WO2008110828A1 - Method for prevention of age-related macular degeneration (amd) - Google Patents

Method for prevention of age-related macular degeneration (amd) Download PDF

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Publication number
WO2008110828A1
WO2008110828A1 PCT/GB2008/000925 GB2008000925W WO2008110828A1 WO 2008110828 A1 WO2008110828 A1 WO 2008110828A1 GB 2008000925 W GB2008000925 W GB 2008000925W WO 2008110828 A1 WO2008110828 A1 WO 2008110828A1
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WIPO (PCT)
Prior art keywords
age
individual
macular degeneration
related macular
polymorphism
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PCT/GB2008/000925
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French (fr)
Inventor
Igor Bendik
Özgür SANCAK
Wolfgang Schalch
Jonas Wittwer
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Dsm Ip Assets B.V.
Wright, Simon, Mark
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Application filed by Dsm Ip Assets B.V., Wright, Simon, Mark filed Critical Dsm Ip Assets B.V.
Publication of WO2008110828A1 publication Critical patent/WO2008110828A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to a novel method for the (treatment and/or) prevention of age related macular degeneration (AMD). It relates to the diagnosis and/or treatment of age-related macular degeneration (or an age-related macular degeneration-related disorder) in an individual (or subject), usually by determining susceptibility of the individual to age- related macular degeneration and, on the basis of that determination, selecting or identifying (and administering) a substance to the individual.
  • AMD age related macular degeneration
  • wet macular degeneration With wet macular degeneration, new weak blood vessels may grow in or under the retina causing fluid and blood to leak into the space under the macula. As a result, wet macular degeneration is sometimes called exudative macular degeneration, or described as choroidal neovascularization.
  • the choroid is the area of blood vessels beneath the retina, and neovascularization refers to growth of new blood vessels in tissue. In choroidal neovascularization, blood vessels from the choroid grow into the macula.
  • wet macular degeneration The classic early symptom of wet macular degeneration is that straight lines appear crooked. This occurs when fluid from the leaking blood vessels gathers and lifts the macula, distorting vision. A small blind spot may also appear in wet macular degeneration, resulting in loss of one's central vision.
  • the present invention relates to the identification of those subjects which can have increased risk for developing (dry and/or wet) AMD and with the treatment and, particularly, prevention of AMD in those subjects suitably at an early as possible point in time.
  • the present invention thus relates to a method for treatment and/or prevention of age- related macular degeneration (AMD), which method comprises:
  • polymorphism detecting one or more polymorphism(s) in a sample taken from a subject (or individual, the terms are used interchangeably), or typing an individual for one or more polymorphisms, wherein the polymorphism is an SNP as shown in Table 1 , such as rsl 1200638 or rs 10490924, or is related to or associated with a (e.g. high temperature requirement) serine protease; and
  • the substance is a (preferably macular) carotenoid, in particular a xanthophyll, such as lutein and/or zeaxanthin and/or vitamin C, vitamin E; beta carotene, zinc and/or copper, and/or or a mixture thereof (the AREDS Cocktail, as described later).
  • a xanthophyll such as lutein and/or zeaxanthin and/or vitamin C, vitamin E; beta carotene, zinc and/or copper, and/or or a mixture thereof (the AREDS Cocktail, as described later).
  • the present invention also provides a method of determining a substance to be administered to an individual, which individual may be susceptible of having age-related macular degeneration AMD (which term includes a (wet or dry) age-related macular degeneration-related disorder or condition unless otherwise specified), the method comprising: a) detecting or typing for one or more polymorphism(s), usually in a sample from an individual, which may indicate the susceptibility of the individual to age-related macular degeneration (AMD), as described earlier; and b) on the basis of the determination in (a), identifying or selecting a substance capable of preventing (and/or treating) age-related macular degeneration (AMD) in that individual.
  • the method may additionally comprise: c) providing (such as administering or communicating) the substance (or its identity) to the individual.
  • the invention further provides: i) means capable of detecting the or each SNP (or haplotype or allelic variant) related or associated with AMD (in a sample from an individual) and means for providing (such as administering or communicating) a substance (or its identity) capable of preventing (or treating) age-related macular degeneration to the individual; ii) a kit for carrying out the method of the invention comprising means for detecting an SNP or haplotype or allelic variant or combination of SNPs and an effective amount of such a substance, e.g.
  • a (preferably macular) carotenoid in particular a xanthophyll, such as lutein and/or (optionally meso-) zeaxanthin and/or vitamin C, vitamin E; beta carotene, zinc and/or copper, and/or or a mixture thereof (the AREDS Cocktail); iii) a method of preparing a customised or personalised composition for an individual which is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), the method comprising:
  • composition suitable for, or tailored to, the individual (as described earlier); iv) a method of providing a customised composition, comprising providing a composition suitable for a subject which is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), wherein the individual has been (e.g. genetically) determined to be susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), using one or more of the polymorphisms disclosed herein; v) a method of identifying a substance for the treatment or prevention (which includes delaying progression or preventing occurrence of) of age-related macular degeneration (or an age-related macular degeneration-related disorder), the method comprising:
  • composition manufacturing apparatus optionally, generating (e.g. electronic) manufacturing instructions to control the operation of composition manufacturing apparatus in accordance with the customised composition formulation;
  • SEQ ID NOs: 1 to 70 show the polynucleotide sequences encompassing the SNPs used in the invention.
  • a method of determining a substance to be administered to an individual comprising: a) detecting one or more polymorphism(s) in a sample taken from an individual, or typing the individual for the presence of one or more polymorphism(s), wherein the polymorphism is rsl 1200638 or rs 10490924, or an SNP shown in Table 1, or is related to or associated with a (e.g. high temperature requirement) serine protease.
  • This detection or typing method is used in many later aspects of the invention.
  • the invention can thus progress to: b) on the basis of the determination in a), identifying a substance capable of preventing and/or treating age-related macular degeneration in that individual.
  • the method may additionally comprise: c) providing (e.g. administering or communicating) the substance (or its identity) to the individual (or subject).
  • Step c) can thus comprise communicating the identity of that substance to the individual, for example proposing, suggesting or recommending that substance. For instance, this may involve supplementing a person's food or diet with said substance.
  • Prevention (of AMD) in this specification includes assisting in preventing AMD, mitigating or alleviating one or more symptoms of AMD, and delaying the progression or preventing occurrence of AMD.
  • AMD includes AMD-related disorders. The detection may thus confirm a diagnosis of AMD by screening for particular molecular markers.
  • the individual may be susceptible to age-related macular degeneration, or an age- related macular degeneration -related disorder. This may mean that they are at risk of, or have a predisposition to, age-related macular degeneration or an age-related macular degeneration-related disorder. That individual may, or may not, in fact, necessarily have an increased risk or susceptibility, depending on the determination.
  • the determination may find that the person has an increased risk, or (on the contrary) a decreased risk, of said disorder.
  • the detection or typing in (a) may thus comprise typing the nucleotide (present in the genome of the individual) at a position equivalent to position 26 in one or more of the sequences identified in the attached sequence listing (eg. any of SEQ ID NOs: 1 to 70).
  • Stage (a) may thus additionally comprise preparing or obtaining a pharmacogenomic, nutrigenomic, nutrigenetic, transcriptomic, epigenetic or proteomic and/or nutritional profile (or identity) of the individual. This may assist in determining the susceptibility (such as risk or predisposition) to a disorder.
  • the determination may comprise: i) conducting or performing a genome or genetic analysis of the individual; and ii) preparing a pharmacogenomic, nutrigenomic, nutrigenetic, transcriptomic, epigenetic or proteomic and/or metabolomic profile and/or identity based on personal and/or clinical information from or about the individual; iii) optionally, performing a test or assay (such as on a biological sample from the individual) that can indicate susceptibility (to age-related macular degeneration).
  • this may involve determining an individual's genotype (for the respective loci). It can comprise identifying an allelic variant, a polymorphism (such as an SNP) a haplotype or combination thereof (e.g. SNP and haplotype) or genetic predisposition (to the relevant disorder). One may therefore be able to draw up a genetic profile of the individual, preferably relevant to the disorder.
  • the determination may, alternatively or in addition to (ii), comprise obtaining relevant information from, or about, that individual. That information may be personal and/or clinical information. The information may relate, directly or indirectly, to age- related macular degeneration (or an age-related macular degeneration-related disorder). Such information may comprise information concerning lifestyle, health, nutritional status, diet.
  • Clinical information may comprise current drug and/or vitamin regimes, current or past treatments, familial data, health risks, family background, medical conditions and/or allergies. It may therefore involve obtaining a patient's medical history or current health status and determining their nutritional profile. The individual may be able to provide this information, for example, by completing a questionnaire.
  • the identification of the individual risk of a subject of developing AMD, or suffering from AMD can be accomplished by genetic (or genome) analysis, more specifically, by determining the presence of gene polymorphism(s) involved in (the development of) or related to AMD.
  • a polymorphism may be present in a gene coding for a serine protease, such as a mammalian (e.g. human) serine protease, or a protease in the enzyme group EC 3.4.21.-.
  • the protease may have a (trypsin like) catalytic or serine protease domain and/or at least one PDZ domain (e.g. at or near the C terminus).
  • the protease may have, alternatively or in addition, an IGF BP binding domain and/or a (e.g. Kazal-type) serpin inhibitor motif (e.g. at or near the N terminus).
  • the serine protease is a high temperature requirement (HTRr, or HTRA) protease, for example: a) HTRA 1 (also known as PRSSl 1 and L56); b) HTRA 2 (also known as PRSS25 and PARKl 3); c) HTRA 3, also known as PRSP; and/or d) HTRA 4, available as GenBank accession number AK075205.1.
