INTRAVITREAL DELIVERY OF A DECORIN POLYPEPTIDE FOR THE TREATMENT OF CHOROIDAL NEOVASCULARISATION
FIELD OF THE INVENTION:
The present invention relates to the intravitreal delivery of a decorin polypeptide for the treatment of choroidal neovascularisation (CNV), in particular CNV resistant to anti-VEGF treatment.
BACKGROUND OF THE INVENTION:
The eye is a sensory organ that collects light from the visible world around us and converts it into nerve impulses. The optic nerve transmits these signals to the brain, which forms an image so thereby providing sight. Human eyes primarily consist of i) the three coating layers: the outer, middle and inner coat, and ii) the inner part of the eyeball: it contains the lens and the vitreous body and is divided into the anterior and the posterior chamber. The eye’s outer layer is made of dense connective tissue, which protects the eyeball and maintains its shape. It is also known as the fibrous tunic. The middle layer of tissue surrounding the eye, also known as the vascular tunic or„uvea“, is formed - from behind forward - by the choroid, the ciliary body, and the iris. The choroid takes up the posterior five-sixths of the bulb and is mainly comprised of blood vessels. Its major functions are oxygen supply and nutrition for the eye. The ciliary body contains a muscle (ciliary muscle), which can change the shape of the lens for adjustment to far or near sight, respectively, thereby controlling the so-called refractive power of the lens (accomodation). Additional functions of the ciliary body are the production, secretion, and outflow of aquaeous humour (the latter via the so-called’’Schlemm’s canal“), a watery fluid that fills both the anterior and the posterior chambers of the eye (see below). The third and inner coat of the eye is the retina, which is responsible for the perception of images - vision. The retina is a light-sensitive layer of nervous tissue composed of multiple sensory cells, so-called light- or photoreceptor cells, as well as associated nerve cells and other types of cells, all working together to make a person see. For vision, there are two types of photoreceptor cells: rods and cones. Rods provide the perception of black-and-white vision, mostly in dim light, whereas cones help to see colors in daylight. The inner part of the eyeball consists of the lens, the vitreous body and the two eye chambers. Bruch's membrane is a pentalaminar structure composed of the RPE basement membrane, inner collagenous layers, middle elastic layer, and outer collagenous layers. This extracellular matrix meshwork between the Retina Pigment Epithelial (RPE) cells and the choroid is 2-4pm is thicknes. The vitreous is a clear gelatinous mass held by collagen fibers. It is situated between lens and retina and comprises about two thirds of the entire eyeball. By pushing the retina towards the choroid, the vitreous promotes keeping the retina in place. Accordingly, the choroid is anatomically separated from the vitreous by at least 3 layers, namely neuroretina, the retinal pigment epithelium monolayer and Bruch's membrane.
Retinal neovascularization and choroidal neovascularization (CNV) are major causes of vision loss. Neovascularization arising from the retinal circulation is seen commonly in diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity (ROP), and sickle cell retinopathy. Neovascularization arising from the choroidal circulation is seen in are age-related macular degeneration (AMD), pathological myopia (PM), choroidits and central serous chorioretinopathy (CSCR). Retinal neovascularization and CNV are thus clinically distinguishable. CNV may cause visual loss due to exudation of intraretinal or subretinal fluid, hemorrhage, or fibrosis at the macula. For instance, AMD is the most frequent cause of social blindness in the elderly population. With a global prevalence of 8%, the projected number of individuals affected in 2020 is 196 million, increasing to 288 million in 2040 h Almost half of early AMD progresses to neovascular AMD (nAMD) also referred as“wet” AMD. Abnormal neovessels growing from the choroid towards the neuroretina underneath the macula, cause macular edema, bleeding, photoreceptors damages and eventually end stage fibrotic scare2. Heredity, diet, smoking, obesity and vascular diseases are involved in the pathogenesis of AMD3 but the exact mechanisms leading to CNV remain imperfectly understood. CNV is not specific to AMD, it complicates other diseases affecting the retinal pigment epithelium (RPE) and the choroid, including high myopia and central serous chorioretinopathy (CSCR). The pathogenesis of CNV is complex and multifactorial. Choroidal vessels ensure nutriments and oxygen supply to the avascular outer retina containing the highly energy demanding photoreceptor cells. Choriocapillaries loss, observed in nAMD eyes4 may cause hypoxia and angiogensis. Growing body of evidence also indicates that low-grade inflammation, activation of the inflammasome5,6 and of the complement alternative pathway play key roles in the pathogenesis of nAMD7. Beside vascular endothelial growth factor (VEGF) family members and their receptors8-10, complement components and pro-inflammatory molecules accumulating in the RPE/choroid complex11 such as cytokines, interleukins12 and angiopoietins13 contribute to the growth of CNV. The down-regulation of anti-angiogenic factors by inflammation and/or hypoxia such as pigment epithelium-derived factor (PEDF), endostatin, and thrombospondin- 1 (TSP-l)14 favor a pro-angiogenic microenvironment. VEGF is an important proangiogenic element that is usually produced by RPE and retinal photoreceptors. In CNV, the RPE boosts atypical neovascularization and the VEGF is the main growth factor responsible for development and progress of new vessels. The idea of inhibiting the activities of VEGF with anti- VEGF drugs has been investigated in the management of ocular neovascular disorders, particularly for the treatment of its consequences i.e. sub retinal and or intraretinal fluid. The current anti- VEGF treatments do not induce a total regression of the CNV requiring repeated injections to maintain vision. Moreover, evidence regarding anti- VEGF therapy resistance suggests a need for alternative medicines for the treatment of CNV.
