EP2694096A1 - Melanocyte-stimulation hormone for suppressing inflammation reactions in hemodialysis - Google Patents

Melanocyte-stimulation hormone for suppressing inflammation reactions in hemodialysis

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Publication number
EP2694096A1
EP2694096A1 EP12710662.3A EP12710662A EP2694096A1 EP 2694096 A1 EP2694096 A1 EP 2694096A1 EP 12710662 A EP12710662 A EP 12710662A EP 2694096 A1 EP2694096 A1 EP 2694096A1
Authority
EP
European Patent Office
Prior art keywords
amsh
linker
conjugate
blood
solid support
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
EP12710662.3A
Other languages
German (de)
French (fr)
Inventor
Jutta PASSLICK-DEET JEN
Rainer Fislage
Anja Kuhn
Sonja Steppan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fresenius Medical Care Deutschland GmbH
Original Assignee
Fresenius Medical Care Deutschland GmbH
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
Application filed by Fresenius Medical Care Deutschland GmbH filed Critical Fresenius Medical Care Deutschland GmbH
Priority to EP12710662.3A priority Critical patent/EP2694096A1/en
Publication of EP2694096A1 publication Critical patent/EP2694096A1/en
Ceased legal-status Critical Current

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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
    • A61K38/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • A61K38/34—Melanocyte stimulating hormone [MSH], e.g. alpha- or beta-melanotropin
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6957—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a device or a kit, e.g. stents or microdevices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/14—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis
    • A61M1/16—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis with membranes
    • 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
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption

Definitions

  • the invention relates to a construct S-L-aMSH comprising a surface S of a solid support, a linker L and melanocyte-stimulation hormone aMSH, wherein aMSH is immobilized on the surface S through the linker L.
  • the invention further relates to a conjugate L-aMSH comprising such a linker L and aMSH.
  • the construct S-L-aMSH as well as the conjugate L-aMSH are useful in dialysis treatment, particularly for the prevention and/or suppression of inflammatory responses and processes.
  • Dialysis is a method of cleansing an individual's blood when the kidneys fail to function properly. Dialysis eliminates the body's extra salt, wastes and water, and assists to control blood pressure.
  • hemodialysis Two types of dialysis are usually distinguished: hemodialysis and peritoneal dialysis.
  • hemodialysis a patient is connected via tubes to an artificial kidney. Blood is pumped out of the body to the artificial kidney which filters the blood, and then the blood is returned to the body.
  • peritoneal dialysis the inside lining of the patient's peritoneal cavity is used as a natural filter. Wastes are removed by means of a fluid, the dialysate, which is washed in and out of the peritoneal cavity in cycles.
  • an artificial kidney In hemodialysis, an artificial kidney (hemodialyzer) is used to remove waste, extra chemicals and excess fluid from the blood.
  • vascular access is required. A common access is made by joining an artery to a vein under the skin and thus creating a fistula. The fistula is then penetrated, allowing access to blood.
  • an access is made by means of a catheter, which is inserted into a large vein in the neck. This type of access may be temporary, but in many instances may be used for long- term treatment.
  • the catheter creates a direct communication between a patient's aseptic internal environment and the outside world. Common complications of this communication are infections, inflammations and thrombotic or infiltrative blockage of the access.
  • Inflammation represents a major medical complication associated with dialysis.
  • the benefits of hemodialysis and peritoneal dialysis are numerous, many of the complications associated with each may be life threatening.
  • Each technique has its own or similar complications including, but not limited to, ultrafiltration failure, compliance, obesity, clearance, and poor fluid compliance.
  • Inflammation is a major factor for high mortality in patients with end stage renal disease (ESRD). It is considered that inflammation plays a primary role in arterial damage in dialysis patients. Although the precise mechanisms leading to this inflammatory state in ESRD are unclear, low-grade infection, repeated exposure to dialysis filters and auto-oxidation products are discussed as likely inciting factors in these patients (cf. C. Zocalli et al., Blood purification, 2003, 21, 29-36; and P. Stenvinkel et al., Semin. Dial., 2002, 15, 329-37).
  • aMSH (melanocyte-stimulation hormone) derives from a precursor hormone, namely propiomelanocortin (POMC).
  • POMC propiomelanocortin
  • aMSH has multiple effects on the host.
  • aMSH controls food-intake and exocrine secretions, modulates fever (cf. D. B. Richards et al., Peptides 1984, 5(4), 815-7) and inflammatory reactions and has an antimicrobial effect (cf. e.g. A. Catania et al., J Leukoc Biol. 2000, 67(2), 233-9).
  • aMSH induces Fos expression in supraoptic oxytocin neurons (N. Sabatier et al., J. Neuroscience, 2003, 23(32), 10351-8).
  • aMSH ( DP-MSH) has been described as a protease-stable aMSH analogue (cf. T. K. Sawyer et al., Proc. Natl. Acad. Sci. USA, 1980, 77(10), 5754-58).
  • US 2005/0130901 and US 2006/0122121 relate to peptides with antimicrobial activity.
  • the peptides are octomeric peptides modified from aMSH.
  • WO 2004/004551 discloses a composition and method for controlling host response to organ and/or tissue transplantation and grafting.
  • aMSH protects organ and tissue after transplantation by controlling factors within the donor, host and of the organ or tissue to be transplanted.
  • aMSH concentrations are elevated in patients on chronic hemodialysis. Concentrations of up to 30 ng/1 are observed whereas healthy patients show concentrations of up to 25 ng/1 only.
  • the concentration of aMSH is elevated particularly when simultaneously an elevated concentration of endotoxin can be observed. Possibly, aMSH release is induced by cytokines in order to limit their effect (cf. L. Airaghi et al., Nephrol Dial Transplant, 2000, 15, 1212-6).
  • WO 03/094990 relates to oligo- and polysaccharides containing the sugar structural element N-acylglucosamine or N-acylgalactosamine, in addition to the use thereof for producing haemocompatible surfaces and methods for coating surfaces with said oligo- and polysaccharides, which constitute the common biosynthetic precursor substances of heparin, heparan sulfates and chitosan.
  • FIGS 1 A and IB summarize the results of the pre-stimulation experiments.
  • FIGS. 2A and 2B summarize the results of the co-incubation experiments.
  • FIG. 3 summarizes the effect of heat treatment of KPV on the inhibition of TNF-a.
  • Figure 4 summarizes the effect of heat treatment of KPV on the inhibition of IL-6.
  • FIG. 5 summarizes the results of the inhibition of TNF-a at different concentrations of
  • aMSH may also be replaced by fractions of aMSH.
  • Fractions may be peptides comprising an amino acid sequence of at least 3 amino acids which is also comprised in aMSH.
  • a preferred peptide of this kind has or comprises the amino acid sequence KPV.
  • a first aspect of the invention relates to a construct S-L-aMSH comprising a surface S of a solid support, a linker L and melanocyte-stimulation hormone aMSH, wherein aMSH is immobilized on the surface S through the linker L for the use in dialysis treatment, preferably hemodialysis or peritoneal dialysis.
  • the construct according to the invention comprises a surface S of a solid support, a linker L and aMSH.
  • the construct S-L-aMSH consists of surface S of a solid support, linker L and aMSH, i.e., does not include further constituents.
  • the surface S of the solid support is not particularly limited, as long as it is capable for immobilization of aMSH through the linker L.
  • the surface S is adapted for being contacted with a body fluid, such as blood, serum and plasma.
  • a body fluid such as blood, serum and plasma.
  • the surface S is hemocompatible.
  • hemocompatibility is preferably defined according to ISO 10993-4 relating to tests concerning the effects of blood contacting the product or compounds on blood or blood components, directly or indirectly during routine use.
  • Hemocompatibility is preferably achieved by providing the solid support with an external coating, e.g., a hemocompatible layer.
