FIELD OF INVENTION

The present invention relates to a method of predicting responsiveness to an inhibitor of the ka llikrei n-kin in pathway, such as plasma kallikrein inhibitors, in subjects with diabetic macular edema.

BACKGROUND OF THE INVENTION

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes mellitus and the leading cause of blindness in working-age adults in the United States, Europe, and increasingly worldwide. Globally, the overall prevalence of DR was estimated at 34.6% of people with diabetes, with a higher prevalence in patients with type 1 diabetes compared to those with type 2 diabetes. Diabetic macular edema, (referred to as DME here on), which is characterised by exudation and accumulation of extracellular fluid in the macula secondary to an increase in vascular permeability, is a major cause of the vision loss associated with DR. The overall prevalence of DME was estimated at approximately 20% of people with DR and between 1.4 - 12.8% of people with diabetes (any type). The prevalence of DME is expected to rise further due to the increasing prevalence of diabetes, ageing of the population and increased life expectancy: the numberof adults aged 20 -79 years with diabetes worldwide was estimated at 463 million in 2019 and is expected to increase to 700 million by 2045.

One of the earlier standard treatment for DME involved focal / grid laser using small, light-intensity laser burns (50-100pm in diameter) to micro-aneurysms / diffuse area of thickening in a grid pattern. However, it has been associated with complications such as loss of central vision, central scotomas and decreased colour vision. In addition, focal laser burns have been observed to expand over time. More recently, subthreshold micropulse laser has been developed as a treatment that avoids damaging the inner neurosensory retina, thereby reducing potential complications. There is emerging evidence to suggest that similar efficacy outcomes can be achieved with micropulse laser as compared to conventional laser. On the other hand, while laser photocoagulation treatment reduces the risk of visual loss and works over a long timescale, it is clear that recovery of vision is much harder to achieve with laser treatment than with other treatments now available.

Some of the pharmacologic agents now available for the treatment of DME include corticosteroids (given intravitreally, intraocularly, peri-ocularly or sustained-release) and anti-Vascular endothelial growth factor (anti-VEGF) agents. For instance, inflammation plays an important role in the pathogenesis of DME. Cytokines and chemokines released by leukocytes in the blood significantly increase vascular permeability leading to more fluid build-up under the retina. Corticosteroid therapies can inhibit inflammatory mediators. Several clinical studies have shown that corticosteroids are effective in decreasing central subfield thickness (CST) and improving vision in DME. However, intravitreal corticosteroids are associated with increased risks of cataract development and elevation of intraocular pressure (IOP). Therefore, the use of intravitreal corticosteroids in patients with DME is reserved as a second-line therapy and is contraindicated in patients with underlying glaucoma.

Since the marketing approval of ranibizumab for the treatment of DME, anti-VEGF agents have become the first-line of treatment for DME. Anti-VEGFs decrease angiogenesis and vascular permeability, leading to reduction of DME. Several clinical studies have shown that anti-VEGF treatment is more effective than focal / grid laser photocoagulation (the previous first-line treatment for DME) at decreasing CST and improving vision in patients with DME. Currently, 3 anti-VEGF agents are in common use for the treatment of DME: aflibercept (Eylea®), ranibizumab (Lucentis®) and bevacizumab (Avastin®). While aflibercept and ranibizumab are approved for the treatment of DME, bevacizumab is used off-label. Ranibizumab (a recombinant humanised monoclonal antibody [mAb] fragment of the immunoglobulin G, isotype 1 [IgGl] kappa sub-class) and bevacizumab (a recombinant humanised IgGl mAb) block all isoforms of VEGF-A. Aflibercept (a recombinant fusion protein containing extracellular VEGF receptor (VEGFR)-l and -2 sequences and the Fc domain of a human IgGl molecule) additionally blocks all isoforms of VEGF-B and PIGF (another member of the VEGF family). Notably, up to 40% DME patients do not adequately respond to anti-VEGF treatment in terms of best-corrected visual acuity (BCVA) and / or CST improvement. This suggests that other pathways, independent of VEGF, can mediate certain cases of DME.

The kallikrein-kinin system (KKS) is a metabolic cascade that, when activated, triggers the release of vasoactive kinins. By virtue of their ability to activate endothelial cells, leading to vasodilation and increased vascular permeability, kinins participate in physiological (e.g. regulation of blood pressure) and pathological (e.g. inflammation) processes. The kallikrein-kinin system is thought to play an important role in the mediation of DME, as substantiated by proteomic analyses and non-clinical experiments. Proteomic analyses identified components of the kallikrein-kinin system, including prekal likrein, in vitreous obtained from patients with advanced DR undergoing pars plana vitrectomy and in vitreous from DME patients. In addition, B1R mRNA levels have been shown to be upregulated in the retina of both diabetic animal models and diabetic patients and non-clinical experiments have shown early B1R upregulation in the rat diabetic retina through a mechanism involving oxidative stress. It has also been shown that intraocular activation of the kallikrein-kinin system increases retinal vascular permeability, retinal thickening, and neovascularisation, and these responses are exacerbated in diabetic animals. In line with this, inhibition of plasma kallikrein (pKal), Bl receptor (B1R), and the B2 receptor (B2R) block enhanced vascular permeability in rodent models.

The serine protease plasma kallikrein is a key player of kallikrein-kinin system, and has been identified as a novel potential target for treatment of DME. Upon activation, plasma kallikrein cleaves its substrate high-molecular-weight kininogen to release bradykinin. Bradykinin activates the B1R and B2R to exert its vasoactive properties. The activity of the kallikrein-kinin system is regulated by protease inhibitors. The primary physiological inhibitors of plasma kallikrein are complement 1 inhibitor (Cl-INH) and a-2 macroglobulin.

While both VEGF and plasma kallikrein levels have been shown to be upregulated in the vitreous of DME patients, their concentrations do not correlate. Moreover, while plasma kallikrein concentrations were upregulated in the vitreous samples of most of the DME patients, vitreous VEGF concentrations displayed a greater range among the different vitreous samples (i.e. VEGF was strongly upregulated in part of the samples, while it was not detected in other samples). The wide variability among the vitreous VEGF concentrations implies that other factor(s) than VEGF play an important role in mediating some cases of DME, and it has been postulated that intraocular plasma kallikrein can mediate DME in a VEGF- independent manner. Interestingly, non-clinical data in diabetic rats has shown that the induction of retinal vascular permeability by plasma kallikrein could be blocked by bradykinin receptor antagonism, but not by anti-VEGF treatment. In addition, bradykinin-induced retinal thickening was not affected by VEGFR- 2 blockage in mice.

It is evident that inhibitors of the kallikrein-kinin system provide an interesting treatment option for DME patients, that is independent of VEGF inhibition. However, information on which patients are more likely to respond to kallikrein-kinin system inhibitor therapy is lacking. Therefore, there is a need to find biomarkers for identifying DME patients who are more likely to respond to kallikrein-kinin system inhibitor therapy.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that specific biomarkers described herein can be used to predict response to treatment of ophthalmic disorders with inhibitors of the kallikrein-kinin pathway. In particular, the biomarker is the presence of a cyst at the retina. Therefore, in a first aspect, the present invention provides a method for predicting whether a subject with an ophthalmic disorder will be nonresponsive to treatment with an inhibitor of the kallikrein-kinin pathway (also termed the kallikrein- kinin system), the method comprising: - determining the presence or absence of a cyst at a retina; wherein the subject is predicted to be nonresponsive if a cyst is present.

In addition to the presence of a cyst, the further presence of an ellipsoid zone disruption and an external limiting membrane disruption may be used to predict response to inhibitors of the kallikrein-kinin pathway. Therefore, in a further embodiment, the method further comprises:

- determining the presence or absence of an ellipsoid zone disruption and an external limiting membrane disruption; wherein the subject is predicted to be nonresponsive if a cyst is present in combination with at least one of an ellipsoid zone disruption and an external limiting membrane disruption. In another embodiment, the presence or absence of ellipsoid zone disruption is determined in the DME patient prior to treatment. In a further embodiment, the presence or absence of external limiting membrane disruption is determined in the DME patient prior to treatment.

