Inhibitors of fxia

Abstract

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

Description

10060061841 Inhibitors of FXIa Cross Reference to the related application

[0001] This application claims the benefit of Australian provisional application no. 2024902088, the entire contents of which are incorporated herein by reference. Field of the invention

[0002] The present disclosure relates to novel cyclic peptide inhibitors of FXIa and functional variants and binding fragments thereof. The disclosure further relates to methods of treatment of diseases or disorders responsive to FXIa inhibition, particularly thrombosis or thrombo-inflammatory conditions. Sequence listing

[0003] A sequence listing in ST.26 format is filed herewith, the entire contents of which are incorporated herein by reference. Background of the invention

[0004] 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 life- threatening bleeding side effects they induce.

[0005] 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.

[0006] Improvements in the treatment of thrombosis and thrombo-inflammatory disorders are needed.

[0007] 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 severe10060061842 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.

[0008] 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.

[0009] There is a continuing need for the development of FXI inhibitors for use in the treatment of thrombosis and thrombo-inflammatory disorders.

[0010] 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. Summary of the invention

[0011] In a first aspect of the invention there is provided a cyclic peptide selected from any one of the following:10060061847or a functional variant or functional fragment thereof.

[0012] In embodiments, the cyclic peptide is selected from any one of SEQ ID No 1-9 or a functional variant or functional fragment thereof: SEQ ID no 1: YPHGLNRHTFC; SEQ ID no 2: YHWKTTRSNYLC; SEQ ID no 3: YHFLNIRSNYDC; SEQ ID no 4: YHDRYIRSNYLC; SEQ ID no 5: YIPASFVNTRLHC; SEQ ID no 6: yAYRIVWPDRTQYEC; SEQ ID no 7: yLLGPYVRIVC; SEQ ID no 8: yRTRIEDIFC; and SEQ ID no 9: yHWRNIRSNYSC.

[0013] 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.10060061848

[0014] 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.

[0015] 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.

[0016] In embodiments there is provided the cyclic peptide as described herein wherein the cyclic peptide comprises any one of SEQ ID No 1-4, or 7-9.

[0017] 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.

[0018] In a further aspect, there is provided a method of inhibiting FXIa, 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.

[0019] In a further aspect there is provided a method of preventing or treating a condition, disease or disorder responsive to inhibiting FXIa, 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.

[0020] In another aspect, there is provided a method of preventing or treating thrombosis, a thrombotic disorder or a thrombo-inflammatory disorder, comprising administering to a subject in need thereof the cyclic peptide, functional variant or functional fragment thereof, or the pharmaceutical composition as described herein.

[0021] In a further aspect, there is provided a method of anticoagulant therapy, comprising administering to a subject in need thereof a cyclic peptide, functional variant or functional fragment thereof, or pharmaceutical composition as described herein.10060061849

[0022] 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 of FXI; and / or treatment of thrombosis, a thrombotic disorder or a thrombo-inflammatory disorder; and / or anti-coagulant therapy.

[0023] 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 of FXI; or treatment of thrombosis, a thrombotic disorder or a thrombo-inflammatory disorder; anti-coagulant therapy.

[0024] 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 of FXI, preferably inhibiting FXIa; as a medicament; as an anti-coagulant; in the treatment of a disease responsive to the inhibition of FXI, preferably FXIa; 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 of FXI, preferably FXIa; or anti-coagulant therapy.100600618410

[0025] 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 of FXI, preferably an inhibitor of FXIa; as a medicament; as an anti-coagulant; in the treatment of a disease responsive to the inhibition of FXI, preferably FXIa; or in the treatment of thrombosis, a thrombotic disorder or a thrombo- inflammatory disorder.

[0026] 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.

[0027] 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. Brief description of the drawings

[0028] Figure 1: Graph showing the prolongation of clotting times in aPTT of cyclic peptides 1-9. Horizontal dashed lines indicate clinically relevant clotting times for inhibitor-less plasma controls in aPTT.

[0029] Figure 2: Graph showing the inhibition of thrombin generation upon triggering the intrinsic pathway of coagulation. Peptide concentrations of 1µM in blue and 0.25 µM in red.

[0030] Figure 3: Graphs showing: 3A - inhibition of fibrin deposition on the microfluidic chip with varying concentrations of peptide 8. Fibrin intensity was quantified using AF555-conjugated fibrinogen.3B - Neutrophil aggregates were quantified by FITC- CD15, and 4C Platelet aggregates were quantified by Vioblue Reaffinitty CD41 / CD61.100600618411

[0031] Figure 4A: Graph showing the IC50 curves for peptides 1-9 against FXIa.

[0032] Figure 4B: Graph showing the IC50 curves for alanine scan analogues 10-18 against FXIa.

[0033] Figure 5: Scatter plot depicting percent inhibition of peptides 1-5 and 7-9 at 100 µM inhibitor concentrations showing the selectivity of peptides against panel of related serine proteases.

[0034] Figure 6A: Thrombinoscope Plots for Peptides 1-9 in human plasma when dosed at 0.25 μM.

[0035] Figure 6B: Thrombinoscope Plots for Peptides 1-9 in human plasma when dosed at 1 μM.

[0036] Figure 7: Graph showing the dose response for peptide 8 = EC5076 nM.

[0037] Figure 8: Graph showing the IC50 determination of inhibitors 2,3,7 and 8 against PK.

