FXI modulators

Abstract

The present disclosure describes cyclic peptide modulators of Factor XI (FXI). Also described are pharmaceutical compositions comprising the cyclic peptides and their use in treating conditions, diseases and / or disorders responsive to FXI modulation, including thrombosis or thrombo-inflammatory conditions.

Description

[0001] FXI modulators

[0002] Cross Reference to the related application

[0003] This application claims the benefit of Australian provisional application no. 2024902087, the entire contents of which are incorporated herein by reference.

[0004] Field of the invention

[0005] The present disclosure relates to novel cyclic peptide modulators of FXI and functional variants and binding fragments thereof. The disclosure further relates to methods of treatment of diseases or disorders responsive to FXI modulation.

[0006] Sequence listing

[0007] A sequence listing in ST. 26 format is filed herewith, the entire contents of which are incorporated herein by reference.

[0008] Background of the invention

[0009] Cardiovascular diseases are the leading cause of death and disability globally and the complex biological processes that underpin these conditions have given rise to a wealth of treatment options. Despite an abundance of anticoagulants in clinical use to treat pathological thrombosis, none have an ideal safety profile due to the lifethreatening bleeding side effects they induce.

[0010] Thrombosis, thrombosis related disorders and thrombo-inflammatory disorders are underlying pathologies associated with cardiovascular diseases such as stroke and ischemic heart disease. To treat these basal conditions, archetypal anticoagulants heparin and warfarin have been administered for decades and although efficacious, present a serious risk of severe bleeding.

[0011] Improvements in the treatment of thrombosis and thrombo-inflammatory disorders are needed.

[0012] An ideal anticoagulant therapy will inhibit thrombus formation without impacting normal haemostatic processes. An attractive approach is the therapeutic targeting of serine protease Factor XI (FXI). Clinical observations conclude that patients with severe FXI deficiency are protected against ischemic stroke and venous thromboembolism. Whilst inversely, high levels of FXI activity correlate with an increased risk of these conditions. Together these observations support the hypothesis that FXI activity is minor in haemostasis, yet a direct and significant contributor to pathological thrombosis in humans.

[0013] For these reasons, in recent years, FXI inhibitors have attracted significant attention as safer anticoagulants compared to archetypal anticoagulants such as heparin and warfarin. FXI inhibition has been shown to prevent pathological thrombosis in animal models with a limited effect on haemostasis. Despite this interest, the development of FXI inhibitors has been slow when compared to FX inhibitors.

[0014] There is a continuing need for the development of FXI modulators for use in the treatment of thrombosis and thrombo-inflammatory disorders.

[0015] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.

[0016] Summary of the invention

[0017] In a first aspect of the invention there is provided a cyclic peptide having the structure selected from:

[0018] ; and or a functional variant or a functional fragment thereof.

[0019] In embodiments there is provided the cyclic peptide of the first aspect, wherein the functional variant or functional fragment has at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the cyclic peptide.

[0020] In embodiments, the cyclic peptide is cyclised via a thioether. The thioether may be formed by reaction of cysteine sulfur atom within the thiol side chain of the amino acid and a chloroacetic acid functionalised N--terminal amino acid. In embodiments there is provided the cyclic peptide or functional variant or functional fragment thereof as described herein, wherein the amino acid sequence has at least 2, at least 3, at least 4, or at least 5 amino acid substitutions. In some embodiments, the cyclic peptide, or functional variant or functional fragment thereof, has at least 1 amino acid substitution. In embodiments there is provided the cyclic peptide as described herein wherein the cyclic peptide comprises any one of the following SEQ ID Nos: 1 to 35:

[0021] SEQ ID No 1 : YRIVWGPDRIEYDC

[0022] SEQ ID No 2: YRIVWGPDRIEY(SO3)DC SEQ ID No 3: YRIIWGDGRTDYNC

[0023] SEQ ID No 4: YRIIWGPDRIEYDC

[0024] SEQ ID No 5: YRI(CHA)WGPDRIEYDC

[0025] SEQ ID No 6: YRI(tBuA)WGPDRIEYDC

[0026] SEQ ID No 7: YRIVWGPDRTEYDC

[0027] SEQ ID No 8: YRIVWGPDRIDYDC

[0028] SEQ ID No 9: YRIVWGPDRIEYNC

[0029] SEQ ID No 10: ARIVWGPDRIEYDC

[0030] SEQ ID No 1 1 : YAIVWGPDRIEYDC

[0031] SEQ ID No 12: YRAVWGPDRIEYDC

[0032] SEQ ID No 13: YRIAWGPDRIEYDC

[0033] SEQ ID No 14: YRIVAGPDRIEYDC

[0034] SEQ ID No 15: YRIVWAPDRIEYDC

[0035] SEQ ID No 16: YRIVWGADRIEYDC

[0036] SEQ ID No 17: YRIVWGPARIEYDC

[0037] SEQ ID No 18: YRIVWGPDAIEYDC

[0038] SEQ ID No 19: YRIVWGPDRAEYDC

[0039] SEQ ID No 20: YRIVWGPDRIAYDC

[0040] SEQ ID No 21 : YRIVWGPDRIEADC

[0041] SEQ ID No 22: YRIVWGPDRIEYAC

[0042] SEQ ID No 23: ARIVWGPDRIEY(SQ3)DC SEQ ID No 24: YAIVWGPDRIEY(SO3)DC

[0043] SEQ ID No 25: YRAVWGPDRIEY(SO3)DC

[0044] SEQ ID No 26: YRIAWGPDRIEY(SO3)DC

[0045] SEQ ID No 27: YRIVAGPDRIEY(SO3)DC

[0046] SEQ ID No 28: YRIVWAPDRIEY(SO3)DC

[0047] SEQ ID No 29: YRIVWGADRIEY(SO3)DC

[0048] SEQ ID No 30: YRIVWGPARIEY(SO3)DC

[0049] SEQ ID No 31 : YRIVWGPDAIEY(SO3)DC

[0050] SEQ ID No 32: YRIVWGPDRAEY(SO3)DC

[0051] SEQ ID No 33: YRIVWGPDRIAY(SO3)DC

[0052] SEQ ID No 34: YRIVWGPDRIEADC

[0053] SEQ ID No 35: YRIVWGPDRIEY(SO3)AC and functional variants and functional fragments thereof.

[0054] In embodiments, the peptide is selected from SEQ ID Nos 1 and 2, or a functional variant or functional fragment thereof.

[0055] In a further aspect, there is provided a pharmaceutical composition comprising the cyclic peptide, functional variant or functional fragment thereof, as described herein and one or more pharmaceutically acceptable excipients.

[0056] In a further aspect, there is provided a method of modulating FXI comprising contacting a cell and / or blood plasma and / or the bloodstream and / or a blood vessel wall and / or a sample with the cyclic peptide, functional variant or functional fragment thereof, or the pharmaceutical composition as described herein.

[0057] In a further aspect there is provided a method of preventing or treating a condition, disease or disorder responsive to inhibiting or modulating FXI, comprising administering to a subject in need thereof the cyclic peptide, functional variant or functional fragment thereof, or the pharmaceutical composition as described herein. In embodiments, the condition, disease or disorder comprise one or more of thrombosis, a thrombotic disorder and a thrombo-inflammatory disorder.

[0058] In a further aspect, there is provided use of the cyclic peptide, functional variant or functional fragment thereof, or pharmaceutical composition as described herein, in the manufacture of a medicament for: treatment of a condition or disease responsive to the inhibition or modulation of FXI; and / or treatment of thrombosis, a thrombotic disorder or a thrombo-inflammatory disorder; and / or anti-coagulant therapy.

