Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
  • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/716—Glucans
  • A61K31/722—Chitin, chitosan
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
  • A61K31/727—Heparin; Heparan
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
• • 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
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • 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
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
  • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
• • 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
  • A61P9/06—Antiarrhythmics
• • 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
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/24—Metalloendopeptidases (3.4.24)
  • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention relates to medicine, namely to the preparation of a pharmaceutical composition comprising a botulinum neurotoxin for use in clinical practice, preferably in cardiology for the treatment of cardiac arrhythmias.
• botulinum neurotoxin - Botulinum neurotoxin type A Botulinum toxin type A
• This neurotoxin is produced during fermentation in the presence of Clostridium botulinum strains.
• botulinum toxin type A such as Botox (Botox), Dysport (Dysport), Kseomin (Xeomin) or Lantoks (Lantox) are used.
• Botox Botox
• Dysport Dysport
• Kseomin Xeomin
• Lantoks Lantoks
• Cardiac arrhythmias are widespread and complex group of cardiac events.
• the only effective and rational surgical treatment of this disease is radiofrequency ablation (burning of arrhythmogenic areas of the heart using high frequency electric current).
• this method is not sufficiently effective (less than 60%) and has a high risk of complications such as hemopericardium, transesophageal fistula, post-ablation, atrial flutter, phrenic nerve paresis, mural thrombus and etc. in more than 30% of the cases (Camm et al, 2010, Guidelines for the management of atrial fibrillation European Heart Journal, 31, 2369-2429).
• compositions comprising a botulinum neurotoxin, selected from the various serotypes A, B, C, D. E, F or G and S botulinum, and polyamine acid, selected from the group comprising polylysine, polyarginine, polyhistidine or polyornithine have been developed (patent application RU 2011125775A; WO 2010/07842).
• compositions comprising a botulinum toxin type A in an amount from 6 pg to 30 ng with a biological activity of approximately 50-250 Units of LD 50 (Lethal Dose, 50%), and additional components, such as buffer pH, excipient, diluent, cryoprotective agent and/ or a stabilizer, selected from of hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol have been developed (patent RU 2453333 C2, WO 2008/000490).
• additional components such as buffer pH, excipient, diluent, cryoprotective agent and/ or a stabilizer, selected from of hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol
• those known compositions do not have a prolonged action, nor increase the therapeutic effect of botulinum toxin and are not intended to treat cardiac arrhythmias.
• those formulations have insufficient exposure in the tissues of the heart for optimal effect, and might have the rapid elimination of the active substance into the systemic circulation.
• a liquid pharmaceutical composition comprising: (a) botulinum neurotoxin complex (type A, B , C, D, E, F or G) or high purity botulinum neurotoxin (type A, B , C , D, E, F or G) at concentration from 50 to 10,000 units LD 50 per 1 ml solution, (b) a stabilizing agent comprising a surfactant (SAS), preferably polysorbate 80 in an amount from 0.005 to 0.02 vol .%, (c) sodium chloride as a crystalline agent in a concentration from 0.15 to 0.3 M, (d) a disaccharide, preferably sucrose, at a concentration 10 - 20 mM, (d) a buffer, mainly histidine, to maintain the pH 5.5-7.5 and water has been also developed (patent RU 2407541 C2, WO 2006/005910).
• those known compositions are not intended to treat disorders of cardiac rhythm, and there are said to induce stabilizing effects on botulinum toxin without any
• the invention relates pharmaceutical compositions of botulinum neurotoxins useful for treating cardiac arrhythmia, in particular having a high therapeutic effect, an increased lasting effect and reduced side effects.
• compositions of the invention achieves increased pharmacological activity of the botulinum toxin type A, a desired therapeutic effect already achieved by a single dose, a prolongation of the botulinum toxin effect, while a reduction of botulinum toxin side effects. Further, compositions of the invention allow preparing compositions with the desired properties for personalized medicine directly into the clinic and present a prolonged activity when maintained in the solution ready to the introduction.
• a first aspect of the invention provides a composition containing botulinum toxin, in particular botulinum toxin type A, and a mucopolysaccharide selected from the group consisting of chitosan and nadroparin, taken in a weight ratio of l :from 10 3 - 10 9 ), preferably 1 : (from 10 6 -10 8 ), and a pharmaceutically acceptable excipient with the following components:
• a second aspect of the invention relates to a pharmaceutical formulation according to the invention for use as a medicament.
