Methods of associating genetic variants with clinical outcome in patients suffering from age-related macular degeneration treated with Anti-vegf

Abstract

To provide a pharmaceutical composition for treating macular degeneration with neovascularization.SOLUTION: A pharmaceutical composition contains Aflibercept and is used for treating age-related macular degeneration with neovascularization in a subject having one or more single nucleotide polymorphisms selected from the group consisting of rs2056688, rs5962084, rs5962087, rs5915722, rs5962095, rs1405303, rs2106124, rs1879796, rs12148845, rs12148100, rs17482885, and rs17629019.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 262,589, filed on Dec. 3, 2015, and U.S. Provisional Patent Application No. 60 / 291,274, filed on Feb. 4, 2016, and the entire contents of both are hereby incorporated by reference herein.

Background Art

[0002] Macular degeneration is a serious medical condition where fluid accumulates in the retina, damaging the retina and potentially leading to loss of vision at the center of the visual field. Macular degeneration can be age - related. “Dry” (non - exudative) and “wet” (“neovascular” or “exudative”) forms of macular degeneration are recognized.

[0003] In neovascular age - related macular degeneration, vision loss can result from abnormal blood vessel growth (choroidal neovascularization). The growth of abnormal blood vessels in the retina is stimulated by vascular endothelial growth factor (VEGF). The new blood vessels are fragile and can lead to leakage of blood and protein under the macula. Hemorrhage, leakage, and scarring due to these blood vessels can ultimately cause irreversible damage to photoreceptors and rapid vision loss.

[0004] Eylea® (aflibercept) injection and Lucentis® (ranibizumab) are biopharmaceuticals approved in the United States and Europe for the treatment of wet - type macular degeneration. Aflibercept and ranibizumab are VEGF inhibitors.

Summary of the Invention

[0005] Disclosed herein is a method of associating a genetic variant with visual acuity, anatomical outcome, or treatment frequency, the method comprising: (i) determining one or more genetic variants in a population of neovascular age - related macular degeneration subjects administered aflibercept or ranibizumab; and (ii) statistically associating the one or more genetic variants with the anatomical outcome in the population of neovascular age - related macular degeneration subjects.

[0006] Disclosed herein is a method of associating a genetic variant with visual acuity, anatomical outcome, or treatment frequency, the method comprising: (a) statistically associating (i) one or more genetic variants of a population of subjects with neovascular age-related macular degeneration who have been administered an intravitreal anti-VEGF agent, with (ii) the anatomical outcome of the population of subjects with neovascular age-related macular degeneration, wherein the one or more genetic variants are associated with the presence of intraretinal cystoid edema (fluid) as compared to the absence of intraretinal cystoid edema (fluid) after one year of treatment.

[0007] Disclosed herein is a method of associating a genetic variant with intraretinal fluid, the method comprising: (a) statistically associating one or more genetic variants of a population of subjects with neovascular age-related macular degeneration with (b) the intraretinal fluid of the population of subjects with neovascular age-related macular degeneration, wherein the one or more genetic variants are associated with a lower level of intraretinal fluid in subjects with neovascular age-related macular degeneration who have been treated with an intravitreal anti-VEGF agent and have one or two copies of the genetic variant allele as compared to the level of intraretinal fluid in subjects with neovascular age-related macular degeneration who have been treated with an intravitreal anti-VEGF agent and do not have a copy of the genetic variant allele.

[0008] The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate some embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0009]

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Mode for Carrying Out the Invention

[0010] Detailed Description The disclosed methods and compositions can be more readily understood by reference to the following detailed description of specific embodiments and the examples contained therein, as well as the drawings and their above and below descriptions.

[0011] It should be understood that, unless otherwise specified, the disclosed methods and compositions are not limited to specific synthesis methods, specific analysis techniques or specific reagents, and can therefore vary. It should also be understood that the technical terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0012] It is understood that the disclosed methods and compositions are not limited to the specific methodologies, protocols, and reagents described, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the appended claims.

[0013] It is never intended, except where expressly so stated, that any method or aspect recited herein be construed as requiring that its steps be performed in a particular order. Accordingly, where a claim reciting a method does not specifically recite in the claim or description that the steps are to be limited to a specific order, no order will ever be implied in any respect. This holds for any possible non-explicit interpretive criteria, including logical matters regarding the ordering of steps or operations flows, simple implications arising from grammatical structure or punctuation, or the number or type of aspects recited in the specification.