[0059] In a further aspect there is provided use of the cyclic peptide, functional variant or functional fragment thereof, or pharmaceutical composition as described herein, for: treatment of a condition or disease responsive to the inhibition or modulation of FXI; or treatment of thrombosis, a thrombotic disorder or a thrombo-inflammatory disorder; anti-coagulant therapy.

[0060] In a further aspect there is provided the cyclic peptide, functional variant or functional fragment thereof, or pharmaceutical composition as described herein for use as an inhibitor or modulator of FXI; as a medicament; as an anti-coagulant; in the treatment of a disease responsive to the inhibition or modulation of FXI; or in the treatment of thrombosis, a thrombotic disorder or a thrombo-inflammatory disorder. in the treatment of a condition, disease or disorder responsive to the inhibition or modulation of FXI; or anti-coagulant therapy.

[0061] In a further aspect there is provided the cyclic peptide, functional variant or functional fragment thereof, or pharmaceutical composition as described herein when used as an inhibitor or modulator of FXI; as a medicament; as an anti-coagulant; in the treatment of a disease responsive to the inhibition or modulation of FXI; or in the treatment of thrombosis, a thrombotic disorder or a thrombo-inflammatory disorder.

[0062] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0063] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

[0064] Brief description of the drawings

[0065] Figure 1 : Graph showing the prolongation of clotting time of peptides Peptide 1 Peptide 2 by in vitro aPTT using human plasma Figure 2: Graphs showing the inhibition of thrombin generation by peptide 1 -9, at 1 pM and at 0.25 pM; A - thrombin concentration (nM) over time, B - peak height for each peptide.

[0066] Detailed description of the embodiments The inventors of the present disclosure have discovered that cyclic peptides having the structures as defined herein are able to inhibit coagulation despite having little or no inhibitory activity of FXIa catalytic activity. The inventors also surprisingly found that these peptides bind to an allosteric site on FXI preventing downstream coagulation. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0067] Peptides of the invention

[0068] of the invention may be isolated, purified, substantially purified, enriched, synthetic or recombinant.

[0069] Definitions

[0070] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0071] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0072] As used herein, the term “and / ot, e.g., “X and / or Y” will be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0073] As used herein, the term “about refers to a quantity, value, dimension, size, or amount that varies by as much as 30%, 25%, 20%, 15% or 10% to a reference quantity, value, dimension, size, or amount.

[0074] As used herein, unless the context requires otherwise, the term “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. As used herein, the term “amino acid’ refers to a compound having an amino group and a carboxylic acid group. The amino acid may be a L- or D- isomer or mixtures thereof. The amino acid may have a naturally occurring side chain (see Table 1 ) or a non-proteinogenic side chain. The amino acid may also be further substituted in the a- position with a group selected from -Ci ealkyl, -(CH2)nCORa, -(CH2)nRb and -PO3H, where n is an integer selected from 1 to 8, Ra is -OH, -NH2, -NHCi salkyl, -OC1 salkyl or - Ci-3alkyl and Rbis -OH, -SH, -SCi-3alkyl, -OCi-3alkyl, -NH2, -NHCi-3alkyl or -NHC(C=NH)NH2 and where each alkyl group may be substituted with one or more groups selected from -OH, -NH2, -NHC1 salkyl, -OC1 salkyl, -SH, -SC1 salkyl, -CO2H, - CO2Ci-3alkyl, -CONH2 and -CONHCi-3alkyl.

[0075] Amino acid structure and single and three letter abbreviations used throughout the specification are defined in Table 1 , which lists the twenty proteinogenic naturally occurring amino acids which occur in proteins as L-isomers.

[0076] Table 1

[0077] As used herein, the term “ non-proteinogenic amino acid’ refers to an amino acid having a side chain that does not occur in the naturally occurring L-a-amino acids recited in Table 1. Examples of non-proteinogenic amino acids and derivatives include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, cyclohexylalanine, ornithine, citrulline, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine, pyridylalanines, substituted variants of the aromatic amino acids tyrosine, phenylalanine and tryptophan and / or D-isomers of natural amino acids.

[0078] As used herein, the code Cha refers to 3-Cyclohexyl alanine and the term tBuA refers to tert-butyl alanine.

[0079] D-isomers of the proteinogenic naturally occurring amino acids may be denoted herein using the corresponding lower case letter of the single letter code included in Table 1 above. For example, in amino acid sequences included herein ‘a’ denotes D- alanine, and ‘y’ denotes D-tyrosine, and so on.

[0080] As used herein, the term “a-amino acid’ refers to an amino acid that has a single carbon atom (the a-carbon atom) separating a carboxyl terminus (C-terminus) and an amino terminus (N-terminus). An a-amino acid includes naturally occurring and non- naturally occurring L-amino acids and their D-isomers and derivatives thereof such as salts or derivatives where functional groups are protected by suitable protecting groups. Unless otherwise stated, the term “amino acid’ as used herein refers to an a-amino acid.

[0081] Similarly, the term “ / 3-amino acid” refers to an amino acid that has a two carbon chain separating a carboxyl terminus (C-terminus) and an amino terminus (N-terminus). Either or both of the carbons between the C- and N-termini may by substituted with any amino acid side-chain described herein. In embodiments, one or more a-amino acids may be substituted by suitable [3-amino acid(s).

[0082] As used herein, the term “hydrophobic amino acid’ refers to an amino acid having a side chain which is non-polar. Examples include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, aminoisobutyric acid, cyclohexylalanine, cyclopentylalanine, norleucine, norvaline, tertbutylglycine and ethylglycine, especially alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan and aminoisobutyric acid.

[0083] As used herein, the term “hydrophilic amino acid’ refers to an amino acid having a side chain which is polar or charged. Examples include, but are not limited to, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine and ornithine.

[0084] As used herein, the term “polar uncharged amino acid’ refers to an amino acid having a side chain that has a dipole moment. Examples include, but are not limited to, serine, threonine, cysteine, tyrosine, asparagine and glutamine.

[0085] As used herein, the term “positively charged amino acid’ refers to an amino acid having a side chain capable of bearing a positive charge. Examples include, but are not limited to, lysine, arginine, histidine and ornithine.

[0086] As used herein, the term “negatively charged amino acid’ refers to an amino acid having a side chain capable of bearing a negative charge. Examples include, but are not limited to, aspartic acid and glutamic acid.

[0087] Those skilled in the art will appreciate that a peptide represents a series of two or more amino acids linked through a covalent bond formed between the carboxyl group of one amino acid and the amino group of another amino acid (i.e. the so-called peptide bond).

[0088] In embodiments, one or more of the amide nitrogen atoms (eg those within the peptide backbone) of the peptides described herein may be substituted with an alkyl group (eg an optionally substituted Ci ealkyl, preferably methyl).

[0089] As used herein, the term “alkyf’ refers to a radical of a straight chain or branched saturated hydrocarbon group. Where appropriate, the alkyl group may have a specified number of carbon atoms, for example, Ci aalkyl which includes alkyl groups having 1 , 2 or 3 carbon atoms in a linear or branched arrangement. Examples of suitable alkyl groups include methyl, ethyl, n-propyl and / -propyl. Alkyl groups described herein may be optionally substituted. Suitable alkyl substituents include one or more (eg 1 , 2, 3, 4, 5 or 6) of -OH, -SH, -NH2, -NHR, -NR2, halo (eg Cl, Br, F and I).

[0090] The present invention also provides a peptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 or 2, or a functional fragment or functional variant thereof, or a pharmaceutically acceptable salt thereof.

[0091] In some embodiments, the amino acid sequence has at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1 or 2. For example, the amino acid sequence may have at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, at least 90% sequence identity to SEQ ID NO:1 or SEQ ID NO 2.

[0092] In some embodiments the amino acid sequence has 100% sequence identity to any one of SEQ ID NO:1 or 2.