• a third aspect of the invention relates a use of a composition according to the invention for the preparation of a pharmaceutical preparation for the prevention and/or treatment of cardiac arrhythmias, in particular atrial fibrillation or arterial hypertension.
• a fourth aspect of the invention relates to a method of preventing and/or treating cardiac arrhythmias, in particular atrial fibrillation, or arterial hypertension in a subject in need thereof, such method comprising administering a pharmaceutical formulation according to the invention in said subject.
• a fifth aspect of the invention relates to a medicinal kit comprising in compartmental form a first compartment or series of compartments comprising a composition according to the invention and a second compartment or series of compartments comprising a syringe for injection with instructions for use.
• a sixth aspect of the invention relates to medicinal kit for the preparation of a composition according to the invention, comprising in compartmental form a first compartment or series of compartments comprising a botulinum toxin solution and a second compartment or series of compartments comprising a chitosan powder and optionally a vial for formulation preparation with instructions for use.
• Figure 1 represents the effect of pharmaceutical compositions of the invention as represented by the change in the electrostimulation threshold (measured in Volts) of rat's femoral muscles as compared to a commercial formulation (Xeomin) and to comparative formulations comprising botulinum toxin and another mucopolysaccharide, as described in Example 9.
• C Comparative formulation N° 11 (— Sodium hyaluronate);
• D Comparative formulation N° 4 (— Heparin);
• Figure 2 represents the effect and its duration of a pharmaceutical composition of the invention as represented by the change in the electrostimulation threshold (measured in Volts) of rat's femoral muscles as compared to a composition containing disaccharide and botulinum toxin A, according to the description of the patent RU 240754 las described in Example 11.
• Figure 3 shows the comparative effects of two pharmaceutical compositions of the invention at different concentrations of chitosan as represented by the change in the electrostimulation threshold (measured in Volts) of rat's femoral muscles with those of a commercial composition (Xeomin), as described in Example 13.
• Xeomin a commercial composition
• Figure 4 shows pictures of a contrast injection [VisipaqueTM (iodixanol)] into the epicardial fat pads containing the anterior right (A), inferior right (B) and superior left GP (C) as described in Example 12. Output needle of catheter into the pericardial space (D).
• VisipaqueTM iodixanol
• Figure 5 shows the threshold of stimulation as measured by the change in the electrostimulation threshold (measured in Volts) of rat's femoral muscles that induced AF as described in Example 11.
• Figure 6 shows an example of administration of a composition of the invention into the visible area of the four major epicardial fat pads.
• the needle tip is positioned manually at several points on the epicardial surface of the fat pads under direct visual control to ensure optimal injection.
• Botulinum toxin refers to any type of Botulinum toxin selected from the types A, B, E, F and G.
• Botulinum toxin acts by blocking the release of acetylcholine from the presynaptic terminal of the neuromuscular junction. Seven distinct antigenic botulinum toxins (BTX-A, B, C, D, E, F, and G) produced by different strains of Clostridium botulinum have been described.
• the human nervous system is susceptible to 5 toxin serotypes (BTX-A, B, E, F, G) and unaffected by 2 (BTX-C, D).
• Botulinum neurotoxins are produced as inactive polypeptides of 150 kDa, which are cleaved by trypsin-like bacterial protease to generate the di-chain active form of the toxin.
• the proportion of single to di-chain toxin is dependent on the toxin's serotype and whether or not the bacterial strain expresses the appropriate protease.
• the 100-kDa heavy (H) chains and the 50-kd light (L) chains are linked together by heat- labile disulfide bonds and noncovalent forces.
• the H and L chains dissociate with heat and boiling, which inactivates the toxin because neurotoxicity requires both H and L chains.
• botulinum toxin type A refers to any commercially available products based on botulinum toxin type A which can be used according to the invention, for example, "BOTOXTM", “DysportTM”, “KseominTM”, “LantoksTM”, etc.
• Botulinum toxin serotype A has typically a molecular weight of about 150 kDa and is a protein in the form of double-chain polypeptide consisting of the heavy chain and light chain which are connected by a disulfide bridge.