  • the hemocompatible layer can be added directly onto the surface of a preferably non hemocompatible solid support or deposited onto other biostable and/or biodegradable layers. Additional biostable and/or biodegradable and/or hemocompatible layers can be present on top of the hemocompatible layer.
  • Hemocompatible materials are known to the person skilled in the art.
  • the surface S of the solid support is rendered hemocompatible inter alia by the presence of aMSH that is immobilized on the surface S through linker L.
  • At least one biostable layer is present under the hemocompatible layer.
  • the hemocompatible layer can be coated totally and/or partially with at least one more, above lying biostable and/or biodegradable layer.
  • biodegradable substances for the biodegradable layer(s) include polyvalero- lactones, polylactonic acid, poly-[epsilon]-decalactones, polyglycolic acid, polylactides, polyglycolides, copolymers of the polylactides and polyglycolides, poly-[epsilon]-caprolac- tone, polyhydroxybutanoic acid, polyhydroxybutyrates, polyhydroxyvalerates, polyhydroxy- butyrate-co-valerates, poly(l,4-dioxane-2,3-diones), poly(l,3-dioxane-2-one), poly-para- dioxanones, polyanhydrides such as polymaleic anhydrides, polyhydroxymethacrylates, fibrin, polycyanoacrylates, polycaprolactonedimethylacrylates, poly-b-maleic acid, polycapro- lactonebutyl-acrylates, multiblock polymers such as e.g.
  • polyether ester multiblock polymers such as e.g. PEG and poly(buty- leneterephtalates), polypivotolactones, polyglycolic acid trimethyl-carbonates, polycapro- lactone-glycolides, poly(g-ethylglutamate), poly(DTH-iminocarbonate), poly(DTE-co-DT- carbonate), poly(bisphenol-A-iminocarbonate), polyiminocarbonates, polyorthoesters, polyglycolic acid trimethyl-carbonates, polytrimethylcarbonates, poly(N-vinyl)-pyrrolidone, poly- vinylalcoholes, polyesteramides, polyphosphoesters, glycolated polyesters, polyphospha- zenes, poly[p-carboxyphenoxy)propane], polyhydroxypentanoic acid, polyanhydrides, poly- (ethyleneoxide-
  • biostable substances for the biostable layer(s) include polyacrylic acid and polyacrylates as polymethylmethacrylate, polybutylmethacrylate, polyacrylamide, polyacrylo- nitriles, polyamides, polyetheramides, polyethylenamine, polyimides, polycarbonates, poly- carbourethanes, polyvinylketones, polyvinylhalogenides, polyvinylidenhalogenides, polyvinylethers, polyisobutylenes, polyvinylaromates, polyvinylesters, polyvinylpyrollidones, polyoxymethylenes, polytetramethyleneoxide, polyethylene, polypropylene, polytetrafluoro- ethylene, polyurethanes, polyetherurethanes, silicone-polyetherurethanes, silicone-poly- urethanes, silicone-polycarbonate-urethanes, polyolefine elastomeres, polyisobutylenes, EPDM gums
  • the surface S contains at least an area with a surface concentration of ctMSH of at least 1.0 nmol/cm 2 , more preferably at least 2.0 nmol/cm 2 , still more preferably at least 5 nmol/cm 2 , yet more preferably at least 10 nmol/cm 2 , most preferably at least 25 nmol/cm and in particular at least 50 nmol/cm .
  • said area is at least 1.0 cm large.
  • the surface S is the surface of a solid support.
  • the solid support is not particularly limited.
  • the solid support is a medical device, e.g. component of an extracorporeal circuit, preferably of a system or component thereof adapted for hemodialysis, hemofiltration, plasmapheresis, apheresis, extracorporeal membrane oxygenation or assisted blood circulation.
  • the surface S of the solid support is suitable for the short- or the longterm contact with blood or blood products.
  • solid supports include medical devices such as prostheses, organs, vessels, aortas, heart valves, tubes, organ spareparts, implants, fibers, hollow fibers, stents, hollow needles, syringes, membranes, tinned goods, blood containers, titrimetric plates, pacemakers, adsorbing media, chromatography media, chromatography columns, dialyzers, connexion parts, sensors, valves, centrifugal chambers, recuperators, endoscopes, filters, and pump chambers. Dialyzers are particularly preferred, especially their components, particularly dialysis membranes.
  • the solid support may be composed of inorganic and/or organic material, metal or polymers. Suitable polymers may be thermoplastic and are known to the skilled artisan.
  • the solid support and the surface S can assume any shape, e.g. plain, convex, concave and the like.
  • the solid support is a dialysis membrane, it may be, e.g., fibrous, tissue-like, a hollow fiber, or a flat membrane.
  • Linkers L that are suitable to immobilize aMSH on a suitable surface S are known to the skilled person. In this regard it can be referred e.g. to G.T. Hermanson, Bioconjugate Techniques, Second Edition, Academic Press 2008; F. Zaragoza Dorwald, Organic Synthesis on Solid Phase: Supports, Linkers, Reactions, Wiley-VCH; 2 edition 2002; J. M. Guisan, Immobilization Of Enzymes And Cells (Methods in Biotechnology), Humana Press; 2 edition 2006; J.A. Camarero, Biopolymers. 2008, 90(3), 450-8.
  • linker L comprises at least one distal end that is faced to aMSH and at least one proximal end that is faced to the surface S.
  • the linker L may have any end-to-end distance from the distal end to the proximal end that is suitable in order to immobilize aMSH on the surface S of the solid support. Typical (average) lengths are in the range of from a few nanometers to several nanometers or more.
  • the linker L is an organic molecule. It may contain an uninterrupted carbon-carbon chain between the distal end and the proximal end. Preferably, however, in between at least some of the carbon atoms of the chain are heteroatoms which are preferably independently selected from Si, N, S and O.
  • linker L is substantially linear.
  • linker L comprises a moiety that is composed of repetition units, like in a polymer or oligomer.
  • the repetition units are -(0-CH 2 CH 2 ) x -.
  • the linker L may be derived from biomolecules, such as peptides, or synthetic, such as polyalkylene glycols, e.g., polyethylene glycols or polypropylene glycols.
  • the linker L may be composed of several segments that differ from one another in chemical nature but are covalently linked with one another.
  • a first segment of linker L may be peptidic and a second segment of linker L that is covalently bound to said first segment may be a polyalkylene glycol or a polyalkylene oxide.
  • the linker L comprises a poly(alkylene glycol) a poly(alkyleneoxide) segment or moiety.
  • the linker L does not include any a-amino acid residue, i.e., the linker is non-peptidic.
  • the overall molecular weight of the linker L is within the range of from 500 g/mol to 2,000,000 g/mol, more preferably 750 g/mol to 1,000,000 g/mol, still more preferably 1000 g/mol to 500,000 g/mol, yet more preferably 1250 g/mol to 100,000 g/mol, most preferably 1500 g/mol to 50,000 g/mol and in particular 2000 g/mol to 10,000 g/mol.
  • the surface S comprises free carboxyl groups (-COQH) which are activated by the use of EDC or other activation compounds. Subsequently, this activated surface S is preferably brought in contact with an Amino-PEG -modified aMSH to yield the corresponding covalent bond.
  • the linker L is preferably covalently bound to the surface S, e.g. via an ester bond, ether bond, amide bond, thioether bond, and the like.
  • Chemoselective immobilizations may be achieved, e.g., by amino group to aldehyde linkage, aminooxyacetyl group to glyoxylyl linkage, cysteine residue to thioester linkage, cyclopentadiene residue to benzoquinone linkage, and the like.
  • Solid supports bearing at their surfaces S reactive functional groups that are capable of reacting with a compatible functional group at the proximal end of liker L are commercially available.
  • the solid supports may bear at their surfaces S reactive functional groups selected from the group consisting of amines, thiols, aldehydes, carboxylic acids, aminooxyacetyl groups, glyoxylyl groups, thioester groups, cyclopentadiene groups, benzoquinones, trialkoxysilanes, and the like.