  • HTRA 1 also known as PRSSl 1 and L56
  • HTRA 2 also known as PRSS25 and PARKl 3
  • HTRA 3 also known as PRSP
  • HTRA 4 available as GenBank accession number AK075205.1.
  • the SNP is at human chromosome 10q26, and is preferably upstream of a transcription start site, such as of the HtrA 1 gene (also known as PRSSl 1, NM_002775).
  • the SNP is present in a conserved AP2-SRF binding element.
  • the risk allele is AA or AG
  • the (3) normal controls carry the normal allele GG (such as for rsl 1200638, SEQ ID NO:2).
  • the risk allele may be TT or TG
  • the (3) normal controls carry the normal allele GG (such as for rsl0490924, SEQ ID NO:1).
  • Preferred SNPs are those in SEQ ID NOs: 1, 2, 68 or 69, or are at position 26 at any of SEQ ID NOs:l to 70.
  • the SNP is present in a serine protease but is expressed in the retina and/or RPE.
  • the SNP resides between two genes on chromosome 10q26.
  • One such gene may be a homology domain containing protein, for example a pleckstrin homology domain containing protein, such PLEKHAl, for example that of GenBank ID 59338.
  • the other (flanking) gene may be a serine protease, such as a heat shock serine protease, for example an HtrA gene.
  • Preferred SNPs are therefore rsl 0490924 and/or rsl 1200638.
  • the SNP rsl 1200638 is located 626 base pairs (bp) upstream of the HTRAl putative translational start site. It is 6096 bp downstream of SNP rsl 0490924.
  • Preferred SNPs are (along with the appropriate SNPs form Table 1): a) associated with ABCAl, e.g. rs2230806, rs28587567, rs35819696, rs9282543, or rs2066718; b) associated with APOB; c) associated with APOE, e.g. rs429358 or rs7412; d) associated with CD36; e) associated with CYPlAl or CYPl A2 e.g. rs2470893, rs2606345, rsl799814, or rs4646903; f) associated with CYP2D6, e.g.
  • rs2854740 or rs2743456 g) associated with ELOVL4, e.g. rs3812153; h) associated with LDLR; i) associated with LRP6, e.g. rs7294695; j) associated with PONl, e.g. rs2237583, rs854560, rs662, or rs854552; k) associated with PPARA, e.g. rsl 800206;
  • PPARG e.g. rsl801282
  • VLDLR VLDLR
  • SNPs are particularly useful for identifying patients with, or who may develop, ' AMD.
  • the SNPs therefore represent a selection that allows the identification of patients that are likely to benefit from, and respond to, treatment or prevention of AMD. It allows the identification of a subset of patients that may be able to respond to AMD treatment, or least have predisposition to AMD, so that this potential condition can be treated early.
  • a polymorphism in a gene for example encoding a protein which is related to the development of AMD, can be determined by methods known in the art. In general, this involves the extraction of genomic DNA by standard procedures (Sambrook J, Fritsch EF, and Maniatis T.: Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Press; 1989) from blood cells, or buccal mucosa cells, hair cells or any other DNA containing human tissues which are easily and usually non-invasively accessible. Alternatively, commercial kits can be used (i.e. QIA AMP blood kit, Qiagen or any other commercially available DNA extraction kit).
  • the characterization of an individual's genotype at a given locus which relates to the determination of the variants or polymorphisms, can be performed according to standard procedures.
  • These classic technologies that relate to the determination of the polymorphisms include DNA sequencing using 96-channel capillary sequencers, single-strand conformation analysis using non-denaturing gel electrophoresis, denaturing gradient gel electrophoresis using the partial melting behaviour of double-stranded DNA, heteroduplex analysis involving denaturing high-performance liquid chromatography, chemical or enzymatic cleavage of mismatch pairing method, and mutation detection by coupled transcription-translation (protein truncation test) procedures involving non-sense stop codons.
  • the more recent techniques are real-time PCR methods like TaqMan, mass spectrometry involving single- stranded PCR fragments generated by the dideoxy-nucleotide PCR methodology, DNA MicroArray technology detecting SNPs provide by commercial suppliers (e.g. Affymetrix Inc., Santa Clara, CA; Illumina, San Diego, CA and Sequenom, San Diego, CA) and many more.
  • Table 1 showing the SNPs used in the present invention, follows. Table 1
  • dbSNP numbers (rs numbers) are given to denote for SNPs.
  • Table 2 below correlates the SEQ ID NOs: in the attached sequence listing with the polymorphisms of Table 1.
  • the determination or identification of the individual risk or susceptibility of a subject of developing AMD or suffering from AMD can be accomplished optically, such as by measuring optical density, e.g. of a macular pigment or other suitable optical measurement in or of the eye (e.g the retina).
  • optical density e.g. of a macular pigment or other suitable optical measurement in or of the eye (e.g the retina).
  • This can comprise measuring the level of a carotenoid, usually a macular carotenoid (such as lutein or zeaxanthin) in the eye of the individual.
  • the (profile of) macular pigment optical density can be measured, e.g. by a technique which is based on the method described by Delori et al. (Delori FC et al., J Opt Soc Am A Opt Image Sci Vis. 2001, 18(6): 1212-30). This technique can record spatial profiles of the density of the yellow macular pigment across the retina.
  • a visual display subjects view a target that alternates between two spectrally different components. One component, the blue light, is absorbed by the macular pigment, whilst the other component, which appears orange to the eye, is not absorbed by the macular pigment.
  • the heterochromatic flicker photometry (HCF) technique uses a visual display and provides a rapid and convenient macular assessment profile (MAP) test.
  • the MAP test is based on the use of an optical notch filter to separate the outputs of the three phosphors of the display into two components, one that is absorbed maximally by the macular pigment (MP) and is derived only from the blue gun (i.e., the test beam) and the other that is based on a combination of red and green phosphor luminances and consists largely of long wavelength light that is not absorbed by the MP (i.e., the reference beam).
  • the luminance of the reference beam is 20 cd m-2 and its modulation depth is fixed at 20%.
  • the MAP test makes full use of the advantages of visual displays to produce stimuli of varying size at a number of randomised locations, to generate counter-phased sinusoidal modulation of the two stimulus beams.
  • the frame rate of the display is 140 Hz and the stimulus modulation frequency is 20 Hz.
  • the high temporal modulation frequency employed ensures that at threshold one isolates the activity of luminance flicker detection mechanisms that rely only on the combined L and M cone signals.
  • the stimulus is presented as a short burst of flicker of approximately 0.5s duration and the subject's task is to report the presence or the absence of perceived flicker.
  • a modified staircase procedure with variable step sizes was then used to measure the mean luminance of the test beam needed to cancel the perception of flicker generated by the reference beam.
  • the MAP test can be used to measure MPOD along any meridian at a number of specified locations from -8° to +8° eccentricity of the visual field.
  • the test stimulus changes from a disc of 0.36 o diameter, when presented at the fovea, to a sector annulus when presented at one of five discrete locations on either side of fixation across the horizontal meridian: ⁇ 8°, ⁇ 6°, ⁇ 4°, ⁇ 2.5°, ⁇ 1.25°, 0°.
  • the width of the test annulus also increases systematically with eccentricity to facilitate the detection and the nulling of luminance flicker.
  • a central spot and radial guides are used to help the subject maintain steady fixation.
  • Five, randomly interleaved, repeat measurements are taken at each spatial location investigated. The test is performed at a viewing distance of 0.7m and the stimulus was presented only to the right eye. Similar measurements made with the left eye can confirm previous findings to show good correlation in MPOD values between the two eyes.
  • An optical density of the macular pigment of lower than 0.2 as determined by HCF can be regarded as evidence for an existing risk of developing AMD or the existence of a macular degeneration.
  • a carotenoid such as lutein, zeaxanthin and/or meso-zeazeanthin, and/or one of the antioxidants or mixture thereof as defined earlier according to step (b) of the method of the present invention
  • the identification of the individual risk of a subject of developing AMD or suffering from known or undetected AMD is accomplished by determining the xanthophyll and/or carotenoid level in a body fluid, such as blood or plasma, and/or skin.
  • the xanthophyll and carotenoid level in plasma and/or skin can be determined by methods known in the art. For example, blood (approx.10 to 15 ml) is collected into pre-cooled Monovettes containing EDTA, and plasma prepared by centrifugation. The preparation of plasma has to be done under appropriate shielding from light. After collection, plasma samples can be stored at -35 0 C in the dark.
  • Xanthophyll and carotenoid plasma levels below 0.25 ⁇ mol/L may be regarded as indicative for an existing risk of developing AMD or of the existence of a macular degeneration, which may require the (or benefit from) administration of lutein or zeaxanthin to the individual.
  • a further method of determining risk or susceptibility to AMD involves assessing visual (which includes contrast) performance (such as acuity.
  • the method may comprise detecting the response or reaction of the individual, or the individual's eye, to a visual stimulus, for example in a certain or specific colour, normally in the visible spectrum, for example blue or green light.
  • the light or visual stimulus may be provided at two or more intensities or contrast levels.
  • the (visible) light may be green or blue and/or may have a wavelength within 495 - 535 run or within 460 - 495 run.
  • the method may involve assessing the level of the pigment, which may be a macular pigment, preferably one that absorbs the visible light.
  • It may comprise detecting a reaction or response of the individual to a visual display or a visual stimulus, for example where two different intensities or contrast levels of the light provided, and the reaction or response to two or more (or each) intensity or contrast level can be assessed.
  • the individual is shown a display with one or more rings, such as Landolt rings, which may be blue in color and/or have a wavelength of from 470 - 800 nm. This method is described in more detail in European Patent Application Number 06255800.2, filed on 13 November 2006 in the name of DSM IP Assets B. V., the contents of which are herein incorporated by reference.
  • the determination may comprise taking (a biological) sample from the individual, such as a body fluid (such as urine, saliva or blood) that may contain cells.