[0038] Figure 9: Peptide 8 was incubated with citrated pooled human plasma (Diagnostica Stago S.A.S., Asnières-sur-Seine, France) for 24 hours. A) Chromatograms of extracted peptide at 1 hr incubation. B) Chromatograms of extracted peptide at 24 hr incubation. C) Structure of cleavage product. Detailed description of the embodiments

[0039] The inventors of the present disclosure have surprisingly discovered that cyclic peptides having the structures as defined herein are able to inhibit FXI. The inventors also surprisingly found that these FXI active cyclic peptides interact with the FXI active site (FXIa).100600618412 Peptides of the invention100600618413100600618414100600618415100600618416and functional variants and / or functional fragments thereof.

[0040] A peptide of the invention may be isolated, purified, substantially purified, enriched, synthetic or recombinant. Definitions

[0041] 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,100600618417 preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0042] 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.

[0043] As used herein, the term “and / or”, 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.

[0044] 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.

[0045] 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.

[0046] 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 ^- position with a group selected from -C1-6alkyl, -(CH2)nCORa, -(CH2)nRb and -PO3H, where n is an integer selected from 1 to 8, Ra is -OH, -NH2, -NHC1-3alkyl, -OC1-3alkyl or - C1-3alkyl and Rb is -OH, -SH, -SC1-3alkyl, -OC1-3alkyl, -NH2, -NHC1-3alkyl or -NHC(C=NH)NH2 and where each alkyl group may be substituted with one or more groups selected from -OH, -NH2, -NHC1-3alkyl, -OC1-3alkyl, -SH, -SC1-3alkyl, -CO2H, - CO2C1-3alkyl, -CONH2 and -CONHC1-3alkyl.

[0047] 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.100600618418 Table 1

[0048] 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-^-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-5-100600618419 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

[0049] 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.

[0050] As used herein, the term “^-amino acid” refers to an amino acid that has a single carbon atom (the ^-carbon atom) separating a carboxyl terminus (C-terminus) and an amino terminus (N-terminus). An ^-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 ^- amino acid.

[0051] Similarly, the term “β-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 be substituted with any amino acid side-chain described herein. In embodiments, one or more ^-amino acids may be substituted by suitable β-amino acid(s).

[0052] 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, tert- butylglycine and ethylglycine, especially alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan and aminoisobutyric acid.

[0053] 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.100600618420

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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).

[0058] 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 C1-6alkyl, preferably methyl).

[0059] As used herein, the term “alkyl” 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, C1-3alkyl 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 i-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).

[0060] The present invention also provides a peptide comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs:1 – 9, or a functional fragment or functional variant thereof, or a pharmaceutically acceptable salt thereof.

[0061] 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 any one of SEQ ID NO:1. For example, the amino acid sequence may have at least 70%, at100600618421 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 any one of SEQ ID NOs:1 to 9.

[0062] In some embodiments, the amino acid sequence of the peptide is any one of SEQ ID NO:1-9, or SEQ ID NO: 1-18, that is, the amino acid sequence has 100% sequence identity to any one of SEQ ID NO:1-9 or SEQ ID NOs 8 or 10-18. Preferably the amino acid sequence of the peptide is any one of SEQ ID NO:1-4 or 7-9.

[0063] In some embodiments , the amino acid sequence of the peptide is SEQ ID no 1, or SEQ ID no 2, or SEQ ID no 3, or SEQ ID no 4, or SEQ ID no 5, or SEQ ID no 6, or SEQ ID no 7, or SEQ ID no 8, or SEQ ID no 9, preferably the amino acid sequence of the peptide is SEQ ID no 1, or SEQ ID no 2, or SEQ ID no 3, or SEQ ID no 4, or SEQ ID no 7, or SEQ ID no 8, or SEQ ID no 9, more preferably the amino acid sequence of the peptide is SEQ ID no 8. The present invention also provides a peptide comprising an amino acid sequence having at least 1 amino acid substitution compared to any one of SEQ ID NOs 1-9, or a pharmaceutically acceptable salt thereof.

[0064] 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 any one of SEQ ID Nos 1-9. 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 IDs NO: 1-9 or SEQ IDs NO: 8, or 10-18.

[0065] 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

[0066] 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 amino100600618422 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.

[0067] 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.

[0068] 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.

[0069] For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: - Ala, Pro, Gly, Gln, Asn, Ser, Thr: - Cys, Ser, Tyr, Thr; - VaI, Ile, Leu, Met, Ala, Phe; - Lys, Arg, His; - Phe, Tyr, Trp, His; and - Asp, Glu.

[0070] Other conservative amino acid substitutions may also be made by another one of the same class, the classes being as follows:100600618423 Non-polar: Ala, Val, Leu, Ile, Pro, Met Phe, Trp Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic: Asp, Glu Basic: Lys, Arg, His

[0071] Other conservative amino acid substitutions may also be made as follows: Aromatic: Phe, Tyr, His Proton Donor: Asn, Gln, Lys, Arg, His, Trp Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln

[0072] 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.

[0073] 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).

[0074] 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 FXIa in a way that inhibits FXI activity.

[0075] 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.

[0076] 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 that100600618424 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 Fcs, etc.

[0077] 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.

[0078] Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.

[0079] 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.

[0080] 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 about100600618425 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.

[0081] 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.

[0082] 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. Cyclic peptides

[0083] 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.

[0084] As molecules that straddle the void between large antibody biologics 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 FXIa active linear peptides.100600618426

[0085] 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.

[0086] 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.

[0087] 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 thioester 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.

[0088] 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 disulfide bond formation. A disulfide stabilizing reagent such as an arsenous acid derivative, dibromopyridazinedione, disubstituted maleimide, dibromoxylene or perfluoroaryl derivatives may be used to improve the stability of a di-sulfide bond by reducing it to a thioether linkage.100600618427

[0089] Further means of cyclisation include but are not limited to biorthogonal reactions, such as Staudinger ligation, α-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 azide-alkyne cycloaddition, enzymatic cyclisation for example cyclisation using a subtiligase, sortase, or asparaginyl endopeptidase. Pharmaceutical compositions

[0090] 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.