[0014] Disclosed are materials, compositions, and components that can be used for, can be used with, can be provided with, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and where combinations, subsets, interactions, groups, etc. of these materials are disclosed, each and every combination and permutation of these complexes, both individually and collectively, is specifically contemplated and described herein even if specific mention of each is not explicitly disclosed. For example, if there is a disclosure and description of a PRR antagonist and a description of some modifications that can be made, every combination and permutation of the PRR antagonist and possible modifications is specifically contemplated unless a contrary specific indication is made. Thus, if a class of molecules A, B, and C is disclosed along with a class of molecules D, E, and F and a combination molecule A-D as an example, each is specifically contemplated both individually and collectively even if each is not individually recited. Thus, in this example, the disclosure of A, B, and C; D, E, and F; and the combination example A-D should be considered to specifically contemplate and disclose each combination A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F. Similarly, any subset or combination of these is specifically contemplated and disclosed. Thus, for example, the disclosure of A, B, and C; D, E, and F; and the combination example A-D should be considered to specifically contemplate and disclose the subgroups A-E, B-F, and C-E. This concept applies to every aspect of this application, including but not limited to the steps of methods of making and using the compositions of the present disclosure. Thus, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered to be disclosed.

[0015] Definitions As used in this specification and the appended claims, the singular forms "a," "an," and "the" include references to the plural unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0016] Throughout this specification and the claims which follow, the term "comprise" and variations of the term, such as "comprising" and "comprises," are to be interpreted as open-ended terms meaning "including, but not limited to." Thus, for example, a method described as "comprising one or more steps" is specifically contemplated to include other additives, components, integers, or steps not recited at that step, unless the step is specifically described as "consisting of" such terms. That is, each step is specifically contemplated to include what is recited (unless the step is described as "consisting of" such terms) and other additives, components, integers, or steps not recited at that step.

[0017] A range can be expressed in this specification as from "about" a particular value and / or to "about" another particular value. When such a range is recited, another embodiment includes from one particular value and / or to the other particular value. Similarly, it will be understood that when a value is recited with the antecedent "about" it forms another embodiment as the approximation of the particular value. Further, it will be understood that the endpoints of each range recite themselves and have meaning independently of the other endpoint. Also, it will be understood that several values are recited in this specification and that each value is disclosed in this specification as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. Further, as will be appreciated by those of ordinary skill in the art, if a particular value is disclosed then the value "less than", "greater than or equal to" that value, and the possible ranges between values are also disclosed. For example, if the value "10" is disclosed, "less than 10" and "greater than or equal to 10" are also disclosed. Further, throughout this application, it will be understood that the data is provided in several different formats and that these data represent ranges for any combination of endpoints, starting points, and data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, then greater than 10 and greater than 15, greater than or equal to 10 and greater than or equal to 15, less than 10 and less than 15, less than or equal to 10 and less than or equal to 15, as well as equal to 10 and equal to 15, and 10 to 15 are considered to be disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0018] "Optional" or "optionally" means that the subsequently recited event or circumstance may or may not occur, and that the description includes both the case where the event or circumstance occurs and the case where it does not.

[0019] As used herein, the term "or" means any one of a particular recited list of members and further includes any combination of that recited list. As used herein, the term "subject" means an individual. In one aspect, the subject is a mammal such as a human. In one aspect, the subject can be a non-human primate. Non-human primates include, by way of example, marmosets, monkeys, chimpanzees, gorillas, orangutans, and macaques. The term "subject" also includes companion animals such as cats and dogs, livestock (e.g., cows (dairy), horses, pigs, sheep, goats, etc.), laboratory animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.), and birds (e.g., chickens, turkeys, ducks, pheasants, pigeons, doves, parrots, parakeets, geese, etc.). The subject can also include, without limitation, fish (e.g., zebrafish, goldfish, tilapia, salmon, and trout), amphibians, and reptiles. As used herein, "subject" is the same as "patient", and the terms can be used interchangeably.

[0020] The term "polymorphism" refers to the occurrence of one or more alternative sequences or alleles that are genetically determined in a population. A "polymorphic site" is the locus at which the sequence change occurs. A polymorphic site has at least one allele. A biallelic polymorphism has two alleles. A triallelic polymorphism has three alleles. A diploid organism can be homozygous or heterozygous for the trait. A polymorphic site can be as few as one base pair. Examples of polymorphic sites include restriction fragment length polymorphisms (RFLPs), variable number tandem repeats (VNTRs), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, and simple sequence repeats. As used herein, reference to a "polymorphism" can include a set of polymorphisms (i.e., a haplotype).

[0021] A "single nucleotide polymorphism (SNP)" may occur at a polymorphic site occupied by a single nucleotide, which is a variant site between allelic sequences. The site may be preceded and followed by highly conserved sequences of the alleles. An SNP may occur by replacement of one nucleotide by another nucleotide at the polymorphic site. The replacement of one purine by another purine, or one pyrimidine by another pyrimidine, is called a transversion. The replacement of a purine by a pyrimidine, or vice versa, is called a transition. A synonymous SNP refers to the replacement of one nucleotide by another nucleotide in the coding region that does not change the amino acid sequence of the encoded polypeptide. A non-synonymous SNP refers to the replacement of one nucleotide by another nucleotide in the coding region that changes the amino acid sequence of the encoded polypeptide. An SNP may also result from the deletion or insertion of one or more nucleotides relative to the reference allele.

[0022] A "set of" polymorphisms means one or more polymorphisms, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or more than 6 polymorphisms.

