WO2014190398A1 - Inhibition of amyloid fibril formation - Google Patents

Inhibition of amyloid fibril formation Download PDF

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Publication number
WO2014190398A1
WO2014190398A1 PCT/AU2014/050058 AU2014050058W WO2014190398A1 WO 2014190398 A1 WO2014190398 A1 WO 2014190398A1 AU 2014050058 W AU2014050058 W AU 2014050058W WO 2014190398 A1 WO2014190398 A1 WO 2014190398A1
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inhibitor
backbone
methacrylate
amyloid
hydrophobic
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PCT/AU2014/050058
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French (fr)
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Anthony Richard BLENCOWE
David Edwin Dunstan
Greg Guanghua Qiao
Innocent Berbelle BEKARD
John Andrew KARAS
Phillip Leigh VAN DER PEET
Sian-Yang OW
Spencer John Williams
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The University Of Melbourne
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Publication of WO2014190398A1 publication Critical patent/WO2014190398A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • 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
    • A61K47/58—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 by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • 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
    • A61K47/595—Polyamides, e.g. nylon
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to compounds and compositions comprising them that are capable of inhibiting the formation of amyloid fibril formation.
  • the invention also relates to methods and uses involving the inhibitor compounds for reducing or preventing amyloid fibril formation in industrial processes and for treating or preventing diseases or disorders associated with amyloid fibril formation.
  • Amyloid fibrils also referred to as amyloid plaques, are a leading cause of many degenerative diseases such as Alzheimer's disease [12], Parkinson's disease [1], type II diabetes, various prion diseases and atherosclerosis, among many others.
  • Alzheimer's disease [12] Parkinson's disease [1]
  • type II diabetes various prion diseases and atherosclerosis
  • Amyloid fibril plaques are resistant to proteolysis [13] and were first observed in the brains of Alzheimer's disease patients in 1963 [11].
  • Amyloid fibril associated diseases such as Alzheimer's disease, are presently incurable.
  • Amyloid fibrils have generic features defining the fibrillar state. These are the misfolding of the protein into a beta sheet form and the assembly of the beta sheets into associated structures where the hydrophobic interfaces of the beta sheets drive the beta sheets together to form the larger fibrillar structures observed in a range of pathologies.
  • the beta sheets are formed through association of peptide sequences via hydrogen bonding.
  • the beta sheets associate through hydrophobic interactions between their faces to form fibrils [1, 5].
  • amyloids on organisms is not well established, it has been argued in the literature that the cytotoxic effects seen in amyloid related pathologies are from the precursors or the free-floating units that make up the amyloids, and that the mature fibrils are inert [14]. However there is evidence that fibrils and precursors grown under different shear conditions exhibit differing cytotoxicity levels [15]. More recently though it has been reported that in vivo the amyloids cause a variety of effects to cell membranes and the combination of these effects is responsible for the various amyloid related pathologies.
  • Amyloid fibril formation also causes problems in industries involving the processing of proteins such as in the food, nutritional supplements, blood products, pharmaceutical, insulin [16] and dairy [17] industries. Some proteins that usually do not form fibrils under physiological conditions may be subject to amyloid fibril formation during industrial processing due to the treatment conditions adopted. In many cases this is undesirable as the presence of amyloids will reduce the quality of product, result in wastage of protein and cause fouling in machine parts [17] leading to production delays and maintenance costs.
  • a number of US patent documents have referred to agents that purport to exhibit some activity in inhibiting amyloid fibril formation, such as US 6794363, US 2002/0042420, US 7166622, US 2011/2075640, US 2005/0054732, US 2010/0204085, US 7288659, US 2008/0004211, US 7790856, and US 6022859.
  • amyloid fibril formation relies on interactions between the exposed hydrophobic surfaces of amyloid fibril precursors [1, 5], surfactants that can bind to these surfaces can inhibit amyloid fibril formation. Indeed, the deployment of amphiphilic surfactants has been shown to result in inhibition of model amyloid systems [24] and specific targeting of residues involved in aggregation, such as lysine, can prevent amyloid formation [25]. There is, however, a pressing need for the development of alternative inhibitors of amyloid fibril formation that can be used to improve the understanding of the mechanism of amyloid fibril formation and which may offer potential in industries associated with the processing of proteins subject to amyloidosis and/or in the therapy / prevention of amyloid associated diseases or disorders.
  • an inhibitor of amyloid fibril formation comprising:
  • pendant chains linked to the backbone, wherein the pendant chains are the same or different and comprise from 1 to about 1000 repeating units that are able to freely rotate about the backbone due to free rotation of the repeating units within the backbone;
  • an inhibitor of amyloid fibril formation comprising:
  • pendant chains linked to the backbone, wherein the pendant chains are the same or different and comprise from about 2 to about 1000 repeating units that are able to freely rotate about the backbone due to free rotation of the repeating units within the backbone;
  • an inhibitor of amyloid fibril formation comprising:
  • pendant chains linked to the backbone, wherein the pendant chains are the same or different and comprise from about 1 to about 1000 repeating units that are able to freely rotate about the backbone due to free rotation of the repeating units within the backbone.
  • an inhibitor of amyloid fibril formation comprising:
  • pendant chains linked to the backbone, wherein the pendant chains are the same or different and comprise from about 2 to about 1000 repeating units that are able to freely rotate about the backbone due to free rotation of the repeating units within the backbone.
  • T, T 1 and T 2 are the same or different and represent a terminal group
  • L, L 1 , L2 and L 3 are the same or different and represent a linker group
  • A represents a hydrophobic backbone unit
  • R represents a hydrophilic pendant chain unit that is bound, optionally via linker group L 2 , to any of A, L or L 1 ;
  • a, a 1 , a2 and a 3 are the same of different and represent the numerals 0, 1, 2 or 3; b represents the numerals 1 to 1000;
  • c represents the numerals 0 to 1000, but represents 1 or greater in at least 25% of cases;
  • T, T 1 and T 2 are the same or different and represent a terminal group
  • L, L 1 , L2 and L 3 are the same or different and represent a linker group
  • A represents a hydrophobic backbone unit
  • R represents a hydrophilic pendant chain unit that is bound, optionally via linker group L 2 , to any of A, L or L 1 ;
  • a, a 1 , a2 and a 3 are the same of different and represent the numerals 0, 1, 2 or 3; b represents the numerals 3 to 500;
  • c represents the numerals 0 to 1000, but represents 2 or greater in at least 50% of cases;
  • the hydrophobic backbone unit can comprise one or more of glycine, alanine, valine, leucine, isoleucine, methionine, selenomethionine, phenylalanine, tryptophan, proline, hydroxyproline, styrene, alpha-methyl styrene, butyl acrylate, butyl methacrylate, amyl methacrylate, hexyl methacrylate, lauryl methacrylate, stearyl methacrylate, ethyl hexyl methacrylate, crotyl methacrylate, cinnamyl methacrylate, oleyl methacrylate, ricinoleyl methacrylate, cholesteryl methacrylates, cholesteryl acrylate, vinyl butyrate, vinyl tert-butyrate, vinyl stearate, vinyl laurate, caprolactide, propylene oxide, divinyl cyclopen
  • the hydrophilic pendant chain unit comprises one or more of one or more of acrylic acid, methacrylic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate, oligo(alkylene glycol)methyl ether (meth)acrylate (OAG(M)A), acrylamide, methacrylamide, hydroxyethyl acrylate, N-methylacrylamide, N,N-dimethylacrylamide and ⁇ , ⁇ -dimethylaminoethyl methacrylate, ⁇ , ⁇ -dimethylaminopropyl methacrylamide, N-hydroxypropyl methacrylamide, norbornenes, 4-acryloylmorpholine, 2-acrylamido-2- methyl-l-propanesulfonic acid, phosphorylcholine methacrylate, N- vinyl pyrolidone, amino ethyl acrylamide, ethylene oxide.
  • acrylic acid methacrylic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate
  • the inhibitor is an arabinogalactan protein (AGP).
  • AGP arabinogalactan protein
  • Another aspect of the invention relates to use of an inhibitor as referred to above in an industrial process where it is desired to inhibit amyloid fibril formation.
  • a further aspect of the invention relates to a method of preventing or reducing amyloid fibril formation in an industrial process in which amyloid fibril formation is prone to occur, which comprises exposing amyloid fibril forming proteins in the process to an inhibitor as outlined above.
  • a still further aspect of the invention relates to use of an inhibitor as referred to above in treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject.
  • Another aspect of the invention relates to use of an inhibitor as outlined above in preparation of a medicament for treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject.
  • a method of treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject which comprises administering to the subject an effective amount of an inhibitor as outlined above.
  • the disease or disorder associated with amyloid fibril formation can be selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, type II diabetes, transmissible spongiform encephalopathy, medullary carcinoma of the thyroid, isolated atrial amyloidosis causing cardiac arrhythmia, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinoma, familial amyloid polyneuropathy, hereditary non-neuropathic systemic amyloidosis, dialysis related amyloidosis, Finnish amyloidosis, lattice corneal dystrophy, cerebral amyloid angiopathy, Icelandic type cerebral amyloid angiopathy, systemic AL amyloidosis and sporadic inclusion body myositis.
  • Alzheimer's disease Parkinson's disease, Huntington's disease, type II diabetes, transmissible spongiform encephalopathy, medullary carcinoma of the thyroid
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an inhibitor as referred to above and one or more pharmaceutically acceptable excipients.
  • Figure 1 shows a schematic image of elements of inhibitors according to one aspect of the invention.
  • Figure 2 shows an image of the chemical structure of an inhibitor according to one aspect of the invention, which is tri-glycosylated 7-hydroxyproline.
  • Figure 3 shows a schematic of the putative binding of the inhibitor AGP to ⁇ -sheet amyloid precursors.
  • Figure 4 shows chemical structures of various synthethic compounds tested against (bovine insulin) BI for effectiveness against amyloid formation.
  • Figure 5 shows chemical structures of various derivative compounds from FA diacid plus the polyhydroxyproline that were tested. Chemicals are referred to as A) PNGE, B) PNGA, C) Oxy-FA and D) glycosylated polyhydroxyproline (PhP), and the variable n represents integers 1 to 1000.
  • Figure 6 shows the chemical structure of a glycosylated polyhydroxyproline molecule with A) 3 sugar chains (idealized), B) a single attached sugar chain and C), D) and E) show glycosylated polyhydroxyproline with hydrophobic amino acid of Phenylalanine, Tyrosine or Leucine where the R group (alkyl or aryl) determines the amino acid.
  • the variables n and m independently represent integers 1 to 1000 and R may for example represent alkyl or aryl.
  • Figure 7 shows a graph of ThT fluorescence intensity against time (hrs) for ⁇ amyloid fibrils grown (line) without inhibitor, ( ) with GA, ( ) with AGP, ( _ . . _ ) 2kDa
  • FIG. 8 shows a graph of ThT fluorescence intensity against time (hrs) for BI amyloid fibrils grown ( _ . _ . ) without inhibitor, ( _ . . _ ) with GA, ( ) with AGP, ( )
  • Figure 9 shows a graph of ThT fluorescence intensity against concentration (mg/ml) at equilibrium (plateau) for various concentrations of GA. Values above 1000 intensity were estimated from fluorescence measured at excitation wavelength of 276nm and an emission wavelength of 470nm. The graph demonstrates that at low concentrations of GA, larger fibrils form, however the amount of fibrils decreases rapidly with increasing GA concentration.
  • Figure 10 shows a graph of lag time (hrs) of BI amyloid fibril formation against concentration (mg/ml) of GA. The graph demonstrates the effectiveness of delaying fibril formation of even low concentrations of GA.
  • Figure 11 shows AFM images of 0.2mg/ml BI incubated with 2mg/ml FA-diacid taken (A) before and (B) after incubation for 90 hours. No amyloid fibrils are observed however protein aggregates are seen both before and after incubation. Each image is ⁇ ⁇ .
  • Figure 12 shows a graph of fluorescence against time (hrs) for 0.2mg/ml BI amyloid fibrils grown under various lOkDa FA diacid concentrations - control (0 mg/ml) is represented by a line, 0.0125 mg/ml is represented by , 0.05 mg/ml is represented by . _ . _ , 2 mg/ml is represented by _ . .
  • FIG. 13 shows a graph of fluorescence intensity against time (hrs) which allows a comparison of the results for the various inhibitors: FA diacid, PNGE, PNGA, GA, AGP and Polyhydroxyproline (PHP).
  • Figure 14 shows a graph of fluorescence intensity against time (hrs) for ThT fluorescence of lysozyme fibrils grown in various conditions. All inhibitors are at 0.2mg/ml concentration except for GA which is at 2mg/ml.
  • Figure 15 shows a graph of fluorescence intensity against time (hrs) for ThT fluorescence of lysozyme fibrils grown in the presence of various inhibitors: GA, PNGE and PNGA
  • Figure 16 shows AFM images of various amyloid fibrils.
  • Top row shows BI amyloid fibrils formed (A) without inhibitor, (B) with GA and (C) with AGP.
  • Second row shows ⁇ amyloid fibrils formed (D) without inhibitor, (E) with GA and (F) with AGP.
  • Non-fibrillar aggregates are seen in the background for (E). All images shown are ⁇ x ⁇ in size.
  • Figure 17 shows AFM images of the lysozyme fibrils formed in the presence of a range of inhibitors.
  • Figure 18 shows chemical structures of examples of compounds of the invention that demonstrate effectiveness against amyloid formation, including (a) poly(valine-ran- (N e -(2,3,4,5,6-pentahydroxyhexanoyl)lysine)), (b) poly(4-(tetrahydro-3,4,5-trihydroxy- 6-(hydroxymethyl)-2H-pyran-2-yloxy)proline)-co-(phenylalanine) and (c) Acetyl- (phenylalaninyl-0-galactosehydroxyprolyline-phenylalaninyl)15-amide.
  • poly(valine-ran- N e -(2,3,4,5,6-pentahydroxyhexanoyl)lysine)
  • Figure 19 shows a graph of fluorescence intensity against time (hrs) for ThT fluorescence of bovine insulin fibrils grown in the presence of Acetyl-(phenylalaninyl-0- galactosehydroxyprolyline-phenylalaninyl)15-amide at concentrations of 0 mg/ml (control - solid line), 0.02mg/ml (dashed line) and 0.2mg/ml (dotted line).
  • the present invention relates to compounds that inhibit amyloid fibril formation (amyloidosis) and to methods and uses involving such compounds.
  • amyloid fibril formation amyloidosis
  • the compounds adsorb to the hydrophobic face of the fibrils and sterically inhibit further proteins from associating to the fibril.
  • the basic structure of the inhibitors is that they include an oligomeric/polymeric backbone of either hydrophobic or hydrophilic species, wherein there is rotational freedom of the bonds in the principle axis of the backbone.
  • pendant hydrophilic or hydrophobic monomers or oligomers bound to the backbone such that when the backbone includes hydrophobic units the pendant chains include hydrophilic units and when the backbone includes hydrophilic units the pendant chains include hydrophobic units. Due to the amphiphilic nature of the inhibitors and free rotation allowed within the backbone the inhibitors are able to adopt "brush" or "comb” three dimensional conformations, depending upon the nature of their local environment.
  • the backbone is comprised of hydrophobic units that are believed to adsorb to ⁇ -sheet protein and the pendant chains comprise hydrophilic units that are believed to act as steric barriers to protein association with the growing fibril.
  • the rotation of the pendant hydrophilic groups is a requirement for physiological solubility and so that upon exposure to the hydrophobic ⁇ -sheet the hydrophobic backbone is adsorbed onto the ⁇ -sheet and the pendant groups rotate to expose the hydrophobic backbone and render the water soluble polymer insoluble.
  • the pendant groups Upon adsorption of the inhibitor to the ⁇ - sheet, the pendant groups act as steric inhibitors to inhibit further association of the protein and any association of the ⁇ -sheets through hydrophobic interactions.
  • a naturally occurring class of compounds known as arabinogalactan proteins have the features required for inhibition and are shown to inhibit fibril growth in vitro.
  • the model inhibitor has a backbone of length approximately equal to the width of the ⁇ -sheet, with water soluble groups attached.
  • the inhibitor is an oligomer of hydroxyproline of four to ten residues with pendant hydrophilic groups attached thereto which act as steric inhibitors of fibril growth.
  • the proposed mechanism of action is depicted graphically in Figures 1 and 2.
  • inhibitors agents that inhibit amyloid fibril formation
  • compounds agents that inhibit amyloid fibril formation
  • agents By reference to inhibition of amyloid fibril formation it is intended to convey that when in association with amyloid fibril forming proteins the agents give rise to a reduction in the extent and/or rate of amyloid fibril formation. This can be in a laboratory setting, on an industrial scale or in a therapeutic setting for treatment or prevention of an amyloid related disease or disorder.
  • the rate or extent of amyloid fibril formation relative to the control situation, where no inhibitor agent is present can be determined by routine experimental techniques, such as atomic force microscopy (AFM), circular dichroism (CD) or use of an amyloid binding dye or marker that can be quantitatively detected via a signal such as fluorescence, radiation, conductivity or the like.
  • AFM atomic force microscopy
  • CD circular dichroism
  • an amyloid binding dye or marker that can be quantitatively detected via a signal such as fluorescence, radiation, conductivity or the like.
  • An example of a suitable marker for amyloid formation is the fluorescent dye Thioflavin T (ThT), which exhibits a characteristic fluorescence peak when bound to amyloid protein.
  • the inhibitors of the invention decrease the rate or extent over a fixed time period of amyloid formation relative to the control situation by at least 5%, at least 10%, at least 20%, at least 40%, at least 50%, at least 80% or at least 90%, 95%, 98% or 99%. Even relatively small percentage reductions in rate and/or or extent of amyloid fibril production can be useful depending upon the context in which reduction in fibril formation is sought.
  • hydrophilic and hydrophobic are generally used in the art to convey interactions between one component relative to another (e.g. attractive or repulsive interactions, or solubility characteristics) and not to quantitatively define properties of a particular component relative to another.
  • a hydrophilic component is more likely to be wetted or solvated by an aqueous medium such as water, whereas a hydrophobic component is less likely to be wetted or solvated by an aqueous medium such as water.
  • a hydrophilic agent, unit, monomer, oligomer, polymer or polymer block is intended to define one that exhibits solubility or miscibility in an aqueous medium, including biological fluids such as blood, plasma, serum, urine, saliva, milk, seminal fluid, vaginal fluid, synovial fluid, lymph fluid, amniotic fluid, sweat, and tears; as well as in an aqueous solution produced by a plant, including, for example, exudates and guttation fluid, xylem, phloem, resin, and nectar; and in an aqueous medium produced or processed in an industrial context such as food or beverage or components thereof such as milk or milk derived material (e.g cheese, cream, yoghurt, whey, ice cream, etc.), pharmaceuticals, nutritional supplements or cosmetics or excipients for inclusion within pharmaceuticals, nutritional supplements or cosmetics, animal feed or supplements or veterinary agents.
  • biological fluids such as blood, plasma, serum, urine, saliva, milk, seminal
  • a hydrophobic polymer or unit, etc. is intended to mean a polymer or unit that exhibits little or no solubility or miscibility in an aqueous medium, such as the various aqueous media referred to above.
  • hydrophilic or hydrophobic in character By being hydrophilic or hydrophobic in character in this context it is meant that upon polymerisation such monomers respectively give rise (directly or indirectly) to the hydrophilic or hydrophobic oligomers or polymers that form the backbone or pendant chains of the inhibitor agents.
  • a hydrophilic polymer that forms part of a pendant chain will generally be prepared by polymerising a monomer composition that comprises hydrophilic monomer.
  • hydrophilic monomers/units include, but are not limited to, acrylic acid, methacrylic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate, oligo(alkylene glycol)methyl ether (meth)acrylate (OAG(M)A), acrylamide, methacrylamide, hydroxyethyl acrylate, N-methylacrylamide, N,N-dimethylacrylamide and ⁇ , ⁇ -dimethylaminoethyl methacrylate, ⁇ , ⁇ -dimethylaminopropyl methacrylamide, N-hydroxypropyl methacrylamide, norbornenes, 4-acryloylmorpholine, 2-acrylamido-2- methyl-l-propanesulfonic acid, phosphorylcholine methacrylate, N- vinyl pyrolidone, amino ethyl acrylamide, ethylene oxide
  • the hydrophilic monomers/units can also include hydrophilic sugar or polysaccharide groups and
  • hydrophobic monomers/units include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, methionine, selenomethionine, phenylalanine, tryptophan, proline, hydroxyproline, styrene, alpha-methyl styrene, butyl acrylate, butyl methacrylate, amyl methacrylate, hexyl methacrylate, lauryl methacrylate, stearyl methacrylate, ethyl hexyl methacrylate, crotyl methacrylate, cinnamyl methacrylate, oleyl methacrylate, ricinoleyl methacrylate, cholesteryl methacrylates, cholesteryl acrylate, vinyl butyrate, vinyl tert-butyrate, vinyl stearate, vinyl laurate, caprolactide, propylene
  • hydrophilic polymer component of the inhibitor agents may therefore be described as comprising the polymerised residues of hydrophilic monomers or units.
  • hydrophobic polymer component of the inhibitor agents can be described as comprising the polymerised residues of hydrophobic monomers or units.
  • hydrophobic components of the inhibitor agents can include monomers or units other than those that are strictly hydrophobic as long as the hydrophobic component (e.g an hydrophobic backbone component of the inhibitor) is generally hydrophobic in character.
  • the hydrophobic nature or character of the backbone can be determined by assessing the ability of the hydrophobic component to adsorb to ⁇ -sheet polymer, which can be detected by the use of ANS dyes which are used to detect hydrophobic domains [43].
  • Another means of determining whether a monomer or polymer component of the inhibitor can generally be classified as hydrophobic is by conducting contact angle analysis on a solid surface of the dried sample.
  • the three phase contact angle the angle made by a droplet of water on a film of the substance, where a hydrophobic surface shows a contact angle of greater than 50-60 degrees. It is also possible to determine hydrophobicity by measuring the miscibility of the molecules in oil or water [44] .
  • the inhibitors described here are amphipathic such they show positive surface excesses at the air water interface.
  • hydrophilic components of the inhibitor agents can include monomers or units other than those that are strictly hydrophilic as long as the hydrophilic component (e.g. a pendant chain bound to the hydrophobic backbone component of the inhibitor) is generally hydrophilic in character.
  • the hydrophilic nature or character of the pendant chain can be determined by assessing the ability of the hydrophilic component to solubilise the inhibitor agent in an aqueous medium, which of course can be readily observed.
  • hydrophobic agents another means of determining whether a monomer or polymer component of the inhibitor can generally be classified as hydrophilic is by conducting contact angle analysis, for example by measuring the three phase contact angle or by measuring the miscibility of the molecules in oil or water, as discussed above.
  • the backbone is hydrophobic and can, for example, comprise hydrophobic amino acid monomer units, including both natural and non-natural amino acids.
  • hydrophobic amino acids that can be included within the backbone include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, methionine, selenomethionine, phenylalanine, tryptophan, proline, hydroxyproline.
  • the hydrophobic backbone comprises an optionally substituted four-, five- or six-membered carbocyclic or heterocyclic ring.
  • ring structures that can be included within the hydrophobic backbone include optionally substitued cyclobutanyl, cyclopentanyl, cyclohexanyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, furanyl, pyranyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, dithiolyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, phenyl, benzyl, pyranyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, triazinyl, oxazinyl, oxathiazinyl
  • the hydrophobic backbone comprises polyhydroxyproline.
  • the polyhydroxyproline backbone has bound thereto a plurality of pendant chains that comprise hydrophilic monosaccharide groups.
  • hydrophilic pendant chains that are bound (either directly or indirectly) to the hydrophobic backbone comprise one or more ring form and/or straight chain monosaccharide.
  • monosaccharides include but are not limited to one of more of fucose, arabinose, arabitol, allose, altrose, glucofuranose, galactopyranose, glucopyranoside, xylanopyranose, fructopyranose, glucose, galactose, gulose, galactosamine, hammelose, xylose, lyxose, mannitol, mannosamine, ribose, rhamnose, threose, talose and substituted derivatives thereof.
  • both the backbone and the pendant chains within the inhibitor compounds of the invention can take the form of copolymers, so that more than one type of monomer can be included within each backbone or pendant chain.
  • Each polymer block in a block co-polymer component of the inhibitor can be a homopolymer block or a copolymer block. Where a polymer block is a copolymer, the copolymer may be a gradient, a random or a statistical copolymer.
  • the backbone and or some or all of the pendant chains can include terminal groups at their ends that are different to the repeating units or monomers throughout the structure.
  • the terminal group may be a simple moiety that terminates polymerisation such as a hydrogen atom, amino group, or the like as may be present in the non-reactive monomer, while in other cases the terminal group may include more complex functionality. The presence of a terminal group may not be discernable in some cases.
  • a terminal group can comprise hydrogen, hydroxyl, halogen, amino, R 1 , - C0 2 H, -OR 1 , - SR 1 , -O 2 CR 1 , -SCOR 1 , and -OCSR 1 ; where the or each R 1 is independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, and an optionally substituted polymer chain.
  • the pendant chains are directly or indirectly linked to the polymer or oligomer backbone, as will be further discussed below. It should be understood, however, that not all monomers or units within the backbone will necessarily have a pendant chain bound thereto. For example there can be a pendant chain attached to at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% of the monomer units within the backbone.
  • the pendant chains can comprise from 1 to about 1000 monomers or repeating units, such as from about 2 to about 500, from about 3 to about 100, from about 4 to about 50, from about 4 to about 20 or about 5, 10 or 12 monomers or repeating units.
  • pendant chains can be covalently coupled directly or indirectly to the backbone. It is also possible for linkages to be present between polymer blocks within the backbone. By being “directly” coupled it is meant that there is only a covalent bond between the pendant chain and the backbone. By being “indirectly” coupled it is meant that there is located between the pendant chain and the backbone (or indeed within the backbone itself) one or more covalently bonded atoms or molecules. Where the pendant chains are indirectly coupled to the support moiety, it is convenient to refer to the pendant chains as being covalent coupled to the backbone through a linker group or groups.
  • each of the pendant chains covalently coupled to the backbone through a linker group.
  • a linker group there is no particular limitation concerning the nature of such a linker group provided it can function to couple the pendant chains to the backbone.
  • linker groups include a divalent form of optionally substituted: oxy (- 0-), disulfide (-S-S-), alkyl, alkenyl, alkynyl, aryl, acyl (including -C(O)-), carbocyclyl, heterocyclyl, heteroaryl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, acyloxy, carbocyclyloxy, heterocyclyloxy, heteroaryloxy, alkylthio, alkenylthio, alkynylthio, arylthio, acylthio, carbocyclylthio, heterocyclylthio, heteroarylthio, alkylalkenyl, alkylalkynyl, alkylaryl, alkylacyl, alkylcarbocyclyl, alkylheterocyclyl, alkylheteroaryl, alkyloxyalkyl, alkenyloxyalkyl,
  • the or each R a may also be independently selected from hydrogen, Ci_i 8 alkyl, Ci_i 8 alkenyl, Ci_i 8 alkynyl, C6-i 8 aryl, C 3 _i 8 carbocyclyl, C 3 -i 8 heteroaryl, C 3 _i 8 heterocyclyl, and C7_i 8 arylalkyl.