[0091] 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.

[0092] The term “pharmaceutically acceptable carrier” 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 carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0093] 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,100600618428 the composition is formulated for oral administration or parenteral administration, especially oral administration or intraperitoneal administration.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.100600618429

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.100600618430

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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 or100600618431 formulated with their agents expected to enhance delivery and retention in the nasal mucosa.

[0109] 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.

[0110] 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).

[0111] 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.

[0112] 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.

[0113] When desired, formulations adapted to give sustained release of the active ingredient may be employed.

[0114] 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 form100600618432 FXIa inhibition

[0115] 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 FXIa inhibitors provide a new means of anti-coagulant therapy, and treatment of thrombosis, thrombotic disorders and thrombo-inflammatory disorders. Thrombosis and Thrombotic disorders

[0116] The cyclic peptides of the invention have been shown to inhibit FXIa, which is useful for the prevention or treatment of thrombosis, thrombotic disorders and / or thrombo-inflammatory disorders.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] As used herein, the term thrombo-inflammatory disorder refers to a disorder related to the loss of the normal antithrombotic and anti-inflammatory functions of100600618433 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, sepsis, Disseminated Intravascular Coagulation (DIC), Pulmonary disseminated coagulation (COVID-19), and bacterial or viral infections.

[0121] 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).

[0122] 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.

[0123] 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., the100600618434 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.

[0124] 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.

[0125] 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.

[0126] 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. Examples

[0127] 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.100600618435

[0128] 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. General Synthetic and Analytical Procedures Reagents and Solvents

[0129] Peptide-grade N,N-dimethylfomamide (DMF) and dichloromethane (DCM) were purchased from RCI Labscan and Merck, respectively. Acetonitrile (MeCN) for chromatography was purchased as ‘gradient grade’ from Sigma-Aldrich and ultrapure water was from a Merck Millipore Direct-Q 5 water purification system. All solvents for chromatography were supplemented with formic acid (FA) purchased from Sigma- Aldrich. All standard Fmoc-protected amino acids were purchased from Mimotopes. Automated SPPS was carried out on Biotage Syro1. Pooled human plasma was purchased from Sigma-Aldrich. General procedure A; Automated Peptide Synthesis (SYRO I peptide synthesizer)

[0130] The resin (164 mg, 100 µmol, 0.61 mmol g-1, 1 eq.) was treated with 40 vol.% piperidine (1.6 mL) in DMF for 3 min, drained, and then treated with 20 vol.% piperidine in DMF for 10 min (1.6 mL), drained, and washed with DMF (4 x 1.6 mL). The resin was then treated with a solution of Fmoc-Xaa-OH (400 µmol, 4 eq.) and Oxyma (57 mg, 400 µmol, 4 eq.) in DMF (800 µL), followed by a solution of DIC (63 µL, 400 µmol, 4 eq.) in DMF (800 µL) and shaken at rt for 1 h. The resin was then drained and washed with DMF (4 x 1.6 mL) before being treated with a solution of 5 vol.% Ac2O and 10 vol.% iPr2NEt in DMF (1.6 mL) for 5 min at rt, drained, washed with DMF (4 x 1.6 mL) and drained.

[0131] Chloroacetic acid coupling: Resin-bound peptide (0.05 mmol) was shaken in a solution of chloroacetic acid (8 eq, 0.5 M), DIC (8 eq., 125 ^L, 0.2 M) and Oxyma (8 eq., 0.5 M) in DMF (4 mL) for 30 min at 75°C. The coupling solution was drained, and the resin washed with DMF (3 x 5 mL), DCM (5 x 4 mL).

[0132] Peptide cleavage from resin: Resin-bound peptide was shaken in a cleavage solution of TFA / TIS / H2O (90:5:5 v / v / v) for 2 h at rt. The crude product was drained, and the resin rinsed with cleavage cocktail (~ 2 mL). These solutions were combined and100600618436 concentrated to <1 mL under nitrogen flow. To precipitate the free peptide, diethyl ether (14 mL) was added to the crude concentrate. The resultant suspension was centrifuged for 4 min at 7000 rcf to pellet the free peptide. The supernatant was then decanted, and the precipitation process repeated once more. The crude peptide was dried under nitrogen flow, then re-dissolved in 50 %v / v aq. MeCN (~6 mL) for cyclisation.

[0133] Linear peptide cyclisation: To a solution of linear peptide in 50 vol.% aq. MeCN (~6 mL), iPr2NEt (300 ^L, 0.3 M) was added and the reaction shaken for 16 h at rt. The cyclic peptide solution was then re-acidified with TFA (240 ^L, 0.5 M) and filtered in preparation for RP-HPLC. Preparative Chromatography

[0134] Reversed-phase high performance liquid chromatography (HPLC) was performed on a Waters 600E multi-solvent delivery system fitted with a Rheodyne 7725i injection valve (5 mL loading loop), a Waters 500 pump and a Waters 490E programmable wavelength detector operating at 214 nm and 230 nm. Preparative reversed-phase HPLC was performed using a Waters semi-preparative Sunfire OED C18 column (5 μm, 19 x 150 mm) at a flow rate of 15 mL min-1. All preparative HPLC used a mobile phase of ultrapure (type 1) water (Solvent A) and MeCN (Solvent B) supplemented with 0.1 vol% formic acid or trifluoroacetic acid (TFA) on gradients as specified. Analytical Chromatography