[0023] As used herein, "nucleic acid", "polynucleotide" or "oligonucleotide" can be a polymeric form of nucleotides of any length, can be DNA or RNA, and can further be single-stranded or double-stranded. Nucleic acids can contain promoters and other regulatory sequences. Oligonucleotides can be prepared by synthetic means. Nucleic acids contain a portion of DNA or its complement that spans or is adjacent to any one polymorphic site. The portion can be 5 to 100 contiguous bases, and can be in the range having a lower limit of 5, 10, 15, 20 or 25 nucleotides and an upper limit of 10, 15, 20, 25, 30, 50 or 100 nucleotides (the upper limit being greater than the lower limit). Nucleic acids of 5 to 10, 5 to 20, 10 to 20, 12 to 30, 15 to 30, 10 to 50, 20 to 50 or 20 to 100 bases are common. The polymorphic site can occur at any position of the portion. A reference to the sequence of one strand of a double-stranded nucleic acid defines the complementary sequence, and, unless otherwise apparent from the context, a reference to a single-stranded nucleic acid also refers to its complement.

[0024] As used herein, "nucleotide" refers to a molecule that forms the individual structural units of nucleic acids RNA and DNA when linked together. A nucleotide consists of a nucleobase (nitrogenous base), a five-carbon sugar (either ribose or 2-deoxyribose), and one phosphate group. "Nucleic acid" is a high-molecular-weight macromolecule made from nucleotide monomers. In DNA, the purine bases are adenine (A) and guanine (G), and the pyrimidines are thymine (T) and cytosine (C). RNA uses uracil (U) instead of thymine (T).

[0025] As used herein, the terms "gene variant" or "variant" refer to a nucleotide sequence that differs from the most predominant sequence in a population of sequences (e.g., differs by only 1 nucleotide in the case of the SNPs described herein). For example, some mutations or substitutions in a nucleotide sequence change the codon such that a different amino acid is encoded, resulting in a gene variant polypeptide. Other non-limiting examples of gene variants include insertions, deletions, indels, frameshift variants, stop codon variants, synonymous variants, non-synonymous variants, and copy number polymorphisms (e.g., deletions and duplications). The term "gene variant" can also refer to a polypeptide that differs from the most predominant sequence in a population of sequences at a position that does not change the amino acid sequence of the encoded polypeptide (i.e., a conserved change). A gene variant polypeptide can be encoded by a risk haplotype, a protective haplotype, or a neutral haplotype. A gene variant polypeptide can be risk-related, protective-related, or neutral.

[0026] "Isolated nucleic acid" or "purified nucleic acid" means DNA that does not contain genes adjacent to the gene in the naturally occurring genome of the organism from which the DNA of the present invention is derived. Thus, the term includes, for example, that incorporated into a vector such as an autonomously replicating plasmid or virus, or incorporated into the genomic DNA of a prokaryote or eukaryote (e.g., a transgene), or existing as a separate molecule (e.g., a cDNA or genomic fragment or cDNA fragment produced by PCR, restriction enzyme digestion or chemical synthesis or in vitro synthesis), recombinant DNA. It also includes recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence. The term "isolated nucleic acid" further refers to RNA, for example, mRNA molecules that are encoded by an isolated DNA molecule or chemically synthesized or separated from or do not contain at least some cellular components (e.g., other types of RNA or polypeptide molecules).

[0027] As used herein, "being treated" or "treating" refers to the medical management of a patient intended to cure, ameliorate, stabilize or prevent a disease, medical condition or disorder. This term includes active treatment, i.e., treatment specifically directed to the improvement of a disease, medical condition or disorder, and further includes causative treatment, i.e., treatment directed to the removal of the cause of the relevant disease, medical condition or disorder. Further, this term includes palliative treatment, i.e., treatment aimed at alleviating symptoms rather than curing a disease, medical condition or disorder, prophylactic treatment, i.e., treatment directed at minimizing or partially or completely suppressing the progression of a relevant disease, medical condition or disorder, and symptomatic treatment, i.e., treatment employed to supplement another specific therapy directed to the improvement of a relevant disease, medical condition or disorder. In various aspects, the term encompasses any treatment of a subject, including mammals (e.g., humans), and also includes (i) prevention of the occurrence of a disease in a patient who is at risk of developing the disease but has not yet been diagnosed as having it, (ii) suppression of a disease, i.e., prevention of its onset, or (iii) alleviation of a disease, i.e., regression of the disease.

[0028] The terms "administering", "being administered" and "administration" refer to any method of providing a pharmaceutical formulation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, sublingual administration, buccal mucosal administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, otic administration, intracerebral administration, intrathecal administration, rectal administration, intraperitoneal administration and parenteral administration by injection (e.g., intravenous administration, arterial administration, intramuscular administration, intradermal administration and subcutaneous administration). Ophthalmic administration can include intraocular administration, including topical administration, subconjunctival administration, sub-Tenon's capsule administration, episcleral administration, retrobulbar administration, intraorbital administration and intravitreal administration. Administration can be continuous or intermittent. In various aspects, the formulation can be administered therapeutically, i.e., administered to treat an existing disease or condition. In further various aspects, the formulation can be administered prophylactically, i.e., administered to prevent a disease or condition.