[0093] The present invention also provides a peptide comprising an amino acid sequence having at least 1 amino acid substitution compared to SEQ ID NO 1 or SEQ ID NO 2, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino acid sequence has at least 1 , at least 2, at least 3, at least 4, at least 5 or at least 6 amino acid substitutions compared to of SEQ ID No 1 or 2. For example, the amino acid sequence may have 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to any one of SEQ ID NO: 1 or 2.

[0094] As used herein, the terms “functional fragment or “biologically active fragment refers to a portion of the peptide of the invention that retains substantially similar functional activity and / or substantially the same biological function or activity as the polypeptide, for example as shown in assays disclosed herein.

[0095] As used herein, the term “biologically active variant or “functional variant” is intended to encompass peptides having an amino acid sequence sufficiently similar to a peptide of the invention. The term “sufficiently similar” means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and / or common functional activity. Preferably, variants will be sufficiently similar to the amino acid sequence of the preferred peptides of the invention. Variants include polypeptides that differ in amino acid sequence due to mutagenesis.

[0096] As used herein, the terms “substantially similar functional activity” and “substantially the same biological function or activity” each mean that the degree of biological activity is within about 50% to 100% or more, within 80% to 100% or more, or within about 90% to 100% or more, of that biological activity demonstrated by the peptide to which it is being compared when the biological activity of each peptide is determined by the same procedure or assay.

[0097] The “similarity’ between two peptides is determined by comparing the amino acid sequence of a first peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M.O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, P. (1989) EMBO J. 8:779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions:

[0098] Ala, Pro, Gly, Gin, Asn, Ser, Thr:

[0099] Cys, Ser, Tyr, Thr;

[0100] Vai, He, Leu, Met, Ala, Phe;

[0101] Lys, Arg, His;

[0102] Phe, Tyr, Trp, His; and

[0103] Asp, Glu.

[0104] Other conservative amino acid substitutions may also be made by another one of the same class, the classes being as follows:

[0105] Non-polar: Ala, Vai, Leu, He, Pro, Met Phe, Trp

[0106] Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gin

[0107] Acidic: Asp, Glu

[0108] Basic: Lys, Arg, His

[0109] Other conservative amino acid substitutions may also be made as follows:

[0110] Aromatic: Phe, Tyr, His

[0111] Proton Donor: Asn, Gin, Lys, Arg, His, Trp

[0112] Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gin

[0113] In embodiments the cyclic peptide of the invention comprises from about 5 amino acids to about 20 amino acids. Preferably the cyclic peptide comprises from about 10 to about 15 amino acids.

[0114] In embodiments, all amino acids of the cyclic peptide are within a core macrocycle of the cyclic peptide. In alternative embodiments, the cyclic peptide may comprise one or more side chains comprising amino acids extending from a core macrocycle of the cyclic peptide. Each side chain may comprise from 1 to 10 amino acids (not including the amino acid forming part of the macrocyclic core).

[0115] Within the scope of the invention is any peptide of the invention as described herein that contains one or more conservative or non-conservative substitutions provided that the peptide retains the ability to bind to FXI in a way that inhibits FXI activity. In particular peptides of the invention retain the ability to bind allosterically to FXI in a way that modulates or inhibits FXI activity.

[0116] Determining whether a peptide of the invention that contains one or more conservative or non-conservative substitutions retains the ability to treat or prevent conditions or disorders responsive to inhibition of FXI can be readily determined empirically, for example by any method described herein.

[0117] In embodiments, the peptides of the invention may be modified, for example to improve their pharmacokinetic profile, including modifications known in the art for increasing a peptide’s half-life following administration. Any suitable modification that does not substantially impact the peptide’s activity may be employed. Suitable modifications include conjugation with albumin binding peptides, polyethylene glycols (PEGs), and fatty acids, and / or incorporation into Fes, etc.

[0118] The peptides of the present invention may be in the form of pharmaceutically acceptable salts. It will be appreciated however that non-pharmaceutically acceptable salts also fall within the scope of the invention since these may be useful as intermediates in the preparation of pharmaceutically acceptable salts or may be useful during storage or transport. The term “pharmaceutically-acceptable salts” refers to those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, trifluoroacetic (TFA), propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicylic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

[0119] Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.

[0120] Basic nitrogen-containing groups may be quaternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

[0121] A peptide of the invention may be isolated, purified, substantially purified, enriched, synthetic or recombinant. The term “isolated’ in the context of the peptide of the invention means the peptide has been identified, separated and / or recovered from a component of its natural environment. As used herein, the term “substantially purified’ in the context of the peptide of the invention means the peptide is substantially free of contaminating agents, for example at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 65%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% free of contaminating agents. As used herein, the term “recombinant’ will be understood to mean the product of artificial genetic recombination. Accordingly, the term “recombinant peptide" in the context of the peptide of the invention is intended to encompass a peptide expressed by artificial recombinant means when it is in an expression system or cell, for example in which it is expressed.

[0122] The peptides of the present invention may be prepared by known chemical methods, including solid-phase and solution-phase peptide synthesis using Fmoc or Boc protected amino acid residues. As shown in Example 1 , the peptides of the invention may advantageously be synthesised by solid-phase peptide synthesis using suitable solid supports, protecting groups and coupling reagents, which allows for facile and rapid synthesis of the peptides.

[0123] The peptides of the present invention may also be prepared by known recombinant DNA technologies, including cell-based and cell-free technologies. In embodiments where the peptides are prepared by recombinant DNA technologies, the peptide may be prepared from a nucleic acid sequence encoding the peptide with or without genetic code reprogramming. Accordingly, the present invention also provides a nucleic acid comprising a nucleotide sequence encoding the peptide of the invention as described herein.

[0124] Cyclic peptides

[0125] Without wishing to be bound by theory, the inventors believe that the cyclic peptides of the invention exhibit higher stability compared to peptide inventions of the prior art. Cyclic peptides are more resistant to proteolysis than their linear counterparts.

[0126] As molecules that straddle the void between large antibody biologies and small molecule inhibitors, macrocyclic peptides have emerged as promising chemotypes owing to their potent and selective inhibitory profiles. More specifically, macrocyclic peptides are uniquely adept at inhibiting protein-protein interactions between shallow or ill-defined surfaces that are otherwise difficult to disrupt with small molecule structures. Furthermore, macrocyclic peptides are typically more resistant to proteolysis than their linear counterparts, which may assist the cyclic peptides of this disclosure overcoming at least some past limitations for linear peptide inhibitors of FXIa.

[0127] Cyclic peptides are polypeptide chains which are formed by cyclisation of amino acids to form a core macrocycle. A polypeptide chain potentially comprises multiple reactive sites, for example head (or left hand terminus), tail (or right hand terminus) or reactive sites on the side chains of certain amino acids. Cyclisation may be through side-chain to side-chain cyclisation, head-to-tail cyclisation, tail-to-side chain cyclisation or head-to-side chain cyclisation.

[0128] The chemical bonds used to form the cyclisation may be an amide bond formation between a carboxylic acid and amine group. Alternatively, the C-terminal -OH of an amino acid may be converted to an acyl halide, acyl azide, anhydride of activated ester and then reacted with an amine group. Protecting groups may be used to prevent side reactions. In embodiments, the chemical bond used to form the cyclisation may be any nucleophilic amino acid side chain, preferably a cysteine sulfur atom. The nucleophile, typically a thiol functionality may react with a suitably electrophilic carbon atom within the peptide, typically the N-terminal -C(O)CH2X group, where X can be any leaving group, typically a halogen such as a chlorine atom. Accordingly, in embodiments, the peptide is cyclized via formation of a thioether group. The thioether may be formed through reaction between an N-terminal chloroacetyl functionality and a sulfhydryl side chain of a downstream Cys residue.