• heavy chain causes fixing with presynaptic cholinergic nerve terminals and cellular uptake of the toxin.
• the light chain is believed to be responsible for the toxic effects, acting as zinc-endopeptidase and splitting specific proteins responsible for membrane fusion. Disrupting the process of membrane fusion within the cell, botulinum toxin prevents the release of acetylcholine in the synaptic cleft.
• Botulinum toxin serotype A can be obtained by purification and isolation from bacterium Clostridium botulinum culture such as described in patent US 7,189,541 (Botulinum toxin production method); patent US 6,818,409 (Isolation and purification of Clostridium botulinum toxins) or recombinantly produced as described in patent US 6,967,088 (Soluble recombinant botulinum toxin proteins).
• chitosan » refers to as a chitin derivatives obtained by partial to substantial alkaline N-deacetylation of chitin also named poly(N-acetyl-D-glucosamine), which is a naturally occurring biopolymer that can be extracted from extracted from the shells of crustaceans, such as shrimp, crab and other sea crustaceans, including Pandalus borealis and cell walls of fungi such as for example described in Kumar, 2000, reactive and Functional Polymers, 46(1), 1-27 and Yogeshkumar, 2013, International Journal of Research in Pharmaceutical and Biomedical Sciences, 4(1), 312-331; Davis et al, 1984, J. Gen. Microbiol, 130(8):2095-102.
• Chitosan as a natural material, has been widely investigated in this field due to its structural similarity to glycosaminoglycans (GAGs), which are the components of the extracellular matrix (ECM).
• GAGs glycosaminoglycans
• ECM extracellular matrix
• chitosans of the invention are of crab shell origin and are obtained after a mechanical comminution and a deacetylation process.
• chitosans of the invention have a deacetylation degree of about 85 % to about 100 %.
• chitosans of the invention have an average molecular weight (Mw) of about 100 kDa to about 000 kDa.
• Heparin refers to a mucopolysaccharide having anticoagulant direct action and has a molecular weight comparable to molecular weight of botulinum toxin. It can be extracted from crushed bovine lung or recombinantly produced as described in Linhardt et al, 2012, Curr. Opin. Pharmacol, 12(2): 217-219.
• Nadroparin refers to a mucopolysaccharide having anticoagulant direct action and is a low-molecular heparin with a molecular weight of more than an order of magnitude less than that of botulinum toxin. It can be isolated from mammalian tissue (US patent 2,884,358 or Synthesized from UDP-sugar precursors as a polymer of alternating D-glucuronic acid and N-acetyl-D-glucosamine residues (Hazardous Substances Data Bank (HSDB®).
• the invention provides pharmaceutical compositions and methods for treating a subject, in particular a mammalian subject, and most particularly a human patient who is suffering from cardiac arrhythmias, in particular atrial fibrillation or a risk of developing cardiac arrhythmias, in particular atrial fibrillation.
• the invention provides pharmaceutical compositions of the invention for use as a medicament.
• compositions of the invention may further comprise one or more pharmaceutically acceptable additional ingredient(s) such as stabilizers, antimicrobial agents, buffers, coloring agents, adjuvants, and the like.
• additional ingredient(s) such as stabilizers, antimicrobial agents, buffers, coloring agents, adjuvants, and the like.
• compositions according to the invention together with a conventionally employed adjuvant, carrier, diluent or excipient may be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as implants or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, in the form of sterile injectable solutions for parenteral (including subcutaneous) use by injection or continuous infusion.
• injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art.
• compositions and unit dosage forms thereof may comprise ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended dosage range to be employed.
• compositions according to the invention are injectable.
• compositions of the invention may optionally include, additional components, such as pH buffering agent, excipient, diluent cryoprotective agent and/or stabilizer.
• compositions of the invention comprise composition according to any one of claims 1 to 4 wherein the weight ratio of botulinum neurotoxin, in particular Botulinum toxin A, to mucopolysaccharide is from about 1 : 1.5 xlO 7 to about l :5xl0 7 , for example 1 :4.4 x 10 7 .
• compositions of the invention comprise composition according to any one of claims 1 to 4 wherein the weight ratio of botulinum neurotoxin, in particular Botulinum toxin A, to mucopolysaccharide is from about 1 : 1.5 xlO 7 to about l :5xl0 7 , for example 1 :76 x 10 7 .