[0029] The calculation of array similarity or array identity (the terms are used interchangeably herein) between arrays is performed as follows. To determine the identity rate between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison). In certain embodiments, the length of the reference sequence to be aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. Thereafter, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0030] The identity rate between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap that needs to be introduced for the optimal alignment of the two sequences.

[0031] The comparison of arrays and the determination of the identity rate between two arrays can be accomplished using a mathematical algorithm. In a preferred embodiment, the identity rate between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (1970, J. Mol. Biol. 48:444-453) incorporated in the GAP program within the GCG software package, using the Blossum62 matrix or the PAM250 matrix, as well as gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6. In another preferred embodiment, the identity rate between two nucleotides is determined using the GAP program within the GCG software package, using the NWSgapdna.CMP matrix, as well as gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6. A particularly preferred (and to be used unless otherwise specified) set of parameters is the Blossum62 score matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0032] The identity rate between two amino acid sequences or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4:11-17) incorporated in the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0033] Unless otherwise defined, all scientific and technical terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of the present invention, particularly useful methods, devices, and materials are described. Publications and materials cited herein are specifically incorporated herein by reference. Nothing in this specification should be construed as an admission that the present invention has no right to antedate such disclosure by virtue of prior invention. The citation of any reference is not an admission that it constitutes prior art. The description of any reference is for the purpose of stating what the author has asserted, and the applicants reserve the right to challenge the accuracy and appropriateness of the cited documents. Although many publications are referred to herein, it will be clearly understood that such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0034] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

[0035] Method A method for associating a gene variant with visual acuity, anatomical outcome, or treatment frequency, the method comprising: (a) (i) statistically associating one or more gene variants of a population of neovascular age-related macular degeneration (AMD) subjects who have received an intravitreal anti-VEGF agent with (ii) the anatomical outcome of the population of neovascular AMD subjects, wherein the one or more gene variants are associated with the presence of intraretinal cystoid edema (fluid) compared to the absence of intraretinal cystoid edema (fluid) after one year of treatment.

[0036] Examples of anti-VEGF agents or intravitreal anti-VEGF agents include, but are not limited to, bevacizumab, ranibizumab, ramucirumab, aflibercept, sunitinib, sorafenib, vandetanib, batranib, tivozanib, axitinib, imatinib or pazopanib.

[0037] Disclosed herein is a method of associating a genetic variant with visual acuity, anatomical outcome or treatment frequency, the method comprising comparing the anatomical outcome of a population of neovascular age-related macular degeneration (AMD) subjects who have received an intravitreal anti-VEGF agent and have one or two copies of a genetic variant allele, with the anatomical outcome of a population of neovascular AMD subjects who have received an intravitreal anti-VEGF agent and do not have a copy of the genetic variant allele, and (i) statistically associating one or more genetic variants of a population of neovascular AMD subjects who have received an intravitreal anti-VEGF agent, with (ii) the anatomical outcome of said population of neovascular AMD subjects.

[0038] Disclosed herein is a method of associating a genetic variant with visual acuity, anatomical outcome or treatment frequency, the method comprising (a) (i) statistically associating one or more genetic variants of a population of neovascular AMD subjects who have received an intravitreal anti-VEGF agent for one year, with (ii) the anatomical outcome of said population of neovascular AMD subjects, wherein the one or more genetic variants are associated with the presence of intraretinal cystoid edema (fluid) in subjects having one or two copies of a genetic variant allele, as compared to the level of intraretinal cystoid edema (fluid) in patients having no copy of the genetic variant allele.

[0039] A method of associating a genetic variant with vision, anatomical outcome, or treatment frequency, the method comprising comparing the anatomical outcomes of a population of neovascular age-related macular degeneration (nAMD) subjects who have received an intravitreal anti-VEGF agent and have one or two copies of a genetic variant allele with the anatomical outcomes of a population of nAMD subjects who have received an intravitreal anti-VEGF agent and do not have copies of the genetic variant allele, and (i) statistically associating one or more genetic variants of a population of nAMD subjects who have received an intravitreal anti-VEGF agent with (ii) the anatomical outcomes of said population of nAMD subjects, wherein genotyping of DNA samples from the subjects is performed prior to the step of statistical association. In some embodiments, the anatomical outcome is an increase of 15 letters (vision). In some embodiments, the treatment frequency can reflect the continued need for active treatment with an intravitreal anti-VEGF agent after annual dosing.

[0040] The statistical associations described herein can include logistic regression analysis, QC of genetic data including Hardy-Weinberg equilibrium testing, identity by state (IBS) assessment, and / or sex confirmation. Population structure can be evaluated using principal component analysis (PCA). The statistical association can include logistic regression using baseline values and any potential population structure variables as covariates of the model.