Decorin is a member of a family of small leucine-rich repeat proteoglycans. Decorin is an approximately lOOkDa proteoglycan consisting of a 40kDa core protein and one chondroitin sulfate or dermatan sulfate glycosaminoglycan chain. Decorin interacts with collagen Type I and II, fibronectin, thrombospondin and TGFP. Decorin functions as a tissue stabilizer and organizer. Decorin is a horseshoe shaped proteoglycan that binds to collagen fibrils in human cornea forming a bidentate ligand attached to two neighboring collagen molecules in the fibril or in adjacent fibrils, helping to stabilize fibrils and orient fibrillogenesis (Scott, JE, Biochemistry, 35: 8795, 1996). Decorin appears to be a ubiquitous component of extracellular matrices linking collagen fibrils at specific binding sites (Scott, JE, et.al, Exp. Cell Res., 243 : 59-66, 1998). Decorin also has a number of biological/physiological characteristics including; inhibition or induction of angiogenesis (Grant, et.al, Oncogene, 21/ 4765-4777, 2002; Sulochana, et.al, J. Biol. Chem. 280:27935-27948, 2005). Indeed, depending on the microenvironment, decorin can exert both pro or anti-angiogenic effects. Accordingly, use of decorin for inhibiting angiogenesis have been suggested in the prior art to be suitable for the treatment of AMD (e.g. W02005116066 and WO2011069046). In particular,
W02011069046 disclosed that intravitreal injection of decorin could be suitable for the treatment of AMD. However the teaching has based on prophetic examples and the documents fails to disclose that intravitreal injection of decorin would be suitable for the treatment of choroidal neovascularisation.
SUMMARY OF THE INVENTION:
The present invention relates to the intravitreal delivery of a decorin polypeptide for the treatment of choroidal neovascularisation (CNV), in particular CNV resistant to anti- VEGF treatment. In particular, the present invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION:
Despite the anatomical separation of choroid from vitreous, the inventors surprisingly found that intravitreal of decorin is suitable for inhibiting choroidal neovascularisation. Accordingly, the first object of the present invention relates to a method of treating choroidal neovascularization in a subject in need thereof comprising delivering a therapeutically effective amount of a decorin polypeptide in the vitreous of the eye.
As used herein, the term "choroidal neovascularization" or“CNV” has its general meaning in the art and refers to new blood vessel growth from the choroid that extends into the subretinal pigment epithelium, or subretinal space, or a combination of both. The standard diagnostic procedures to characterize CNV are typically based on Fluorescein Angiography (FA) and Optical Coherence Tomography (SD-OCT) investigations. With these techniques, different types of CNV are typically defined: type 1 CNV (within the sub-RPE space, typically corresponding to angiographically occult CNV), type 2 CNV (within the subretinal space, typically corresponding to angiographically classic CNV) and type 3 NV (intraretinal retinal angiomatous proliferation). The method of the present invention is particularly suitable for the treatment of type 1 CNV.
As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
Representative diseases involving CNV include age-related macular degeneration, myopic choroidal neovascularization, idiopathic choroidal neovascularization, CNV associated with central serous chorioretinopathy, polypoidal chorioretinopathy associated or not to central serous chorioretinoapthy and some inflammatory conditions, can also induce this pathological condition. In the present invention, diseases involving CNV are not limited to the diseases described above, and also include diseases involving CNV that are caused by other diseases that result in damage at the level of Bruch's membrane and retinal pigment epithelia and subsequent inflammatory choroidal neovascularization, such as uveitis posterior, traumatic choroidal rupture, angioid streaks, and ocular histoplasmosis syndrome.