• compositions of the invention comprise a dosage of about 20 to about 200 UI of Botulinum toxin (typically 50UI), in particular Botulinum toxin A, for 1 injection procedure.
• compositions of the invention are in the form of a dosage of injection procedure volumes of 2 ⁇ 00 ⁇ ,.
• compositions of this invention may be administered in any manner including, in particular, in the epicardiac area (notably in the visible area of the major epicardiac fat pads) in case of treatment atrial fibrillation.
• a combination of different administration routes may also be used.
• Methods such as intramyocardial administration by injection endovascular catheters, intravascular infusion into the artery that feeds the target organ (heart, kidney) can be used for the administration of compositions of the invention.
• composition compositions are readily determined by one of skill in the art depending upon a variety of factors, including pharmacokinetic properties, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired.
• compositions of the invention are administered before or at the beginning of the coronary artery bypass graft surgery.
• compositions of the invention is injected into the entire visible area of the four major epicardial fat pads (such as illustrated on Figure 6).
• compositions of the invention are administered at a botulinum toxin dose of about 50 U/l mL in at least one in at least one epicardial fat pad, preferably in at each fat pad.
• pharmaceutical formulations of the invention can be administered alone or in combination with a co-agent useful in the prevention and/or treatment of cardiac arrhythmias, in particular atrial fibrillation or arterial hypertension e.g. for example a co-agent selected from an antiarrhythmic substance of I, II, III classes (such as procainamide, amidaron, sotalol).
• a co-agent selected from an antiarrhythmic substance of I, II, III classes (such as procainamide, amidaron, sotalol).
• the invention encompasses the administration of a pharmaceutical formulation to an individual prior to, simultaneously or sequentially with other therapeutic/prophylactic regimens or co-agents in the prevention or treatment of cardiac arrhythmias, in particular atrial fibrillation or arterial hypertension (e.g. combined regimen), in a therapeutically effective amount.
• a pharmaceutical formulation that is administered simultaneously with said co-agents can be administered in the same or different composition(s) and by the same or different route(s) of administration.
• patients according to the invention are patients suffering from a disorder selected from cardiac arrhythmia, in particular atrial fibrillation, and arterial hypertension.
• patients according to the invention are patients at risk of suffering from a disorder selected from cardiac arrhythmia, in particular atrial fibrillation, for example patients undergoing coronary artery bypass graft (CABG) surgery, heart valve surgery or other open heart surgery, which are associated with a 30% risk of atrial fibrillation in the early postoperative period (Filardo et al, 2009, Circ. Cardiovasc. Qual. Outcomes, 2:164-169).
• a disorder selected from cardiac arrhythmia in particular atrial fibrillation
• CABG coronary artery bypass graft
• patients according to the invention are patients suffering from cardiac arrhythmia.
• patients according to the invention are patients suffering from arterial hypertension.
• a process for inducing a decrease in cardiac arrhythmias, in particular in atrial fibrillation in a subject by use of a formulation or a combination as described herein.
• a process for inducing a decrease in the release of renin and/or blood pressure in a subject in need thereof by use of a formulation or a combination as described herein.
• the formulation or combination according to the invention is administered in an amount and in accordance with a dosage regimen that is effective for inducing a decrease in cardiac arrhythmias and/or blood pressure.
• a method for preventing, repressing or treating cardiac arrhythmias, in particular in atrial fibrillation, or arterial hypertension comprising administering in a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation according to the invention.
• formulations of the inventions could be used in different areas of medicine, where botulinum toxin only is used, i.e. not only for heart rhythm disorders treatment, since the main effect achieved by formulations of the invention is a blockage of neuromediator release from presynaptic terminal of nervous system and there are no principal histologic and cytologic differences between these synaptic compositions in other zones on human body.
• the formulations of the inventions are to be administered into intramyo cardial GPs and epicardial fat pads.
• compositions of the invention can be prepared by mixing aqueous solutions of the components in predetermined proportions.
• a formulation of the invention is that it can be prepared from pure botulinum toxin type A or from a commercially available product based on botulinum toxin type A that allows the preparation of the required solution from the available components directly into clinical practice.
