Plasma kal likrein inhibitor therapy for anti-VEGF sensitization

FIELD OF THE INVENTION

The present invention relates to the use of plasma kallikrein inhibitors for sensitizing patients having an ophthalmic disorder to anti-VEGF therapy and to increase the effect of anti-VEGF therapy.

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. DME, which is characterized 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 number of 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. There is thus an urgent need for therapies for treating DME in patients that do not adequately respond to anti-VEGF therapies.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that plasma kallikrein inhibitor treatment allows to sensitize subjects having an ophthalmic disorder, which do not adequately respond to anti-VEGF therapy, to such anti-VEGF therapies. Therefore, in a first aspect, the present invention provides a plasma kallikrein inhibitor for use in the treatment of a subject having an ophthalmic disorder, the treatment comprising:

- administering a plasma kallikrein inhibitor, and

- subsequently administering an anti-VEGF agent.

In a particular embodiment, the plasma kallikrein inhibitor is a direct plasma kallikrein inhibitor that is selected from the group consisting of an antibody, a peptide, a nucleotide, or a chemical molecule. In another embodiment, wherein the plasma kail ikrein inhibitor has an inhibition constant that is at least 100 times lower for plasma kalli krein than for tissue kalli krein and transmembrane protease, serine 2 (TMPRSS2).

In a further embodiment, the plasma kail ikrein inhibitor is selected from the group consisting of ecallantide, lanadelumab, berotralstat, Cl inhibitor, a compound of formula A 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 another embodiment, the plasma kallikrein inhibitor 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. In a further embodiment, the plasma kallikrein inhibitor is 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. In another further embodiment, the plasma kallikrein inhibitor is a compound comprising [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 the molecular scaffold.

In a particular embodiment, the anti-VEGF agent administered after administration of the plasma kallikrein inhibitor is a compound that binds to and inhibits the activity of vascular endothelial growth factor (VEGF) or a VEGF receptor. In a further embodiment, the anti-VEGF agent is selected from bevacizumab, ranibizumab, brolucizumab, conbercept, and aflibercept.

In yet another particular embodiment, the plasma kallikrein inhibitor is administered from one to ten times before the anti-VEGF agent is administered. In a second aspect, the present invention provides a method for sensitizing a subject having an ophthalmic disorder to an anti-VEGF agent, the method comprising administering a plasma kallikrein inhibitor.

In another aspect, the present invention provides a method for providing an increased effect of anti-VEGF therapy in a subject having an ophthalmic disorder, the method comprising administering a plasma kallikrein inhibitor.

The present invention is particularly useful for a subject that has no or a poor response to anti- vascular endothelial growth factor (anti-VEGF) therapy before administration of the plasma kallikrein inhibitor. The present invention is further useful for a subject who was previously treated with anti-VEGF therapy and still has persistent edema.

In a 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.

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

Fig. 3 Change in BCVA from baseline in first subject. Syringe indicates administration of plasma kallikrein inhibitor; V indicates administration of anti-VEGF agent. BL: study visit at baseline; Mx: study visit at month x.

Fig. 4 Change in CST from baseline in first subject. Syringe indicates administration of plasma kallikrein inhibitor; V indicates administration of anti-VEGF agent. BL: study visit at baseline; Mx: study visit at month x. Dotted line shows data after administration of the anti-VEGF agent.

Fig. 5 Change in BCVA from baseline in second subject. Syringe indicates administration of plasma kalli krein inhibitor; V indicates administration of anti-VEGF agent. BL: study visit at baseline; Mx: study visit at month x. Dotted line shows data after administration of the anti-VEGF agent. Fig. 6 Change in CST from baseline in second subject. Syringe indicates administration of plasma kallikrein inhibitor; V indicates administration of anti-VEGF agent. BL: study visit at baseline; Mx: study visit at month x. Dotted line shows data after administration of the anti-VEGF agent.

Fig. 7 Change in BCVA from baseline in third subject. Syringe indicates administration of plasma kallikrein inhibitor; V indicates administration of anti-VEGF agent. BL: study visit at baseline; Mx: study visit at month x. Dotted line shows data after administration of the anti-VEGF agent.

Fig. 8 Change in CST from baseline in third subject. Syringe indicates administration of plasma kallikrein inhibitor; V indicates administration of anti-VEGF agent. BL: study visit at baseline; Mx: study visit at month x. Dotted line shows data after administration of the anti-VEGF agent.

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 prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or 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 mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

The term "sensitizing" or "sensitization" refers to increasing the response of a subject to a particular treatment.

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.