[0129] Alternative means of cyclization include native chemical ligation (NCL) which is an effective method of linking two unprotected peptide fragments. In this context, NCL involves cyclisation of a C-terminal thio-ester and an N-terminal cysteine residue. Alternatively, the cysteine residue may be replaced with a non-natural thio-containing amino acids. Advantageously native chemical ligation typically does not require the use of protecting groups, and the reaction can proceed in aqueous conditions and neutral pH and tolerates the presence of chaotropic reagents, and reducing agents.

[0130] A further means of cyclisation is formation of disulfide bonds from cysteine residues present in the peptide. When the peptide comprises more than two cysteine residues orthogonal protecting groups may be used to facilitate regioselective di-sulfide bond formation. A di-sulfide stabilizing reagent such as an arsenous acid derivative, dibromopyridazinedione, dihaloxylenes, disubstituted maleimide, or perfluoroaryl derivatives may be used to improve the stability of a di-sulfide bond by reducing it to a thioether linkage.

[0131] Further means of cyclisation include but are not limited to biorthogonal reactions, such as Staudinger ligation, a-ketoacid-hydroxylamine ligation (KAHA ligation), serine / threonine ligation, 1 ,2-aminothiol and 2-((alkylthio)(aryl)methylene)malononitrile (TAMM) ligation, strain promoted azide-alkyne cycloaddition, metal catalysed cyclisation such as copper-catalysed azide-alkyne cycloaddition or ruthenium catalyzed azidealkyne cycloaddition, enzymatic cyclisation for example cyclisation using a subtiligase, sortase, or asparaginyl endopeptidase.

[0132] The present invention also provides pharmaceutical compositions comprising, consisting essentially of, or consisting of the peptide of the invention as described herein, and at least one pharmaceutically acceptable carrier. As used herein, the term “consisting essentially of’ or “consisting of’ in the context of the pharmaceutical composition will be understood to imply that the composition does not comprise any additional active agents other than those specified in the composition.

[0133] The term “pharmaceutically acceptable carried as used herein refers to a solid or liquid filler, diluent, excipient, solvent or encapsulating substance that may be safely used in topical or systemic administration. The camer(s) must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0134] The pharmaceutical composition may be suitably formulated for administration by a particular route. Suitable routes of administration include oral, transmucosal, transdermal, and parenteral administration. In some embodiments, the composition is formulated for oral administration, topical administration such as buccal or sublingual administration or administration by transdermal patch, nasal administration, transdermal administration, or parenteral administration such as subcutaneous, intradermal, intramuscular, intraperitoneal or intravenous administration. In some embodiments, the composition is formulated for oral administration, transdermal administration including administration by transdermal patch, or parenteral administration including subcutaneous, intradermal and intravenous administration. In preferred embodiments, the composition is formulated for oral administration or parenteral administration, especially oral administration or intraperitoneal administration.

[0135] Pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual) or parenteral (including intramuscular, subcutaneous, intradermal and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The peptides of the invention, together with a conventional adjuvant, carrier, excipient, or diluent, may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. The peptides of the present invention can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a peptide of the invention or a pharmaceutically acceptable salt or derivative of the peptide of the invention.

[0136] Pharmaceutical formulations may include formulations of the peptides of the inventions as nano-formulations, nanoparticles or encapsulations. For example, as described in Thimmiah et al., Appl. Sci. 2022, 12(24), 12777.

[0137] For preparing pharmaceutical compositions from the peptides of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

[0138] In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.

[0139] In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.

[0140] The powders and tablets preferably contain from five or ten to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term “preparation" is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

[0141] For preparing suppositories, a low melting wax, such as admixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.

[0142] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

[0143] The peptides according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

[0144] Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing and thickening agents, as desired.

[0145] Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.

[0146] Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[0147] For topical administration to the epidermis, the peptides according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and / or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or colouring agents.

[0148] Formulations suitable for topical administration in the mouth include lozenges comprising active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0149] Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in single or multidose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomizing spray pump. To improve nasal delivery and retention, the peptides according to the invention may be encapsulated with cyclodextrins, or formulated with their agents expected to enhance delivery and retention in the nasal mucosa.

[0150] Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example, dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve. Alternatively, the active ingredient may be provided in the form of a dry powder, for example a powder mix of the active compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).

[0151] Conveniently, the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.

[0152] In formulations intended for administration to the respiratory tract, including intranasal formulations, the active compound will generally have a small particle size for example of the order of 1 to 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronisation.

[0153] When desired, formulations adapted to give sustained release of the active ingredient may be employed.

[0154] The pharmaceutical preparations can be in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0155] FXI modulation

[0156] An attractive approach for new treatments for anticoagulant therapies is the therapeutic targeting of serine protease Factor XI (FXI). Clinical observations conclude that patients with severe FXI deficiency are protected against ischemic stroke and venous thromboembolism. Whilst inversely, high levels of FXI activity correlate with an increased risk of these conditions. Together these observations indicate that FXI activity is minor in haemostasis, yet a direct and significant contributor to pathological thrombosis in humans and that FXI modulators or inhibitors provide a new means of anti-coagulant therapy, and treatment of thrombosis, thrombotic disorders and thrombo- inflammatory disorders.

[0157] Thrombosis and Thrombotic disorders

[0158] The cyclic peptides of the invention have been shown to modulate the activity of FXI, which is useful for the prevention or treatment of thrombosis, thrombotic disorders and / or thrombo-inflammatory disorders.

[0159] As used herein the term thrombosis refers to local coagulation or clotting of the blood in a part of the circulatory system. The thrombosis may be a venous thrombosis or venous thromboembolism, arterial thrombosis, or coronary thrombosis.

[0160] As used herein, references to “thrombotic disorders” are intended to refer to conditions which interfere with haemostasis, the natural process of blood clotting. When haemostasis functions normally, blood clots, or thrombi, form when there is damage to a blood vessel, but do not produce clots that block normal vessels. Abnormalities can result in the inability to form clots or in the excessive formation of clots, both of which can cause serious damage and may even be fatal.

[0161] Examples of thrombotic disorders include, but are not limited to, heart attack, myocardial infarction, acute ischemic stroke, transient ischemic attack (TIA), Deep vein thrombosis, pulmonary embolism, and phlebitis. Cyclic peptides of the invention may also be useful for treatment of subjects requiring cardiac bypass, extracorporeal membrane oxygenation (ECMO) or kidney dialysis.

[0162] As used herein, the term thrombo-inflammatory disorder refers to a disorder related to the loss of the normal antithrombotic and anti-inflammatory functions of endothelial cells, leading to dysregulation of coagulation, complement, platelet activation, and leukocyte recruitment in the microvasculature. Non-limiting examples of thrombo-inflammatory disorders include antiphospholipid syndrome, preeclampsia, sickle cell disease and bacterial or viral infections, sepsis, disseminated intravascular coagulopathy and Pulmonary disseminated coagulation (COVID-19).

[0163] As used herein, the terms “preventing or “prevention" are intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a condition, disorder or disease (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).

[0164] As used herein, the term “therapeutically effective amount is generally intended to refer to an amount of an active agent, such as a peptide of the invention, that (i) treats the particular condition, disorder or disease, (ii) attenuates, ameliorates or eliminates one or more symptoms of the particular condition, disorder or disease, or (iii) delays the onset of one or more symptoms of the particular condition, disorder or disease as described herein. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[0165] Suitable dosages of a peptide of the invention will vary depending on the specific the condition to be treated and / or the subject being treated. It is within the ability of a skilled physician to determine a suitable dosage, for example by commencing with a sub-optimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, to determine an appropriate dosage for treatment / prophylaxis, data from cell culture assays or animal studies may be used, wherein a suitable dose is within a range of circulating concentrations that include the ED50 of the active compound with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A therapeutically / prophylactically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration or amount of the compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high performance liquid chromatography. The peptide of the invention as described herein or the pharmaceutical composition as described herein may be administered, or formulated for administration by, any route described herein. As used herein, the term “administered’ means administration of a therapeutically effective dose of the peptide of the invention to the subject. As used herein, the term “formulated for administration" means a therapeutically effective dose of the peptide of the invention is formulated in such a way that is suitable for the route of administration. In preferred embodiments, the peptide of the invention (or pharmaceutical composition) is administered orally or parenterally, especially orally. In other preferred embodiments, the peptide of the invention (or pharmaceutical composition) is formulated for oral or parenteral administration, especially oral administration.