  • the linker L and the surface S of the solid support are linked with one another only by covalent bonds so that between aMSH and surface S there is a continuous chain of covalent bonds, which chain may be linear or branched.
  • the linker L is non-covalently bound to the surface S, e.g. by biotin- (strept)avidin interaction and the like.
  • the solid support bears at its surface S, e.g., biotin molecules that are attached to the surface S by conventional methods.
  • the proximal end of the linker L in turn bears a compatible (strept)avidine group so that a molecular complex can be formed thereby immobilizing the linker L at the surface S.
  • a compatible (strept)avidine group so that a molecular complex can be formed thereby immobilizing the linker L at the surface S.
  • Biotin-labelling can be easily achieved, e.g., by biotin- pegylation.
  • the linker L is preferably covalently bound to aMSH, e.g. via an ester bond, ether bond, amide bond, thioether bond, and the like. Such a linkage may be achieved, e.g., by amino group to aldehyde linkage, cysteine residue to thioester linkage, amide linkage and the like.
  • Linkers L bearing at their distal ends reactive functional groups that are capable of reacting with a compatible functional group at aMSH are commercially available.
  • the linkers L may bear at their distal ends reactive functional groups selected from the group consisting of amines, thiols, alcohols, aldehydes, carboxylic acids, aminooxyacetyl groups, glyoxylyl groups, thioester groups and the like.
  • linker L is non-covalently bound to aMSH, e.g. by biotin- (strept)avidin interaction and the like. Under these circumstances the linker L bears at its distal end, e.g., biotin molecules that are attached to the linker L by conventional methods.
  • the aMSH in turn bears a compatible (strept)avidine group so that a molecular complex can be formed thereby attaching aMSH to the distal end of the linker L.
  • a skilled person recognizes that the location of the biotine and the (strept)avidine at aMSH and linker L can be exchanged.
  • every linker L bears one molecule of aMSH. It is also possible however, that more than one molecule of aMSH is linked to a single linker L. This can be achieved, for example, by means of branched linkers L that bear one proximal end attached to the surface S of the solid support and more than one distal end, e.g. 2, 3 or 4 distal ends, linked to one molecule of aMSH each.
  • the construct S-L-aMSH according to the invention comprises a sub-structure -L-aMSH according to general formula (I)
  • R' is -C 1-6 -alkyl or -0-Ci -6 -alkyl
  • n is an integer of from 0 to 250, preferably 1 to 25, more preferably 2 to 10; and m is an integer of from 0 to 12, preferably 1, 2 or 3.
  • a further aspect of the invention relates to a conjugate L-aMSH comprising a linker L as defined above covalently bound to aMSH.
  • linker L comprises a polyalkylene glycol moiety, particularly preferred a polyethylene glycol moiety (peg) such that the conjugate L-aMSH preferably can be regarded as a-MSH that is pegylated by the polyethylene glycol moiety of linker L.
  • peg polyethylene glycol moiety
  • the conjugate L-aMSH according to the invention has a structure according to general formula (II)
  • K is a bond or a functional group selected from -C 1- -alkylene-, -NH-, -0-, -S-,
  • n is an integer of from 0 to 250;
  • n is an integer of from 0 to 12;
  • the conjugate L-aMSH according to the invention may be employed as an intermediate in the preparation of the construct S-L-aMSH according to the invention by attaching the conjugate L-aMSH to the surface S of the solid support be appropriate methods. Alternatively, however, the conjugate L-aMSH according to the invention can be used as medicament as such.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the conjugate L-aMSH according to the invention and a physiologically acceptable carrier.
  • the pharmaceutical composition according to the invention can be solid, pasty or liquid. It may contain the conjugate L-aMSH within the range of from 0.001 to 99.999 wt.-%, based on the total weight of the composition.
  • the pharmaceutical composition is formulated for parenteral administration.
  • Parenteral administration can be carried out, for example, subcutaneously, intravenously, intramuscularly, intraarterially, intraperitoneally, intracutaneously, intraarticularly, intrathecally, intracardially, intravitreally, retrobulbarly, intrapulmonarily and intraosseously.
  • injectable pharmaceuticals When administration is parenteral, injectable pharmaceuticals may be prepared in conventional forms, as aqueous or non-aqueous solutions or suspensions; as solid forms suitable for solution or suspension in liquid prior to injection; or as emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • injectable organic esters such as ethyl oleate.
  • suitable excipients are water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cystine hydrochloride, or the like.
  • the injectable pharmaceutical compositions may contain minor amounts of non-toxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption-enhancing preparations (e. g., liposomes) may be used.
  • the pharmaceutical composition is formulated for administration via an extracorporeal circuit, such as a dialysis circuit.
  • the pharmaceutical composition is added e.g. into the circuit at the dialyzer and administered to the subject through the extracorporeal circuit.
  • Suitable pharmaceutical compositions which are suitable for injection or infusion are known to the person skilled in the art. In this connection, for example, reference can be made in its entirety to K. H. Bauer et al., Lehrbuch der Pharmazeutica Technologie [Textbook of Pharmaceutical Technology], WVG Stuttgart 1999.
  • compositions which are suitable for injection are customarily sterile solutions, emulsions or suspensions, which are prepared by dissolving, emulsifying or suspending the active substance and optionally further excipients in water, in a suitable nonaqueous liquid which does not have to be sterile if this is justified, or in a mixture of these vehicles.
  • compositions which are suitable for infusion are customarily sterile, aqueous solutions or emulsions with water as the continuous phase.
  • compositions for injection or infusion can optionally contain further excipients.
  • Excipients of this type are preferably solubilizers, substances for isotonicization, buffers, antioxidants, chelating agents, preservatives and emulsifiers.
  • compositions include locking solutions, catheter care and wound site infection gels and dialysates.
  • a locking solution is a fluid used in a catheter or other desired patient access to the body. It contains a substance that is chosen for its ability to resist clotting, blockage and other impairments.
  • a dialysate is a fluid used for fluid exchange in peritoneal dialysis. Diuresis refers to an osmotic gradient created for the purpose of limiting reabsorption of water.
  • the dialysates preferably contain glucose, sodium chloride, calcium chloride, magnesium chloride, glucose and/or the conjugate L-aMSH in at least about the millimolar to nanomolar range such as 1 x 10 " to 1 x 10 " mol/1 and at least about 2 x 10 "3 to 2 x 10 " 11 mo/1 and concentrations in between. It is important to note that glucose concentration may vary with an individual patient's needs.
  • the conjugate L-aMSH is preferably supplied in lyophilized sterile vials and is added to the fluid through standard aseptic techniques such as re-suspension of the lyophilized peptide and with sterile water, withdrawing the mixture into an appropriate size syringe and injecting the mixture into the fluid.
  • the conjugate L-aMSH may be kept dry until the time of use. It is also contemplated that the dialysate may contain the conjugate L-aMSH in solution ready for peritoneal dialysis.
  • the peritoneal dialysate is typically administered hyperosmolar relative to plasma to create an osmotic gradient that favors net movement of water into the peritoneal cavity; this is the nature of the ability of peritoneal dialysis to accomplish cleansing.
  • the sugar glucose serves as the osmotic agent that enhances ultrafiltration. Available concentrations range from 1.5 wt.-% to 4.25 wt.-% dextrose or glucose.
  • the conjugate L-aMSH may be used as an adjunct to commercially available dialysates wherein the conjugate L-aMSH may be added to the dialysate.
  • Locking solutions are also commercially available. "Heparin locks" are the most common locking solution in catheters in general and heparin is a common component of locking solutions in dialysis.
  • a locking solution containing anti-microbial and anti-inflammatory peptides is used for both peritoneal dialysis and hemodialysis.
  • Parenteral anti-coagulants such as heparin, danaparoid and lepirudin are contemplated for use in the invention.