  • alkyl used either alone or in compound words denotes straight chain, branched or cyclic alkyl, preferably Ci_ 2 o alkyl, e.g. Ci_io or Ci_ 6.
  • straight chain and branched alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl, i-butyl, n-pentyl, 1,2-dimethylpropyl, 1,1 -dimethyl-prop yl, hexyl, 4-methylpentyl, 1- methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3- dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2- trimethylpropyl, heptyl, 5-methylhexyl
  • cyclic alkyl examples include mono- or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like. Where an alkyl group is referred to generally as "propyl", butyl” etc, it will be understood that this can refer to any of straight, branched and cyclic isomers where appropriate. An alkyl group may be optionally substituted by one or more optional substituents as herein defined.
  • alkenyl denotes groups formed from straight chain, branched or cyclic hydrocarbon residues containing at least one carbon to carbon double bond including ethylenically mono-, di- or polyunsaturated alkyl or cycloalkyl groups as previously defined, preferably C2-20 alkenyl (e.g.
  • alkenyl examples include vinyl, allyl, 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3- decenyl, 1,3-butadienyl, 1,4-pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4- hexadienyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-cycloheptadienyl
  • alkynyl denotes groups formed from straight chain, branched or cyclic hydrocarbon residues containing at least one carbon-carbon triple bond including ethylenically mono-, di- or polyunsaturated alkyl or cycloalkyl groups as previously defined. Unless the number of carbon atoms is specified the term preferably refers to C2-20 alkynyl (e.g. C 2 -io or C 2 -6)- Examples include ethynyl, 1-propynyl, 2-propynyl, and butynyl isomers, and pentynyl isomers. An alkynyl group may be optionally substituted by one or more optional substituents as herein defined.
  • halogen denotes fluorine, chlorine, bromine or iodine (fluoro, chloro, bromo or iodo).
  • aryl denotes any of single, polynuclear, conjugated and fused residues of aromatic hydrocarbon ring systems(e.g. C 6 - 24 or C 6 -i8)- ⁇
  • aryl include phenyl, biphenyl, terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, idenyl, azulenyl, chrysenyl.
  • aryl include phenyl and naphthyl.
  • An aryl group may or may not be optionally substituted by one or more optional substituents as herein defined.
  • arylene is intended to denote the divalent form of aryl.
  • carbocyclyl includes any of non-aromatic monocyclic, polycyclic, fused or conjugated hydrocarbon residues, preferably C3-20 (e.g. C3-10 or C 3-8 ).
  • the rings may be saturated, e.g. cycloalkyl, or may possess one or more double bonds (cycloalkenyl) and/or one or more triple bonds (cycloalkynyl).
  • Particularly preferred carbocyclyl moieties are 5- 6-membered or 9-10 membered ring systems.
  • Suitable examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopentenyl, cyclohexenyl, cyclooctenyl, cyclopentadienyl, cyclohexadienyl, cyclooctatetraenyl, indanyl, decalinyl and indenyl.
  • a carbocyclyl group may be optionally substituted by one or more optional substituents as herein defined.
  • the term "carbocyclylene" is intended to denote the divalent form of carbocyclyl.
  • heteroatom refers to any atom other than a carbon atom which may be a member of a cyclic organic group.
  • heteroatoms include nitrogen, oxygen, sulfur, phosphorous, boron, silicon, selenium and tellurium, more particularly nitrogen, oxygen and sulfur.
  • heterocyclyl when used alone or in compound words includes any of monocyclic, polycyclic, fused or conjugated hydrocarbon residues, preferably C3-20 (e.g. C 3 _io or C 3-8 ) wherein one or more carbon atoms are replaced by a heteroatom so as to provide a non-aromatic residue.
  • Suitable heteroatoms include O, N, S, P and Se, particularly O, N and S. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms.
  • the heterocyclyl group may be saturated or partially unsaturated, i.e. possess one or more double bonds. Particularly preferred heterocyclyl are 5-6 and 9-10 membered heterocyclyl.
  • heterocyclyl groups may include azridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 2H-pyrrolyl, pyrrolidinyl, pyrrolinyl, piperidyl, piperazinyl, morpholinyl, indolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, thiomorpholinyl, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrrolyl, tetrahydrothiophenyl, pyrazolinyl, dioxalanyl, thiazolidinyl, isoxazolidinyl, dihydropyranyl, oxazinyl, thiazinyl, thiomorpholinyl, oxathianyl, dithi
  • heteroaryl includes any of monocyclic, polycyclic, fused or conjugated hydrocarbon residues, wherein one or more carbon atoms are replaced by a heteroatom so as to provide an aromatic residue.
  • Preferred heteroaryl have 3-20 ring atoms, e.g. 3-10.
  • Particularly preferred heteroaryl are 5-6 and 9-10 membered bicyclic ring systems.
  • Suitable heteroatoms include, O, N, S, P and Se, particularly O, N and S. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms.
  • heteroaryl groups may include pyridyl, pyrrolyl, thienyl, imidazolyl, furanyl, benzothienyl, isobenzothienyl, benzofuranyl, isobenzofuranyl, indolyl, isoindolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, quinolyl, isoquinolyl, phthalazinyl, 1,5-naphthyridinyl, quinozalinyl, quinazolinyl, quinolinyl, oxazolyl, thiazolyl, isothiazolyl, isoxazolyl, triazolyl, oxadialzolyl, oxatriazolyl, triazinyl, and furazanyl.
  • a heteroaryl group may be optionally substituted by one or more optional substituents as
  • Preferred acyl includes C(0)-R e , wherein R e is hydrogen or an alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl residue.
  • R e is hydrogen or an alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl residue.
  • Examples of acyl include formyl, straight chain or branched alkanoyl (e.g.
  • Ci_2o such as acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2- dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl and icosanoyl; cycloalkylcarbonyl such as cyclopropylcarbonyl cyclobutylcarbonyl, cyclopentylcarbonyl and cyclohexylcarbonyl; aroyl such as benzoyl, toluoyl and naphthoyl; aralkanoyl
  • phenylacetyl phenylpropanoyl, phenylbutanoyl, phenylisobutylyl, phenylpentanoyl and phenylhexanoyl
  • naphthylalkanoyl e.g. naphthylacetyl, naphthylpropanoyl and naphthylbutanoyl]
  • aralkenoyl such as phenylalkenoyl (e.g.
  • phenylpropenoyl e.g., phenylbutenoyl, phenylmethacryloyl, phenylpentenoyl and phenylhexenoyl and naphthylalkenoyl (e.g.
  • aryloxyalkanoyl such as phenoxyacetyl and phenoxypropionyl
  • arylthiocarbamoyl such as phenylthiocarbamoyl
  • arylglyoxyloyl such as phenylglyoxyloyl and naphthylglyoxyloyl
  • arylsulfonyl such as phenylsulfonyl and napthylsulfonyl
  • heterocycliccarbonyl heterocyclicalkanoyl such as thienylacetyl, thienylpropanoyl, thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl and tetrazolylacetyl
  • sulfoxide refers to a group -S(0)R wherein R is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl.
  • R is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl.
  • R is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl.
  • R is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl.
  • Examples of preferred R include Ci_ 2 oalkyl, phenyl and benzyl.
  • sulfonyl refers to a group S(0) 2 -R , wherein R is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl and aralkyl. Examples of preferred R include Ci_ 2 oalkyl, phenyl and benzyl.
  • sulfonamide refers to a group S(0)NR f R f wherein each R is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl.
  • R is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl.
  • preferred R include Ci_ 2 oalkyl, phenyl and benzyl.
  • at least one R is hydrogen.
  • both R are hydrogen.
  • amino is used here in its broadest sense as understood in the art and includes groups of the formula NR a R b wherein R a and R b may be any independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl, heterocyclyl, arylalkyl, and acyl.
  • R a and R b together with the nitrogen to which they are attached, may also form a monocyclic, or polycyclic ring system e.g. a 3-10 membered ring, particularly, 5-6 and 9- 10 membered systems.
  • Examples of "amino” include NH 2 , NHalkyl (e.g.
  • Ci_ 2 oalkyl NHaryl (e.g. NHphenyl), NHaralkyl (e.g. NHbenzyl), NHacyl (e.g. NHC(O)Ci_ 20 alkyl, NHC(O)phenyl), Nalkylalkyl (wherein each alkyl, for example Ci_ 2 o, may be the same or different) and 5 or 6 membered rings, optionally containing one or more same or different heteroatoms (e.g. O, N and S).
  • NHaryl e.g. NHphenyl
  • NHaralkyl e.g. NHbenzyl
  • NHacyl e.g. NHC(O)Ci_ 20 alkyl, NHC(O)phenyl
  • Nalkylalkyl wherein each alkyl, for example Ci_ 2 o, may be the same or different
  • 5 or 6 membered rings optionally containing one or more same or different heteroatoms (e.g
  • amido is used here in its broadest sense as understood in the art and includes groups having the formula C(0)NR a R b , wherein R a and R b are as defined as above.
  • Examples of amido include C(0)NH 2 , C(0)NHalkyl (e.g. Ci_ 20 alkyl), C(0)NHaryl (e.g. C(O)NHphenyl), C(0)NHaralkyl (e.g. C(O)NHbenzyl), C(0)NHacyl (e.g.
  • C(O)NHC(O)Ci- 20 alkyl C(0)NHC(0)phenyl
  • C(0)Nalkylalkyl wherein each alkyl, for example Ci_ 2 o, may be the same or different
  • 5 or 6 membered rings optionally containing one or more same or different heteroatoms (e.g. O, N and S).
  • carboxy ester is used here in its broadest sense as understood in the art and includes groups having the formula C0 2 R g , wherein R g may be selected from groups including alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl, heterocyclyl, aralkyl, and acyl.
  • R g may be selected from groups including alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl, heterocyclyl, aralkyl, and acyl.
  • Examples of carboxy ester include C0 2 Ci_ 2 oalkyl, C0 2 aryl (e.g.. C0 2 phenyl), C0 2 aralkyl (e.g. C0 2 benzyl).
  • aryloxy refers to an "aryl” group attached through an oxygen bridge.
  • aryloxy substituents include phenoxy, biphenyloxy, naphthyloxy and the like.
  • acyloxy refers to an "acyl” group wherein the "acyl” group is in turn attached through an oxygen atom.
  • acyloxy examples include hexylcarbonyloxy (heptanoyloxy), cyclopentylcarbonyloxy, benzoyloxy, 4-chlorobenzoyloxy, decylcarbonyloxy (undecanoyloxy), propylcarbonyloxy (butanoyloxy), octylcarbonyloxy (nonanoyloxy), biphenylcarbonyloxy (eg 4-phenylbenzoyloxy), naphthylcarbonyloxy (eg 1-naphthoyloxy) and the like.
  • alkyloxycarbonyl refers to a "alkyloxy” group attached through a carbonyl group.
  • alkyloxycarbonyl groups include butylformate, sec- butylformate, hexylformate, octylformate, decylformate, cyclopentylformate and the like.
  • arylalkyl refers to groups formed from straight or branched chain alkanes substituted with an aromatic ring. Examples of arylalkyl include phenylmethyl (benzyl), phenylethyl and phenylpropyl.
  • alkylaryl refers to groups formed from aryl groups substituted with a straight chain or branched alkane. Examples of alkylaryl include methylphenyl and isopropylphenyl.
  • a group may or may not be substituted or fused (so as to form a condensed polycyclic group) with one, two, three or more of organic and/or inorganic groups, including those selected from: alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, heteroaryl, acyl, aralkyl, alkaryl, alkheterocyclyl, alkheteroaryl, alkcarbocyclyl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, halocarbocyclyl, haloheterocyclyl, haloheteroaryl, haloacyl, haloaryalkyl, hydroxy, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxycarbocyclyl, hydroxyaryl,
  • Optional substitution may also be taken to refer to where a -CH 2 - group in a chain or ring is replaced by a group selected from -0-, -S-, -NR a -, -C(O)- (i.e. carbonyl), -C(0)0- (i.e. ester), and -C(0)NR a - (i.e. amide), where R a is as defined herein.
  • Preferred optional substituents include alkyl, (e.g. Ci_ 6 alkyl such as methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), hydroxyalkyl (e.g.
  • alkoxyalkyl e.g. methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl etc
  • alkoxy e.g.
  • Ci_6 alkoxy such as methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy), halo, trifluoromethyl, trichloromethyl, tribromomethyl, hydroxy, phenyl (which itself may be further substituted e.g., by C 1-6 alkyl, halo, hydroxy, hydroxyCi-6 alkyl, C 1-6 alkoxy, haloCi-6alkyl, cyano, nitro OC(0)Ci_6 alkyl, and amino), benzyl (wherein benzyl itself may be further substituted e.g., by Ci_ 6 alkyl, halo, hydroxy, hydroxyCi_6alkyl, Ci_ 6 alkoxy, haloCi_6 alkyl, cyano, nitro OC(0)Ci_6 alkyl, and amino), phenoxy (wherein phenyl itself may be further substituted e.g., by C 1-6 alky
  • Ci_ 6 alkyl such as methylamino, ethylamino, propylamino etc
  • dialkylamino e.g. C 1-6 alkyl, such as dimethylamino, diethylamino, dipropylamino
  • acylamino e.g.
  • NHC(0)CH 3 NHC(0)CH 3
  • phenylamino wherein phenyl itself may be further substituted e.g., by Ci_ 6 alkyl, halo, hydroxy, hydroxyCi_6 alkyl, Ci_ 6 alkoxy, haloCi_6 alkyl, cyano, nitro OC(0)Ci_6 alkyl, and amino
  • nitro, formyl, -C(0)-alkyl e.g. C 1-6 alkyl, such as acetyl
  • 0-C(0)-alkyl e.g.
  • Ci_ 6 alkyl such as acetyloxy
  • benzoyl wherein the phenyl group itself may be further substituted e.g., by Ci_ 6 alkyl, halo, hydroxy hydroxyCi_6 alkyl, Ci_ 6 alkoxy, haloCi_6 alkyl, cyano, nitro OC(0)C 1-6 alkyl, and amino
  • Ci-6 alkyl such as methyl ester, ethyl ester, propyl ester, butyl ester
  • C0 2 phenyl wherein phenyl itself may be further substituted e.g., by C 1-6 alkyl, halo, hydroxy, hydroxyl Ci_ 6 alkyl, Ci_ 6 alkoxy, halo Ci_ 6 alkyl, cyano, nitro OC(0)Ci_6 alkyl, and amino
  • CONH 2 CONHphenyl (wherein phenyl itself may be further substituted e.g., by Ci_ 6 alkyl, halo, hydroxy, hydroxyl C 1-6 alkyl, C 1-6 alkoxy, halo C 1-6 alkyl, cyano, nitro OC(0)C 1-6 alkyl, and amino)
  • CONHbenzyl wherein benzyl itself may be further substituted e.g., by Ci_6 alkyl, halo, hydroxy hydroxyl Ci_ 6
  • Ci_ 6 alkyl such as methyl ester, ethyl ester, propyl ester, butyl amide) CONHdialkyl (e.g. Ci_ 6 alkyl) aminoalkyl (e.g., HN Ci_ 6 alkyl-, Ci_ 6 alkylHN-Ci_6 alkyl- and (Ci_ 6 alkyl) 2 N-Ci_ 6 alkyl-), thioalkyl (e.g., HS Ci_ 6 alkyl-), carboxyalkyl (e.g., H0 2 CCi_6 alkyl-), carboxyesteralkyl (e.g., Ci_ 6 alkyl0 2 CCi_6 alkyl-), amidoalkyl (e.g., H 2 N(0)CCi_ 6 alkyl-, H(Ci_ 6 alkyl)N(0)CCi_ 6 alkyl-), formylalkyl (e.g., OHCCi_ 6 alkyl-),
  • the inhibitor compounds have the structure as provided in Formula I, as follows:
  • T, T 1 and T 2 are the same or different and represent a terminal group, for example as defined hereinbefore;
  • L, L 1 , L2 and L 3 are the same or different and represent a linker group, for example as defined hereinbefore;
  • A represents a hydrophobic backbone unit, for example as defined
  • R represents a hydrophilic pendant chain unit, for example as defined hereinbefore, that is bound, optionally via linker group L , to any of A, L or L 1 ; a, a 1 , a2 and a 3 are the same of different and represent the numerals 0, 1, 2 or 3;
  • b represents the numerals 1 to 1000;
  • c represents the numerals 0 to 1000, but represents 1 or greater in at least 25% of cases;
  • b represents the numerals 5 to 10.
  • c represents the numerals 3 to 20 in at least 50% of cases, which implies that at least 50% of backbone monomer units have a pendant chain bound thereto and that these pendant chains are 3 to 20 units in length.
  • c represents the numerals 4 to 10 or about 5 in at least 80% of cases, which implies that at least 80% of backbone monomer units have a pendant chain bound thereto and that these pendant chains are 3 to 20, 4 to 10 or 5 units in length.
  • polystyrene resin poly(ca/?ro-lactide) (PCL) backbone and polyethylene oxide side chians.
  • PCL poly(ca/?ro-lactide)
  • This polymer may be synthesized by the formation of functionalized PCL followed by attachment of PEO side chians on the backbone.
  • a further example of the polymer with hydrophobic backbone and hydrophilic side chains is a copolymer with PPO backbone and PEO side chains.
  • This polymer may be synthesized by initial formation of linear PEO with only one end having hydroxyl functionality which is then coupled with an epoxy group. The copolymerization of this long chain monomer with normal PO monomer forms the copolymer with hydrophobic PPO backbone and hydrophilic PEO side chains.
  • polymer with hydrophilic backbone and hydrophobic side chains is a copolymer with polyethylene oxide (PEO) backbone and polypropylene oxide (PPO) side chians.
  • PEO polyethylene oxide
  • PPO polypropylene oxide
  • This polymer may be synthesized by initial formation of PPO side chains with only one end having hydroxyl functionality which is then coupled with an epoxy group.
  • the copolymerization of this long chain monomer with normal PEO monomer forms the copolymer with h drophilic PEO backbone and hydrophobic PPO side chains.
  • a further example of the polymer with hydrophilic backbone and hydrophobic side chains is a copolymer with polyacrylamide (PAA) backbone and polypropylene oxide (PPO) side chians.
  • PAA backbone can be functionalised with amine functionality by copolymerization of acrylamide with amino ethyl acrylamide.
  • Amine functionalized PAA can then coupled with the hydrophobic side chian with epoxy functionality.
  • PPO with epoxy functionality at one chain end is one example.
  • Naturally occurring precursors may be used to form such copolymer structures.
  • polymannose chain blocks which can be derived from, for example, guar.
  • the hydrophobic polymannose can then be coupled to a hydrophilic polymer backbone with functional group along the chians.
  • the coupling between the backbone and side chains may through an epoxy-amine coupling or NPC coupling with amines.
  • PAA with amine functionality along the backbone can be used to couple multiple polymannose side chians.
  • variables a, x, y and z independently represent the integers 1 to 1000.
  • Guar, containing polymannose backbone and glactose side chains may be further modified to form comb shaped polymers with improved performance.
  • the modification may be carried out by a periodate reaction to convert the glycol moieties of the glactose to a aldehyde functionality which can then linked to a hydrophilic polymer with an amine functionality in one polymer chain end.
  • Such hydrophilic polymer chains may be PEO based or functionalised with charged groups to improve the hydrophilicity.
  • the inhibitors of the invention may be prepared by any suitable means.
  • the process of preparing the inhibitors comprises the polymerisation of ethylenically unsaturated monomers.
  • Polymerisation of the ethylenically unsaturated monomers is preferably conducted using a living polymerisation technique, but other approaches such as using the step-growth technique (e.g. PHP) and solid phase peptide synthesis (e.g. glycosylated PHP and GAA) are also possible.
  • step-growth technique e.g. PHP
  • solid phase peptide synthesis e.g. glycosylated PHP and GAA
  • Living polymerisation is generally considered in the art to be a form of chain polymerisation in which irreversible chain termination is substantially absent.
  • An important feature of living polymerisation is that polymer chains will continue to grow while monomer and reaction conditions to support polymerisation are provided.
  • Polymer chains prepared by living polymerisation can advantageously exhibit a well defined molecular architecture, a predetermined molecular weight and narrow molecular weight distribution or low polydispersity.
  • living polymerisation examples include ionic polymerisation, radical polymerisation (RP) and controlled radical polymerisation (CRP).
  • RP and CRP include, but are not limited to, iniferter polymerisation, stable free radical mediated polymerisation (SFRP), atom transfer radical polymerisation (ATRP), and reversible addition fragmentation chain transfer (RAFT) polymerisation. Ring opening metathesis polymerisation is also a possible synthesis route.
  • living polymerisation agent a compound that can participate in and control or mediate the living polymerisation of one or more ethylenically unsaturated monomers so as to form a living polymer chain (i.e. a polymer chain that has been formed according to a living polymerisation technique).
  • Living polymerisation agents include, but are not limited to, those which promote a living polymerisation technique selected from ionic polymerisation and CRP.
  • the branched polymer is prepared using ionic polymerisation.
  • Living ionic polymerisation is a form of addition polymerisation whereby the kinetic-chain carriers are ions or ion pairs.
  • the polymerisation proceeds via anionic or cationic kinetic- chain carriers.
  • the propagating species will either carry a negative or positive charge, and as such there will also be an associated counter cation or counter anion, respectively.
  • the living polymerisation agent might be represented as ⁇ + , where I represents an organo-anion (e.g.
  • the living polymerisation agent might be represented as I + M " , where I represents an organo-cation (e.g. an optionally substituted alkyl cation) and M represents an associated counteranion.
  • Suitable agents for conducting anionic and cationic living polymerisation include, but are not limited to, aprotonic acids (e.g. aluminium trichloride, boron trifluoride), protonic (Bronstead) acids, stable carbenium-ion salts, organometallic compounds (e.g. N-butyl lithium, cumyl potassium) and Ziegler-Natta catalysts (e.g. triethyl aluminium and titanium tetrachloride).
  • the branched polymer is prepared using CRP.
  • the branched polymer is prepared using iniferter polymerisation.
  • Iniferter polymerisation is a well known form of CRP, and is generally understood to proceed by a mechanism illustrated below in Scheme 1.
  • Scheme 1 General mechanism of controlled radical polymerisation with iniferters.
  • the iniferter agent AB dissociates chemically, thermally or photochemically to produce a reactive radical species A and generally a relatively stable radical species B (for symmetrical iniferters the radical species B will be the same as the radical species A) (step a).
  • the radical species A can initiate polymerisation of monomer M (in step b) and may be deactivated by coupling with radical species B (in step c). Transfer to the iniferter (in step d) and/or transfer to dormant polymer (in step e) followed by termination (in step f) characterise iniferter chemistry.
  • Suitable iniferter agents are well known to those skilled in the art, and include, but are not limited to, dithiocarbonate, disulphide, and thiuram disulphide compounds.
  • the branched polymer is prepared using SFRP.
  • this mode of radical polymerisation involves the generation of a stable radical species as illustrated below in Scheme 2.
  • SFRP agent CD dissociates to produce an active radical species C and a stable radical species D.
  • the active radical species C reacts with monomer M, which resulting propagating chain may recombine with the stable radical species D.
  • SFRP agents do not provide for a transfer step.
  • Suitable agents for conducting SFRP are well known to those skilled in the art, and include, but are not limited to, moieties capable of generating phenoxy and nitroxy radicals. Where the agent generates a nitroxy radical, the polymerisation technique is more commonly known as nitroxide mediated polymerisation (NMP).
  • SFRP agents capable of generating phenoxy radicals include those comprising a phenoxy group substituted in the 2 and 6 positions by bulky groups such as tert-alkyl (e.g. t-butyl), phenyl or dimethylbenzyl, and optionally substituted at the 4 position by an alkyl, alkyloxy, aryl, or aryloxy group or by a heteroatom containing group (e.g. S, N or O) such as dimethylamino or diphenylamino group.
  • a heteroatom containing group e.g. S, N or O
  • Thiophenoxy analogues of such phenoxy containing agents are also contemplated.
  • SFRP agents capable of generating nitroxy radicals include those comprising the substituent R 1 R2 N-0-, where R 1 and R2 are tertiary alkyl groups, or where R 1 and R2 together with the N atom form a cyclic structure, preferably having tertiary branching at the positions a to the N atom.
  • nitroxy substituents include 2,2,5,5- tetraalkylpyrrolidinoxyl, as well as those in which the 5-membered hetrocycle ring is fused to an alicyclic or aromatic ring, hindered aliphatic dialkylaminoxyl and iminoxyl substituents.
  • a common nitroxy substituent employed in SFRP is 2,2,6, 6-tetramethyl-l- piperidinyloxy.
  • polymer components of the inhibitors are prepared using ATRP.
  • ATRP generally employs a transition metal catalyst to reversibly deactivate a propagating radical by transfer of a transferable atom or group such as a halogen atom to the propagating polymer chain, thereby reducing the oxidation state of the metal catalyst as illustrated below in Scheme 3.
  • a transferable group or atom (X , e.g. halide, cyanato, thiocyanato or azido) is transferred from the organic compound (E-X) to a transition metal catalyst (M t , e.g. copper, iron, palladium, cobalt, rhenium, rhodium, ruthenium, molybdenum, niobium, or nickel) having oxidation number (n), upon which a radical species is formed that initiates polymerisation with monomer (M).
  • M transition metal catalyst
  • M t e.g. copper, iron, palladium, cobalt, rhenium, rhodium, ruthenium, molybdenum, niobium, or nickel
  • polymer components of the inhibitor are prepared using RAFT polymerisation.
  • RAFT polymerisation is well known in the art and is believed to operate through the mechanism outlined below in Scheme 4.
  • RAFT polymerisation is believed to proceed through initial reaction sequence (a) that involves reaction of a RAFT agent (1) with a propagating radical.
  • This radical can then promote polymerisation of monomer (M), thereby reinitiating polymerisation.
  • the propagating polymer chain can then react with the dormant polymer species (3) to promote the reaction sequence (b) that is similar to reaction sequence (a).
  • a labile intermediate radical (4) is formed and subsequently fragments to form again a dormant polymer species together with a radical which is capable of further chain growth.
  • a polymer formed by RAFT polymerisation may conveniently be referred to as a RAFT polymer.
  • RAFT polymer By virtue of the mechanism of polymerisation, such polymers will comprise residue of the RAFT agent that facilitated polymerisation of the monomer.
  • RAFT agents suitable for use in accordance with the invention comprise a thiocarbonylthio group (which is a divalent moiety represented by: -C(S)S-).
  • RAFT agents are described in Moad G.; Rizzardo, E; Thang S, H. Polymer 2008, 49, 1079-1131and Aust. J. Chem., 2005, 58, 379-410; Aust. J. Chem., 2006, 59, 669-692; Aust. J. Chem., 2009, 62, 1402-1472 (the entire contents of which are incorporated herein by reference) and include xanthate, dithioester, dithiocarbamate and trithiocarbonate compounds.