In a particular embodiment, the cyst as described herein is located in a central 1mm circle of an EDTRS (Early Treatment Diabetic Retinopathy Study) grid. In another embodiment, the cyst occupies and disrupts all retinal layers.

As will be understood by the skilled person, determining the presence or absence of one or more biomarkers at a retina of a subject, such as a retinal cyst, an ellipsoid zone disruption or an external limiting membrane disruption, is performed using previously obtained data of a retina of the subject. In particular, one or more images of a retina of the subject will have been obtained before performing the determination of the biomarker is present or absent. Therefore, in a particular embodiment, determining the presence or absence of the biomarkers disclosed herein uses one or more previously obtained images of a retina of the subject.

In a particular embodiment, the one or more biomarkers described herein are determined by optical coherence tomography (OCT).

In another particular embodiment, the ophthalmic disorder is selected from the group consisting of wet age-related macular degeneration, macular edema, diabetic retinopathy, and retinal vein occlusion. In a preferred embodiment, the subject has diabetic macular edema (DME). Thus, in a particular embodiment, the present invention provides a method for predicting whether a subject with diabetic macular edema will be nonresponsive to an inhibitor of the kallikrein-kinin pathway, the method comprising determining the presence or absence of one or more biomarkers as described herein; wherein the subject is predicted to be nonresponsive if the biomarker is present. The present invention further provides inhibitors of the kallikrein-kinin pathway for use in treating a subject with an ophthalmic disorder, wherein the method comprises determining the presence or absence of a biomarker as described herein; and if the biomarker is absent, administering the inhibitor of the kallikrein-kinin pathway to the subject.

The present inventors have further found that plasma kallikrein inhibitors are particularly effective for use in a method of treatment of DME, comprising the steps for determining the presence or absence of cyst at retina. Therefore, in a particular embodiment, the presence or absence of a cyst at the retina is determined in the DME patient prior to treatment. In another embodiment, the presence or absence of cyst a retina in the central 1 mm is determined in the DME patient prior to treatment. In a further embodiment, the presence or absence of cyst occupying all retinal layers is determined in the DME patient prior to treatment.

In a preferred embodiment, the inhibitor of the kallikrein-kinin pathway is a plasma kallikrein inhibitor. The plasma kallikrein inhibitor for use in the invention is preferably a direct plasma kallikrein inhibitor. It may for example be selected from the group consisting of an antibody (such as lanadelumab), a peptide (such as ecallantide or Cl inhibitor), a nucleotide (such as a silencing RNA), or a chemical molecule (such as berotralstat or a compound of Formula A, Formula B, or Formula C), Formula B,

Formula C.

Additionally, the plasma kallikrein inhibitor for use in the invention is preferably a selective inhibitor for plasma kallikrein that shows no or only weak inhibition of other serine proteases. In a particular embodiment, the plasma kallikrein inhibitor has an inhibition constant that is at least 100 times lower for plasma kallikrein than for tissue kallikrein and transmembrane protease, serine 2 (TMPRSS2).

Particular plasma kallikrein inhibitors that may be used in the invention are selected from the group consisting of ecallantide, lanadelumab, Cl inhibitor, a compound of Formula A, Formula B, and Formula C, or a compound comprising a polypeptide sequence comprising at least three cysteine residues separated by two peptide loops, wherein the at least three cysteine residues are covalently attached to a molecular scaffold and wherein each peptide loop independently comprises from 2 to 10 amino acids.

In a further embodiment, the plasma kallikrein inhibitor for use in the invention is a compound comprising a polypeptide sequence comprising at least three cysteine residues separated by two peptide loops, wherein the at least three cysteine residues are covalently attached to a molecular scaffold, wherein each peptide loop independently comprises from 2 to 10 amino acids, and wherein the first peptide loop comprises the sequence SFPYR (SEQ ID NO: 1), wherein optionally P is replaced with azetidine carboxylic acid and/or R is replaced with homoarginine or N-methylarginine. For example, the plasma kallikrein inhibitor may be a compound comprising [Cl]SF(Aze)Y(HArg)[C2]VYYPDI[C3] (SEQ ID NO: 2), wherein Aze is azetidine carboxylic acid, HArg is homo-arginine, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to the molecular scaffold. Alternatively, the plasma kallikrein inhibitor may be a compound comprising [Cl]SF(Aze)Y(HArg)[C2](Ala(4jCH2NH))HQDL[C3] (SEQ ID NO: 3) wherein Aze is azetidine carboxylic acid, HArg is homo-arginine, Ala(QjCH2NH) is alanine wherein the carbonyl is reduced to methylene, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to the molecular scaffold.

The present invention further provides pharmaceutical compositions comprising a plasma kallikrein inhibitor as defined herein and a pharmaceutically acceptable carrier, for use in a method of treating an ophthalmic disorder, in particular for treating DME, in a subject following determining the presence or absence of biomarkers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Chemical structure of cpdl, referred to as Ac-(06-34-18) Phe2 Aze3 Tyr4 Harg5 Ala (IJJ CH ₂ N H )6 in International Patent Application Publication WO 2014/167122 Al

FIG. 2 Chemical structure of cpd2, referred to as Ac-(06-550)-Sar ₃ -(DArg ₂ ) Aze3HArg5 in International Patent Application Publication WO 2015/063465 A2

FIG. 3 shows the mean change in best-corrected visual acuity (BCVA) in subjects treated with cpdl for DME. Subjects without the biomarker of the invention (full bullets, bold line) showed a marked improvement in the BCVA upon plasma kallikrein therapy, while subjects having the biomarker of the invention (empty bullets, black line) did not respond to the therapy.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a human, and includes relieving the disease, i.e., causing regression of the disease.

The term "inhibition" or "inhibitor" refers to a reduction in the biological activity of a given molecule, such as a reduction of the enzymatic activity of the molecule, a reduction of the expression or amount of the molecule, or a reduction of the receptor signaling activity of the molecule. Inhibitors in the context of the invention thus also refer to antagonists. In a preferred embodiment, inhibitor refers to a molecule that binds to and inhibits the proteolytic activity of the target molecule.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. The terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e. an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include scFV and dcFV fragments, Fab and F (a b') 2 fragments which can be generated by treating the antibody with an enzyme such as papain or pepsin, respectively. The antibody can be a polyclonal, monoclonal, recombinant, e.g. a chimeric or humanized, fully human, non-human, e.g. murine, or single chain antibody.

The term "specifically binds," refers to an antibody or a ligand, which recognizes and binds with a cognate binding partner protein, but the antibody or ligand does not substantially recognize or bind other molecules in the sample.

The term "subject", refers to any living organisms which can have an ophthalmic disorder. In a preferred embodiment, the subject is a mammal, preferably a human.

The term "atrophy" refers to any decrease in size or thinning or degeneration of cells, tissues, or organs.

As used herein, the term "therapeutically effective amount" is used herein to refer to an amount of therapeutic agent either as an individual compound or in combination with other compounds that is sufficient to induce a therapeutic effect on the ailment which the compound is applied to. This phrase should not be understood to mean that the dose must completely eradicate the ailment. What constitutes a therapeutically effective amount will vary depending on, inter alia, the biopharmacological properties of the compound used in the methodology, the condition being treated, the frequency of administration, the mode of delivery, characteristics of the individual to be treated the severity of the disease and the response of the patient. These are the types of factors that a skilled pharmaceutical chemist will be aware of and will be able to account for when formulating compositions for a treatment as described herein.

As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity.

As used herein, the terms "comprising" and "including" are inclusive or open-ended and do not exclude additional unrecited elements, compositional components, or method steps. Accordingly, the terms "comprising" and "including" encompass the more restrictive terms "consisting essentially of" and "consisting of".

Inhibitors

Compounds for use in the invention are inhibitors or antagonists of the kallikrein-kinin pathway, also referred to herein as the kallikrein-kinin system, in particular inhibitors of plasma kallikrein. As mentioned herein before, an inhibitor refers to any compound that leads to a reduction in the biological activity of a given molecule, such as a reduction of the enzymatic activity of the molecule, a reduction of the expression or amount of the molecule, or a reduction of the receptor signaling activity of the molecule. For example, a plasma kallikrein inhibitor may be a molecule that reduces the expression of the preka llikrein protein, that reduces the conversion of the zymogen prekal likrein to the active molecule plasma kallikrein, that inhibits the enzymatic activity of plasma kallikrein, or that increases the degradation of plasma kallikrein. Examples of inhibitors include an antibody, a peptide, a nucleotide, or a chemical molecule (also referred to in the art as a small molecule or inorganic molecule) as well as synthetic derivatives thereof.