[0041] In some embodiments, the anatomical outcome is the presence of intraretinal cystoid edema, improvement in vision / visual acuity, or reduction of intraretinal fluid. Further anatomical outcomes that can be used include, but are not limited to, for example, a reduction in central retinal thickness measured by optical coherence tomography (OCT), complete dissipation of both intraretinal and subretinal fluid, reduction in the choroidal neovascularization (CNV) area, reduction in the overall size of the neovascular lesion measured by fluorescein angiography, and reduction of subretinal hyperreflective (SHM) material measured by OCT.

[0042] In some embodiments, the statistical relevance can be measured as a p-value. For example, different types of p-values can be obtained, namely, the simple t-test p-values for the raw data and the log-transformed data assuming equal variances on both sides, and the Chebyshev checker p-values. These p-values can be represented not only on an individual basis but also by taking into account multiple comparisons. The mix-o-matic method can be applied to provide further information about these p-values. In some embodiments, the p-value of the relevance is 1×10 -5 or less, 1×10 -6 or less, 1×10 -7 or less, 1×10 -8 or less, etc. In some embodiments, the p-value of the relevance is 1×10 -5 i.e., suggestive statistical significance and 1×10 -8 i.e., equal to or less than the experimental statistical significance.

[0043] In some embodiments, the magnitude of the effect of the statistical relevance can be measured as an odds ratio. For example, the magnitude of the effect of the statistical relevance can be measured as the ratio of the odds of the presence of intraretinal cystoid edema (fluid) in neovascular age-related macular degeneration subjects treated with intravitreal anti-VEGF agents and having no copies of the allele to the ratio of the odds of the presence of intraretinal cystoid edema (fluid) in neovascular age-related macular degeneration subjects treated with intravitreal anti-VEGF agents and having one or two copies of the allele. In some embodiments, the odds ratio is 0.2 or less, 0.3 or less, 0.4 or less, 0.5 or less, 0.6 or less, 0.7 or less, 0.8 or less, or 0.9 or less. Having one copy of the allele will have a smaller effect than an individual having two copies of the allele.

[0044] In some embodiments, the statistical relevance can be measured as the ratio of the odds of a 15-letter (visual acuity) increase in neovascular age-related macular degeneration subjects treated with intravitreal anti-VEGF agents and having no copies of the allele, to the odds of a 15-letter increase in neovascular age-related macular degeneration subjects treated with intravitreal anti-VEGF agents and having one or two copies of the allele. In some embodiments, the odds ratio is 2.4 or greater, 2.5 or greater, 2.6 or greater, 2.7 or greater, 2.8 or greater, or 2.9 or greater.

[0045] In some embodiments, the statistical relevance can be measured as the ratio of the odds of neovascular age-related macular degeneration subjects treated with intravitreal anti-VEGF agents and having no copies of the allele, who have a lower need for continued aggressive treatment with intravitreal anti-VEGF agents, to the odds of neovascular age-related macular degeneration subjects treated with intravitreal anti-VEGF agents and having one or two copies of the allele, who have a higher need for continued aggressive treatment with intravitreal anti-VEGF agents. In some embodiments, the odds ratio is 4.0 or less, 3.9 or less, 3.8 or less, 3.7 or less, 3.6 or less, 3.5 or less, 3.4 or less, 3.3 or less, or 3.2 or less.

[0046] In some embodiments, the method can be used to associate a gene variant with visual acuity, anatomical outcome, or treatment frequency. In some embodiments, the gene variant can be one or more single nucleotide polymorphisms.

[0047] Disclosed herein is a method of associating a gene variant with visual acuity, anatomical outcome, or treatment frequency, the method comprising: (a) (i) statistically associating one or more gene variants of a population of neovascular age-related macular degeneration subjects administered an intravitreal anti-VEGF agent, with (ii) the anatomical outcome of the population of neovascular age-related macular degeneration subjects, wherein the one or more gene variants are associated with a decrease in the level of presence of intraretinal cystoid edema (fluid) after one year of treatment.

[0048] A method of associating a gene variant with visual acuity, anatomical outcome, or treatment frequency, the method comprising: (a) (i) statistically associating one or more gene variants of a population of neovascular age-related macular degeneration (AMD) subjects who have received an intravitreal anti-VEGF agent, with (ii) the anatomical outcomes of the population of neovascular AMD subjects, wherein the one or more gene variants are associated with a decrease in the level of intraretinal fluid in subjects having one or two copies of the gene variant allele, compared to the level of intraretinal fluid in neovascular AMD subjects who have received an intravitreal anti-VEGF agent and do not have a copy of the gene variant allele.

[0049] A method of associating a gene variant with visual acuity, anatomical outcome, or treatment frequency, the method comprising: (a) (i) statistically associating one or more gene variants of a population of neovascular age-related macular degeneration (AMD) subjects who have received an intravitreal anti-VEGF agent, with (ii) the anatomical outcomes of the population of neovascular AMD subjects, wherein the one or more gene variants are associated with the presence of intraretinal cystoid edema (fluid), compared to the absence of intraretinal cystoid edema (fluid) after one year of treatment.

