increase of tolerance to changes in body position). The smallest difference between the maximum and minimum heart rate in the breath test confirmed a better balance of the two divisions of ANS after receiving a one-day composition ULD anti-S100 + anti-eNOS (25.1 ± 2.66 beats / min, p≤ 0.05). By the end of week course of therapy the stabilizing effect on the balance of ANS after the CCEAC test (with orthostatic and breath test) is also noticed in the group receiving the composition ULD anti-S100 + anti-eNOS (Tables 10 and 1 1 ).
Table 0
The HRV parameters of participants of the study
at orthostatic test before and after kinetic action
Figure imgf000050_0001
sec.
Placebo group (M±SD)
Exercise reaction, 1.30±0.04 1.30±0.04 1.40 ± 0.06 1.30±0.06 c.u.
Reaction time, sec. 9.5±1.28 8.1±0.90 10.4 ± 1.58 8.8±1.09
Stabilization time, 18.3±0.94 16.811.09 18.0 ± 1.37 16.5±1.1 1 sec.
Note: * the significant difference in comparison with placebo, p<0.05);
χ the significant difference in comparison with ULD anti-S100, p<0.05.
Table 11
The HRV parameters of participants of the study
at breath test before and after kinetic action
Figure imgf000051_0001
max HR - rrrin HR, 27.7±2.68 27.2±2.40 25.7±2.24 26.9±2.67 beats/min.
ULD anti-eNOS (M±SD) Group
Corellation
max HR / min HR, 1.5±0.05 1.5±0.04 1.5±0.06 1.6±0.05 c.u.
Difference
max HR - min HR, 26.7±2.44 26.2±2.04 27.7±2.47 27.3±2.12 beats/min.
Placebo group (M±SD)
Corellation
max HR / min HR, 1.6±0.07 1.6±0.06 1.5±0.05 1 .6±0.05 c.u.
Difference
max HR - min HR, 31.2±3.06 28.2±2.50 27.7±2.37 29.2±2.44 beats/min.
Note: * the significant difference in comparison with placebo, p<0,05
The results of self-esteem of functional state (well-being, activity, mood) of the subjects which was conducted by the participants of the study after the simulation of motion sickness (CCEAC tests) at the beginning and at the end of therapy showed that the subjects of all the groups have given 'average* points for each of the parameters (Table 12). Thus, on the background of drugs intake the CCEAC tolerance was satisfactory. The highest growth rates compared with data of the placebo group by the end of the 7th day of intake (more than 10%) was observed in the group of composition of ULD anti-S100 + anti-eNOS.
Table 12
The dynamics of parameters of self-esteem of functional condition (well-being-activity-mood) of study participants
Figure imgf000053_0001
The safety analysis included data from all the subjects who participated in the study. During the observation period a well tolerance of studied preparations was noticed. No adverse events associated with drug administration identified. All the subjects of studied groups completed treatment in the terms established by the study protocol; there was not persons early dropped out.
According to the results of physical examination including indicators of heart rate, systolic and diastolic blood pressure and according to the Harvard step test data the subjects were not recorded as with any abnormalities during the study (Table 13). All identified changes were not beyond the normal range. In this case, subjectively all subjects reported satisfactory well-being.
Table 13
The dynamics of physical parameters and exercise tolerance of study participants before and after kinetic action
Figure imgf000054_0001
Step-test index - 50.6±1.71 - 53.0±1.63
ULD anti-eNOS (M±SE) Group
HR (beats/min) 76.5+2.59 67.3±1.98 77.3±2.02 70.1±3.23
Systolic blood 127.3±3.14 131.5±5.16 123.5±3.06 129.3±4.13 pressure
(mmhg.)
Diastolic blood 75.2±2.24 80.3±2.66 73.9+2.83 81.0±3.22 pressure
(mmhg.)
Step-test index — 51.8±2.12 — 51.2±2.21
Placebo group (M±SE)
HR (beats/min) 74.5±2.78 68.9±3.46 73.9±3.23 72.3±3.58
Systolic blood 125.3±3.30 133.3±4.73 124.3±2.83 126.9±3.95 pressure
(mmhg.)
Diastolic blood 76.2±2.15 81.7±2.83 75.4±1.86 79.7±3.03 pressure
(mmhg.)
Step-test index — 50.0±2.03 — 50.1±1.99
In addition to the hemodynamic parameters, for evaluation of the safety of studied drugs and its possible negative impact on the central nervous functions, the following physiological parameters were examined in subjects: (RMO (reaction on moving object), SMRT (simple motor reaction time), RA (range of attention), attention span (AS), and attention stability factor (ASF)). In addition, the Stange's test was conducted to assess tolerance to hypoxia.
According to received results (Table 9) neither one-day or course drug intake had a significant effect on the estimated parameters. Indexes of sensory motor coordination (SMRT, RMO) did not differ from the results of the placebo group before and after the CCEAC test at both visits. Study data of such complicated functions like volume and stability of attention showed that the studied drugs both before and after the CCEAC test did not change the degree of concentration and shift in attention not being different from the placebo group.
The analysis of standard exercise tests with breath holding showed a tendency to increase of the tolerance of hypoxia by the subjects (Table 14). When holding the breath the duration of Stange's test grew after taking all study drugs. However, only intake of the combination composition ULD anti-S100 + anti-eNOS showed significantly longer time in the holding of the breath after the kinetic effect (68.1 ± 18.8 sec. at baseline and 91.7 ± 27.4 sec. after the CCEAC test; p <0.05). The increase of tolerance of hypoxia was also noted when the Gench's test (Stange's test) (breath holding at expiration, P> 0.05) was used.
Table 14
The dynamics of parameters of psycho-physiological
state of study participants before and after kinetic action
Figure imgf000056_0001
Gench's test 47.1±4.03 50.1±3.94 46.7±3.28 48.114.52
ULD anti-S100 (M±SE) Group
SMRT 258.9±9.95 282.4±13.56 268.4±11.37 279.119.20
RMO, c.u. 58.1 ±6.40 57.5±6.34 55.115.06 53.815.02
RMO, % of target 3.7±1.50 2.0±0.82 2.3±0.83 5.011.69 hit
AS, sec. 6.0±0.40 6.4±0.52 6.2±0.42 6.010.41
Range of 42.6±2.68 42.1±2.27 42.7±2.30 41.912.52 attention, sec.
ASF 14.5±1.16 14.9±1.26 15.3±1.13 15.411.18
Stange's test 59.0±4.09 72.6±6.19 64.5±4.93 75.915.67
Gench's test 47.114.48 49.4±4.69 48.3±4.30 48.814.14
ULD anti-eNOS (M±SE) group
SMRT 257.7±8.49 279.4±14.23 266.7±13.19 275.5111.44
RMO, c.u. 48.3±3.67 51.9±4.39 52.5±4.79 49.614.22
RMO, % of target 2.3±0.83 2.0±0.82 3.3±1.26 5.711.68 hit
AS, sec. 5.9±0.25 6.0±0.34 5.5+0.24 5.910.33
Range of 41.9±2.10 43.8±2.39 41.3±2.00 42.512.22 attention, sec.
ASF 13.7±1.34 14.8±1.31 15.6±1.24 14.111.40
Stange's test 62.5±5.49 69.5±5.09 56.7±3.34 73.117.98
Gench's test 43.1l3.51 45.7±3.15 43.413.77 45.814.03
Placebo group (M±SE)
SMRT 267.6±7.64 290.1±11.33 281.119.78 263.316.85
RMO, c.u. 60.7±8.31 54.1±5.57 51.113.69 52.615.38
RMO, % of target 3.7±1.03 3.7±1.24 3.310.93 4.311.61 hit
AS, sec. 6.1±0.71 5.7±0.36 5.510.32 5.910.71 Range of 41.9±2.09 42.4±2.81 41.3+2.18 39.612.26 attention, sec.
ASF 14.5±1.64 14.511.79 15.3+1.55 15.911.58
Stange's test 63.7±4.71 s. 67.916.90 64.815.94 83.0112.24
Gench's test 44.7±2.52 47.113.30 43.712.71 47.813.78
Thus, the study using an experimental motion sickness demonstrated the effectiveness of the combination composition ULD anti-S100 + anti-eNOS and monocomponent preparation ULD-S100. The studied drugs increase the stability of the subjects to the kinetic effect after simulation of the clinical and physiological effects of motion sickness contributing to more mild clinical process of motion sickness and earlier recovery of the subjects after cessation of treatment. In addition, it was shown that the anti motion sickness effect of the combination composition (compositions ULD anti-S100 + anti-eNOS) increases the efficiency of individual components. The effectiveness of the combination composition ULD anti-S100 + anti-eNOS in the control of the vestibular- autonomic and sensory reactions of a body in experimental motion sickness increases at course intake. It should be noted that ULD anti-eNOS in the form of monopreparation does not have a protective effect against motion sickness but when combined with ULD anti-S100 significantly enhances the anti motion sickness effect of the last one which manifests itself as at one-day so at short course intake of the drug. The best ability to adjust the transient processes that is to influence to the reactivity of the parasympathetic and sympathetic parts of ANS as well as adaptive capabilities of ANS in a state of motion sickness (to increase the tolerance to sudden changes in a body position) was observed in the composition ULD anti-S100 + anti-eNOS which is an important component of anti motion sickness properties of the drug. Composition ULD anti-S100 + anti-eNOS and monocomponent preparation ULD anti-S100 when using them as anti motion sickness preparation including when performing an operator functions are safe and do not adversely impact on the physical and psycho- physiological parameters. Combination composition ULD anti-S100 + anti-eNOS and ULD anti-S100 can be recommended for the prophylaxis and relief of kinesia in motion disease (including sea, air and car sicknesses) to persons with low and moderate degree of stability. The combination composition has high safety and no adverse effects on the quality of professional activity.
Example 9.
To study the properties of the combination pharmaceutical composition of the present application for the treatment of psychoorganic syndrome, tablets with weight of 300 mg were used. The tablets were impregnated with pharmaceutical composition containing water-alcohol solutions (6 mg/tab.) of activated-potentiated forms of polyclonal affinity purified rabbit brain-specific proteins antibodies S-100 (anti-S100) and to endothelial NO-synthase (anti-eNOS) in ultra low doses (ULD) obtained by super dilution of initial solution (with concentration of 2.5 mg/ml) in 10012, 10030, 100200 times, of equivalent mixture of centesimal homeopathic dilutions C12, C30, C200 ("ULD anti- Si 00 + anti-eNOS").
The control group patients received 300 mg tablets impregnated with pharmaceutical composition containing water-alcohol solutions (6 mg/tablet) of activated-potentiated forms of polyclonal affinity purified rabbit brain-specific proteins antibodies S-100 (anti-S100) in ultra low doses (ULD) obtained by super dilution of initial solution (with concentration of 2.5 mg/ml) in 10012, 10030, 100200 times.