  • a RAFT agent suitable for use in accordance with the invention may be represented by general formula (I) or (II):
  • x is an integer > 3; and y is an integer > 3.
  • R* and Z* may represent a core moiety (CM).
  • R and R* will typically be an optionally substituted organic group that function as a free radical leaving group under the polymerisation conditions employed and yet, as a free radical leaving group, retain the ability to reinitiate polymerisation.
  • Z* is a y-valent group, with y being an integer > 2. Accordingly, Z* may be di-valent, tri-valent or of higher valency. Generally, y will be an integer ranging from 2 to about 20, for example from about 2 to about 10, or from 2 to about 5.
  • R in RAFT agents used in accordance with the invention include optionally substituted, and in the case of R* in RAFT agents used in accordance with the invention include a x-valent form of optionally substituted, alkyl, alkenyl, alkynyl, aryl, acyl, carbocyclyl, heterocyclyl, heteroaryl, alkylthio, alkenylthio, alkynylthio, arylthio, acylthio, carbocyclylthio, heterocyclylthio, heteroarylthio, alkylalkenyl, alkylalkynyl, alkylaryl, alkylacyl, alkylcarbocyclyl, alkylheterocyclyl, alkylheteroaryl, alkyloxyalkyl, alkenyloxyalkyl, alkynyloxyalkyl, aryloxyalkyl, alkylacyloxy, alkylcarbocyclyloxy
  • alkyl, alkenyl etc is intended to mean each group such as alkyl and alkenyl is optionally substituted.
  • R in RAFT agents used in accordance with the invention also include optionally substituted, and in the case of R* in RAFT agents used in accordance with the invention also include an x-valent form of optionally substituted, alkyl; saturated, unsaturated or aromatic carbocyclic or heterocyclic ring; alkylthio; dialkylamino; an organometallic species; and a polymer chain.
  • R in RAFT agents used in accordance with the invention include optionally substituted, and in the case of R in RAFT agents used in accordance with the invention include an x-valent form of optionally substituted, Ci-Cis alkyl, C 2 -Ci 8 alkenyl, C 2 -C 18 alkynyl, C 6 -Ci 8 aryl, Ci-Cis acyl, C 3 -C 18 carbocyclyl, C 2 -C 18 heterocyclyl, C 3 -C 18 heteroaryl, Q-Cis alkylthio, C 2 -Ci 8 alkenylthio, C 2 -Ci 8 alkynylthio, C 6 -Ci 8 arylthio, Q-Cis acylthio, C 3 -C 18 carbocyclylthio, C 2 -Ci 8 heterocyclylthio, C 3 -C 18 heteroarylthio, C 3 -C 18 alkylalkenyl, C
  • polystyrene More specific examples include polystyrene, polyacrylamide, poly(methyl acrylate), poly(methyl methacrylate), poly(n-butyl acrylate), poly (tert-butyl acrylate), poly(acrylic acid), poly (vinyl acetate), poly(vinyl pyrrolidone), poly(N- isopropyl acrylamide), polystyrene-block-poly(tert-butyl acrylate), polystyrene-block- poly(acrylic acid), poly (para-acetoxystryene), poly(para-hydroxystyrene), poly(N,N- dimethyl acrylamide, poly(hydroxyethyl acrylate), poly(oligoethylene glycol acrylate), poly(N,N-dimethylaminoethyl methacrylate), poly(N-acryloylmorpholine), poly(methyl methacrylate)-block-poly(styrene), poly(ethyleneoxide)-block
  • R in RAFT agents used in accordance with the invention include, and in the case of R* in RAFT agents used in accordance with the invention include an x-valent form of, an optionally substituted polymer chain
  • the polymers chain may be formed by any suitable polymerisation process such as radical, ionic, coordination, step-growth or condensation polymerisation.
  • Living polymerisation agents that comprise a polymer chain are commonly referred to in the art as "macro" living polymerisation agents.
  • Such "macro" living polymerisation agents may conveniently be prepared by polymerising one or more ethylenically unsaturated monomers under the control of a given living polymerisation agent.
  • such a polymer chain is formed by polymerising ethylenically unsaturated monomer under the control of a RAFT agent.
  • Z in RAFT agents used in accordance with the invention include optionally substituted, and in the case of Z* in RAFT agents used in accordance with the invention include a y-valent form of optionally substituted: F, CI, Br, I, alkyl, aryl, acyl, amino, carbocyclyl, heterocyclyl, heteroaryl, alkyloxy, aryloxy, acyloxy, acylamino, carbocyclyloxy, heterocyclyloxy, heteroaryloxy, alkylthio, arylthio, acylthio, carbocyclylthio, heterocyclylthio, heteroarylthio, alkylaryl, alkylacyl, alkylcarbocyclyl, alkylheterocyclyl, alkylheteroaryl, alkyloxyalkyl, aryloxyalkyl, alkylacyloxy, alkylcarbocyclyloxy, alkylheterocyclyloxy,
  • Z in RAFT agents used in accordance with the invention include optionally substituted, and in the case of Z* in RAFT agents used in accordance with the invention include a y-valent form of optionally substituted: F, CI, Ci-Cis alkyl, C 6 -Ci8 aryl, Ci-Cis acyl, amino, C3-C18 carbocyclyl, C2-C18 heterocyclyl, C3-C18 heteroaryl, Q-Cis alkyloxy, C 6 -Ci 8 aryloxy, Q-Cis acyloxy, C 3 -C 18 carbocyclyloxy, C 2 - Ci 8 heterocyclyloxy, C 3 -C 18 heteroaryloxy, Q-Cis alkylthio, C 6 -Ci 8 arylthio, Q-Cis acylthio, C 3 -C 18 carbocyclylthio, C 2 -Cis heterocyclylthio, C 3 -C 18 heteroarylthi
  • R k is selected from optionally substituted Ci-Ci 8 alkyl, optionally substituted C 6 -Ci 8 aryl, optionally substituted C 2 -Ci 8 heterocyclyl, and optionally substituted C 7 -C 24 alkylaryl, cyano (i.e. -CN), and -S-R, where R is as defined in respect of formula (II).
  • the RAFT agent used in accordance with the invention is a trithiocarbonate RAFT agent and Z or Z* is an optionally substituted alkylthio group.
  • MacroRAFT agents suitable for use in accordance with the invention may obtained commercially, for example see those described in the SigmaAldrich catalogue (w w w . si gmaaldrich .com).
  • RAFT agents that can be used in accordance with the invention include those described in WO201083569 and Benaglia et al, Macromolecules. (42), 9384-9386, 2009, the entire contents of which are incorporated herein by reference.
  • the polymerisation will usually require initiation from a source of free radicals.
  • a source of initiating radicals can be provided by any suitable means of generating free radicals, such as the thermally induced homolytic scission of suitable compound(s) (thermal initiators such as peroxides, peroxyesters, or azo compounds), the spontaneous generation from monomers (e.g. styrene), redox initiating systems, photochemical initiating systems or high energy radiation such as electron beam, X- or gamma-radiation.
  • suitable compound(s) thermal initiators such as peroxides, peroxyesters, or azo compounds
  • suitable compound(s) such as peroxides, peroxyesters, or azo compounds
  • spontaneous generation from monomers e.g. styrene
  • redox initiating systems e.g. styrene
  • photochemical initiating systems e.g. X- or gamma-radiation.
  • Thermal initiators are generally chosen to have an appropriate half life at the temperature of polymerisation. These initiators can include one or more of the following compounds:
  • Photochemical initiator systems are generally chosen to have an appropriate quantum yield for radical production under the conditions of the polymerisation. Examples include benzoin derivatives, benzophenone, acyl phosphine oxides, and photo-redox systems.
  • Redox initiator systems are generally chosen to have an appropriate rate of radical production under the conditions of the polymerisation; these initiating systems can include, but are not limited to, combinations of the following oxidants and reductants: oxidants: potassium, peroxydisulfate, hydrogen peroxide, t-butyl hydroperoxide. reductants: iron (II), titanium (III), potassium thiosulfite, potassium bisulfite.
  • Initiators that are more readily solvated in hydrophilic media include, but are not limited to, 4,4-azobis(cyanovaleric acid), 2,2'-azobis ⁇ 2-methyl-N-[l,l-bis(hydroxymethyl)-2- hydroxyethyl]propionamide ⁇ , 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis(N,N'-dimethyleneisobutyramidine), 2,2'-azobis(N,N'- dimethyleneisobutyramidine) dihydrochloride, 2,2'-azobis(2-amidinopropane) dihydrochloride, 2,2'-azobis ⁇ 2-methyl-N- [1,1 -bis(hydroxymethyl)-2-ethyl]propionamide ⁇ , 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis(isobutyramide) dihydrate, and derivatives thereof.
  • Initiators that are more readily solvated in hydrophobic media include azo compounds exemplified by the well known material 2,2'-azobisisobutyronitrile.
  • Other suitable initiator compounds include the acyl peroxide class such as acetyl and benzoyl peroxide as well as alkyl peroxides such as cumyl and t-butyl peroxides. Hydroperoxides such as t-butyl and cumyl hydroperoxides are also widely used.
  • the present invention also relates to methods and uses involving the inhibitor agents to inhibit amyloid fibril formation that can be conducted in an industrial context in relation to the production or processing of proteins that are prone to the formation of amyloid fibril structures.
  • Such methods comprise the introduction of an effective amount (that can readily be titrated by a skilled person for the particular circumstances encountered) of one or more inhibitor compounds into the processing or production environment in order that the proteins are brought into contact or exposed to the inhibitor agents, for example by simple mixing and dissolution of the agents into a batch or continuous production stream.
  • the invention also relates to methods of treatment or prevention of subjects suffering from or prone to suffer from amyloid related diseases or disorders, which involve administering an effective amount of the inhibitor agent to the subject.
  • Example of diseases or disorders that are associated with amyloid fibril formation include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, type II diabetes, transmissible spongiform encephalopathy, medullary carcinoma of the thyroid, isolated atrial amyloidosis causing cardiac arrhythmia, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinoma, familial amyloid polyneuropathy, hereditary non- neuropathic systemic amyloidosis, dialysis related amyloidosis, Finnish amyloidosis, lattice corneal dystrophy, cerebral amyloid angiopathy, Icelandic type cerebral amyloid angiopathy, systemic AL amyloidosis and sporadic inclusion body myositis.
  • subject means an animal, preferably mammalian and including a human, who is suffering from or who is prone to or suspected of suffering from an amyloid associated disease.
  • Animal subjects include primates, livestock animals (including cows, horses, sheep, pigs and goats), companion animals (including dogs, cats, rabbits and guinea pigs), captive wild animals (including those commonly found in a zoo environment), and aquatic animals (including freshwater and saltwater animals such as fish and crustaceans).
  • Laboratory animals such as rabbits, mice, rats, guinea pigs and hamsters are also contemplated as they may provide a convenient test system.
  • the subject is a human subject.
  • administration of the inhibitors to a subject is meant that the agent or composition is presented such that it can be or is transferred to the subject.
  • mode of administration There is no particular limitation on the mode of administration, but this will generally be by way of the oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intrathecal, and intraspinal), inhalation (including nebulisation), rectal or vaginal modes.
  • compositions such as veterinary and pharmaceutical compositions comprising the inhibitors of the present invention.
  • composition will comprise the nucleic inhibitors of the present invention and one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • a preferred agent for use in the methods, uses and compositions of the invention is arabinogalactan protein (AGP), which can be derived from naturally occurring gum Arabic (GA), which itself can also be used as an inhibitor of the invention.
  • AGP arabinogalactan protein
  • GA naturally occurring gum Arabic
  • the inhibitors are typically formulated for administration in an effective amount.
  • effective amount and therapeutically effective amount typically mean a sufficient amount of the agent to provide in the course the desired therapeutic or prophylactic effect in at least a statistically significant number of subjects. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner would typically balance the potential benefits against the potential risks in determining what is an appropriate "effective amount", taking into account the general medical condition, species, age, weight, sex, pregnancy status, and ethnic background of the patient as well as the mode of administration, as appropriate.
  • the exact amount required may also vary from subject to subject, so that dosages can be optimized with individual patients over time. Thus, it may not be possible to specify an exact "effective amount”. However, an appropriate "effective amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.
  • an effective amount for a human subject lies in the range of about O. lng/kg body weight/dose to lg/kg body weight/dose. In some embodiments, the range is about ⁇ g to lg, about lmg to lg, lmg to 500mg, lmg to 250mg, lmg to 50mg, or ⁇ g to lmg/kg body weight/dose. Dosage regimes are adjusted to suit the exigencies of the situation and may be adjusted to produce the optimum therapeutic or prophylactic dose. For example, several doses may be provided daily, weekly, monthly or other appropriate time intervals.
  • the time and conditions sufficient for treatment can be determined by one skilled such as a medical practitioner who is able to specify a therapeutically or prophylactively effective amount.
  • pharmaceutically acceptable carrier excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable; that is, the material may be administered to a subject along with the complex of the present invention without causing any or a substantial adverse reaction.
  • Carriers may include excipients and other additives such as diluents, detergents, colouring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
  • Example 1 Preparation of amyloid fibril formation inhibitory agents and determination of amyloid fibril formation inhibitory activity
  • Arabinogalactan Protein is a natural surfactant found in Gum Arabic (GA). GA has been widely used in the food industry for its emulsifying capabilities [26] and consists of 88.4% by mass Arabinogalactan which is a 3.8 x 10 5 Da polysaccharide, 10.4% AGP with a Mw of 1.45 x 10 6 Da, and 1.24% Glycoprotein with a Mw of 2.5 x 10 5 Da [27]. The exact molecular weights of the components, especially AGP vary in the literature [28, 29].
  • AGP is believed to either exist as a "wattle blossom” structure or as a “twisted hairy rope” [30] or a structure with both properties [31, 32].
  • the inventors developed the hypothesis that the hydrophobic core and hydrophilic side chains of AGP could give rise to activity in inhibiting amyloid fibril formation by a putative mechanism involving interaction with exposed hydrophobic surfaces of amyloid intermediates.
  • This proposed mechanism of action of AGP as an amyloid inhibitor is illustrated in Figure 4.
  • the hydrophilic carbohydrate groups will prevent further aggregation of the amyloid by physically preventing more intermediates from binding to the exposed hydrophobic surface of a growing fibril, as illustrated in Figure 3.
  • ThT Thioflavin T
  • Fa-diacid IUPAC name poly-3,5-divinylcyclopentane-l,2-dicarboxilicacid
  • Fa-diacid has a similar structure to the AGP molecule in that it has a hydrophobic 5-membered ring backbone similar to the hydroxyproline rich backbone of AGP, and freely rotating hydrophilic side chains.
  • amphiphillic surfactants that have a hydrophobic backbone and hydrophilic side- chain were also tested. The structures of these compounds are shown in Figure 4.
  • the carboxylic acid side chains of the Fa-diacid compound present opportunities to attach hydrophilic groups such as sugar moieties to produce compounds with structures analogous to that of AGP. It was speculated that such modification could give rise to improved inhibition of amyloid fibril formation.
  • Sugar substituted derivatives of the Fa- diacid compound produced and tested included PNGE, which has a cyclic sugar attached to it and PNGA, which includes an open-chain sugar group side chain.
  • PNGE which has a cyclic sugar attached to it
  • PNGA which includes an open-chain sugar group side chain.
  • a modified version of the Fa-diacid compound that includes an oxygen group in the backbone was also produced and tested to investigate the effect on inhibitor activity of variations in backbone structure. The structures of these compounds are shown in Figure 5.
  • the solution was then filtered (0.45 ⁇ filter), concentrated in vacuo to ca. 3 mL in volume and precipitated into acetone (40 mL).
  • the precipitate was collected via centrifugation and redissolved in water (3 mL) and precipitated once again into acetone (40 mL).
  • the precipitate was isolated via centrifugation, dissolved in the minimum amount of water and then dialysed (2 kDa MWCO) against water for 48 h.
  • the solution was then concentrated in vacuo to afford FA- diacid as a pale brown crystalline solid, 0.17 g.
  • Different molecular weight FA-diacids were prepared by variation of the monomer to catalyst ratio.
  • oxoFA-diacid was prepared from exo- 3,6-epoxy-l,2,3,6-tetrahydrophthalic anhydride 3b via the polymeric anhydride 4b.
  • a solid-phase Fmoc synthesis strategy was employed, which was performed on a CEM Liberty Microwave peptide synthesiser using Tentagel amide resin (0.22mmol/g, 455mg) on a O.lmmol scale.
  • Fmoc-Hyp(Gal-Ac4)-OH (3 eq., 0.3mmol) was activated with HATU (3 eq., 0.3mmol) and catalysed with DIPEA (6 eq., 0.6mmol) in DMF.
  • 20 minute microwave couplings were employed (25W at 75°C).
  • Fmoc cleavage was effected by a 20% piperidine solution in DMF for 3 minutes under microwave conditions (45W at 75°C).
  • the peptide was cleaved from the resin by treatment with a solution of TFA/H 2 0 (97.5/2.5) for 2 hours. The reaction mixture was then filtered and aspirated with a stream of nitrogen until all TFA had evaporated, then dissolved in 50% acetonitrile/H 2 0 and lyophilised. A mass yield of 160mg was recovered.
  • the crude peptide was analysed via RP-HPLC on an Agilent 1100 HPLC system using a Phenomenex Gemini 5u C18 (4.6 x 150mm) column.
  • the molecular weight was confirmed via ESI-MS on an Agilent Accurate-Mass TOF LC/MS.
  • the acetyl-protected peptide was then treated with a catalytic amount of NaOMe in methanol for 48 hours.
  • the clean fractions were pooled and lyophilised, giving a final mass yield of 12.7mg, and the correct molecular weight of 1943.8 was confirmed via ESI-MS.
  • n and m independently represent the integers 1 to 1000.
  • N-Hydroxysuccinimide (1.1 g, 9.2 mmol), irans-4-hydroxy-L-proline (0.20 g, 1.5 mmol) and DMAP (77 mg, 0.63 mmol) were weighed into a Schlenk tube and N,N- dimethylformamide (DMF) (5 mL) and water (5 mL) were added.
  • DMF N,N- dimethylformamide
  • N,N-diisopropylethylamine (0.26 g, 2.0 mmol) and N-(3-dimethylaminopropyl)- N'-ethylcarbodiimide hydrochloride (EDCI) (385, 2.0 mmol) were added, the flask was sealed, and the mixture was stirred for 24 h to afford a homogeneous solution. The solution was then heated at 100 °C for 7 days. After cooling to room temperature the solution was precipitated into acetone (100 mL) and the precipitate was isolated via centrifugation.
  • DIPEA DIPEA
  • EDCI N-(3-dimethylaminopropyl)- N'-ethylcarbodiimide hydrochloride
  • This compound was synthesised using solid state peptide addition with appropriate capping agents in order to direct the reaction.
  • the final product was analysed using mass spec and shown to be the desired product .
  • the structure of this compound is shown in Figure 18(c).
  • Bovine Insulin (BI) from Sigma Aldrich was weighed and dissolved in 1.5ml of 0.1% (ph 1.5) solution of HC1 that was filtered through a 0.2um syringe filter.
  • the BI concentration was determined spectroscopically with a Varian Cary 3E UV-visible Spectrophotometer using an extinction coefficient at 280nm of 5730 M ⁇ cm "1 [41, 42] and a Mw of 5734 g/mol.
  • ThT was added to the solution as well as a volume of any inhibitor used to make a final solution of 0.2mg/ml BI, 50um ThT and inhibitor in 0.1% HC1.
  • Inhibitors added were either 2mg/ml GA, 2mg/ml AGP, 0.0125mg/ml Fa-diacid (lOkDa) or 0.0025mg/ml Fa- diacid (2kDa).
  • concentrations of the Fa-diacid were the same molar concentration as that of 2mg/ml AGP (0.00125 ⁇ /ml).
  • Amyloid Beta ( ⁇ ) 1-40 from Yale University Keck Laboratory was weighed out and dissolved in filtered (0.2um nylon membrane filter) 0.5M, pH 7.1 phosphate buffer of an appropriate concentration. This was left 18 hours overnight at 4°C in the refrigerator to solubilise the protein before concentration was verified spectroscopic ally. Tht and any inhibitor were then added from stock solutions to obtain a final solution of 0.277mg/ml ⁇ , 50um ThT in Phosphate buffer. Inhibitors added were either 2mg/ml GA, 2mg/ml AGP, 0.0125mg/ml Fa-diacid (lOkDa) or 0.0025mg/ml Fa- diacid (2kDa). 3ml of this solution was incubated in a 1cm x 1cm quartz cuvette with a stirrer bar at 37°C, 550RPM stirring speed and the fluorescence monitored using kinetics program as with insulin.
  • Hen egg white lysozyme from Sigma- Aldrich, L7651, 3X crystallized, dialyzed and lyophilized, used without further treatment
  • the tube was then left in the refrigerator at 4°C to settle for 15 minutes. Following that the sample was centrifuged for 10 minutes at 10,000G to remove dust and insoluble materials.
  • the absorbance at 280nm wavelength of the stock was then analyzed in a Cary 3E Absorbance spectrophotometer and the mass calculated using a Mw of HSOOgmol "1 and a extinction coefficient of 37895M "1 cm “1 .
  • AGP was used to inhibit BI and AB fibril growth in vitro in order to show proof of principle. For BI a significant increase in the induction (lag) time for fibril formation (reduction in fibril formation rate) was observed while for AB a marked decrease in the ThT fluorescence intensity was observed. AGP was shown to inhibit fibril formation and growth as detected by ThT fluorescence. Without wishing to be bound by theory the inventors believe that the mechanism of stabilisation is via both binding to the hydrophobic faces of the beta sheets and through stabilisation of the alpha helical structures in the protein/peptide systems.
  • the Fa-diacid (at both molecular weights), AGP and GA all showed inhibitory effects relative to the control in which no inhibitor was present.
  • the inhibitors increased the lag time by varying degrees and also decreased the final fibril concentration formed.
  • Figure 8 shows the effect of the addition of GA, purified AGP, and 2 molecular weights of the Fa-diacid to BI.
  • the formation of BI amyloid fibrils as seen in Figure 8 was reduced significantly by the addition of the inhibitors.
  • the addition of 2mg/ml of GA to 0.2mg/ml of BI increased the lag time of amyloid fibril formation from 5 hours to 24 hours.
  • Using 2mg/ml of purified AGP further increased the lag time to approximately 45 hours as seen in Figure 8.
  • Figure 19 shows the concentration dependent effect of variable concentrations (0.02mg/ml and 0.2mg/ml) of Acetyl-(phenylalaninyl-0-galactosehydroxyprolyline- phenylalaninyl)15-amide in inhibiting bovine insulin fibril formation.
  • AFM imaging showed that larger but fewer fibrils were formed when BI and ⁇ were incubated with AGP and GA than without any inhibitor (Figure 14), which may account for the higher final intensity as seen from AGP and GA.
  • Large fibrils from BI appear to be a rope-like bundle of several smaller amyloid fibrils to form a large superstructure.
  • BI fibrils grown in the presence of Fa-diacid tend to form fibrils with similar morphology to those grown without any inhibitor.
  • the lower fibril density seen in ⁇ amyloid fibrils incubated with GA and AGP may account for the lower fluorescent intensity seen.
  • ⁇ fibrils formed in the presence of an inhibitor appear to be much longer than those formed without inhibitors. The actual density of fibrils is difficult to quantify for ⁇ due to the fact that fibrils often form aggregates.
  • CD scans at various time points show that when BI is incubated with GA, there is a preservation of the alpha-helical structure of the BI until elongation occurs. This is in contrast to when BI is incubated alone, where the conversion of the alpha-helical structure to the beta-sheet occurs near the start of the reaction, as shown below in Table 1. a-helix (%) ?-sheet (%)
  • Table 1 Total a-helical and ⁇ -sheet structural content from CD scans of BI fibrils grown with AGP, showing preservation of a-helical structure when incubated with GA. Analysis was done using web-based software Dichroweb, CDSSTR algorithm [38, 39]. CD scans of BI with GA were using a blank of 2mg/ml GA in HCL and ThT.
  • CD scans of ⁇ incubated with AGP show that the structure changes from random coil to a mixture of random coil and beta-sheet, while ⁇ incubated without AGP resulted in predominantly ⁇ -sheet CD spectra.
  • This effect may be due to the fact that AGP has a random coil structure and contributes to the effects seen.
  • due to the contribution to the signal and overall protein mass made by the AGP as well as the noise of the signal it was difficult to accurately deconvolute using Dichroweb.
  • Natural GA was also tested at various concentrations against 0.2mg/ml of BI and the results show that at 2mg/ml and above the lag time remains constant at approximately 22 hours. However, the final intensity seems to peak between 0-lmg/ml and then rapidly decreases at higher concentrations of GA. This is shown in Figures 9 and 10.
  • FA-diacid which was the most effective of the experimental compounds from Figure 4, was subject to further testing. When the concentration was increased to 2mg/ml, no fibril formation was observed. AFM images, shown in Figure 11, were taken at the end of that experiment of show disordered aggregates before and after the experiment, with no fibril formation observed.
  • FA diacid (lOkDa) was tested over a range of concentrations as shown in Figure 12 and a concentration dependent increase in lag time was found for the amount of Fa-diacid added to the system.
  • this compound there appears to be an optimal concentration for inhibition of between 0.05mg/ml and 0.3mg/ml, as higher concentrations of Fa-diacid (i.e. 0.3488mg/ml and above) led to flocculation of the protein.
  • a modified version of Fa-diacid including an oxygen atom in the backbone 5-carbon ring and also a 10-monomer polyhydroxyproline molecule was shown to bind to and inhibit the amyloid fibril formation of BI more effectively than Fa-diacid, even at a concentration of 0.0875mg/ml. This was confirmed using AFM, which revealed very few, small fibrils in the AFM image of the sample. However, the oxy-fa compound did cause flocculation in the mixture, as observed in high concentrations of Fa- diacid.
  • polyhydroxyproline compound exhibited some inhibitory ability, causing a slight increase in lag time to 10 hours at 2mg/ml inhibitor with a final intensity of about 140.
  • AFM imaging showed that the fibrils formed were similar to those formed without inhibitor. This can be seen in Figure 13.
  • polyhydroxyproline does not appear to be as effective an inhibitor as 2mg/ml of GA or AGP.
  • Atomic force microscopy images taken of the fibrils are seen in Figure 17. They show that there is a slight change in fibrillar morphology when the fibrils are grown in an inhibitor. The fibrils appear to be slightly thicker than that of the control and this effect is most pronounced in GA. It is notable that the AFM is unable to determine fibril density due to the fact that the fibrils tend to clump together during the preparation of the AFM slide.
  • Circular Dichroism shows that there is an increase in the beta-sheet content of the protein after fibril formation occurs, (note: data not shown)
  • AGP may stabilise the native alpha-helical structure of the protein, as suggested from Circular Dichroism measurements as seen in Table 1. By stabilising the native protein conformation, the AGP will increase the lag time to amyloid fibril formation due to prevention of protein denaturation.
  • AGP exhibits a preference for stabilising alpha-helical structures over random coil structures, resulting in better binding to the protein and hence improved inhibition.