Preferred inhibitors for use in the invention are specific inhibitors, referring to molecules that have a higher specificity for the target molecule than for another molecule outside of the kallikrein-kinin pathway, particularly than for another serine protease. For example, a specific plasma kallikrein inhibitor refers to a molecule having an inhibition constant that is lowerfor plasma kallikrein than for another serine protease, in particular than for tissue kallikrein and/or transmembrane protease, serine 2 (TMPRSS2). In a particular embodiment, a specific inhibitor refers to an inhibitor that has an inhibition constant that is at least 10 times, in particular at least 100 times, more in particular at least 1000 times lower for the target molecule in the kallikrein-kinin pathway than for a serine protease outside of said pathway. In a particular embodiment, a specific inhibitor refers to an inhibitor that has an inhibition constant that is at least 10 times, in particular at least 100 times, more in particular at least 1000 times lower for the target molecule in the kallikrein-kinin pathway than for tissue kallikrein. In another embodiment, a specific inhibitor refers to an inhibitor that has an inhibition constant that is at least 10 times, in particular at least 100 times, more in particular at least 1000 times lower for the target molecule in the kallikrein-kinin pathway than for transmembrane protease, serine 2 (TMPRSS2). In a preferred embodiment, the inhibitor has an inhibition constant that is at least 10 times, in particular at least 100 times, more in particular at least 1000 times lower for the target molecule in the kallikrein-kinin pathway than for tissue kallikrein and transmembrane protease, serine 2 (TMPRSS2). Assays to determine inhibition constants (K,) are well-known in the art. We determining inhibition constants for different targets in order to compare specificity, the assay features are kept as identical as possible and the assays are preferably ran simultaneously in parallel.

In a particular embodiment, the inhibitor for use in the invention has an inhibition constant for the target in the kallikrein-kinin pathway of 50 pM or less, in particular 10 pM or less, more in particular 1 pM or less. In preferred embodiment, the inhibitor has a inhibition constant for the target in the kallikrein- kinin pathway of 500 nM or less, in particular 100 nM or less, preferably 50 nM or less.

In another preferred embodiment, the inhibitor is a direct inhibitor, meaning that it directly binds to and interferes with the target protein, or the gene or mRNA from which it is derived. In a further preferred embodiment, the inhibitor binds to and interferes with the target protein. In an even further embodiment, the inhibitor binds to and reduces the enzymatic activity of its target protein.

Plasma kallikrein inhibitors

As is evident from the above, preferred molecules (herein also referred to as compounds) for the methods of the invention are plasma kallikrein inhibitors. In the context of the invention, suitable plasma kallikrein inhibitors reduce the expression of prekallikrein, reduce the conversion of prekallikrein to kallikrein, or inhibit the enzymatic activity of plasma kallikrein. Among these, direct plasma kallikrein inhibitors that bind to plasma kallikrein and reduce its enzymatic activity are preferred.

Suitable, preferred, plasma kallikrein inhibitors for the invention have, for example, been developed by Bicycle Therapeutics and are disclosed in W02013050616 Al, WO2014167122 Al, and WO2015063465 Al, which are hereby incorporated by reference. These plasma kallikrein inhibitors will also be referred to as bicyclic peptide or bicyclic plasma kallikrein inhibitors herein. These inhibitors comprise a polypeptide sequence comprising at least three cysteine residues separated by two peptide loops, wherein the at least three cysteine residues are covalently attached to a molecular scaffold and wherein each peptide loop independently comprises from 2 to 10 amino acids. Amino acids refer to naturally occurring as well as non-natural amino acids and peptide bonds may be chemically modified. The compounds can be denoted as comprising a peptide sequence [Cl] - Loopl - [C2] - Loop2 - [C3], wherein [Cl] to [C3] denote a cysteine which is covalently attached to the molecular scaffold and wherein Loop 1 and Loop2 denote the first and second loop. For full clarity, in the nomenclature used in the context of the invention, [Cl] to [C3] are not considered to be part of the first and second peptide loop in these bicyclic compounds. The chemical scaffold is preferably trisbromomethylbenzene (TBMB). Therefore, in a particular embodiment, the plasma kallikrein inhibitor for use in the invention is a compound comprising a polypeptide sequence comprising at least three cysteine residues separated by two peptide loops, wherein the at least three cysteine residues are covalently attached to a molecular scaffold and wherein each peptide loop independently comprises from 2 to 10 amino acids. In a further embodiment, the peptidic compound of the invention comprises two peptide loops, wherein each loop comprises 4, 5 or 6 amino acids; preferably 5 or 6 amino acids. In yet another particular embodiment, the peptide loops in the peptidic compounds of the invention comprise in total of at least 5, preferably at least 8, more preferably at least 10 amino acids, which are optionally chemically modified. In a further embodiment, the peptide loops comprise in total from 5 to 25 amino acids; in particular from 8 to 20 amino acids; more in particular from 10 to 15 amino acids; even more in particular 10 or 11 amino acids.

In a particular embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of W02013050616 Al, WO2014167122 Al, or WO2015063465 Al, particularly those bicyclic peptides for which the tables show a Ki value towards human (plasma) kallikrein of 100 nM or less. In a further embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of W02013050616 Al, WO2014167122 Al, or WO2015063465 Al for which the table shows a Ki value towards human (plasma) kallikrein of 50 nM or less, preferably 25 nM or less.

In a further embodiment, the first peptide loop comprises the sequence SFPYR (SEQ ID NO: 1), wherein optionally P is replaced with azetidine carboxylic acid and/or R is replaced with homoarginine or N-methylarginine. The presence of this motif in the peptidic compounds described herein imparts plasma kallikrein inhibitory activity to the molecule. In a particularly preferred embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of W02013050616 Al, WO2014167122 Al, or WO2015063465 Al wherein the first peptide loop comprises the sequence SFPYR (SEQ ID NO: 1), wherein optionally P is replaced with azetidine carboxylic acid and/or R is replaced with homoarginine or N- methylarginine. In particular, those bicyclic peptides for which the tables show a Ki value towards human (plasma) kallikrein of 100 nM or less. In a further embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of WQ2013050616 Al, WO2014167122 Al, or WQ2015063465 Al wherein the first peptide loop comprises the sequence SFPYR (SEQ ID NO: 1), wherein optionally P is replaced with azetidine carboxylic acid and/or R is replaced with homoarginine or N-methylarginine, and for which the table shows a Ki value towards human (plasma) kal likrein of 50 nM or less, preferably 25 nM or less.

In a further embodiment, the first peptide loop comprises the sequence SF(Aze)Y(HArg) (SEQ ID NO: 4) wherein Aze is azetidine carboxylic acid, HArg is homo-arginine. In a particularly preferred embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of W02013050616 Al, WO2014167122 Al, or WO2015063465 Al wherein the first peptide loop comprises the sequence SF(Aze)Y(HArg) (SEQ ID NO: 4) wherein Aze is azetidine carboxylic acid, HArg is homo-arginine. In particular, those bicyclic peptides for which the tables show a Ki value towards human (plasma) kallikrein of 100 nM or less. In a further embodiment, the plasma kallikrein inhibitor is a bicyclic peptide disclosed in the tables of WQ2013050616 Al, WO2014167122 Al, or WQ2015063465 Al wherein the first peptide loop comprises the sequence SF(Aze)Y(HArg) (SEQ ID NO: 4) wherein Aze is azetidine carboxylic acid, HArg is homo-arginine, and for which the table shows a Ki value towards human (plasma) kallikrein of 50 nM or less, preferably 25 nM or less.