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 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 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".

Plasma kallikrein

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 KKS 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 KKS, including plasma kallikrein (pKal), 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 KKS increases retinal vascular permeability, retinal thickening, and neovascularisation, and these responses are exacerbated in diabetic animals. In line with this, inhibition of pKal, Bl receptor (B1R), and the B2 receptor (B2R) block enhanced vascular permeability in rodent models.

The serine protease pKal is a key player of KKS, and has been identified as a novel potential target for treatment of DME. Upon activation, pKal 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 KKS is regulated by protease inhibitors. The primary physiological inhibitors of pKal are complement 1 inhibitor (Cl-INH) and a-2 macroglobulin.

While both VEGF and pKal levels have been shown to be upregulated in the vitreous of DME patients, their concentrations do not correlate. Moreover, while pKal 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 pKal 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 pKal 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.

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, papilloph lebitis, 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.

As will be understood from the descriptions herein, the ophthalmic disorder is a disorder that is treatable with an anti-VEGF agent. 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

Subjects

As is evident from the disclosures herein, subjects have an ophthalmic disorder and may also be referred to herein as patients. The present invention is particularly useful to sensitize patients to anti-VEGF therapy, i.e. treatment with an anti-VEGF agent. The present invention is further useful to increase the effect of anti-VEGF therapy in patients. Although the subject may have been determined to be a nonresponder or poor responder to anti-VEGF therapy or been pretreated with anti-VEGF therapy and still have persistent edema, this is not a requirement for performing the invention. Indeed, the present invention can also be used to increase the chance that the patient will responds to anti-VEGF therapy. For example, the plasma kallikrein inhibitor may be administered as a first line treatment to the subject having the ophthalmic disorder, whereafter the anti-VEGF agent is administered. This ensures that a larger population of patients will respond to the anti-VEGF agent compared to first-line treatment with the anti- VEGF agent.

In a particular embodiment, the subject has no or low response to anti-VEGF therapy. It is well- known to the skilled person how to determine no or low response to anti-VEGF therapy. In particular, the subject may receive one or multiple administrations of an anti-VEGF agent after which treatment response is determined. If there is no or low response, the subject may receive treatment with a plasma kal likrein inhibitor and subsequent treatment with an anti-VEGF agent. In a particular embodiment, the subject has been diagnosed as having no or a low response to anti-VEGF therapy. In a further embodiment, the subject has no or a low response to anti-VEGF therapy as determined after at least three administrations of an anti-VEGF agent, particularly after at least five administrations, more particularly after at least seven administrations. In another particular embodiment, the subject has previously received treatment with an anti-VEGF agent and has an insufficient response to the anti-VEGF therapy as determined by either a best corrected visual acuity (BCVA) letter score or central subfield thickness (CST). The latter can preferably be determined by optical coherence tomography (OCT), particularly spectral domain optical coherence tomography (SD-OCT). In a particular embodiment, no or low response to anti-VEGF therapy is determined by an improvement of the BCVA letter score of less than 10, such as less than 9, less than 8, or less than 7 due to the anti-VEGF therapy. Preferably less than 6, particularly preferably less than 5. In another particular embodiment, no or low response to anti-VEGF therapy is determined by a CST reduction of less than 130 pm, less than 120 pm, less than 110 pm, particularly less than 100 pm due to anti-VEGF therapy. In a further embodiment, less than 90 pm, less than 80 pm, preferably less than 70 pm.

In another particular embodiment, the subject has previously received treatment with an anti- VEGF agent and has either a best corrected visual acuity (BCVA) letter score of less than 40 or has a central subfield thickness (CST) of 305 pm or more, such as preferably determined by optical coherence tomography (OCT), particularly spectral domain optical coherence tomography (SD-OCT). In a further embodiment, the subject has previously received treatment with an anti-VEGF agent and has a best corrected visual acuity (BCVA) letter score of less than 40 and a central subfield thickness (CST) of 305 pm or more.

Inhibitors

Compounds for use in the invention are inhibitors or antagonists of the kallikrein-kinin pathway, in particular inhibitors of plasma kail ikrein. 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 preka llikrein 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 lower for 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 Therepeutics 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 WQ2013050616 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 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. 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) kallikrein 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 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. 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(4jCH 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 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 He;

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

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 lie Phe Thr Arg Gin Cys Glu Glu Phe lie 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.