  • a sample may comprise buccal and/or skin cells, for example taken from the mouth using for example a swab.
  • the genetic analysis may be performed using a microarray (one or more genes on a chip) or a multiwell plate, for example in a laboratory. It may thus involve the use of a gene/DNA chip, or a strip, test tube or other solid surface suitably comprising one or more
  • SNPs or genetic polymorphism(s) that may, or may not, cover one or more genes, but are found in a genetic locus of interest.
  • the nature or identity of the substance can be communicated either to the individual, or their doctor, optician, physician, guardian, dietician or (genetic) counselor.
  • the communication may be electronically, for example via a computer (a personal computer or a laptop), portable computer or mobile phone or using the internet (e.g. via email, webpage and/or blog). Alternatively, it may be communicated on paper, for example in a booklet or information pack.
  • the communication of the nature or identity of the substance may be provided through a handheld and/or portable device such as a bracelet, necklace or watch-type device, personal computer, cell or mobile phone, personal digital assistant (PDA), a device which may be attached to or integral with a shopping cart or trolley, a terminal (eg. to an on-line service, which may be in an outlet or retail store, such as a super-or hypermarket, pharmacy or health store), for example through the internet, a telephone with voice communication, kiosk or centralised computer system.
  • a handheld and/or portable device such as a bracelet, necklace or watch-type device, personal computer, cell or mobile phone, personal digital assistant (PDA), a device which may be attached to or integral with a shopping cart or trolley, a terminal (eg. to an on-line service, which may be in an outlet or retail store, such as a super-or hypermarket, pharmacy or health store), for example through the internet, a telephone with voice communication, kiosk or centralised computer system.
  • PDA personal digital assistant
  • the identification or determination of the risk of an individual may have been undetected, or indeed an increase or decrease to risk may not been known to that individual before the determination. It can be accomplished by genome and/or genetic analysis or, preferably, by determining the presence (or absence) of a gene polymorphism, for example involved in the development of age-related macular degeneration (or age-related macular degeneration -related disorders).
  • the presence of a polymorphism in genes can be determined by methods known in the art. In general, this will usually involve the extraction of genomic DNA by standard procedures, for example from blood cells, or buccal mucosa cells, hair cells or any other DNA containing tissue, which is suitably easily, and usually non-invasively, accessible. Alternatively, commercially available DNA extraction kits can be employed.
  • the characterisation of an individual's genotype at a given locus can be performed according to standard procedures.
  • This technology can involve the use of DNA sequencing apparatus, for example using a 96-channel capillary sequencer, a single strand confirmation analysis using non-denaturing gel electrophoresis, denaturing gradient gel electrophoresis (using the partial melting behaviour of double stranded DNA), heteroduplex analysis involving denaturing HPLC, chemical or enzymatic cleavage of mismatch pairing method and/or mutation detection by coupled transcription-translation (protein truncation test) procedures, for example involving nonsense mutations and/or variations.
  • the most recent techniques are real-time PCR methods like TaqMan mass spectrometry involving a single-stranded PCR fragments, for example generated by the dideoxy-nucleotide PCR methodology, DNA microarray technology for detecting SNPs, as provided by commercial suppliers (such as Affymetrix Inc., Santa Clara, California (CA), Illumina, San Diego, CA and Sequenom, San Diego, CA). Substances and compositions to be provided to the individual
  • the substance or composition may comprise a compound, such as an active ingredient, a drug, pharmaceutical or nutraceutical.
  • the substance may be edible and/or comprise a food, foodstuff or feed, for example a (dietary) supplement, or pharmaceutical composition.
  • the substance may be in any form, for example suitable for oral administration, such as in solid form such as tablets, including effervescent tablets, soft or hard-shell capsules, or in liquid form such as solutions or suspensions, such as an oily suspension.
  • the preparation may contain one or more conventional (eg pharmaceutical) carrier materials, additives and adjuvants, for example, including one or more of gelatine, vegetable gum, sugar, vegetable oil, polyalkylene glycol, flavouring agent, preservative, stabilizer, an emulsifying agent and/or a buffer.
  • the substance, if a medicament, can be a controlled (or delayed) release formulation.
  • the (therapeutic) substance may be administered in various manners such as orally, intracranially, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, and subcutaneously.
  • the pharmaceutical compositions that contain the therapeutic agent will normally be formulated with an appropriate pharmaceutically acceptable carrier or diluent depending upon the particular mode of administration being used.
  • parenteral formulations are usually injectable fluids that use pharmaceutically and physiologically acceptable fluids such as physiological saline, balanced salt solutions, or the like as a vehicle.
  • Oral formulations may be solids, for example tablets or capsules, or liquid solutions or suspensions, hi a preferred embodiment, the therapeutic agent is administered to the individual in their diet, for example in a drink or food.
  • the present invention may thus provide an optimisation of diet and or nutritional supplementation and or pharmaceutical administration, based on the determination of susceptibility to the relevant disorder.
  • the optimisation for example of nutrition or nutritional supplementation, may be for a group of individuals, usually related ones, such as a family. If the substance is a nutritional supplement, this may include foods, capsules, pills, powders, gums and liquids or other oral dosage forms. Also encompassed are nutritional supplements that can be delivered for example to the digestive system, or intravenously, as well as supplements that can be administered through other routes, such as mucous membranes.
  • the individual supplements may comprise excipients, impurities or other components other than the substance of interest.
  • the individual's susceptibility has been determined, one can optimise the nutritional intake, in particular of the substance or composition.
  • the amount, dosage and the form in which it is ingested or administered can be tailored to that individual, so that the substance is personalised for that particular individual.
  • the result of the examination may include a proposal to reduce intake of supplement, macronutrient or foodstuff, as well as increasing or adding a substance or other nutritional substances.
  • a (preferably macular) carotenoid such as lutein and/or zeaxanthin and/or the AREDS cocktail (a component thereof) can be suggested or administered.
  • the substance can be a xanthophyll (for example, a carotenoid possessing one or more oxygen atoms, such as an - OH or hydroxy group).
  • an effective amount of the carotenoid can be used.
  • this is lutein, meso- zeaxanthin, and/or zeaxanthin and/or "the AREDS cocktail" (vitamin C, vitamin E, beta carotene, zinc and/or copper, AREDS Report No. 8, Arch. Ophthalmol. 2001;l 19:1417- 1436, referred to as "AREDS Cocktail", also at HKJ Ophthalmol. Vol. 4, Nr. 1, (2000), p. 31 -42) and/or one of the components of the AREDS cocktail.
  • this can be e.g., within the range of from 0.001 mg per kg body weight to about 20 mg per kg body weight.
  • the carotenoid, e.g. lutein and/or (optionally meso-)zeaxanthin are administered at a dosage of from 1 or 5 to 15, 30 or 50 mg/day, such as from 8 or 10 to 12, 15 or 20mg/day and may be present in compositions at that (daily) dosage.
  • Preferred compositions can contain from 8 to 12mg lutein or zeaxanthin (and preferably both within this range).
  • vitamin C is 1 to about 10 mg per kg body weight, for beta- carotene 0.1 to about 0.3 mg per kg body weight, for vitamin E HU to about 10 IU per kg body weight, for zinc 0.1 mg per kg body weight to about 1.5 mg kg body weight, and for copper 0.01 mg per kg body weight to about 0.05 mg per kg body weight.
  • Zinc is preferably used as zinc oxide and copper as cupric oxide.
  • Preferable daily dosages and/or amount in an oral (e.g. daily) formulation, such as a tablet are as follows.
  • the formulation may comprise an antioxidant. This may be vitamin C (such as at from 200 to 800mg, 400 to 600mg, such as 450 to 550mg). There may be 1, 2 or 3 antioxidants present.
  • vitamin E another antioxidant is vitamin E. This may be present at a dosage of from 100 to 700 IU, such as from 200 to 600 IU, preferably from 300 to 500 IU.
  • the zinc may be present as zinc oxide, and can be an amount of from 20 to 140mg, such as from 60 to lOOmg, preferably from 70 to 90mg.
  • the copper may be present at from 1 to 2mg.
  • treatment is started with an initial dosage of 0.5 - l.Omg of carotenoid (eg. xanthophyll), such as lutein and/or zeaxanthin, per kg body weight per day for 1 -2 months whereupon the dosage may be lower to secure a macular pigment optical density of three times the threshold value, i.e. 0.6.
  • carotenoid eg. xanthophyll
  • two or more xanthophylls are present, such as a combination of lutein and zeaxanthin.
  • these compounds are preferably used in a ratio of 0.1-1.0 : 1.0- 0.1 parts (by weight), such as from 0.5-1.0:1.0-0.5, especially 0.9-1.1 :0.9-1.1, to each other.
  • the substance such as lutein and/or zeaxanthin and/or the "AREDS Cocktail” or its individual components can be provided in any appropriate form, suitably for oral administration, e.g. as a pharmaceutical composition, or in food or beverage.
  • the term "providing” as used herein is to be understood as denoting the act of collecting the desired active ingredients and processing them into a suitable administration form, as well as the direction for use and/or administration to the subject involved. Higher dosages and amounts can be provided to individuals who appear to be at greater risk, for example one or more polymorphisms associated or related to AMD, and so one can correlate higher dosages with greater risk (or more polymorphisms).
  • the invention relates to the use of a (preferably macular) carotenoid, e.g. xanthophyll, such as lutein, meso-zeaxanthin and/or zeaxanthin and/or a vitamin C, beta-carotene, vitamin E, zinc and copper or a mixture thereof in the manufacture of a medicament for the treatment and/or prevention of age-related macular degeneration (AMD) in a subject which has been identified as being at risk of developing AMD, or as suffering from AMD, especially by one of the methods (of the invention) identified above.