In one preferred embodiment, the plasma kallikrein inhibitor comprises [Cl]SF(Aze)Y(HArg)[C2](Ala(4jCH ₂ NH))HQDL[C3] (SEQ ID NO: 3) wherein Aze is azetidine carboxylic acid, HArg is homo-arginine, Ala(QjCH ₂ NH) is alanine wherein the carbonyl is reduced to methylene, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to a molecular scaffold. In particular, the molecular scaffold being TBMB. In a most preferred embodiment, the plasma kallikrein inhibitor for use in the invention is cpdl, presented in FIG. 1.

In another preferred embodiment, the plasma kallikrein inhibitor comprises [Cl]SF(Aze)Y(HArg)[C2]VYYPDI[C3] (SEQ ID NO: 2), wherein Aze is azetidine carboxylic acid, HArg is homoarginine, and wherein the cysteine residues [Cl] to [C3] are attached via thioether linkages to the molecular scaffold. In particular, the molecular scaffold being TBMB. In a most preferred embodiment, the plasma kallikrein inhibitor for use in the invention is cpd2, presented in FIG. 2.

Other plasma kallikrein inhibitors suitable for use in the treatments disclosed herein include Kunitz domain plasma kallikrein inhibitors, such as those developed by Dyax and disclosed in US5786328, US6333402, US6010880, and US9107928, which references are incorporated by reference herein. These kallikrein inhibitors comprise the consensus sequence amino acid sequence Xaal Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 XaalO Xaall Gly Xaal3 Cys Xaal5 XaalG Xaal7 Xaal8 Xaal9 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO: 5) with Xaa being each independently from one another any amino acid. In a particular embodiment, the plasma kallikrein inhibitor for use in the invention comprises the amino acid sequence of SEQ ID NO: 5, wherein

Xaal, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 are, independently from one another, any amino acid or absent;

XaalO is an amino acid selected from the group consisting of Asp and Glu;

Xaall is an amino acid selected from the group consisting of Asp, Gly, Ser, Vai, Asn, He, Ala and Thr;

Xaal3 is an amino acid selected from the group consisting of Arg, His, Pro, Asn, Ser, Thr, Ala, Gly, Lys and

Gin;

Xaal5 is an amino acid selected from the group consisting of Arg, Lys, Ala, Ser, Gly, Met, Asn and Gin;

XaalG is an amino acid selected from the group consisting of Ala, Gly, Ser, Asp and Asn;

Xaal7 is an amino acid selected from the group consisting of Ala, Asn, Ser, He, Gly, Vai, Gin and Thr;

Xaal8 is an amino acid selected from the group consisting of His, Leu, Gin and Ala;

Xaal9 is an amino acid selected from the group consisting of Pro, Gin, Leu, Asn and lie;

Xaa21 is an amino acid selected from the group consisting of Trp, Phe, Tyr, His and lie;

Xaa22 is an amino acid selected from the group consisting of Tyr and Phe;

Xaa23 is an amino acid selected from the group consisting of Tyr and Phe;

Xaa31 is an amino acid selected from the group consisting of Glu, Asp, Gin, Asn, Ser, Ala, Vai, Leu, lie and Thr;

Xaa32 is an amino acid selected from the group consisting of Glu, Gin, Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly and Vai;

Xaa34 is an amino acid selected from the group consisting of Thr, lie, Ser, Vai, Ala, Asn, Gly and Leu;

Xaa35 is an amino acid selected from the group consisting of Tyr, Trp and Phe;

Xaa39 is an amino acid selected from the group consisting of Glu, Gly, Ala, Ser and Asp;

Xaa40 is an amino acid selected from the group consisting of Gly and Ala;

Xaa43 is an amino acid selected from the group consisting of Asn and Gly;

Xaa45 is an amino acid selected from the group consisting of Phe and Tyr;

Xaa6, Xaa7, Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41, Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and Xaa54 are, independently from one another, any amino acid. In another particular embodiment, the plasma kalli krein inhibitor for use in the present invention is a peptide having the amino acid sequence of any of SEQ ID NO: 2 to SEQ ID NO: 43 of US9107928 B2. In a further particular embodiment, the plasma kallikrein inhibitor for use in the present invention is a peptide comprising the amino acid sequence Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn He Phe Thr Arg Gin Cys Glu Glu Phe He Tyr Gly Gly Cys Glu Gly Asn Gin Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO: 6). In a further particular embodiment, the plasma kallikrein inhibitor for use in the present invention is ecallantide, commercially available under the trade name Kalbitor.

In yet another particular embodiment, the plasma kallikrein inhibitor for use in the present invention is aprotinin. This kunitz domain polypeptide is also known under its commercial name Trasylol.

Another suitable plasma kallikrein inhibitor for use in the present invention is Cl-inhibitor (also known as Cl-INH or Cl esterase inhibitor). Cl-inhibitor is a naturally occurring plasma kallikrein inhibitor and often considered to the most important physiological inhibitor of plasma kallikrein.

Other plasma kallikrein inhibitors for use in the invention are anti-kall ikrein antibodies or anti- preka llikrein antibodies that prevent conversion of prekalli krein to kallikrein. As defined herein, antibodies include antigen-binding fragments of full-length antibodies. Anti-kallikrein antibodies may bind both preka llikrein and kallikrein. In another embodiment, the anti-kallikrein antibodies bind to kallikrein but do not bind to preka Hi krei n. Anti-prekall ikrei n and anti-kallikrein antibodies are available commercially or can easily be obtained using the general knowledge in the art. Suitable anti-plasma kallikrein antibodies for use in the present invention include the antibodies disclosed in WQ2011085103 A2 and WQ2012094587 Al, both reference are herewith incorporated herein. When using an antibody as plasma kallikrein inhibitor, lanadelumab is preferred.

Other plasma kallikrein inhibitors for use in the invention are chemical molecules (also referred to in the art as a small molecules or inorganic molecules). Suitable chemical molecules with plasma kallikrein inhibitory activity are known in the art. For example, suitable plasma kallikrein inhibitors include those disclosed in WQ2017072020 Al, WQ2017072021 Al and WO2018192866 Al developed by Boehringer Ingelheim International, as well as those disclosed in WQ2019028362 Al developed by Dyax, and those disclosed in WQ03076458 A2 and WQ2013005045 Al developed by Kalvista Pharmaceuticals, all reference herewith being incorporated by reference. In a particular embodiment, the plasma kallikrein inhibitor for use in the present invention is a compound selected from the compounds listed in claim 26 of WQ2019028362 Al, berotralstat, and a compound of Formula A, Formula B, and Formula C,

In a further embodiment, the plasma kallikrein inhibitor for use in the present invention is berotralstat or a compound of Formula A, Formula B, or Formula C, Formula A,

Formula C.

In another embodiment, the plasma kail ikrein inhibitor is an inhibitory nucleic acid molecules that targets prekallikrein RNA, e.g., antisense, siRNA, ribozymes, and aptamers, are used. In some embodiments, the inhibitory nucleic acid targets prekallikrein mRNA.

RNAi is a process whereby double-stranded RNA (dsRNA, also referred to herein as si RNAs or ds siRNAs, for double-stranded small interfering RNAs,) induces the sequence-specific degradation of homologous mRNA in animals and plant cells (Hutvagner and Zamore, Curr. Opin. Genet. Dev. 12:225-232 (2002); Sharp, Genes Dev., 15:485-490 (2001)). In mammalian cells, RNAi can be triggered by 21- nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al., Mol. Cell. 10:549-561 (2002); Elbashir et al., Nature 411 :494-498 (2001)), or by micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which are expressed in vivo using DNA templates with RNA polymerase III promoters (Zeng et al., Mol. Cell 9:1327-1333 (2002); Paddison et al., Genes Dev. 16:948-958 (2002); Lee et al., Nature Biotechnol. 20:500-505 (2002); Paul et al., Nature Biotechnol. 20:505-508 (2002); Tuschl, Nature Biotechnol. 20:440-448 (2002); Yu et al., Proc. Natl. Acad. Sci. USA 99(9): 6047-6052 (2002); McManus et al., RNA 8:842-850 (2002); Sui et al., Proc. Natl. Acad. Sci. USA 99(6):5515-5520 (2002)).

The nucleic acid molecules or constructs can include dsRNA molecules comprising 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the mRNA, and the other strand is complementary to the first strand. The dsRNA molecules can be chemically synthesized, or can transcribed be in vitro from a DNA template, or in vivo from, e.g., shRNA. The dsRNA molecules can be designed using any method known in the art; a number of algorithms are known, and are commercially available. Gene walk methods can be used to optimize the inhibitory activity of the siRNA.