[0135] Liquid Chromatography-Mass Spectrometry (UPLC) was performed on a Shimadzu 2020 UPLC instrument with a Nexera X2 LC-30AD pump, Nexera X2 SPD- M30A UV / Vis diode array detector and a Shimadzu 2020 (ESI) mass spectrometer operating in either positive or negative mode. Separations were performed on a Waters Acquity BEH3001.7 ^m, 2.1 x 50 mm (C18) column at a flow rate of 0.6 mL min-1. All separations were performed using a mobile phase of 0.1 vol% formic acid in water (Solvent A) and 0.1 vol% formic acid in MeCN (Solvent B) using gradients as specified. Analytical reversed-phase HPLC was performed on a Waters Acquity UPLC system equipped with a PDA ^ detector (^ = 210 – 400 nm). Separations were performed on a Waters Acquity BEH3001.7 ^m, 2.1 x 50 mm (C18) column at a flow rate of 0.6 mL100600618437 min-1. All separations were performed using a mobile phase of 0.1 vol% TFA in water (Solvent A) and 0.1 vol% TFA in MeCN (Solvent B) using gradients as specified. High Res Mass Spectrometry

[0136] High resolution mass spectra were recorded on a Bruker-Daltronics Apex Ultra 7.0 T Fourier transform (FTICR) mass spectrometer. Assays of human FIIa, FXIIa, Fxa, FXIa, and PK activity

[0137] The inhibition of human FIIa (Haematologic Technologies) amidolytic activity was followed spectrophotometrically using Tos-Gly-Pro-Arg-p-nitroanilide (Chomozym TH, Roche) as chromogenic substrate. The assays were performed in 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mg / mL BSA with 0.2 nM FIIa, 100 μM substrate and 33 µM of corresponding inhibitor. All reactions were initiated by the addition of the protease and measurements were recorded.

[0138] The inhibition of human FXIa or FXa (Haematologic Technologies) was determined in 25 mM HEPES / MOPS sodium salt pH 7.5, 100 mM NaCl, 5 mM CaCl2 and 1 mg / mL BSA with 0.5 nM of protease, 200 µM of fluorescence substrate SN45 or SN7 (Haematologic Technologies) and 33 µM of corresponding synthetic inhibitor. All the reactions were initiated by the addition of the protease and carried out at 37 °C in 96-well black flat bottom microtiter plates. Fluorescence was recorded for 60 min using a tungsten lamp with an excitation filter of 360 ± 40 nm and emission filter of 460 ± 40 nm. Microplate reader (Synergy2, BioTek) was set up with Top400 configuration and a sensitivity of 35. Inhibition of FXa was assayed in 25 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM CaCl2 and 1 mg / mL BSA with 0.5 nM of FXa, 500 µM of methoxycarbonyl- D-Nle-Gly-Arg-pNA substrate (L-1565, Bachem) and varied inhibitor concentrations (0 to 500 µM). All reactions were initiated by the addition of the protease and measurements were recorded and analyzed. Thrombin generation

[0139] Thrombin generation was measured in normal pool plasma with the calibrated automated thrombogram (CAT) method. Thrombin generation was followed continuously with fluorogenic substrate I-1140, as described previously.35,36Measurements were acquired in duplicate, and the lag-time, peak height and100600618438 endogenous thrombin potential (ETP) were calculated from averaged thrombin generation curves with CAT software provided by Thrombinoscope BV (Maastricht, The Netherlands). EC50 values were derived from peak height data fitted to a dose response function (Graphpad 8.3.0). Trigger via Intrinsic pathway

[0140] Normal pool plasma (80 µL) was incubated for 5 min at 37 °C with ellagic acid (53 nM), 4 µM phospholipid vesicles (20:60:20 DOPS / DOPC / DOPE, mol / mol / mol) and 0.5 or 2 µM of FXIa inhibitor. Subsequently, coagulation was initiated with the addition of 16 mM CaCl2 and 300 µM I-1140 (final concentrations in a total volume of 125 µL). Trigger via Extrinsic pathway

[0141] Normal pool or FXI deficient plasma (George King Biomedical, Inc) (80 µL) was incubated for 7 min at 37 °C in presence of 0-8 µM FXIa inhibitor. Next, tissue factor (14 pM) and phospholipid vesicles (4 µM, 20:60:20 DOPS / DOPC / DOPE, mol / mol / mol) were introduced. Subsequently, coagulation was initiated by the addition of 16 mM CaCl2 and 300 µM I-1140 (final concentrations in a total volume of 125 µL). aPTT / PT