[0166] Although the peptide of the invention finds application in humans, it will be understood that the invention may also be useful for veterinary purposes. Thus, in all aspects, the methods described herein may be for domestic animals such as cattle, sheep, horses and poultry; for companion animals such as cats and dogs; and for zoo animals. Therefore, the general term “subject or “subject to be / being treated' will be understood to include all animals (such as humans, apes, dogs, cats, horses, and cows) in need of treatment.

[0167] Examples

[0168] The invention will be further described by way of non-limiting examples. It will be understood to persons skilled in the art of the invention that modifications may be made without departing from the spirit and scope of the invention.

[0169] The practice of the present invention employs, unless otherwise indicated, conventional synthesis, analysis and molecular characterisation techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. Example 1 - Synthesis of Peptides

[0170] Peptide 1

[0171] 1 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence / V-YRIVWGPDRIEYDC-C’was generated by automated SPPS on Rink amide resin (102 mg, 50 pmol, capacity: 0.49 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin and cyclisation were performed as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 40 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 1 as a white solid (14.4 mg, 16%). UPLC: Rt = 4.57 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, A = 214 nm). LRMS (ESI+): m / z = 1823.8 [M + H]+. HRMS (ESI+): m / z calcd. for: C83H118N22O23S1Na2, [M+2Na] 2+ 934.41141 , found 934.41227 1 H NMR (500 MHz, DMSO-d6) 5 10.65 (d, J = 59.5 Hz, 1 H), 9.41 - 8.99 (m, 2H), 8.80 - 8.17 (m, 10H), 7.95 (d, J = 84.3 Hz, 3H), 7.59 - 6.74 (m, 21 H), 6.59 (dd, J = 44.1 , 8.0 Hz, 4H), 5.04 - 4.07 (m, 14H), 4.07 - 2.47 (m, 78H), 2.47 - 2.32 (m, 1 H), 2.16 (dt, J = 16.7, 10.2 Hz, 2H), 2.09 - 1 .97 (m, 1 H), 1 .97 - 1 .08 (m, 21 H), 1 .07 - 0.87 (m, 2H), 0.87 - 0.75 (m, 7H), 0.71 (q, J = 7.7 Hz, 6H), 0.66 - 0.44 (m, 6H). Peptide 2

[0172] Access to the sulfated analogue 2 was achieved using Fmoc SPPS, with the incorporation of the sulfotyrosine building block, containing a neopentyl-protected sulfonate ester on the phenol of Fmoc-L-Tyr-OH [FmocTyr(OS03nP)-OH], Upon completion of the peptide sequence, the resin was cleaved and the linear peptide cyclised by treatment with Hunig’s base in DMF. Next, the neopentyl sulfate ester was deprotected upon treatment with sodium azide (10 eq.) in DMF for 14 h at 80 °C.

[0173] Peptide 3

[0174] 3 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence / V-YRIIWGDGRTDYNC-C’was generated by automated SPPS on Rink amide resin (85 mg, 50 pmol, capacity: 0.57 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin and cyclisation were performed as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 3 as a white solid (12.1 mg, 14%). UPLC: Rt = 4.18 min. (0 to 70 vol.% B over 5 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1771.9 [M + H]+. HRMS (ESI+): m / z calcd. for : C77H109N23O23S1 [M+2H]2+885.90447, found 885.90369.

[0175] Peptide 4 4 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence / V -YRIIWGPDRIEYDC-C’ was generated by automated SPPS on Rink amide resin (85 mg, 50 pmol, capacity: 0.57 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin and cyclisation were performed as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 4 as a white solid (8 mg, 9%). UPLC: Rt = 4.19 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, = 214 nm). LRMS (ESI+): m / z = 1839.0 [M + H]+. HRMS (ESI+): m / z calcd. for: C84H120N22O23S1 [M+2H]2+919.43815, found 919.43806. Peptide 5

[0176] 5 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence A / -YRI(CHA)WGPDRIEYDC-C’ was generated by automated SPPS on Rink amide resin (85 mg, 50 pmol, capacity: 0.57 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin and cyclisation were performed as described in the general methods. The crude cyclic peptide was purified by semipreparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 5 as a white solid (6.4 mg, 7%).

[0177] UPLC: Rt = 4.43 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1879.1 [M + H]+. HRMS (ESI+): m / z calcd. for: C87H124N22O23S! [M+2H]2+939.45380, found 939.45308.

[0178] Peptide 6

[0179] 6 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence A / -YRI(tBuAla)WGPDRIEYDC-C’was generated by automated SPPS on Rink amide resin (85 mg, 50 pmol, capacity: 0.57 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin and cyclisation were performed as described in the general methods. The crude cyclic peptide was purified by semipreparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 6 as a white solid (8.6 mg, 9%). UPLC: Rt = 4.19 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1839.0 [M + H]+. HRMS (ESI+): m / z calcd. for: C84Hi2oN22023S[M+2H]2+919.43815, found 919.43721.

[0180] Peptide 7 7 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence / V-YRIVWGPDRTEYDC-C’was generated by automated SPPS on Rink amide resin (85 mg, 50 pmol, capacity: 0.57 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin and cyclisation were performed as described in the general methods. The crude cyclic peptide was purified by semipreparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 7 as a white solid (12 mg, 13%). UPLC: Rt= 3.89 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1812.9 [M + H]+. HRMS (ESI+): m / z calcd. for: C8iHii4N22O24Si[M+2H]2+906.41213, found 906.41090.

[0181] Peptide 8

[0182] 8 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence / V-YRIVWGPDRIDYDC-C’was generated by automated SPPS on Rink amide resin (85 mg, 50 pmol, capacity: 0.57 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin and cyclisation were performed as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 8 as a white solid (8 mg, 14%). UPLC: Rt= 4.53 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1810.9 [M + H]+. HRMS (ESI+): m / z calcd. for: C82H116N22O23S1 [M+2H]2+905.42250, found 905.42221. Peptide 9

[0183] 9 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence / V-YRIVWGPDRIEYNC-C’was generated by automated SPPS on Rink amide resin (85 mg, 50 pmol, capacity: 0.57 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin and cyclisation were performed as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 9 as a white solid (8.2 mg, 9%). UPLC: Rt= 4.51 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, = 214 nm). LRMS (ESI+): m / z = 1823.9 [M + H]+. HRMS (ESI+): m / z calcd. for: C82H116N22O23S1 [M+2H]2+911 .93831 , found 911.93858.

[0184] Peptide 10 Molecular Weight: 1731.95 10 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence A / -ARIVWGPDRIEYDC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 10 as a white solid (6 mg, 13%). HPLC: Rt= 10.23 min. (0 to 60 vol.% B over 30 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1739.6[M + H]+. HRMS (ESI+): m / z calcd. for: C77H114N22O22S [M+H]+1731 .82753, found 1731 .82715.

[0185] Peptide 11

[0186] 11 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence A / -YAIVWGPDRIEYDC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 11 as a white solid (11 mg, 25%).

[0187] HPLC: Rt = 19.33 min. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, I = 214 nm). LRMS (ESI+): m / z = 1739.6[M + H]+. HRMS (ESI+): m / z calcd. for: C80H111 N19O23S [M+H]+1738.78194, found 1738.78937.