• the formulations of the invention allow achieving an increase in the effect of botulinum toxin type A, while reducing side effects of systemic effect and produce immunoresistance (Substance of invention is using mucopolysaccharides to create mechanic protection of molecule and delay distribution of botulinum toxin molecule from targeted location).
• Region of invention is using mucopolysaccharides to create mechanic protection of molecule and delay distribution of botulinum toxin molecule from targeted location).
• Molecules of chitosan are neutral and mechanically cover immunoreactive centers of botulinum toxin (Katherine Bowman et al, 2006, Int. J. Nanomedicine, 1(2): 117-128).
• botulinum toxin composition of the invention is that -it would allow the reducing the necessary therapeutic dose, while increasing duration of drug effect as well as reduced side effects as undesirable denervation of unused muscles and development of systemic effects.
• Treatment of atrial fibrillation requires epicardial administration of the composition, where the priority property of chitosan is elongation release of botulinum toxin (botulinum toxin type A and chitosan in ratio of 1 to 4.4 x 10 7 by weight).
• Treatment of arterial hypertension requires renal intraarterial infusion of the composition, where the priority property of chitosan is an increase the adhesive properties of botulinum toxin for juxtaglomerular membrane (botulinum toxin type A and chitosan in ratio of 1 to 4x 10 5 by weight).
• botulinum toxin type A/mucopoly saccharide of the invention With increasing ratio of active components (botulinum toxin type A/mucopoly saccharide of the invention) above 1 : 10 9 by weight, there are difficulties with preparing therapeutically acceptable injectable preparations. For example, at a ratio of botulinum toxin type A/chitosan equal to 1 : 10 9 , and 40 U of botulinum toxin activity (equivalent to 1 ng), the necessary amount of chitosan is 1 gram that leads to an injection volume of 50 ml for clinically relevant concentrations of chitosan in 2% injection. This amount is excessive for therapy.
• a medicinal kit comprising in compartmental form a first compartment or series of compartments comprising a composition of the invention and a second compartment or series of compartments comprising a syringe for injection with instructions for use. Examples illustrating the invention will be described hereinafter in a more detailed manner and by reference to the embodiments represented in the Figures.
• Example 1 Preparation of pharmaceutical composition (M I) containing botulinum toxin type A and chitosan in ratio of 1 to 4.4 x 10 7 by weight
• botulinum toxin type A To prepare the solution of botulinum toxin type A in all the experiments, the commercial preparations of botulinum toxin type A "KseominTM” produced by MERZ PHARMA GmbH & Co., KGaA (Germany), registration number LSR-004746/08, auxiliaries, sucrose and human serum albumin, or "LantoxsTM” produced by Lanzhou Institute of Biological Products, (China), registration number LSR-001587/08, auxiliaries: gelatin, dextran and sucrose, were used.
• the pharmaceutical composition was prepared by mixing in 1 ml syringe equipped with a system Luer-Lok Tip, 100 ⁇ ⁇ of botulinum toxin type A solution (containing 10 U or 0.25 ng of botulinum toxin type A) and 400 ⁇ ⁇ of 2.75% chitosan solution (containing 11 mg of chitosan).
• the result was a liquid pharmaceutical composition comprising the following components:
• botulinum toxin type A 20 IU / ml (0.5 ng / ml)
• Example 2 Preparation of pharmaceutical composition (JYs 2) containing botulinum toxin type A and chitosan in ratio of 1 to 1.76 x 10 7 by weight
• the pharmaceutical composition was prepared by mixing in 2 ml syringe.
• the 100 units solution of botulinum toxin in 400 ⁇ ⁇ of saline was prepared and it was used (it contains 100 units, or 2.5 ng of botulinum toxin type A) for mixing with 1600 ⁇ _, of 2.75% chitosan solution (containing 44 mg of chitosan).
• botulinum toxin type A 50 IU / ml (1.25 ng / ml)
• Example 3 Preparation of pharmaceutical composition (JYs 3) containing botulinum toxin type A and heparin in the ratio of 1 to 1.6 * 10 7 by weight f comparative formulation not of the invention)
• Heparin solution with a concentration of 5000 IU/ml was used (production of Synthesis company, Kurgan, Russia, drug registration number P N000116/01).