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-kalli krein antibodies may bind both prekalli krein and kallikrein. In another embodiment, the anti-kalli krein antibodies bind to kallikrein but do not bind to prekal likrein . Anti-preka llikrein and a nti-kall ikrein 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 W02011085103 A2 and WO2012094587 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 W02017072020 Al, W02017072021 Al and WO2018192866 Al developed by Boehringer Ingelheim International, as well as those disclosed in WO2019028362 Al developed by Dyax, those disclosed in WO03076458 A2, W02013005045 Al, and W02019106361 Al developed by Kalvista Pharmaceuticals, and those disclosed in W02021007189 Al developed by Rezolute, 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 WO2019028362 Al, berotralstat, and a compound of Formula A, Formula B, and Formula C, Formula B,

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 C. In another embodiment, the plasma ka llikrein 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 kalli krein, 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 preka llikrein 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 A,

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, and formula C. In a preferred embodiment, the plasma kallikrein inhibitor is cpdl or cpd2, more preferably cpdl.

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 an ophthalmic disorder, wherein the treatment is as described herein. In another particular embodiment, the present invention provides the use of a plasma kallikrein inhibitor for the manufacture of a medicament to sensitize a subject that has an ophthalmic disorder to an anti-VEGF agent. In yet another particular embodiment, the present invention provides the use of a plasma kallikrein inhibitor for the manufacture of a medicament to increase the effect of anti-VEGF therapy in a subject that has an ophthalmic disorder.

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.

As will furthermore be understood by the skilled person, the plasma kal likrein inhibitor and the anti-VEGF agent may be administered by the same or by different routes of administration. In a particular embodiment, the anti-VEGF agent is administered by intraocular injection, particularly intravitreal injection. In another particular embodiment, the plasma kallikrein inhibitor is administered by oral administration or by intraocular injection. In a preferred embodiment, the plasma kallikrein inhibitor is administered by intravitreal injection. In another preferred embodiment, the anti-VEGF agent is administered by intravitreal injection and the plasma kallikrein inhibitor is administered by intravitreal injection.

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.

In a particular embodiment, the plasma kallikrein inhibitor and the anti-VEGF agent are optionally administered in alternation. In a further embodiment, one or multiple doses of the plasma kallikrein inhibitor are administered to the subject, subsequently one or multiple doses of the anti-VEGF agent are administered to the subject, and this alternating administration is continued thereafter. Phrased otherwise, in a particular embodiment, the treatment of the present invention optionally comprises alternating administration of a plasma kallikrein inhibitor and an anti-VEGF agent, wherein during each alternation one or multiple doses of the plasma kallikrein inhibitor are administered and one or multiple doses of the anti-VEGF agent are administered. In a particular embodiment, the plasma kallikrein inhibitor is administered over a course of 1 to 8 months prior to administration of the anti-VEGF agent, more in particular over a course of 1 to 5 months, even more in particular over a course of 2 to 4 months, preferably over a course of three months. In another embodiment, 1 to 5 monthly doses of plasma kallikrein inhibitor are administered to the subject, followed by a 6 to 24 month treatment with an anti-VEGF agent, and this alternating administration is continued thereafter. In a preferred embodiment, 1 to 3 monthly doses of plasma kal likrein inhibitor are administered to the subject, followed by a 6 to 24 month treatment with an anti-VEGF agent, and this alternating administration is continued thereafter. In another preferred embodiment, three monthly dose of plasma kallikrein inhibitor is administered to the subject, followed by a 6 to 24 month treatment with an anti-VEGF agent, and this alternating administration is continued thereafter. As just disclosed, in particular embodiments of alternating treatment, the treatment with an anti-VEGF agent may be a 6 to 24 month treatment, particular a 6 to 18 month treatment, such as a 6 to 12 month treatment. The duration between anti-VEGF administration during such treatment will be dependent on the anti-VEGF agent and its formulation. Usually, the anti-VEGF agent will be administered every one or every two months.

In another embodiment, there is a loading phase priorto initiating the aforementioned alternating administration, comprising of administering 1 to 3 monthly doses of plasma kallikrein inhibitor.