  • AMD age-related macular degeneration
  • the nature of the substance (or its dosage for example) may be modified over time.
  • the substance and/or dosage may therefore change, depending on the individual's response to the treatment, to the subtance, or the nature or condition of AMD.
  • the individual may be able to measure his/her response or reaction to the substance, for example the benefit of the substance administered, or of on going supplements.
  • the individual, patient, physician, optician or other person may therefore change the substance, medicament or other composition, in view of variables such as increasing age, susceptibility to AMD and the progression (or otherwise) of AMD.
  • the individual may therefore be able to self- monitor his/her response to the prevention or treatment that is ocurring.
  • a database can be provided with information concerning available nutritional supplements, including contents, price and dosage form.
  • a further database may include information, including risks and benefits, about constituency of nutritional supplements, for example information concerning the substance.
  • the invention can further include apparatus for formulating the substance, for example in a food or in a nutritional supplement, usually based on the determination of susceptibility.
  • a specific formulation may then be provided or communicated to the individual, which may or may not be standard dosage form.
  • the invention thus additionally contemplates a vending machine or point of sale dispensing machine which can formulate, or combine, pre-prepared dosage forms of nutritional supplements, based on the opposed nutritional supplementation or the substance to be taken by the individual.
  • an interface may be provided, for example a touch screen.
  • an individual may be interviewed, optionally in the presence of a trained professional, with the data inputted or accepted in an appropriate format.
  • a trained professional such as doctor, nurse, chiropractor, social worker or nutritionist may assist in the input in medical information, etc.
  • the detection of allelic variants or SNPs may comprise contacting a polynucleotide or protein of the individual with a specific binding agent for an AMD variant (related to or associated with AMD), and determining whether the agent binds to the polynucleotide or protein.
  • the binding of the agent can indicate the presence of the age-related macular degeneration variant, and lack of binding of the agent may indicate the absence of the age-related macular degeneration variant.
  • the method is generally carried out in vitro on a sample from the individual.
  • the sample typically comprises a body fluid and/or cells of the individual and may, for example, be obtained using a swab, such as a mouth or buccal swab.
  • the sample may be a blood, saliva, skin, buccal mucosa (or cheek) cell or hair root sample.
  • the sample is typically processed before the method is carried out, for example DNA extraction may be carried out.
  • the polynucleotide or protein in the sample may be cleaved either physically or chemically, for example using a suitable enzyme.
  • any one or more methods may comprise determining the presence or absence of one or more age-related macular degeneration variants in the individual.
  • the age-related macular degeneration variant is typically detected by directly determining the presence of the polymorphic sequence in a polynucleotide or protein of the individual.
  • Such a polynucleotide is typically genomic DNA, mRNA or cDNA.
  • the allelic variant may be detected by any suitable method such as those mentioned below.
  • a specific binding agent is an agent that binds with preferential or high affinity to the protein or polypeptide having the allelic variant but does not bind or binds with only low affinity to other polypeptides or proteins.
  • the specific binding agent may be a probe or primer.
  • the probe may be a protein (such as an antibody) or an oligonucleotide.
  • the probe may be labelled or may be capable of being labelled indirectly.
  • the binding of the probe to the polynucleotide or protein may be used to immobilise either the probe or the polynucleotide or protein.
  • determination of the binding of the agent to the age-related macular degeneration variant can be carried out by determining the binding of the agent to the polynucleotide or protein from the individual.
  • the agent is also able to bind the corresponding wild-type sequence, for example by binding the nucleotides or amino acids which flank the allelic variant position, although the manner of binding to the wild-type sequence will be detectably different to the binding of a polynucleotide or protein containing the allelic variant.
  • the method may be based on an oligonucleotide ligation assay in which two oligonucleotide probes are used. These probes can bind to adjacent areas on the polynucleotide which contains the allelic variant, allowing after binding the two probes to be ligated together by an appropriate ligase enzyme. However the presence of single mismatch within one of the probes may disrupt binding and ligation. Thus ligated probes will only occur with a polynucleotide that contains the allelic variant, and therefore the detection of the ligated product may be used to determine the presence of the allelic variant.
  • the probe is used in a heteroduplex analysis based system.
  • a heteroduplex analysis based system when the probe is bound to polynucleotide sequence containing the allelic variant it forms a heteroduplex at the site where the allelic variant occurs and hence does not form a double strand structure.
  • a heteroduplex structure can be detected by the use of single or double strand specific enzyme.
  • the probe is an RNA probe, the heteroduplex region is cleaved using RNAase H and the allelic variant is detected by detecting the cleavage products.
  • the method may be based on fluorescent chemical cleavage mismatch analysis which is described for example in PCR Methods and Applications 3, 268-71 (1994) and Proc. Natl. Acad. Sci. 85, 4397-4401 (1998).
  • a PCR primer is used that primes a PCR reaction only if it binds a polynucleotide containing the allelic variant, for example a sequence- or allele-specific PCR system, and the presence of the allelic variant may be determined by the detecting the PCR product.
  • the region of the primer which is complementary to the allelic variant is at or near the 3' end of the primer.
  • the presence of the allelic variant may be determined using a fluorescent dye and quenching agent-based PCR assay such as the Taqman PCR detection system.
  • one or more of the probes and/or primers are used in a Taqman assay to detect an allelic variant.
  • the specific binding agent may be capable of specifically binding the amino acid sequence encoded by a variant sequence.
  • the agent may be an antibody or antibody fragment.
  • the detection method may be based on an ELISA system.
  • the method may be an RFLP based system. This can be used if the presence of the allelic variant in the polynucleotide creates or destroys a restriction site that is recognised by a restriction enzyme.
  • the presence of the allelic variant may be determined based on the change it makes to the mobility of the polynucleotide or protein during gel electrophoresis.
  • SSCP single-stranded conformation analysis
  • DDGE denaturing gradient gel electrophoresis
  • a polynucleotide comprising the polymorphic region is sequenced across the region that contains the allelic variant to determine the presence of the allelic variant.
  • Detection kit The invention also provides a kit that comprises means for determining the presence or absence of one or more age-related macular degeneration allelic variant(s) in an individual, hi particular, such means may include a specific binding agent, probe, primer pair or combination of primers, or antibody, including an antibody fragment, as defined herein which is capable of detecting or aiding detection of an age-related macular degeneration allelic variant.
  • the primer pair or combination of primers may be sequence specific primers which only cause PCR amplification of a polynucleotide sequence comprising the age-related macular degeneration variant(s) to be detected, as discussed herein.
  • the kit may also comprise a specific binding agent, probe, primer pair or combination of primers, or antibody that is capable of detecting the absence of the allelic variant.
  • the kit may further comprise buffers or aqueous solutions.
  • the kit may additionally comprise one or more other reagents or instruments that enable any of the embodiments of the method mentioned above to be carried out.
  • reagents or instruments may include one or more of the following: a means to detect the binding of the agent to the allelic variant, a detectable label such as a fluorescent label, an enzyme able to act on a polynucleotide, typically a polymerase, restriction enzyme, ligase, RNAse H or an enzyme which can attach a label to a polynucleotide, suitable buffer(s) or aqueous solutions for enzyme reagents, PCR primers which bind to regions flanking the allelic variant, a positive and/or negative control, a gel electrophoresis apparatus, a means to isolate DNA from sample, a means to obtain a sample from the individual, such as swab or an instrument comprising a needle, or a support comprising wells on which detection reactions can be carried out.
  • the kit may be, or include, an array such as a polynucleotide array comprising the specific binding agent, preferably a probe, of the invention.
  • the kit may additionally comprise a substance (or composition) for administration to the individual, as discussed before.
  • the kit typically includes a set of instructions for using the kit.
  • the invention provides a method for identifying a substance useful for the treatment of age-related macular degeneration, which method comprises contacting a variant age-related macular degeneration polypeptide or a polynucleotide with a test agent and determining whether the agent is capable of binding to the polypeptide or modulating the activity or expression of the polypeptide or polynucleotide.
  • Any suitable binding assay format can be used to determine whether the age-related macular degeneration variant binds the test agent, such as the formats discussed below.
  • the method may be carried out in vitro, either inside or outside a cell, or in vivo.
  • the method is carried out on a cell, cell culture or cell extract that comprises a variant age-related macular degeneration protein or polynucleotide.
  • the cell may be any suitable cell, and is typically a cell in which the product is naturally expressed.
  • modulate includes any of the ways mentioned herein in which the agent is able to modulate activity of an age-related macular degeneration variant polypeptide or polynucleotide.
  • This may be determined by contacting the polypeptide or polynucleotide with the test agent under conditions that permit activity of the polypeptide or polynucleotide, and then determining whether the test agent is able to modulate the activity of the polypeptide or polynucleotide.
  • the test agent is a food ingredient.
  • the invention relates to a method of screening food ingredients to determine whether they contribute to or aggravate age-related macular degeneration in susceptible individuals, or if they prevent or alleviate age-related macular degeneration.
  • the present invention also provides an agent identified by a screening method of the invention.
  • An agent identified in the screening method of the invention may be used in the therapeutic treatment of AMD.
  • Such an agent may be formulated and administered in any means or amounts as discussed below.
  • Customised composition eg. food
  • the invention relates to a customised diet for an individual that is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder).
  • the present invention enables the preparation of a customised composition (or diet) suitable for an individual which is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), wherein the customised composition or diet comprises one or more ingredient(s) that can prevent or alleviate age-related macular degeneration (or age-related macular degeneration-related disorders) and/or does not comprise components that contribute to or aggravate age-related macular degeneration (or age-related macular degeneration-related disorders).
  • Such ingredients may be any of those known in the art to prevent or alleviate age-related macular degeneration.
  • screening methods as discussed herein may identify such ingredients.