• One unit of heparin is equal to 0.0077 mg of the International Standard heparin, i.e. 1 mg contains 130 IU (Pershyn GN, Gvozdeva EI Textbook of Pharmacology - Moscow: Medgiz, 1961 - s.405).
• the pharmaceutical composition was prepared by mixing 100 ⁇ ⁇ of botulinum toxin type A solution (containing 10 U or 0,25 ng of botulinum toxin type A), 104 ⁇ ⁇ of heparin solution containing 4 mg of heparin ( ⁇ 520 IU) and 500 ⁇ ⁇ of saline.
• the pharmaceutical composition comprising:
• botulinum toxin type A 20 IU / ml (0.5 ng / ml)
• Example 4 Preparation of pharmaceutical composition (JT° 4) containing botulinum toxin type A and heparin in the ratio of 1 to 5.3 x l(f by weight (comparative formulation not of the invention)
• Example 3 Analogously to Example 3 to obtain the pharmaceutical composition 300 iL of botulinum toxin type A solution (containing 30 U or 0,75 ng of botulinum toxin type A), 104 iL of heparin solution, containing 4 mg of heparin ( ⁇ 520 IU) and 500 iL of saline were mixed.
• the pharmaceutical composition comprising:
• botulinum toxin type A 60 IU / ml (1.5 ng / ml)
• Example 5 Preparation of pharmaceutical composition (NQ 5) containing botulinum toxin type A and nadroparin in ratio of 1 to 8 x l(f by weight
• fraxiparine (nadroparin), (Sanofi Winthrop Industry, France, registration number P N012486/01) with a concentration of 9500 IU per mL was used for the experiment.
• One unit of action of low molecular heparin - nadroparin (average molecular weight - 4000-7000 Da) has been accepted by us equal to one unit of heparin, i.e. 0.0077 mg.
• nadroparin solution containing 2 mg of nadroparin ( ⁇ 260 IU) and saline up to 500 iL were mixed.
• the pharmaceutical composition comprising:
• botulinum toxin type A 20 IU / ml (0.5 ng / ml)
• nadroparin 520 IU / mL (4 mg / ml)
• Example 6 Preparation of pharmaceutical composition (M 6) containing botulinum toxin type A and nadroparin in ratio of 1 to 2.67 x l(f by weight
• nadroparin solution containing 2 mg nadroparin ( ⁇ 260 IU) and 500 iL of saline were mixed.
• a pharmaceutical composition comprising:
• botulinum toxin type A 60 IU / ml (1.5 ng / ml)
• nadroparin 520 IU / mL (4 mg / ml) saline 0.5 ml.
• Example 7 Preparation of a pharmaceutical composition (JYs 10) containing botulinum toxin type A and chitosan ratio of 1 to 2 * 10 s by weight
• Example 2 Analogously to Example 1, a 5% solution of chitosan in saline was prepared. 10 ml of this solution was used to dissolve 100 U botulinum toxin type A (containing 100 ng or 2.5 units of botulinum toxin type A and 500 mg of chitosan). The result was a liquid pharmaceutical composition comprising the following components:
• botulinum toxin type A 10 IU / ml (0.25 ng / ml)
• the pharmaceutical composition was prepared by mixing in 1 ml syringe equipped with a system Luer-Lok Tip, 100 ⁇ of botulinum toxin type A solution (containing 20 U or 0,5 ng of botulinum toxin type A) and 400 ⁇ . of 1% sodium hyaluronate (containing 4 mg of sodium hyaluronate, Sigma-aldrich cat# 53747).
• the result was a liquid pharmaceutical composition comprising the following components:
• botulinum toxin type A 20 U/ml (0.5 ng/ml)
• physiological saline make up to volume 0.5 ml.
• compositions of the invention were performed in comparison with solutions of commercial preparations of botulinum toxin type A in saline and with pharmaceutical compositions comprising botulinum toxin type A and another mucopolysaccharide than chitosan or heparin (N° 3 and 4) at the same dose, expressed in units.
• Injection volume 0.5 ml in each thigh.
• Method of administration intramuscularly, three injection points (back, medial, lateral surface of the thigh), 0.16 ml in each.