  • AGP also binds to exposed hydrophobic surfaces of beta-sheets and in so doing, prevents aggregation, as depicted in Figure 3.
  • AGP inhibits fibril formation even when added after BI has reached the elongation stage (data not shown), where the insulin is largely in beta-sheet form [37]. This may explain why AGP is able to inhibit ⁇ amyloid fibril formation, as well as explaining the difference in kinetics between the proteins.
  • GA seems to increase in lag time and has a further reduction in final fluorescence intensity as compared with AGP for ⁇ , indicating that GA is a more effective inhibitor than AGP. This is likely either due to a contribution from the AGP fraction or because 2mg/ml of AGP is too concentrated, and AGP loses its effectiveness beyond a certain concentration ratio to that of the amyloid. Alternatively this may be due to a change in fibril morphology when exposed to pure AGP, as opposed to when exposed to the other components of GA.
  • Fa-diacid was shown to possess similar inhibitory ability to AGP towards BI and ⁇ , although it does not cause an increase in the lag time for ⁇ but does so for BI.
  • lOkDa Fa-diacid is a more effective inhibitor than the 2kDa version.
  • the lOkDa Fa-diacid gives rise to a larger decrease in intensity in both BI and ⁇ , but is less effective than AGP.
  • the difference in efficacy between the two molecular weight samples also supports the hypothesis that the inhibitor works by binding to multiple beta- sheet precursors of the amyloid fibrils, and hence the total mass of the inhibitor may be more important than the total number of inhibitor molecules.
  • the Fa-diacid follows a different inhibitory pathway to GA by causing the BI to form amorphous aggregates and hence preventing amyloid fibril formation. This effect seems to be more pronounced with the Oxy-Fa compound.
  • PNGA, PNGE and GA are shown to be effective at reducing the amount of lysozyme amyloid fibrils formed as well as increasing the amount of time that the fibrils take to form.
  • GHP also does show some inhibitory activity, however it is less significant as compared to the other 3 inhibitors tested, while of the inhibitors tested, PNGA has the highest inhibitory activity per mass, followed by PNGE then GA.
  • Those inhibitors also appear to reduce the amount of fibrils formed as supported by evidence from ThT fluorescence as well as the ultracentrifugation results.
  • lysozyme appears to be more susceptible to inhibition than BI and ⁇ , which may be due to either the fact that lysozyme is larger or more stable than BI or ⁇ as it requires a harsher condition to form fibrils. Additionally, in lieu of the results obtained from BI, it is shown that GA, PNGE and PNGA are effective as a generic amyloid fibril inhibitors.
  • Polyhydroxyproline appears to be a less effective inhibitor than AGP. This is likely the case as polyhydroxyproline lacks a large hydrophilic steric group, or a carbohydrate/sugar side chain, so it may be simply be incorporated into the amyloid fibril structure over time, as it cannot block further aggregation as effectively with only a lone OH group.
  • a sugar- modified version of this polyhydroxyproline is expected to exhibit improved inhibition of amyloid fibril formation.
  • Amyloid fibril formation is associated with a large variety of diseases in the human body as well as problems in the manufacture of some proteins.
  • AFM and CD techniques AGP from GA as well as the similarly structured Fa-diacid, PNGE and PNGA were shown to be able to inhibit formation of amyloid fibrils in BI and ⁇ . It is proposed that the inhibitors work by preserving native alpha-helical structures as well as binding to exposed hydrophobic surfaces of the precursors to prevent amyloid fibril formation.

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Abstract

An inhibitor of amyloid fibril formation comprising: (a) a backbone that is hydrophobic or hydrophilic and comprises from 1 to about 1000 repeating units; (b) one or more hydrophobic or hydrophilic pendant chains linked to the backbone, wherein the pendant chains are the same or different and comprise from 1 to about 1000 repeating units that are able to freely rotate about the backbone due to free rotation of the repeating units within the backbone; wherein when the backbone is hydrophobic the pendant chains are hydrophilic and when the backbone is hydrophilic the pendant chains are hydrophobic.

Description

Inhibition of Amyloid Fibril Formation
Field of the Invention
The present invention relates to compounds and compositions comprising them that are capable of inhibiting the formation of amyloid fibril formation. The invention also relates to methods and uses involving the inhibitor compounds for reducing or preventing amyloid fibril formation in industrial processes and for treating or preventing diseases or disorders associated with amyloid fibril formation.
Background of the Invention
Amyloid fibrils, also referred to as amyloid plaques, are a leading cause of many degenerative diseases such as Alzheimer's disease [12], Parkinson's disease [1], type II diabetes, various prion diseases and atherosclerosis, among many others. In fact, there are at least 40 known diseases associated with amyloidosis [1, 2] and a table of amyloid associated diseases and the corresponding amyloid protein is provided in reference 2, the disclosure of which is included herein in its entirety by way of reference. Amyloid fibril plaques are resistant to proteolysis [13] and were first observed in the brains of Alzheimer's disease patients in 1963 [11]. Amyloid fibril associated diseases, such as Alzheimer's disease, are presently incurable.
Amyloid fibrils have generic features defining the fibrillar state. These are the misfolding of the protein into a beta sheet form and the assembly of the beta sheets into associated structures where the hydrophobic interfaces of the beta sheets drive the beta sheets together to form the larger fibrillar structures observed in a range of pathologies. The beta sheets are formed through association of peptide sequences via hydrogen bonding. The beta sheets associate through hydrophobic interactions between their faces to form fibrils [1, 5].
The effect of amyloids on organisms is not well established, it has been argued in the literature that the cytotoxic effects seen in amyloid related pathologies are from the precursors or the free-floating units that make up the amyloids, and that the mature fibrils are inert [14]. However there is evidence that fibrils and precursors grown under different shear conditions exhibit differing cytotoxicity levels [15]. More recently though it has been reported that in vivo the amyloids cause a variety of effects to cell membranes and the combination of these effects is responsible for the various amyloid related pathologies.
Amyloid fibril formation also causes problems in industries involving the processing of proteins such as in the food, nutritional supplements, blood products, pharmaceutical, insulin [16] and dairy [17] industries. Some proteins that usually do not form fibrils under physiological conditions may be subject to amyloid fibril formation during industrial processing due to the treatment conditions adopted. In many cases this is undesirable as the presence of amyloids will reduce the quality of product, result in wastage of protein and cause fouling in machine parts [17] leading to production delays and maintenance costs.
There have been many attempts at developing inhibitors against amyloid fibril formation. Treatment with chelators has been found to improve the condition of patients suffering from Alzheimer's disease [18, 19] as the chelators bind to the free copper and iron ions that play an important role in Αβ fibril formation. In 1996 the Elan company conducted a phase 2 clinical trial [21] in respect of the use of antibodies raised against the Αβ [20] amyloid fibres. However the trial was halted after some patients developed meningoencephalitis. Other methods of inhibiting amyloid fibril formation that have been attempted include using synthetic peptides that bind to fibrils [10, 22] and using small ligands to stabilise native AB [23].
A number of US patent documents have referred to agents that purport to exhibit some activity in inhibiting amyloid fibril formation, such as US 6794363, US 2002/0042420, US 7166622, US 2011/2075640, US 2005/0054732, US 2010/0204085, US 7288659, US 2008/0004211, US 7790856, and US 6022859.
As amyloid fibril formation relies on interactions between the exposed hydrophobic surfaces of amyloid fibril precursors [1, 5], surfactants that can bind to these surfaces can inhibit amyloid fibril formation. Indeed, the deployment of amphiphilic surfactants has been shown to result in inhibition of model amyloid systems [24] and specific targeting of residues involved in aggregation, such as lysine, can prevent amyloid formation [25]. There is, however, a pressing need for the development of alternative inhibitors of amyloid fibril formation that can be used to improve the understanding of the mechanism of amyloid fibril formation and which may offer potential in industries associated with the processing of proteins subject to amyloidosis and/or in the therapy / prevention of amyloid associated diseases or disorders.
Summary of the Invention
According to one aspect of the present invention there is provided an inhibitor of amyloid fibril formation comprising:
(a) a backbone that is hydrophobic or hydrophilic and comprises from 1 to about 1000 repeating units;
(b) one or more hydrophobic or hydrophilic pendant chains linked to the backbone, wherein the pendant chains are the same or different and comprise from 1 to about 1000 repeating units that are able to freely rotate about the backbone due to free rotation of the repeating units within the backbone;
wherein when the backbone is hydrophobic the pendant chains are hydrophilic and when the backbone is hydrophilic the pendant chains are hydrophobic.
According to another aspect of the present invention there is provided an inhibitor of amyloid fibril formation comprising:
(a) a backbone that is hydrophobic or hydrophilic and comprises from about 3 to about 500 repeating units;
(b) a plurality of hydrophobic or hydrophilic pendant chains linked to the backbone, wherein the pendant chains are the same or different and comprise from about 2 to about 1000 repeating units that are able to freely rotate about the backbone due to free rotation of the repeating units within the backbone;
wherein when the backbone is hydrophobic the pendant chains are hydrophilic and when the backbone is hydrophilic the pendant chains are hydrophobic. According to another aspect of the invention there is provided an inhibitor of amyloid fibril formation comprising:
(a) a backbone that is hydrophobic and comprises from 1 to about 1000 repeating units;
(b) one or more hydrophilic pendant chains linked to the backbone, wherein the pendant chains are the same or different and comprise from about 1 to about 1000 repeating units that are able to freely rotate about the backbone due to free rotation of the repeating units within the backbone.
According to another aspect of the invention there is provided an inhibitor of amyloid fibril formation comprising:
(a) a backbone that is hydrophobic and comprises from about 3 to about 500 repeating units;
(b) a plurality of hydrophilic pendant chains linked to the backbone, wherein the pendant chains are the same or different and comprise from about 2 to about 1000 repeating units that are able to freely rotate about the backbone due to free rotation of the repeating units within the backbone.
According to a further aspect of the invention there is provided an inhibitor of amyloid fibril formation of Formula I:
Figure imgf000005_0001
Figure imgf000005_0002
I
T2
Formula I wherein:
T, T1 and T2 are the same or different and represent a terminal group;
L, L 1 , L2 and L 3 are the same or different and represent a linker group;
A represents a hydrophobic backbone unit;
R represents a hydrophilic pendant chain unit that is bound, optionally via linker group L 2 , to any of A, L or L 1 ;
a, a 1 , a2 and a 3 are the same of different and represent the numerals 0, 1, 2 or 3; b represents the numerals 1 to 1000;
c represents the numerals 0 to 1000, but represents 1 or greater in at least 25% of cases;
and wherein free rotation is allowed within and/or between adjacent A units.
According to a further aspect of the invention there is provided an inhibitor of amyloid fibril formation of Formula I:
Figure imgf000006_0001
L3 a3
I
T2
Formula I
wherein:
T, T1 and T2 are the same or different and represent a terminal group;
L, L 1 , L2 and L 3 are the same or different and represent a linker group;
A represents a hydrophobic backbone unit;
R represents a hydrophilic pendant chain unit that is bound, optionally via linker group L 2 , to any of A, L or L 1 ;
a, a 1 , a2 and a 3 are the same of different and represent the numerals 0, 1, 2 or 3; b represents the numerals 3 to 500;
c represents the numerals 0 to 1000, but represents 2 or greater in at least 50% of cases;
and wherein free rotation is allowed within and/or between adjacent A units.
For example, the hydrophobic backbone unit can comprise one or more of glycine, alanine, valine, leucine, isoleucine, methionine, selenomethionine, phenylalanine, tryptophan, proline, hydroxyproline, styrene, alpha-methyl styrene, butyl acrylate, butyl methacrylate, amyl methacrylate, hexyl methacrylate, lauryl methacrylate, stearyl methacrylate, ethyl hexyl methacrylate, crotyl methacrylate, cinnamyl methacrylate, oleyl methacrylate, ricinoleyl methacrylate, cholesteryl methacrylates, cholesteryl acrylate, vinyl butyrate, vinyl tert-butyrate, vinyl stearate, vinyl laurate, caprolactide, propylene oxide, divinyl cyclopentane and mannose.
In another example, the hydrophilic pendant chain unit comprises one or more of one or more of acrylic acid, methacrylic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate, oligo(alkylene glycol)methyl ether (meth)acrylate (OAG(M)A), acrylamide, methacrylamide, hydroxyethyl acrylate, N-methylacrylamide, N,N-dimethylacrylamide and Ν,Ν-dimethylaminoethyl methacrylate, Ν,Ν-dimethylaminopropyl methacrylamide, N-hydroxypropyl methacrylamide, norbornenes, 4-acryloylmorpholine, 2-acrylamido-2- methyl-l-propanesulfonic acid, phosphorylcholine methacrylate, N- vinyl pyrolidone, amino ethyl acrylamide, ethylene oxide.
In one aspect of the invention the inhibitor is an arabinogalactan protein (AGP).
Another aspect of the invention relates to use of an inhibitor as referred to above in an industrial process where it is desired to inhibit amyloid fibril formation.
A further aspect of the invention relates to a method of preventing or reducing amyloid fibril formation in an industrial process in which amyloid fibril formation is prone to occur, which comprises exposing amyloid fibril forming proteins in the process to an inhibitor as outlined above.
A still further aspect of the invention relates to use of an inhibitor as referred to above in treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject.
Another aspect of the invention relates to use of an inhibitor as outlined above in preparation of a medicament for treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject.
In another aspect of the invention there is provided a method of treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject, which comprises administering to the subject an effective amount of an inhibitor as outlined above.
For example, the disease or disorder associated with amyloid fibril formation can be selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, type II diabetes, transmissible spongiform encephalopathy, medullary carcinoma of the thyroid, isolated atrial amyloidosis causing cardiac arrhythmia, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinoma, familial amyloid polyneuropathy, hereditary non-neuropathic systemic amyloidosis, dialysis related amyloidosis, Finnish amyloidosis, lattice corneal dystrophy, cerebral amyloid angiopathy, Icelandic type cerebral amyloid angiopathy, systemic AL amyloidosis and sporadic inclusion body myositis.
The invention also relates to a pharmaceutical composition comprising an inhibitor as referred to above and one or more pharmaceutically acceptable excipients.
Brief Description of the Figures
The invention will be further described with reference to the following non-limiting figures, wherein: Figure 1 shows a schematic image of elements of inhibitors according to one aspect of the invention.
Figure 2 shows an image of the chemical structure of an inhibitor according to one aspect of the invention, which is tri-glycosylated 7-hydroxyproline.
Figure 3 shows a schematic of the putative binding of the inhibitor AGP to β-sheet amyloid precursors.
Figure 4 shows chemical structures of various synthethic compounds tested against (bovine insulin) BI for effectiveness against amyloid formation. Molecular weights of each of the polymers tested were as follows: P(GAMA) = 330Kda, P(Am-co-VPD25%) = 624KDa, FA-diacid (poly-3,5-divinylcyclopentane-l,2-dicarboxilicacid) = lOKDa, FA-NH3 + = 90KDa.
Figure 5 shows chemical structures of various derivative compounds from FA diacid plus the polyhydroxyproline that were tested. Chemicals are referred to as A) PNGE, B) PNGA, C) Oxy-FA and D) glycosylated polyhydroxyproline (PhP), and the variable n represents integers 1 to 1000.
Figure 6 shows the chemical structure of a glycosylated polyhydroxyproline molecule with A) 3 sugar chains (idealized), B) a single attached sugar chain and C), D) and E) show glycosylated polyhydroxyproline with hydrophobic amino acid of Phenylalanine, Tyrosine or Leucine where the R group (alkyl or aryl) determines the amino acid. The variables n and m independently represent integers 1 to 1000 and R may for example represent alkyl or aryl.
Figure 7 shows a graph of ThT fluorescence intensity against time (hrs) for Αβ amyloid fibrils grown (line) without inhibitor, ( ) with GA, ( ) with AGP, ( _ . . _ ) 2kDa
FA di-acid and ( _ . _ . ) lOkDa FA di-acid. The graph demonstrates the inhibitory effect of AGP, GA and FA di-acid of two different molecular masses on Αβ fibril formation. Figure 8 shows a graph of ThT fluorescence intensity against time (hrs) for BI amyloid fibrils grown ( _ . _ . ) without inhibitor, ( _ . . _ ) with GA, ( ) with AGP, ( )
2kDa FA di-acid and (line) lOkDa FA di-acid. The graph demonstrates the inhibitory effect of AGP, GA and FA di-acid of two different molecular masses on BI fibril formation.
Figure 9 shows a graph of ThT fluorescence intensity against concentration (mg/ml) at equilibrium (plateau) for various concentrations of GA. Values above 1000 intensity were estimated from fluorescence measured at excitation wavelength of 276nm and an emission wavelength of 470nm. The graph demonstrates that at low concentrations of GA, larger fibrils form, however the amount of fibrils decreases rapidly with increasing GA concentration.
Figure 10 shows a graph of lag time (hrs) of BI amyloid fibril formation against concentration (mg/ml) of GA. The graph demonstrates the effectiveness of delaying fibril formation of even low concentrations of GA.
Figure 11 shows AFM images of 0.2mg/ml BI incubated with 2mg/ml FA-diacid taken (A) before and (B) after incubation for 90 hours. No amyloid fibrils are observed however protein aggregates are seen both before and after incubation. Each image is ΙΟμιη χΐθμιη.
Figure 12 shows a graph of fluorescence against time (hrs) for 0.2mg/ml BI amyloid fibrils grown under various lOkDa FA diacid concentrations - control (0 mg/ml) is represented by a line, 0.0125 mg/ml is represented by , 0.05 mg/ml is represented by . _ . _ , 2 mg/ml is represented by _ . . _ and 0.3488 mg/ml is represented by The graph demonstrates that the inhibitory ability of FA diacid increases with increasing concentration of diacid, however flocculation and formation of disordered aggregates in place of amyloid formation is observed for concentrations of 2mg/ml and 0.3488mg/ml FA, which account for the unusual graph shape. Figure 13 shows a graph of fluorescence intensity against time (hrs) which allows a comparison of the results for the various inhibitors: FA diacid, PNGE, PNGA, GA, AGP and Polyhydroxyproline (PHP).
Figure 14 shows a graph of fluorescence intensity against time (hrs) for ThT fluorescence of lysozyme fibrils grown in various conditions. All inhibitors are at 0.2mg/ml concentration except for GA which is at 2mg/ml.
Figure 15 shows a graph of fluorescence intensity against time (hrs) for ThT fluorescence of lysozyme fibrils grown in the presence of various inhibitors: GA, PNGE and PNGA
Figure 16 shows AFM images of various amyloid fibrils. Top row shows BI amyloid fibrils formed (A) without inhibitor, (B) with GA and (C) with AGP. Second row shows Αβ amyloid fibrils formed (D) without inhibitor, (E) with GA and (F) with AGP. Non-fibrillar aggregates are seen in the background for (E). All images shown are ΙΟμιη x ΙΟμιη in size.
Figure 17 shows AFM images of the lysozyme fibrils formed in the presence of a range of inhibitors. A. PNGE. B. PNGA. C. GHP. D. GA and E. without inhibitors. All images shown are ΙΟμιη x ΙΟμιη in size.
Figure 18 shows chemical structures of examples of compounds of the invention that demonstrate effectiveness against amyloid formation, including (a) poly(valine-ran- (Ne-(2,3,4,5,6-pentahydroxyhexanoyl)lysine)), (b) poly(4-(tetrahydro-3,4,5-trihydroxy- 6-(hydroxymethyl)-2H-pyran-2-yloxy)proline)-co-(phenylalanine) and (c) Acetyl- (phenylalaninyl-0-galactosehydroxyprolyline-phenylalaninyl)15-amide.
Figure 19 shows a graph of fluorescence intensity against time (hrs) for ThT fluorescence of bovine insulin fibrils grown in the presence of Acetyl-(phenylalaninyl-0- galactosehydroxyprolyline-phenylalaninyl)15-amide at concentrations of 0 mg/ml (control - solid line), 0.02mg/ml (dashed line) and 0.2mg/ml (dotted line). Detailed Description of the Invention
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
As used herein, the singular forms "a", "and" and "the" are intended to include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell as well as two or more cells; reference to "an agent" includes one agent, as well as two or more agents; and so forth.
The full disclosures of publications referred to within this specification are taken to be included herein in their entirety by way of reference, unless it is clear from the context in which the publication is referred to that this is not the intention.
In its broadest sense the present invention relates to compounds that inhibit amyloid fibril formation (amyloidosis) and to methods and uses involving such compounds. Without wishing to be bound by theory, it is understood that the compounds adsorb to the hydrophobic face of the fibrils and sterically inhibit further proteins from associating to the fibril. Thus the growth of the fibrils is inhibited by the presence of the inhibiting agents. The basic structure of the inhibitors is that they include an oligomeric/polymeric backbone of either hydrophobic or hydrophilic species, wherein there is rotational freedom of the bonds in the principle axis of the backbone. There are also pendant hydrophilic or hydrophobic monomers or oligomers bound to the backbone, such that when the backbone includes hydrophobic units the pendant chains include hydrophilic units and when the backbone includes hydrophilic units the pendant chains include hydrophobic units. Due to the amphiphilic nature of the inhibitors and free rotation allowed within the backbone the inhibitors are able to adopt "brush" or "comb" three dimensional conformations, depending upon the nature of their local environment.
In one aspect of the invention the backbone is comprised of hydrophobic units that are believed to adsorb to β-sheet protein and the pendant chains comprise hydrophilic units that are believed to act as steric barriers to protein association with the growing fibril. The rotation of the pendant hydrophilic groups is a requirement for physiological solubility and so that upon exposure to the hydrophobic β-sheet the hydrophobic backbone is adsorbed onto the β-sheet and the pendant groups rotate to expose the hydrophobic backbone and render the water soluble polymer insoluble. Upon adsorption of the inhibitor to the β- sheet, the pendant groups act as steric inhibitors to inhibit further association of the protein and any association of the β-sheets through hydrophobic interactions.
A naturally occurring class of compounds known as arabinogalactan proteins (AGPs) have the features required for inhibition and are shown to inhibit fibril growth in vitro. The model inhibitor has a backbone of length approximately equal to the width of the β-sheet, with water soluble groups attached. In one specific embodiment the inhibitor is an oligomer of hydroxyproline of four to ten residues with pendant hydrophilic groups attached thereto which act as steric inhibitors of fibril growth. The proposed mechanism of action is depicted graphically in Figures 1 and 2.
Throughout this specification the agents that inhibit amyloid fibril formation are referred to generally as "inhibitors", "compounds" or "agents" according to the invention. By reference to inhibition of amyloid fibril formation it is intended to convey that when in association with amyloid fibril forming proteins the agents give rise to a reduction in the extent and/or rate of amyloid fibril formation. This can be in a laboratory setting, on an industrial scale or in a therapeutic setting for treatment or prevention of an amyloid related disease or disorder. Depending upon the context, the rate or extent of amyloid fibril formation relative to the control situation, where no inhibitor agent is present, can be determined by routine experimental techniques, such as atomic force microscopy (AFM), circular dichroism (CD) or use of an amyloid binding dye or marker that can be quantitatively detected via a signal such as fluorescence, radiation, conductivity or the like. An example of a suitable marker for amyloid formation is the fluorescent dye Thioflavin T (ThT), which exhibits a characteristic fluorescence peak when bound to amyloid protein. For example, the inhibitors of the invention decrease the rate or extent over a fixed time period of amyloid formation relative to the control situation by at least 5%, at least 10%, at least 20%, at least 40%, at least 50%, at least 80% or at least 90%, 95%, 98% or 99%. Even relatively small percentage reductions in rate and/or or extent of amyloid fibril production can be useful depending upon the context in which reduction in fibril formation is sought.
Terms such as "hydrophilic" and "hydrophobic" are generally used in the art to convey interactions between one component relative to another (e.g. attractive or repulsive interactions, or solubility characteristics) and not to quantitatively define properties of a particular component relative to another. For example, a hydrophilic component is more likely to be wetted or solvated by an aqueous medium such as water, whereas a hydrophobic component is less likely to be wetted or solvated by an aqueous medium such as water.
In the context of the present invention a hydrophilic agent, unit, monomer, oligomer, polymer or polymer block is intended to define one that exhibits solubility or miscibility in an aqueous medium, including biological fluids such as blood, plasma, serum, urine, saliva, milk, seminal fluid, vaginal fluid, synovial fluid, lymph fluid, amniotic fluid, sweat, and tears; as well as in an aqueous solution produced by a plant, including, for example, exudates and guttation fluid, xylem, phloem, resin, and nectar; and in an aqueous medium produced or processed in an industrial context such as food or beverage or components thereof such as milk or milk derived material (e.g cheese, cream, yoghurt, whey, ice cream, etc.), pharmaceuticals, nutritional supplements or cosmetics or excipients for inclusion within pharmaceuticals, nutritional supplements or cosmetics, animal feed or supplements or veterinary agents.
In contrast, a hydrophobic polymer or unit, etc. is intended to mean a polymer or unit that exhibits little or no solubility or miscibility in an aqueous medium, such as the various aqueous media referred to above.
When discussing the types of monomers or units that can be used to prepare the backbone or pendant chains included within the inhibitors, it is convenient to refer to the monomers or units as being hydrophilic or hydrophobic in character. By being hydrophilic or hydrophobic in character in this context it is meant that upon polymerisation such monomers respectively give rise (directly or indirectly) to the hydrophilic or hydrophobic oligomers or polymers that form the backbone or pendant chains of the inhibitor agents. For example, a hydrophilic polymer that forms part of a pendant chain will generally be prepared by polymerising a monomer composition that comprises hydrophilic monomer.
As a guide only, examples of hydrophilic monomers/units include, but are not limited to, acrylic acid, methacrylic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate, oligo(alkylene glycol)methyl ether (meth)acrylate (OAG(M)A), acrylamide, methacrylamide, hydroxyethyl acrylate, N-methylacrylamide, N,N-dimethylacrylamide and Ν,Ν-dimethylaminoethyl methacrylate, Ν,Ν-dimethylaminopropyl methacrylamide, N-hydroxypropyl methacrylamide, norbornenes, 4-acryloylmorpholine, 2-acrylamido-2- methyl-l-propanesulfonic acid, phosphorylcholine methacrylate, N- vinyl pyrolidone, amino ethyl acrylamide, ethylene oxide The hydrophilic monomers/units can also include hydrophilic sugar or polysaccharide groups and hydrophilic amino acid sequences forming peptides or proteins.
As a guide only, examples of hydrophobic monomers/units include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, methionine, selenomethionine, phenylalanine, tryptophan, proline, hydroxyproline, styrene, alpha-methyl styrene, butyl acrylate, butyl methacrylate, amyl methacrylate, hexyl methacrylate, lauryl methacrylate, stearyl methacrylate, ethyl hexyl methacrylate, crotyl methacrylate, cinnamyl methacrylate, oleyl methacrylate, ricinoleyl methacrylate, cholesteryl methacrylates, cholesteryl acrylate, vinyl butyrate, vinyl tert-butyrate, vinyl stearate, vinyl laurate, caprolactide, propylene oxide, divinyl cyclopentane and mannose.