In another embodiment, an antisense nucleic acid is used that is complementary to the sense nucleic acid encoding prekallikrein and plasma kallikrein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to a TEF mRNA sequence. The antisense nucleic acid can be complementary to the entire coding strand of the target sequence, or to only a portion thereof. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence (e.g., the 5' and 3' untranslated regions). An antisense nucleic acid can be designed such that it is complementary to the entire coding region of a target prekallikrein mRNA, but can also be an oligonucleotide that is antisense to only a portion of the coding or noncoding region of the target mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the target mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length. Based upon the known sequences of prekallikrein and plasma kallikrein and the non-coding neighboring regions, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention.

Prekallikrein gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoters and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See generally, Helene, Anticancer Drug Des. 6:569-84 (1991); Helene, Ann. N.Y. Acad. Sci. 660:27-36 (1992); and Maher, Bioassays 14:807-15 (1992).

Ribozymes are a type of RNA that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. By cleaving the target RNA, ribozymes inhibit translation, thus preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme genes can be introduced into cells through gene-delivery mechanisms known in the art. A ribozyme having specificity for a prekallikrein nucleic acid can include one or more sequences complementary to the nucleotide sequence of prekallikrein cDNA, and a sequence having known catalytic sequence responsible for mRNA cleavage (see US5093246 or Haselhoff and Gerlach Nature 334:585-591 (1988)). Alternatively, a target mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak, Science 261:1411-1418 (1993).

In a particular embodiment of the present invention, the plasma kallikrein inhibitor for use in the invention is selected from the group consisting of ecallantide, lanadelumab, berotralstat, Cl inhibitor, cpdl, cpd2, and a compound of Formula A, Formula B, and Formula C,

Formula C.

In a further particular embodiment, the plasma kallikrein inhibitor is a specific plasma kallikrein inhibitor and is selected from the group consisting of ecallantide, lanadelumab, berotralstat, cpdl, cpd2, and a compound of Formula A, formula B, or Formula C. In a preferred embodiment, the plasma kallikrein inhibitor is cpdl or cpd2, and more preferably cpdl.

Other kallikrein-kinin pathway inhibitors Factor XII and Factor XI la inhibitors are known in the art. These are for example disclosed in Kenne and Renne (Drug Discovery Today 2014, 19:1459-1464) and Larsson et al. (Sci Transl Med 2014, 222:222ral7).

Bradykinin receptor antagonists are also known in the art. In a particular embodiment, the bradykinin receptor antagonist is a Bl receptor antagonist. In another particular embodiment, the bradykinin receptor antagonist is a B2 receptor antagonist. If a bradykinin receptor antagonist is used for the invention, it is preferably a bradykinin Bl receptor antagonist, as the inventors have experimentally identified a significantly increased expression of the bradykinin Bl receptor in patients admitted to the hospital for SARS-CoV-2 infections. Suitable antagonists for use in the methods of the invention include, but are not limited to, the bradykinin Bl receptor antagonists disclosed in W02010097372 A, W02011104203 Al and WO2012022795 Al, which are herewith incorporated by reference. In a further particular embodiment, the small molecule bradykinin receptor antagonist for use in the invention is selected from the group consisting of:

In another further particular embodiment, the bradykinin receptor antagonist is selected from the group consisting of:

In an even further particular embodiment, the bradykinin receptor antagonist is Treatment of diabetic macular edema

As described above, Diabetic macular edema (or DME) is characterised by exudation and accumulation of extracellular fluid in the macula secondary to an increase in vascular permeability, and is typically associated with diabetic retinopathy, specifically in patients with diabetes mellitus.

The present inventors have found that determining the presence or absence of particular biomarkers in DME patients is important for guiding their treatment with inhibitors of the kail ikrein-kinin pathway, also known as kallikrein-kinin system (KKS). As mentioned earlier, the serine protease plasma kallikrein is a key player of the kallikrein-kinin system, a metabolic cascade that, when activated, triggers the release of vasoactive kinins. By virtue of their ability to activate endothelial cells, leading to vasodilation and increased vascular permeability, kinins participate in physiological (e.g. regulation of blood pressure) and pathological (e.g. inflammation) processes. Upon activation, plasma kallikrein cleaves its substrate high-molecular-weight kininogen to release bradykinin. Bradykinin activates the Bl receptor (B1R) and the B2 receptor (B2R) to exert its vasoactive properties. The activity of the kallikrein-kinin system is regulated by protease inhibitors. The primary physiological inhibitors of plasma kallikrein are complement 1 inhibitor (Cl-INH) and a-2 macroglobulin.

The inventors have identified that inhibition of the kallikrein-kinin pathway is able to treat DME and especially by its use in a method involving determining the presence or absence of subfoveal atrophy and cyst at retina biomarkers. Therefore, in a first aspect, the present invention provides an inhibitor of the kallikrein-kinin pathway for treating DME, wherein the subject does not have one or more biomarkers as described herein. As the skilled person will understand, inhibition of the pathway can be obtained through inhibition of one of its components, in particular through inhibition of plasma kallikrein, antagonism of bradykinin, or inhibition of bradykinin receptor Bl or B2.

Therefore, in a particular embodiment, the present invention provides an inhibitor of prekallikrein for use in a method of treatment of DME. In another particular embodiment, the present invention provides an inhibitor of plasma kallikrein for use in a method of treatment of DME. In yet another particular embodiment, the present invention provides a bradykinin antagonist for use in a method of treatment of DME. In yet another particular embodiment, the present invention provides an inhibitor of bradykinin receptor Bl for use in a method of treatment of DME. In yet another particular embodiment, the present invention provides an inhibitor of bradykinin receptor B2 for use in a method of treatment of DME. Particularly preferred is the inhibition of plasma kallikrein. Biomarkers

It has been identified that specific biomarkers govern the effectiveness of treatment of ophthalmic disorders with inhibitors of the kail ikrein-ki nin pathway. In a particular embodiment, the first biomarker is the presence or absence of cyst at retina. In a further embodiment, the cyst is an intraretinal cyst. In another embodiment, the cyst is graded as mild, moderate or severe; in particular as moderate or severe; more preferably as severe. In another embodiment, the first biomarker is a cyst at a retina in the central 1mm. In another embodiment, the first biomarker is determined based on cyst at retina occupying and disrupting all retinal layers. In yet another particular embodiment, the second biomarker is the presence of at least one of an ellipsoid zone (EZ) disruption and an external limiting membrane (ELM) disruption. Disruption of the EZ and/or ELM refers to an EZ and/or ELM status of disruption or absence. In a particular embodiment, the EZ and/or ELM status is graded as being disrupted or absent, preferably as being absent. Suitable grading systems for cysts and EZ/ELM disruption are e.g. described in Panozzo et al. (European Journal of Ophthalmology 2020 (30):8-18; doi: 10.1177/1120672119880394). In another particular embodiment, the second biomarker is the presence or absence of subfoveal atrophy. In another embodiment, subfoveal atrophy is determined based on ellipsoid zone disruption. In another embodiment, subfoveal atrophy is determined based on external limiting membrane disruption. In yet another embodiment, subfoveal atrophy is determined based on both ellipsoid zone and external limiting membrane disruption.

As will be understood by the skilled person, determining the presence or absence of one or more biomarkers at a retina of a subject, such as a retinal cyst, an ellipsoid zone disruption or an external limiting membrane disruption, is performed using previously obtained data of a retina of the subject. In particular, one or more images of a retina of the subject will have been obtained before performing the determination of the biomarker is present or absent. Therefore, in a particular embodiment, determining the presence or absence of the biomarkers disclosed herein uses one or more previously obtained images of a retina of the subject. Phrased otherwise, particularly, the methods disclosed herein comprise:

- acquiring one or more previously obtained images of a retina of a subject; and

- determining the presence or absence of a biomarker as disclosed herein using the previously obtained images.