The study included patients diagnosed with psychoorganic syndrome of posttraumatic origin. Psychoorganic syndrome is characterized by the following triad of signs: weakness of memory, loop of intelligence, incontinence of affect (Walther Buel triad).
The study was an open-label randomized comparative parallel group clinical trial of efficacy and safety of the therapy in patients with psychoorganic syndrome of posttraumatic origin (the first group of patients took the preparation of ULD anti-S100, the second group of patients - the preparation of ULD anti-S100+anti-eNOS). The study included 6 patients aged 35 to 90 years old (mean age 70.83 ± 21 .95) diagnosed with psychoorganic syndrome.
Compliance of patients to following inclusion and exclusion criteria was checked: Inclusion criteria:
1 . Patients diagnosed with posttraumatic encephalopathy with psychoorganic syndrome or with encephalopathy of complex etiology (vascular, posttraumatic) with psychoorganic syndrome, confirmed by medical history, neurological examinations and medical records.
2. Patient without change in concomitant therapy within at least one month prior to Visit 1.
3. No need for change in concomitant therapy for the whole observation period.
4. No need for immunomodulatory drugs prescription for the next 6 months.
5. Patients with a level of education sufficient to adequately communicate with the researcher and study coordinator.
6. Patients assessed by the researcher as reliable and ready to perform all scheduled clinical visits, tests and procedures stipulated in the protocol.
7. Patients having a valid home address.
Exclusion criteria:
1 . Any brain surgery in medical history.
2. Acute myocardial infarction.
3. Hemorrhagic stroke.
4. The diagnosis of psychosis, bipolar disorder or schizoaffective disorder in medical history.
5. Major depressive disorder according to criteria of depression module of international neuropsychiatric mini-interview (MINI).
6. Factors/conditions of medical or another character which in the opinion of the researcher may affect to the test results for patients in the study. Answers "2A", "2B", "2C" or "3" in the section Ί" of Beck Depression questionnaire (active suicidal ideation with some intent to act, without a specific plan, or active suicidal ideation with a specific plan and intent).
Autoimmune disease in medical history.
Acute damage of liver or severe cirrhosis (class C by Child-Pugh).
Non-corrected disorder of thyroid gland function.
Decompensated arterial hypertension in medical history.
Serious or decompensated cardiovascular disease, liver disease, kidney disease, metabolic, respiratory or hematological disease, symptomatic peripheral vascular disease or another medical or psychiatric condition which in the opinion of the researcher, may affect the patient's participation in the study or could lead to prolonged hospitalization or re-hospitalization during the study.
Diseases and conditions which in the opinion of researcher may prevent patient from the participation in the study.
The intake of the drug containing ULD anti-eNOS or the drug containig ULD anti- Si 00 before inclusion in the study.
The intake of antidepressants of any group including plant and homeopathic preparations.
The intake of anxiolytics of any group including plant and homeopathic preparations.
The intake of immunomodulators including plant and homeopathic preparations. The treatment with systemic steroids within 1 month before Visit 0.
The participation in the study of the drug containing ULD anti-eNOS or the drug containing ULD anti-S100 if patients took at least one doze of preparation.
Participation in other clinical studies within 1 month before within 1 month before being enrolled in this study.
Pregnancy, breast feeding, impossibility to use an adequate contraception during the study period and within 1 month after the last intake of the studied drug.
The presence of allergy/intolerance of any component of drugs including lactose intolerance. 23. Patients taking narcotic drugs and neuroleptics, alcoholic dependence, psychiatric diseases in patients.
24. Patients are the staff of the center which directly related to the conducted study and/or are family members of the research center staff's which directly associated with the ongoing study. The "family members" are a husband (wife), parents, children, brothers (sisters).
25. Participation in the trial or presumable receiving of compensation or participation in the judicial process in the opinion of a researcher.
After the determination of patient conformity to inclusion and exclusion criteria the patients were randomized into two study groups: a group of patients receiving ULD anti- S100 (3 patients, women - 33.33%, men - 66.66%, mean age - 71.33 ± 16.25 years old), a group of patients receiving ULD anti-S100 + anti-eNOS (3 patients, women - 66.66 % men - 33.33 %, mean age - 70.33 ± 30.66 years old).
During this study the five visits were carried out. Treatment phase lasted from Visit 1 to Visit 4 for 84 ± 5 days on average. Visit 4 (Day 84 ± 5) was the first endpoint of the study followed by a follow-up observation. Follow-up phase continued from Visit 4 to Visit 5 (Day 168 ± 5 on average).
In the safety analysis the data of all patients participating in the study (n = 6) was included. During the study good tolerance of the drug was recorded. No adverse events were registered. All patients of studied groups have completed the treatment according to the protocol; no early dropouts.
The effect of ULD anti-S100 + anti-eNOS preparation on the main clinical signs and symptoms of psychoorganic syndrome (NPI neuropsychiatric inventory, Intensity section), on the intensity of concomitant distress of the person attending to the patient (NPI Neuropsychiatric Inventory, Distress section) as well as the on patient's cognitive functions (The Mini Mental State Examination, MMSE) were assessed. An improvement was found in the key symptoms of psychoorganic syndrome such as statistically significant reduction of the intensity section of NPI neuropsychiatnc inventory (from 91.0±15.13 to 69.0±+6.24, p<0.05), decrease of distress section score of NPI neuropsychiatnc inventory (from 44.33±17.78 to 36.33±3.21 , p<0.05) at Visit 4 (Table 15).
In the group of patients receiving ULD anti-S100 alone no clinical improvement was recorded.
At that, a difference between the groups of patients in the total score of the Intensity section of NPI neuropsychiatnc inventory at the end of therapy was statistically significant at p<0.05.
Table 15.
Figure imgf000063_0001
* - p from baseline <0.05; # - p from control <0.05
Thus, in the conducted clinical study a positive effect of combined pharmaceutical composition ULD anti-S100 + anti-eNOS on the main clinical signs and symptoms of psychoorganic syndrome and tendency to effect cognitive functions with psychoorganic syndrome. In addition, good drug tolerability was confirmed. No drug-related adverse events were registered.
Example 10.
To study the properties of the combination pharmaceutical composition of the present application for the treatment of Alzheimer's disease, tablets with weight of 300 mg were used. The tablets were impregnated with pharmaceutical composition containing water-alcohol solutions (6 mg/tablet.) of activated-potentiated forms of polyclonal affinity purified rabbit brain-specific proteins antibodies S-100 (anti-S100) and to endothelial NO-synthase (anti-eNOS) in ultra low doses (ULD) obtained by super dilution of initial solution (with concentration of 2.5 mg/ml) in 10012, 10030, 100200 times, of equivalent mixture of centesimal homeopathic dilutions C12, C30, C200 (ratio: 1 :1 ) ("ULD anti-S100 + anti-eNOS").
The control group patients received 300 mg tablets impregnated with pharmaceutical composition containing water-alcohol solutions (3 mg/tablet) of activated-potentiated forms of polyclonal affinity purified rabbit brain-specific proteins antibodies S-100 (anti-S100) in ultra low doses (ULD) obtained by super dilution of initial solution (with concentration of 2.5 mg/ml) in 10012, 10030, 100200 times, of equivalent mixture of centesimal homeopathic dilutions C12, C30, C200
The study included patients diagnosed with Alzheimer's disease. Alzheimer's disease is characterized by dementia (acquired dementia, stable impairment of cognitive activity with certain loss of previously acquired knowledge and practical skills, difficulties or impossibility to gain new knowledge).
The study was an open-label randomized comparative clinical trial of efficiency and safety of the therapy in two parallel groups (preparations of ULD anti-S100 and ULD anti-S100+anti-eNOS) in the treatment of patients with mild to moderate Alzheimer's disease. The study included 6 patients aged 55 - 64 years old (mean age 59.0 ± 3.58) diagnosed with mild to moderate Alzheimer's disease.
Compliance of patients to following inclusion and exclusion criteria was checked:
Inclusion criteria are as follows:
1. Patients with mild to moderate Alzheimer's disease, confirmed by medical history, neurological examinations and medical records.
2. Patient without change in concomitant therapy within at least one month prior to Visit 1.
3. No need for change in concomitant therapy for the whole observation period.
4. No need for immunomodulatory drugs prescription for the next 6 months.
5. Patients with a level of education sufficient to adequately communicate with, the researcher and study coordinator.
6. Patients assessed by the researcher as reliable and ready to perform all scheduled clinical visits, tests and procedures stipulated in the protocol.
7. Patients having a valid home address.
Exclusion criteria are as follows:
1. Any brain surgery in medical history.
2. Acute myocardial infarction.
3. Hemorrhagic stroke.
. The diagnosis of psychosis, bipolar disorder or schizoaffective disorder in medical history.
5. Major depressive disorder according to criteria of depression module of international neuropsychiatric mini-interview (MINI).
. Factors/conditions of medical or another character which in the opinion of the researcher may affect to the test results for patients in the study. Answers "2A", "2B", "2C" or "3" in the section "I" of Beck Depression questionnaire (active suicidal ideation with some intent to act, without a specific plan, or active suicidal ideation with a specific plan and intent).