As described herein before, the inventors have surprisingly identified that plasma kallikrein inhibitors can be used for sensitizing patients to anti-VEGF therapy, and to increase the effect of anti-VEGF therapy. In methods for sensitizing a subject, the plasma kallikrein inhibitor may be administered before the anti-VEGF agent is administered or it may be administered in combination with the anti-VEGF agent. Therefore, in a particular embodiment, the present invention provides a plasma kallikrein inhibitor for use in the treatment of a subject that has an ophthalmic disorder, the treatment comprising administering a plasma kallikrein inhibitor in combination with an anti-VEGF agent. Whenever reference is made to a combination of active ingredients, the active ingredients can be provided in a single composition or in separate composition. In addition, a combination of active ingredients also encompasses active ingredients that are linked non-covalently or covalently. Thus, in a particular embodiment, the present invention provides a composition for use in the treatment of a subject that has an ophthalmic disorder, wherein the composition comprises a plasma kallikrein inhibitor and an anti-VEGF agent. In another particular embodiment, the present invention provides a plasma kallikrein inhibitor for use in the treatment of a subject that has an ophthalmic disorder, wherein the treatment comprises coadministration of a plasma kallikrein inhibitor and an anti-VEGF agent. In one further embodiment, the plasma kallikrein inhibitor and the anti-VEGF agent are present in the same pharmaceutical composition. In one other further and preferred embodiment, the plasma kallikrein inhibitor and the anti-VEGF agent are present in different pharmaceutical compositions. As is evident from the disclosures herein, it is a further object of the invention to provide methods for treatments as described herein. For example, the prevent invention provides a method for treating a subject that has an ophthalmic disorder, the method comprising:

- administering a plasma kallikrein inhibitor to said subject, and

- subsequently administering an anti-VEGF agent to said subject.

In another particular embodiment, the present invention provides a method to sensitize a subject that has an ophthalmic disorder to an anti-VEGF agent, the method comprising administering a plasma kallikrein inhibitor to said subject.

In yet another particular embodiment, the present invention provides a method to increase the effect of anti-VEGF therapy in a subject that has an ophthalmic disorder, the method comprising administering a plasma kallikrein inhibitor to said subject.

EXAMPLES

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

EXAMPLE 1 - Study evaluating anti-VEGF sensitization

Study setup

Data on the use of plasma kallikrein inhibitor therapy to sensitize ophthalmic patients to anti-VEGF therapy and to provide an increased effect of anti-VEGF therapy was obtained in a masked study in human patients. To evaluate the impact, DME patients were carefully selected to be no or poor responders to anti-VEGF therapy by relying on key inclusion criteria that required multiple prior injections with anti-VEGF agents, but no or poor response as determined by a low BCVA ETDRS letter score (indicating poor vision) combined with high CST values (indicating presence of significant edema).

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 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. A number of patients in the study received an intravitreal injection with an anti-VEGF agent (bevacizumab or aflibercept) as rescue treatment after plasma kallikrein inhibitor treatment. Throughout the study and up to 6 months, the subjects were assessed to determine BCVA and CST.

Results

Four subjects with DME who were selected for poor or no response to anti-VEGF therapy as described above received plasma kallikrein inhibitor and subsequently anti-VEGF agent. One patient received bevacizumab, but exited the study and no follow-up data on BCVA and CST are available. Three other subjects received aflibercept.

In particular, a first subject received three doses of 0.01 mg of plasma kallikrein inhibitor in one month intervals (baseline, study visit month 1 and study visit month 2). In month 3, the subject received the anti-VEGF agent. BCVA and CST results are shown in figures 3 and 4, respectively. As can also be seen in the figure, although the patient was selected to have no or poor response to anti-VEGF, after plasma kallikrein inhibitor treatment, the patient responded well to anti-VEGF therapy. This is evidenced by both a marked increase in the BCVA letter score and a decrease in CST.

A second subject received two doses of 0.04 mg of the plasma kail ikrein inhibitor in one month intervals (baseline and study visit month 1). In month 2, the subject received the anti-VEGF agent. BCVA and CST results are shown in figures 5 and 6, respectively. As can also be seen in the figure, although the patient was selected to have no or poor response to anti-VEGF, after plasma kail ikrein inhibitor treatment, the patient responded well to anti-VEGF therapy. This is evidenced by both a marked increase in the BCVA letter score and a decrease in CST.

A third subject received three doses of 0.01 mg of the plasma kal likrein inhibitor in one month intervals (baseline, study visit month 1 and study visit month 2). In month 4, the subject received the anti- VEGF agent. BCVA and CST results are shown in figures 7 and 8, respectively. As can also be seen in the figure, although the patient was selected to have no or poor response to anti-VEGF, after plasma kallikrein inhibitor treatment, the patient responded well to anti-VEGF therapy. This is evidenced by both a marked increase in the BCVA letter score and a decrease in CST. In summary, patients were selected to have no or poor response to anti-VEGF treatment.

However, after treatment with plasma kallikrein inhibitors, patients responded well to anti-VEGF therapy. This was in each case evidenced by both a marked increase in the BCVA letter score (indicating improved vision) and a decrease in CST (indicating reduced edema).