[0188] Peptide 12 12 was synthesised via Fmoc-strategy SPPS as specified in general methods.

[0189] The linear sequence A / -YRAVWGPDRIEYDC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 12 as a white solid (8 mg, 14%). UPLC: Rt = 16.13 min. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1782.6 [M + H]+. HRMS (ESI+): m / z calcd. for: C80H112N22O23S [M+H]+1781 .80716, found 1781 .80642.

[0190] Peptide 13

[0191] 13 was synthesised via Fmoc-strategy SPPS as specified in general methods.

[0192] The linear sequence A / -YRIAWGPDRIEYDC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 13 as a white solid (8 mg, 14%).

[0193] UPLC: Rt= 17.63 min. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1796.6 [M + H]+. HRMS (ESI+): m / z calcd. for: C81H114N22O23S [M+H]+1795.82212, found 1795.82207.

[0194] Peptide 14 14 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence A / -YRIVAGPDRIEYDC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 14 as a white solid (13 mg, 19%). HPLC: Rt = 15.83 min. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1709.6 [M + H]+. HRMS (ESI+): m / z calcd. for: C75H113N21O23S [M+H]+1708.81130, found 1708.81117.

[0195] Peptide 15

[0196] 15 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence A / -YRIVWAPDRIEYDC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 15 as a white solid (12 mg, 16%).

[0197] HPLC: Rt = 17.5 min. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1838.75 [M + H]+. HRMS (ESI+): m / z calcd. for: C84H120N22O23S [M+H]+1837.86921 , found 1837.86902. Peptide 16

[0198] 16 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence A / -YRIVWGADRIEYDC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 16 as a white solid (8 mg, 14%). UPLC: Rt 18.23 min. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1798.6 [M + H]+. HRMS (ESI+): m / z calcd. for: C81H116N22O23S [M+H]+1797.84533, found 1797.83772. Peptide 17 17 was synthesised via Fmoc-strategy SPPS as specified in general methods.

[0199] The linear sequence A / -YRIVWGPARIEYDC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 17 as a white solid. The peptide was further purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % TFA over 40 min). The appropriate fractions were combined and lyophilised to afford 17 as a white solid. (10.2 mg, 23%) based on original resin loading. UPLC: Rt= 19.89 min. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1780.6 [M + H]+. HRMS (ESI+): m / z calcd. for: C82H118N22O21S [M+H]+1779.86410, found 1779.86354.

[0200] Peptide 18

[0201] 18 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence A / -YRIVWGPDAIEYDC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 18 as a white solid (12 mg, 19%). UPLC: Rt = 19.44 min. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1739.6 [M + H]+. HRMS (ESI+): m / z calcd. for: C80H111 N19O23S [M+H]+1738.79282, found 1738.78937

[0202] Peptide 19

[0203] 19 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence A / -YRIVWGPDRAEYDC-C’ was generated by automated SPPS on Rink amide resin (86 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 19 as a white solid (9 mg, 15433pZA_))%). UPLC: Rt= 15.73 min. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, I = 214 nm). LRMS (ESI+): m / z = 1782.6 [M + H]+. HRMS (ESI+): m / z calcd. for: C80H112N22O23S [M+H]+1781.79746, found 1781.80642.

[0204] Peptide 20

[0205] 20 was synthesised via Fmoc-strategy SPPS as specified in general methods.

[0206] The linear sequence A / -YRIVWGPDRIAYDC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 20 as a white solid (8.2 mg, 18%). UPLC: Rt= 18.26 min. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, X = 214 nm). LRMS (ESI+): m / z = 1767.6 [M + H]+. HRMS (ESI+): m / z calcd. for: C81H116N22O21S [M+H]+1765.84274, found 1765.84789.

[0207] Peptide 21

[0208] 21 was synthesised via Fmoc-strategy SPPS as specified in general methods.

[0209] The linear sequence A / -YRIVWGPDRIEADC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 21 as a white solid (8 mg, 14%). HPLC: Rt= 18.43 min. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, X = 214 nm).

[0210] LRMS (ESI+): m / z = 1732.6 [M + H]+. HRMS (ESI+): m / z calcd. for: C77H114N22O22S [M+H]+1731 .81987, found 1731 .82715.

[0211] Peptide 22

[0212] 22 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence A / -YRIVWGPDRIEYAC-C’ was generated by automated SPPS on Rink amide resin (163 mg, 50 pmol, capacity: 0.33 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin were performed as described in the general methods. A 25 pmol portion of crude linear peptide was dissolved in DMSO and cyclised as described in the general methods. The crude cyclic peptide was purified by semi-preparative RP-HPLC (0 to 50 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford 22 as a white solid (8 mg, 16%). UPLC: Rt = 18.43 mins. (0 to 50 vol.% B over 30 min, 0.1 vol.% TFA, I = 214 nm). LRMS (ESI+): m / z = 1780.7 [M + H]+. HRMS (ESI+): m / z calcd. for: C82H118N22O21S [M+H]+1779.86367, found 1779.86354. Peptide 23

[0213] 23 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence / V-YAIVWGPDRIEY(SO3)DC-C’ was generated by manual SPPS on Rink amide resin (1 .4074 g, 355 pmol, capacity: 0.252 mmolg-1). [FmocTyr(OS03nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was generated by automated SPPS (25 pmol). Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. A portion of crude linear peptide (17.5 mg, 8.6 pmol) underwent cyclisation and neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at RT for 68 h. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 34 as a white solid (0.62 mg, 4%). HPLC: Rt= 19.43 min. (1 to 60 % B over 30 min, 0.1 % TFA, I = 214 nm). LRMS (ESI+): m / z = 1739.2 [M + H]+ S03, 1817.8 [M - H]-.

[0214] Peptide 23

[0215] 23 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence A / -YRIVWGPDRIEY(SO3)AC-C’ was generated by manual SPPS on Rink amide resin (84 mg, 50 pmol, capacity: 0.594 mmolg-1). [FmocTyr(OS03nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was then generated by automated SPPS. Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. Cyclisation was affected by the dissolution of crude peptide in 10% v / v DIPEA / DMSO. Crude cyclic 23 was purified by super-preparative RP-HPLC (0 to 40 % B + 0.1 % Formic over 35 min). The appropriate fractions were combined and lyophilised to afford FXI L02sulf Ala-1 as neopentyl protected sulfotyrosine. A portion of the pure cyclic peptide (6.22 mg, 3.1 pmol) underwent neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at RT over 36 h. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 23 as a white solid (0.85 mg, 14.9 %). HPLC: Rt= 19.12 min. (1 to 60 % B over 30 min, 0.1 % TFA, X = 214 nm). LRMS (ESI+): m / z = 1860.2 [M + H]+. Peptide 24

[0216] 24 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence / V-YAIVWGPDRIEY(SO3)DC-C’ was generated by manual SPPS on Rink amide resin (1 .4074 g, 355 pmol, capacity: 0.252 mmolg-1). [FmocTyr(OS03nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was generated by automated SPPS (25 pmol). Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. A portion of crude linear peptide (17.5 mg, 8.6 pmol) underwent cyclisation and neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at RT for 68 h. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 24 as a white solid (0.62 mg, 4%). HPLC: Rt= 19.43 min. (1 to 60 % B over 30 min, 0.1 % TFA, I = 214 nm). LRMS (ESI+): m / z = 1739.2 [M + H]+ S03, 1817.8 [M - H]-.

[0217] Peptide 25

[0218] 25 was synthesised via Fmoc-strategy SPPS as specified in general methods.