  • the preparation of customised food may be carried out using electronic means, for example by using a computer system.
  • the composition may be formulated to alter the profile of food proteins in order to minimise the potential for secondary dietary sensitivity.
  • the customised food may be hypoallergenic and/or may exclude ingredients that are poorly tolerated or cause allergies, for example gluten-containing grains such as wheat, particular protein sources such as animal proteins, milk (lactose), eggs, soy, peanuts, shellfish, fruits or tree nuts.
  • the (customised) composition may be formulated to include functional or active ingredients that help prevent or alleviate age-related macular degeneration (or an age-related macular degeneration-related disorder).
  • the present invention also relates to a method of providing a composition (eg. food) suitable for an individual who has been determined to be susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder) such as by a method of the invention.
  • a composition eg. food
  • an individual who has been determined to be susceptible to age-related macular degeneration or an age-related macular degeneration-related disorder
  • the customised composition can be made to an inventory and supplied from inventory, i.e. is pre-manufactured rather than being made to order. Therefore the composition may not be specifically designed for one particular individual but may be suitable for a relative of the individual that may also be susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder). Alternatively, the composition may be suitable for a group of individuals that are susceptible to an age- related macular degeneration-related disorder, such as members of a family. In preferred embodiment, the composition is personalised or customised to meet the nutritional requirements of a specific individual.
  • the sequences of the age-related macular degeneration variants or SNPs may be stored in an electronic format, for example in a computer database. Accordingly, the invention provides a database comprising information relating to age-related macular degeneration allelic variant sequences, which may include further information about the allelic variant, for example the level of association of the allelic variant with an age-related macular degeneration-related disorder or the frequency of the allelic variant in the population.
  • the database can comprise information regarding the substance(s), which are suitable and/or not suitable for individuals (e.g. who may possess a particular allelic variant of age-related macular degeneration).
  • a database may be used to determine the susceptibility of an individual to age-related macular degeneration (or an age-related macular degeneration-related disorder). Such a determination may be carried out by electronic means, for example by using a computer system (such as a PC). Typically, the determination will be carried out by inputting genetic data from the individual to a computer system; comparing the genetic data to a database comprising information relating to age-related macular degeneration allelic variants; and on the basis of this comparison, determining the susceptibility of the individual to an age-related macular degeneration-related disorder.
  • the invention also provides a computer program comprising program code means for performing all the steps of a method of the invention when said program is run on a computer. Also provided is a computer program product comprising program code means stored on a computer readable medium for performing a method of the invention when said program is run on a computer. A computer program product comprising program code means on a carrier wave that, when executed on a computer system, instruct the computer system to perform a method of the invention is additionally provided.
  • the invention also provides an apparatus arranged to perform a method according to the invention.
  • the apparatus typically comprises a computer system, such as a PC.
  • the computer system comprises: means for receiving genetic data from the individual; a module for comparing the data with a database comprising information relating to age-related macular degeneration allelic variants; and means for determining on the basis of said comparison the susceptibility of the individual to an age-related macular degeneration-related disorder.
  • composition/food manufacturing the manufacture of a customised composition may be controlled electronically.
  • information relating to the age-related macular degeneration allelic variant(s) present in an individual may be processed electronically to generate a customised composition.
  • the customised composition may then be used to generate electronic manufacturing instructions to control the operation of composition manufacturing apparatus.
  • the apparatus used to carry out these steps will typically comprise a computer system, such as a PC, which comprises means for processing the nutritional information to generate a customised composition; means for generating electronic manufacturing instructions to control the operation of composition manufacturing apparatus; and a composition manufacturing apparatus.
  • the composition manufacturing apparatus may comprise a packaging apparatus.
  • the packaging apparatus typically packages the composition into a container (such as a plastic or paper bag or box).
  • the apparatus may also comprise means for labelling the composition, typically after packaging.
  • the label may provide information such as: ingredient list; nutritional information; date of manufacture; best before date; weight; and types of individual(s) for which the composition is suitable.
  • Example 1 The invention is illustrated further by the Examples given below which should not be construed as being limiting.
  • Example 1 The invention is illustrated further by the Examples given below which should not be construed as being limiting.
  • a buccal (cheek) swab using a fibre brush or a Q-tip is taken.
  • the buccal (cheek) swab is stored at 4°C until analysis.
  • the buccal mucosa cells derived from this swab are used for DNA analysis and determination of the genotypes.
  • DNA extraction is performed according to commercial suppliers (e.g. Qiagen Ltd, 8634 Hombrechtikon, Switzerland) using standardized protocols e.g. "Isolation of DNA from buccal cells using the EZl DNA Tissue Kit (Qiagen Ltd, 8634 Hombrechtikon, Switzerland)". This protocol is designed for the isolation of total genomic and mitochondrial DNA from buccal cells.
  • the genotype analysis can be performed involving diverse technologies which are known to a skilled person and which are available through commercial services.
  • the following genotypes as a risk factor is included in the analysis: a) The SNP rs 11200638 ! , located 626bp upstream of the translational start site of the HTRAl gene (also known as PRSSl 1, NM 002775); b) The SNP rsl0490924 2 , residing between two genes on chromosome 10q26;
  • the SNP rs 12006381 is located 626bp upstream of the HTRAl putative translational start site and 6096bp downstream of SNP rs 10490924.
  • SEQ. ID No. 1 shows a sequence that includes the SNP rs 10490924, which is shown at position 26 (GfT).
  • the total length of the sequence is 51 bp, so the sequence includes 25bp flanking either side of the SNP.
  • SEQ ID NO:2 shows the SNP rsl 1200638 at position 26, the total length of the sequence being 51bp. For this second SNP, therefore, the sequence also gives 25bp flanking either side of the SNP.
  • step (a) of the claimed method thus involved one or more of the individual methods discussed above, i.e., by genome or genetic analysis and/or determining the macular pigment optical density and/or xanthophyll and carotenoid plasma or cellular level.
  • Example 2
  • a buccal (cheek) swab is taken from an individual (adult man, aged 60, different from the individual in Example 1) and DNA extracted as described in Example 1.
  • the DNA is analysed for the SNP rsl 1200638, (SEQ ID NO:2) located 626 base pairs (bp) upstream of the translational start site of the HTRAl gene 1 .
  • the adult having this polymorphism and is recommended a course of zeaxanthin at a dosage of 12mg/day, reducing to 6mg/day after one month.
  • Example 3 A buccal (cheek) swab was taken from an individual (adult man, aged 56) and DNA extracted as described in Example 1. The DNA is analysed for the SNP rsl 0490924 (SEQ ID NO:1), located 6722 base pairs (bp) upstream of the translational start site of the HTRAl gene 2 . The adult having this polymorphism, is recommended a course of zeaxanthin at a dosage of 12mg/day, reducing to 6mg/day after one month.
  • SNP rsl 0490924 SEQ ID NO:1
  • Soft gelatin capsules to be administered to an individual determined to be susceptible to AMD are described in the following Examples. These capsules were prepared comprising the following ingredients: Ingredient Amount per Capsule
  • One or more capsules may be taken, suitably with breakfast.
  • Soft gelatin capsules were prepared comprising the following ingredients: Ingredient Amount per Capsule
  • Soy bean oil 200 mg Examples 8 and 9
  • Soft gelatin capsules were prepared comprising the following ingredients: Ingredient Amount per Capsule
  • Example 10 Soft gelatin capsules were prepared comprising the following ingredients:
  • Soft gelatin capsules was prepared comprising the following ingredients:
  • Soft gelatin capsules were prepared comprising the following ingredients: Ingredient Amount per Capsule

Abstract

A method of prevention of age-related macular degeneration (AMD) involves, in a first step, taking a sample from an individual and detecting the presence of a polymorphism (rsl 1200638 or rs 10490924, or an SNP related to or associated with a (high temperature requirement) serine protease). In a second step the individual is provided with a substance that can prevent AMD, or can mitigate or alleviate symptoms of AMD. The substance is a medication comprising lutein and/or zeaxanthin and/or certain antioxidants (or a mixture thereof) is tailored to that individual. The invention also provides a method of determining the substance to be administered to an individual, which individual may be susceptible of having age-related macular degeneration (or an age-related macular degeneration-related disorder) by detecting the presence of the polymorphism and identifying a substance capable of preventing or treating age-related macular degeneration in that individual. The method may additionally comprise providing (such as administering or communicating) the substance (or its identity) to the individual.

Description

METHOD FOR PREVENTION OF AGE-RELATED MACULAR DEGENERATION (AMD)
Field of the invention The present invention relates to a novel method for the (treatment and/or) prevention of age related macular degeneration (AMD). It relates to the diagnosis and/or treatment of age-related macular degeneration (or an age-related macular degeneration-related disorder) in an individual (or subject), usually by determining susceptibility of the individual to age- related macular degeneration and, on the basis of that determination, selecting or identifying (and administering) a substance to the individual.
Background of the invention
As the most common cause of vision loss among people over the age of 60, macular degeneration impacts millions of older adults every year. The disease affects central vision and can sometimes make it difficult to read, drive or perform other activities requiring fine, detailed vision. When the macula is damaged, the eye loses its ability to see detail, such as small print, facial features or small objects. The damaged parts of the macula often cause scotomas, or localized areas of vision loss.
There are two types of the disease: dry macular degeneration and wet macular degeneration. Ninety percent of people who have macular degeneration have the dry form of the condition. In dry macular degeneration or atrophic macular degeneration, waste products may accumulate in the tissues underneath the macula forming yellowish deposits called drusen. The continued presence of drusen interferes with the blood flow to the retina and, in particular, to the macula. Less blood flow reduces the nourishment to the macula causing its light sensitive cells to stop working efficiently, or atrophy.
