Classifications

• • 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
  • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
• • 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
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

• the present invention relates to a new collagen peptide fraction (CPF) consisting of several peptide species which can among others be used for the stabilisation of protein and peptide drugs during long term infusion, of liquid forms of protein and peptide drugs and of freeze dried respectively lyophilised proteins and peptides or freeze dried respectively lyophilised drugs containing proteins and peptides for topical, nasal or transdermal application.
• CPF collagen peptide fraction
• Bioly active proteins in their natural environment are stabilized within a balanced system of biopolymers, carbohydrates and electrolytes. Maintenance of stability during handling and storage of highly purified proteins and polypeptides for therapeutic and other uses requires special procedures.
• Lyophilized pharmaceutical preparations in general may contain bulking agents (eg amino acids, carbohydrates or polyalcohols) , inorganic or organic buffer substances, electrolytes and bacteriostatics. Every single component of a freeze dried preparation has to be compatible with each other and the total composition has to provide an optimum environment for the stability of the active ingredient in the freeze dried state.
• bulking agents eg amino acids, carbohydrates or polyalcohols
• inorganic or organic buffer substances e.g electrolytes and bacteriostatics.
• a composition that provides stability in a freeze dried state is not necessarily best for drug stability in solution after reconstitution. This is of minor importance if the preparation is injected immediately after reconstitution. It may cause major problems, however, if the reconstituted drug has to be continuously infused during a prolonged time period.
• the composition of the respective pharmaceutical preparation has to guarantee maximum stability in the freeze dried state as well as after reconstitution.
• Loss of protein activity in solution may be caused by sub- optimal pH and ionic strength, autocatalytic degradation, heat, oxygen, surface denaturation, adsorbant surfaces, shear forces, high pressure, irradiation, etc.
• HSA human serum albumin
• BSA bovine serum albumin
• ovalbumin are strong antigens and can therefore not be used for the stabilization of injectable drugs.
• Human serum albumin is not antigenic to humans but it can bear a risk of viral (HIV, hepatitis) and mycoplasma contamination.
• a further disadvantage of HSA as a stabilizing agent involves the possible contamination with other biologically active materials eg proteinases or proteinase inhibitors that may interact with the protein to be stabilized (see M.C.E. Van Dam-Mieras, A.D.
• HSA is similarly disadvantageous when used in topical applications.
• solubilized collagen cannot be used in injectable preparations because of its activating effect on platelet aggregation.
• a further disadvantage of solubilized collagen is its high viscosity in solution and its instability. Heating over 40°C causes denaturation of collagen and subsequent gel formation upon cooling to room temperature. Addition of phosphate anions to collagen causes the formation of an insoluble gel.
• collagen following partial acid hydrolysis into lower molecular peptides, could still exhibit protein stabilising properties, did not cause platelets to aggregate, formed low viscosity solutions and was compatible with current buffering substances including phosphates. This was named as a collagen peptide fraction (CPF) .
• CPF collagen peptide fraction
• CPF was capable of saturating or blocking protein adsorbing or covalently binding sites of glass and synthetic or natural polymers in preparative, analytical, diagnostic and medical devices such as microtiter plates, blotting membranes, filters, tubings etc.
• a treatment by rinsing or incubation with CPF solution can therefore be used to prevent unspecific antibody or antigen binding in immunological techniques such as enzyme linked immuno adsorption (ELISA), immunoblotting and related procedures.
• ELISA enzyme linked immuno adsorption
• Incubation with CPF can also be used to saturate excessive active groups in activated supports for affinity chro atography, and washing with CPF of filter material, glass or plastic ware will prevent adsorption of proteins from solutions during processing.
• CPF collagen peptide fraction
• CPF collagen peptide fraction
• CPF collagen peptide fraction
• CPF collagen peptide fraction
• Collagen suitable for the production of a collagen peptide fraction (CPF) according to the present invention is obtainable from animal tissues, preferably pig skin.
• Pepsin solubilized type I collagen can be prepared by conventional techniques eg according to the method of N D Light, 1985.
• Collagen in Skin Preparation and Analysis, in: Methods in Skin Research (D Skerrow and C J Skerrow, Eds) J Wiley and Sons Ltd. p 559-585.
• Controlled hydrolysis of type I collagen can be performed by heating an aqueous collagen suspension at low pH for a defined time period.
• a collagen peptide fraction (CPF) according to the invention is obtainable by heating a collagen suspension at pH 2.5-4.0 for 30-90 minutes at 100 -150°C in an autoclave.
• the parameters (pH, temperature, time) can be varied to a wide extent. E.g. low pH and high temperature will reduce the heating time.
• a collagen peptide fraction (CPF) consists of several peptide species, >70%, especially >80%, of which have an average molecular weight of 8-30 kDa, especially 8 to 25 kDa, more especially 10-20 kDa, most especially 20 kDa (estimated by gel permeation chromatography), a hydroxyproline content of 15-19%, a proline content of 18-22% and a glycine content of 27-33% (according to D.H. Spackman et al., Anal. Chem. 30, 1190-1206, (1958)).
• a mixture of equal volumes of 10% aqueous CPF solution and 10% trichloro- -acetic acid does not form any protein precipitate while albumin or gelatin, under similar conditions form strong precipitates.
• a CPF solution in 5% ammonia after heating to 95°C with 1 ml silver nitrate, 0.1 M, does not show any brownish colouration, whereas gelatin under similar conditions, due to its content of reducing carbohydrates, shows a dark brown colour.
• reducing carbohydrates originating from glucosaminoglycan degradation produce a strong orcinol colour reaction whereas CPF shows a very weak reaction only.
• Type I collagen from pig skin was purified according to Light (see ref. above). Freshly frozen pig skin was ground and defatted by solvent extraction. The resulting skin fibre pulp was treated with pepsin to solubilize type I collagen. Insoluble material was removed by filtration, collagen was precipitated from the filtrate at pH 7.5, dissolved in saline and further purified by salt fractionation and ion exchange treatments. Precipitated type I collagen was suspended in water, the pH adjusted to 3.5 with hydrochloric acid, the acidified suspension was heated in an autoclave for 60 min at 145°C, the concentration was adjusted with water to 10 ⁇ 1% solids.
• Figure 2 shows that the activity of TPA which is commonly used in therapy as an infusion also suffered less loss of activity when incubated with CPF rather than HSA at 37°C.
• the loss in the activities during lyophilisation was approximately similar for CPF and HSA and the potencies of the enzymes lyophilized from CPF solutions were similar to those lyophilized from HSA solutions following storage for 12 weeks over a wide range of temperatures. Only at a severely elevated temperature (e.g.

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• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Organic Chemistry (AREA)
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• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Inorganic Chemistry (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicinal Preparation (AREA)

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• 1996
  • 1996-06-06 WO PCT/EP1996/002453 patent/WO1996041817A1/en not_active Ceased
  • 1996-06-06 JP JP9502592A patent/JPH11507918A/ja active Pending
  • 1996-06-06 AU AU62228/96A patent/AU6222896A/en not_active Abandoned
  • 1996-06-06 EP EP96920798A patent/EP0837882A1/de not_active Withdrawn
  • 1996-06-07 ZA ZA9604825A patent/ZA964825B/xx unknown

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