Thus, the present invention provides a method for predicting whether a subject with an ophthalmic disorder will be nonresponsive to plasma kail ikrein inhibitor therapy, the method comprising:

- acquiring one or more previously obtained images of a retina of a subject; and - determining the presence or absence of a biomarker as disclosed herein using the previously obtained images; wherein the subject is predicted to be nonresponsive if the one or more biomarkers are present. In a further embodiment, the subject has diabetic macular edema.

Additionally, the present invention thus provides a plasma kalli krein inhibitor for use in a method of treating a subject with an ophthalmic disorder, wherein the method comprises:

- acquiring one or more previously obtained images of a retina of a subject;

- determining the presence or absence of a biomarker as disclosed herein using the previously obtained images; and

- if the one or more biomarkers are absent, administering to the subject the plasma kallikrein inhibitor.

It thus follows that the present invention also provides a plasma kallikrein inhibitor for use in a method of treating a subject with diabetic macular edema, wherein the method comprises:

- acquiring one or more previously obtained images of a retina of a subject;

- determining the presence or absence of a biomarker as disclosed herein using the previously obtained images; and

- if the one or more biomarkers are absent, administering to the subject the plasma kallikrein inhibitor.

In a particular embodiment, the one or more biomarkers described herein are determined by optical coherence tomography (OCT). In a further embodiment, the one or more biomarkers described herein are determined by spectral domain optical coherence tomography (SD-OCT).ln a particular embodiment, plasma kallikrein inhibitors are used in the method of treatment.

Subjects

As is evident from the disclosures herein, subjects have an ophthalmic disorder and may also be referred to herein as patients. As will be understood by the skilled person from the disclosures herein, the present invention relates to treatment of patients having an ophthalmic disorder, such as DME. In a preferred embodiment, the present invention relates to the treatment of patients that may have shown poor or no response to treatment with anti-VEGF. Although the subject may have been determined to be a non-responder or poor responder to anti-VEGF therapy, this is not a requirement for performing the invention. In a preferred embodiment, the age of the patients to be treated using the product of the invention is 18 years or above. In preferred embodiments, the patients to be treated have been diagnosed with DME and have type 1 or 2 diabetes. In a further preferred embodiment, the patients have study eye International Clinical Diabetic Retinopathy Disease Severity Scale (ETDRS) letter score < 62 and > 23.

In preferred embodiments, the patients to be treated do not have macular edema due to causes other than DME or any concurrent disease in the study eye, other than DME, that could compromise BCVA or require medical or surgical intervention during the treatment period or could confound interpretation of the results. In another preferred embodiment, the patients to be treated do not have any condition that could confound the ability to detect a change in CST in the study eye, any previous confounding treatments or procedures, or their planned or expected use during the treatment period. In further embodiments, the patients to be treated do not have presence of neovascularization at the disc (NVD) or uncontrolled glaucoma in the study eye, or any active ocular / intra-ocular infection or inflammation in either eye, or poorly controlled diabetes mellitus, or uncontrolled hypertension.

In a preferred embodiment, the patients to be treated do not have subfoveal atrophy. In another preferred embodiment, patients to be treated do not have ellipsoid zone disruption. In yet another preferred embodiment, the patients to be treated do not have external limiting membrane disruption. In a further embodiment, the patients to be treated do not have either ellipsoid zone disruption or external limiting membrane disruption.

In a particular embodiment, the patients to be treated to not have one or more of the biomarkers as disclosed herein. In a preferred embodiment, the patients to be treated do not have a cyst at the retina. In a further embodiment, the patients to be treated do not have a cyst in the central 1mm. In yet another preferred embodiment, the patients to be treated do not have cyst occupying and disrupting all retinal layers.

As will be understood by the skilled person from the disclosures herein, the present invention provides plasma kallikrein inhibitors for use in the treatment of subjects having an ophthalmic disorder, wherein the subjects have previously been determined not to have one or more of the biomarkers disclosed herein. In a particularly preferred embodiment, the present invention relates to the treatment of subjects which have been previously determined not to have an ellipsoid zone or external limiting membrane disruption and not to have a cyst at a retina. In particular, which have been previously determined not to have an ellipsoid zone or external limiting membrane disruption and not to have a severe cyst at a retina. More in particular, which have previously been determined not to have an ellipsoid zone or external limiting membrane disruption and not to have a severe cyst in the central 1 mm occupying and disrupting all retinal layers. Thus, for example, the present invention also provides plasma kalli krein inhibitors for use in the treatment of a subject having diabetic macular edema, wherein the subject has previously been diagnosed as not to have an ellipsoid zone or external limiting membrane disruption and not to have a severe cyst in the central 1 mm occupying and disrupting all retinal layers.

Ophthalmic disorder

Examples of suitable "ophthalmic disorders" (including exudative and/or inflammatory ophthalmic disorders, disorders related to impaired retinal vessel permeability and/or integrity, disorders related to retinal microvessel rupture leading to focal hemorrhages, back of the eye diseases, retinal diseases and front of the eye diseases) include but are not limited to: age related macular degeneration (ARMD) , exudative macular degeneration (also known as "wet" or neovascular age-related macular degeneration (wet-AMD), macular oedema, aged disciform macular degeneration, cystoid macular oedema, palpebral oedema, retinal oedema, diabetic retinopathy, acute macular neuroretinopathy, central serous chorioretinopathy, chorioretinopathy, choroidal neovascularization, neovascular maculopathy, neovascular glaucoma, obstructive arterial and venous retinopathies (e.g. retinal venous occlusion or retinal arterial occlusion) , central retinal vein occlusion, disseminated intravascular coagulopathy, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal arterial microaneurysms, Coat's disease, parafoveal telangiectasis, hemi-retinal vein occlusion, papillophlebitis, central retinal artery occlusion, branch retinal artery occlusion, carotid artery disease (CAD) , frosted branch angitis, sickle cell retinopathy and other hemoglobinopathies, angioid streaks, macular oedema occuring as a result of aetiologies such as disease (e.g. diabetic macular oedema), eye injury or eye surgery; retinal ischemia or degeneration produced for example by injury, trauma or tumours, uveitis, iritis, retinal vasculitis, endophthalmitis, panophthalmitis, metastatic ophthalmia, choroiditis, retinal pigment epithelitis, conjunctivitis, cyclitis, scleritis, episcleritis, optic neuritis, retrobulbar optic neuritis, keratitis, blepharitis, exudative retinal detachment, corneal ulcer, conjunctival ulcer, chronic nummular keratitis, thygeson keratitis, progressive mooren's ulcer, an ocular inflammatory disease caused by bacterial or viral infection, and by an ophthalmic operation, an ocular inflammatory disease caused by a physical injury to the eye, a symptom caused by an ocular inflammatory disease including itching, flare, oedema and ulcer, erythema, erythema exsudativum multiforme, erythema nodosum, erythema annulare, scleroedema, dermatitis, angioneurotic oedema, laryngeal oedema, glottic oedema, subglottic laryngitis, bronchitis, rhinitis, pharyngitis, sinusitis, laryngitis or otitis media. References herein to "back-of-eye diseases" include diseases affecting among other the retina, macular, fovea in the posterior region of the eye. Examples of suitable "back-of-eye diseases" include but are not limited to: macular oedema such as clinical macular oedema or angiographic cystoid macular oedema arising from various aetiologies such as diabetes, exudative macular degeneration and macular oedema arising from laser treatment of the retina, age-related macular degeneration, retinopathy of prematurity (also known as retrolental fibroplasia), retinal ischemia and choroidal neovascularization, retinal diseases (diabetic retinopathy, diabetic retinal oedema, retinal detachment, senile macular degeneration due to sub-retinal neovascularization, myopic retinopathy); inflammatory diseases; uveitis associated with neoplasms such as retinoblastoma or pseudoglioma; neovascularization following vitrectomy; vascular diseases (retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis, retinopathies resulting from carotid artery ischemia); and neovascularization of the optic nerve.