[0050] A method for associating a genetic variant with visual acuity, anatomical outcome, or treatment frequency, comprising: (a) (i) statistically associating one or more genetic variants of a population of neovascular age-related macular degeneration (AMD) subjects administered an intravitreal anti-VEGF agent with (ii) the anatomical outcome of the above population of neovascular AMD subjects, wherein the one or more genetic variants are associated with a decrease in intraretinal fluid as compared to the level of intraretinal fluid in neovascular AMD subjects not treated with an intravitreal anti-VEGF agent, and the genetic variant is a single nucleotide polymorphism selected from the group consisting of rs2056688, rs5962084, rs5962087, rs5915722, and rs5962095. In some embodiments, the genetic variant is a single nucleotide polymorphism selected from the group consisting of rs2056688, rs5962084, rs5962087, rs5915722, rs5962095, rs2106124, rs1879796, rs12148845, rs12148100, rs17482885, and rs17629019.

[0051] A method for associating a genetic variant with visual acuity, anatomical outcome, or treatment frequency, comprising: (a) (i) statistically associating one or more genetic variants of a population of neovascular age-related macular degeneration (AMD) subjects administered an intravitreal anti-VEGF agent with (ii) the anatomical outcome of the above population of neovascular AMD subjects, wherein the one or more genetic variants are associated with the presence of intraretinal cystoid edema (fluid) as compared to the absence of intraretinal cystoid edema (fluid) after one year of treatment.

[0052] A method for associating a genetic variant with intraretinal fluid, comprising: (a) statistically associating one or more genetic variants of a population of subjects with neovascular age-related macular degeneration (AMD) with (b) the intraretinal fluid of said population of subjects with neovascular AMD, wherein the one or more genetic variants are associated with a decrease in intraretinal fluid in subjects with neovascular AMD treated with intravitreal anti-VEGF agents compared to the level of intraretinal fluid in subjects with neovascular AMD not treated with intravitreal anti-VEGF agents, and the decrease in intraretinal fluid is an improvement in visual acuity in subjects with neovascular AMD treated with intravitreal anti-VEGF agents compared to the level of intraretinal fluid in subjects with neovascular AMD not treated with intravitreal anti-VEGF agents. The method is disclosed.

[0053] A method for associating a genetic variant with visual acuity, anatomical outcome, or treatment frequency, comprising: (a) statistically associating (i) one or more genetic variants of a population of subjects with neovascular age-related macular degeneration (AMD) who have received intravitreal anti-VEGF agents with (ii) the anatomical outcome of said population of subjects with neovascular AMD, wherein the one or more genetic variants are associated with the presence of intraretinal cystoid edema (fluid) compared to the absence of intraretinal cystoid edema (fluid) after one year of treatment. The method is disclosed.

[0054] A method for associating a genetic variant with intraretinal fluid, comprising: (a) statistically associating one or more genetic variants of a population of subjects with neovascular age-related macular degeneration (AMD) with (b) the intraretinal fluid of said population of subjects with neovascular AMD, wherein the one or more genetic variants are associated with a decrease in intraretinal fluid in subjects with neovascular AMD treated with intravitreal anti-VEGF agents compared to the level of intraretinal fluid in subjects with neovascular AMD not treated with intravitreal anti-VEGF agents, and the p-value of the association is 1×10 -6 The following. The method is disclosed.

[0055] A method of associating a gene variant with visual acuity, anatomical outcome, or treatment frequency, the method comprising: (a) (i) statistically associating one or more gene variants of a population of neovascular age-related macular degeneration subjects administered an intravitreal anti-VEGF agent with (ii) the anatomical outcome of the above population of neovascular age-related macular degeneration subjects, wherein the one or more gene variants are associated with the presence of intraretinal cystoid edema (fluid) compared to the absence of intraretinal cystoid edema (fluid) after one year of treatment. The method is disclosed.

[0056] A method of associating a gene variant with intraretinal fluid, the method comprising: (a) statistically associating one or more gene variants of a population of neovascular age-related macular degeneration subjects with (b) the intraretinal fluid of the above population of neovascular age-related macular degeneration subjects, wherein the one or more gene variants are associated with a decrease in intraretinal fluid in neovascular age-related macular degeneration subjects treated with an intravitreal anti-VEGF agent compared to the level of intraretinal fluid in neovascular age-related macular degeneration subjects not treated with an intravitreal anti-VEGF agent, and the odds ratio of the decrease in intraretinal fluid in neovascular age-related macular degeneration subjects treated with an intravitreal anti-VEGF agent to the decrease in intraretinal fluid in neovascular age-related macular degeneration subjects not treated with an intravitreal anti-VEGF agent is 0.5 or less. The method is disclosed.