[0219] The C-terminal tripeptide of the linear sequence / V-YRAVWGPDRIEY(S03)DC-C’ was generated by manual SPPS on Rink amide resin (1 .4074 g, 355 pmol, capacity: 0.252 mmolg-1). [FmocTyr(OS03nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was generated by automated SPPS (25 pmol). Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. A portion of crude linear peptide (31.9 mg, 14.5 pmol) underwent cyclisation and neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at RT for 68 h. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 25 as a white solid (6.55 mg, 24.2%). HPLC: Rt = 17.67 min. (1 to 60 % B over 30 min, 0.1 % TFA, I = 214 nm). LRMS (ESI+): m / z = 1862.2 [M + H]+.

[0220] Peptide 26 26 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence A / -YRIAWGPDRIEY(SO3)DC-C’ was generated by manual SPPS on Rink amide resin (1.4074 g, 355 pmol, capacity: 0.252 mmolg-1). [FmocTyr(OS03nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was generated by automated SPPS (25 pmol). Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. A portion of crude linear peptide (13.2 mg, 6 pmol) underwent cyclisation and neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at RT for 68 h. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 26 as a white solid (9.32 mg, 83.2%). HPLC: Rt = 18.38 min. (1 to 60 % B over 30 min, 0.1 % TFA, I = 214 nm). LRMS (ESI+): m / z = 1876.2 [M + H]+.

[0221] Peptide 27

[0222] 27 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence A / -YRIVAGPDRIEY(SO3)DC-C’ was generated by manual SPPS on Rink amide resin (1.4074 g, 355 pmol, capacity: 0.252 mmolg-1). [FmocTyr(OSG3nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was generated by automated SPPS (25 pmol). Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. A portion of crude linear peptide (27.1 mg, 12.8 pmol) underwent cyclisation and neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at RT for 68 h. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 27 as a white solid (2.88 mg, 12.6%). HPLC: Rt= 16.18 min. (1 to 60 % B over 30 min, 0.1 % TFA, I = 214 nm). LRMS (ESI+): m / z = 1789.2 [M + H]+.

[0223] Peptide 28

[0224] 28 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence A / -YRIVWAPDRIEY(SO3)DC-C’ was generated by manual SPPS on Rink amide resin (1.4074 g, 355 pmol, capacity: 0.252 mmolg-1). [FmocTyr(OS03nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was generated by automated SPPS (25 pmol). Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. A portion of the crude linear peptide (51.5 mg, 22.9 pmol) underwent cyclisation and neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at 40 °C for 2 h, followed by 22 h at RT. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 30 as a white solid (4.2 mg, 9.6%). HPLC: Rt = 18.63 min. (1 to 60 % B over 30 min, 0.1 % TFA, I = 214 nm). LRMS (ESI+): m / z = 1917.9 [M + H]+. Peptide 29

[0225] 29 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence / V -YRIVWGADRIEY(SO3)DC-C’ was generated by manual SPPS on Rink amide resin (1 .4074 g, 355 pmol, capacity: 0.252 mmolg-1). [FmocTyr(OS03nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was generated by automated SPPS (25 pmol). Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. A portion of the crude linear peptide (44 mg, 19.9 pmol) underwent cyclisation and neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at RT for 68 h. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 29 as a white solid (1.59 mg, 4.3%). HPLC: Rt= 18.93 min. (1 to 60 % B over 30 min, 0.1 % TFA, I = 214 nm). LRMS (ESI+): m / z = 1860.1 [M + H]+.

[0226] Peptide 30

[0227] 30 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence / V-YRIVWGPARIEY(SO3)DC-C’ was generated by manual SPPS on Rink amide resin (1 .4074 g, 355 pmol, capacity: 0.252 mmolg-1). [FmocTyr(OS03nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was generated by automated SPPS (25 pmol). Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. A portion of the crude linear peptide (30.9 mg, 14.1 pmol) underwent cyclisation and neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at RT for 68 h. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 30 as a white solid (1.97 mg, 7.5%). HPLC: Rt= 19.88 min. (1 to 60 % B over 30 min, 0.1 % TFA, I = 214 nm). LRMS (ESI+): m / z = 1860.1 [M + H]+.

[0228] Peptide 31 31 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence A / -YRIVWGPDAIEY(SO3)DC-C’ was generated by manual SPPS on Rink amide resin (1.4074 g, 355 pmol, capacity: 0.252 mmolg-1). [FmocTyr(OS03nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was generated by automated SPPS (25 pmol). Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. A portion of the crude linear peptide (28.8 mg, 14.1 pmol) underwent cyclisation and neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at 40 °C for 1 .5 h, followed by 68 h at RT. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 31 as a white solid (2.82 mg, 22%). HPLC: F?f= 19.8 min. (1 to 60 % B over 30 min, 0.1 % TFA, I = 214 nm). LRMS (ESI+): m / z = 1819.7 [M + H]+.

[0229] Peptide 32

[0230] 32 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence A / -YRIVWGPDRAEY(SO3)DC-C’ was generated by manual SPPS on Rink amide resin (1.4074 g, 355 pmol, capacity: 0.252 mmolg-1). [FmocTyr(OSG3nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was generated by automated SPPS (25 pmol). Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. The crude linear peptide was purified by super-preparative RP-HPLC (0 to 40 % B + 0.1 % Formic over 35 min). The appropriate fractions were combined and lyophilised to afford 32 as neopentyl protected sulfotyrosine. A portion of the pure linear peptide (4.7 mg, 2.1 pmol) underwent cyclisation and neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at RT for 36 h. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 32 as a white solid (0.1 mg, 2.5%). HPLC: Rt= 18.25 min. (1 to 60 % B over 30 min, 0.1 % TFA, I = 214 nm). LRMS (ESI+): m / z = 1862.2 [M + H]+.

[0231] Peptide 33

[0232] 33 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence A / -YRIVWGPDRIAY(SO3)DC-C’ was generated by manual SPPS on Rink amide resin (1.4074 g, 355 pmol, capacity: 0.252 mmolg-1). [FmocTyr(OS03nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was generated by automated SPPS (25 pmol). Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. The crude linear peptide was purified by super-preparative RP-HPLC (0 to 40 % B + 0.1 % Formic over 35 min). The appropriate fractions were combined and lyophilised to afford 33 as neopentyl protected sulfotyrosine. A portion of the pure linear peptide (5.5 mg, 2.5 pmol) underwent cyclisation and neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at RT for 36 h. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 33 as a white solid (1.15 mg, 24.7%). HPLC: Rt = 18.67 min. (1 to 60 % B over 30 min, 0.1 % TFA, X = 214 nm). LRMS (ESI+): m / z = 1846.4 [M + H]+. Peptide 34

[0233] 34 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence A / -YRIVWGPDRIEADC-C’ was generated by automated SPPS on Rink amide resin (84 mg, 50 pmol, capacity: 0.594 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin and cyclisation were performed as described in the general methods. Cyclisation was affected by the dissolution of crude peptide in 10% v / v DIPEA / DMSO. The crude cyclic peptide was purified by superpreparative RP-HPLC (0 to 40 % B + 0.1 % formic acid over 35 min). The appropriate fractions were combined and lyophilised to afford peptide 34 as a white solid (4.9 mg, 5.7%). HPLC: Rt= 19.97 min. (1 to 60 % B over 30 min, 0.1 % TFA, X = 214 nm). LRMS (ESI+): m / z = 1732.4 [M + H]+.