With wet macular degeneration, new weak blood vessels may grow in or under the retina causing fluid and blood to leak into the space under the macula. As a result, wet macular degeneration is sometimes called exudative macular degeneration, or described as choroidal neovascularization. The choroid is the area of blood vessels beneath the retina, and neovascularization refers to growth of new blood vessels in tissue. In choroidal neovascularization, blood vessels from the choroid grow into the macula.
The most common early sign of dry macular degeneration is blurred vision. As fewer cells in the macula are able to function, people will see details less clearly in front of them, such as faces or words in a book. If the loss of these light-sensing cells becomes great, people may see a small - but growing - blind spot in the middle of their vision.
The classic early symptom of wet macular degeneration is that straight lines appear crooked. This occurs when fluid from the leaking blood vessels gathers and lifts the macula, distorting vision. A small blind spot may also appear in wet macular degeneration, resulting in loss of one's central vision.
Regular eye exams are necessary for early detection of macular degeneration since symptoms may or may not be present in people who have the disease. Early drusen can be seen in an eye exam before symptoms develop. The disease typically develops over an extended period of time and becomes apparent mostly not before it has reached an advanced stage. Further, while the etiology so far has remained largely unclear it has been reported that susceptibility for AMD may inter alia be genetically predetermined.
The present invention relates to the identification of those subjects which can have increased risk for developing (dry and/or wet) AMD and with the treatment and, particularly, prevention of AMD in those subjects suitably at an early as possible point in time.
Summary of the invention The present invention thus relates to a method for treatment and/or prevention of age- related macular degeneration (AMD), which method comprises:
(a) detecting one or more polymorphism(s) in a sample taken from a subject (or individual, the terms are used interchangeably), or typing an individual for one or more polymorphisms, wherein the polymorphism is an SNP as shown in Table 1 , such as rsl 1200638 or rs 10490924, or is related to or associated with a (e.g. high temperature requirement) serine protease; and
(b) providing an effective amount of a substance to the individual, which substance can, or is able to, prevent or treat AMD or mitigate or alleviate symptoms of AMD.
Suitably the substance is a (preferably macular) carotenoid, in particular a xanthophyll, such as lutein and/or zeaxanthin and/or vitamin C, vitamin E; beta carotene, zinc and/or copper, and/or or a mixture thereof (the AREDS Cocktail, as described later).
The present invention also provides a method of determining a substance to be administered to an individual, which individual may be susceptible of having age-related macular degeneration AMD (which term includes a (wet or dry) age-related macular degeneration-related disorder or condition unless otherwise specified), the method comprising: a) detecting or typing for one or more polymorphism(s), usually in a sample from an individual, which may indicate the susceptibility of the individual to age-related macular degeneration (AMD), as described earlier; and b) on the basis of the determination in (a), identifying or selecting a substance capable of preventing (and/or treating) age-related macular degeneration (AMD) in that individual. The method may additionally comprise: c) providing (such as administering or communicating) the substance (or its identity) to the individual.
The invention further provides: i) means capable of detecting the or each SNP (or haplotype or allelic variant) related or associated with AMD (in a sample from an individual) and means for providing (such as administering or communicating) a substance (or its identity) capable of preventing (or treating) age-related macular degeneration to the individual; ii) a kit for carrying out the method of the invention comprising means for detecting an SNP or haplotype or allelic variant or combination of SNPs and an effective amount of such a substance, e.g. a (preferably macular) carotenoid, in particular a xanthophyll, such as lutein and/or (optionally meso-) zeaxanthin and/or vitamin C, vitamin E; beta carotene, zinc and/or copper, and/or or a mixture thereof (the AREDS Cocktail); iii) a method of preparing a customised or personalised composition for an individual which is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), the method comprising:
(a) detecting a polymorphism (in a sample from an individual), which may indicate whether the individual is susceptible to age-related macular degeneration, or age-related macular degeneration-related disorder, in the method of the invention; and
(b) preparing a composition suitable for, or tailored to, the individual (as described earlier); iv) a method of providing a customised composition, comprising providing a composition suitable for a subject which is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), wherein the individual has been (e.g. genetically) determined to be susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), using one or more of the polymorphisms disclosed herein; v) a method of identifying a substance for the treatment or prevention (which includes delaying progression or preventing occurrence of) of age-related macular degeneration (or an age-related macular degeneration-related disorder), the method comprising:
(a) contacting an age-related macular degeneration haplotype (or allelic variant) polypeptide or a polynucleotide related or associated with AMD, or which encodes an age- related macular degeneration allelic variant related or associated with AMD, or with a test agent; and
(b) determining whether the substance is capable of binding to the polypeptide or modulating the activity or expression of the polypeptide or polynucleotide, and providing (such as administering or communicating) the substance (or its identity) to an individual; vi) use of a compound which is therapeutic for age-related macular degeneration (or an age-related macular degeneration-related disorder), optionally in the manufacture of a medicament, for the prevention or treatment of age-related macular degeneration (or an age-related macular degeneration-related disorder) in a individual that has been identified as being susceptible to age-related macular degeneration by a method of the invention; vii) a method of treating an individual for, or preventing, age-related macular degeneration (or an age-related macular degeneration-related disorder), the method comprising administering to the individual an (effective amount of a) therapeutic substance (or compound) which can prevent or treat AMD or a related disorder, wherein the individual has been identified as being susceptible to age-related macular degeneration, or an age-related macular degeneration-related disorder, by a method of the invention; viii) a database comprising information relating to age-related macular degeneration haplotypes or allelic variants and optionally their association with age-related macular degeneration related disorder(s) and/or substances capable of preventing or treating age- related macular degeneration; ix) a method for determining whether an individual is susceptible to age-related macular degeneration, or an age-related macular degeneration-related disorder, the method comprising:
(a) inputting data of one or more haplotype(s), allelic variant(s) and/or SNPs (or combination thereof) present in the subject to a computer system; (b) comparing the data to a computer database, which database comprises information relating to haplotype(s) and/or allelic variants related or associated with AMD and the age-related macular degeneration related disorder susceptibility associated with the haplotype(s) and/or variants; and (c) determining, on the basis of the comparison, whether the individual is susceptible to age-related macular degeneration or a related disorder; x) a computer program comprising program code means that, when executed on a computer system, instructs the computer system to perform a method according to the invention; xi) a computer program product comprising a computer-readable storage medium having recorded thereon a computer program according to the invention; xii) a computer program product comprising program code means on a carrier wave, which program code means, when executed on a computer system, instructs the computer system to perform a method according to the invention; xiii) a computer system arranged to perform a method according to the invention, such as comprising:
(a) means for receiving data of the one or more haplotype(s) and/or allelic variant(s) and/or SNPs (or combination thereof) related to or associated with AMD present in the individual; (b) a module for comparing the data with a database comprising information relating to haplotype(s), allelic variants and/or SNPs (or combination thereof) related to or associated with AMD and the age-related macular degeneration related disorder susceptibility associated with the haplotype(s) and/or variants; and
(c) means for determining, on the basis of said comparison whether the individual is susceptible to age-related macular degeneration (or an age-related macular degeneration- related disorder); xiv) a method of preparing a customised composition for an individual which is susceptible to age-related macular degeneration (or an age-related macular degeneration- related disorder), the method comprising: (a) determining whether the individual is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder) by a method of the invention; (b) determining (such as electronically generating) a customised composition suitable for the individual;
(c) optionally, generating (e.g. electronic) manufacturing instructions to control the operation of composition manufacturing apparatus in accordance with the customised composition formulation; and
(d) manufacturing the customised food (according to the manufacturing instructions); or xiv) use of a computer system of the invention to make a customized composition product.
Brief Description of the Sequences
SEQ ID NOs: 1 to 70 show the polynucleotide sequences encompassing the SNPs used in the invention.
Detailed description of the invention
According to the first aspect of the present invention, there is provided a method of determining a substance to be administered to an individual (or subject, the terms are used interchangeably), the method comprising: a) detecting one or more polymorphism(s) in a sample taken from an individual, or typing the individual for the presence of one or more polymorphism(s), wherein the polymorphism is rsl 1200638 or rs 10490924, or an SNP shown in Table 1, or is related to or associated with a (e.g. high temperature requirement) serine protease. This detection or typing method is used in many later aspects of the invention.
The invention can thus progress to: b) on the basis of the determination in a), identifying a substance capable of preventing and/or treating age-related macular degeneration in that individual. The method may additionally comprise: c) providing (e.g. administering or communicating) the substance (or its identity) to the individual (or subject). Step c) can thus comprise communicating the identity of that substance to the individual, for example proposing, suggesting or recommending that substance. For instance, this may involve supplementing a person's food or diet with said substance. Prevention (of AMD) in this specification includes assisting in preventing AMD, mitigating or alleviating one or more symptoms of AMD, and delaying the progression or preventing occurrence of AMD. In addition, AMD includes AMD-related disorders. The detection may thus confirm a diagnosis of AMD by screening for particular molecular markers.
Determination of susceptibility
The individual may be susceptible to age-related macular degeneration, or an age- related macular degeneration -related disorder. This may mean that they are at risk of, or have a predisposition to, age-related macular degeneration or an age-related macular degeneration-related disorder. That individual may, or may not, in fact, necessarily have an increased risk or susceptibility, depending on the determination. The determination may find that the person has an increased risk, or (on the contrary) a decreased risk, of said disorder. The detection or typing in (a) may thus comprise typing the nucleotide (present in the genome of the individual) at a position equivalent to position 26 in one or more of the sequences identified in the attached sequence listing (eg. any of SEQ ID NOs: 1 to 70).