  • a locking solution may contain the conjugate L-P preferably in an amount in the range of millimolar to nanomolar amounts.
  • the conjugate L-aMSH can be mixed in a composition with a non-toxic, biologically compatible carrier prior to administration.
  • a non-toxic, biologically compatible carrier prior to administration.
  • this will be an aqueous solution, such as normal saline or phosphate-buffered saline (PBS), Ringer's solution, Ringer's lactate or any isotonic physiologically acceptable solution for administration by the chosen means.
  • PBS normal saline or phosphate-buffered saline
  • Ringer's solution Ringer's lactate or any isotonic physiologically acceptable solution for administration by the chosen means.
  • the solution is manufactured and packaged under current Good Manufacturing Processes (GMP's) as approved by the FDA.
  • GMP's Good Manufacturing Processes
  • the pharmaceutical composition is formulated for oral a ⁇ iministration.
  • the pharmaceutical composition is an oral administration form selected from the group consisting of tablets, powders, pellets, granules, sugar-coated tablets, syrups, juices, solutions, effervescent powders, effervescent granules, effervescent tablets, lyophilizates and capsules.
  • Suitable adininistration forms which are suitable for oral administration are known to the person skilled in the art.
  • Suitable excipients for the formulation of oral administration forms are known to the person skilled in the art.
  • a further aspect of the invention relates to a process for the manufacture of the construct S-L- aMSH according to the invention comprising the step of linking aMSH as defined above to a surface S of a solid support as defined above through a linker L as defined above.
  • the process according to the invention comprises the step of linking a conjugate L-aMSH as defined above to a surface S of a solid support as defined above.
  • the proximal end of linker L bears a functional group that is adapted for covalent or non-covalent immobilization of linker L at surface S.
  • surface S typically bears a functional group that is compatible with the functional group at the proximal end of linker L so that a stable linkage can be achieved, optionally after chemical activation of one or both of the functional groups.
  • S-L-aMSH and the conjugate L-aMSH according to the invention are useful for dialysis such as hemodialysis and peritoneal dialysis, hemofiltration, plasmapheresis, apheresis, extracorporeal membrane oxygenation or assisted blood circulation.
  • they are used to prevent and/or suppress inflammation, preferably in patients in the course of dialysis, hemofiltration, plasmapheresis, apheresis, extracorporeal membrane oxygenation or assisted blood circulation, preferably in patients suffering from ESRD.
  • a further aspect of the invention relates to a conjugate L-aMSH as described above for the use in
  • ERD end stage renal disease
  • PBMCs peripheral blood mononuclear cells
  • PB Cs from healthy male volunteers were isolated and stimulated as indicated below to mimic an uremic and inflammatory setting comparable to patients (LPS pre-incubation for 3 hours)
  • a first goal of this experiment was to test whether the three amino acid antiinflammatory peptide KPV (unpegylated and pegylated version "monopeptide”) will retain its activity under conditions of steam sterilization at 125°C for 15 min used in the production process of dialysis modules.
  • a second goal was to study the anti-inflammatory effect of aMSH and the peptides in uremic patients, which represent the real clinical situation.
  • TNF-a and IL-6 ELISA showed that the heat treatment did not influence the activity of the peptide.
  • the average inhibition for the tested concentrations of KPV for TNF-a was around 30% and for and IL-6 around 20%.

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Abstract

The invention relates to a construct S-L-aMSH comprising a surface S of a solid support, a linker L and melanocyte-stimulation hormone aMSH, wherein aMSH is immobilized on the surface S through the linker L for the use in dialysis treatment, preferably hemodialysis treatment.

Description

MELANOCYTE-STIMULATION HORMONE FOR SUPPRESSING INFLAMMATION REACTIONS IN HEMODIALYSIS
Technical Field
The invention relates to a construct S-L-aMSH comprising a surface S of a solid support, a linker L and melanocyte-stimulation hormone aMSH, wherein aMSH is immobilized on the surface S through the linker L. The invention further relates to a conjugate L-aMSH comprising such a linker L and aMSH. Further, the construct S-L-aMSH as well as the conjugate L-aMSH are useful in dialysis treatment, particularly for the prevention and/or suppression of inflammatory responses and processes.
Background of the Invention
Dialysis is a method of cleansing an individual's blood when the kidneys fail to function properly. Dialysis eliminates the body's extra salt, wastes and water, and assists to control blood pressure.
Two types of dialysis are usually distinguished: hemodialysis and peritoneal dialysis. In hemodialysis, a patient is connected via tubes to an artificial kidney. Blood is pumped out of the body to the artificial kidney which filters the blood, and then the blood is returned to the body. In peritoneal dialysis, the inside lining of the patient's peritoneal cavity is used as a natural filter. Wastes are removed by means of a fluid, the dialysate, which is washed in and out of the peritoneal cavity in cycles.
In hemodialysis, an artificial kidney (hemodialyzer) is used to remove waste, extra chemicals and excess fluid from the blood. To transfer a dialysis patient's blood into the artificial kidney, vascular access is required. A common access is made by joining an artery to a vein under the skin and thus creating a fistula. The fistula is then penetrated, allowing access to blood. Occasionally, an access is made by means of a catheter, which is inserted into a large vein in the neck. This type of access may be temporary, but in many instances may be used for long- term treatment. As with any vascular access or catheter treatment, the catheter creates a direct communication between a patient's aseptic internal environment and the outside world. Common complications of this communication are infections, inflammations and thrombotic or infiltrative blockage of the access.
Inflammation represents a major medical complication associated with dialysis. In fact, although the benefits of hemodialysis and peritoneal dialysis are numerous, many of the complications associated with each may be life threatening. Each technique has its own or similar complications including, but not limited to, ultrafiltration failure, compliance, obesity, clearance, and poor fluid compliance.
Inflammation is a major factor for high mortality in patients with end stage renal disease (ESRD). It is considered that inflammation plays a primary role in arterial damage in dialysis patients. Although the precise mechanisms leading to this inflammatory state in ESRD are unclear, low-grade infection, repeated exposure to dialysis filters and auto-oxidation products are discussed as likely inciting factors in these patients (cf. C. Zocalli et al., Blood purification, 2003, 21, 29-36; and P. Stenvinkel et al., Semin. Dial., 2002, 15, 329-37).
There is a demand for methods that cause less inflammation in hemodialysis patients. aMSH (melanocyte-stimulation hormone) derives from a precursor hormone, namely propiomelanocortin (POMC). The post-translational processing of POMC yields in peptide hormones such as ACTH, aMSH, gMSH and b-endorphin. aMSH has multiple effects on the host.
The stimulatory effect of aMSH in pigment cells has been known for over 50 years. More recent findings indicate that aMSH controls food-intake and exocrine secretions, modulates fever (cf. D. B. Richards et al., Peptides 1984, 5(4), 815-7) and inflammatory reactions and has an antimicrobial effect (cf. e.g. A. Catania et al., J Leukoc Biol. 2000, 67(2), 233-9). aMSH induces Fos expression in supraoptic oxytocin neurons (N. Sabatier et al., J. Neuroscience, 2003, 23(32), 10351-8).
[NIe4-d-Phe7] aMSH ( DP-MSH) has been described as a protease-stable aMSH analogue (cf. T. K. Sawyer et al., Proc. Natl. Acad. Sci. USA, 1980, 77(10), 5754-58). US 2005/0130901 and US 2006/0122121 relate to peptides with antimicrobial activity. The peptides are octomeric peptides modified from aMSH.
WO 2004/004551 discloses a composition and method for controlling host response to organ and/or tissue transplantation and grafting. aMSH protects organ and tissue after transplantation by controlling factors within the donor, host and of the organ or tissue to be transplanted.