[0057] A method of associating a gene variant with visual acuity, anatomical outcome, or treatment frequency, the method comprising: (a) (i) statistically associating one or more gene variants of a population of neovascular age-related macular degeneration subjects administered an intravitreal anti-VEGF agent with (ii) the anatomical outcome of the above population of neovascular age-related macular degeneration subjects, wherein the one or more gene variants are associated with the presence of intraretinal cystoid edema (fluid) compared to the absence of intraretinal cystoid edema (fluid) after one year of treatment. The method is disclosed.

[0058] A method for associating a genetic variant with intraretinal fluid, comprising: (a) statistically associating one or more genetic variants of a population of subjects with neovascular age-related macular degeneration (AMD) with (b) the intraretinal fluid of said population of subjects with neovascular AMD, wherein the one or more genetic variants are associated with a decrease in intraretinal fluid in subjects with neovascular AMD treated with intravitreal anti-VEGF agents compared to the level of intraretinal fluid in subjects with neovascular AMD not treated with intravitreal anti-VEGF agents, and the genetic variant is a single nucleotide polymorphism, is disclosed.

[0059] A method for associating a genetic variant with visual acuity, anatomical outcome, or treatment frequency, comprising: (a) statistically associating (i) one or more genetic variants of a population of subjects with neovascular AMD treated with intravitreal anti-VEGF agents with (ii) the anatomical outcome of said population of subjects with neovascular AMD, wherein the one or more genetic variants are associated with the presence of intraretinal cystoid edema (fluid) compared to the absence of intraretinal cystoid edema (fluid) after one year of treatment.

[0060] A method for associating a genetic variant with intraretinal fluid, comprising: (a) statistically associating one or more genetic variants of a population of subjects with neovascular AMD with (b) the intraretinal fluid of said population of subjects with neovascular AMD, wherein the one or more genetic variants are associated with a decrease in intraretinal fluid in subjects with neovascular AMD treated with intravitreal anti-VEGF agents compared to the level of intraretinal fluid in subjects with neovascular AMD not treated with intravitreal anti-VEGF agents, the genetic variant is a single nucleotide polymorphism, and the single nucleotide polymorphism is selected from the group consisting of rs2056688, rs5962084, rs5962087, rs5915722, and rs5962095.

[0061] Kit Furthermore, kits for utilizing the methods described herein are described herein. The kits described herein can include one or more assays for detecting one or more genetic variants in a sample of interest.

Example

[0062] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods described in the claims of this specification are made and evaluated, and are merely intended as examples of the invention and are not intended to limit the scope of what the inventors regard as the invention. Although efforts have been made to ensure the accuracy of numbers (e.g., amounts, temperatures, etc.), some errors and variations should be accounted for. Unless otherwise indicated, parts are by weight, temperature is in °C or ambient temperature, and pressure is at or near atmospheric pressure.

[0063] VIEW1 and VIEW2 were Phase III clinical trials of neovascular age-related macular degeneration (AMD) (VEGF Trap-Eye: Investigation of Efficacy and Safety in Wet AMD), in which subjects received intravitreal injection of aflibercept (Heier JS, et al., Am. Acad. Opthalmol. 119:2537 (2012)).

[0064] The objective of this statistical study was to identify genetic variants associated with anti-VEGF drug response as measured by visual acuity, anatomical outcomes and treatment frequency in the VIEW1 trial. The outline of the VIEW1 trial is shown in Figure 1. In the VIEW1 trial, the efficacy and safety of intravitreal injection (IAI) of aflibercept were evaluated compared to ranibizumab in the treatment of neovascular AMD.

[0065] In the 52nd week, all IAI groups demonstrated that the improvement in all visual acuity endpoints was comparable to that of Rq4. The incidence of ocular adverse events was comparable in all treatment groups, and the adverse events occurring in more than 10% of patients were conjunctival hemorrhage, eye pain, retinal hemorrhage, and decreased visual acuity.

[0066] A genome-wide association study (GWAS) was conducted on 362 VIEW1 patients. Genotyping of DNA samples was performed using the Illumina Omni Express Exome chip. Logistic regression using baseline values was carried out to establish the association between gene variants and efficacy variables. GWAS analysis was performed on approximately one million variants. The association between gene variants and efficacy variables was determined using logistic regression with baseline values. All treatment groups were combined. For each SNP, genotypes were coded according to the additive model of genetic traits. Variants associated with an increase of 15 or more ETDRS letters at week 52, the presence of intraretinal cystoid edema (fluid measured by time-domain optical coherence tomography (TD-OCT)) at week 52, and the frequency of treatment at week 96 were evaluated. Also, variants were associated with treatment burden. Specifically, patients who required more than 7 injections from week 52 to week 96 [the second year of the study] were analyzed. Furthermore, variants were associated with the presence of intraretinal cystoid edema (defined as fluid) at week 52. Also, the patient demographics and baseline characteristics of VIEW1 were identified. (See Figure 2). Quality control measures were applied to SNPs on the chip to generate the final sample set. (See Figures 3 and 4).