[0142] Prothrombin time (PT) and activated partial thromboplastin time (aPTT) assays were performed on a Siemens BCS XP system according to manufacturer’s instructions using Innovin and actin FS reagents, respectively. Example 1 - Chemical Synthesis of Peptides 1-9 of the L and D Series Peptide 1100600618439 Peptide 1 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-YPHGLNRHTFC-C’ was generated by automated SPPS on Rink amide resin (102 mg, 50 µmol, 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 30 % B + 0.1 % Formic over 30 min). The appropriate fractions were combined and lyophilised to afford 158 as a white solid (12.6 mg, 20%). UPLC: Rt = 3.64 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1383.6 [M + H]+. HRMS (ESI+): m / z calcd. for: C₆₂H₈₆N₂₀O₁₅S₁ [M+2H]2+692.32198, found 692.32239. Peptide 2Peptide 2 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-YHWKTTRSNYLC -C’ was generated by automated SPPS on Rink amide resin (102 mg, 50 µmol, 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 Peptide 2 as a white solid (18.1 mg, 22%). UPLC: Rt= 3.85 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1611.3 [M + H]+. HRMS (ESI+): m / z calcd. For: C₇₃H₁₀₃N₂₁O₁₉S₁Na2, [M+H+2Na]2+827.86153, found 827.86221.100600618440 Peptide 3Peptide 3 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-YHFLNIRSNYDC -C’ was generated by automated SPPS on Rink amide resin (102 mg, 50 µmol, 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 Peptide 3 as a white solid (12.0 mg, 15%). UPLC: Rt = 4.50 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1584.6 [M + H]+. HRMS (ESI+): m / z calcd. for C₇₁H₉₈N₂₀O₂₀S₁Na1 [M+H+Na]2+803.34683, found 803.34760. Peptide 4Peptide 4 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-YIPASFVNTRLHC -C’ was generated by automated SPPS on Rink amide resin (102 mg, 50 µmol, capacity: 0.49 mmolg-1). Coupling of100600618441 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 Peptide 4 as a white solid (11.9 mg, 15%). UPLC: Rt = 4.31 min (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). (LRMS (ESI+): m / z = 1560.4 [M + H]+. HRMS (ESI+): m / z calcd. for C₇₁H₁₀₆N₂₀O₁₈S₁Na1, [M+H+Na]2+791.38398, found 791.38334. Peptide 5Peptide 5 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-YHDRYIRSNYLC -C’ was generated by automated SPPS on Rink amide resin (102 mg, 50 µmol, 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 peptide 5 as a white solid (6.2 mg, 7%). UPLC: Rt= 4.86 min. (0 to 70 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1642.5 [M + H]+. HRMS (ESI+): m / z calcd. for C73H104N22O20S1, [M+2H]2+821.38317, found 821.38297.100600618442 Peptide 6Peptide 6 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yAYRIVWPDRTQYEC -C’ was generated by automated SPPS on Rink amide resin (102 mg, 50 µmol, 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 Peptide 6 as a white solid (8.9 mg, 9%). UPLC: Rt= 4.69 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1001.95 [M + 2H]+. HRMS (ESI+): m / z calcd. for C92H128N24O25S1, [M+2H]2+1001.46692, found 1001.46743. Peptide 7Peptide 7 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yLLGPYVRIVC -C’ was generated by automated SPPS on Rink amide resin (102 mg, 50 µmol, 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-100600618443 preparative RP-HPLC (0 to 40 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford Peptide 7 as a white solid (13.3 mg, 20%). UPLC: Rt= 5.22 min. (0 to 70 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1335.6 [M + H]. HRMS (ESI+): m / z calcd. for C64H99N15O14S1Na [M+H+Na]2+678.85908, found 678.85834. Peptide 8Peptide 8 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yRTRIEDIFC -C’ was generated by automated SPPS on Rink amide resin (186 mg, 100 µmol, 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 40 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford Peptide 8 as a white solid (24 mg, 19%). UPLC: Rt = 4.41 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1355.7 [M + H]+. HRMS (ESI+): m / z calcd. for C₆₀H₉₁N₁₇O₁₇S₁Na1, [M+H+Na]2+688.82323, found 688.822731H NMR (500 MHz, D2O / CD3CN 1:1 v / v) δ 7.28 – 7.19 (m, 4H), 7.19 – 7.14 (m, 1H), 7.02 (d, J = 8.5 Hz, 2H), 6.71 (d, J = 8.6 Hz, 2H), 4.58 (tapt, J = 6.7 Hz, 1H), 4.50 (dd, J = 10.8, 5.0 Hz, 1H), 4.40 (dd, J = 9.0, 7.0 Hz, 2H), 4.34 (dd, J = 8.9, 4.8 Hz, 4H), 4.28 – 4.26 (m, 10H), 4.23 – 4.18 (m, 2H), 4.14 (dd, J = 9.1, 5.3 Hz, 1H), 4.09 (dd, J = 9.9, 4.4 Hz, 1H), 3.98 (d, J = 7.4 Hz, 1H), 3.95 (d, J = 5.7 Hz, 1H), 3.23 (d, J = 15.0 Hz, 1H), 3.18 (d, J = 15.1 Hz, 1H), 3.17 (dd, J = 14.0, 5.0 Hz, 1H), 3.07 (tapt, J = 7.1 Hz, 2H), 2.97 – 2.73 (m, 9H), 2.33 (tapt, J = 7.6 Hz, 2H), 2.07 – 1.98 (m, 1H), 1.95 – 1.86 (m, 1H), 1.81 – 1.60 (m, 5H), 1.53 – 1.32 (m, 4H), 1.16 – 1.01 (m, 7H), 0.97 – 0.74 (m, 8H), 0.62 (tapt, J = 7.4 Hz, 3H), 0.55 (d, J = 6.8 Hz, 3H).100600618444 Peptide 9Peptide 9 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yHWRNIRSNYSC -C’ was generated by automated SPPS on Rink