It is known that aMSH concentrations are elevated in patients on chronic hemodialysis. Concentrations of up to 30 ng/1 are observed whereas healthy patients show concentrations of up to 25 ng/1 only. The concentration of aMSH is elevated particularly when simultaneously an elevated concentration of endotoxin can be observed. Possibly, aMSH release is induced by cytokines in order to limit their effect (cf. L. Airaghi et al., Nephrol Dial Transplant, 2000, 15, 1212-6).
WO 03/094990 relates to oligo- and polysaccharides containing the sugar structural element N-acylglucosamine or N-acylgalactosamine, in addition to the use thereof for producing haemocompatible surfaces and methods for coating surfaces with said oligo- and polysaccharides, which constitute the common biosynthetic precursor substances of heparin, heparan sulfates and chitosan.
These compositions, however, are not satisfactory in every respect and there is a demand for further improvements of hemodialysis.
Brief Description of the Figures
Figures 1 A and IB summarize the results of the pre-stimulation experiments.
Figures 2A and 2B summarize the results of the co-incubation experiments.
Figure 3 summarizes the effect of heat treatment of KPV on the inhibition of TNF-a.
Figure 4 summarizes the effect of heat treatment of KPV on the inhibition of IL-6.
Figure 5 summarizes the results of the inhibition of TNF-a at different concentrations of
KPV. Detailed Description of the Invention
It is an object of the invention to provide improvements for hemodialysis and other methods where body fluids are guided through extracorporeal circuits, particularly with respect to the occurrence of inflammation.
This object is achieved by the subject-matter of the patent claims.
It has been surprisingly found that surfaces that are modified with aMSH are particularly useful in hemodialysis with respect to the prevention and/or suppression of inflammation.
For the purpose of this invention aMSH may also be replaced by fractions of aMSH. Fractions may be peptides comprising an amino acid sequence of at least 3 amino acids which is also comprised in aMSH. A preferred peptide of this kind has or comprises the amino acid sequence KPV.
A first aspect of the invention relates to a construct S-L-aMSH comprising a surface S of a solid support, a linker L and melanocyte-stimulation hormone aMSH, wherein aMSH is immobilized on the surface S through the linker L for the use in dialysis treatment, preferably hemodialysis or peritoneal dialysis.
The construct according to the invention comprises a surface S of a solid support, a linker L and aMSH. Preferably, the construct S-L-aMSH consists of surface S of a solid support, linker L and aMSH, i.e., does not include further constituents.
The surface S of the solid support is not particularly limited, as long as it is capable for immobilization of aMSH through the linker L.
Preferably, the surface S is adapted for being contacted with a body fluid, such as blood, serum and plasma.
In a particularly preferred embodiment, the surface S is hemocompatible. For the purpose of the specification hemocompatibility is preferably defined according to ISO 10993-4 relating to tests concerning the effects of blood contacting the product or compounds on blood or blood components, directly or indirectly during routine use.
Hemocompatibility is preferably achieved by providing the solid support with an external coating, e.g., a hemocompatible layer. The hemocompatible layer can be added directly onto the surface of a preferably non hemocompatible solid support or deposited onto other biostable and/or biodegradable layers. Additional biostable and/or biodegradable and/or hemocompatible layers can be present on top of the hemocompatible layer.
Hemocompatible materials are known to the person skilled in the art. Preferably, the surface S of the solid support is rendered hemocompatible inter alia by the presence of aMSH that is immobilized on the surface S through linker L.
It is preferred that at least one biostable layer is present under the hemocompatible layer. In addition the hemocompatible layer can be coated totally and/or partially with at least one more, above lying biostable and/or biodegradable layer.
Examples of biodegradable substances for the biodegradable layer(s) include polyvalero- lactones, polylactonic acid, poly-[epsilon]-decalactones, polyglycolic acid, polylactides, polyglycolides, copolymers of the polylactides and polyglycolides, poly-[epsilon]-caprolac- tone, polyhydroxybutanoic acid, polyhydroxybutyrates, polyhydroxyvalerates, polyhydroxy- butyrate-co-valerates, poly(l,4-dioxane-2,3-diones), poly(l,3-dioxane-2-one), poly-para- dioxanones, polyanhydrides such as polymaleic anhydrides, polyhydroxymethacrylates, fibrin, polycyanoacrylates, polycaprolactonedimethylacrylates, poly-b-maleic acid, polycapro- lactonebutyl-acrylates, multiblock polymers such as e.g. from oligocaprolactonedioles and oligodioxanonedioles, polyether ester multiblock polymers such as e.g. PEG and poly(buty- leneterephtalates), polypivotolactones, polyglycolic acid trimethyl-carbonates, polycapro- lactone-glycolides, poly(g-ethylglutamate), poly(DTH-iminocarbonate), poly(DTE-co-DT- carbonate), poly(bisphenol-A-iminocarbonate), polyiminocarbonates, polyorthoesters, polyglycolic acid trimethyl-carbonates, polytrimethylcarbonates, poly(N-vinyl)-pyrrolidone, poly- vinylalcoholes, polyesteramides, polyphosphoesters, glycolated polyesters, polyphospha- zenes, poly[p-carboxyphenoxy)propane], polyhydroxypentanoic acid, polyanhydrides, poly- (ethyleneoxide-propyleneoxide), soft polyurethanes, polyurethanes with amino acid residues in the backbone, polyether esters such as polyethyleneoxide, polyalkeneoxalates, polyortho- esters as well as their copolymers, carrageenans, lipides, fibrinogen, starch, collagen, protein based polymers, polyamino acids, synthetic polyamino acids, zein, modified zein, poly- hydroxyalkanoates, actinic acid, pectic acid, modified and non modified fibrin and casein, carboxymethylsulphate, albumin, moreover hyaluronic acid, chitosan and its derivatives, heparansulphates and its derivatives, heparins, chondroitinsulphate, dextran, b-cyclodextrins, copolymers with PEG and polypropyleneglycol, gummi arabicum, guar, gelatine, collagen, collagen-N-hydroxysuccinimide, lipids, phospholipids, modifications and copolymers and/or mixtures of the afore mentioned substances.
Examples of biostable substances for the biostable layer(s) include polyacrylic acid and polyacrylates as polymethylmethacrylate, polybutylmethacrylate, polyacrylamide, polyacrylo- nitriles, polyamides, polyetheramides, polyethylenamine, polyimides, polycarbonates, poly- carbourethanes, polyvinylketones, polyvinylhalogenides, polyvinylidenhalogenides, polyvinylethers, polyisobutylenes, polyvinylaromates, polyvinylesters, polyvinylpyrollidones, polyoxymethylenes, polytetramethyleneoxide, polyethylene, polypropylene, polytetrafluoro- ethylene, polyurethanes, polyetherurethanes, silicone-polyetherurethanes, silicone-poly- urethanes, silicone-polycarbonate-urethanes, polyolefine elastomeres, polyisobutylenes, EPDM gums, fluorosilicones, carboxymethylchitosanes, polyaryletheretherketones, poly- etheretherketones, polyethylenterephthalate, polyvalerates, carboxymethylcellulose, cellulose, rayon, rayontriacetates, cellulosenitrates, celluloseacetates, hydroxyethylcellulose, cellulosebutyrates, celluloseacetatebutyrates, ethylvinylacetate copolymers, polysulphones, epoxy resins, ABS resins, EPDM gums, silicones as polysiloxanes, polydimethylsiloxanes, polyvinylhalogenes and copolymers, celluloseethers, cellulosetriacetates, chitosanes and copolymers and/or mixtures of these substances.
In a preferred embodiment, the surface S contains at least an area with a surface concentration of ctMSH of at least 1.0 nmol/cm2, more preferably at least 2.0 nmol/cm2, still more preferably at least 5 nmol/cm2, yet more preferably at least 10 nmol/cm2, most preferably at least 25 nmol/cm and in particular at least 50 nmol/cm . Preferably, said area is at least 1.0 cm large.