[0067] The anatomical response, namely the X chromosome SNP (rs2056688), showed the highest association with anatomical outcomes, with an odds ratio (OR) of 0.2578 and a point-wise association with the presence of intraretinal fluid (p-value 7.27×10 -7 ) demonstrated at week 52. (See Figure 5).

[0068] Four neighboring SNPs (rs5962084, rs5962087, rs5915722, rs5962095) showed comparable ORs (0.3151 - 0.3461) and per-point associations (5.48×10 -6 ~8.59×10 -6 ). rs2056688 is located in a non-coding region, and the nearest associated functional gene (protein kinase X-related (PRK-X)) was mapped approximately 400 kb upstream of the putative variant. (See Figure 6). Additional SNPs with lower significance were found to be associated with the proportion of patients with an increase of 15 or more ETDRS letters in vision at week 52 and the frequency of treatment at week 96.

[0069] Additional neighboring SNPs showed a dose effect. It was found that as the number of variant copies increased from 0 -> 1 -> 2, the likelihood of the presence of fluid at week 52 decreased from approximately 50% to approximately 25% and then to approximately 10%. (See Figure 7). Figure 8 summarizes the SNAs identified in the study.

[0070] Conclusion: A suggestive association between the gene variant and the presence of intraretinal fluid measured by TD-OCT at week 52 was identified by GWAS in neovascular AMD patients receiving anti-VEGF treatment in the VIEW1 trial. The variant was located on the X chromosome in the vicinity of the gene for PRK-X, a serine / threonine protein kinase involved in angiogenesis.

[0071] [Appendix 1] Use of a vascular endothelial growth factor (VEGF) inhibitor in the manufacture of a medicament for treating a patient with age-related macular degeneration, wherein the patient has been previously administered a VEGF inhibitor for about one year and has one or more genetic variants determined by performing or having performed a genotyping assay on a DNA sample obtained from the patient, and the one or more genetic variants are single nucleotide polymorphisms selected from the group consisting of rs2056688, rs5962084, rs5962087, rs5915722, rs5962095, rs2106124, rs1879796, rs12148845, rs12148100, rs17482885 and rs17629019.

[0072] [Appendix 2] The use according to Appendix 1, wherein the one or more genetic variants are single nucleotide polymorphisms selected from the group consisting of rs2056688, rs5962084, rs5962087, rs5915722 and rs5962095.

[0073] [Appendix 3] The use according to Appendix 1, wherein the one or more genetic variants are single nucleotide polymorphisms selected from the group consisting of rs2106124, rs1879796, rs12148845 and rs12148100.

[0074] [Appendix 4] The use according to Appendix 1, wherein the one or more genetic variants are single nucleotide polymorphisms selected from the group consisting of rs17482885 and rs17629019.

[0075] [Appendix 5] The use according to Appendix 1, wherein the patient is heterozygous for the one or more genetic variants.

[0076] [Appendix 6] The use according to Appendix 1, wherein the patient is homozygous for the one or more genetic variants.

[0077] [Appendix 7] The use according to Supplementary Note 1, wherein the gene variant is rs2056688. [Supplementary Note 8] The use according to any one of Supplementary Notes 1 to 4, wherein the VEGF inhibitor is in an amount of about 2 mg every three months for the treatment of the patient with macular degeneration.

[0078] [Supplementary Note 9] The use according to any one of Supplementary Notes 1 to 5, wherein the VEGF inhibitor is aflibercept.

Claims

1. 1. A pharmaceutical composition for treating age-related macular degeneration with neovascularization in a subject having one or more single nucleotide polymorphisms selected from the group consisting of rs2056688, rs5962084, rs5962087, rs5915722, rs5962095, rs1405303, rs2106124, rs1879796, rs12148845, rs12148100, rs17482885, and rs17629019, comprising: A pharmaceutical composition containing aflibercept.

2. 1. A pharmaceutical composition for the treatment of age-related macular degeneration with neovascularization in a subject, wherein the subject has one or more single nucleotide polymorphisms selected from the group consisting of rs2056688, rs5962084, rs5962087, rs5915722, rs5962095, rs1405303, rs2106124, rs1879796, rs12148845, rs12148100, rs17482885, and rs17629019, as determined by performing or having performed a genotype assay on a DNA sample obtained from the subject; A pharmaceutical composition containing aflibercept.

3. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs2056688.

4. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs5962084.

5. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs5962087.

6. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs5915722.

7. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs5962095.

8. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs1405303.

9. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs2106124.

10. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs1879796.

11. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs12148845.

12. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs12148100.

13. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs17482885.

14. The pharmaceutical composition of claim 1 or 2, wherein the single nucleotide polymorphism comprises rs17629019.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the subject is heterozygous for the one or more single nucleotide polymorphisms.

16. The pharmaceutical composition according to any one of claims 1 to 14, wherein the subject is homozygous for the one or more single nucleotide polymorphisms.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the aflibercept is administered intravitreally in an amount of 2 mg per dose.

18. 18. The pharmaceutical composition of claim 17, wherein the aflibercept is administered once every 4 or 8 weeks.

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