amide resin (85 mg, 50 µmol, 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 40 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford Peptide 9 as a white solid (10.9 mg, 13%). UPLC: Rt= 4.22 min. (0 to 60 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1638.9 [M + H]+. HRMS (ESI+): m / z calcd. for C72H100N24O19S1, [M+2H]2+819.37314, found 819.37399. Peptide 10Peptide 10 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-ARTRIEDIFC -C’ was generated by automated SPPS on Rink amide resin (85 mg, 50 µmol, capacity: 0.57 mmolg-1). Coupling of chloroacetic acid to the N-terminus, cleavage from resin and cyclisation were performed as100600618445 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 peptide 10 as a white solid (6.7 mg, 11%). UPLC: Rt= 4.51 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1263.1 [M + H]+. HRMS (ESI+): m / z calcd. for C₅₄H₈₇N₁₇O₁₆S₁Na, [M+H+Na]2+642.81012, found 642.80964. Peptide 11Peptide 11 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yATRIEDIFC -C’ was generated by automated SPPS on Rink amide resin (85 mg, 50 µmol, 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 40 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford Peptide 11 as a white solid (4.7 mg, 7%). UPLC: Rt= 4.92 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1269.7 [M + H]+. HRMS (ESI+): m / z calcd. for C₅₇H₈₄N₁₄O₁₇S₁Na2 [M+2Na]2+657.28220, found 657.28247. Peptide 12100600618446 Peptide 12 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yRARIEDIFC -C’ was generated by automated SPPS on Rink amide resin (85 mg, 50 µmol, 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 40 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford Peptide 12 as a white solid (7.5 mg, 12%). UPLC: Rt = 4.71 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1325.3 [M + H]+. HRMS (ESI+): m / z calcd. for C₅₉H₈₉N₁₇O₁₆S₁Na1 [M+H+Na]2+673.81794, found 673.81799. Peptide 13Peptide 13 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yRTAIEDIFC -C’ was generated by automated SPPS on Rink amide resin (85 mg, 50 µmol, 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 40 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford Peptide 13 as a white solid (4.9 mg, 8%). UPLC: Rt = 4.42 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1269.7 [M + H]+. HRMS (ESI+): m / z calcd. for C₅₇H₈₄N₁₄O₁₇S₁Na2 [M+2Na]2+657.28220, found 657.28172.100600618447 Peptide 14Peptide 14 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yRTRAEDIFC -C’ was generated by automated SPPS on Rink amide resin (85 mg, 50 µmol, 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 40 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford Peptide 14 as a white solid (8.6 mg, 13%). UPLC: Rt = 4.40 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1313.3 [M + H]+. HRMS (ESI+): m / z calcd. for C₅₇H₈₅N₁₇O₁₇S₁Na1 [M+H+Na]2+667.79975, found 667.79982. Peptide 15Peptide 15 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yRTRIADIFC -C’ was generated by automated SPPS on Rink amide resin (85 mg, 50 µmol, 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 40 % B + 0.1 % Formic over 40 min). The appropriate100600618448 fractions were combined and lyophilised to afford Peptide 15 as a white solid (9.2 mg, 14%). UPLC: Rt = 4.86 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1297.3 [M + H]+. HRMS (ESI+): m / z calcd. for C₅₈H₈₉N₁₇O₁₅S1Na1 [M+H+Na]2+659.82049, found 659.82057. Peptide 16Peptide 16 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yRTRIEAFC-C’ was generated by automated SPPS on Rink amide resin (85 mg, 50 µmol, 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 40 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford Peptide 16 as a white solid (5.5 mg, 8%). UPLC: Rt= 4.32 min. (0 to 70 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1311.4 [M + H]+. HRMS (ESI+): m / z calcd. for C59H91N17O15S1 [M+2H]2+655.83734, found 655.83638. Peptide 17Peptide 17 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yRTRIEDAFC-C’ was generated by automated SPPS100600618449 on Rink amide resin (85 mg, 50 µmol, 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 40 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford peptide 17 as a white solid (6.9 mg, 11%). UPLC: Rt = 4.18 min. LRMS (ESI+): m / z = 1313.3 [M + H]+. HRMS (ESI+): m / z calcd. for C₅₇H₈₅N₁₇O₁₇S Na1 [M+H+Na]2+667.79975, found 667.79989. Peptide 18Peptide 18 was synthesised via Fmoc-strategy SPPS as specified in general methods. The linear sequence N’-yRTRIEDIAC-C’ was generated by automated SPPS on Rink amide resin (85 mg, 50 µmol, 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 40 % B + 0.1 % Formic over 40 min). The appropriate fractions were combined and lyophilised to afford Peptide 18 as a white solid (8.3 mg, 13%). UPLC: Rt = 5.13 min. (0 to 50 vol.% B over 5 min, 0.1 vol.% TFA, ^ = 214 nm). LRMS (ESI+): m / z = 1279.7 [M + H]+. HRMS (ESI+): m / z calcd. for C₅₄H₈₇N₁₇O₁₇S Na1 [M+H+Na]2+650.80808, found 650.80758.