The surface S is the surface of a solid support. The solid support is not particularly limited. Preferably, the solid support is a medical device, e.g. component of an extracorporeal circuit, preferably of a system or component thereof adapted for hemodialysis, hemofiltration, plasmapheresis, apheresis, extracorporeal membrane oxygenation or assisted blood circulation.
In a preferred embodiment the surface S of the solid support is suitable for the short- or the longterm contact with blood or blood products. Examples of solid supports include medical devices such as prostheses, organs, vessels, aortas, heart valves, tubes, organ spareparts, implants, fibers, hollow fibers, stents, hollow needles, syringes, membranes, tinned goods, blood containers, titrimetric plates, pacemakers, adsorbing media, chromatography media, chromatography columns, dialyzers, connexion parts, sensors, valves, centrifugal chambers, recuperators, endoscopes, filters, and pump chambers. Dialyzers are particularly preferred, especially their components, particularly dialysis membranes.
The solid support may be composed of inorganic and/or organic material, metal or polymers. Suitable polymers may be thermoplastic and are known to the skilled artisan.
The solid support and the surface S, respectively, can assume any shape, e.g. plain, convex, concave and the like. When the solid support is a dialysis membrane, it may be, e.g., fibrous, tissue-like, a hollow fiber, or a flat membrane.
Linkers L that are suitable to immobilize aMSH on a suitable surface S are known to the skilled person. In this regard it can be referred e.g. to G.T. Hermanson, Bioconjugate Techniques, Second Edition, Academic Press 2008; F. Zaragoza Dorwald, Organic Synthesis on Solid Phase: Supports, Linkers, Reactions, Wiley-VCH; 2 edition 2002; J. M. Guisan, Immobilization Of Enzymes And Cells (Methods in Biotechnology), Humana Press; 2 edition 2006; J.A. Camarero, Biopolymers. 2008, 90(3), 450-8.
The chemical nature of the linker L is not particularly limited. Typical linkers L comprise at least one distal end that is faced to aMSH and at least one proximal end that is faced to the surface S.
The linker L may have any end-to-end distance from the distal end to the proximal end that is suitable in order to immobilize aMSH on the surface S of the solid support. Typical (average) lengths are in the range of from a few nanometers to several nanometers or more. Preferably, the linker L is an organic molecule. It may contain an uninterrupted carbon-carbon chain between the distal end and the proximal end. Preferably, however, in between at least some of the carbon atoms of the chain are heteroatoms which are preferably independently selected from Si, N, S and O.
Preferably, linker L is substantially linear. Preferably, linker L comprises a moiety that is composed of repetition units, like in a polymer or oligomer. For example, when linker L comprises a polyethylene glycol moiety, the repetition units are -(0-CH2CH2)x-.
The linker L may be derived from biomolecules, such as peptides, or synthetic, such as polyalkylene glycols, e.g., polyethylene glycols or polypropylene glycols.
The linker L may be composed of several segments that differ from one another in chemical nature but are covalently linked with one another. For example, a first segment of linker L may be peptidic and a second segment of linker L that is covalently bound to said first segment may be a polyalkylene glycol or a polyalkylene oxide.
Preferably, the linker L comprises a poly(alkylene glycol) a poly(alkyleneoxide) segment or moiety.
In a preferred embodiment the linker L does not include any a-amino acid residue, i.e., the linker is non-peptidic.
In a preferred embodiment, the overall molecular weight of the linker L is within the range of from 500 g/mol to 2,000,000 g/mol, more preferably 750 g/mol to 1,000,000 g/mol, still more preferably 1000 g/mol to 500,000 g/mol, yet more preferably 1250 g/mol to 100,000 g/mol, most preferably 1500 g/mol to 50,000 g/mol and in particular 2000 g/mol to 10,000 g/mol.
In a preferred embodiment, the surface S comprises free carboxyl groups (-COQH) which are activated by the use of EDC or other activation compounds. Subsequently, this activated surface S is preferably brought in contact with an Amino-PEG -modified aMSH to yield the corresponding covalent bond. The linker L is preferably covalently bound to the surface S, e.g. via an ester bond, ether bond, amide bond, thioether bond, and the like. Chemoselective immobilizations may be achieved, e.g., by amino group to aldehyde linkage, aminooxyacetyl group to glyoxylyl linkage, cysteine residue to thioester linkage, cyclopentadiene residue to benzoquinone linkage, and the like.
Solid supports bearing at their surfaces S reactive functional groups that are capable of reacting with a compatible functional group at the proximal end of liker L are commercially available. For example, the solid supports may bear at their surfaces S reactive functional groups selected from the group consisting of amines, thiols, aldehydes, carboxylic acids, aminooxyacetyl groups, glyoxylyl groups, thioester groups, cyclopentadiene groups, benzoquinones, trialkoxysilanes, and the like.
Methods for introducing said reactive functional groups to the surfaces S of solid supports are also known to the person skilled in the art.
Preferably, the linker L and the surface S of the solid support are linked with one another only by covalent bonds so that between aMSH and surface S there is a continuous chain of covalent bonds, which chain may be linear or branched.
It is also possible that the linker L is non-covalently bound to the surface S, e.g. by biotin- (strept)avidin interaction and the like. Under these circumstances the solid support bears at its surface S, e.g., biotin molecules that are attached to the surface S by conventional methods. The proximal end of the linker L in turn bears a compatible (strept)avidine group so that a molecular complex can be formed thereby immobilizing the linker L at the surface S. A skilled person recognizes that the location of the biotine and the (strept)avidine at surface S and linker L can be exchanged. Biotin-labelling can be easily achieved, e.g., by biotin- pegylation.
The linker L is preferably covalently bound to aMSH, e.g. via an ester bond, ether bond, amide bond, thioether bond, and the like. Such a linkage may be achieved, e.g., by amino group to aldehyde linkage, cysteine residue to thioester linkage, amide linkage and the like. Linkers L bearing at their distal ends reactive functional groups that are capable of reacting with a compatible functional group at aMSH are commercially available. For example, the linkers L may bear at their distal ends reactive functional groups selected from the group consisting of amines, thiols, alcohols, aldehydes, carboxylic acids, aminooxyacetyl groups, glyoxylyl groups, thioester groups and the like.
It is also possible that the linker L is non-covalently bound to aMSH, e.g. by biotin- (strept)avidin interaction and the like. Under these circumstances the linker L bears at its distal end, e.g., biotin molecules that are attached to the linker L by conventional methods. The aMSH in turn bears a compatible (strept)avidine group so that a molecular complex can be formed thereby attaching aMSH to the distal end of the linker L. A skilled person recognizes that the location of the biotine and the (strept)avidine at aMSH and linker L can be exchanged.
Preferably, every linker L bears one molecule of aMSH. It is also possible however, that more than one molecule of aMSH is linked to a single linker L. This can be achieved, for example, by means of branched linkers L that bear one proximal end attached to the surface S of the solid support and more than one distal end, e.g. 2, 3 or 4 distal ends, linked to one molecule of aMSH each.
In a preferred embodiment, the construct S-L-aMSH according to the invention comprises a sub-structure -L-aMSH according to general formula (I)
-K-[CH2-CH2-0]n-[CH2]m-Q-aMSH (I) wherein
Q is a bond or a functional group selected from -0-, -NH-, -C(=0)-, -C(=S)-, - C(=NH)-, -NH-C(=0)-, -NH-C(=S)- and -NH-C(=NH)-;
K is a bond or a functional group selected from -NH-, -0-, -S-, -C(=0)-, -C(=S)-, -C(=NH)-, -NH-C(=0)-, -NH-C(=S)-, -NH-C(=NH)-, -NH-C(=0)-0-, -NH-C(=0)- NH-,
-0-C(=0)-0- or -Si(R')2-, wherein R' is -C1-6-alkyl or -0-Ci-6-alkyl;
n is an integer of from 0 to 250, preferably 1 to 25, more preferably 2 to 10; and m is an integer of from 0 to 12, preferably 1, 2 or 3. A further aspect of the invention relates to a conjugate L-aMSH comprising a linker L as defined above covalently bound to aMSH.