An hydrophilic polymer component of the inhibitor agents may therefore be described as comprising the polymerised residues of hydrophilic monomers or units. Similarly, the hydrophobic polymer component of the inhibitor agents can be described as comprising the polymerised residues of hydrophobic monomers or units. There exist a range of amphiphilic monomers which show both hydrophobic and hydrophilic properties and can be incorporated into either chain.
It will be understood that the hydrophobic components of the inhibitor agents can include monomers or units other than those that are strictly hydrophobic as long as the hydrophobic component (e.g an hydrophobic backbone component of the inhibitor) is generally hydrophobic in character. In this case the hydrophobic nature or character of the backbone can be determined by assessing the ability of the hydrophobic component to adsorb to β-sheet polymer, which can be detected by the use of ANS dyes which are used to detect hydrophobic domains [43]. Another means of determining whether a monomer or polymer component of the inhibitor can generally be classified as hydrophobic is by conducting contact angle analysis on a solid surface of the dried sample. For example by measuring the three phase contact angle (the angle made by a droplet of water on a film of the substance, where a hydrophobic surface shows a contact angle of greater than 50-60 degrees). It is also possible to determine hydrophobicity by measuring the miscibility of the molecules in oil or water [44] . The inhibitors described here are amphipathic such they show positive surface excesses at the air water interface.
It will similarly be understood that the hydrophilic components of the inhibitor agents can include monomers or units other than those that are strictly hydrophilic as long as the hydrophilic component (e.g. a pendant chain bound to the hydrophobic backbone component of the inhibitor) is generally hydrophilic in character. In this case the hydrophilic nature or character of the pendant chain can be determined by assessing the ability of the hydrophilic component to solubilise the inhibitor agent in an aqueous medium, which of course can be readily observed. As for hydrophobic agents, another means of determining whether a monomer or polymer component of the inhibitor can generally be classified as hydrophilic is by conducting contact angle analysis, for example by measuring the three phase contact angle or by measuring the miscibility of the molecules in oil or water, as discussed above.
In some aspects of the invention the backbone is hydrophobic and can, for example, comprise hydrophobic amino acid monomer units, including both natural and non-natural amino acids. Examples of hydrophobic amino acids that can be included within the backbone include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, methionine, selenomethionine, phenylalanine, tryptophan, proline, hydroxyproline.
In other aspects of the invention the hydrophobic backbone comprises an optionally substituted four-, five- or six-membered carbocyclic or heterocyclic ring. Examples of such ring structures that can be included within the hydrophobic backbone include optionally substitued cyclobutanyl, cyclopentanyl, cyclohexanyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, furanyl, pyranyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, dithiolyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, phenyl, benzyl, pyranyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, triazinyl, oxazinyl, oxathiazinyl and morpholinyl, each of which can be optionally substituted.
In one aspect of the invention the hydrophobic backbone comprises polyhydroxyproline. In a preferred embodiment the polyhydroxyproline backbone has bound thereto a plurality of pendant chains that comprise hydrophilic monosaccharide groups.
In another aspect of the invention the hydrophilic pendant chains that are bound (either directly or indirectly) to the hydrophobic backbone comprise one or more ring form and/or straight chain monosaccharide. Examples of monosaccharides include but are not limited to one of more of fucose, arabinose, arabitol, allose, altrose, glucofuranose, galactopyranose, glucopyranoside, xylanopyranose, fructopyranose, glucose, galactose, gulose, galactosamine, hammelose, xylose, lyxose, mannitol, mannosamine, ribose, rhamnose, threose, talose and substituted derivatives thereof.
It will be understood that both the backbone and the pendant chains within the inhibitor compounds of the invention can take the form of copolymers, so that more than one type of monomer can be included within each backbone or pendant chain. Each polymer block in a block co-polymer component of the inhibitor can be a homopolymer block or a copolymer block. Where a polymer block is a copolymer, the copolymer may be a gradient, a random or a statistical copolymer. The backbone and or some or all of the pendant chains can include terminal groups at their ends that are different to the repeating units or monomers throughout the structure. In some cases the terminal group may be a simple moiety that terminates polymerisation such as a hydrogen atom, amino group, or the like as may be present in the non-reactive monomer, while in other cases the terminal group may include more complex functionality. The presence of a terminal group may not be discernable in some cases.
For example a terminal group can comprise hydrogen, hydroxyl, halogen, amino, R1, - C02H,
Figure imgf000018_0001
-OR1, - SR1, -O2CR1, -SCOR1, and -OCSR1; where the or each R1 is independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, and an optionally substituted polymer chain.
Within the inhibitor compounds the pendant chains are directly or indirectly linked to the polymer or oligomer backbone, as will be further discussed below. It should be understood, however, that not all monomers or units within the backbone will necessarily have a pendant chain bound thereto. For example there can be a pendant chain attached to at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% of the monomer units within the backbone. In a broad aspect of the invention the pendant chains can comprise from 1 to about 1000 monomers or repeating units, such as from about 2 to about 500, from about 3 to about 100, from about 4 to about 50, from about 4 to about 20 or about 5, 10 or 12 monomers or repeating units.
As noted above the pendant chains can be covalently coupled directly or indirectly to the backbone. It is also possible for linkages to be present between polymer blocks within the backbone. By being "directly" coupled it is meant that there is only a covalent bond between the pendant chain and the backbone. By being "indirectly" coupled it is meant that there is located between the pendant chain and the backbone (or indeed within the backbone itself) one or more covalently bonded atoms or molecules. Where the pendant chains are indirectly coupled to the support moiety, it is convenient to refer to the pendant chains as being covalent coupled to the backbone through a linker group or groups.
In one embodiment, each of the pendant chains covalently coupled to the backbone through a linker group. There is no particular limitation concerning the nature of such a linker group provided it can function to couple the pendant chains to the backbone.
Examples of suitable linker groups include a divalent form of optionally substituted: oxy (- 0-), disulfide (-S-S-), alkyl, alkenyl, alkynyl, aryl, acyl (including -C(O)-), carbocyclyl, heterocyclyl, heteroaryl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, acyloxy, carbocyclyloxy, heterocyclyloxy, heteroaryloxy, alkylthio, alkenylthio, alkynylthio, arylthio, acylthio, carbocyclylthio, heterocyclylthio, heteroarylthio, alkylalkenyl, alkylalkynyl, alkylaryl, alkylacyl, alkylcarbocyclyl, alkylheterocyclyl, alkylheteroaryl, alkyloxyalkyl, alkenyloxyalkyl, alkynyloxyalkyl, aryloxyalkyl, alkylacyloxy, alkyloxyacylalkyl, alkylcarbocyclyloxy, alkylheterocyclyloxy, alkylheteroaryloxy, alkylthioalkyl, alkenylthioalkyl, alkynylthioalkyl, arylthioalkyl, alkylacylthio, alkylcarbocyclylthio, alkylheterocyclylthio, alkylheteroarylthio, alkylalkenylalkyl, alkylalkynylalkyl, alkylarylalkyl, alkylacylalkyl, arylalkylaryl, arylalkenylaryl, arylalkynylaryl, arylacylaryl, arylacyl, arylcarbocyclyl, arylheterocyclyl, arylheteroaryl, alkenyloxyaryl, alkynyloxyaryl, aryloxyaryl, arylacyloxy, arylcarbocyclyloxy, arylheterocyclyloxy, arylheteroaryloxy, alkylthioaryl, alkenylthioaryl, alkynylthioaryl, arylthioaryl, arylacylthio, arylcarbocyclylthio, arylheterocyclylthio, and arylheteroarylthio, wherein where present the or each -CH2- group in any alkyl chain may be replaced by a divalent group independently selected from -0-, -OP(0)2-, -OP(0)20-, -S-, -S(O)-, -S(0)20-, -OS(0)20-, -N=N-, -OSi(ORa)20-, -Si(ORa)20-, -OB(ORa)0-, -B(ORa)0-, -NRa-, -C(O)-, -C(0)0-, -OC(0)0-, -OC(0)NRa- and -C(0)NRa-, where the or each Ra may be independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl, heterocyclyl, arylalkyl, and acyl. The or each Ra may also be independently selected from hydrogen, Ci_i8alkyl, Ci_i8alkenyl, Ci_i8alkynyl, C6-i8aryl, C3_i8carbocyclyl, C3-i8heteroaryl, C3_i8heterocyclyl, and C7_i8arylalkyl.
As used herein, the term "alkyl", used either alone or in compound words denotes straight chain, branched or cyclic alkyl, preferably Ci_2o alkyl, e.g. Ci_io or Ci_6. Examples of straight chain and branched alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl, i-butyl, n-pentyl, 1,2-dimethylpropyl, 1,1 -dimethyl-prop yl, hexyl, 4-methylpentyl, 1- methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3- dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2- trimethylpropyl, heptyl, 5-methylhexyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3- dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4- dimethyl-pentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6- methylheptyl, 1-methylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-, 5-, 6- or 7- methyloctyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-, 2- or 3-propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and 8-methylnonyl, 1-, 2-, 3-, 4-, 5- or 6-ethyloctyl, 1-, 2-, 3- or 4-propylheptyl, undecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-methyldecyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-ethylnonyl, 1-, 2-, 3-, 4- or 5-propyloctyl, 1-, 2- or 3-butylheptyl, 1-pentylhexyl, dodecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-ethyldecyl, 1-, 2-, 3-, 4-, 5- or 6-propylnonyl, 1-, 2-, 3- or 4-butyloctyl, 1-2-pentylheptyl and the like. Examples of cyclic alkyl include mono- or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like. Where an alkyl group is referred to generally as "propyl", butyl" etc, it will be understood that this can refer to any of straight, branched and cyclic isomers where appropriate. An alkyl group may be optionally substituted by one or more optional substituents as herein defined.
The term "alkenyl" as used herein denotes groups formed from straight chain, branched or cyclic hydrocarbon residues containing at least one carbon to carbon double bond including ethylenically mono-, di- or polyunsaturated alkyl or cycloalkyl groups as previously defined, preferably C2-20 alkenyl (e.g. C2-10 or C2-6)- Examples of alkenyl include vinyl, allyl, 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3- decenyl, 1,3-butadienyl, 1,4-pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4- hexadienyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-cycloheptadienyl, 1,3,5- cycloheptatrienyl and 1,3,5,7-cyclooctatetraenyl. An alkenyl group may be optionally substituted by one or more optional substituents as herein defined.
As used herein the term "alkynyl" denotes groups formed from straight chain, branched or cyclic hydrocarbon residues containing at least one carbon-carbon triple bond including ethylenically mono-, di- or polyunsaturated alkyl or cycloalkyl groups as previously defined. Unless the number of carbon atoms is specified the term preferably refers to C2-20 alkynyl (e.g. C2-io or C2-6)- Examples include ethynyl, 1-propynyl, 2-propynyl, and butynyl isomers, and pentynyl isomers. An alkynyl group may be optionally substituted by one or more optional substituents as herein defined.
The term "halogen" ("halo") denotes fluorine, chlorine, bromine or iodine (fluoro, chloro, bromo or iodo).
The term "aryl" (or "carboaryl") denotes any of single, polynuclear, conjugated and fused residues of aromatic hydrocarbon ring systems(e.g. C6-24 or C6-i8)- · Examples of aryl include phenyl, biphenyl, terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, idenyl, azulenyl, chrysenyl. Preferred aryl include phenyl and naphthyl. An aryl group may or may not be optionally substituted by one or more optional substituents as herein defined. The term "arylene" is intended to denote the divalent form of aryl.
The term "carbocyclyl" includes any of non-aromatic monocyclic, polycyclic, fused or conjugated hydrocarbon residues, preferably C3-20 (e.g. C3-10 or C3-8). The rings may be saturated, e.g. cycloalkyl, or may possess one or more double bonds (cycloalkenyl) and/or one or more triple bonds (cycloalkynyl). Particularly preferred carbocyclyl moieties are 5- 6-membered or 9-10 membered ring systems. Suitable examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopentenyl, cyclohexenyl, cyclooctenyl, cyclopentadienyl, cyclohexadienyl, cyclooctatetraenyl, indanyl, decalinyl and indenyl. A carbocyclyl group may be optionally substituted by one or more optional substituents as herein defined. The term "carbocyclylene" is intended to denote the divalent form of carbocyclyl.
The term "heteroatom" or "hetero" as used herein in its broadest sense refers to any atom other than a carbon atom which may be a member of a cyclic organic group. Particular examples of heteroatoms include nitrogen, oxygen, sulfur, phosphorous, boron, silicon, selenium and tellurium, more particularly nitrogen, oxygen and sulfur.
The term "heterocyclyl" when used alone or in compound words includes any of monocyclic, polycyclic, fused or conjugated hydrocarbon residues, preferably C3-20 (e.g. C3_io or C3-8) wherein one or more carbon atoms are replaced by a heteroatom so as to provide a non-aromatic residue. Suitable heteroatoms include O, N, S, P and Se, particularly O, N and S. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms. The heterocyclyl group may be saturated or partially unsaturated, i.e. possess one or more double bonds. Particularly preferred heterocyclyl are 5-6 and 9-10 membered heterocyclyl. Suitable examples of heterocyclyl groups may include azridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 2H-pyrrolyl, pyrrolidinyl, pyrrolinyl, piperidyl, piperazinyl, morpholinyl, indolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, thiomorpholinyl, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrrolyl, tetrahydrothiophenyl, pyrazolinyl, dioxalanyl, thiazolidinyl, isoxazolidinyl, dihydropyranyl, oxazinyl, thiazinyl, thiomorpholinyl, oxathianyl, dithianyl, trioxanyl, thiadiazinyl, dithiazinyl, trithianyl, azepinyl, oxepinyl, thiepinyl, indenyl, indanyl, 3H-indolyl, isoindolinyl, 4H-quinolazinyl, chromenyl, chromanyl, isochromanyl, pyranyl and dihydropyranyl. A heterocyclyl group may be optionally substituted by one or more optional substituents as herein defined. The term "heterocyclylene" is intended to denote the divalent form of heterocyclyl.
The term "heteroaryl" includes any of monocyclic, polycyclic, fused or conjugated hydrocarbon residues, wherein one or more carbon atoms are replaced by a heteroatom so as to provide an aromatic residue. Preferred heteroaryl have 3-20 ring atoms, e.g. 3-10. Particularly preferred heteroaryl are 5-6 and 9-10 membered bicyclic ring systems. Suitable heteroatoms include, O, N, S, P and Se, particularly O, N and S. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms. Suitable examples of heteroaryl groups may include pyridyl, pyrrolyl, thienyl, imidazolyl, furanyl, benzothienyl, isobenzothienyl, benzofuranyl, isobenzofuranyl, indolyl, isoindolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, quinolyl, isoquinolyl, phthalazinyl, 1,5-naphthyridinyl, quinozalinyl, quinazolinyl, quinolinyl, oxazolyl, thiazolyl, isothiazolyl, isoxazolyl, triazolyl, oxadialzolyl, oxatriazolyl, triazinyl, and furazanyl. A heteroaryl group may be optionally substituted by one or more optional substituents as herein defined. The term "heteroarylene" is intended to denote the divalent form of heteroaryl.
The term "acyl" either alone or in compound words denotes a group containing the moiety C=0 (and not being a carboxylic acid, ester or amide). Preferred acyl includes C(0)-Re, wherein Re is hydrogen or an alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl residue. Examples of acyl include formyl, straight chain or branched alkanoyl (e.g. Ci_2o) such as acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2- dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl and icosanoyl; cycloalkylcarbonyl such as cyclopropylcarbonyl cyclobutylcarbonyl, cyclopentylcarbonyl and cyclohexylcarbonyl; aroyl such as benzoyl, toluoyl and naphthoyl; aralkanoyl such as phenylalkanoyl (e.g. phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutylyl, phenylpentanoyl and phenylhexanoyl) and naphthylalkanoyl (e.g. naphthylacetyl, naphthylpropanoyl and naphthylbutanoyl] ; aralkenoyl such as phenylalkenoyl (e.g. phenylpropenoyl, phenylbutenoyl, phenylmethacryloyl, phenylpentenoyl and phenylhexenoyl and naphthylalkenoyl (e.g. naphthylpropenoyl, naphthylbutenoyl and naphthylpentenoyl); aryloxyalkanoyl such as phenoxyacetyl and phenoxypropionyl; arylthiocarbamoyl such as phenylthiocarbamoyl; arylglyoxyloyl such as phenylglyoxyloyl and naphthylglyoxyloyl; arylsulfonyl such as phenylsulfonyl and napthylsulfonyl; heterocycliccarbonyl; heterocyclicalkanoyl such as thienylacetyl, thienylpropanoyl, thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl and tetrazolylacetyl; heterocyclicalkenoyl such as heterocyclicpropenoyl, heterocyclicbutenoyl, heterocyclicpentenoyl and heterocyclichexenoyl; and heterocyclicglyoxyloyl such as thiazolyglyoxyloyl and thienylglyoxyloyl. The Re residue may be optionally substituted as described herein.
The term "sulfoxide", either alone or in a compound word, refers to a group -S(0)R wherein R is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl. Examples of preferred R include Ci_2oalkyl, phenyl and benzyl.
The term "sulfonyl", either alone or in a compound word, refers to a group S(0)2-R , wherein R is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl and aralkyl. Examples of preferred R include Ci_2oalkyl, phenyl and benzyl.
The term "sulfonamide", either alone or in a compound word, refers to a group S(0)NR f R f wherein each R is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl. Examples of preferred R include Ci_ 2oalkyl, phenyl and benzyl. In one embodiment at least one R is hydrogen. In another embodiment, both R are hydrogen.
The term, "amino" is used here in its broadest sense as understood in the art and includes groups of the formula NRaRb wherein Ra and Rb may be any independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl, heterocyclyl, arylalkyl, and acyl. Ra and Rb, together with the nitrogen to which they are attached, may also form a monocyclic, or polycyclic ring system e.g. a 3-10 membered ring, particularly, 5-6 and 9- 10 membered systems. Examples of "amino" include NH2, NHalkyl (e.g. Ci_2oalkyl), NHaryl (e.g. NHphenyl), NHaralkyl (e.g. NHbenzyl), NHacyl (e.g. NHC(O)Ci_20alkyl, NHC(O)phenyl), Nalkylalkyl (wherein each alkyl, for example Ci_2o, may be the same or different) and 5 or 6 membered rings, optionally containing one or more same or different heteroatoms (e.g. O, N and S).
The term "amido" is used here in its broadest sense as understood in the art and includes groups having the formula C(0)NRaRb, wherein Ra and Rb are as defined as above. Examples of amido include C(0)NH2, C(0)NHalkyl (e.g. Ci_20alkyl), C(0)NHaryl (e.g. C(O)NHphenyl), C(0)NHaralkyl (e.g. C(O)NHbenzyl), C(0)NHacyl (e.g. C(O)NHC(O)Ci-20alkyl, C(0)NHC(0)phenyl), C(0)Nalkylalkyl (wherein each alkyl, for example Ci_2o, may be the same or different) and 5 or 6 membered rings, optionally containing one or more same or different heteroatoms (e.g. O, N and S).
The term "carboxy ester" is used here in its broadest sense as understood in the art and includes groups having the formula C02Rg, wherein Rg may be selected from groups including alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl, heterocyclyl, aralkyl, and acyl. Examples of carboxy ester include C02Ci_2oalkyl, C02aryl (e.g.. C02phenyl), C02aralkyl (e.g. C02 benzyl).
As used herein, the term "aryloxy" refers to an "aryl" group attached through an oxygen bridge. Examples of aryloxy substituents include phenoxy, biphenyloxy, naphthyloxy and the like. As used herein, the term "acyloxy" refers to an "acyl" group wherein the "acyl" group is in turn attached through an oxygen atom. Examples of "acyloxy" include hexylcarbonyloxy (heptanoyloxy), cyclopentylcarbonyloxy, benzoyloxy, 4-chlorobenzoyloxy, decylcarbonyloxy (undecanoyloxy), propylcarbonyloxy (butanoyloxy), octylcarbonyloxy (nonanoyloxy), biphenylcarbonyloxy (eg 4-phenylbenzoyloxy), naphthylcarbonyloxy (eg 1-naphthoyloxy) and the like.
As used herein, the term "alkyloxycarbonyl" refers to a "alkyloxy" group attached through a carbonyl group. Examples of "alkyloxycarbonyl" groups include butylformate, sec- butylformate, hexylformate, octylformate, decylformate, cyclopentylformate and the like. As used herein, the term "arylalkyl" refers to groups formed from straight or branched chain alkanes substituted with an aromatic ring. Examples of arylalkyl include phenylmethyl (benzyl), phenylethyl and phenylpropyl.
As used herein, the term "alkylaryl" refers to groups formed from aryl groups substituted with a straight chain or branched alkane. Examples of alkylaryl include methylphenyl and isopropylphenyl.
In this specification "optionally substituted" is taken to mean that a group may or may not be substituted or fused (so as to form a condensed polycyclic group) with one, two, three or more of organic and/or inorganic groups, including those selected from: alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, heteroaryl, acyl, aralkyl, alkaryl, alkheterocyclyl, alkheteroaryl, alkcarbocyclyl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, halocarbocyclyl, haloheterocyclyl, haloheteroaryl, haloacyl, haloaryalkyl, hydroxy, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxycarbocyclyl, hydroxyaryl, hydroxyheterocyclyl, hydroxyheteroaryl, hydroxyacyl, hydroxyaralkyl, alkoxyalkyl, alkoxyalkenyl, alkoxyalkynyl, alkoxycarbocyclyl, alkoxyaryl, alkoxyheterocyclyl, alkoxyheteroaryl, alkoxyacyl, alkoxyaralkyl, alkoxy, alkenyloxy, alkynyloxy, aryloxy, carbocyclyloxy, aralkyloxy, heteroaryloxy, heterocyclyloxy, acyloxy, haloalkoxy, haloalkenyloxy, haloalkynyloxy, haloaryloxy, halocarbocyclyloxy, haloaralkyloxy, haloheteroaryloxy, haloheterocyclyloxy, haloacyloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, nitroheteroayl, nitrocarbocyclyl, nitroacyl, nitroaralkyl, amino (NH2), alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, aralkylamino, diaralkylamino, acylamino, diacylamino, heterocyclamino, heteroarylamino, carboxy, carboxyester, amido, alkylsulphonyloxy, arylsulphenyloxy, alkylsulphenyl, arylsulphenyl, thio, alkylthio, alkenylthio, alkynylthio, arylthio, aralkylthio, carbocyclylthio, heterocyclylthio, heteroarylthio, acylthio, sulfoxide, sulfonyl, sulfonamide, aminoalkyl, aminoalkenyl, aminoalkynyl, aminocarbocyclyl, aminoaryl, aminoheterocyclyl, aminoheteroaryl, aminoacyl, aminoaralkyl, thioalkyl, thioalkenyl, thioalkynyl, thiocarbocyclyl, thioaryl, thioheterocyclyl, thioheteroaryl, thioacyl, thioaralkyl, carboxyalkyl, carboxyalkenyl, carboxyalkynyl, carboxycarbocyclyl, carboxyaryl, carboxyheterocyclyl, carboxyheteroaryl, carboxyacyl, carboxyaralkyl, carboxyesteralkyl, carboxyesteralkenyl, carboxyesteralkynyl, carboxyestercarbocyclyl, carboxyesteraryl, carboxyesterheterocyclyl, carboxyesterheteroaryl, carboxyesteracyl, carboxyesteraralkyl, amidoalkyl, amidoalkenyl, amidoalkynyl, amidocarbocyclyl, amidoaryl, amidoheterocyclyl, amidoheteroaryl, amidoacyl, amidoaralkyl, formylalkyl, formylalkenyl, formylalkynyl, formylcarbocyclyl, formylaryl, formylheterocyclyl, formylheteroaryl, formylacyl, formylaralkyl, acylalkyl, acylalkenyl, acylalkynyl, acylcarbocyclyl, acylaryl, acylheterocyclyl, acylheteroaryl, acylacyl, acylaralkyl, sulfoxidealkyl, sulfoxidealkenyl, sulfoxidealkynyl, sulfoxidecarbocyclyl, sulfoxidearyl, sulfoxideheterocyclyl, sulfoxideheteroaryl, sulfoxideacyl, sulfoxidearalkyl, sulfonylalkyl, sulfonylalkenyl, sulfonylalkynyl, sulfonylcarbocyclyl, sulfonylaryl, sulfonylheterocyclyl, sulfonylheteroaryl, sulfonylacyl, sulfonylaralkyl, sulfonamidoalkyl, sulfonamidoalkenyl, sulfonamidoalkynyl, sulfonamidocarbocyclyl, sulfonamidoaryl, sulfonamidoheterocyclyl, sulfonamidoheteroaryl, sulfonamidoacyl, sulfonamidoaralkyl, nitroalkyl, nitroalkenyl, nitroalkynyl, nitrocarbocyclyl, nitroaryl, nitroheterocyclyl, nitroheteroaryl, nitroacyl, nitroaralkyl, cyano, sulfate, phosphate, triarylmethyl, triarylamino, oxadiazole, and carbazole groups. Optional substitution may also be taken to refer to where a -CH2- group in a chain or ring is replaced by a group selected from -0-, -S-, -NRa-, -C(O)- (i.e. carbonyl), -C(0)0- (i.e. ester), and -C(0)NRa- (i.e. amide), where Ra is as defined herein. Preferred optional substituents include alkyl, (e.g. Ci_6 alkyl such as methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), hydroxyalkyl (e.g. hydroxymethyl, hydroxyethyl, hydroxypropyl), alkoxyalkyl (e.g. methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl etc) alkoxy (e.g. Ci_6 alkoxy such as methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy), halo, trifluoromethyl, trichloromethyl, tribromomethyl, hydroxy, phenyl (which itself may be further substituted e.g., by C1-6 alkyl, halo, hydroxy, hydroxyCi-6 alkyl, C1-6 alkoxy, haloCi-6alkyl, cyano, nitro OC(0)Ci_6 alkyl, and amino), benzyl (wherein benzyl itself may be further substituted e.g., by Ci_6 alkyl, halo, hydroxy, hydroxyCi_6alkyl, Ci_6 alkoxy, haloCi_6 alkyl, cyano, nitro OC(0)Ci_6 alkyl, and amino), phenoxy (wherein phenyl itself may be further substituted e.g., by C1-6 alkyl, halo, hydroxy, hydroxyCi-6 alkyl, C1-6 alkoxy, haloCi-6 alkyl, cyano, nitro OC(0)C1-6 alkyl, and amino), benzyloxy (wherein benzyl itself may be further substituted e.g., by Ci_6 alkyl, halo, hydroxy, hydroxyCi_6 alkyl, Ci_6 alkoxy, haloCi_6 alkyl, cyano, nitro OC(0)Ci_6 alkyl, and amino), amino, alkylamino (e.g. Ci_6 alkyl, such as methylamino, ethylamino, propylamino etc), dialkylamino (e.g. C1-6 alkyl, such as dimethylamino, diethylamino, dipropylamino), acylamino (e.g. NHC(0)CH3), phenylamino (wherein phenyl itself may be further substituted e.g., by Ci_6 alkyl, halo, hydroxy, hydroxyCi_6 alkyl, Ci_6 alkoxy, haloCi_6 alkyl, cyano, nitro OC(0)Ci_6 alkyl, and amino), nitro, formyl, -C(0)-alkyl (e.g. C1-6 alkyl, such as acetyl), 0-C(0)-alkyl (e.g. Ci_ 6alkyl, such as acetyloxy), benzoyl (wherein the phenyl group itself may be further substituted e.g., by Ci_6 alkyl, halo, hydroxy hydroxyCi_6 alkyl, Ci_6 alkoxy, haloCi_6 alkyl, cyano, nitro OC(0)C1-6alkyl, and amino), replacement of CH2 with C=0, C02H, C02alkyl (e.g. Ci-6 alkyl such as methyl ester, ethyl ester, propyl ester, butyl ester), C02phenyl (wherein phenyl itself may be further substituted e.g., by C1-6 alkyl, halo, hydroxy, hydroxyl Ci_6 alkyl, Ci_6 alkoxy, halo Ci_6 alkyl, cyano, nitro OC(0)Ci_6 alkyl, and amino), CONH2, CONHphenyl (wherein phenyl itself may be further substituted e.g., by Ci_6 alkyl, halo, hydroxy, hydroxyl C1-6 alkyl, C1-6 alkoxy, halo C1-6 alkyl, cyano, nitro OC(0)C1-6 alkyl, and amino), CONHbenzyl (wherein benzyl itself may be further substituted e.g., by Ci_6 alkyl, halo, hydroxy hydroxyl Ci_6 alkyl, Ci_6 alkoxy, halo Ci_6 alkyl, cyano, nitro OC(0)Ci_6 alkyl, and amino), CONHalkyl (e.g. Ci_6 alkyl such as methyl ester, ethyl ester, propyl ester, butyl amide) CONHdialkyl (e.g. Ci_6 alkyl) aminoalkyl (e.g., HN Ci_6 alkyl-, Ci_6alkylHN-Ci_6 alkyl- and (Ci_6 alkyl)2N-Ci_6 alkyl-), thioalkyl (e.g., HS Ci_6 alkyl-), carboxyalkyl (e.g., H02CCi_6 alkyl-), carboxyesteralkyl (e.g., Ci_6 alkyl02CCi_6 alkyl-), amidoalkyl (e.g., H2N(0)CCi_6 alkyl-, H(Ci_6 alkyl)N(0)CCi_6 alkyl-), formylalkyl (e.g., OHCCi_6alkyl-), acylalkyl (e.g., Ci_6 alkyl(0)CCi_6 alkyl-), nitroalkyl (e.g., 02NCi_6 alkyl-), sulfoxidealkyl (e.g., R(0)SCi_6 alkyl, such as Ci_6 alkyl(0)SCi_6 alkyl-), sulfonylalkyl (e.g., R(0)2SCi_6 alkyl- such as Ci_6 alkyl(0)2SCi_6 alkyl-), sulfonamidoalkyl (e.g., 2HRN(0)SCi_6 alkyl, H(Ci_6 alkyl)N(0)SCi_6 alkyl-), triarylmethyl, triarylamino, oxadiazole, and carbazole.