In some embodiments, the method of the present invention is particularly suitable for the treatment of CNV secondary to age-related macular degeneration (AMD) (also called age- related maculopathy). AMD is a progressive disease of the eye and is associated with choroidal neovascularization. AMD causes progressive damage to the macula, the central and most vital area of the retina, resulting in gradual loss of central vision. It is the most common cause of irreversible loss of central vision in the elderly. It is equally common among men and women.There are two forms of AMD: dry (atrophic) and wet (neo vascular or exudative) AMD. Ninety percent of those with macular degeneration have the dry type. However, 90% of the blindness caused by macular degeneration occurs in the 10% of people who have the wet form.
In some embodiments, the method of the present invention is particularly suitable for the treatment of CNV secondary to pathological myopia. Myopic choroidal neovascularization is indeed the most common disease causing visual impairment in persons with pathologic myopia. When vessels are newly generated in the macular area, pigmented fibrous scars, which result in scotoma in the center of vision, are often formed. Excessive myopia, which is common in the Japanese people, is caused by the abnormal extension of the antero-posterior length of the eye (the eye’s axis). As a result, various myopia-specific eyeground lesions, such as CNV, are developed at the posterior pole of eyeground, which can cause visual disorders.
In some embodiments, the method of the present invention is particularly suitable for the treatment of idiopathic choroidal neovascularisation. Idiopathic choroidal neovascularization often develops in one eye in young women, and can be diagnosed when uveitis, injury, collagen disease, infection, and the like can be negated. Tiny newly generated vascular tissues, hemorrhages and exudative changes, such as edema, may be found under the retina. The involvement of inflammation has been suggested in this disease, since it eases after a few months of treatment with anti-inflammatory steroids.
In some embodiments, the method of the present invention is particularly suitable for the treatment of polypoidal choroidal vasculopathy (PCV). PCV is a form of choroidal vasculoathy classified as a specific form of choroidal neovascularization but which genetic favouringfactors are different from classical AMD and which present as aneurysmal dilations and leaky branching vessels and which has a more important resistance to classical anti VEGF treatments. PCV can be associated or not to central serous chorioretinopathy.
As used herein, the term“decorin” or“DCN” has its general meaning in the art and refers to a member of a family of small leucine-rich repeat proteoglycans. Decorin is an approximately lOOkDa proteoglycan consisting of a 40kDa core protein and one chondroitin sulfate or dermatan sulfate glycosaminoglycan chain. A human exemplary amino acid sequence is represented by SEQ ID NO: 1.
SEQ ID NO : 1 >sp | P07585 | PGS2_HUMAN Decorin OS=Homo sapiens OX=9606
GN=DCN PE=1 SV=1
MKATI ILLLLAQVSWAGPFQQRGLFDFMLEDEASGIGPEVPDDRDFEPSLGPVCPFRCQC HLRWQCSDLGLDKVPKDLPPDTTLLDLQNNKITEIKDGDFKNLKNLHALILVNNKISKV SPGAFTPLVKLERLYLSKNQLKELPEKMPKTLQELRAHENEITKVRKVTFNGLNQMIVIE LGTNPLKSSGIENGAFQGMKKLSYIRIADTNITSIPQGLPPSLTELHLDGNKISRVDAAS LKGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLDNNKLTRVPGGLAEHKYIQWYLH NNNISWGSSDFCPPGHNTKKASYSGVSLFSNPVQYWEIQPSTFRCVYVRSAIQLGNYK
In some embodiments, the decorin polypeptide comprises an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: l . According to the invention a first amino acid sequence having at least 70% identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the second amino acid sequence. Amino acid sequence identity is typically determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990).
According to the invention, the decorin polypeptide of the invention is produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art. The decorin polypeptides of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols as described in Stewart and Young; Tam et al, 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. The decorin polypeptides of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a Model 433A from Applied Biosystems Inc. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art. As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides. A variety of expression vector/host systems may be utilized to contain and express the peptide or protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al, 1999); insect cell systems infected with virus expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al., 2000); or animal cell systems. Those of skill in the art are aware of various techniques for optimizing mammalian expression of proteins, see e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the peptide substrates or fusion polypeptides in bacteria, yeast and other invertebrates are known to those of skill in the art and a briefly described herein below. Mammalian host systems for the expression of recombinant proteins also are well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein. In some embodiments, it is contemplated that the decorin polypeptides of the invention used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase half-life, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. A strategy for improving drug viability is the utilization of macro molecules such as colloids or water-soluble polymers. Various water- soluble polymers have been shown to modify bio distribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel bio materials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
In some embodiments, the delivery of the decorin polypeptide in the vitreous is performed by the direct injection of the decorin polypeptide in the vitreous.