Autoimmune disease in medical history.
Acute damage of liver or severe cirrhosis (class C by Child-Pugh).
Non-corrected disorder of thyroid gland function.
Decompensated arterial hypertension in medical history.
Serious or decompensated cardiovascular disease, liver disease, kidney disease, metabolic, respiratory or hematological disease, symptomatic peripheral vascular disease or another medical or psychiatric condition which in the opinion of the researcher, may affect the patient's participation in the study or could lead to prolonged hospitalization or re-hospitalization during the study.
Diseases and conditions which in the opinion of researcher may prevent patient from the participation in the study.
The intake of the drug containing ULD anti-eNOS or the drug containig ULD anti- Si 00 before inclusion in the study.
The intake of antidepressants of any group including plant and homeopathic preparations.
The intake of anxiolytics of any group including plant and homeopathic preparations.
The intake of immunomodulators including plant and homeopathic preparations. The treatment with systemic steroids within 1 month before Visit 0.
The participation in the study of the drug containing ULD anti-eNOS or the drug containing ULD anti-S100 if patients took at least one doze of preparation.
Participation in other clinical studies within 1 month before within 1 month before being enrolled in this study.
Pregnancy, breast feeding, impossibility to use an adequate contraception during the study period and within 1 month after the last intake of the studied drug.
The presence of allergy/intolerance of any component of drugs including lactose intolerance. 23. Patients taking narcotic drugs and neuroleptics, alcoholic dependence, psychiatric diseases in patients.
24. Patients are the staff of the center which directly related to the conducted study and/or are family members of the research center staff's which directly associated with the ongoing study. The "family members" are a husband (wife), parents, children, brothers (sisters).
25. Participation in the trial or presumable receiving of compensation or participation in the judicial process in the opinion of a researcher.
After the determination of patient conformity to inclusion and exclusion criteria the patients were randomized into two study groups: a group of patients receiving ULD anti- Si 00 (3 patients, women - 100%, men - 0%, mean age - 59.0 ± 3.6 years old), a group of patients receiving ULD anti-S100 + anti-eNOS (3 patients, women - 66.66 % men - 33.33 %, mean age - 59.0 ± 4.36 years old).
During this study the five visits were carried out. Treatment phase lasted from Visit 1 to Visit 4 for 84 ± 5 days on average. Visit 4 (Day 84 ± 5) was the first endpoint of the study followed by a follow-up observation. Follow-up phase continued from Visit 4 to Visit 5 (Day 168 ± 5 on average).
In the safety analysis the data of all patients participating in the study (n = 6) was included. During the study good tolerance of the drug was recorded. No adverse events were registered. All patients of studied groups have completed the treatment according to the protocol; no early dropouts.
The effect of ULD anti-S100 + anti-eNOS preparation on the main clinical signs and symptoms of Alzheimer's disease (NPI neuropsychiatric inventory, Intensity section), on the intensity of concomitant distress of the person attending to the patient (NPI Neuropsychiatric Inventory, Distress section) as well as the on patient's cognitive functions (The Mini Mental State Examination, MMSE) were assessed. An improvement was found in the key symptoms of Alzheimer's disease such as statistically significant reduction of the intensity section of NPI neuropsychiatric inventory (from 24.33± 4.73 to 12.0±3.46, p<0.05) at Visit 4 (Table 16).
A tendency for reduction of distress of the person attending to the patient was also found as well as for the reduction in activity of the patient's everyday life at the end of therapy (however, without any statistically significant difference, possibly due to the small number of patients included in the study).
Besides, a tendency for improvement of cognitive functions was found, manifested by increase of MMSE score from 23.66±3.21 to 26.66±1.53 points, however, the difference also failed to reach statistically significant values at the end of therapy, which may also be related to the small sample size.
The same endpoints in the group of patients receiving ULD anti-S100, showed no trend for improvement, except a statistically insignificant improvement of MMSE score from 22.66±0.58 to 23.33±0.58 points.
At that, a difference between the groups of patients in the total MMSE score at the end of therapy was statistically significant at p<0.05.
Table 16.
NPI (intensity) NPI ADCS-ADL MMSE
(distress)
ULD anti- 24.33±4.73 9.66±1.53 71.0±6.56 23.66±3.21
Si 00+anti-eNOS
before treatment
ULD anti- 12.0±3.46 * 5.0±3.61 74.33±2.51 26.66±1.53#
Si 00+anti-eNOS
after treatment
ULD anti-S100 35.6615.50 22.33±5.50 61.66±5.13 22.66±0.58 before treatment
ULD anti-S100 38.33±8.5 23.0±5.0 61.33±5.86 23.33±0.58 after treatment
- p from baseline < 0.05; # - p from control <0.05 Thus, in the conducted clinical study a positive effect of combined pharmaceutical composition ULD anti-S100 + anti-eNOS on the main clinical signs and symptoms of Alzheimer's disease and tendency to effect cognitive functions with Alzheimer's disease. In addition, good drug tolerability was confirmed. No drug-related adverse events were registered.
Example 11.
Group 1 - the active drug group was given 300 mg tablets impregnated with an aqueous-alcohol solutions (6 mg/tab) of activated-potentiated form of polyclonal rabbit antibodies to brain specific S-100 protein (anti-S-100), and to endothelial NO-synthase (anti-eNOS) in ultra low dose (ULD anti-S-100 + ULD anti-eNOS), purified on antigen, obtained by super dilution of initial solution (with concentration of 2.5 mg/ml) in 10012, 10030, 100200 time, equivalent to mixture of centesimal homeopathic dilutions C12, C30, C200;
Group 2 - the comparison group was given 300 mg tablets impregnated with an aqueous-alcohol solution (3 mg/tab) of activated-potentiated forms of polyclonal rabbit antibodies to brain-specific S-100 protein purified on antigen in ultra low dose (ULD anti- S100) obtained by super dilution of initial solution in 10012, 10030, 10050 times, of equivalent mixture homeopathic dilutions C12, C30, C50.
Group 3 - the control group (placebo) was given of 300 mg tablets having excipients (lactose monohydrate - 267 mg, microcrystal cellulose - 30 mg, magnesium stearate - 3 mg).
The effectiveness of the active drug ULD anti-S100 + anti-eNOS in the treatment of patients with syndrome of attention deficit and hyperactivity disorder (ADHD) was conducted in comparative double blind placebo-controlled study in 146 children from 6 to 12 years old (mean age 9.3±0.24 years old) who were randomized into three groups depending on prescribed therapy. Within 12 weeks the patients of group No.1 (n = 46) received the composition ULD anti-S100 + anti-eNOS, 2 tablets twice a day; the comparison group 2 members (n = 50) received ULD anti-S100, 2 tablets twice a day; the control group 3 members (n = 50) received 2 tablets twice a day. All the patients included in the study had clinically marked presentations of ADHD which was confirmed by high points on ADHD symptoms assessing scale (ADHDRS-IV-Home Version): 33.8 ± 0.92 in group 1 ; 32.5 ± 1 .14 in group 2 and 33.6 ± 0.91 in group 3. Most of the children were characterized by a moderate degree of severity of ADHD according to the CGI-ADHD-Severity questionnaire. The total score on this scale was 4.0 ± 0.02 points in the group 1 , 4.0 ± 0.03 points in the group 2, and 4.0 ± 0.00 points in the group 3. Thus, initially the patients of the three groups had comparable indicators of the severity of ADHD. According to the results of neurological, clinical - laboratory and instrumental examination at the time of enrollment to the study no abnormalities in any patient was detected. Over the 12 weeks of treatment, patients were seen six times by a doctor. During which the physician-researcher recorded the dynamics of intensity of clinical presentations of ADHD (total score on a scale ADHDRS-IV-Home Version) and disease severity (on the CGI-ADHD-Severity), supervised the prescriptions and administration of treatment and evaluated the safety of the treatment.
The analysis of the effectiveness of 12 weeks of therapy in the three groups showed a decrease of more than 25% from the initial total score on a scale ADHDRS-IV- Home Version in 75% (n = 36) of children treated with the composition ULD anti-S100 + anti-eNOS; in 66% (n = 33) of patients treated with ULD anti-S100 and in 56% (n = 28) of children receiving placebo. Differences of efficiency between the groups showing a more detailed assessment, taking into account the three-level grading of improvement of condition (reduction of total score on a scale ADHDRS-IV for <25%, 25-49.9% or > 50% from the baseline), are presented in Table 17. Significant improvement with a reduction in total score on 50% or more from the baseline was noted in 52% of children in group 9 who were taking ULD anti-S100 + anti-eNOS, and in 34% of children in group 2 who were taking ULD anti-S100 (vs. 8% of patients in group 3 with placebo).
Significant reduction (p <0.001 ) of clinical implications of ADHD in comparison with the initial state is already occurred after 2 weeks of therapy in all three groups of observation. Positive dynamics was more significant in patients of groups 9 and 2 as the significant differences were identified in them between total scores ADHDRS-IV-Home Version, not only in relation to the screening visit but when compared with the indexes of the group 3 with placebo. In subsequent weeks of treatment the efficacy of treatment with composition ULD anti-S100 + anti-eNOS and monocomponent preparation ULD- S100 started to grow, the most significantly in the active drug group (p <0.05). The resulting decrease in total score on a scale ADHDRS-IV-Home Version in children of the group 9 with ULD anti-S100 + anti-eNOS was 16.5 points, in patients of the group 2 with ULD anti-S100 - 12.4 points (compared to 6.3 points in the group 3 with placebo). As a result of 12-week of treatment the intensity of clinical implications of ADHD in children treated with the composition ULD anti-S100 + anti-eNOS decreased by almost in half (- 48.8%) and in patients treated with ULD anti-S100 more than in one-third (-38.2%) compared with the baseline.