Stage (a) may thus additionally comprise preparing or obtaining a pharmacogenomic, nutrigenomic, nutrigenetic, transcriptomic, epigenetic or proteomic and/or nutritional profile (or identity) of the individual. This may assist in determining the susceptibility (such as risk or predisposition) to a disorder. Thus, the determination may comprise: i) conducting or performing a genome or genetic analysis of the individual; and ii) preparing a pharmacogenomic, nutrigenomic, nutrigenetic, transcriptomic, epigenetic or proteomic and/or metabolomic profile and/or identity based on personal and/or clinical information from or about the individual; iii) optionally, performing a test or assay (such as on a biological sample from the individual) that can indicate susceptibility (to age-related macular degeneration).
In (i), this may involve determining an individual's genotype (for the respective loci). It can comprise identifying an allelic variant, a polymorphism (such as an SNP) a haplotype or combination thereof (e.g. SNP and haplotype) or genetic predisposition (to the relevant disorder). One may therefore be able to draw up a genetic profile of the individual, preferably relevant to the disorder. The determination may, alternatively or in addition to (ii), comprise obtaining relevant information from, or about, that individual. That information may be personal and/or clinical information. The information may relate, directly or indirectly, to age- related macular degeneration (or an age-related macular degeneration-related disorder). Such information may comprise information concerning lifestyle, health, nutritional status, diet. Other personal information of relevance can include age, sex, weight and/or ethnic background. Clinical information may comprise current drug and/or vitamin regimes, current or past treatments, familial data, health risks, family background, medical conditions and/or allergies. It may therefore involve obtaining a patient's medical history or current health status and determining their nutritional profile. The individual may be able to provide this information, for example, by completing a questionnaire.
Determining risk/susceptibility (i) Genetics In one embodiment of the present invention the identification of the individual risk of a subject of developing AMD, or suffering from AMD (which may have been undetected so far) can be accomplished by genetic (or genome) analysis, more specifically, by determining the presence of gene polymorphism(s) involved in (the development of) or related to AMD. Such a polymorphism may be present in a gene coding for a serine protease, such as a mammalian (e.g. human) serine protease, or a protease in the enzyme group EC 3.4.21.-. The protease may have a (trypsin like) catalytic or serine protease domain and/or at least one PDZ domain (e.g. at or near the C terminus). Preferably the protease may have, alternatively or in addition, an IGF BP binding domain and/or a (e.g. Kazal-type) serpin inhibitor motif (e.g. at or near the N terminus). Suitably the serine protease is a high temperature requirement (HTRr, or HTRA) protease, for example: a) HTRA 1 (also known as PRSSl 1 and L56); b) HTRA 2 (also known as PRSS25 and PARKl 3); c) HTRA 3, also known as PRSP; and/or d) HTRA 4, available as GenBank accession number AK075205.1.
Preferably the SNP is at human chromosome 10q26, and is preferably upstream of a transcription start site, such as of the HtrA 1 gene (also known as PRSSl 1, NM_002775). Preferably the SNP is present in a conserved AP2-SRF binding element. Suitably the risk allele is AA or AG, whereas the (3) normal controls carry the normal allele GG (such as for rsl 1200638, SEQ ID NO:2). However, the risk allele may be TT or TG, whereas the (3) normal controls carry the normal allele GG (such as for rsl0490924, SEQ ID NO:1). Preferred SNPs are those in SEQ ID NOs: 1, 2, 68 or 69, or are at position 26 at any of SEQ ID NOs:l to 70.
Preferably, the SNP is present in a serine protease but is expressed in the retina and/or RPE.
Preferably, the SNP resides between two genes on chromosome 10q26. One such gene may be a homology domain containing protein, for example a pleckstrin homology domain containing protein, such PLEKHAl, for example that of GenBank ID 59338. The other (flanking) gene may be a serine protease, such as a heat shock serine protease, for example an HtrA gene. Preferred SNPs are therefore rsl 0490924 and/or rsl 1200638. The SNP rsl 1200638 is located 626 base pairs (bp) upstream of the HTRAl putative translational start site. It is 6096 bp downstream of SNP rsl 0490924. Preferred SNPs are (along with the appropriate SNPs form Table 1): a) associated with ABCAl, e.g. rs2230806, rs28587567, rs35819696, rs9282543, or rs2066718; b) associated with APOB; c) associated with APOE, e.g. rs429358 or rs7412; d) associated with CD36; e) associated with CYPlAl or CYPl A2 e.g. rs2470893, rs2606345, rsl799814, or rs4646903; f) associated with CYP2D6, e.g. rs2854740 or rs2743456; g) associated with ELOVL4, e.g. rs3812153; h) associated with LDLR; i) associated with LRP6, e.g. rs7294695; j) associated with PONl, e.g. rs2237583, rs854560, rs662, or rs854552; k) associated with PPARA, e.g. rsl 800206;
1) associated with PPARG, e.g. rsl801282; and/or m) associated with VLDLR.
The applicant has found that these particular SNPs are particularly useful for identifying patients with, or who may develop,' AMD. The SNPs therefore represent a selection that allows the identification of patients that are likely to benefit from, and respond to, treatment or prevention of AMD. It allows the identification of a subset of patients that may be able to respond to AMD treatment, or least have predisposition to AMD, so that this potential condition can be treated early.
The presence of a polymorphism in a gene, for example encoding a protein which is related to the development of AMD, can be determined by methods known in the art. In general, this involves the extraction of genomic DNA by standard procedures (Sambrook J, Fritsch EF, and Maniatis T.: Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Press; 1989) from blood cells, or buccal mucosa cells, hair cells or any other DNA containing human tissues which are easily and usually non-invasively accessible. Alternatively, commercial kits can be used (i.e. QIA AMP blood kit, Qiagen or any other commercially available DNA extraction kit).
The characterization of an individual's genotype at a given locus, which relates to the determination of the variants or polymorphisms, can be performed according to standard procedures. These classic technologies (Cotton RGH, Mutation detection, Oxford, Oxford University Press, 1997) that relate to the determination of the polymorphisms include DNA sequencing using 96-channel capillary sequencers, single-strand conformation analysis using non-denaturing gel electrophoresis, denaturing gradient gel electrophoresis using the partial melting behaviour of double-stranded DNA, heteroduplex analysis involving denaturing high-performance liquid chromatography, chemical or enzymatic cleavage of mismatch pairing method, and mutation detection by coupled transcription-translation (protein truncation test) procedures involving non-sense stop codons. The more recent techniques are real-time PCR methods like TaqMan, mass spectrometry involving single- stranded PCR fragments generated by the dideoxy-nucleotide PCR methodology, DNA MicroArray technology detecting SNPs provide by commercial suppliers (e.g. Affymetrix Inc., Santa Clara, CA; Illumina, San Diego, CA and Sequenom, San Diego, CA) and many more.
Table 1, showing the SNPs used in the present invention, follows. Table 1
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
where available, dbSNP numbers (rs numbers) are given to denote for SNPs.
Table 2 below correlates the SEQ ID NOs: in the attached sequence listing with the polymorphisms of Table 1.
Table 2
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
(H) Optically
In a further embodiment of the invention, the determination or identification of the individual risk or susceptibility of a subject of developing AMD or suffering from AMD can be accomplished optically, such as by measuring optical density, e.g. of a macular pigment or other suitable optical measurement in or of the eye (e.g the retina). This can comprise measuring the level of a carotenoid, usually a macular carotenoid (such as lutein or zeaxanthin) in the eye of the individual.
This can be accomplished by different methods (Berendschot T and Norren Dv, , Arch Biochem Biophys 2004, 430:149-155, Trieschmann et al, Graefe's Arch. Clin. Exp. Ophthalmol., DOI 10.1007, 2006, Springer- Verlag), for example Heterochromatic Flicker ( (Delori FC, J Opt Soc Am A Opt Image Sci Vis. 2001, 18(6):1212-30)), Scanning Laser Ophthalmoscopy (SLO), Fundus Refiectometry, Raman Spectroscopy (Ermakov I, J Biomed Opt. 2004 Jan-Feb;9(l): 139-48) or US 5,873,831 (Bernstein/University of Utah).
(a) In a preferred embodiment of the invention, the (profile of) macular pigment optical density (MPOD) can be measured, e.g. by a technique which is based on the method described by Delori et al. (Delori FC et al., J Opt Soc Am A Opt Image Sci Vis. 2001, 18(6): 1212-30). This technique can record spatial profiles of the density of the yellow macular pigment across the retina. On a visual display, subjects view a target that alternates between two spectrally different components. One component, the blue light, is absorbed by the macular pigment, whilst the other component, which appears orange to the eye, is not absorbed by the macular pigment. This differential absorption causes an imbalance between the luminance of these two components and in turn causes the test stimulus to appear to be flickering. Flicker can be eliminated by increasing the luminance of the blue component to compensate for the absorption by the macular pigment, and the lowest luminance just required for this condition is a quantitative measure of MPOD. For the construction of MPOD profiles these measurements are done with the test target presented at different points across the retina.
(b) The heterochromatic flicker photometry (HCF) technique uses a visual display and provides a rapid and convenient macular assessment profile (MAP) test. The MAP test is based on the use of an optical notch filter to separate the outputs of the three phosphors of the display into two components, one that is absorbed maximally by the macular pigment (MP) and is derived only from the blue gun (i.e., the test beam) and the other that is based on a combination of red and green phosphor luminances and consists largely of long wavelength light that is not absorbed by the MP (i.e., the reference beam). The luminance of the reference beam is 20 cd m-2 and its modulation depth is fixed at 20%. The MAP test makes full use of the advantages of visual displays to produce stimuli of varying size at a number of randomised locations, to generate counter-phased sinusoidal modulation of the two stimulus beams. The frame rate of the display is 140 Hz and the stimulus modulation frequency is 20 Hz. The high temporal modulation frequency employed ensures that at threshold one isolates the activity of luminance flicker detection mechanisms that rely only on the combined L and M cone signals. The stimulus is presented as a short burst of flicker of approximately 0.5s duration and the subject's task is to report the presence or the absence of perceived flicker.