In a particular aspect of the invention the inhibitor compounds have the structure as provided in Formula I, as follows:
Figure imgf000029_0001
Figure imgf000029_0002
I
T2
Formula I
wherein:
T, T 1 and T 2 are the same or different and represent a terminal group, for example as defined hereinbefore;
L, L 1 , L2 and L 3 are the same or different and represent a linker group, for example as defined hereinbefore;
A represents a hydrophobic backbone unit, for example as defined
hereinbefore;
R represents a hydrophilic pendant chain unit, for example as defined hereinbefore, that is bound, optionally via linker group L , to any of A, L or L1 ; a, a 1 , a2 and a 3 are the same of different and represent the numerals 0, 1, 2 or 3;
b represents the numerals 1 to 1000;
c represents the numerals 0 to 1000, but represents 1 or greater in at least 25% of cases;
and wherein free rotation is allowed within and/or between adjacent A units. In some embodiments b represents the numerals 5 to 10.
In some embodiments c represents the numerals 3 to 20 in at least 50% of cases, which implies that at least 50% of backbone monomer units have a pendant chain bound thereto and that these pendant chains are 3 to 20 units in length. In other embodiments c represents the numerals 4 to 10 or about 5 in at least 80% of cases, which implies that at least 80% of backbone monomer units have a pendant chain bound thereto and that these pendant chains are 3 to 20, 4 to 10 or 5 units in length.
In addition to compounds of the invention shown in Figures 2 and 4 to 6, other specific examples of inhibitors of the invention are provided below:
One example of the polymer with hydrophobic backbone and hydrophilic side chains is a copolymer with poly(ca/?ro-lactide) (PCL) backbone and polyethylene oxide side chians. This polymer may be synthesized by the formation of functionalized PCL followed by attachment of PEO side chians on the backbone.
Figure imgf000030_0001
A further example of the polymer with hydrophobic backbone and hydrophilic side chains is a copolymer with PPO backbone and PEO side chains. This polymer may be synthesized by initial formation of linear PEO with only one end having hydroxyl functionality which is then coupled with an epoxy group. The copolymerization of this long chain monomer with normal PO monomer forms the copolymer with hydrophobic PPO backbone and hydrophilic PEO side chains.
Figure imgf000031_0001
One example of the polymer with hydrophilic backbone and hydrophobic side chains is a copolymer with polyethylene oxide (PEO) backbone and polypropylene oxide (PPO) side chians. This polymer may be synthesized by initial formation of PPO side chains with only one end having hydroxyl functionality which is then coupled with an epoxy group. The copolymerization of this long chain monomer with normal PEO monomer forms the copolymer with h drophilic PEO backbone and hydrophobic PPO side chains.
Figure imgf000031_0002
A further example of the polymer with hydrophilic backbone and hydrophobic side chains is a copolymer with polyacrylamide (PAA) backbone and polypropylene oxide (PPO) side chians. PAA backbone can be functionalised with amine functionality by copolymerization of acrylamide with amino ethyl acrylamide. Amine functionalized PAA can then coupled with the hydrophobic side chian with epoxy functionality. PPO with epoxy functionality at one chain end is one example.
Figure imgf000032_0001
Naturally occurring precursors may be used to form such copolymer structures. One example is the use of polymannose chain blocks which can be derived from, for example, guar. The hydrophobic polymannose can then be coupled to a hydrophilic polymer backbone with functional group along the chians. The coupling between the backbone and side chains may through an epoxy-amine coupling or NPC coupling with amines. PAA with amine functionality along the backbone can be used to couple multiple polymannose side chians.
Figure imgf000032_0002
In the structures provided above the variables a, x, y and z independently represent the integers 1 to 1000.
Guar, containing polymannose backbone and glactose side chains may be further modified to form comb shaped polymers with improved performance. The modification may be carried out by a periodate reaction to convert the glycol moieties of the glactose to a aldehyde functionality which can then linked to a hydrophilic polymer with an amine functionality in one polymer chain end. Such hydrophilic polymer chains may be PEO based or functionalised with charged groups to improve the hydrophilicity.
The inhibitors of the invention may be prepared by any suitable means. In one embodiment, the process of preparing the inhibitors comprises the polymerisation of ethylenically unsaturated monomers. Polymerisation of the ethylenically unsaturated monomers is preferably conducted using a living polymerisation technique, but other approaches such as using the step-growth technique (e.g. PHP) and solid phase peptide synthesis (e.g. glycosylated PHP and GAA) are also possible.
Living polymerisation is generally considered in the art to be a form of chain polymerisation in which irreversible chain termination is substantially absent. An important feature of living polymerisation is that polymer chains will continue to grow while monomer and reaction conditions to support polymerisation are provided. Polymer chains prepared by living polymerisation can advantageously exhibit a well defined molecular architecture, a predetermined molecular weight and narrow molecular weight distribution or low polydispersity.
Examples of living polymerisation include ionic polymerisation, radical polymerisation (RP) and controlled radical polymerisation (CRP). Examples of RP and CRP include, but are not limited to, iniferter polymerisation, stable free radical mediated polymerisation (SFRP), atom transfer radical polymerisation (ATRP), and reversible addition fragmentation chain transfer (RAFT) polymerisation. Ring opening metathesis polymerisation is also a possible synthesis route.
Equipment, conditions, and reagents for performing living polymerisation are well known to those skilled in the art.
Where ethylenically unsaturated monomers are to be polymerised by a living polymerisation technique, it will generally be necessary to make use of a so-called living polymerisation agent. By "living polymerisation agent" is meant a compound that can participate in and control or mediate the living polymerisation of one or more ethylenically unsaturated monomers so as to form a living polymer chain (i.e. a polymer chain that has been formed according to a living polymerisation technique).
Living polymerisation agents include, but are not limited to, those which promote a living polymerisation technique selected from ionic polymerisation and CRP.
In one embodiment of the invention, the branched polymer is prepared using ionic polymerisation.
Living ionic polymerisation is a form of addition polymerisation whereby the kinetic-chain carriers are ions or ion pairs. The polymerisation proceeds via anionic or cationic kinetic- chain carriers. In other words, the propagating species will either carry a negative or positive charge, and as such there will also be an associated counter cation or counter anion, respectively. For example, in the case of anionic polymerisation, the living polymerisation agent might be represented as ΓΜ+, where I represents an organo-anion (e.g. an optionally substituted alkyl anion) and M represents an associated countercation, or in the case of living cationic polymerisation, the living polymerisation agent might be represented as I+M", where I represents an organo-cation (e.g. an optionally substituted alkyl cation) and M represents an associated counteranion. Suitable agents for conducting anionic and cationic living polymerisation are well known to those skilled in the art and include, but are not limited to, aprotonic acids (e.g. aluminium trichloride, boron trifluoride), protonic (Bronstead) acids, stable carbenium-ion salts, organometallic compounds (e.g. N-butyl lithium, cumyl potassium) and Ziegler-Natta catalysts (e.g. triethyl aluminium and titanium tetrachloride).
In one embodiment of the invention, the branched polymer is prepared using CRP.
In a further embodiment of the invention, the branched polymer is prepared using iniferter polymerisation.
Iniferter polymerisation is a well known form of CRP, and is generally understood to proceed by a mechanism illustrated below in Scheme 1.
a) AB ^ Α· + ·Β
b) Α· + M ► ·"™"·
c) Α^™"· + ·Β ^ A^B d) A*™" + AB ^ A^B + ·Α
e) * B A ^ A^^1 B "t" * ^"""Ά
Scheme 1: General mechanism of controlled radical polymerisation with iniferters.
With reference to Scheme 1, the iniferter agent AB dissociates chemically, thermally or photochemically to produce a reactive radical species A and generally a relatively stable radical species B (for symmetrical iniferters the radical species B will be the same as the radical species A) (step a). The radical species A can initiate polymerisation of monomer M (in step b) and may be deactivated by coupling with radical species B (in step c). Transfer to the iniferter (in step d) and/or transfer to dormant polymer (in step e) followed by termination (in step f) characterise iniferter chemistry. Suitable iniferter agents are well known to those skilled in the art, and include, but are not limited to, dithiocarbonate, disulphide, and thiuram disulphide compounds.
In a further embodiment of the invention, the branched polymer is prepared using SFRP. As suggested by its name, this mode of radical polymerisation involves the generation of a stable radical species as illustrated below in Scheme 2.
CD C •D
M
Figure imgf000036_0001
Scheme 2: General mechanism of controlled radical polymerisation with stable free radical mediated polymerisation.
With reference to Scheme 2, SFRP agent CD dissociates to produce an active radical species C and a stable radical species D. The active radical species C reacts with monomer M, which resulting propagating chain may recombine with the stable radical species D. Unlike iniferter agents, SFRP agents do not provide for a transfer step. Suitable agents for conducting SFRP are well known to those skilled in the art, and include, but are not limited to, moieties capable of generating phenoxy and nitroxy radicals. Where the agent generates a nitroxy radical, the polymerisation technique is more commonly known as nitroxide mediated polymerisation (NMP).
Examples of SFRP agents capable of generating phenoxy radicals include those comprising a phenoxy group substituted in the 2 and 6 positions by bulky groups such as tert-alkyl (e.g. t-butyl), phenyl or dimethylbenzyl, and optionally substituted at the 4 position by an alkyl, alkyloxy, aryl, or aryloxy group or by a heteroatom containing group (e.g. S, N or O) such as dimethylamino or diphenylamino group. Thiophenoxy analogues of such phenoxy containing agents are also contemplated.
SFRP agents capable of generating nitroxy radicals include those comprising the substituent R 1 R2 N-0-, where R 1 and R2 are tertiary alkyl groups, or where R 1 and R2 together with the N atom form a cyclic structure, preferably having tertiary branching at the positions a to the N atom. Examples of such nitroxy substituents include 2,2,5,5- tetraalkylpyrrolidinoxyl, as well as those in which the 5-membered hetrocycle ring is fused to an alicyclic or aromatic ring, hindered aliphatic dialkylaminoxyl and iminoxyl substituents. A common nitroxy substituent employed in SFRP is 2,2,6, 6-tetramethyl-l- piperidinyloxy.
In another embodiment of the invention, polymer components of the inhibitors are prepared using ATRP.
ATRP generally employs a transition metal catalyst to reversibly deactivate a propagating radical by transfer of a transferable atom or group such as a halogen atom to the propagating polymer chain, thereby reducing the oxidation state of the metal catalyst as illustrated below in Scheme 3.
E-X + Mt n Ε· + Mt nX
M + Mt n+1X
Figure imgf000037_0001
Scheme 3: General mechanism of controlled radical polymerisation with atom transfer radical polymerisation.
With reference to Scheme 3, a transferable group or atom (X , e.g. halide, cyanato, thiocyanato or azido) is transferred from the organic compound (E-X) to a transition metal catalyst (Mt, e.g. copper, iron, palladium, cobalt, rhenium, rhodium, ruthenium, molybdenum, niobium, or nickel) having oxidation number (n), upon which a radical species is formed that initiates polymerisation with monomer (M). As part of this process, the metal complex is oxidised (Mt n+1X). A similar reaction sequence is then established between the propagating polymer chain and the dormant X end-capped polymer chains.
In a further embodiment of the invention, polymer components of the inhibitor are prepared using RAFT polymerisation.
RAFT polymerisation is well known in the art and is believed to operate through the mechanism outlined below in Scheme 4.
Figure imgf000038_0001
Scheme 4: General mechanism of controlled radical polymerisation with reversible addition fragmentation chain transfer polymerisation.
With reference to Scheme 4, RAFT polymerisation is believed to proceed through initial reaction sequence (a) that involves reaction of a RAFT agent (1) with a propagating radical. The labile intermediate radical species (2) that is formed fragments to form a temporarily deactivated dormant polymer species (3) together a radical (R) derived from the RAFT agent. This radical can then promote polymerisation of monomer (M), thereby reinitiating polymerisation. The propagating polymer chain can then react with the dormant polymer species (3) to promote the reaction sequence (b) that is similar to reaction sequence (a). Thus, a labile intermediate radical (4) is formed and subsequently fragments to form again a dormant polymer species together with a radical which is capable of further chain growth.
A polymer formed by RAFT polymerisation may conveniently be referred to as a RAFT polymer. By virtue of the mechanism of polymerisation, such polymers will comprise residue of the RAFT agent that facilitated polymerisation of the monomer.
RAFT agents suitable for use in accordance with the invention comprise a thiocarbonylthio group (which is a divalent moiety represented by: -C(S)S-). Examples of RAFT agents are described in Moad G.; Rizzardo, E; Thang S, H. Polymer 2008, 49, 1079-1131and Aust. J. Chem., 2005, 58, 379-410; Aust. J. Chem., 2006, 59, 669-692; Aust. J. Chem., 2009, 62, 1402-1472 (the entire contents of which are incorporated herein by reference) and include xanthate, dithioester, dithiocarbamate and trithiocarbonate compounds.
A RAFT agent suitable for use in accordance with the invention may be represented by general formula (I) or (II):
Figure imgf000039_0001
(I) (II) where Z and R are groups, and R* and Z* are x-valent and y-valent groups, respectively, that are independently selected such that the agent can function as a RAFT agent in the polymerisation of one or more ethylenically unsaturated monomers; x is an integer > 1; and y is an integer > 2.
In one embodiment, x is an integer > 3; and y is an integer > 3. In that case, R* and Z* may represent a core moiety (CM).
In order to function as a RAFT agent in the polymerisation of one or more ethylenically unsaturated monomers, those skilled in the art will appreciate that R and R* will typically be an optionally substituted organic group that function as a free radical leaving group under the polymerisation conditions employed and yet, as a free radical leaving group, retain the ability to reinitiate polymerisation. Those skilled in the art will also appreciate that Z and Z* will typically be an optionally substituted organic group that function to give a suitably high reactivity of the C=S moiety in the RAFT agent towards free radical addition without slowing the rate of fragmentation of the RAFT-adduct radical to the extent that polymerisation is unduly retarded.
In formula (I), R* is a x-valent group, with x being an integer > 1. Accordingly, R* may be mono-valent, di-valent, tri-valent or of higher valency. For example, R* may be a C2o alkyl chain, with the remainder of the RAFT agent depicted in formula (I) presented as multiple substituent groups pendant from the chain. Generally, x will be an integer ranging from 1 to about 20, for example from about 2 to about 10, or from 1 to about 5. In one embodiment, x = 2.
Similarly, in formula (II), Z* is a y-valent group, with y being an integer > 2. Accordingly, Z* may be di-valent, tri-valent or of higher valency. Generally, y will be an integer ranging from 2 to about 20, for example from about 2 to about 10, or from 2 to about 5.
Examples of R in RAFT agents used in accordance with the invention include optionally substituted, and in the case of R* in RAFT agents used in accordance with the invention include a x-valent form of optionally substituted, alkyl, alkenyl, alkynyl, aryl, acyl, carbocyclyl, heterocyclyl, heteroaryl, alkylthio, alkenylthio, alkynylthio, arylthio, acylthio, carbocyclylthio, heterocyclylthio, heteroarylthio, alkylalkenyl, alkylalkynyl, alkylaryl, alkylacyl, alkylcarbocyclyl, alkylheterocyclyl, alkylheteroaryl, alkyloxyalkyl, alkenyloxyalkyl, alkynyloxyalkyl, aryloxyalkyl, alkylacyloxy, alkylcarbocyclyloxy, alkylheterocyclyloxy, alkylheteroaryloxy, alkylthioalkyl, alkenylthioalkyl, alkynylthioalkyl, arylthioalkyl, alkylacylthio, alkylcarbocyclylthio, alkylheterocyclylthio, alkylheteroarylthio, alkylalkenylalkyl, alkylalkynylalkyl, alkylarylalkyl, alkylacylalkyl, arylalkylaryl, arylalkenylaryl, arylalkynylaryl, arylacylaryl, arylacyl, arylcarbocyclyl, arylheterocyclyl, arylheteroaryl, alkenyloxyaryl, alkynyloxyaryl, aryloxyaryl, alkylthioaryl, alkenylthioaryl, alkynylthioaryl, arylthioaryl, arylacylthio, arylcarbocyclylthio, arylheterocyclylthio, arylheteroarylthio, and a polymer chain.
For avoidance of any doubt reference herein to "optionally substituted", alkyl, alkenyl etc, is intended to mean each group such as alkyl and alkenyl is optionally substituted.
Examples of R in RAFT agents used in accordance with the invention also include optionally substituted, and in the case of R* in RAFT agents used in accordance with the invention also include an x-valent form of optionally substituted, alkyl; saturated, unsaturated or aromatic carbocyclic or heterocyclic ring; alkylthio; dialkylamino; an organometallic species; and a polymer chain.
More specific examples of R in RAFT agents used in accordance with the invention include optionally substituted, and in the case of R in RAFT agents used in accordance with the invention include an x-valent form of optionally substituted, Ci-Cis alkyl, C2-Ci8 alkenyl, C2-C18 alkynyl, C6-Ci8 aryl, Ci-Cis acyl, C3-C18 carbocyclyl, C2-C18 heterocyclyl, C3-C18 heteroaryl, Q-Cis alkylthio, C2-Ci8 alkenylthio, C2-Ci8 alkynylthio, C6-Ci8 arylthio, Q-Cis acylthio, C3-C18 carbocyclylthio, C2-Ci8 heterocyclylthio, C3-C18 heteroarylthio, C3-C18 alkylalkenyl, C3-C18 alkylalkynyl, C7-C24 alkylaryl, C2-Cis alkylacyl, C4-Ci8 alkylcarbocyclyl, C3-Ci8 alkylheterocyclyl, C4-Ci8 alkylheteroaryl, C2- Cis alkyloxyalkyl, C3-Ci8 alkenyloxyalkyl, C3-Ci8 alkynyloxyalkyl, C7-C24 aryloxyalkyl, C2-Ci8 alkylacyloxy, C2-Ci8 alkylthioalkyl, C3-Ci8 alkenylthioalkyl, C3-Ci8 alkynylthioalkyl, C7-C24 arylthioalkyl, C2-Ci8 alkylacylthio, C4-Ci8 alkylcarbocyclylthio, C3-C18 alkylheterocyclylthio, C4-Ci8 alkylheteroarylthio, C4-Ci8 alkylalkenylalkyl, C4-Ci8 alkylalkynylalkyl, C8-C24 alkylarylalkyl, C3-Ci8 alkylacylalkyl, Ci3-C24 arylalkylaryl, C14- C24 arylalkenylaryl, Ci4-C24 arylalkynylaryl, Ci3-C24 arylacylaryl, C7-Ci8 arylacyl, C9-Ci8 arylcarbocyclyl, C8-Ci8 arylheterocyclyl, C9-Ci8 arylheteroaryl, C8-Ci8 alkenyloxyaryl, C8- Cis alkynyloxyaryl, Ci2-C24 aryloxyaryl, alkylthioaryl, C8-Ci8 alkenylthioaryl, C8-Ci8 alkynylthioaryl, Ci2-C24 arylthioaryl, C7-Ci8 arylacylthio, C9-Ci8 arylcarbocyclylthio, C8- Cis arylheterocyclylthio, C9-Ci8 arylheteroarylthio, and a polymer chain having a number average molecular weight in the range of about 500 to about 80,000, for example in the range of about 500 to about 30,000.
More specific examples of a polymer chain include polystyrene, polyacrylamide, poly(methyl acrylate), poly(methyl methacrylate), poly(n-butyl acrylate), poly (tert-butyl acrylate), poly(acrylic acid), poly (vinyl acetate), poly(vinyl pyrrolidone), poly(N- isopropyl acrylamide), polystyrene-block-poly(tert-butyl acrylate), polystyrene-block- poly(acrylic acid), poly (para-acetoxystryene), poly(para-hydroxystyrene), poly(N,N- dimethyl acrylamide, poly(hydroxyethyl acrylate), poly(oligoethylene glycol acrylate), poly(N,N-dimethylaminoethyl methacrylate), poly(N-acryloylmorpholine), poly(methyl methacrylate)-block-poly(styrene), poly(ethyleneoxide)-block-poly(methyl methacrylate, poly(ethyleneoxide)-block-poly(N-isopropyl acrylamide), and poly(ethyleneoxide)-block- poly styrene-block-poly(acrylic acid) .
Where R in RAFT agents used in accordance with the invention include, and in the case of R* in RAFT agents used in accordance with the invention include an x-valent form of, an optionally substituted polymer chain, the polymers chain may be formed by any suitable polymerisation process such as radical, ionic, coordination, step-growth or condensation polymerisation.
Living polymerisation agents that comprise a polymer chain are commonly referred to in the art as "macro" living polymerisation agents. Such "macro" living polymerisation agents may conveniently be prepared by polymerising one or more ethylenically unsaturated monomers under the control of a given living polymerisation agent. In one embodiment, such a polymer chain is formed by polymerising ethylenically unsaturated monomer under the control of a RAFT agent.
Examples of Z in RAFT agents used in accordance with the invention include optionally substituted, and in the case of Z* in RAFT agents used in accordance with the invention include a y-valent form of optionally substituted: F, CI, Br, I, alkyl, aryl, acyl, amino, carbocyclyl, heterocyclyl, heteroaryl, alkyloxy, aryloxy, acyloxy, acylamino, carbocyclyloxy, heterocyclyloxy, heteroaryloxy, alkylthio, arylthio, acylthio, carbocyclylthio, heterocyclylthio, heteroarylthio, alkylaryl, alkylacyl, alkylcarbocyclyl, alkylheterocyclyl, alkylheteroaryl, alkyloxyalkyl, aryloxyalkyl, alkylacyloxy, alkylcarbocyclyloxy, alkylheterocyclyloxy, alkylheteroaryloxy, alkylthioalkyl, arylthioalkyl, alkylacylthio, alkylcarbocyclylthio, alkylheterocyclylthio, alkylheteroarylthio, alkylarylalkyl, alkylacylalkyl, arylalkylaryl, arylacylaryl, arylacyl, arylcarbocyclyl, arylheterocyclyl, arylheteroaryl, aryloxyaryl, arylacyloxy, arylcarbocyclyloxy, arylheterocyclyloxy, arylheteroaryloxy, alkylthioaryl, arylthioaryl, arylacylthio, arylcarbocyclylthio, arylheterocyclylthio, arylheteroarylthio, dialkyloxy- , diheterocyclyloxy- or diaryloxy- phosphinyl, dialkyl-, diheterocyclyl- or diaryl- phosphinyl, cyano (i.e. -CN), and -S-R, where R is as defined in respect of formula (II).
More specific examples of Z in RAFT agents used in accordance with the invention include optionally substituted, and in the case of Z* in RAFT agents used in accordance with the invention include a y-valent form of optionally substituted: F, CI, Ci-Cis alkyl, C6-Ci8 aryl, Ci-Cis acyl, amino, C3-C18 carbocyclyl, C2-C18 heterocyclyl, C3-C18 heteroaryl, Q-Cis alkyloxy, C6-Ci8 aryloxy, Q-Cis acyloxy, C3-C18 carbocyclyloxy, C2- Ci8 heterocyclyloxy, C3-C18 heteroaryloxy, Q-Cis alkylthio, C6-Ci8 arylthio, Q-Cis acylthio, C3-C18 carbocyclylthio, C2-Cis heterocyclylthio, C3-C18 heteroarylthio, C7-C24 alkylaryl, C2-Cis alkylacyl, C4-Ci8 alkylcarbocyclyl, C3-Ci8 alkylheterocyclyl, C4-Ci8 alkylheteroaryl, C2-Ci8 alkyloxyalkyl, C7-C24 aryloxyalkyl, C2-Ci8 alkylacyloxy, C4-Ci8 alkylcarbocyclyloxy, C3-Ci8 alkylheterocyclyloxy, C4-Ci8 alkylheteroaryloxy, C2-Ci8 alkylthioalkyl, C7-C24 arylthioalkyl, C2-Ci8 alkylacylthio, C4-Ci8 alkylcarbocyclylthio, C3- Ci8 alkylheterocyclylthio, C4-Ci8 alkylheteroarylthio, C8-C24 alkylarylalkyl, C3-Ci8 alkylacylalkyl, Ci3-C24 arylalkylaryl, Ci3-C24 arylacylaryl, C7-Ci8 arylacyl, C9-Ci8 arylcarbocyclyl, C8-Ci8 arylheterocyclyl, C9-Ci8 arylheteroaryl, Ci2-C24 aryloxyaryl, C7- Cis arylacyloxy, C9-Ci8 arylcarbocyclyloxy, C8-Ci8 arylheterocyclyloxy, C9-Ci8 arylheteroaryloxy, C7-Ci8 alkylthioaryl, Ci2-C24 arylthioaryl, C7-Ci8 arylacylthio, C9-Ci8 arylcarbocyclylthio, C8-Ci8 arylheterocyclylthio, C9-Ci8 arylheteroarylthio, dialkyloxy- , diheterocyclyloxy- or diaryloxy- phosphinyl (i.e. -P(=0)ORk 2), dialkyl-, diheterocyclyl- or diaryl- phosphinyl (i.e. -P(=0)Rk 2), where Rk is selected from optionally substituted Ci-Ci8 alkyl, optionally substituted C6-Ci8 aryl, optionally substituted C2-Ci8 heterocyclyl, and optionally substituted C7-C24 alkylaryl, cyano (i.e. -CN), and -S-R, where R is as defined in respect of formula (II).