The intake of composition ULD anti-S100 + anti-eNOS or ULD anti-S100 influenced on both clusters of symptoms of ADHD which was confirmed by dynamics of assessments by two sections of the scale with ADHDRS-IV-Home Version. Moreover, the treatment with the composition ULD anti-S100 + anti-eNOS was significantly higher than the effectiveness of therapy with monopreparation ULD anti-S100 in the degree of influence on the intensity of implications and attention deficit and hyperactivity/impulsivity.
The positive therapeutic effect of the active drug ULD anti-S100 + anti-eNOS and drug of comparison ULD-S100 was shown in evaluating of patients' treatment results on a scale of ADHD severity assessment (CGI-ADHR-Severity) (Table 17). Almost the fourth part of the patients in ULD anti-S100 + anti-eNOS group the severity of disease was decreased from moderate to mild and even to minimal as confirmed by a decrease in mean value on a scale CGI-ADHR-Severity on 15% after 3 months of therapy (from 4.0 ± 0.02 to 3.4 ± 0.06; p <0.001 ). The effect of therapy with monopreparation ULD anti-S100 was slightly lower and indicated -10% on a scale CGI-ADHR-Severity over 3 months (vs. 5% in the placebo group). The safety analysis included data of all the patients participating in the study. During the whole period of monitoring there was both, well comparable to placebo, the tolerance of active drug ULD anti-S100 + anti-eNOS and preparation of comparison ULD-S100. Adverse events were reported in one patient of the group with ULD anti-S100 (subside during the fourth week of the study headaches) and in one patient of the placebo group (sleepwalking during the second month of observation). These adverse events were not connected with the therapy. In addition, during the treatment the single cases of acute respiratory disease were observed which also are not associated with the therapy. All the patients of studied groups completed the treatment to schedule established by the study protocol; no early dropouts. The absence of pathological changes according to physical examination of the patients and in the course of repeated analysis of laboratory parameters confirmed the safety of studied therapy.
According to the results of physical examination (heart rate, SBP, DBP, body temperature) in patients any pathological alterations during treatment were not registered. Differences in analyzing rates according to visits and in the compared groups did not reach the statistical significance and do not exceed the limits of physiologically-allowable deviations. High rates of adherence to therapy additionally evidenced as about effectiveness so as about the safety of studied preparations. By the end of the third month of treatment the adherence was 99.8 ± 1.15% and 98.8 ± 2.25% in the group 9 with ULD anti-S100 + anti-eNOS and in the group 2 with ULD anti-S100 respectively (versus 74.6 ± 2.54% in the group 3 with placebo).
Thus, the study demonstrated the efficacy and safety of the compositions ULD anti-S100 + anti-eNOS and of monocomponent preparation ULD-S100 in the treatment of children with ADHD. The most pronounced therapeutic effect in the 12-week course was observed in complex drug (ULD anti-S100 + anti-eNOS) which was manifested by positive dynamics of clinical symptoms in the majority (75%) of children. The composition ULD anti-S100 + anti-eNOS had correcting influence to both of the clusters of symptoms of ADHD and as a result, the significant reduction of attention disorders and hyperactivity in patients with ADHD was noted. Table 17. The dynamics of total score by the scale ADHDRS-IV-Home
Version by the end of 12 weeks of therapy
Figure imgf000073_0001
The difference is significant in comparison with the placebo group: ** p<0.01.
Table 18. The dynamics of evidence of clinical implications of ADHD by the scale
ADHDRS-IV-Home Version
Figure imgf000073_0002
*** # ***
22.6 -33.1% 22.7 29.9
4 weeks ±0.98 *** ± 1.23 -30.2 % ± 1.06 -11.0%
##
***
19.4 -42.6% 20.8 29.0
6 weeks ±0.95 "* ± 1.06 -36.0 % ± 1.25 -13.7 %
##
*** ## ***
18.9 -44.1% 20.9 27.6
8 weeks ±0.94 *** ± 1.30 -35.7 % ± 1.35 -17.9%
um
*** ### ***
17.3 -48.8% 20.1 27.3
12 weeks ±0.96 *** ± 1.21 -38.2 % ± 1.48 -18.8 %
###&
*** ### ***
Attention disorders
18.4 17.4 18.4
Screening
±0.55 ± 0.57 ± 0.43
12.8 -30.4% 13.7 16.1
2 weeks ±0.57 ± 0.68 -21.3% ±0.66 -12.5 %
*** #
***
11.6 -37.0% 12.9 16.4
4 weeks ±0.56 ± 0.79 -25.9 % ±0.57 -10.9 %
*** # ##
***
10.7 -41.8% 11.9
16.0 ±
6 weeks ±0.54 ± 0.64 *** -31.6 % -13.0%
*** ### 0.70 ***
10.3 -44.0%
11.5 15.1
8 weeks -33.9 % -17.9%
±0.53
*** ### ±0.70 ± 0.76 *** ### ***
9.7 -47.3% 11.4 14.9
12 weeks ±0.55 ±0.68 -34.5 % ±0.78 -19.0%
★ ** # # &
*** ## ***
Hyperactivity /impulsion
15.4 15.1 15.2
Screening
±0.61 ±0.77 ±0.62
11.3 -26.6% 12.7
11.4
2 weeks ±0.63 -24.5 % ±0.74 -16.4 %
*** ± 0.61 ***
***
11.0 -28.6% 9.8 13.5
4 weeks ±0.62 ±0.64 -35.1 % ± 0.67 -11.2 %
*** ### *** ### **
8.7 -43.5% 8.9 12.9
6 weeks ±0.59 ±0.64 -41.1 % ±0.73 -15.1 %
*** ### **
8.6 -44.2% 9.5 12.5
8 weeks ±0.60 ±0.76 -37.1 % ±0.81 -17.8%
*** ## *** ## ***
7.6 -50.6% 8.7 12.5
12 weeks ±0.57 ±0.70 -42.4 % ± 0.82 -17.8%
***
Note. The difference is significant in comparison with baseline parameter:
* p<0.05, ** p<0.01, *** p<0.001.
The difference is significant in comparison with placebo group:
# p<0.05, p<0.0 , p<0.00 . The difference is significant in comparison with the group of ULDanti-S100:&p<0.05.
Table 19. The dynamics of severity level of ADHD by the scale
CGI-ADHD-Severity
Figure imgf000077_0001
The difference is significant in comparison with the baseline parameter: ** p<0.01 , *** p<0.001.
Example 12.
Double blind, placebo-controlled clinical study of a combination of activated potentiated forms of antibodies to the C-terminal fragment of the angiotensin II AT1- receptor, in a mixture of homeopathic dilutions of C12, C30, C200, with activated potentiated form of antibodies to endothelial NO-synthase, in a mixture of homeopathic dilutions of C12, C30, C200, in human patients with chronic heart failure to evaluate key parameters of the CHF pathology.
80 patients (CHF of ll-IV functional class (FC), left ventricular ejection fraction (LVEF) less than 40%) were divided in 4 equal treatment and control groups for a 6 months study. The background therapy was not discontinued (bisoprolol β-blocker, ACE inhibitor enalapril, aspirin (unless contraindicated); administration of diuretics, nitrates, digoxin was also admitted). Group 1 received the activated potentiated form of antibodies to a C-terminal fragment of the angiotensin II AT1 -receptor (mixture of homeopathic dilutions C12, C30, C200) (3 tablets/day, n=20). Group 2 received the activated potentiated form of antibodies to endothelial NO-synthase (mixture of homeopathic dilutions C12, C30, C200) (3 tablets/day, n=20). Group 3 received the combination pharmaceutical composition comprising both activated potentiated form of antibodies to a C-terminal fragment of angiotensin II AT1 -receptor (mixture of homeopathic dilutions C12, C30, C200) and activated potentiated form of antibodies to endothelial NO-synthase (mixture of homeopathic dilutions C12, C30, \ C200) (3 tablets/day, n=20). Group 4 received placebo (3 tablets/day, n=20). The groups were comparable in the initial study parameters: in age and sex, and severity (class of CHF and LVEF) and duration of the disease.
Before and after treatment, the patients were evaluated for the effect of the administered medications on vascular remodeling and endothelium dysfunction that is important for the CHF process and progression. The effects of the medications on the processes of vascular remodeling were evaluated by pulse wave velocity (PWV) ("Colson" system) in the carotid-femoral (CF) (elastic type) and carotid-radial (CR) (muscle type) segments of arteries.
Table 20 shows the dynamics in the rates of pulse wave velocity in the carotid- femoral (CF) (elastic type) and carotid-radial (CR) (muscle type) segments of arteries.
Table 20
Figure imgf000078_0001
angiotensin II of angiotensine II
and ULD of Abs
to endothelial
NO-synthase
& Δ% & & & Δ%
CF, m/c 9, 8± -14.8* 10 9.8± - 10. 8.6 - 8. 8.2±0. 0.1
7± 0.6 .1 0.4 2.9 8± ±0. 20.3* . 2± 5
0. ±0 7 0.3 6 0.
5 .5 4
CR, m/c 8. 8.9 2.9 8. 8.3± - 8.9 7.6 - 9. 9.7±0. 6.4*
6± ±0. 8± 0.3 5.7 ±0. ±0. 15.6* 1± 3
0. 3 0. 5 7 #$ 0.
2 1 3
(Λ) denotes initial value
(&) denotes 6 month after beginning of administration
(*) denotes difference from initial value is verifiable with p value < 0.05.
(#) denotes difference from the group receiving ULDs of Abs to C-terminal fragment AT1 receptor angiotensin II with verifiable difference in p value of < 0.05.
($) denotes difference from the group receiving ULDs of Abs to endothelial NO- synthase with verifiable difference in p value of < 0.05.
(1 ) ULD denotes ultra-low doses.
(2) Abs denotes antibodies.
After 6 months of treatment, only group 3 showed a proven effect of the claimed pharmaceutical composition on the stiffness of muscular type arteries. Group 1 which received ULD of antibodies to a C-terminal fragment of angiotensin II AT1 -receptor, and group 3 which received the combination pharmaceutical composition of the invention showed a proven increase in the stiffness of elastic type arteries.
Example 13.