References herein to "front-of-eye diseases" refers to diseases affecting predominantly the tissues at the front-of-eye, such as the cornea, iris, ciliary body, conjunctiva etc. Examples of suitable "front-of- eye diseases" include but are not limited to: corneal neovascularization (due to inflammation, transplantation, developmental hypoplasia of the iris, corneal diseases or opacifications with an exudative or inflammatory component, neovascularization due to penetration of the eye or contusive ocular injury; chronic uveitis; anterior uveitis; inflammatory conditions resulting from surgeries such as LASIK, LASEK, refractive surgery, IOL implantation; irreversible corneal oedema as a complication of cataract surgery; oedema as a result of insult or trauma (physical, chemical, pharmacological, etc); inflammation; conjunctivitis (e.g. persistent allergic, giant papillary, seasonal intermittent allergic, perennial allergic, toxic, conjunctivitis caused by infection by bacteria, viruses or Chlamydia); keratoconjunctivitis (vernal, atopic, sicca) ; iridocyclitis; iritis; scleritis; episcleritis; infectious keratitis; superficial punctuate keratitis; keratoconus; posterior polymorphous dystrophy; Fuch's dystrophies (corneal and endothelial); aphakic and pseudophakic bullous keratopathy; corneal oedema; scleral disease; ocular cicatrcial pemphigoid; pars planitis; Posner Schlossman syndrome; Behcet's disease; Vogt-Koyanagi-Harada syndrome; hypersensitivity reactions; ocular surface disorders; conjunctival oedema; toxoplasmosis chorioretinitis; inflammatory pseudotumor of the orbit; chemosis; conjunctival venous congestion; periorbital cellulitis; acute dacryocystitis; non-specific vasculitis; sarcoidosis; and cytomegalovirus infection.

Examples of suitable "disorders associated with excessive vascular permeability and/or edema in the eye", e.g. in the retina or vitreous, include, but are not limited to, age-related macular degeneration (AMD), retinal edema, retinal hemorrhage, vitreous hemorrhage, macular edema (ME), diabetic macular edema (DME), proliferative diabetic retinopathy (PDR) and non-proliferative diabetic retinopathy (DR), radiation retinopathy, telangiectasis, central serous retinopathy, and retinal vein occlusions. Retinal edema is the accumulation of fluid in the intraretinal space. DME is the result of retinal microvascular changes that occur in patients with diabetes. This compromise of the blood-retinal barrier leads to the leakage of plasma constituents into the surrounding retina, resulting in retinal edema. Other disorders of the retina include retinal vein occlusions (e.g. branch or central vein occlusions), radiation retinopathy, sickle cell retinopathy, retinopathy of prematurity, Von Hippie Lindau disease, posterior uveitis, chronic retinal detachment, Irvine Gass Syndrome, Eals disease, retinitis, and/or choroiditis.

In a particular embodiment, the ophthalmic disorder is a back-of-eye disease associated with excessive vascular permeability and/or edema in the eye. . In yet another particular embodiment, the ophthalmic disorder is selected from the group consisting of wet age-related macular degeneration, diabetic macular edema, proliferative diabetic retinopathy, visual impairment due to macular edema secondary to retinal vein occlusion, visual impairment due to choroidal neovascularization, and retinopathy of prematurity. In another particular embodiment, the ophthalmic disorder is selected from the group consisting of wet age-related macular degeneration, macular edema, diabetic retinopathy, and retinal vein occlusion. In another particular embodiment, the ophthalmic disorder is selected from wat age-related macular degeneration and macular edema; particularly from age-related macular degeneration and diabetic macular edema; preferably diabetic macular edema

Compositions

The present invention further relates to a pharmaceutical composition, the composition comprising a compound as defined above and as defined in any one of the embodiments presented herein.

In the rest of the text, the expression "compound " or "compound according to the invention" is understood, for the purposes of the present invention, both in the plural and the singular, that is to say that the inventive composition may comprise one or more than one "compound according to the invention".

In a particular embodiment, the present invention provides a pharmaceutical composition comprising the compound according to any one of the claims, and one or more pharmaceutically acceptable carriers. Examples of pharmaceutically acceptable formulations as well as methods for making them can be found, e.g., in Remington's Pharmaceutical Sciences (e.g. 20th Edition; Lippincott, Williams & Wilkins, 2000) or in any Pharmacopeia handbook (e.g. US-, European- or International Pharmacopeia). In another embodiment, the present invention provides a composition comprising an aqueous buffer wherein a compound of the invention has been dissolved. In a further embodiment, the present invention provides a pharmaceutical composition comprising an aqueous buffer wherein a compound of the invention and one or more pharmaceutically acceptable carriers have been dissolved. In another further embodiment, the present invention provides a pharmaceutical composition consisting of an aqueous buffer, a compound of the invention and one or more pharmaceutically acceptable carriers.

In a particular embodiment, the present invention therefore provides the use of a plasma kallikrein inhibitor for the manufacture of a medicament for the treatment of DME, wherein the method of treatment is as described herein. In another particular embodiment, the present invention provides the use of biomarkers for the manufacture of a medicament for the treatment of DME, wherein the method of treatment is as described herein.

Administration

For performing the administration, the compounds or the compositions as described herein may be administered to a subject by any method that leads to delivery of the therapeutic agent to the site of the ophthalmic condition, such as by administration to the eye. In another embodiment, the use, treatment and/or prevention comprises contacting the vitreous and/or aqueous humour with an effective amount of a composition comprising a compound of the invention. Administration may be by an ocular route, such as topical, subconjunctival, sub-Tenon, intraocular, ocular implants, etcetera. Topical administration may comprise administration of one or a few drops of a composition comprising a compound of the invention to the eye. Delivery to areas within the eye, in situ can be accomplished by injection, cannula or other invasive device designed to introduce precisely metered amounts of a desired ophthalmic composition to a particular compartment or tissue within the eye (e.g. posterior chamber or retina). An intraocular injection may be into the vitreous (intravitreal), or under the conjunctiva (subconjunctival), or behind the eye (retrobulbar), into the sclera, or under the Capsule of Tenon (subTenon). Other intraocular routes of administration and injection sites and forms are also contemplated and are within the scope of the invention. In a preferred embodiment, the treatment and/or prevention comprises administration of the compound by intravitreal injection. Preferably this is performed through self-sealing gauge needles or other any suitably calibrated delivery device. Injection into the eye may be through the pars plana via the self-sealing needle. When administering the composition by intravitreal injection, the active agents should be concentrated to minimize the volume for injection. Preferably, the volume for injection is less than about 5 mL. Volumes such as this may require compensatory drainage of the vitreous fluid to prevent increases in intraocular pressure and leakage of the injected fluid through the opening formed by the delivery needle. More preferably, the volume injected is between about 10 and 200 pL. Most preferably, the volume for injection is between 30 and 100 pL, in particular about 50 pL.

With regard to the preferred routes of administration, the pharmaceutically acceptable salt, pharmaceutically acceptable solvate, isomer or mixture thereof is also an ophthalmically acceptable salt, ophthalmically acceptable solvate, isomer or mixture thereof. In a preferred embodiment, the pharmaceutical composition comprises the compound and one or more ophthalmically acceptable carriers. Interestingly, compounds of the invention were found to have a high aqueous solubility of over 100 mg/ml, while prior art compounds have single digit mg/ml or lower aqueous solubilities. In a preferred embodiment, a composition of the invention as disclosed herein is an aqueous solution comprising a compound of the invention. In another embodiment, the present invention provides a kit of parts comprising a container comprising a compound of the invention and another container comprising an aqueous buffer for dissolving the compound of the invention.

As will be understood to the skilled person, a composition as disclosed herein is preferably a sterile composition. In a particular embodiment, the present invention provides a sterile container comprising a compound or composition described herein. In a further embodiment, the sterile container is a vial comprising a compound or composition described herein. The vial may comprise the compound as a powder or as a solution, preferably an aqueous solution. In a more particular embodiment, the present invention provides a sterile vial comprising an aqueous solution of the compound described herein. In an even more particular embodiment, a vial as described herein comprises a part that is designed to be pierceable by a syringe needle. In alternative embodiment, the present invention provides a kit comprising a vial with the compound described herein in a powder form and a container comprising an aqueous solution, particularly an aqueous buffer solution. In another embodiment, the sterile container is a syringe prefilled with a pharmaceutical composition described herein, particularly an aqueous solution comprising the compound described herein. In yet another embodiment, the sterile container is a container comprising at least one tablet comprising a pharmaceutical composition described herein. In particular a blister package or bottle comprising multiple tablets comprising a compound described herein and one or more pharmaceutically acceptable carriers. In one particular embodiment, a vial as described comprises less than 5 mL, in particular less than 4 mL, more in particular less than 3 mL of a solution comprising the compound described herein.