• Comparison of the formulations was carried out by assessing the reduction of thigh muscles in response to electrical stimulation. Threshold minimum change of intramuscular electrostimulation was compared in the course of time. Measurement of electrostimulation threshold was carried out intramuscularly using two sterile steel microelectrodes and ERA 300 device (Biotronic, USA). These microelectrodes, for the period of measurement, were temporarily inserted intramuscularly to a depth of about 4 mm in outer area of the mouse thigh, 10 mm away from each other.
• the physiological effect of the composition N° 1 at the time of measurement is increased by 2 times compared with a commercial preparation of botulinum toxin.
• compositions N° 3-6 the maximum effect was not observed during the first week after injection but was found during the second week; but the absolute value of the differences from the control was not as great as in the case of chitosan.
• N°3.Maximum thresholds of intramuscular electrical stimulation during the second week were 1.8 ⁇ 0.1 V (control 2.3 ⁇ 0.1 V, 1.0 ⁇ 0.1 initially V), and for composition JVTs5: 2.2 ⁇ 0.1 V (control 1.5 ⁇ 0.1 V, initially 1.2 ⁇ 0.1 V) and comparative composition N° 11 : 1.5 ⁇ 0.1 V (control 2.6 ⁇ 0.2 V, initially 1 ⁇ 0.1 V).
• botulinum toxin type A Probably heparin quite firmly holds botulinum toxin type A, preventing its biological effects as compared with commercial preparations, whereas low molecular nadroparin, as opposed, enhances the action of botulinum toxin. It is further supported by the absence of significant effects for comparative formulation 4 as compared to formulation of the invention N° 6.
• compositions of the invention were assessed for potential toxicity by histo- morphological study through histological examination of internal organs of experimental animals. Upon microscopic analysis of formalin-fixed and paraffin embedded tissue samples of liver, kidney, spleen, heart, skeletal muscle and brain, no morphological signs of pathological changes were found. Therefore, it was concluded, to the absence of damaging influence of botulinum toxin type A and pharmaceutical compositions of the invention at the testes doses, on tissues and organs of experimental animals.
• Example 10 Investigation of suppressing induction of atrial fibrillation by injection of a pharmaceutical composition containing botulinum toxin type A into epicardial fat pad
• the test was conducted on a group of 10 dogs using a pharmaceutical composition N° 2.
• Epicardial fat pads containing the right-center ganglion plexus of the left atrium, were allocated through the right lateral thoracotomy.
• Electrophysiological effects were evaluated after 1, 2, 3 and 4 weeks after injection, with and without cervical vagus nerve stimulation. Atrial fibrillation was achieved in both groups of dogs through cervical vagus nerve stimulation. This effect was blocked by the administration of the test and control solutions of botulinum toxin in the above described doses and areas in both groups. In the control group, the blockage of vagal effect disappeared on the 8 th day after injection (the observation period). In the group of experience, the blockage of vagal effect persisted for more than 30 days after injection.
• Comparative formulation N° 11 contains:
• botulinum neurotoxin type A (20 U/ml (0.5 ng / ml))
• sucrose at a concentration 20 mM
• physiological saline saline of sodium chloride 0,9%
• Thresholds of intramuscular electrical stimulation of rat thigh muscle after injection was measured as described above.
• Results are represented on Figure 2: Maximum thresholds of intramuscular electrical stimulation were for the Group 1 : (9.8 ⁇ 0.3 V) vs Group 2: (2.6 ⁇ 0.1 V); Initially 1.2 ⁇ 0.1 V vs 1.0 ⁇ 0.1 V, respectively.
• Group 1 10.0 ⁇ 0.4 V vs Group 2:2.5 ⁇ 0.1 V.
• Group 1 5.0 ⁇ 0.2 V vs Group 2: 2.0 ⁇ 0.1 V.
• Group 1 3.3 ⁇ 0.2 V
• stimulation threshold in the Group 2 decreased to close to the initial (1.5 ⁇ 0.1 V).
• formulations of the invention comprising chitosan and botulinum toxin showed better efficacy and longer lasting effect than the registered commercial composition comprising disaccharide and botulinum toxin as described in patent RU 2407541.
• botulinum toxin injection into the epicardial fat pads can suppress atrial fibrillation (AF) inducibility.