Double blind, placebo-controlled clinical study of a combination of activated potentiated forms of antibodies to the C-terminal fragment of angiotensin II AT1 - receptor, in a mixture of homeopathic dilutions of C12, C30, C200, with activated potentiated form of antibodies to endothelial NO-synthase, in a mixture of homeopathic dilutions of C12, C30, C200, in human patients with chronic heart failure to evaluate key measurement of quality of life. 80 patients (CHF of ll-IV functional class (FC), left ventricular ejection fraction (LVEF) less than 40%) were divided in 4 equal treatment and control groups for a 6 months study. The background therapy was not discontinued (bisoprolol β-blocker, ACE inhibitor enalapril, aspirin (unless contraindicated); administration of diuretics, nitrates, digoxin was also admitted). Group 1 received the activated potentiated form of antibodies to a C-terminal fragment of angiotensin II AT1 -receptor (mixture of homeopathic dilutions C12, C30, C200) (3 tablets/day, n=20). Group 2 received the activated potentiated form of antibodies to endothelial NO-synthase (mixture of homeopathic dilutions C12, C30, C200) (3 tablets/day, n=20). Group 3 received the combination pharmaceutical composition comprising both activated potentiated form of antibodies to a C-terminal fragment of angiotensin II AT1 -receptor (mixture of homeopathic dilutions C12, C30, C200) and activated potentiated form of antibodies to endothelial NO-synthase (mixture of homeopathic dilutions C12, C30, \ C200) (3 tablets/day, n=20). Group 4 received placebo (3 tablets/day, n=20). The groups were comparable in the initial study parameters: in age and sex, and severity (class of CHF and LVEF) and duration of the disease. Before and after treatment, the patients were evaluated for the life quality (Minnesota and Kansas questionnaires), morphological parameters of the heart, and tolerance to physical exercise.
Table 3 shows the results of the study in the form of dynamics in the basic parameters of the treatment efficacy.
After 6 months of treatment, the patients in group 1 treated with ULD of antibodies to a C-terminal fragment of angiotensin II AT1 -receptor showed a significant improvement of the life quality, improvement of the left ventricular systolic function, and an increased tolerance to physical exercise. Group 2 showed a proven decrease in the anxiety and depression levels and in the life quality, which were evaluated using the Kansas questionnaire. The study confirmed that the maximum therapeutic effect was achieved with the combination pharmaceutical composition of the invention in combination with the standard CHF therapy, which was administered to patients from group 3 that showed a proven positive dynamics in all parameters under study. The combination of activated (potentiated) forms of antibodies to a C-terminal fragment of angiotensin II AT1 -receptor and to endothelial nitric oxide synthase (NO- synthase) in the pharmaceutical composition of the invention (combination drug) provides an unexpected synergistic therapeutic effect implying an enhanced influence on vascular remodeling and endothelium dysfunction that is critical for the CHF process and progression, as also on the improvement of the patients' life quality, on morphological parameters of the heart and tolerance to physical exercise, which is confirmed by clinical trials.
The results are set forth in Table 21.
Figure imgf000081_0001
Figure imgf000082_0001
*, **, *** - p values < 0.05, 0.01 and 0.001, respectively
#- difference from group receiving ULDs of Abs to C-terminal fragment ATI of angiotensin receptor II with verifiable with p value < 0.05
$, $$ - difference from the group receiving ULDs of Abs to endothelial NO-synthase is verifiable at p values of 0.05 and 0.01, respectively.
(1) -ULD means ultra low doses
(2) Abs means antibodies
(3) "Minnesota" denotes Minnesota Questionnaire (4) "Kansas" denotes Kansas Questionnaire
(5) HADS denotes HADS total score
(6) FC CHF denotes patients with chronic heart failure, functional class
(7) FF LV denotes fraction of functioning of left vertical.
Example 14.
To study properties of the proposed pharmaceutical composition in the treatment of patients with a benign prostatic hyperplasia, 300 mg pills were used, saturated with the pharmaceutical composition containing water-alcohol solutions (6 mg/pill) of activated - potentiated rabbit polyclonal affinity purified antibodies to prostate specific antigen (anti-PSA) and endothelial NO synthase (anti-eNOS) in ultra low doses (ULD), produced by ultra dilution of the initial matrix solution 10012, 10030, 100200 times, equivalent to the mixture of centesimal homeopathic dilutions C12, C30, C200 (ULD anti-PSA +anti-eNOS), and 300 mg pills, saturated with the pharmaceutical composition containing water-alcohol solutions (3 mg/pill) of activated potentiated rabbit polyclonal affinity purified antibodies to prostate specific antigen in ultra low doses (ULD), obtained by an ultra dilution of the initial matrix solution 10012, 10030, 100200 times, equivalent to the mixture of centesimal homeopathic dilutions C12, C30, C200 (ULD anti-PSA).
Benign prostatic hyperplasia (BPH) is one of the most frequently occurring disorders in males (Bruskewitz R.C., 2003; Rosen R., 2003): on the one hand, epidemiological studies, carried out in Russia, point to a gradual increase in frequency of BPH from 11.3% in 40-49 year olds to 81.4% in 80 year olds (Gorilovskiy, L.M., 1999); on the other hand, demographic studies conducted by WHO confirm a significant increase in the population over 60 years old, surpassing any other age group growth.
The main symptoms of benign prostatic hyperplasia are lower urinary tract symptoms, which can cause significant discomfort and decrease quality of life (Bruskewitz R.C., 2003; Lepor H., 2004; O'Leary M.P., 2005). In severe cases, the disease can be accompanied by complications, such as acute urinary retention, urinary tract infection, erythruria, kidney failure (Stepanov, V.N., 1999; Jacobsen S.J., 1997; Lepor H., 2004). BPH is also associated with development of erectile dysfunction in patients (Bruskewitz R.C., 2003; Daly MP, 2005).
An open-label comparative parallel group study of efficacy and safety the of pharmaceutical compositions containing ULD anti-PSA + ULD anti-eNOS and ULD anti- PSA in ameliorating urinary disturbances to caused by benign prostatic hyperplasia (BPH), included 40 patients selected in accordance with inclusions/exclusions criteria. Patients were randomized in 2 groups, one group received 1 pill 3 times per day during 12 weeks (n=21 ) of a ULD anti-PSA +anti-eNOS, and another one 1 pill 3 times per day during 12 weeks (n=19) of a ULD anti-PSA. The groups were comparable in age, severity of BPH symptoms, urination parameters and prostate volume.
The study included patients over 45 years old with a history of BPH with corresponding symptoms of lower urinary tract for no less than 6 months, IPSS≥13, prostate volume according to transrectal ultrasonography >30 cm3, with maximum urinary flow speed of≥4 ml/s and≤15 ml/sc and minimum residual urine volume equal to 125 ml, with PSA level <4 ng/ml. A necessary inclusion criterion was absence of intake of the following medications in the medical records: finasteride, dutasteride, or other experimental drug 6 months prior to inclusion in the study, a1 -adrenoreceptor blockers and herbal medications 4 weeks prior to the inclusion into the study, any inhibitors of phosphodiesterase type 5 and other erectile dysfunction treatments 4 weeks prior to the inclusion into the study.
The study did not include patients undergone invasive methods of treatment of BPH, including transurethral prostatic resection, thermotherapy, transurethral needle ablation, stent angioplasty and other; with malignant oncological disease, acute urination delay, bladder stones, urethral stricture, Marion's disease, genitourinary system infections in the phase of active inflammation and others.
Clinical efficacy of pharmaceutical compositions was assessed by the improvement of clinical symptoms of lower urinary tract, evaluated using IPSS questionnaire (International Prostate Symptom Score), urination parameters (maximum and average urinary flow speed, urination volume, volume of residual urine) and prostate volume based on the data of transurethral ultrasound (TU), and also erectile function was evaluated based on the data obtained from IIEF questionnaire (International Index of Erectile Function). Results of the study are shown in tables 22 and 23.
Table 22.
Figure imgf000086_0001
or is total number of patients in the study (N). Table 23.
Dynamics of subscales of obstructive and irritative symptoms, and question 7 of
IPSS questionnaire
Figure imgf000087_0001
* - p<0.05 vs baseline; ** - p<0.01 vs vaseline; *** - p<0.001 vs baseline
## - p<0,01 vs ULD anti-PSA
2 - shows decrease compared to the baseline in %, average group value
The given data confirm that both ULD anti-PSA, and ULD anti-PSA + ULD anti- eNOS were used to effectively treat symptoms of lower urinary tract, increase average and maximum urinary flow speed, improve quality of life of patients (Table 22). The course of the was not long (12 weeks), therefore, a decrease in prostate volume was not observed in any study group. ULD anti-PSA did not effect the volume of urination, which increased only in 52.6% patients, on average the group showed some statistically insignificant decrease of urination volume by 1 1.8 ml (5.4%) compared to the baseline values. At the same time, patients, treated with ULD anti-PSA + ULD anti-eNOS, showed an increase in urination volume in 71 .4%, and on average, an increase in volume was 48.3 ml (23.7%) compared to the baseline.
An analysis of dynamics of obstructive and irritative symptoms according to IPSS subscales as well as nucturia evidence (question 7 of IPSS) showed that both pharmaceutical compositions contributed to a decrease of obstruction and irritative symptoms, and also a decrease of nucturia symptoms. At the same time, a ULD anti- PSA +anti-eNOS was more effective compared to a ULD anti-PSA in decreasing irritative symptoms of lower urinary tract (28.2% vs. 40.3%, p<0.05) and nighttime urination urges (2.0% vs. 37.7%,).
It should be noted, that ULD anti-PSA + ULD anti-eNOS is also more effective compared to ULD anti-PSA in improving erectile function in patients. In ULD anti-PSA + ULD anti-eNOS group, the total IIEF (International Index of Erectile Dysfunction) score increased by 19% in patients (in ULD anti-PSA group by 10.5%), an average increase of IIEF score in ULD anti-PSA + ULD anti-eNOS group was 8% vs 4.5% in a ULD anti-PSA group.
The pharmaceutical compositions showed excellent safety profile, no adverse effects related to the administered medications were observed in the course of study.
Therefore, ULD anti-PSA + ULD anti-eNOS showed better efficacy compared to that of ULD anti-PSA in treating urination problems caused by benign prostatic hyperplasia. In addition, a greater positive effect of ULD anti-PSA + ULD anti-eNOS on erectile function of patients compared to ULD anti-PSA was revealed.