In one embodiment, the RAFT agent used in accordance with the invention is a trithiocarbonate RAFT agent and Z or Z* is an optionally substituted alkylthio group.
MacroRAFT agents suitable for use in accordance with the invention may obtained commercially, for example see those described in the SigmaAldrich catalogue (w w w . si gmaaldrich .com).
Other RAFT agents that can be used in accordance with the invention include those described in WO201083569 and Benaglia et al, Macromolecules. (42), 9384-9386, 2009, the entire contents of which are incorporated herein by reference.
Where a free radical polymerisation technique is to be used in polymerising one or more ethylenically unsaturated monomers so as to form at least part of inhibitors of the invention, the polymerisation will usually require initiation from a source of free radicals.
A source of initiating radicals can be provided by any suitable means of generating free radicals, such as the thermally induced homolytic scission of suitable compound(s) (thermal initiators such as peroxides, peroxyesters, or azo compounds), the spontaneous generation from monomers (e.g. styrene), redox initiating systems, photochemical initiating systems or high energy radiation such as electron beam, X- or gamma-radiation.
Thermal initiators are generally chosen to have an appropriate half life at the temperature of polymerisation. These initiators can include one or more of the following compounds:
2,2'-azobis(isobutyronitrile), 2,2'-azobis(2-cyanobutane), dimethyl 2,2'-azobis(isobutyrate), 4,4'-azobis(4-cyanovaleric acid), l,l'-azobis(cyclohexanecarbonitrile), 2-(t-butylazo)-2- cyanopropane, 2,2'-azobis { 2-methyl-N- [1,1 -bis(hydroxymethyl)-2- hydroxyethyl]propionamide}, 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis(N,N'-dimethyleneisobutyramidine) dihydrochloride, 2,2'-azobis(2- amidinopropane) dihydrochloride, 2,2'-azobis(N,N'-dimethyleneisobutyramidine), 2,2'- azobis { 2-methyl-N- [1,1 -bis(hydroxymethyl)-2-hydroxyethyl]propionamide } , 2,2'- azobis { 2-methyl-N- [1,1 -bis(hydroxymethyl)-2-ethyl]propionamide } , 2,2'-azobis [2-methyl- N-(2-hydroxyethyl)propionamide], 2,2'-azobis(isobutyramide) dihydrate, 2,2'-azobis(2,2,4- trimethylpentane), 2,2'-azobis(2-methylpropane), t-butyl peroxyacetate, t-butyl peroxybenzoate, t-butyl peroxyneodecanoate, t-butylperoxy isobutyrate, t-amyl peroxypivalate, t-butyl peroxypivalate, diisopropyl peroxydicarbonate, dicyclohexyl peroxydicarbonate, dicumyl peroxide, dibenzoyl peroxide, dilauroyl peroxide, potassium peroxydisulfate, ammonium peroxydisulfate, di-t-butyl hyponitrite, dicumyl hyponitrite. This list is not exhaustive.
Photochemical initiator systems are generally chosen to have an appropriate quantum yield for radical production under the conditions of the polymerisation. Examples include benzoin derivatives, benzophenone, acyl phosphine oxides, and photo-redox systems.
Redox initiator systems are generally chosen to have an appropriate rate of radical production under the conditions of the polymerisation; these initiating systems can include, but are not limited to, combinations of the following oxidants and reductants: oxidants: potassium, peroxydisulfate, hydrogen peroxide, t-butyl hydroperoxide. reductants: iron (II), titanium (III), potassium thiosulfite, potassium bisulfite.
Other suitable initiating systems are described in commonly available texts. See, for example, Moad and Solomon "The Chemistry of Free Radical Polymerisation", Pergamon, London, 1995, pp 53-95.
Initiators that are more readily solvated in hydrophilic media include, but are not limited to, 4,4-azobis(cyanovaleric acid), 2,2'-azobis{2-methyl-N-[l,l-bis(hydroxymethyl)-2- hydroxyethyl]propionamide}, 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis(N,N'-dimethyleneisobutyramidine), 2,2'-azobis(N,N'- dimethyleneisobutyramidine) dihydrochloride, 2,2'-azobis(2-amidinopropane) dihydrochloride, 2,2'-azobis { 2-methyl-N- [1,1 -bis(hydroxymethyl)-2-ethyl]propionamide } , 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis(isobutyramide) dihydrate, and derivatives thereof.
Initiators that are more readily solvated in hydrophobic media include azo compounds exemplified by the well known material 2,2'-azobisisobutyronitrile. Other suitable initiator compounds include the acyl peroxide class such as acetyl and benzoyl peroxide as well as alkyl peroxides such as cumyl and t-butyl peroxides. Hydroperoxides such as t-butyl and cumyl hydroperoxides are also widely used.
The present invention also relates to methods and uses involving the inhibitor agents to inhibit amyloid fibril formation that can be conducted in an industrial context in relation to the production or processing of proteins that are prone to the formation of amyloid fibril structures. Such methods comprise the introduction of an effective amount (that can readily be titrated by a skilled person for the particular circumstances encountered) of one or more inhibitor compounds into the processing or production environment in order that the proteins are brought into contact or exposed to the inhibitor agents, for example by simple mixing and dissolution of the agents into a batch or continuous production stream. The invention also relates to methods of treatment or prevention of subjects suffering from or prone to suffer from amyloid related diseases or disorders, which involve administering an effective amount of the inhibitor agent to the subject. Example of diseases or disorders that are associated with amyloid fibril formation include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, type II diabetes, transmissible spongiform encephalopathy, medullary carcinoma of the thyroid, isolated atrial amyloidosis causing cardiac arrhythmia, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinoma, familial amyloid polyneuropathy, hereditary non- neuropathic systemic amyloidosis, dialysis related amyloidosis, Finnish amyloidosis, lattice corneal dystrophy, cerebral amyloid angiopathy, Icelandic type cerebral amyloid angiopathy, systemic AL amyloidosis and sporadic inclusion body myositis.
The term "subject", as used herein, means an animal, preferably mammalian and including a human, who is suffering from or who is prone to or suspected of suffering from an amyloid associated disease. Animal subjects include primates, livestock animals (including cows, horses, sheep, pigs and goats), companion animals (including dogs, cats, rabbits and guinea pigs), captive wild animals (including those commonly found in a zoo environment), and aquatic animals (including freshwater and saltwater animals such as fish and crustaceans). Laboratory animals such as rabbits, mice, rats, guinea pigs and hamsters are also contemplated as they may provide a convenient test system. In some embodiments, the subject is a human subject.
By "administration" of the inhibitors to a subject is meant that the agent or composition is presented such that it can be or is transferred to the subject. There is no particular limitation on the mode of administration, but this will generally be by way of the oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intrathecal, and intraspinal), inhalation (including nebulisation), rectal or vaginal modes.
The present invention is also directed to compositions, such as veterinary and pharmaceutical compositions comprising the inhibitors of the present invention. In some embodiments, such composition will comprise the nucleic inhibitors of the present invention and one or more pharmaceutically acceptable carriers, diluents and/or excipients.
A preferred agent for use in the methods, uses and compositions of the invention is arabinogalactan protein (AGP), which can be derived from naturally occurring gum Arabic (GA), which itself can also be used as an inhibitor of the invention.
In the compositions of the present invention, the inhibitors are typically formulated for administration in an effective amount. The terms "effective amount" and "therapeutically effective amount" of the inhibitors as used herein typically mean a sufficient amount of the agent to provide in the course the desired therapeutic or prophylactic effect in at least a statistically significant number of subjects. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner would typically balance the potential benefits against the potential risks in determining what is an appropriate "effective amount", taking into account the general medical condition, species, age, weight, sex, pregnancy status, and ethnic background of the patient as well as the mode of administration, as appropriate. The exact amount required may also vary from subject to subject, so that dosages can be optimized with individual patients over time. Thus, it may not be possible to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.
In some embodiments, an effective amount for a human subject lies in the range of about O. lng/kg body weight/dose to lg/kg body weight/dose. In some embodiments, the range is about ^g to lg, about lmg to lg, lmg to 500mg, lmg to 250mg, lmg to 50mg, or ^g to lmg/kg body weight/dose. Dosage regimes are adjusted to suit the exigencies of the situation and may be adjusted to produce the optimum therapeutic or prophylactic dose. For example, several doses may be provided daily, weekly, monthly or other appropriate time intervals. Thus, the time and conditions sufficient for treatment can be determined by one skilled such as a medical practitioner who is able to specify a therapeutically or prophylactively effective amount. By "pharmaceutically acceptable" carrier, excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable; that is, the material may be administered to a subject along with the complex of the present invention without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, colouring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
The invention will now be further described with reference to the following non-limiting examples.
Example 1 - Preparation of amyloid fibril formation inhibitory agents and determination of amyloid fibril formation inhibitory activity
Background
Arabinogalactan Protein (AGP) is a natural surfactant found in Gum Arabic (GA). GA has been widely used in the food industry for its emulsifying capabilities [26] and consists of 88.4% by mass Arabinogalactan which is a 3.8 x 105 Da polysaccharide, 10.4% AGP with a Mw of 1.45 x 106 Da, and 1.24% Glycoprotein with a Mw of 2.5 x 105 Da [27]. The exact molecular weights of the components, especially AGP vary in the literature [28, 29].
AGP is believed to either exist as a "wattle blossom" structure or as a "twisted hairy rope" [30] or a structure with both properties [31, 32]. There exists a mix of aribinoside and aribinogalactan polysaccharide attachment sites in a fairly repetitive, hydroxyproline-rich protein sequence [32-34]. It is also widely believed that this molecule will fold such that the hydrophobic protein backbone interacts with hydrophobic surfaces, while leaving the carbohydrate groups in the hydrophilic solvent [35].
The inventors developed the hypothesis that the hydrophobic core and hydrophilic side chains of AGP could give rise to activity in inhibiting amyloid fibril formation by a putative mechanism involving interaction with exposed hydrophobic surfaces of amyloid intermediates. This proposed mechanism of action of AGP as an amyloid inhibitor is illustrated in Figure 4. Once bound to the amyloid fibrils, it is hypothesized that the hydrophilic carbohydrate groups will prevent further aggregation of the amyloid by physically preventing more intermediates from binding to the exposed hydrophobic surface of a growing fibril, as illustrated in Figure 3.
In this study, AGP and a number of synthetic polymers having similar structure were investigated for possible inhibitory effects to amyloid fibril formation using Bovine Insulin (BI) and Amyloid βι_4ο (Αβ) as model protein systems. Thioflavin T (ThT) was used as a marker for amyloid fibril formation as ThT is a specific extrinsic dye that fluoresces when bound to multimeric beta sheets such as amyloid fibrils [6, 7] when excited at 450nm, with an emission peak at 482nm. ThT does not interfere with fibril aggregation.
Materials and methods
Preparation of inhibitor compounds
Using the design of an inhibitor based upon the structure of AGP, a synthethic polymer, Fa-diacid (IUPAC name poly-3,5-divinylcyclopentane-l,2-dicarboxilicacid), with a structure shown in Figure 1, was chosen for the tests. Fa-diacid has a similar structure to the AGP molecule in that it has a hydrophobic 5-membered ring backbone similar to the hydroxyproline rich backbone of AGP, and freely rotating hydrophilic side chains. Several other amphiphillic surfactants that have a hydrophobic backbone and hydrophilic side- chain were also tested. The structures of these compounds are shown in Figure 4.
Initial testing of the four synthetic compounds revealed that the Fa-diacid compound was the most effective inhibitor. This compound hence became the subject of further investigation and modifications.
The carboxylic acid side chains of the Fa-diacid compound present opportunities to attach hydrophilic groups such as sugar moieties to produce compounds with structures analogous to that of AGP. It was speculated that such modification could give rise to improved inhibition of amyloid fibril formation. Sugar substituted derivatives of the Fa- diacid compound produced and tested included PNGE, which has a cyclic sugar attached to it and PNGA, which includes an open-chain sugar group side chain. A modified version of the Fa-diacid compound that includes an oxygen group in the backbone was also produced and tested to investigate the effect on inhibitor activity of variations in backbone structure. The structures of these compounds are shown in Figure 5.
Finally using AGP as a model, a 10-monomer long polyhydroxyproline molecule was also synthesized and tested to investigate the effectiveness of amyloid fibril inhibitory activity. A generic structure of a glycosylated form of the polyhydroxyproline (PHP) compound is shown in Figure 6.
General scheme for ROMP of norbornene derivatives
Figure imgf000051_0001
Structure of Grubbs Is and 2n generation catalysts 1 and 2, respectively.
Figure imgf000051_0002
General scheme for synthesis ofFA-diacid and oxoFA-diacid
Figure imgf000052_0001
X = CH2; 3a X = CH2; 4a χ = CH2; FA-diacid
X = O; 3b X = O; 4b χ = Q; oxoFA-diacid
Synthesis of poly(3,5-divinylcyclopentane-l ,2-dicarboxylic acid) FA-diacid
cw-5-Norbomene-e«iio-2,3-dicarboxylic anhydride 3a (0.25 g, 1.5 mmol) was dissolved in anhydrous THF (3 mL) under argon. Separately, catalyst 2 (36 mg, 25 μηιοΐ) was dissolved in anhydrous THF (3 mL) and added rapidly to 3a. The mixture was stirred at room temperature for 2 h and then ethyl vinyl ether (EVE) was added and stirring continued for 1 h to afford 4a. 2 M NaOH (5 mL) was added and the mixture was stirred rapidly for 16 h before adjusting to pH 1 with 0.25 M HCl. The solution was then filtered (0.45 μιη filter), concentrated in vacuo to ca. 3 mL in volume and precipitated into acetone (40 mL). The precipitate was collected via centrifugation and redissolved in water (3 mL) and precipitated once again into acetone (40 mL). The precipitate was isolated via centrifugation, dissolved in the minimum amount of water and then dialysed (2 kDa MWCO) against water for 48 h. The solution was then concentrated in vacuo to afford FA- diacid as a pale brown crystalline solid, 0.17 g. Different molecular weight FA-diacids were prepared by variation of the monomer to catalyst ratio. 1H NMR (400 MHz, D20) 5H 7.19 (br s, Ph end group), 5.42 (br s, =CH), 3.16-2.75 (m, CH), 2.06-0.63 (m, CH2) ppm.
Synthesis ofpoly(tetrahydro-2,5-divinylfuran-3,4-dicarboxylic acid) oxoFA-diacid
Using the same procedure reported for FA-diacid, oxoFA-diacid was prepared from exo- 3,6-epoxy-l,2,3,6-tetrahydrophthalic anhydride 3b via the polymeric anhydride 4b. 1H NMR (400 MHz, D20) δΗ 5.70-5.48 (m, =CH), 4.88 (br s, CHO), 2.96 (br s, CH) ppm. Scheme for synthesis ofpoly( acrylamide-ran-( 1 -vinylpyrrolidin-2-one ) )
Figure imgf000053_0001
Synthesis of poly( acrylamide-ran-( 1 -vinylpyrrolidin-2-one ) )
Acrylamide (12 g, 170 mmol), l-vinylpyrrolidin-2-one (6.3 g, 56 mmol) and 2,2'-azobis(2- methylpropionamidine) dihydrochloride (AMPA) (2.0 g, 7.5 mmol) were dissolved in water (270 mL) under argon and heated at 100 °C for 2 h. After cooling to room temperature the mixture was diluted with water (130 mL) and precipitated into acetone (4.0 L). The precipitate was collected as a spongy solid mass, which was compressed to remove entrapped solvent, and then dried in vacuo to afford a colourless solid, 18.7 g. 1H NMR (400 MHz, D20), 3.81-3.70 (m, CHN), 3.07 (br s, CH2N), 2.50-1.20 (m, CH2 & CH) ppm. From NMR, 25 % of the monomer units are l-vinylpyrrolidin-2-one.
Scheme for synthesis of poly(2,3,4,5,6-pentahydroxy-N-(2-methacryloyloxyethyl) hexanamide )
Figure imgf000053_0002
Synthesis of poly ( 2,3,4,5, 6-pentahydroxy-N-(2-methacryloyloxyethyl)hexanamide )
2,3,4,5, 6-Pentahydroxy-N-(2-methacryloyloxyethyl)hexanamide (0.25 g, 0.81 mmol), and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AMPA) (11 mg, 40 μιηοΐ) were dissolved in water (1.6 mL) under argon and heated at 100 °C for 2 h. After cooling to room temperature the mixture was precipitated into acetone (30 mL). The precipitate was collected via centrifugation and dried in vacuo to afford a crystalline colourless solid, 240 mg. 1H NMR (400 MHz, D20), 4.36-3.51 (m, CHOH, CH2OH, CH20 & CH2N), 2.24-1.58 (m, CH & CH2), 1.29-0.85 (m, CH3) ppm.
Peptide Synthesis and Purification of [Hyp(Gal)]y amide
A solid-phase Fmoc synthesis strategy was employed, which was performed on a CEM Liberty Microwave peptide synthesiser using Tentagel amide resin (0.22mmol/g, 455mg) on a O.lmmol scale. Fmoc-Hyp(Gal-Ac4)-OH (3 eq., 0.3mmol) was activated with HATU (3 eq., 0.3mmol) and catalysed with DIPEA (6 eq., 0.6mmol) in DMF. 20 minute microwave couplings were employed (25W at 75°C). Fmoc cleavage was effected by a 20% piperidine solution in DMF for 3 minutes under microwave conditions (45W at 75°C). The peptide was cleaved from the resin by treatment with a solution of TFA/H20 (97.5/2.5) for 2 hours. The reaction mixture was then filtered and aspirated with a stream of nitrogen until all TFA had evaporated, then dissolved in 50% acetonitrile/H20 and lyophilised. A mass yield of 160mg was recovered.
The crude peptide was analysed via RP-HPLC on an Agilent 1100 HPLC system using a Phenomenex Gemini 5u C18 (4.6 x 150mm) column. Buffer A = 0.1% TFA in H20 and Buffer B = 0.1% TFA in acetonitrile; the gradient was as follows: 0-80% Buffer B over 40 minutes, linear; flow rate = 1.0 niL/min, detection wavelength = 220nm. The molecular weight was confirmed via ESI-MS on an Agilent Accurate-Mass TOF LC/MS. Purification was performed on a Phenomenex Luna 5u C18(2) (150 x 21.2mm) column, using the same buffer systems as above, and with a gradient of 10-90% Buffer B over 80 minutes, linear; flow rate = 8.0mL/min; detection wavelength = 220nm. The clean fractions were pooled and lyophilised, with a mass yield of 38mg.
The acetyl-protected peptide was then treated with a catalytic amount of NaOMe in methanol for 48 hours. The volume was reduced in vacuo and the peptide was desalted using a Phenomenex Hydro-RP 4u C18 (40 x 21.2mm) column using a gradient of 0-20% Buffer B over 40 minutes, linear; flow rate = 8.0mL/min; detection wavelength = 220nm. The clean fractions were pooled and lyophilised, giving a final mass yield of 12.7mg, and the correct molecular weight of 1943.8 was confirmed via ESI-MS.
Scheme for synthesis of poly (4 -hydroxy Iproline) via step-growth polymerisation
Figure imgf000055_0001
poly(4-hydroxyproline)
PHP
In the structures provided above the variables n and m independently represent the integers 1 to 1000.
Synthesis of poly(4-hydroxyproline )
N-Hydroxysuccinimide (NHS) (1.1 g, 9.2 mmol), irans-4-hydroxy-L-proline (0.20 g, 1.5 mmol) and DMAP (77 mg, 0.63 mmol) were weighed into a Schlenk tube and N,N- dimethylformamide (DMF) (5 mL) and water (5 mL) were added. After stirring for 2 h, N,N-diisopropylethylamine (DIPEA) (0.26 g, 2.0 mmol) and N-(3-dimethylaminopropyl)- N'-ethylcarbodiimide hydrochloride (EDCI) (385, 2.0 mmol) were added, the flask was sealed, and the mixture was stirred for 24 h to afford a homogeneous solution. The solution was then heated at 100 °C for 7 days. After cooling to room temperature the solution was precipitated into acetone (100 mL) and the precipitate was isolated via centrifugation. The residue was dissolved in MeOH:water (1: 1 v/v, 5 mL) and propylamine (0.2 mL) was added. The flask was sealed and the solution was stirred for 4 h at room temperature. The solvent was removed in vacuo, the residue was dissolved in MeOH (4 mL) and precipitated into acetone (40 mL). The precipitate was isolated via centrifugation, dissolved in MeOH (4 mL) and precipitated into DEE (40 mL). The precipitate was isolated via centrifugation and dried in vacuo (60 °C, 0.1 mbar, 24 h) to afford PHP as a very hygroscopic cream solid, 67 mg (34 %). 1H NMR (400 MHz, D20) δΗ 4.45-4.38 (m), 3.75-3.11 (m), 2.90-2.00 (m), 1.70 (m, CH2 end group), 1.10 (t, CH3 end group) ppm ( n(NMR) = 1.4 kDa). GPC (H20): n = 2.4 kDa, PDI = 1.26.
Synthesis of Acet l-(phenylalaninyl-0-galactosehydroxyprolyline-phenylalaninyl)l 5 -amide (15mer:(Ac-FX15-NH2) where X = hyp(gal) is and F is pheny alanine)
This compound was synthesised using solid state peptide addition with appropriate capping agents in order to direct the reaction. The final product was analysed using mass spec and shown to be the desired product . The structure of this compound is shown in Figure 18(c).
Preparation of and testing of insulin fibrils
Bovine Insulin (BI) from Sigma Aldrich was weighed and dissolved in 1.5ml of 0.1% (ph 1.5) solution of HC1 that was filtered through a 0.2um syringe filter. The BI concentration was determined spectroscopically with a Varian Cary 3E UV-visible Spectrophotometer using an extinction coefficient at 280nm of 5730 M^cm"1 [41, 42] and a Mw of 5734 g/mol. ThT was added to the solution as well as a volume of any inhibitor used to make a final solution of 0.2mg/ml BI, 50um ThT and inhibitor in 0.1% HC1. Inhibitors added were either 2mg/ml GA, 2mg/ml AGP, 0.0125mg/ml Fa-diacid (lOkDa) or 0.0025mg/ml Fa- diacid (2kDa). The concentrations of the Fa-diacid were the same molar concentration as that of 2mg/ml AGP (0.00125 μιηοΐ/ml).
3 ml of the solution was then incubated in a 1cm x 1cm quartz cuvette with a stirrer bar (Varian Cary Eclipse Fluorescence Spectrophotometer, temperature controlled by a Cary single-cell peltier accessory with an external water bath) fluorometer at 60°C, 550 RPM stirring speed and fluorescence at various wavelengths was monitored using the kinetics program at 5min intervals. 3.5ml of solution was used instead if samples were to be taken for CD (circular dichroism) measurements or AFM (atomic force microscopy) imaging during the experiment.
All circular dichroism readings were performed using a Jasco J-815 circular dichroism machine and all AFM images of samples were taken using an Asylum Research MFP3Dtm atomic force microscope. Preparation and testing of 'Αβ Fibrils
0.7 - 0.8mg of Amyloid Beta (Αβ) 1-40 from Yale University Keck Laboratory was weighed out and dissolved in filtered (0.2um nylon membrane filter) 0.5M, pH 7.1 phosphate buffer of an appropriate concentration. This was left 18 hours overnight at 4°C in the refrigerator to solubilise the protein before concentration was verified spectroscopic ally. Tht and any inhibitor were then added from stock solutions to obtain a final solution of 0.277mg/ml Αβ, 50um ThT in Phosphate buffer. Inhibitors added were either 2mg/ml GA, 2mg/ml AGP, 0.0125mg/ml Fa-diacid (lOkDa) or 0.0025mg/ml Fa- diacid (2kDa). 3ml of this solution was incubated in a 1cm x 1cm quartz cuvette with a stirrer bar at 37°C, 550RPM stirring speed and the fluorescence monitored using kinetics program as with insulin.
Preparation of Lysozyme Fibrils
Hen egg white lysozyme (from Sigma- Aldrich, L7651, 3X crystallized, dialyzed and lyophilized, used without further treatment) was weighed out into a centrifuge tube and dissolved via vortexing or disruption in 1.5ml of 0.1% HCL (pH 1-5-1.6, filtered through a 0.2μιη syringe filter). The tube was then left in the refrigerator at 4°C to settle for 15 minutes. Following that the sample was centrifuged for 10 minutes at 10,000G to remove dust and insoluble materials. The absorbance at 280nm wavelength of the stock was then analyzed in a Cary 3E Absorbance spectrophotometer and the mass calculated using a Mw of HSOOgmol"1 and a extinction coefficient of 37895M"1cm"1.
Each test sample was then made in a Starna magnetic stirrer quartz cell with a stirrer bar to 3ml with a final concentration of 0.2mg/ml Lysozyme in 0.1%HC1, (pH 1.5-1.6) 50μ1 ThT and any inhibitor from the stock lysozyme solution. The solutions were then incubated in a multicell holder (varian) at a block temperature of 70°C at a stirring speed of 840RPM. The fluorescence was monitored at an excitation value of 450nm and an emission value of 482nm using the kinetics programme of the Varian Cary eclipse fluorescence spectrophotometer. Purification and Preparation of AGP
5.0134g of Gum Arabic (GA) from Sigma Aldrich was weighed out and dissolved in milliQ water to obtain a solution of 200mg/ml. This was then centrifuged at 9400g for 20 minutes to pellet out undissolved particulates. 25ml of the supernatant was obtained and diluted with MilliQ water to obtain a 50mg/ml solution of GA. Sodium sulphate (AR grade, approximately 38.94g) was added to the solution slowly with constant stirring until a brown precipitate formed that floated to the surface. This is adapted from a method in the literature to extract proteinaceous components from the solution [40]. The precipitate was then recovered, redissolved in MilliQ water and dialyzed in a 14000kda dialysis membrane against 5L of MilliQ water 5 times over 3 days until conductance of water reached ΙμΜΗΟ.