In a particular embodiment, the plasma kallikrein inhibitor administration comprises administration of at least 2 doses of the plasma kallikrein inhibitor, such as at least 3 doses of the plasma kallikrein inhibitor. In another particular embodiment, plasma kallikrein inhibitor administration comprises administration of from 1 to 10 doses of the plasma kallikrein inhibitor, in particular from 1 to 7 doses of the plasma kallikrein inhibitor, such as from 1 to 5 doses of the plasma kallikrein inhibitor or from 1 to 4 doses of the plasma kallikrein inhibitor. In a preferred embodiment, the plasma kallikrein inhibitor administration comprises administration of from 1 to 3 doses of the plasma kallikrein inhibitor, such as 2 or 3 doses of the plasma kallikrein inhibitor.

In a particular embodiment, administration of plasma kallikrein inhibitor comprises administration with an interval of at least 1 week between doses of the plasma kallikrein inhibitor, in particular at least 2 weeks, more in particular at least three weeks. In a further embodiment, administration of plasma kallikrein inhibitor comprises administration with an interval of at least 4 weeks, in particular at least one month between doses of the plasma kallikrein inhibitor. In another particular embodiment, administration of plasma kallikrein inhibitor comprises administration with an interval of at most 4 months between doses of the plasma kallikrein inhibitor, such as at most 3 months between doses of the plasma kallikrein inhibitor, in particular at most 2 months between doses of the plasma kallikrein inhibitor. In yet another particular embodiment, administration of plasma kallikrein inhibitor comprises administration with an interval of from 1 week to 4 months between doses of the plasma kallikrein inhibitor, in particular from 2 weeks to 3 months, more in particular from 3 weeks to 2 months. In a preferred embodiment, administration of plasma kallikrein inhibitor comprises administration of multiple doses of the plasma kallikrein inhibitor with an interval of about 1 month between the doses.

Determining suitable dosages of the plasma kallikrein inhibitor for use in the invention is within the ambit of the skilled person. In a particular embodiment, the plasma kallikrein inhibitor is administered in a dose of from 0.001 to 10 mg per eye, such as from 0.001 to 5 mg per eye, or from 0.001 to 3 mg per eye. In another particular embodiment from 0.005 to 3 mg per eye, particularly 0.005 to 1 mg per eye, more particularly 0.005 to 0.5 mg per eye, preferably from 0.005 to 0.2 mg per eye. In a further preferred embodiment, the plasma kallikrein inhibitor is administered in a dose from about 0.01 mg to about 0.13 mg/eye. In another preferred embodiment, the plasma kallikrein inhibitor is administered in a dose of about 0.10 to 0.16 mg per eye, preferably about 0.13 mg per eye. As described herein before, in a preferred embodiment, the plasma kallikrein inhibitor is a bicyclic peptide as described herein, such as cpdl or cpd 2. In a particular embodiment, the bicyclic peptide is administered in a dose of from 0.001 to 10 mg per eye, such as from 0.001 to 5 mg per eye, or from 0.001 to 3 mg per eye. In another particular embodiment from 0.005 to 3 mg per eye, particularly 0.005 to 1 mg per eye, more particularly 0.005 to 0.5 mg per eye, preferably from 0.005 to 0.2 mg per eye. In a further preferred embodiment, the bicyclic peptide, such as cpdl or cpd2 is administered in a dose from about 0.01 mg to about 0.13 mg/eye. In another preferred embodiment, the plasma kallikrein inhibitor is administered in a dose of about 0.10 to 0.16 mg per eye, preferably about 0.13 mg per eye. Evidently from the description of the invention herein, embodiments pertaining to doses, intervals and preferred kallikrein inhibitors can be combined to obtain particular and preferred embodiments according to the invention. As a mere example, in a preferred embodiment, administration of plasma kallikrein inhibitor comprises administration of multiple doses from about 0.01 mg to about 0.13 mg/eye of a bicyclic peptide as described herein, such as cpdl or cpd2, with an interval of about 1 month between the doses.

Successful treatment of a subject in accordance with the invention may result in the inducement of a reduction or alleviation of symptoms in a subject afflicted with medical or biological disorder to, for example, halt the further progression of the disorder.

In another particular embodiment, the present invention provides a method for the treatment of an ophthalmic disorder, such as DME, as disclosed herein, the method comprising determining the presence or absence of biomarkers and, in the absence of the biomarkers, administering a subject in need thereof an inhibitor of the kallikrein-kinin pathway. In a further embodiment, the present invention provides a method for the treatment of an ophthalmic disorder, such as DME, as disclosed herein, the method comprising determining the presence or absence of one or more biomarkers as described herein and, in the absence of the biomarkers, administering a subject in need thereof an inhibitor of the kallikrein- kinin pathway, such as a plasma kallikrein inhibitor. In another embodiment, the present invention provides a method for inhibiting a plasma kallikrein in a subject in need thereof, wherein the method comprises administering a plasma kallikrein inhibitor as disclosed herein and wherein the subject has an ophthalmic disorder and does not have one or more biomarkers as described herein. EXAMPLES

The following examples are provided in order to demonstrate certain preferred embodiments and aspects of the present invention and are not construed as limiting the scope thereof.

Example 1 Study evaluating use of biomarkers in DME therapy

Study setup

Data on the use of biomarkers to identify subjects with an ophthalmic disorder that are more likely to respond to inhibitors of the kail ikrein-kini n pathway was obtained in a masked study in human patients.

In particular, subjects had to provide written informed consent prior to screening procedures and subjects selected were male or female subjects with type 1 or 2 diabetes, aged 18 or above, with central involved DME (CI-DME). In the study eye, subjects had (a) a BCVA ETDRS letter score < 73 and > 39 and (b) central involved DME (CI-DME) with CST of > 320pm in men or > 305pm in women, as determined by analysis of images obtained from spectral domain optical coherence tomography (SD-OCT). The BCVA ETDRS letter score had to be > 34 in the fellow eye. Furthermore, subjects had to have received > 5 anti- VEGF injections (any kind) for the treatment of CI-DME, and the subject's first anti-VEGF injection in the study eye occurred < 36 months prior to screening and the last 2 injections were aflibercept 2mg, administered within < 6 months prior to screening, with the last injection administered at 3 - 8 weeks prior to screening.

The selected subjects did not have macular edema due to causes other than DME or any concurrent disease in the study eye, other than DME, that could compromise BCVA or require medical or surgical intervention during the study period or could confound interpretation of the results. Furthermore, selected subjects did not have any condition that could confound the ability to detect a change in CST in the study eye, any previous confounding treatments or procedures, or their planned or expected use during the study period. The selected subjects also did not have presence of neovascularization at the disc (NVD) or uncontrolled glaucoma in the study eye, or any active ocular / intra-ocular infection or inflammation in either eye. Additionally, poorly controlled diabetes mellitus or uncontrolled hypertension were also used to exclude possible subjects from the study.

Subjects were randomized and received 0.01 mg, 0.04 mg or 0.13 mg of a plasma kallikrein inhibitor (cpdl) in three monthly intravitreal injections. Throughout the study and up to 6 months, the subjects were assessed to determine BCVA and CST.

Results Eight subjects with DME were selected as described above and assigned to receive 0.13 mg dosage of cpdl. Six of these subjects did not have the described biomarkers, while two of them had at least one cyst and showed an ellipsoid zone (EZ) disruption and/or an external limiting membrane (ELM) disruption in the study eye. Cysts were graded as severe (3) and EZ and/or ELM status as absent (2) in accordance with the grading system as described by Panozzo et al. (European Journal of Ophthalmology 2020 (30):8- 18; doi: 10.1177/1120672119880394).

Subjects without the biomarkers showed clear improvement in best-corrected visual acuity (BCVA). In contrast, subjects with the biomarkers did not respond to the plasma kallikrein inhibitor therapy, but continued to decline (FIG. 3). The biomarkers as described herein thus allow to predict response to treatment with inhibitors of the kalli krein-kini n pathway.