Claims

What is claimed is:
1. A method of increasing the effect of an activated-potentiated form of an antibody to an endogenous biological molecule, said method comprising combining said endogenous biological molecule with an activated-potentiated form of an antibody to endothelial NO-synthase.
2. The method of claim 1 , further comprising administering said combination to a patient in need of treatment with said activated-potentiated form of an antibody.
3. The method of claim 2, wherein said activated-potentiated form of an antibody to an endogenous biological molecule is an antibody to S-100 protein.
4. The method of claim 2, wherein said activated-potentiated form of an antibody to an endogenous biological molecule is an antibody to prostate specific antigen.
5. The method of claim 2, wherein said activated-potentiated form an antibody to an endogenous biological molecule is an antibody to insulin receptor.
6. The method of claim 2, wherein said activated-potentiated form an antibody to an endogenous biological molecule is an antibody to antigiotensin receptor II.
7. A pharmaceutical composition comprising a) an activated-potentiated form of an antibody to an endogenous biological molecule, and b) an activated-potentiated form of an antibody to NO synthase.
8. The pharmaceutical composition of claim 7, further comprising pharmaceutically acceptable solid carrier.
9. The pharmaceutical composition of claim 8, wherein said activated- potentiated form of an antibody to endothelial NO-synthase is in the form of a mixture of of C12, C30, and C200 homeopathic dilutions impregnated onto said solid carrier.
10. The pharmaceutical composition of claim 7, wherein said activated- potentiated form of an antibody to an endogenous biological molecule is a monoclonal, monoclonal, or natural antibody.
11. The pharmaceutical composition of claim 10, wherein said antibody to an endogenous biological molecule is a polyclonal antibody.
12. The pharmaceutical composition of claim 7, wherein said activated- potentiated form of an antibody to an endogenous biological molecule is prepared by successive centesimal dilutions coupled with shaking of every dilution.
13. The pharmaceutical composition of claims 7, wherein said antibody to endothelial NO-synthase is monoclonal, polyclonal or natural antibody.
14. The pharmaceutical composition of claim 8, wherein said antibody to endothelial NO-synthase is a polyclonal antibody.
15. The pharmaceutical composition of claim 9, the activated-potentiated form of an antibody to endothelial NO-synthase is prepared by successive centesimal dilutions coupled with shaking of every dilution.
PCT/IB2011/002350 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody WO2012007845A2 (en)

Priority Applications (21)

Application Number Priority Date Filing Date Title
SG2013002324A SG187038A1 (en) 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody
MYPI2013000107A MY171206A (en) 2011-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody
SE1350184A SE1350184A1 (en) 2010-07-15 2011-07-15 PROCEDURE TO INCREASE THE EFFECT OF AN ACTIVATED, POTENTIZED FORM OF AN ANTIBODY
UAA201300112A UA112753C2 (en) 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody
CN2011800443324A CN103108657A (en) 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody
DE112011102358T DE112011102358T5 (en) 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody
GB1302649.7A GB2495884B (en) 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody
NZ606767A NZ606767A (en) 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody
CA2804966A CA2804966A1 (en) 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody
BR112013000840A BR112013000840A2 (en) 2010-07-15 2011-07-15 method for enhancing the effect of potentiated activated form of antibody to biological endogenous molecule, pharmaceutical composition and use of combination of endogenous biological molecule with potentiated activated form of antibody to endothelial synthase.
JP2013519175A JP2013538186A (en) 2010-07-15 2011-07-15 Method for increasing the effect of activation-enhancing antibodies
ES201390006A ES2440393R1 (en) 2010-07-15 2011-07-15 A METHOD FOR INCREASING THE EFFECT OF A POTENTIATED ACTIVATED FORM OF AN ANTIBODY
EA201300126A EA030566B1 (en) 2010-07-15 2011-07-15 Method of increasing therapeutic effectiveness of an activated-potentiated form of an antibody to an endogenous biological molecule and pharmaceutical composition
EEP201300006A EE201300006A (en) 2010-07-15 2011-07-15 A pharmaceutical composition for enhancing the effect of an activated and potentiated form of an antibody
AU2011278038A AU2011278038B2 (en) 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody
EP11775838.3A EP2593140A2 (en) 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody
KR1020137003861A KR20140014059A (en) 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody
NO20130222A NO20130222A1 (en) 2010-07-15 2013-02-08 Process for increasing the effect of an activated-potentiated form of an antibody
DKPA201370079A DK201370079A (en) 2010-07-15 2013-02-14 A method of increasing the effect of an activated-potentiated form of an antibody
FI20135143A FI20135143L (en) 2010-07-15 2013-02-15 A method of increasing the potency of an activated potentiated form of an antibody
FI20135152A FI20135152L (en) 2010-07-21 2013-02-21 PHARMACEUTICAL COMBINATION COMPOSITION FOR METHODS OF TREATMENT OF DIABETES AND METABOLIC DISEASES

Applications Claiming Priority (36)

Application Number Priority Date Filing Date Title
RU2010129291/15A RU2525156C2 (en) 2010-07-15 2010-07-15 Method of treatment and prevention of arterial hypertension and pharmaceutical composition for treatment of arterial hypertension
RU2010129291 2010-07-15
RU2010129294 2010-07-15
RU2010129292 2010-07-15
RU2010129298 2010-07-15
RU2010129295/15A RU2531049C2 (en) 2010-07-15 2010-07-15 Therapeutic agent for treating prostatic diseases and method of treating prostatic diseases
RU2010129298/15A RU2526153C2 (en) 2010-07-15 2010-07-15 Method for increase of pharmacological activity of active agent of drug preparation and pharmaceutical composition
RU2010129290 2010-07-15
RU2010129294/15A RU2542414C2 (en) 2010-07-15 2010-07-15 Medication for treating erectile dysfunctions and method of treating erectile dysfunctions
RU2010129295 2010-07-15
RU2010129292/15A RU2523557C2 (en) 2010-07-15 2010-07-15 Method of treating vegetovascular dystonia and pharmaceutical composition for treating vegetovascular dystonia
RU2010129290/15A RU2525155C2 (en) 2010-07-15 2010-07-15 Method of treating chronic heart failure and pharmaceutical composition for complex therapy of chronic heart failure
RU2010130358 2010-07-21
RU2010130353 2010-07-21
RU2010130353/15A RU2542445C2 (en) 2010-07-21 2010-07-21 Medication for treating alzheimer's disease and method of treating alzheimer's disease
RU2010130355 2010-07-21
RU2010130348/15A RU2531048C2 (en) 2010-07-21 2010-07-21 Drug substance for reducing insulin resistance and treating diabetes mellitus and method for providing higher therapeutic effectiveness in diabetes mellitus with insulin and/or antihyperglycemic medications
RU2010130356 2010-07-21
RU2010130358/15A RU2519695C2 (en) 2010-07-21 2010-07-21 Medication for treating attention deficit disorder and method of treating attention deficit disorder
RU2010130355/15A RU2530638C2 (en) 2010-07-21 2010-07-21 Medication and method of treating organic diseases of nervous system, psychoorganic syndrome and encephalopathies of different genesis
RU2010130356/15A RU2542453C2 (en) 2010-07-21 2010-07-21 Therapeutic agent and method of treating vegetative-vascular dystonia, dizziness syndrome of various origins, and kinetosis
RU2010130348 2010-07-21
RU2011110106/15A RU2523451C2 (en) 2011-03-17 2011-03-17 Method of treating chronic cardiac failure and pharmaceutical composition for treating chronic cardiac failure
RU2011110106 2011-03-17
RU2011127051 2011-06-01
RU2011127052/15A RU2503462C2 (en) 2011-07-01 2011-07-01 Method of treating vertigo of various origins, kinetosis and vegetative-vascular dystonia (versions) and drug preparation
RU2011127053 2011-07-01
RU2011127052 2011-07-01
RU2011127058 2011-07-01
RU2011127055 2011-07-01
RU2011127051/15A RU2509572C2 (en) 2011-07-01 2011-07-01 Drug for reducing insulin resistance and for treating diabetes, method of reducing insulin resistance, method of treating diabetes and method of treating diabetes with insulin and/or hypoglycemic preparations
RU2011127058/15A RU2536232C2 (en) 2011-07-01 2011-07-01 Therapeutic agent for alzheimer's disease and method of treating alzheimer's disease
RU2011127059 2011-07-01
RU2011127055/15A RU2536230C2 (en) 2011-07-01 2011-07-01 Therapeutic agent for treating neurological behaviour disorders and method of treating neurological behaviour development disorders
RU2011127053/15A RU2565400C2 (en) 2011-07-01 2011-07-01 Medication for treating genitourinary system diseases and method of treating genitourinary system diseases
RU2011127059/15A RU2536234C2 (en) 2011-07-01 2011-07-01 Neurotropic drug and method of treating structural diseases of nervous system, psychoorganic syndrome and encephalopathies of various origins

Publications (2)

Publication Number Publication Date
WO2012007845A2 true WO2012007845A2 (en) 2012-01-19
WO2012007845A3 WO2012007845A3 (en) 2012-03-29

Family

ID=44863151

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2011/002350 WO2012007845A2 (en) 2010-07-15 2011-07-15 A method of increasing the effect of an activated-potentiated form of an antibody

Country Status (25)

