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
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
• • 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
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
• • 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)
• • 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
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
• • 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
  • A61P3/06—Antihyperlipidemics
• • 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
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Milk from domestic animals has been used as a source of proteins and other products for the food and pharmaceutical industries for many years, and a variety of techniques are known for isolating these products.
• Milk is a colloidal suspension composed primarily of fats, lactose and proteins in water.
• milk contains an average of 30 to 140 grams of protein per litre, or about 4-17% by weight, depending on the species.
• the bulk of these proteins are caseins, which are complexed with calcium and phosphate in supramolecular structures known as micelles.
• the other major class of milk proteins is whey proteins, predominantly comprised of beta-lactoglobulin and alpha-lactalbumin, but also including lactoferrin, immunoglobulins, and serum albumin.
• Lactoferrin is an 80 kD iron-binding glycoprotein found naturally in biological fluids such as saliva, bile, bronchial mucus, gastrointestinal fluids, cervico-vaginal mucus, seminal fluid, and milk.
• the richest source of lactoferrin is mammalian milk and colostrum.
• the concentration of lactoferrin in bovine skimmed milk is usually small, typically between 80-200 mg/ml depending on factors including the pasteurisation and other pre-treatment history of the skimmed milk. After precipitation of the casein present in milk the concentration of lactoferrin in bovine whey is typically 10-100 mg/ml depending on the physical and chemical pre-treatment of the whey.
• Milk fractions enriched for RNAse 5 and growth factors have multiple postulated biological roles including promoting muscle growth, neuroprotection, promoting osteogenesis and treating neurological diseases or disorders, spinal injuries or diseases, bone diseases or disorders, diseases involving glucose homeostasis, wound healing, or for providing nervous system functional support and managing metabolic diseases,
• the inventors When purifying lactoferrin from milk using membrane filtration with a 30 kD or 50 kD cut off the inventors found that the lactoferrin retentate was contaminated with growth factors and RNAses. As these growth factors and RNAses have a molecular weight of less than 30 kD they should have passed through the membrane and into the permeate and not be present as an impurity in the retentate lactoferrin fraction. Accordingly the discovery of growth factors and RNAses in the retentate was surprising. The inventors found that the addition of a large amount of salt to the milk prior to filtration removed the growth factors and RNAses from the retentate and concentrated them in the permeate.
• the inventors propose that under normal conditions the RNAses and growth factors in milk aggregate or otherwise form a mass that is larger than their individual molecular weights. It is proposed that the salt treatment causes the RNAses and growth factors to disaggregate or disassociate.
• process of the first aspect provides a process for purifying
• RNAses and growth factors from milk or lactoferrin comprising subjecting the milk or lactoferrin to filtration to separate it into a retentate fraction comprising lactoferrin and a permeate fraction comprising growth factors and/or RNAses, wherein prior to and/or during filtration the milk or lactoferrin is subjected to salt treatment capable of disaggregating or disassociating any mass of RNAses or growth factors such that growth factors and/or RNAses flow into the permeate.
• the inventors alternatively propose that under conditions of low ionic strength, protein aggregates may become associated with the membrane, thereby forming a layer with a smaller apparent pore size to that of the membrane, which prevents RNAses and growth factors in milk passing through the membrane. It is proposed that the salt treatment prevents formation of or removes the protein layer, allowing any RNAses and growth factors to pass through the membrane into the permeate.
• the process of the first aspect provides a process for for purifying RNAses and growth factors from milk or alctoferrin, the process comprising subjecting the milk or lactoferrin to membrane filtration to separate it into a retentate fraction comprising lactoferrin and a permeate fraction comprising growth factors and/or RNAses, wherein prior to and/or during filtration the milk or lactoferrin is subjected to salt treatment capable of separating any RNAses or growth factors from the membrane such that growth factors and/or RNAses flow into the permeate.
• a second aspect provides RNAses and growth factors obtained from the process of the first aspect.
• RNAses and growth factors of the second aspect are subjected to further purification.
• a third aspect provides use of RNAses and growth factors of the second aspect for the treatment of diseases caused by viruses, bacteria, or fungi and their toxins, to target pathogens which cause infections of human mucosal surfaces, to promote angiogenesis, for treating a disorder characterised by elevated myostatin, for treating disorders where the interaction between follistatin and angiogenin can be used to improve function in tissues, for promoting muscle growth, for improving recovery of muscle from injury or use, for improving muscle strength, for improving exercise tolerance, for increasing the proportion of muscle, for decreasing fat, for decreasing an individual's fat to muscle ratio, for treating neurological diseases or disorders, for treating spinal injuries or diseases, for treating bone diseases or disorders, for treating diseases involving glucose homeostasis, for wound healing, or for providing neuroprotection, nervous system functional support, managing metabolic diseases and/or increasing the bone density of an individual, for treating inflammation, to treat cancer, to treat cancer cachexia, to treat periodontitis and in all other uses of growth factors and RNAses known to persons skilled in the art.
• Figure 1 shows ultrafiltration of a mixture of cationic milk proteins through a 50 kDa membrane, in the presence of sodium chloride (100mS), selectively allows the transmission of RNAses and other growth factors.
• Figure 2 shows ultrafiltration of a mixture of cationic milk proteins through a 50 kDa membrane, in the presence of sodium chloride (20 or 40 mS), selectively allows the
• RNAses and other growth factors which do not permeate in the absence of sodium chloride (0 mS).
• Figure 3 shows increased transmission of growth factors, such as the RNAses. Growth factor transmission increases quickly between 0 mS and 20 mS.