Preferably, linker L comprises a polyalkylene glycol moiety, particularly preferred a polyethylene glycol moiety (peg) such that the conjugate L-aMSH preferably can be regarded as a-MSH that is pegylated by the polyethylene glycol moiety of linker L.
In a preferred embodiment, the conjugate L-aMSH according to the invention has a structure according to general formula (II)
H-K-[CH2-CH2-0]n-[CH2]m-Q-a-MSH (II) wherein
Q is a bond or a functional group selected from -0-, -NH-, -C(=0)-, -C(=S)-, - C(=NH , -NH-C(=0)-, -NH-C(=S)- and -NH-C(=NH)-;
K is a bond or a functional group selected from -C1- -alkylene-, -NH-, -0-, -S-,
-C(=0)-, or -C(=0)0-;
n is an integer of from 0 to 250; and
m is an integer of from 0 to 12;
For the purpose of the specification, bivalent functional groups that have two possibilities of being incorporated in a general formula in two different directions, e.g. -NH-C(:=0)-, can be incorporated in both directions alternatively.
In a particularly preferred embodiment Q is -C(=0)-, m is 2, n is 3 and K is -NH-, so that the conjugate L-P has the following structure (III)
H2N-CH2-CH2-0-CH2-CH2-0-CH2-CH2-0-CH2-CH2-C(=0)-aMSH (III).
The conjugate L-aMSH according to the invention may be employed as an intermediate in the preparation of the construct S-L-aMSH according to the invention by attaching the conjugate L-aMSH to the surface S of the solid support be appropriate methods. Alternatively, however, the conjugate L-aMSH according to the invention can be used as medicament as such.
Therefore, a further aspect of the invention relates to a pharmaceutical composition comprising the conjugate L-aMSH according to the invention and a physiologically acceptable carrier.
The pharmaceutical composition according to the invention can be solid, pasty or liquid. It may contain the conjugate L-aMSH within the range of from 0.001 to 99.999 wt.-%, based on the total weight of the composition.
In a preferred embodiment, the pharmaceutical composition is formulated for parenteral administration. Parenteral administration can be carried out, for example, subcutaneously, intravenously, intramuscularly, intraarterially, intraperitoneally, intracutaneously, intraarticularly, intrathecally, intracardially, intravitreally, retrobulbarly, intrapulmonarily and intraosseously.
When administration is parenteral, injectable pharmaceuticals may be prepared in conventional forms, as aqueous or non-aqueous solutions or suspensions; as solid forms suitable for solution or suspension in liquid prior to injection; or as emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Other suitable excipients are water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cystine hydrochloride, or the like. In addition, the injectable pharmaceutical compositions may contain minor amounts of non-toxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption-enhancing preparations (e. g., liposomes) may be used.
In another preferred embodiment, the pharmaceutical composition is formulated for administration via an extracorporeal circuit, such as a dialysis circuit. In this embodiment, the pharmaceutical composition is added e.g. into the circuit at the dialyzer and administered to the subject through the extracorporeal circuit. Suitable pharmaceutical compositions which are suitable for injection or infusion are known to the person skilled in the art. In this connection, for example, reference can be made in its entirety to K. H. Bauer et al., Lehrbuch der Pharmazeutischen Technologie [Textbook of Pharmaceutical Technology], WVG Stuttgart 1999. Pharmaceutical compositions which are suitable for injection are customarily sterile solutions, emulsions or suspensions, which are prepared by dissolving, emulsifying or suspending the active substance and optionally further excipients in water, in a suitable nonaqueous liquid which does not have to be sterile if this is justified, or in a mixture of these vehicles.
Pharmaceutical compositions which are suitable for infusion are customarily sterile, aqueous solutions or emulsions with water as the continuous phase.
Pharmaceutical compositions for injection or infusion can optionally contain further excipients. Excipients of this type are preferably solubilizers, substances for isotonicization, buffers, antioxidants, chelating agents, preservatives and emulsifiers.
Particularly preferred pharmaceutical compositions include locking solutions, catheter care and wound site infection gels and dialysates. A locking solution is a fluid used in a catheter or other desired patient access to the body. It contains a substance that is chosen for its ability to resist clotting, blockage and other impairments. A dialysate is a fluid used for fluid exchange in peritoneal dialysis. Diuresis refers to an osmotic gradient created for the purpose of limiting reabsorption of water.
In different embodiments of the invention, the dialysates preferably contain glucose, sodium chloride, calcium chloride, magnesium chloride, glucose and/or the conjugate L-aMSH in at least about the millimolar to nanomolar range such as 1 x 10" to 1 x 10" mol/1 and at least about 2 x 10"3 to 2 x 10" 11 mo/1 and concentrations in between. It is important to note that glucose concentration may vary with an individual patient's needs. The conjugate L-aMSH is preferably supplied in lyophilized sterile vials and is added to the fluid through standard aseptic techniques such as re-suspension of the lyophilized peptide and with sterile water, withdrawing the mixture into an appropriate size syringe and injecting the mixture into the fluid. For purposes of preservation, the conjugate L-aMSH may be kept dry until the time of use. It is also contemplated that the dialysate may contain the conjugate L-aMSH in solution ready for peritoneal dialysis.
To meet the ultrafiltration requirements of patients on peritoneal dialysis, the peritoneal dialysate is typically administered hyperosmolar relative to plasma to create an osmotic gradient that favors net movement of water into the peritoneal cavity; this is the nature of the ability of peritoneal dialysis to accomplish cleansing. In commercially available peritoneal dialysates, the sugar glucose serves as the osmotic agent that enhances ultrafiltration. Available concentrations range from 1.5 wt.-% to 4.25 wt.-% dextrose or glucose.
In one embodiment of the invention, the conjugate L-aMSH may be used as an adjunct to commercially available dialysates wherein the conjugate L-aMSH may be added to the dialysate.
Locking solutions are also commercially available. "Heparin locks" are the most common locking solution in catheters in general and heparin is a common component of locking solutions in dialysis. In another embodiment of the invention, a locking solution containing anti-microbial and anti-inflammatory peptides is used for both peritoneal dialysis and hemodialysis. Parenteral anti-coagulants such as heparin, danaparoid and lepirudin are contemplated for use in the invention. A locking solution may contain the conjugate L-P preferably in an amount in the range of millimolar to nanomolar amounts.
For catheter care, the conjugate L-aMSH can be mixed in a composition with a non-toxic, biologically compatible carrier prior to administration. Usually, this will be an aqueous solution, such as normal saline or phosphate-buffered saline (PBS), Ringer's solution, Ringer's lactate or any isotonic physiologically acceptable solution for administration by the chosen means. Preferably, the solution is manufactured and packaged under current Good Manufacturing Processes (GMP's) as approved by the FDA.
In another preferred embodiment, the pharmaceutical composition is formulated for oral a<iministration. Preferably, the pharmaceutical composition is an oral administration form selected from the group consisting of tablets, powders, pellets, granules, sugar-coated tablets, syrups, juices, solutions, effervescent powders, effervescent granules, effervescent tablets, lyophilizates and capsules.
Suitable adininistration forms which are suitable for oral administration (oral medicaments) are known to the person skilled in the art. In this connection, reference can be made in its entirety, for example, to K. H. Bauer et al., Lehrbuch der Pharmazeutischen Technologie [Textbook of Pharmaceutical Technology], WVG Stuttgart 1999. Suitable excipients for the formulation of oral administration forms are known to the person skilled in the art. In this connection, reference can be made, for example, to H. P. Fiedler, Lexikon der Hilfstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete [Encyclopedia of excipients for pharmacy, cosmetics and related areas], Editio Cantor Aulendorf, 2001.