Country Link
US (3) US9308275B2 (en)
EP (1) EP2593140A2 (en)
JP (2) JP2013538186A (en)
KR (1) KR20140014059A (en)
AR (1) AR082247A1 (en)
AU (1) AU2011278038B2 (en)
BR (1) BR112013000840A2 (en)
CA (1) CA2804966A1 (en)
CZ (1) CZ2013105A3 (en)
DE (1) DE112011102358T5 (en)
DK (1) DK201370079A (en)
EA (1) EA030566B1 (en)
EE (1) EE201300006A (en)
ES (1) ES2440393R1 (en)
FI (1) FI20135143L (en)
FR (1) FR2962656A1 (en)
GB (1) GB2495884B (en)
IT (1) ITTO20110639A1 (en)
LT (1) LT5988B (en)
MX (1) MX2013000543A (en)
NO (1) NO20130222A1 (en)
NZ (1) NZ606767A (en)
PE (1) PE20130398A1 (en)
SG (2) SG10201505561TA (en)
WO (1) WO2012007845A2 (en)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2962655A1 (en) * 2010-07-15 2012-01-20 Oleg Iliich Epshtein PHARMACEUTICAL ASSOCIATION COMPOSITION AND USES THEREOF IN METHODS OF TREATMENT OF GENITAL URINARY SYSTEM DISORDERS
CZ2013105A3 (en) * 2010-07-15 2013-05-22 Iliich Epshtein@Oleg Method of increasing effect of antibody activated potentiated form
WO2012010966A2 (en) * 2010-07-21 2012-01-26 Oleg Iliich Epshtein A combination pharmaceutical composition and methods of treating diabetes and metabolic disorders
JP2013536174A (en) * 2010-07-21 2013-09-19 イリイチ・エプシテイン オレグ Combination pharmaceutical composition and method of treating rotational dizziness, motion sickness and autonomic neurovascular dystonia
EP2450375A1 (en) 2010-11-09 2012-05-09 Sandoz Gmbh Cell culture medium and process for protein expression, said medium and process comprising a PAM inhibitor
RU2013111961A (en) * 2013-03-18 2014-09-27 Олег Ильич Эпштейн METHOD FOR DETERMINING THE EXPRESSION OF MODIFICATION ACTIVITY ASSOCIATED WITH A CARRIER
RU2013111962A (en) * 2013-03-18 2014-09-27 Олег Ильич Эпштейн METHOD FOR DETERMINING THE EXPRESSION OF MODIFICATION ACTIVITY ASSOCIATED WITH A CARRIER

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4311897A (en) 1979-08-28 1982-01-19 Union Carbide Corporation Plasma arc torch and nozzle assembly
US7229648B2 (en) 2003-03-14 2007-06-12 Dreyer Lee R Homeopathic formulations useful for treating pain and/or inflammation
US7572441B2 (en) 2002-08-02 2009-08-11 Oleg Iliich Epshtein Media and method for treating pathological syndrome
US7582294B2 (en) 2002-08-02 2009-09-01 Oleg Oliich Epshtein Medicament for treating prostate diseases
US7700096B2 (en) 2002-08-02 2010-04-20 Oleg Iliich Epshtein Medicinal agent for treating erectile dysfunction

Family Cites Families (77)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB750155A (en) 1953-03-17 1956-06-13 Nat Res Dev Substituted alanines
US3134718A (en) 1963-12-12 1964-05-26 Schering Corp Pregna-1,4-dienes and compositions containing same
US3901967A (en) 1973-09-10 1975-08-26 Union Corp Sustained release of atropine
SE393532B (en) 1974-05-02 1977-05-16 Draco Ab MAKE A PREPARATION OF A PHARMACEUTICAL ZINC PREPARATION FOR THE SUBSTANCE OF A TASTEFUL, PERTAINABLE, PERORAL ZINC SOLUTION CONTAINING A ZINC COMPLEX
US4963367A (en) 1984-04-27 1990-10-16 Medaphore, Inc. Drug delivery compositions and methods
US4839341A (en) 1984-05-29 1989-06-13 Eli Lilly And Company Stabilized insulin formulations
SU1331508A1 (en) 1985-08-14 1987-08-23 Киевский Кабельный Завод "Укркабель" Apparatus for pneumatic massaging of manъs urogenital sphere
FI101344B (en) 1988-03-31 1998-06-15 Tanabe Seiyaku Co The method comprises preparing a preparation from which a pharmaceutically active substance is released in a controlled manner
US4987127A (en) 1989-01-31 1991-01-22 Dal Sirany Method of treating a virus outbreak
SU1730144A1 (en) 1989-02-24 1992-04-30 Научный Центр По Разработке И Внедрению Современных Методов Молекулярной Диагностики Method of viruses reproduction suppression
US5733572A (en) 1989-12-22 1998-03-31 Imarx Pharmaceutical Corp. Gas and gaseous precursor filled microspheres as topical and subcutaneous delivery vehicles
US5698195A (en) 1991-03-18 1997-12-16 New York University Medical Center Methods of treating rheumatoid arthritis using chimeric anti-TNF antibodies
RU2007989C1 (en) 1991-11-12 1994-02-28 Акционерное общество "Трейдис" Method for selecting homeopathic preparations and their single dose
US5741488A (en) 1992-10-08 1998-04-21 The Kennedy Institute For Rheumatology Treatment of rheumatoid arthritis with anti-CD4 antibodies in conjunction with anti-TNF antibodies
DK140992D0 (en) 1992-11-24 1992-11-24 Ole Buchardt PROCEDURE FOR THE PRODUCTION OF ANTIBODIES AGAINST HAPTENES AND OTHER B-CELL ANTIGENS, ANTIBODIES OBTAINED BY THE PROCEDURE AND USE OF THESE ANTIBODIES FOR THE PREPARATION OF VACCINS
US5879677A (en) 1992-12-09 1999-03-09 The Scripps Research Institute Method for inhibition of cerebral tissue factor mediated reperfusion damage
WO1994022846A1 (en) 1993-03-30 1994-10-13 Pfizer Inc. Compounds enhancing antitumor activity of other cytotoxic agents
RU2033784C1 (en) 1993-05-28 1995-04-30 Индивидуальное частное предприятие "Диалог" Device for reproducing homeopathic and isopathic preparations
US5895783A (en) 1993-07-16 1999-04-20 Schering Aktiengesellschaft Treatment of preeclampsia and preterm labor with combination of progestational agent and a nitric oxide synthase substrate and/or donor
IT1261849B (en) 1993-09-02 1996-06-03 Avantgarde Spa MEDICAL DEVICE FOR THE ADMINISTRATION OF ACTIVE INGREDIENTS OR VERY LOW DOSAGE DRUGS, IN PARTICULAR HOMEOPATHIC DRUGS.
EP0652014A1 (en) 1993-11-10 1995-05-10 National Institute Of Immunology Treatment of prostatic hypertrophy
NZ278607A (en) 1994-02-07 1999-05-28 Knoll Ag Use of tnf antagonists for treating disorders involving elevated serum levels of il-6 wherein the serum levels are 500pg/ml or above
ATE372966T1 (en) 1994-03-25 2007-09-15 Isotechnika Inc IMPROVED EFFECTIVENESS OF DRUGS THROUGH DEUTERATION
US5629286A (en) 1994-03-31 1997-05-13 Brewitt; Barbara Homeopathic dilutions of growth factors
IL110035A0 (en) 1994-06-16 1994-10-07 Tapuach Natural Technologies 1 Homeopathic formulations
RU2137483C1 (en) 1995-08-02 1999-09-20 Божедомов Владимир Александрович Method of treatment of patients with urogenital chlamydium, ureaplasma and mycoplasma infection
US6379669B1 (en) 1995-08-04 2002-04-30 Akhouri A. Sinha Targeting of organs by immunoconjugates
DE69601765T2 (en) 1995-09-07 1999-07-08 Oreal Iridaceae extract and compositions containing it
IL125743A (en) 1996-02-12 2003-09-17 Epshtein Oleg Iliich Medicinal preparation containing an active medical substance with homeopathic potentiation
IL118096A0 (en) 1996-05-01 1996-09-12 Yeda Res & Dev Antibodies against interferon alpha/beta receptor
US6150500A (en) 1996-07-12 2000-11-21 Salerno; John C. Activators of endothelial nitric oxide synthase
SE9604341D0 (en) 1996-11-26 1996-11-26 Ferring Bv Hepta-peptide oxytocin analogue
RU2104032C1 (en) 1997-03-11 1998-02-10 Общество с ограниченной ответственностью "Снежный барс" Method of potentiation of medicinal agent curative effect
US5849528A (en) 1997-08-21 1998-12-15 Incyte Pharmaceuticals, Inc.. Polynucleotides encoding a human S100 protein
DE19746868A1 (en) 1997-10-23 1999-04-29 Knoll Ag Use of tumour necrosis factor antagonists
WO1999026657A1 (en) 1997-11-25 1999-06-03 Musc Foundation For Research Development Inhibitors of nitric oxide synthase
RU2122858C1 (en) 1997-12-29 1998-12-10 Яковлева Людмила Борисовна Homeopathic drug showing sedative effect
DK1062952T3 (en) 1998-03-11 2003-11-24 Grelan Pharmaceutical Co Effervescent enteric preparations
RU2187334C2 (en) 1998-05-22 2002-08-20 Эпштейн Олег Ильич Method of correction of impaired immune homeostasis and medicinal agent
US6933272B1 (en) 1998-09-22 2005-08-23 Erik Helmerhorst Use of non-peptidyl compounds for the treatment of insulin related ailments
RU2144370C1 (en) 1999-01-06 2000-01-20 Титиева Наталья Михайловна Homeopathic drug for treatment of patient with prostate hypertrophy
RU2156621C1 (en) 1999-03-04 2000-09-27 Эпштейн Олег Ильич Neurotropic drug
RU2181297C2 (en) 2000-06-20 2002-04-20 Эпштейн Олег Ильич Method of treatment of pathological syndrome and medicinal agent
RU2177795C1 (en) 2001-02-15 2002-01-10 Эпштей Олег Ильич Homeopathic preparation for treatment and prophylaxis of juvenile adenoviral infections
RU2192882C1 (en) 2001-04-18 2002-11-20 Эпштейн Олег Ильич Medicinal agent and method of treatment of pathological syndrome caused by hemopoiesis impairment
RU2201255C1 (en) 2001-12-26 2003-03-27 Эпштейн Олег Ильич Medicinal agent and method of regulation of vascular tonus
RU2199345C1 (en) 2001-12-26 2003-02-27 Эпштейн Олег Ильич Medicinal agent and method of regulation of carbohydrate and lipid metabolism
UA76640C2 (en) 2002-08-02 2006-08-15 Олєг Ільіч Епштєйн Method for correcting pathological immune responses and homeopathic medicinal agent
RU2253478C1 (en) 2003-10-01 2005-06-10 Эпштейн Олег Ильич Agent for potentiation therapeutical effects: enhancing effect of drug
WO2005067928A1 (en) * 2004-01-20 2005-07-28 Astellas Pharma Inc. Method for treating erectile dysfunction
BRPI0514903A (en) * 2004-09-03 2008-06-24 Hansens Lab vegetable proteins or fermented milk comprising receptor ligand and use thereof
RU2309732C1 (en) 2006-03-13 2007-11-10 Олег Ильич Эпштейн Pressed solid oral formulation of medicinal preparation and method for preparing solid oral formulation of medicinal preparation
NZ571673A (en) * 2006-04-04 2011-08-26 Dong A Pharm Co Ltd A pyrazolopyrimidinone derivative for the prevention and treatment of prostatic hyperplasia
JP5687425B2 (en) 2006-06-06 2015-03-18 オレグ イリッチ エプシュテイン Drugs for the treatment of obesity, diabetes mellitus and diseases with impaired glucose tolerance
RU2438707C2 (en) 2006-06-06 2012-01-10 Олег Ильич Эпштейн Medication for per oral treatment of diabetes mellitus and other diseases accompanied by disturbance of tolerance to glucose, and method of obtaining hard drug form for per oral therapy of diabetes mellitus and other diseases, accompanied by disturbance of tolerance to glucose
EP2040756A4 (en) * 2006-07-13 2013-01-09 Mazence Inc Composition containing metal-acidic amino acid chelate accelerating absorption of metal
GB0812019D0 (en) 2008-07-02 2008-08-06 Asterion Ltd Insulin
US20130189707A1 (en) 2010-01-26 2013-07-25 Svetlana Alexandrovna Sergeeva Method of determination of autoantibody level by means of enzyme immunoassay
MX2013000544A (en) 2010-07-15 2013-10-28 Oleg Iliich Epshtein A combination pharmaceutical composition and methods of treating diseases or conditions associated with the cardiovascular system.
CZ2013105A3 (en) * 2010-07-15 2013-05-22 Iliich Epshtein@Oleg Method of increasing effect of antibody activated potentiated form
GB2496794B (en) 2010-07-15 2017-11-01 Iliich Epshtein Oleg Combination pharmaceutical composition and methods of treating functional diseases or conditions of gastrointestinal tract
FR2962652A1 (en) 2010-07-15 2012-01-20 Oleg Iliich Epshtein PHARMACEUTICAL ASSOCIATION COMPOSITION AND ITS USE IN METHODS FOR TREATING DISEASES OR DISORDERS ASSOCIATED WITH NEURODEGENERATIVE DISEASES
FR2962655A1 (en) * 2010-07-15 2012-01-20 Oleg Iliich Epshtein PHARMACEUTICAL ASSOCIATION COMPOSITION AND USES THEREOF IN METHODS OF TREATMENT OF GENITAL URINARY SYSTEM DISORDERS
CN103124742A (en) 2010-07-15 2013-05-29 奥列格·伊里奇·爱泼斯坦 Pharmaceutical compositions and methods of treatment
NZ606970A (en) * 2010-07-21 2015-08-28 Oleg Iliich Epshtein A method of treating attention deficit hyperactivity disorder
JP2013536174A (en) * 2010-07-21 2013-09-19 イリイチ・エプシテイン オレグ Combination pharmaceutical composition and method of treating rotational dizziness, motion sickness and autonomic neurovascular dystonia
JP2013533269A (en) 2010-07-21 2013-08-22 イリイチ・エプシテイン オレグ Combination pharmaceutical compositions and methods of treating diseases and conditions associated with respiratory diseases or conditions
WO2012010966A2 (en) * 2010-07-21 2012-01-26 Oleg Iliich Epshtein A combination pharmaceutical composition and methods of treating diabetes and metabolic disorders
WO2012014078A2 (en) * 2010-07-21 2012-02-02 Olge Iliich Epshtein Method of treatment of organic diseases of nervous system, psychoorganic syndrome and encephalopathy
JP2013535445A (en) * 2010-07-21 2013-09-12 イリイチ・エプシテイン オレグ How to treat Alzheimer's disease
RU2535033C2 (en) 2010-08-06 2014-12-10 Олег Ильич Эпштейн Therapeutic agent and method for prevention of hiv infection and treatment of hiv-caused or hiv-associated diseases, including aids
RU2535034C2 (en) 2010-08-06 2014-12-10 Олег Ильич Эпштейн Medication and method of preventing hiv infection, prevention and treatment of hiv-induced or hiv-associated diseases, including aids
US20120263725A1 (en) 2010-08-06 2012-10-18 Epshtein Oleg Iiiich Pharmaceutical composition and methods of treating and preventing the diseases caused by HIV or associated with HIV
RU2517084C2 (en) 2010-08-06 2014-05-27 Олег Ильич Эпштейн Method and means for inhibiting production or enhancing protein p24 elimination
CZ2013159A3 (en) 2010-08-06 2013-06-12 Iliich Epshtein@Oleg Combined pharmaceutical composition and methods of treating and prevention of infectious diseases
US20130171161A1 (en) 2010-12-23 2013-07-04 Oleg Iliich Epshtein Method and composition for the treatment of diseases caused by or associated with hiv
JP5418549B2 (en) * 2011-07-06 2014-02-19 アイシン精機株式会社 Vehicle door outer handle device