[0143] Cyclic peptide targets from the L- and D- libraries 1-9 were synthesised via Fmoc-strategy solid-phase peptide synthesis (Fmoc-SPPS). Specifically, Rink amide- functionalized polystyrene resin was loaded with the C-terminal amino acid of each target sequence, followed by peptide elongation through iterative deprotection and coupling cycles. Following Fmoc-deprotection of the N-terminal L-Tyr or D-Tyr of each peptide, chloroacetic acid was coupled. The full-length linear peptides were then100600618450 cleaved from solid support with concomitant side chain deprotection by treatment with a trifluoroacetic acid-based cocktail. The peptides were then cyclised with 5 vol.% Hünig’s base in 1:1 v / v water:MeCN and purified with reversed-phase high-performance liquid chromatography to afford 1-9 in 8-24% yield (based on the original resin loading). Determining the Inhibitory Activity of Novel Peptide Binders 1-9

[0144] The FXIa inhibitory activity of the novel peptide binders was assessed via a continuous colorimetric assay. As FXIa is a serine protease, a fluorogenic tripeptide substrate containing 6-amino-1-naphthalenesulfonamide (ANSN or L-EGR-ANSNH- C3H7 • 2 HBr) that is selective for and sensitive to the enzyme’s activity was employed. This artificial substrate was used in excess and upon hydrolysis by FXIa, liberated ANSN that was quantified by UV-vis absorbance at 405 nm. Compounds that inhibit FXIa activity directly reduce the amount of ANSN liberated by the protease. Therefore, by examining enzyme activity over a diverse range of inhibitor concentrations, the relationship between inhibitor concentration and FXIa inhibition can be fitted and an IC50 determined. Pleasingly of the nine peptides screened, 1-4 and 7-9 had IC50 values < 250 nM. Of this subsample, peptides 2-4, 7 and 8 were the most potent with IC50 values <100 nM (table 3). Interestingly, peptide 6 had little inhibitory activity against the FXIa catalytic site, implying that this peptide may bind to alternative sites on FXIa. Table 3 Sequences and inhibitory activity of FXIa of discovered peptides. SPR = surface plasmon resonance against FXIa to measure binding affinities. PK = plasma kallikrein.100600618451Determining the Selectivity of the Lead Inhibitors 1-4, and 7-9

[0145] To gauge the selectivity of the FXIa inhibitors, select active candidates were counter-screened against a panel of related and functionally important serine proteases from the coagulation cascade. When profiled against FXIIa, FIIa and FXa at an inhibitor concentration of 100 µM, peptides 1, 4, 7 and 8 displayed excellent selectivity for FXIa and only weakly inhibited the other serine proteases (0 to < 50% inhibition). However, when screened against PK at the same inhibitor concentration, peptides 3, 4, 8 displayed some inhibitory activity (>65 % inhibition); only catalytic inhibitor 1 was completely selective for FXIa over FXIIa, FIIa, FXa and PK. Given the selectivity assay was conducted at an inhibitor concentration much higher than that of a realistic therapeutic range, it was sought to establish inhibitory constants against PK for peptides 3, 4, 8 which displayed some activity against the protease. This would allow the quantification of the selectivity of these peptides with greater precision and determine whether the off-target activity towards PK posed a pharmacological risk. Pleasingly upon determining the IC50 of the lead peptides there was a >1000-fold selectivity for FXIa over PK (see table 3). In vitro Evaluation of Intrinsic Coagulation by Potent FXIa Inhibitors

[0146] Activated partial thromboplastin time (aPTT) and prothrombin time (PT) are routine blood tests carried out by physicians that measure the activity of the intrinsic and extrinsic pathways of coagulation, respectively. A potent and selective FXIa inhibitor will increase the time of coagulation in the aPTT, whilst sparing an increase in PT. Encouraged by the potency of the discovered peptide leads, next, the performance of peptides were evaluated in aPTT at a range of inhibitor concentrations (Figure 2). Peptides of the invention followed a general trend towards potency against FXIa. In particular peptide 8 performed the best in this assay and showed a clinically relevant100600618452 prolongation of aPTT at a concentration of 2.5 µM, (Figure 2). Peptides 5, 6 and 9 did not have a significant effect on aPTT prolongation, despite a high screening concentration of 25 uM. Example 2 - Microfluidic Thrombosis Model

[0147] VenaT4 biochips were coated with 100 µg / mL fibrillar collagen and incubated overnight at 4°C. Immediately before loading 30 µL of whole blood, 1 µL of 1 M NaOH was added to bring the pH to 7.4 and placed in an incubator for 60 minutes at 37°C to allow the collagen gel to polymerize. After the gel solidified, the biochip was placed in the VenaFlux Pro microfluidics system. Whole blood from healthy donors was pre- incubated in the absence or presence of peptide 8, and Vioblue Reaffinitty CD41 / CD61 (stains platelets), FITC-CD15 (for neutrophils) and AF555-conjugated fibrinogen. Samples were reconstituted with 7.5 mM calcium chloride (CaCl2, final concentration) and 3.7 mM magnesium chloride (final) and 50 µl were immediately perfused into microchannels and maintained under a venous shear rate of 67 dyne / cm2. Kinetic data utilized a region of interest (ROI) based upon the final thrombus size. Fluorescence intensity over time for each channel was measured and normalized to initial fluorescent intensity at time point t=0. Final images (20x) at t=30 min were used for endpoint thrombus size measurements. Quantitation of neutrophil and platelet rolling, adhesion and aggregation over time, as well as total thrombus area were determined using InstallShield Wizard software (Cellix). Data presentation and analysis

[0148] Statistical analysis was performed with GraphPad Prism version 8 or IBM SPSS Statistics version 26 software packages.

[0149] Collectively, the in vitro data placed head-to-tail cyclic peptide 8 as the lead candidate; peptide 8 was the most potent inhibitor of FXIa catalysis (table 3), induced the most significant prolongation of the aPTT and was most effective at inhibiting thrombin generation (figure 2 and figure 3). Next peptide 8 was profiled in an ex vivo thrombosis model. In brief microfluidic chips were coated with collogen and human blood was perfused through the channels under venous shear. Upon exposure to the collogen channels blood clots form and fibrin, platelet and neutrophil aggregates can be quantified. Incubated human blood was dosed with varied concentrations of peptide 8100600618453 and perfused these samples through the microfluidic channels. After quantification of the fibrin signal and platelet and neutrophils it was discovered that peptide 8 potently inhibited thrombus formation in this model, as characterised by almost complete inhibition of fibrin deposition at as low as 10 nM (Figure 4). In addition, platelet and neutrophil aggregates were also lower in peptide 8 treated blood, presumably since the levels of active thrombin in the system were reduced compared to the control (Figure 4).