The solution was then dried via a rotary evaporator and 1.3727g of precipitate was recovered. This was redissolved in MilliQ water to obtain a 50mg/ml solution. Sodium sulphate (AR, approximately 10.62g) was added slowly until a brown precipitate formed. Precipitate was recovered, and redissolved in MilliQ water, dialyzed and dried in same method as outlined above.
This solution was then redissolved with 0.5M (AR grade) sodium chloride solution and fractionated using the fraction collector of a GPC with Waters ultrahydrogel columns (500, 2500 and guard columns) and monitored using UV Absorbance at 280nm, MALLs, QUELS and RI detector using the ASTRA 4 software. Two fractions were obtained and the higher molecular weight fraction corresponded to the AGP. GP collected from chromatographic fractionation of GA was analyzed through the GPC. A single peak was observed on all detectors with an estimated Mw value of 1.647 x 106 g/mol as calculated by the ASTRA 4 software. The GPC traces obtained (data not shown) were similar to traces from size-exclusion chromatography as recorded in the literature [30, 32].
The purity of the AGP was checked by running the sample through the GPC. The two precipitation steps were required to remove the AG component and concentrate the other two components to allow for more accurate fractionation. Results
Purified AGP was used to inhibit BI and AB fibril growth in vitro in order to show proof of principle. For BI a significant increase in the induction (lag) time for fibril formation (reduction in fibril formation rate) was observed while for AB a marked decrease in the ThT fluorescence intensity was observed. AGP was shown to inhibit fibril formation and growth as detected by ThT fluorescence. Without wishing to be bound by theory the inventors believe that the mechanism of stabilisation is via both binding to the hydrophobic faces of the beta sheets and through stabilisation of the alpha helical structures in the protein/peptide systems.
The other compounds tested also demonstrated effectiveness in inhibiting amyloid fibril formation in the two protein systems used, as further explained below.
Amyloid β— protein
Αβ incubated with AGP had a reduced fluorescent intensity at the plateau at 20 hours as seen in Figure 7 to about 1/3 of that when Αβ was incubated without inhibitor. When incubated with GA, an increase in lag time was observed and the final intensity was less than that observed when Αβ was incubated with AGP. Fa-diacid in the same molar concentration as AGP was observed to cause a reduction in fluorescence intensity. Although the lOkDa Fa-diacid caused a larger reduction in fluorescence intensity, it did slightly increase the rate of fibril formation. This is illustrated in Figure 7.
The Fa-diacid (at both molecular weights), AGP and GA all showed inhibitory effects relative to the control in which no inhibitor was present. The inhibitors increased the lag time by varying degrees and also decreased the final fibril concentration formed.
Bovine insulin
Figure 8 shows the effect of the addition of GA, purified AGP, and 2 molecular weights of the Fa-diacid to BI. The formation of BI amyloid fibrils as seen in Figure 8 was reduced significantly by the addition of the inhibitors. The addition of 2mg/ml of GA to 0.2mg/ml of BI increased the lag time of amyloid fibril formation from 5 hours to 24 hours. Using 2mg/ml of purified AGP further increased the lag time to approximately 45 hours as seen in Figure 8. Incubation with Fa-diacid in the same molar concentration as AGP showed a reduction in ThT fluorescence intensity as well as an increase in lag time to about 10 hours, with this effect being more pronounced in the lOkDa Fa-diacid compared to the 2kDa Fa-diacid.
Figure 19 shows the concentration dependent effect of variable concentrations (0.02mg/ml and 0.2mg/ml) of Acetyl-(phenylalaninyl-0-galactosehydroxyprolyline- phenylalaninyl)15-amide in inhibiting bovine insulin fibril formation.
AFM imaging showed that larger but fewer fibrils were formed when BI and Αβ were incubated with AGP and GA than without any inhibitor (Figure 14), which may account for the higher final intensity as seen from AGP and GA. Large fibrils from BI appear to be a rope-like bundle of several smaller amyloid fibrils to form a large superstructure. BI fibrils grown in the presence of Fa-diacid tend to form fibrils with similar morphology to those grown without any inhibitor. The lower fibril density seen in Αβ amyloid fibrils incubated with GA and AGP may account for the lower fluorescent intensity seen. It is also notable that Αβ fibrils formed in the presence of an inhibitor appear to be much longer than those formed without inhibitors. The actual density of fibrils is difficult to quantify for Αβ due to the fact that fibrils often form aggregates.
CD scans at various time points show that when BI is incubated with GA, there is a preservation of the alpha-helical structure of the BI until elongation occurs. This is in contrast to when BI is incubated alone, where the conversion of the alpha-helical structure to the beta-sheet occurs near the start of the reaction, as shown below in Table 1. a-helix (%) ?-sheet (%)
Time (hrs)
BI without GA BI with GA BI without GA BI with GA
0 52 54 31 30
2.5 13 - 54 -
6.5 - 51 - 32
24 6 40 61 35
63 9 10 58 64
Table 1: Total a-helical and β-sheet structural content from CD scans of BI fibrils grown with AGP, showing preservation of a-helical structure when incubated with GA. Analysis was done using web-based software Dichroweb, CDSSTR algorithm [38, 39]. CD scans of BI with GA were using a blank of 2mg/ml GA in HCL and ThT.
CD scans of Αβ incubated with AGP show that the structure changes from random coil to a mixture of random coil and beta-sheet, while Αβ incubated without AGP resulted in predominantly β-sheet CD spectra. This effect may be due to the fact that AGP has a random coil structure and contributes to the effects seen. However, due to the contribution to the signal and overall protein mass made by the AGP as well as the noise of the signal it was difficult to accurately deconvolute using Dichroweb.
Natural GA was also tested at various concentrations against 0.2mg/ml of BI and the results show that at 2mg/ml and above the lag time remains constant at approximately 22 hours. However, the final intensity seems to peak between 0-lmg/ml and then rapidly decreases at higher concentrations of GA. This is shown in Figures 9 and 10.
FA-diacid, which was the most effective of the experimental compounds from Figure 4, was subject to further testing. When the concentration was increased to 2mg/ml, no fibril formation was observed. AFM images, shown in Figure 11, were taken at the end of that experiment of show disordered aggregates before and after the experiment, with no fibril formation observed.
FA diacid (lOkDa) was tested over a range of concentrations as shown in Figure 12 and a concentration dependent increase in lag time was found for the amount of Fa-diacid added to the system. However, with this compound there appears to be an optimal concentration for inhibition of between 0.05mg/ml and 0.3mg/ml, as higher concentrations of Fa-diacid (i.e. 0.3488mg/ml and above) led to flocculation of the protein.
To investigate the effect of modifications to the polymer backbone on BI amyloid fibril formation, two other synthethic compounds were tested: a modified version of Fa-diacid including an oxygen atom in the backbone 5-carbon ring and also a 10-monomer polyhydroxyproline molecule. The modified FA-diacid, which is referred to as "oxy-fa" was shown to bind to and inhibit the amyloid fibril formation of BI more effectively than Fa-diacid, even at a concentration of 0.0875mg/ml. This was confirmed using AFM, which revealed very few, small fibrils in the AFM image of the sample. However, the oxy-fa compound did cause flocculation in the mixture, as observed in high concentrations of Fa- diacid.
The polyhydroxyproline compound exhibited some inhibitory ability, causing a slight increase in lag time to 10 hours at 2mg/ml inhibitor with a final intensity of about 140. AFM imaging showed that the fibrils formed were similar to those formed without inhibitor. This can be seen in Figure 13. However polyhydroxyproline does not appear to be as effective an inhibitor as 2mg/ml of GA or AGP.
Further, two derivatives of Fa-diacid with glucose molecules attached to the carboxylic acid groups via ester-linkages were also tested and the results are shown in Figure 13. The compounds, PNGE and PNGA (with amide linkages derived from amino norborene monomer) were found to exhibit similar effectiveness in reducing the fluorescence intensity as the unmodified versions (when tested at the same molar concentration), though not as effective at increasing lag times. The latter result may be due to the increased size of the molecule making it harder to interact with the exposed hydrophobic ends of the amyloid fibrils. However both are far more soluble in the HC1 buffer than the Fa-diacid and do not flocculate the BI at 2mg/ml. When tested at 2mg/ml both compounds appear to have a similar lag time as 2mg/ml AGP while exhibiting a far lower intensity, suggesting that they may be a more potent inhibitors than AGP. Lysozyme fibrils grown in the presence of GA, PNGA, PNGE and GHP all show a reduction in ThT fluorescence intensity, as seen in Figure 15 and 16. This effect is more pronounced in GA, PNGE and PNGA, with over 20 time's lower intensity in GA and much lower in PNGA and PNGE. The lag times are also increased from 1 hour for lysozyme grown without inhibitor to 4 hours with GA and approximately 5 hours for PNGE and PNGA, while there is little difference in the lag time of amyloid fibril formation due to the addition of GHP.
Atomic force microscopy images taken of the fibrils are seen in Figure 17. They show that there is a slight change in fibrillar morphology when the fibrils are grown in an inhibitor. The fibrils appear to be slightly thicker than that of the control and this effect is most pronounced in GA. It is notable that the AFM is unable to determine fibril density due to the fact that the fibrils tend to clump together during the preparation of the AFM slide.
Circular Dichroism shows that there is an increase in the beta-sheet content of the protein after fibril formation occurs, (note: data not shown)
Ultracentrifugation experiments show that there are fewer fibrils formed for PNGE, PNGA and GA than without inhibitor as there is a larger amount of protein detected in the supernatant as inferred from a higher absorbance at 280nm. (full data set not complete yet due to complication in weighing pellet).
Discussion
It is possible that AGP may stabilise the native alpha-helical structure of the protein, as suggested from Circular Dichroism measurements as seen in Table 1. By stabilising the native protein conformation, the AGP will increase the lag time to amyloid fibril formation due to prevention of protein denaturation.
The difference between the inhibitory effects observed between BI and Αβ due to AGP may be because AGP exhibits a preference for stabilising alpha-helical structures over random coil structures, resulting in better binding to the protein and hence improved inhibition. However, AGP also binds to exposed hydrophobic surfaces of beta-sheets and in so doing, prevents aggregation, as depicted in Figure 3. We believe this to be the case because AGP inhibits fibril formation even when added after BI has reached the elongation stage (data not shown), where the insulin is largely in beta-sheet form [37]. This may explain why AGP is able to inhibit Αβ amyloid fibril formation, as well as explaining the difference in kinetics between the proteins. Additionally, as GA's AGP component is shown to undergo hydrolysis under the acidic environment in which testing was conducted (data not shown), it is likely that hydrolysis of the sugar groups attached to AGP causes the eventual loss of inhibition seen in BI over extended periods of time, while the inhibitory effects in Αβ was maintained due to the neutral incubation environment.
It is further noted that GA seems to increase in lag time and has a further reduction in final fluorescence intensity as compared with AGP for Αβ, indicating that GA is a more effective inhibitor than AGP. This is likely either due to a contribution from the AGP fraction or because 2mg/ml of AGP is too concentrated, and AGP loses its effectiveness beyond a certain concentration ratio to that of the amyloid. Alternatively this may be due to a change in fibril morphology when exposed to pure AGP, as opposed to when exposed to the other components of GA.
Fa-diacid was shown to possess similar inhibitory ability to AGP towards BI and Αβ, although it does not cause an increase in the lag time for Αβ but does so for BI. For the same molar concentration, lOkDa Fa-diacid is a more effective inhibitor than the 2kDa version. The lOkDa Fa-diacid gives rise to a larger decrease in intensity in both BI and Αβ, but is less effective than AGP. These findings support the hypothesis that Fa-diacid works in a similar manner to that of AGP due to its similar physical structure and amphiphilic properties. The difference in efficacy between the two molecular weight samples also supports the hypothesis that the inhibitor works by binding to multiple beta- sheet precursors of the amyloid fibrils, and hence the total mass of the inhibitor may be more important than the total number of inhibitor molecules. However when BI was incubated with 2mg/ml Fa-diacid, no fibrils were formed after 4 days but flocculation of the protein was observed instead. It is also likely that the Fa-diacid follows a different inhibitory pathway to GA by causing the BI to form amorphous aggregates and hence preventing amyloid fibril formation. This effect seems to be more pronounced with the Oxy-Fa compound.
Based on the results obtained, PNGA, PNGE and GA are shown to be effective at reducing the amount of lysozyme amyloid fibrils formed as well as increasing the amount of time that the fibrils take to form. GHP also does show some inhibitory activity, however it is less significant as compared to the other 3 inhibitors tested, while of the inhibitors tested, PNGA has the highest inhibitory activity per mass, followed by PNGE then GA. Those inhibitors also appear to reduce the amount of fibrils formed as supported by evidence from ThT fluorescence as well as the ultracentrifugation results.
It is notable that there is a tendency for larger fibrils to be present when the amyloid is grown in the presence of an inhibitor. The mechanism for this is not fully understood at the moment but it is hypothesized that this is due to the inhibitor promoting the association of fibrils with each other, causing them to twist together like a rope to form a larger fibril. This may have implications for various nanotechnological applications of amyloid fibrils. Centrifugation data (not presented) also shows that the mass of fibrils formed in the presence of the inhibitors is significantly less than for the controls for the three proteins studied.
It is also notable that for the same mass of inhibitor, lysozyme appears to be more susceptible to inhibition than BI and Αβ, which may be due to either the fact that lysozyme is larger or more stable than BI or Αβ as it requires a harsher condition to form fibrils. Additionally, in lieu of the results obtained from BI, it is shown that GA, PNGE and PNGA are effective as a generic amyloid fibril inhibitors.
Polyhydroxyproline appears to be a less effective inhibitor than AGP. This is likely the case as polyhydroxyproline lacks a large hydrophilic steric group, or a carbohydrate/sugar side chain, so it may be simply be incorporated into the amyloid fibril structure over time, as it cannot block further aggregation as effectively with only a lone OH group. A sugar- modified version of this polyhydroxyproline is expected to exhibit improved inhibition of amyloid fibril formation.
It has also been observed that compounds with a hydrophobic five-membered ring structure backbone appear to be better inhibitors than compounds with just a hydrophobic backbone. This leads to the hypothesis that the ring backbone structure of the inhibitors may be correlated to important amyloid fibril inhibitor activity. It is possible that the five- membered ring structure is of significance for binding to the amyloid surface, while the hydrophilic sugar side chains are involved in preventing aggregation.
Amyloid fibril formation is associated with a large variety of diseases in the human body as well as problems in the manufacture of some proteins. Using fluorescence, AFM and CD techniques, AGP from GA as well as the similarly structured Fa-diacid, PNGE and PNGA were shown to be able to inhibit formation of amyloid fibrils in BI and Αβ. It is proposed that the inhibitors work by preserving native alpha-helical structures as well as binding to exposed hydrophobic surfaces of the precursors to prevent amyloid fibril formation.
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Claims

Claims
1. An inhibitor of amyloid fibril formation comprising:
(a) a backbone that is hydrophobic or hydrophilic and comprises from 1 to about 1000 repeating units;
(b) one or more hydrophobic or hydrophilic pendant chains linked to the backbone, wherein the pendant chains are the same or different and comprise from 1 to about 1000 repeating units that are able to freely rotate about the backbone due to free rotation of the repeating units within the backbone;
wherein when the backbone is hydrophobic the pendant chains are hydrophilic and when the backbone is hydrophilic the pendant chains are hydrophobic.
2. An inhibitor of amyloid fibril formation comprising:
(a) a backbone that is hydrophobic and comprises from 1 to about 1000 repeating units;
(b) one or more hydrophilic pendant chains linked to the backbone, wherein the pendant chains are the same or different and comprise from 1 to about 1000 repeating units that are able to freely rotate about the backbone due to free rotation of the repeating units within the backbone.
3. The inhibitor of either claim 1 or claim 2 wherein the backbone comprises from about 5 to about 10 repeating units.
4. The inhibitor of any one of claims 1 to 3 wherein the pendant chains comprise from about 3 to about 20 repeating units.
5. The inhibitor of any one of claims 1 to 4 wherein the backbone and/or the pendant chains is/are copolymers.
6. The inhibitor of any one of claims 1 to 5 wherein the pendant chains are the same.
7. The inhibitor of any one of claims 1 to 6 wherein there is a pendant chain linked to at least about 50% of the repeating units within the backbone.
8. The inhibitor of any one of claims 1 to 6 wherein there is a pendant chain linked to any or all of the repeating units within the backbone.
9. The inhibitor of any one of claims 1 to 6 wherein there is a pendant chain linked to each of the repeating units within the backbone.
10. The inhibitor of any one of claims 1 to 9 wherein repeating units within the hydrophobic backbone or pendant chains comprise one or more of glycine, alanine, valine, leucine, isoleucine, methionine, selenomethionine, phenylalanine, tryptophan, proline, hydroxyproline, styrene, alpha-methyl styrene, butyl acrylate, butyl methacrylate, amyl methacrylate, hexyl methacrylate, lauryl methacrylate, stearyl methacrylate, ethyl hexyl methacrylate, crotyl methacrylate, cinnamyl methacrylate, oleyl methacrylate, ricinoleyl methacrylate, cholesteryl methacrylates, cholesteryl acrylate, vinyl butyrate, vinyl tert-butyrate, vinyl stearate, vinyl laurate, caprolactide, propylene oxide, divinyl cyclopentane and mannose.
11. The inhibitor of any one of claims 1 to 10 wherein repeating units within the hydrophilic backbone or pendant chains comprise one or more of acrylic acid, methacrylic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate, oligo(alkylene glycol)methyl ether (meth)acrylate (OAG(M)A), acrylamide, methacrylamide, hydroxyethyl acrylate, N-methylacrylamide, N,N- dimethylacrylamide and Ν,Ν-dimethylaminoethyl methacrylate, N,N- dimethylaminopropyl methacrylamide, N-hydroxypropyl methacrylamide, norbornenes, 4-acryloylmorpholine, 2-acrylamido-2-methyl- 1 -propanesulfonic acid, phosphorylcholine methacrylate, N-vinyl pyrolidone, amino ethyl acrylamide and ethylene oxide.
12. The inhibitor of any one of claims 1 to 10 wherein repeating units within the hydrophilic backbone or pendant chains comprise one or more ring form and/or straight chain monosaccharide.
13. The inhibitor of claim 12 wherein the monosaccharide comprises one or more of fucose, arabinose, arabitol, allose, altrose, glucofuranose, galactopyranose, glucopyranoside, xylanopyranose, fructopyranose, glucose, galactose, gulose, galactosamine, hammelose, xylose, lyxose, mannitol, mannosamine, ribose, rhamnose, threose, talose and substituted derivatives thereof.
14. The inhibitor of claim 2 or any one of claims 3 to 13 when dependent on claim 2, wherein the hydrophobic backbone unit comprises an optionally substituted four-, five- or six-membered carbocyclic or heterocyclic ring.
15. The inhibitor of claim 2 or any one of claims 3 to 13 when dependent on claim 2, wherein the hydrophobic backbone comprises polyhydroxyproline.
16. An inhibitor of amyloid fibril formation of Formula I:
Figure imgf000074_0001
Figure imgf000074_0002
I
T2
Formula I
wherein:
T, T1 and T2 are the same or different and represent a terminal group; L, L 1 , L2 and L 3 are the same or different and represent a linker group;
A represents a hydrophobic backbone unit;
R represents a hydrophilic pendant chain unit that is bound, optionally via linker group L2, to any of A, L or L1 ;
a, a 1 , a2 and a 3 are the same of different and represent the numerals 0, 1, 2 or 3;
b represents the numerals 3 to 500;
c represents the numerals 0 to 1000, but represents 2 or greater in at least 50% of cases;
and wherein free rotation is allowed within and/or between adjacent A units.
17. The inhibitor of claim 16 wherein the terminal group comprises hydrogen, hydroxyl, halogen, amino, R1, -C02H, -CO2R1, -COR1, -CSR1, -CSOR1, -COSR1, - CONH2, -CONHR1, -CONR^, -OR1, -SR1, -O2CR1, -SCOR1, and -OCSR1; where the or each R1 is independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, and an optionally substituted polymer chain.
18. The inhibitor of either claim 16 or claim 17 wherein the linker group comprises a divalent form of optionally substituted: oxy (-0-), disulfide (-S-S-), alkyl, alkenyl, alkynyl, aryl, acyl (including -C(O)-), carbocyclyl, heterocyclyl, heteroaryl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, acyloxy, carbocyclyloxy, heterocyclyloxy, heteroaryloxy, alkylthio, alkenylthio, alkynylthio, arylthio, acylthio, carbocyclylthio, heterocyclylthio, heteroarylthio, alkylalkenyl, alkylalkynyl, alkylaryl, alkylacyl, alkylcarbocyclyl, alkylheterocyclyl, alkylheteroaryl, alkyloxyalkyl, alkenyloxyalkyl, alkynyloxyalkyl, aryloxyalkyl, alkylacyloxy, alkyloxyacylalkyl, alkylcarbocyclyloxy, alkylheterocyclyloxy, alkylheteroaryloxy, alkylthioalkyl, alkenylthioalkyl, alkynylthioalkyl, arylthioalkyl, alkylacylthio, alkylcarbocyclylthio, alkylheterocyclylthio, alkylheteroarylthio, alkylalkenylalkyl, alkylalkynylalkyl, alkylarylalkyl, alkylacylalkyl, arylalkylaryl, arylalkenylaryl, arylalkynylaryl, arylacylaryl, arylacyl, arylcarbocyclyl, arylheterocyclyl, arylheteroaryl, alkenyloxyaryl, alkynyloxyaryl, aryloxyaryl, arylacyloxy, arylcarbocyclyloxy, arylheterocyclyloxy, arylheteroaryloxy, alkylthioaryl, alkenylthioaryl, alkynylthioaryl, arylthioaryl, arylacylthio, arylcarbocyclylthio, arylheterocyclylthio, and arylheteroarylthio, wherein where present the or each -CH2- group in any alkyl chain may be replaced by a divalent group independently selected from -0-, -OP(0)2-, -OP(0)20-, -S-, -S(O)-, -S(0)20-, -OS(0)20-, -N=N-, -OSi(OR2)20-, -Si(OR2)20-, -OB(OR2)0-, - B(OR2)0-, -NR2-, -C(O)-, -C(0)0-, -OC(0)0-, -OC(0)NR2- and -C(0)NR2-, where the or each R may be independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl, heterocyclyl, arylalkyl, and acyl.
19. The inhibitor of any one of claims 16 to 18 wherein the hydrophobic backbone unit comprises one or more of glycine, alanine, valine, leucine, isoleucine, methionine, selenomethionine, phenylalanine, tryptophan, proline, hydroxyproline, styrene, alpha-methyl styrene, butyl acrylate, butyl methacrylate, amyl methacrylate, hexyl methacrylate, lauryl methacrylate, stearyl methacrylate, ethyl hexyl methacrylate, crotyl methacrylate, cinnamyl methacrylate, oleyl methacrylate, ricinoleyl methacrylate, cholesteryl methacrylates, cholesteryl acrylate, vinyl butyrate, vinyl tert-butyrate, vinyl stearate, vinyl laurate, caprolactide, propylene oxide, divinyl cyclopentane and mannose.
20. The inhibitor of any one of claims 16 to 18 wherein the hydrophobic backbone unit comprises an optionally substituted four-, five- or six-membered carbocyclic or heterocyclic ring.
21. The inhibitor of any one of claims 16 to 18 wherein the hydrophobic backbone unit comprises an optionally substituted five-membered carbocyclic or heterocyclic ring.
22. The inhibitor of any one of claims 16 to 21 wherein the hydrophilic pendant chain unit comprises one or more of one or more of acrylic acid, methacrylic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate, oligo(alkylene glycol)methyl ether (meth)acrylate (OAG(M)A), acrylamide, methacrylamide, hydroxyethyl acrylate, N-methylacrylamide, Ν,Ν-dimethylacrylamide and N,N- dimethylaminoethyl methacrylate, Ν,Ν-dimethylaminopropyl methacrylamide, N- hydroxypropyl methacrylamide, norbornenes, 4-acryloylmorpholine, 2-acrylamido- 2-methyl-l-propanesulfonic acid, phosphorylcholine methacrylate, N-vinyl pyrolidone, amino ethyl acrylamide and ethylene oxide.
23. The inhibitor of any one of claims 16 to 22 wherein repeating units within the hydrophilic backbone or pendant chains comprise one or more ring form and/or straight chain monosaccharide.
24. The inhibitor of claim 23 wherein the monosaccharide comprises one or more of fucose, arabinose, arabitol, allose, altrose, glucofuranose, galactopyranose, glucopyranoside, xylanopyranose, fructopyranose, glucose, galactose, gulose, galactosamine, hammelose, xylose, lyxose, mannitol, mannosamine, ribose, rhamnose, threose, talose and substituted derivatives thereof.
25. The inhibitor of any one of claims 16 to 24 wherein b represents the numerals 5 to 10.
26. The inhibitor of any one of claims 16 to 25 wherein c represents the numerals 4 to 20 in at least 50% of cases.
The inhibitor of any one of claims 16 to 25 wherein c represents the numerals 4 to 20 in at least 80% of cases.
28. Use of an inhibitor of any one of claims 1 to 27 in an industrial process where it is desired to inhibit amyloid fibril formation.
29. Use of an arabinogalactan protein (AGP) in an industrial process where it is desired to inhibit amyloid fibril formation.
30. A method of preventing or reducing amyloid fibril formation in an industrial process in which amyloid fibril formation is prone to occur, which comprises exposing amyloid fibril forming proteins in the process to an inhibitor of any one of claims 1 to 27.
31. A method of preventing or reducing amyloid fibril formation in an industrial process in which amyloid fibril formation is prone to occur, which comprises exposing amyloid fibril forming proteins in the process to an arabinogalactan protein (AGP).
32. Use of an inhibitor of any one of claims 1 to 27 in treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject.
33. Use of an arabinogalactan protein (AGP) in treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject.
34. Use of an inhibitor of any one of claims 1 to 27 in preparation of a medicament for treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject.
35. Use of an arabinogalactan protein (AGP) in preparation of a medicament for treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject.
36. A method of treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject, which comprises administering to the subject an effective amount of an inhibitor of any one of claims 1 to 27.
37. A method of treatment or prevention of a disease or disorder associated with amyloid fibril formation in a mammalian subject, which comprises administering to the subject an effective amount of an arabinogalactan protein (AGP).
38. The use of any one of claims 31 to 35 or the method of either claim 36 or claim 37 wherein the disease or disorder associated with amyloid fibril formation is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, type II diabetes, transmissible spongiform encephalopathy, medullary carcinoma of the thyroid, isolated atrial amyloidosis causing cardiac arrhythmia, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinoma, familial amyloid polyneuropathy, hereditary non-neuropathic systemic amyloidosis, dialysis related amyloidosis, Finnish amyloidosis, lattice corneal dystrophy, cerebral amyloid angiopathy, Icelandic type cerebral amyloid angiopathy, systemic AL amyloidosis and sporadic inclusion body myositis.
39. A pharmaceutical composition comprising as active ingredient an inhibitor of any one of claims 1 to 27 and one or more pharmaceutically acceptable excipients.
40. A pharmaceutical composition comprising as active ingredient an arabinogalactan protein (AGP) and one or more pharmaceutically acceptable excipients.
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