By a "therapeutically effective amount" is meant a sufficient amount of decorin polypeptide for the treatment of CNV at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. Treatment of the patient is usually not a single event. Rather, the decorin polypeptide will likely be injected on multiple occasions, that may be, depending on the results obtained, several days apart, several weeks apart, or several months apart, or even several years apart. Indeed, chronically intravitreal injections of the decorin polypeptide may be needed to reach a therapeutic effect in a long term manner.
In some embodiments, the decorin polypeptide is delivered in the vitreous in combination with an anti-VEGF agent.
As used herein an "anti-VEGF agent" refers to a molecule that inhibits VEGF -mediated angiogenesis. For example, an anti-VEGF therapeutic may be an antibody to or other antagonist of VEGF. An "anti-VEGF antibody" is an antibody that binds to VEGF with sufficient affinity and specificity to be useful in a method of the invention. An anti-VEGF antibody will usually not bind to other VEGF homologues such as VEGF- B or VEGF-C, or other growth factors such as P1GF, PDGF or bFGF. A preferred anti- VEGF antibody is a monoclonal antibody that binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC® HB 10709 and is a high-affinity anti-VEGF antibody. A "high-affinity anti- VEGF antibody" has at least lO-fold better affinity for VEGF than the monoclonal anti-VEGF antibody A4.6.1. Preferably the anti-VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody fragment generated according to WO 98/45331, including an antibody comprising the CDRs or the variable regions of Y0317. More preferably, anti-VEGF antibody is the antibody fragment known as ranibizumab (FUCENTIS ®). The anti-VEGF antibody ranibizumab is a humanized, affinity-matured anti-human VEGF Fab fragment. Ranibizumab is produced by standard recombinant technology methods in E. coli expression vector and bacterial fermentation. Ranibizumab is not glycosylated and has a molecular mass of -48,000 daltons. See W098/45331 and U.S. 2003/0190317. Anti-VEGF agents include but are not limited to bevacizumab (rhuMab VEGF, Avastin®, Genentech, South San Francisco Calif.), ranibizumab (rhuFAb V2, Fucentis®, Genentech), pegaptanib (Macugen®, Eyetech Pharmaceuticals, New York N.Y.), sunitinib maleate (Sutent®, Pfizer, Groton Conn.). In some embodiments, the anti-VEGF agent is a dimeric fusion protein capable of binding VEGF with a high affinity composed of two receptor-Fc fusion protein consisting of the , principal ligand binding portions of the human VEGFR1 or VEGFR2 receptor extracellular domains fused to the Fc portion of human IgGI (termed a "VEGF trap"). Specifically, the VEGF trap consists of Ig domain 2 from VEGFR1 , which is fused to Ig domain 3 from VEGFR2, which in turn is fused to the Fc domain of IgGI.
In some embodiments, the method of the present invention is particularly suitable for the treatment of a subject refractory to an anti-VEGF treatment.
As used herein, the expression "subject refractory to an anti-VEGF treatment" applies to a subject who is non responder to the treatment with an anti-VEGF agent. By "non responder", it is meant that subject does not recover, ameliorate, or stabilize his condition with the anti-VEGF agent. For example, a subject refractory to anti-VEGF treatment is a subject which has been unsuccessfully treated with an anti-VEGF agent or a subject known to be unable to successfully respond to a treatment based on an anti-VEGF agent.
In some embodiments, the method of the present invention is particularly suitable for the treatment of type 1 CNV in a subject refractory to an anti-VEGF treatment.
In some embodiments, the method of the present invention is particularly suitable for the treatment of CNV associated with central serous chorioretinopathy in a subject refractory to an anti-VEGF treatment.