A further aspect of the invention relates to a process for the manufacture of the construct S-L- aMSH according to the invention comprising the step of linking aMSH as defined above to a surface S of a solid support as defined above through a linker L as defined above.
In a preferred embodiment, the process according to the invention comprises the step of linking a conjugate L-aMSH as defined above to a surface S of a solid support as defined above.
In the process according to the invention the proximal end of linker L bears a functional group that is adapted for covalent or non-covalent immobilization of linker L at surface S. For that purpose, surface S typically bears a functional group that is compatible with the functional group at the proximal end of linker L so that a stable linkage can be achieved, optionally after chemical activation of one or both of the functional groups.
Suitable functional groups and activation methods are known to the skilled person and have been described above in connection with linker L and surface S, respectively.
The construct S-L-aMSH and the conjugate L-aMSH according to the invention are useful for dialysis such as hemodialysis and peritoneal dialysis, hemofiltration, plasmapheresis, apheresis, extracorporeal membrane oxygenation or assisted blood circulation. In a preferred embodiment, they are used to prevent and/or suppress inflammation, preferably in patients in the course of dialysis, hemofiltration, plasmapheresis, apheresis, extracorporeal membrane oxygenation or assisted blood circulation, preferably in patients suffering from ESRD.
A further aspect of the invention relates to a conjugate L-aMSH as described above for the use in
- preventing and/or suppressing inflammation in dialysis patients;
- preventing and/or treating end stage renal disease (ESRD);
- preventing and/or treating autoimmune disease;
- preventing and/or treating sepsis;
- preventing and/or treating fever in dialysis patients; and/or
- preventing and/or treating complications after transplantation.
Examples
The following examples further illustrate the invention but are not to be construed as limiting its scope.
Example 1 :
Ex vivo stimulation of human peripheral blood mononuclear cells (PBMCs)
PB Cs from healthy male volunteers (n=5) were isolated and stimulated as indicated below to mimic an uremic and inflammatory setting comparable to patients (LPS pre-incubation for 3 hours)
Material and Methods
For the stimulation of PBMCs the following concentrations were chosen:
• LPS 1 ng/ml alone
• (xMSH
o ΙμΜ; ΙΟμΜ; ΙΟΟμΜ
• Peg-KPV (pegylated tripeptide)
o ΙμΜ; ΙΟμΜ; ΙΟΟμΜ; ΙΟΟΟμΜ
The stimulation procedure was as described below:
All cells were incubated for 24h and centrifuged at 1700 rpm and supematants were used for TNF-a ELISA measurement according to manufacturer's protocols. Results
The results of the pre-stimulation are summarized in the following table and are additionally depicted in Figures 1 A and IB:
The results of the co-incubation are summarized in the following table and are additionally depicted in Figures 2 A and 2B:
The data indicate an inhibitory effect for KPV and peg-KPV (provided by Invitrogen Life Technolgies) as well as aMSH for LPS-induced TNF-a release ("inflammation") when the inflammatory process has already started (pre-stimulation) or if the peptides and inflammatory stimulus is given simultaneously (co-incubation). The pre-incubation of LPS was used as a model to mimic an uremic situation in patients, in which inflammatory processes are ongoing already. Example 2:
Whole blood studies with blood from uremic patients including heat sterilization experiments
A first goal of this experiment was to test whether the three amino acid antiinflammatory peptide KPV (unpegylated and pegylated version "monopeptide") will retain its activity under conditions of steam sterilization at 125°C for 15 min used in the production process of dialysis modules. A second goal was to study the anti-inflammatory effect of aMSH and the peptides in uremic patients, which represent the real clinical situation.
Since peptides tend to lose activity when heated the experiment was designed to mimic the dialysis production process with the known parameters.
To perform this experiment whole blood samples were taken from voluntary uremic patients (n=10) and subsequently stimulated ex vivo with lipopoly saccharide (LPS) with or without aMSH, KPV or pegylated KPV (non heated and heated) and TNF-a and IL-6 release was determined by ELISA.
Material and Methods
25 ml blood from 10 uremic patients (dialysis patients treated at the hospital in Konstanz, Germany) was drawn and blood values were analyzed immediately. Blood was 1 :10 diluted with PBS buffer and each stimulation condition was performed in triplicates and incubated at 200 μΐ in 96 well plates.
The following conditions were chosen:
• Unstimulated blood
• Blood stimulated with LPS 1 ng/ml alone (maximal stimulation of proinflammatory cytokine TNF-a.
• Blood stimulated with LPS 1 ng/ml and 4 different concentrations of aMSH
o 0.1 μΜ; ΙμΜ; 10 μΜ; ΙΟΟμΜ
• Blood stimulated with LPS 1 ng/ml and 5 different concentrations of
o KPV (0.1 μΜ; 1 μΜ; ΙΟρΜ; ΙΟΟμΜ; 1000μΜ)
o KPV-peg (0.1 μΜ; ΙμΜ; 10μΜ; ΙΟΟμΜ; ΙΟΟΟμΜ)
• Blood stimulated with LPS 1 ng/ml and 5 different concentrations of
o KPV heated(0.^M; ΙμΜ; 10μΜ; ΙΟΟμΜ; 1000μΜ)
ο KPV-peg heated (0.1 μΜ; ΙμΜ; 10μΜ; ΙΟΟμΜ; ΙΟΟΟμΜ)
Blood was incubated for 24 h, centrifiiged at 1700 rpm and serum supematants were used for TNF-a and IL-6 ELISA measurement according to manufacturer's protocols.
Results
TNF-a and IL-6 ELISA showed that the heat treatment did not influence the activity of the peptide. The average inhibition for the tested concentrations of KPV for TNF-a was around 30% and for and IL-6 around 20%.
The results are further depicted in Figures 3, 4 and 5.

Claims

Claims
1. A construct S-L-aMSH comprising a surface S of a solid support, a linker L and melanocyte-stimulation hormone aMSH, wherein aMSH is immobilized on the surface S through the linker L for the use in dialysis treatment, preferably hemodialysis treatment.
2. The construct according to claim 1, wherein the linker L does not include any a-amino acid residue.
3. The construct according to claim 1 or 2, wherein the linker L comprises a poly(alkylene- oxide) moiety.
4. The construct according to any of the preceding claims, wherein the surface S is adapted for being contacted with a body fluid.
5. The construct according to any of the preceding claims, wherein the solid support is a component of an extracorporeal circuit.
6. A conjugate L-aMSH according to the general formula
H-K-[CH2-CH2-0]n-[CH2]m-Q-aMSH
wherein
Q is a bond or a functional group selected from -0-, -NH-, -C(=0)-, -C(=S)-, - C(=NH)-, -NH-C(=0)-, -NH-C(=S)- and -NH-C(=NH)-;
K is a bond or a functional group selected from -Ci-8-alkylene-, -NH-, -0-, -S-,
-C(=0)-, or -C(=0)0-;
n is an integer of from 0 to 250; and
m is an integer of from 0 to 12;
7. The conjugate L-aMSH as defined in claim 6 for the use in dialysis treatment, preferably hemodialysis treatment.
8. The conjugate L-aMSH according to claim 7, wherein the patients suffer from end stage renal disease (ESRD).
EP12710662.3A 2011-04-07 2012-03-17 Melanocyte-stimulation hormone for suppressing inflammation reactions in hemodialysis Ceased EP2694096A1 (en)

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WO2004046166A2 (en) 2002-11-14 2004-06-03 Zengen, Inc. Modified alpha-msh peptides and derivatives thereof
US7135548B2 (en) 2002-11-14 2006-11-14 Zengen, Inc. Modified α-MSH peptides and derivatives thereof
US20050074485A1 (en) * 2003-05-14 2005-04-07 Lipton James M. Anti-inflammatory/anti-microbial peptides for use in dialysis
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