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4311897A (en) 1979-08-28 1982-01-19 Union Carbide Corporation Plasma arc torch and nozzle assembly
US7572441B2 (en) 2002-08-02 2009-08-11 Oleg Iliich Epshtein Media and method for treating pathological syndrome
US7582294B2 (en) 2002-08-02 2009-09-01 Oleg Oliich Epshtein Medicament for treating prostate diseases
US7700096B2 (en) 2002-08-02 2010-04-20 Oleg Iliich Epshtein Medicinal agent for treating erectile dysfunction
US7229648B2 (en) 2003-03-14 2007-06-12 Dreyer Lee R Homeopathic formulations useful for treating pain and/or inflammation

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
G. FRIMEL, M.: "Meditsyna", IMMUNOTECHNIQUES, 1987, pages 9 - 33
HABIB ET AL.: "Nitric Oxide Measurement From Blood To Lungs, Is There A Link?", PAK J PHYSIOL, vol. 3, no. 1, 2007
LAFFLY E., SODOYER R., HUM. ANTIBODIES. MONOCLONAL AND RECOMBINANT ANTIBODIES, vol. 14, no. 1-2, 2005, pages 33 - 55
See also references of EP2593140A2
V. SCHWABE, HOMEOPATHIC MEDICINES, 1967, pages 14 - 29

Also Published As

Publication number Publication date
JP2016216483A (en) 2016-12-22
LT2013016A (en) 2013-11-25
GB201302649D0 (en) 2013-04-03
NO20130222A1 (en) 2013-03-22
DE112011102358T5 (en) 2013-04-25
PE20130398A1 (en) 2013-04-10
GB2495884A (en) 2013-04-24
EE201300006A (en) 2013-08-15
CZ2013105A3 (en) 2013-05-22
EP2593140A2 (en) 2013-05-22
WO2012007845A3 (en) 2012-03-29
AU2011278038A1 (en) 2013-03-07
AR082247A1 (en) 2012-11-21
ITTO20110639A1 (en) 2012-01-16
FR2962656A1 (en) 2012-01-20
ES2440393A2 (en) 2014-01-28
US20160251448A1 (en) 2016-09-01
MX2013000543A (en) 2013-10-28
KR20140014059A (en) 2014-02-05
US9308275B2 (en) 2016-04-12
EA030566B1 (en) 2018-08-31
US20160244531A1 (en) 2016-08-25
CA2804966A1 (en) 2012-01-19
SG10201505561TA (en) 2015-08-28
EA201300126A1 (en) 2013-12-30
ES2440393R1 (en) 2014-10-10
GB2495884B (en) 2017-05-31
NZ606767A (en) 2015-08-28
JP2013538186A (en) 2013-10-10
BR112013000840A2 (en) 2016-06-07
DK201370079A (en) 2013-02-14
FI20135143L (en) 2013-02-15
AU2011278038B2 (en) 2017-02-02
LT5988B (en) 2014-01-27
SG187038A1 (en) 2013-02-28
US20140079696A1 (en) 2014-03-20

Similar Documents

Publication Publication Date Title
AU2011281248B2 (en) Combination pharmaceutical compositions and method of treatment of vertigo, kinetosis and vegetative-vascular dystonia
US20160244531A1 (en) Method of increasing the effect of an activated-potentiated form of an antibody
US8987206B2 (en) Method of treating attention deficit hyperactivity disorder
US20130058982A1 (en) Method of treating Alzheimer&#39;s disease
US20120258146A1 (en) Method of treating organic diseases of nervous system, pschoorganic syndrome and encephalopathy
KR20140009110A (en) A combination pharmaceutical composition and methods of treating diseases or conditions associated with the cardiovascular system
SE1350184A1 (en) PROCEDURE TO INCREASE THE EFFECT OF AN ACTIVATED, POTENTIZED FORM OF AN ANTIBODY

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201180044332.4

Country of ref document: CN

ENP Entry into the national phase

Ref document number: 2804966

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2013519175

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 224212

Country of ref document: IL

Ref document number: MX/A/2013/000543

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 000076-2013

Country of ref document: PE

Ref document number: P201390006

Country of ref document: ES

Ref document number: 1120111023588

Country of ref document: DE

Ref document number: 112011102358

Country of ref document: DE

Ref document number: 12013500107

Country of ref document: PH

WWE Wipo information: entry into national phase

Ref document number: 2011775838

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 1302649

Country of ref document: GB

Kind code of ref document: A

Free format text: PCT FILING DATE = 20110715

WWE Wipo information: entry into national phase

Ref document number: 1302649.7

Country of ref document: GB

Ref document number: PV2013-105

Country of ref document: CZ

Ref document number: 201300126

Country of ref document: EA

Ref document number: 2013016

Country of ref document: LT

ENP Entry into the national phase

Ref document number: 20137003861

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: A201300112

Country of ref document: UA

Ref document number: 20135143

Country of ref document: FI

WWE Wipo information: entry into national phase

Ref document number: 2013/02316

Country of ref document: TR

ENP Entry into the national phase

Ref document number: 2011278038

Country of ref document: AU

Date of ref document: 20110715

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112013000840

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 112013000840

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20130114