In some embodiments, the decorin polypeptide is administered in the form a pharmaceutical composition compatible with an intravitreal injection. Typically, the pharmaceutical compositions of the present invention include a pharmaceutically or physiologically acceptable carrier such as saline, sodium phosphate, etc., although this need not always be the case. Suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphates, alginate, tragacanth, gelatin, calcium silicate, micro crystalline cellulose, polyvinylpyrrolidone, celluose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, mineral oil, etc. The formulations can also include lubricating agents, wetting agents, emulsifying agents, preservatives, buffering agents, etc.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1. Effects of anti-angiogenic decorin (DCN) protein in CNV. (A) On western- blot, the DCN level decreases significantly in the rat retinal pigment epithelium (RPE)-choroid at various time points (day 3 and 7) after laser induction compared to the normal rat RPE- choroid (ctrl) and slowly recovered to normal levels at day 15. (B) Intravitreal injection (IVT) of recombinant DCN protein in rat eyes inhibits choroidal vascular leakage on fluorescein angiography (FA); DCN 10 pg/mF significantly reduces the CNV angiographic score, whereas DCN at both 1 pg/mF and 10 pg/mL both decrease the size of CNV induced by laser. Data are expressed as mean ± SEM. Non parametric Kruskal- Wallis test was used. *, P<0.05.
Figure 2: Kinetics of DCN protein expression in the rat RPE-choroid after CNV induction. Quantification of Western blots showed that DCN was reduced significantly at D3 and D7 after laser compared to DO (non-laser treated controls). DCN protein level was then recovered from D7 to D10. Data are expressed as relative intensity compared to actin. n=5-8 eyes.
Figure 3: Effects of decorin and iron in CNV. . Quantification showed that mouse photoreceptor cells were reduced significantly when exposed to iron. A significant rescue of photoreceptor cells was observed when decorin was added.
EXAMPLE:
Methods
Laser-induced CNV
After anesthesia and dilation of the pupils, coverslips were positioned on the cornea as a contact glass. For rats, six to eight bums were performed 2 to 3 optic disc diameters away from the optic nerve with an Argon laser (532 nm) mounted on a slit lamp (l75mW, 0.1 s and 50 pm). For mice, four laser bums were induced at the 3, 6, 9 and 12 o’clock positions around the optic disc (250 mW, 0.05 s and 50 pm). The presence of a bubble witnessed the rupture of Bruch’s membrane and confirmed a successful laser impact.
Intravitreal injection (IVT) of recombinant DCN protein
We used recombinant mouse DCN protein (R&D Systems) that shares 87% amino-acid sequence identity with rat DCN. IVT of DCN was performed after laser induction at a final concentration of 1 pg/mF or 10 pg/mF in the rat vitreous. FA was performed at day 14, and CNV quantification on choroidal flat-mounts at day 16.
Fluorescein angiography (FA)
FA was performed 14 days (in rats) or 10 days (in mice) after laser induction. After pupils dilatation, fluorescein (0.2 mL of 10% fluorescein in saline) was injected intravenously in the tail of rats, or intraperitoneal (0.1 ml) in mice. Early and late phase angiograms were recorded 1-3 and 5-7 min respectively after fluorescein injection. For each laser- induced lesion, fluorescein leakage was graded qualitatively by evaluating the increase in size/intensity of dye between the early and late phases. Angiographic scores were established by 2blinded observers according to the following criteria: grade 0, no hyperfluorescence; grade 1, slight hyperfluorescence with no increase in intensity nor in size; grade 2, hyperfluorescence increasing in intensity but not in size; grade 3, hyperfluorescence increasing both in intensity and size; grade 4, hyperfluorescence size increase more than 2-diameter of the initial laser bum.
RPE-choroid flat-mounts and CNV quantifications
Two days after FA examination (time necessary for fluorescein elimination), eyes were enucleated, fixed in 4% PFA for 15 min at room temperature and sectioned at the limbus; the cornea and lens were discarded. The retina was separated from the RPE-choroid complex. Eight radial incisions were made on the RPE-choroid, which was then flat-mounted and post-fixed with acetone for 15 min at -20°C. After washing with 0.1% Triton xlOO in PBS, FITC-GSL I- Isolectin B4 (1 :200, Vector, AbCys, Paris, France) was applied over night at -4°C. After washing with PBS, the RPE-choroid was flat-mounted and observed with a confocal microscope (Zeiss LSM710, Le Pecq, France). Images of the CNV were captured with a digital video camera coupled to a computer system. Horizontal optical sections (at 1 pm intervals) were obtained from the CNV surface. The deepest focal plane in which the surrounding choroidal vascular network connecting to the lesion could be identified was judged to be the floor of the CNV lesion. The area of CNV-related fluorescence on each horizontal section was measured using the ImageJ software. The summation of the entire fluorescent area on z-stack images from the top to the bottom of the CNV was used as an index for the CNV volume.