[0150] Cyclic peptides are known to be more resistant to proteolysis than their linear counterparts. Peptide 8 demonstrated excellent plasma stability, with greater than 95% peptide remaining after 24 hours. By contrast Peptide 7 was less stable. However, this still represents favourable plasma stability since in vivo peptides would likely be cleared within 2 hours via glomerular filtration. Example 3 - Alanine Scan

[0151] An alanine scan of the peptide 8 sequence by systematically replacing each residue for an alanine was conducted. These alanine mutants were accessed with Fmoc-SPPS and their FXIa inhibitory activity quantified by the same continuous colorimetric assay as described in the previous example. The results of the inhibitory activity quantification is shown in table 4. Table 4 Experimentally derived IC50 measurements of alanine scan mutants 10-18. NI = No Inhibition. The bracket (red) signifies the cyclic thioether bridge between the side chain thiol of cysteine and N-terminus.100600618454Example 4 - In-vivo rabbit study to assess prolongation of clotting time following administration of peptides of the invention

[0152] Rabbits are assessed and monitored during a two week acclimitisation period to the study venue. The rabbits are housed together.

[0153] The rabbits are sedated and then anaesthetised and recovered. Each rabbit undergoes a minimum of four and maximum of six procedures with 7 days of recovery in between procedures. They are monitored once a day in group housing (if they are deemed fit, well and socially compatible) during this recovery period.

[0154] The aim is to perform all procedures on the same day in sequential order for the 4 rabbits at the start of the week to allow for monitoring throughout the week. However, if need be, the rabbit procedures may be split across multiple days.

[0155] On day 1 after the acclimatisation period, the first procedure is performed. The rabbits are moved into individual housing cages in the procedure room. They will not be fasted prior to the procedure. Each rabbit receives a physical exam to confirm that they are healthy and suitable to undergo the procedure. Then, they will be premedicated and have local anaesthetic cream applied to the skin over the auricular vein to be catheterised. Once adequately sedated, an indwelling intravenous catheter is inserted and the rabbit is anaesthetised. Baseline blood samples are collected (t=0) from a peripheral vein using a 23 - 25 G hypodermic needle and a 3 ml single use syringe. Pressure is applied to the venepuncture site for up to 30 minutes to prevent bruising and haematoma formation. The test substance 10 mg / kg Investigational Peptide is administered via single IV bolus through the indwelling catheter. Blood is collected at the following timepoints: t= 15, 30, 45, 60, 90 mins. Each timepoint sample is collected from a different venipuncture site for successful sampling. Following the 90-minute timepoint blood collection, rabbits are recovered from anaesthesia and placed back in individual cages for approximately 24hrs. Vital signs are monitored at approximately one100600618455 hour following the procedure and repeated at the end of the day. Food and water intake are monitored and recorded appropriately.

[0156] Following the 7-day recovery period, the second procedure is performed on day 7. This will mimic the first procedure, the dosage and route of administration will remain the same.

[0157] Following a further 7-day recovery period, the third procedure is performed on day 14. This mimics the first procedure, the dosage and route of administration remains the same and an additional timepoint (t=24hrs) for blood collection. In order to collect blood for this timepoint, the rabbits are gently restrained to allow successful venipuncture. If necessary, a short sedation and appropriate monitoring is provided to reduce animal stress.

[0158] Following a further 7-day recovery period, a fourth procedure is performed on day 21. This mimics the third procedure. One endpoint is when all rabbits have received the appropriate dose of a FXIa investigational peptide suitable to prolong aPTT (clotting time) between 2-3-fold. Each animal will have a maximum of 6 doses. Once this has been achieved the animal's involvement in the study will end and they will be rehomed, anticipated to occur on day 28.

[0159] Clotting protocol: Measurements are performed on BFT II Analyser using the manufacturer’s instructions. In brief, aPTT reagents (ACTIN FS & calcium chloride Siemens Dade). Rabbit plasma, are incubated for 30 mins at 37oC. The rabbit plasma is pre-warmed to 37 °C.50 μL of aPTT-ACTIN FS are added, mixed well with 50 μL plasma in a stirred reaction vessel and incubated for 3 min at 37 °C. Next, 50 μL aPTT- calcium chloride is added to the mixture to initiate the reaction. The time taken for the mixture to turn from transparent to opaque / white web-like precipitate as an indication of fibrin formation is recorded. The entire aPTT is performed in duplicate and semi- automated by the BFT II Analyzer by employing a turbodensitometric detection technique.

Claims

100600618456 CLAIMS 1. A cyclic peptide selected from:100600618460or a functional variant or functional fragment thereof.

2. The cyclic peptide of claim 1, selected from any one of SEQ ID No 1-9 or a functional variant or functional fragment thereof: SEQ ID no 1: YPHGLNRHTFC; SEQ ID no 2: YHWKTTRSNYLC; SEQ ID no 3: YHFLNIRSNYDC; SEQ ID no 4: YHDRYIRSNYLC; SEQ ID no 5: YIPASFVNTRLHC; SEQ ID no 6: yAYRIVWPDRTQYEC; SEQ ID no 7: yLLGPYVRIVC;100600618461 SEQ ID no 8: yRTRIEDIFC; and SEQ ID no 9: yHWRNIRSNYSC.

3. 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.

4. The cyclic peptide of any one of claims 1-3, or functional variant or functional fragment thereof, wherein the peptide is cyclised via formation of a thioether group through reaction between an N-terminal chloroacetyl functionality and a sulfhydryl side chain of a downstream Cys residue.

5. The cyclic peptide of any one of claims 1-4, 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.

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

7. The cyclic peptide of any one of claims 1-6, or a functional variant or functional fragment thereof, wherein the cyclic peptide is selected from any one of SEQ ID No 1-4, or 7-9.

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 inhibiting FXIa, 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 8.100600618462 10. A method of preventing or treating a condition, disease or disorder responsive to inhibiting FXIa, 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 1-7, or a functional variant or functional fragment thereof, in the manufacture of a medicament for the treatment of a condition, disease or disorder responsive to the inhibition of FXIa.

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 1-7 in the manufacture of an anti- coagulant.

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