• the present invention provides improved methods for purifying RNAses and growth factors from milk or lactoferrin or enriching milk or lactoferrin for such products.
• the inventors have recognised the need for a process which allows the preparation of enriched RNAse and growth factor fractions in an efficient manner.
• milk includes whole milk, skim milk, buttermilk, whey (such as acid or cheese/renneted whey) or a whey derivative (such as whey protein concentrate or whey protein isolate flow through), and colostrum. It also includes milk fractions, for example fractions that have been subjected to purification steps such as cation exchange chromatography. Such fractions include milk basic protein and fractions containing lactoferrin.
• the milk may be obtained from any lactating animal, e.g. ruminants such as cows, sheep, buffalos, goats, and deer, non-ruminants including primates such as a human, and monogastrics such as pigs. It is preferred that skim milk which is derived from whole cow's milk is used in the process of the present invention.
• lactating animal e.g. ruminants such as cows, sheep, buffalos, goats, and deer
• non-ruminants including primates such as a human
• monogastrics such as pigs.
• skim milk which is derived from whole cow's milk is used in the process of the present invention.
• the filtration may involve ultrafiltration or diafiltration or both.
• Conductivity is generally measured using a conductivity meter for example a Hach Sension 5. Persons skilled in the art would be aware of suitable alternative means to measure conductivity. There is a generally linear relationship between sodium ion concentration and conductivity.
• the salt used in the salt treatment is not limited and alternatives to NaCI would be known to the person skilled in the art.
• any soluble, non-toxic buffer can be used such as the soluble sodium, potassium, calcium, magnesium or lithium salts of chloride, citrate, phosphate, acetate, sulphate, bicarbonate, hydroxide, imidazole, or maleate.
• Synthetic zwitterion buffers such as Trizma, HEPES or tricine may also be used.
• the ionic strength of the milk or lactoferrin must be at least 0.2 M (1 .1 %) NaCI or equivalent or more prior to the filtration step.
• the ionic strength of the milk is increased by adding 1 .1 %, salt, 1.5% salt, 2% salt, 2.5% salt, 3% salt, 3.5% salt, 4% salt, 4.5% salt, 5% salt, 5.5% salt, 6% salt or more.
• the salt treatment is carried out at 4-10 degrees.
• the salt treatment is carried out at 30-50 degrees.
• the filtration step is carried out at 10-30 degrees.
• the filtration step is carried out at a transmembrane pressure less than 2.5 Bar per membrane, more likely less than 2.0 Bar, more likely again less than 1.5 Bar but ideally 1 .0-1 .4 Bar, although a pressure of 0.0-1 .0 Bar could also work acceptably but have a lower transmembrane flux.
• Treating covers any treatment of, or prevention of a condition in a vertebrate, a mammal, particularly a human.
• Preventing refers to keeping from occurring, or to hinder, defend from, or protect from the occurrence of a condition, disease, disorder, or phenotype, including an abnormality or symptom.
• a subject in need of prevention may be prone to develop the condition.
• ameliorate or “amelioration” refers to a decrease, reduction or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.
• a subject in need of treatment may already have the condition, or may be prone to have the condition or may be in whom the condition is to be prevented.
• a pharmaceutical composition is one which is suitable for administration to humans.
• a veterinary composition is one that is suitable for administration to animals. Generally such compositions will contain purified RNAses or growth factors or at the very least all components of the composition will be verifiable.
• the pharmaceutical or veterinary composition can be administered in one dose, or at intervals such as once daily, once weekly, and once monthly.
• the pharmaceutical or veterinary composition is administered as a bolus dose, to maximize the circulating levels of active agent for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.
• the nutraceutical composition may be in the form of a soluble powder, a liquid or a ready-to-drink formulation.
• the nutritional composition may be in solid form as a food; for example in the form of a ready-to-eat bar or breakfast cereal.
• Various flavours, fibres, sweeteners, and other additives may also be present.
• the nutraceutical preferably has acceptable sensory properties (such as acceptable smell, taste and palatability), and may further comprise vitamins and/or minerals selected from at least one of vitamins A, B1 , B2, B3, B5, B6, B1 1 , B12, biotin, C, D, E, H and K and calcium, magnesium, potassium, zinc and iron.

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• Gastroenterology & Hepatology (AREA)
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• 2013
  • 2013-04-12 AU AU2013204850A patent/AU2013204850B2/en not_active Ceased
  • 2013-10-08 BR BR112015007675A patent/BR112015007675A2/pt not_active IP Right Cessation
  • 2013-10-08 JP JP2015534885A patent/JP2016500662A/ja active Pending
  • 2013-10-08 WO PCT/AU2013/001154 patent/WO2014056026A1/en not_active Ceased
  • 2013-10-08 CN CN201380055327.2A patent/CN104736560A/zh active Pending
  • 2013-10-08 CA CA 2887003 patent/CA2887003A1/en not_active Abandoned
  • 2013-10-08 US US14/434,272 patent/US20150232823A1/en not_active Abandoned
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  • 2013-10-08 KR KR1020157010221A patent/KR20150079618A/ko not_active Withdrawn
  • 2013-10-08 EP EP13844822.0A patent/EP2904007A4/en not_active Withdrawn
  • 2013-10-08 RU RU2015116931A patent/RU2015116931A/ru not_active Application Discontinuation
• 2015
  • 2015-04-09 IN IN2968DEN2015 patent/IN2015DN02968A/en unknown

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