Mice were perfused with FITC-dextran (molecular weight 2,000,000, Sigma-Aldrich, St-Quentin Fallavier, France) before enucleation. After RPE-choroidal flat-mounting, the FITC-dextran perfused CNV was examined and analyzed as previously described.
Decorin and iron
Mouse photoreceptor cell line were exposed to 100mM Fe3+ that is toxic for the photoreceptor cells.
Results
In the rat CNV model, DCN protein expression was reduced in the RPE-choroid as early as 3 days after CNV induction (Figure 1A and Figure 2). The DCN levels partly recovered from day 7 to day 10, but remained below the level in the RPE-choroid of control eyes (Figure 1A and Figure 2), suggesting that decreased DCN might contribute to the pro-angiogenic balance in this CNV model. To analyze the anti-angiogenic effect of DCN in CNV, we used a recombinant mouse DCN protein that shares 87% amino-acid sequence identity with rat DCN. Intravitreous injection of recombinant mDCN was performed after laser induction at an estimated final concentration of 1 pg/ml or 10 pg/ml in rat vitreous. At day 14, eyes treated with recombinant mDCN at 10 lig/ml showed reduced choroidal vascular leakage (Figure IB). The CNV volume was decreased significantly by DCN at bothl lig/ml and 10 pg/ml (Figure IB), confirming a major role of DCN in inhibiting CNV proliferation and leakage.
When cells lines were exposed to iron, at 24 hours, about 80% of cells survive. When decorin lpg/ml was added to the iron, a significant rescue of photoreceptor cells was observed demonstrating that decorin is neuroprotective against iron-mediated oxidative stress in photoreceptor cells (Figure 3).
Conclusions:
Anti-VEGF therapy has revolutionized the visual prognosis of CNV, in particular in nAMD patients, from rapid progression to central blindness to central vision stabilized or even improved for several years1, demonstrating the key role of VEGF in the regulation of hydro - ionic retinal homeostasis and vascular permeability2. However not all nAMD patients respond well to anti- VEGF treatment, particularly when CNV develops underneath the detached retinal pigment epithelium, which represents more than 50% of CNV in AMD cases (Type 1 CNV)3,4and complicates 30-40% of chronic CSCR cases. Moreover, in around 50% of nAMD patients, depending on the drug and regimens, fluid retention persists in the macula despite optimal anti- VEGF treatments5. Anti-platelet-derived-growth-factor -(PDGF) therapies failed to demonstrate synergistic effects with anti-VEGFs in 2 phase-3 clinical trials6, and no other therapeutic options are currently available for nAMD patients not responding optimally to anti- VEGFs. No animal model recapitulates all nAMD features, and the common laboratory animals have no macula. However laser- induced CNV models in rodents and primate are validated in screening anti-angiogenic drugs for nAMD40. Here, we showed an anti-angiogenic effect of DCN on CNV. Importantly, laser-induced CNV decreased DCN expression in the RPE- Choroid.
In the healthy human eye, DCN is strongly expressed in the cornea, where it intervenes in fibrillogenesis and tissue repair7. In the retina, DCN has been located in all layers but was particularly abundant in Bruch’s membrane, which separates the RPE from the choroid, and in the choroid itself8. As a component of the extracellular matrix, DCN can modulate angiogenesis through interaction with numerous molecules9. In corneal wound healing models and in inflammatory angiogenesis, DCN is anti-angiogenic10,11. This effect results from the sequestration of growth factors such as TGF-b, an antagonism of tyrosine kinase family receptors including the VEGF-R29,12,13, and stimulation of angiostatic molecules such as thrombospondin and tissue inhibitor of metalloproteinase 3 (TIMP3)14. Under hypoxic conditions, DCN reduced choroidal endothelial cell proliferation induced by the RPE in culture15. In addition, DCN exerts an anti-inflammatory effect through a decrease in macrophage proliferation16 and recruitment, in turn mediated by MCP-l downregulation17. DCN expression in relation to AMD has not been studied specifically; however, a quantitative proteomic analysis of Bruch’s membrane/choroid from AMD eyes showed significantly decreased DCN levels compared to those in healthy controls18. In addition, DCN expression was reduced by approximately 20% in advanced wet as compared to advanced dry AMD eyes18, supporting a role for DCN in wet AMD.
Moreover, as choroidal neovascularization is associated with bleeding and iron- mediated toxicity for photoreceptor cells, the experiment suggests that decorin and ion addition reduce choroidal neovascularization and protect photoreceptor cells from death.
REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
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