• the aim of the present study was to compare the efficacy and safety of endocardial botulinum toxin injection into epicardial fat pads and intramyocardial left atrial ganglionated plexi (GP) for preventing AF using chitosan+botulinum toxin type A (1 : 1.76x 10 ) (formulation of the invention (N° 2) and commercial formulations of Botulinum toxin.
• transvenous catheters were passed into the left atrium. Sites where vagal reflexes were evoked by high-frequency stimulation (HFS) were tagged on an electroanatomic mapping system and then designated for injection. Intramyocardial injections (10 U/0.2 mL at each) of botulinum toxin were administered at 7 sites per dog. In addition, 3 injections per dog were made into the epicardial fat pads containing the anterior right, inferior right and superior left GP (50 U/l mL at each) also by endocardial approach (Figure 4).
• HFS high-frequency stimulation
• vagal reflexes by HFS and AF inducibility were evaluated before injections and then every 2 weeks until the return of all changes to baseline by precise catheter reposition and stimulation over the GP sites marked on the previously recorded map.
• 15 of 30 dogs were injected by chitosan+botulinum toxin composition (l : 1.76x l0 7 ), other 15 of 30 dogs were injected by botulinum toxin (Xeomin, Germany).
• the aim of the present study was to compare the efficacy and safety of botulinum toxin infusion into renal arteries for preventing arterial hypertension using formulations of the invention, in particular chitosan+botulinum toxin type A (formulation of the invention N° 1, 1 :4.4 ⁇ 10 7 ), chitosan+botulinum toxin type A (formulation of the invention N° 2, 1 : 1.76 ⁇ 10 7 ) and commercial formulations of Botulinum toxin (Xeomin, Germany).
• transvenous catheters were consequentially passed into the left and right renal artery.
• Intrarenal infusion (50 U/l mL) of botulinum toxin was administered at each pig's kidney (for composition N° 1 : 3 pigs; composition N° 2: 3 pigs; commercial formulation of Botulinum toxin: 3 pigs).
• 3 pigs were included in Placebo group with 1 ml of physiological saline (saline of sodium chloride 0.9%) infusion into each renal artery. All protocols were approved by the Institutional Animal Care and Use Committee in accord with the Guide for the Care and Use of Laboratory Animals. At 1 week after the procedure, all the kidneys were explanted. Kidneys were decapsulated and homogenized.
• the obtained cells were incubated in solution which contained 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 5% fetal calf serum at 37°C/5% C0 2 in poly-D-lysine-coated plates (0.1 mg/ml).
• Cells were serum deprived for 2 h by replacing the medium with serum-free solution which contained 100 U/ml penicillin and 100 ⁇ g/ml streptomycin. Renin release was stimulated by increasing intracellular levels of cAMP with forskolin (10 ⁇ ) plus 3-isobutyl-l-methylxanthine (0.5 mM) for 1 h. Following treatment, the medium was centrifuged to remove cellular debris. Supernatants were collected in fresh tubes and stored at -20°C until processing. Analysis of the stimulated renin content in the supernatant was performed by using a reagent Renin ELISA kit (R&D Systems, USA).
• Renin concentration % were measured in the supernatant samples: formulation of the invention JVfe 1, (1 :4.4x l0 7 ) - 2.8+0.5%; formulation of the invention JVfe 2, (l : 1.76x l0 7 ) - 1.5 ⁇ 0.2%; commercial formulations of Botulinum toxin (Xeomin, Germany) - 4.2 ⁇ 1.2%; placebo group (saline of sodium chloride 0.9%) - 4.8 ⁇ 1.4%. No procedure- related complications occurred.
• composition when administered into such target areas as renal artery and ventricular myocardium, where there is direct contact with the blood supply system, it is require achieving rapidly a therapeutic concentration and an increase of the exposure to botulinum toxin. This requirement is satisfied by reducing the quantitative ratio of chitosan, which in turn leads to prevailing adhesion function of the composition.
• botulinum toxin When endomyocardial injection is performed, botulinum toxin is more diluted as compared with botulinum toxin injection in epicardial fat pads. Accordingly, epicardial injections of chitosan+botulinum toxin need another concentration ratio (tends to l :4.4x l0 7 ), that allows to get good effectiveness and reduces risks of serious adverse events, whereas an endomyocardial injection needs a concentration ratio tending to 1 : 1.76x 10 7 that allows getting therapeutic concentration in targeted location.

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