[0234] Peptide 35

[0235] 35 was synthesised via Fmoc-strategy SPPS as specified in general methods. The C-terminal tripeptide of the linear sequence A / -YRIVWGPDRIEY(SO3)AC-C’ was generated by manual SPPS on Rink amide resin (84 mg, 50 pmol, capacity: 0.594 mmolg-1). [FmocTyr(OS03nP)-OH (1 .2 eq.) was manually coupled with DIC (2 eq.) and Oxyma (2 eq.) over 16 h. The remainder of the peptide was then generated by automated SPPS. Coupling of chloroacetic acid to the N-terminus and cleavage from resin were performed as described in the general methods. Cyclisation was affected by the dissolution of crude peptide in 10% v / v DIPEA / DMSO. Crude cyclic 35 was purified by super-preparative RP-HPLC (0 to 40 % B + 0.1 % Formic over 35 min). The appropriate fractions were combined and lyophilised to afford 35 as neopentyl protected sulfotyrosine. A portion of the pure cyclic peptide (6.22 mg, 3.1 pmol) underwent neopentyl deprotection in basic ligation buffer (6 M guanidine, 0.1 M HEPES, pH 8.5) at RT over 36 h. Upon completion as determined by UPLC, the crude peptide was purified by super-preparative RP-HPLC (1 to 40 % B + 0.1 % formic acid over 40 min). The appropriate fractions were combined and lyophilised to afford 35 as a white solid (0.85 mg, 14.9 %). HPLC: F?f= 19.12 min. (1 to 60 % B over 30 min, 0.1 % TFA, X = 214 nm). LRMS (ESI+): m / z = 1860.2 [M + H]+. Table 2: Binding of compounds 10 to 35 to human FXIa as measured by surface plasmon resonance (SPR) Example 2- Inhibitory activity of peptide 1

[0236] The inhibitory activity of peptide 1 was assessed using a standard continuous colorimetric assay.

[0237] In this assay, a chromogenic tripeptide substrate D-Pro-Phe-Arg-p-nitroanilide is used in excess, and upon hydrolysis by FXIa, p-nitroaniline is liberated and can be quantified by UV-vis absorbance at 405 nm. Compounds that inhibit FXIa activity will therefore reduce the amount of p-nitroaniline liberated by the protease. By examining enzyme activity over a diverse range of inhibitor concentrations, the relationship between inhibitor concentration and FXI inhibition can be fitted and an IC50 determined.

[0238] Surprisingly peptide 1 had little or no inhibitory activity against the active site of FXI, implying that this peptide binds to a non-catalytic site of FXI. This peptide may serve as an interesting inhibitor beyond the inhibition of the catalytic domain.

[0239] Example 3- Inhibition of Thrombin generation (TG)

[0240] To further investigate the effect of the FXI inhibitor on coagulation, a thrombin generation assay was performed. A calibrated automated thrombogram (CAT) was used which employs a fluorogenic thrombin substrate, thus allowing the measurement of formation of thrombin in plasma in real-time. The intrinsic pathway was triggered using ellagic acid and resulted in thrombin generation with a delayed lag-time and maximum concentration of thrombin of approximately 300 nM. Addition of FXI inhibitor peptide 1 at 1 pM led to a decrease of thrombin generation and increase in lag-time (Figure 1 ).

[0241] Peptide 2 was highly effective at inhibiting thrombin generation (Figure 1 ). At 1 pM 2 completely inhibited thrombin generation (Figure 1) In addition, 2 inhibited thrombin generation by 54% at inhibitor concentration 0.25 uM.

[0242] These results suggest the suitability of the cyclic peptides described herein for treating or preventing thrombosis, and thrombosis related disorders.

Claims

CLAIMS1. A cyclic peptide having the structure:or a functional variant or functional fragment thereof.

2. The cyclic peptide according to claim 1 or 2, wherein the functional variant or functional fragment has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the cyclic peptide.

3. The cyclic peptide of claim 1 or 2, or functional variant or functional fragment thereof, wherein the peptide is cyclised via formation of a thioether group.

4. The cyclic peptide of any one of claims 1 -3, or functional variant or functional fragment thereof, wherein the amino acid sequence has at least 2, at least 3, at least 4, or at least 5 amino acid substitutions.

5. The cyclic peptide of any one of claims 1 -3, or functional variant or functional fragment thereof, wherein the amino acid sequence has at least 1 amino acid substitution.

6. The cyclic peptide of any one of claims 1 to 5 wherein the cyclic peptide comprises any one of the following SEQ ID Nos: 1 to 35:SEQ ID No 1 : YRIVWGPDRIEYDCSEQ ID No 2: YRIVWGPDRIEY(SQ3)DCSEQ ID No 3: YRIIWGDGRTDYNCSEQ ID No 4: YRIIWGPDRIEYDCSEQ ID No 5: YRI(CHA)WGPDRIEYDCSEQ ID No 6: YRI(tBuA)WGPDRIEYDCSEQ ID No 7: YRIVWGPDRTEYDCSEQ ID No 8: YRIVWGPDRIDYDCSEQ ID No 9: YRIVWGPDRIEYNCSEQ ID No 10 ARIVWGPDRIEYDCSEQ ID No 1 1 YAIVWGPDRIEYDCSEQ ID No 12 YRAVWGPDRIEYDCSEQ ID No 13 YRIAWGPDRIEYDCSEQ ID No 14 YRIVAGPDRIEYDCSEQ ID No 15 YRIVWAPDRIEYDCSEQ ID No 16 YRIVWGADRIEYDCSEQ ID No 17 YRIVWGPARIEYDCSEQ ID No 18 YRIVWGPDAIEYDCSEQ ID No 19 YRIVWGPDRAEYDCSEQ ID No 20 YRIVWGPDRIAYDCSEQ ID No 21 YRIVWGPDRIEADCSEQ ID No 22 YRIVWGPDRIEYACSEQ ID No 23: ARIVWGPDRIEY(SO3)DCSEQ ID No 24: YAIVWGPDRIEY(SO3)DC SEQ ID No 25: YRAVWGPDRIEY(SO3)DC SEQ ID No 26: YRIAWGPDRIEY(SO3)DC SEQ ID No 27: YRIVAGPDRIEY(SO3)DC SEQ ID No 28: YRIVWAPDRIEY(SO3)DC SEQ ID No 29: YRIVWGADRIEY(SO3)DC SEQ ID No 30: YRIVWGPARIEY(SO3)DC SEQ ID No 31 : YRIVWGPDAIEY(SO3)DC SEQ ID No 32: YRIVWGPDRAEY(SO3)DC SEQ ID No 33: YRIVWGPDRIAY(SO3)DC SEQ ID No 34: YRIVWGPDRIEADC SEQ ID No 35: YRIVWGPDRIEY(SO3)AC and functional variants and functional fragments thereof.

7. The peptide of any one of claims 1 to 6 wherein the peptide is selected from SEQ ID Nos 1 and 2, or a functional variant or functional fragment thereof.

8. A pharmaceutical composition comprising the cyclic peptide of any one of claims 1 to 7, or functional variant or functional fragment thereof, and one or more pharmaceutically acceptable excipients.

9. A method of modulating FXI comprising contacting a cell and / or blood plasma and / or the bloodstream and / or a blood vessel wall and / or a sample with the cyclic peptide of any one of claims 1 to 7, or functional variant or functional fragment thereof, or the pharmaceutical composition of claim 8.

10. A method of preventing or treating a condition, disease or disorder responsive to modulating FXI comprising administering to a subject in need thereof the cyclic peptide of any one of claims 1 to 7 or the pharmaceutical composition of claim 8.11 . The method of claim 10 wherein the condition or disorder is thrombosis, thrombotic disorders or a thrombo-inflammatory disorder.

12. A method of anticoagulant therapy, comprising administering to a subject in need thereof a cyclic peptide of any one of claims 1 to 7, or a functional variant or functional fragment thereof, or the pharmaceutical composition of claim 8.

13. Use of the cyclic peptide of any one of claims 1 to 7 in the manufacture of a medicament for the treatment of a condition, disease or disorder responsive to modulation of FXI.

14. The use of claim 13 wherein the condition or disease is thrombosis, a thrombotic disorder or a thrombo-inflammatory disorder.

15. Use of the peptide of any one of claims 1 to 7 in the manufacture of an anticoagulant.

Images