A modified staircase procedure with variable step sizes was then used to measure the mean luminance of the test beam needed to cancel the perception of flicker generated by the reference beam. The MAP test can be used to measure MPOD along any meridian at a number of specified locations from -8° to +8° eccentricity of the visual field. The test stimulus changes from a disc of 0.36 o diameter, when presented at the fovea, to a sector annulus when presented at one of five discrete locations on either side of fixation across the horizontal meridian: ±8°, ±6°, ±4°, ±2.5°, ±1.25°, 0°. The width of the test annulus also increases systematically with eccentricity to facilitate the detection and the nulling of luminance flicker. A central spot and radial guides are used to help the subject maintain steady fixation. Five, randomly interleaved, repeat measurements are taken at each spatial location investigated. The test is performed at a viewing distance of 0.7m and the stimulus was presented only to the right eye. Similar measurements made with the left eye can confirm previous findings to show good correlation in MPOD values between the two eyes. (c) An optical density of the macular pigment of lower than 0.2 as determined by HCF can be regarded as evidence for an existing risk of developing AMD or the existence of a macular degeneration. This may lead to the administration of a carotenoid, such as lutein, zeaxanthin and/or meso-zeazeanthin, and/or one of the antioxidants or mixture thereof as defined earlier according to step (b) of the method of the present invention
(Aleman TS, Duncan JL, Bieber ML, et al. Macular pigment and lutein supplementation in retinitis pigmentosa and Usher syndrome; Invest. Ophthalmol. Vis. Sci. 2001 ;42(8): 1873- 81; Curran-Celentano J, Hammond BR, Ciulla TA, et al. Relation between dietary intake, serum concentrations, and retinal concentrations of lutein and zeaxanthin in adults in a Midwest population, Am J Clin Nutr 2001 ;74(6):796-802; Koh HH, Murray IJ, Nolan D, et al. Plasma and macular responses to lutein supplement in subjects with and without age- related maculopathy: a pilot study. Exp. Eye Res. 2004;79(l):21-27).
(d) In still another embodiment of the present invention the identification of the individual risk of a subject of developing AMD or suffering from known or undetected AMD is accomplished by determining the xanthophyll and/or carotenoid level in a body fluid, such as blood or plasma, and/or skin. The xanthophyll and carotenoid level in plasma and/or skin can be determined by methods known in the art. For example, blood (approx.10 to 15 ml) is collected into pre-cooled Monovettes containing EDTA, and plasma prepared by centrifugation. The preparation of plasma has to be done under appropriate shielding from light. After collection, plasma samples can be stored at -35 0C in the dark. Analyses of xanthophylls and carotenoids can be performed by high pressure liquid chromatography according to published protocols (Hartmann D, et al., Am J Clin Nutr 2004;79:410 -7, Aebischer CP, et al., Methods Enzymol 1999;299:348-62.)
Xanthophyll and carotenoid plasma levels below 0.25 μmol/L, measured as described above, may be regarded as indicative for an existing risk of developing AMD or of the existence of a macular degeneration, which may require the (or benefit from) administration of lutein or zeaxanthin to the individual.
(e) A further method of determining risk or susceptibility to AMD involves assessing visual (which includes contrast) performance (such as acuity. The method may comprise detecting the response or reaction of the individual, or the individual's eye, to a visual stimulus, for example in a certain or specific colour, normally in the visible spectrum, for example blue or green light. The light or visual stimulus may be provided at two or more intensities or contrast levels. The (visible) light may be green or blue and/or may have a wavelength within 495 - 535 run or within 460 - 495 run. The method may involve assessing the level of the pigment, which may be a macular pigment, preferably one that absorbs the visible light. It may comprise detecting a reaction or response of the individual to a visual display or a visual stimulus, for example where two different intensities or contrast levels of the light provided, and the reaction or response to two or more (or each) intensity or contrast level can be assessed. In a preferred embodiment the individual is shown a display with one or more rings, such as Landolt rings, which may be blue in color and/or have a wavelength of from 470 - 800 nm. This method is described in more detail in European Patent Application Number 06255800.2, filed on 13 November 2006 in the name of DSM IP Assets B. V., the contents of which are herein incorporated by reference.
Sampling
The determination may comprise taking (a biological) sample from the individual, such as a body fluid (such as urine, saliva or blood) that may contain cells. Preferably, a sample may comprise buccal and/or skin cells, for example taken from the mouth using for example a swab.
The genetic analysis may be performed using a microarray (one or more genes on a chip) or a multiwell plate, for example in a laboratory. It may thus involve the use of a gene/DNA chip, or a strip, test tube or other solid surface suitably comprising one or more
SNPs or genetic polymorphism(s) that may, or may not, cover one or more genes, but are found in a genetic locus of interest.
Communication of substance The nature or identity of the substance can be communicated either to the individual, or their doctor, optician, physician, guardian, dietician or (genetic) counselor. The communication may be electronically, for example via a computer (a personal computer or a laptop), portable computer or mobile phone or using the internet (e.g. via email, webpage and/or blog). Alternatively, it may be communicated on paper, for example in a booklet or information pack.
The communication of the nature or identity of the substance may be provided through a handheld and/or portable device such as a bracelet, necklace or watch-type device, personal computer, cell or mobile phone, personal digital assistant (PDA), a device which may be attached to or integral with a shopping cart or trolley, a terminal (eg. to an on-line service, which may be in an outlet or retail store, such as a super-or hypermarket, pharmacy or health store), for example through the internet, a telephone with voice communication, kiosk or centralised computer system.
Genetic determination of susceptibility
The identification or determination of the risk of an individual may have been undetected, or indeed an increase or decrease to risk may not been known to that individual before the determination. It can be accomplished by genome and/or genetic analysis or, preferably, by determining the presence (or absence) of a gene polymorphism, for example involved in the development of age-related macular degeneration (or age-related macular degeneration -related disorders). The presence of a polymorphism in genes can be determined by methods known in the art. In general, this will usually involve the extraction of genomic DNA by standard procedures, for example from blood cells, or buccal mucosa cells, hair cells or any other DNA containing tissue, which is suitably easily, and usually non-invasively, accessible. Alternatively, commercially available DNA extraction kits can be employed.
The characterisation of an individual's genotype at a given locus, which may relate to the determination of variance of polymorphisms, can be performed according to standard procedures. This technology can involve the use of DNA sequencing apparatus, for example using a 96-channel capillary sequencer, a single strand confirmation analysis using non-denaturing gel electrophoresis, denaturing gradient gel electrophoresis (using the partial melting behaviour of double stranded DNA), heteroduplex analysis involving denaturing HPLC, chemical or enzymatic cleavage of mismatch pairing method and/or mutation detection by coupled transcription-translation (protein truncation test) procedures, for example involving nonsense mutations and/or variations. The most recent techniques are real-time PCR methods like TaqMan mass spectrometry involving a single-stranded PCR fragments, for example generated by the dideoxy-nucleotide PCR methodology, DNA microarray technology for detecting SNPs, as provided by commercial suppliers (such as Affymetrix Inc., Santa Clara, California (CA), Illumina, San Diego, CA and Sequenom, San Diego, CA). Substances and compositions to be provided to the individual
The substance or composition may comprise a compound, such as an active ingredient, a drug, pharmaceutical or nutraceutical. The substance may be edible and/or comprise a food, foodstuff or feed, for example a (dietary) supplement, or pharmaceutical composition.
The substance (or composition) may be in any form, for example suitable for oral administration, such as in solid form such as tablets, including effervescent tablets, soft or hard-shell capsules, or in liquid form such as solutions or suspensions, such as an oily suspension. Besides any active ingredient, the preparation may contain one or more conventional (eg pharmaceutical) carrier materials, additives and adjuvants, for example, including one or more of gelatine, vegetable gum, sugar, vegetable oil, polyalkylene glycol, flavouring agent, preservative, stabilizer, an emulsifying agent and/or a buffer. The substance, if a medicament, can be a controlled (or delayed) release formulation.
The (therapeutic) substance may be administered in various manners such as orally, intracranially, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, and subcutaneously. The pharmaceutical compositions that contain the therapeutic agent will normally be formulated with an appropriate pharmaceutically acceptable carrier or diluent depending upon the particular mode of administration being used. For instance, parenteral formulations are usually injectable fluids that use pharmaceutically and physiologically acceptable fluids such as physiological saline, balanced salt solutions, or the like as a vehicle. Oral formulations, on the other hand, may be solids, for example tablets or capsules, or liquid solutions or suspensions, hi a preferred embodiment, the therapeutic agent is administered to the individual in their diet, for example in a drink or food. The present invention may thus provide an optimisation of diet and or nutritional supplementation and or pharmaceutical administration, based on the determination of susceptibility to the relevant disorder. The optimisation, for example of nutrition or nutritional supplementation, may be for a group of individuals, usually related ones, such as a family. If the substance is a nutritional supplement, this may include foods, capsules, pills, powders, gums and liquids or other oral dosage forms. Also encompassed are nutritional supplements that can be delivered for example to the digestive system, or intravenously, as well as supplements that can be administered through other routes, such as mucous membranes. The individual supplements may comprise excipients, impurities or other components other than the substance of interest.
Once the individual's susceptibility has been determined, one can optimise the nutritional intake, in particular of the substance or composition. In this sense the identity of the substance itself, the amount, dosage and the form in which it is ingested or administered can be tailored to that individual, so that the substance is personalised for that particular individual. The result of the examination may include a proposal to reduce intake of supplement, macronutrient or foodstuff, as well as increasing or adding a substance or other nutritional substances. Once the individual has been identified as having (a risk of developing) AMD, or is suffering from known or undetected AMD, an effective amount of a (preferably macular) carotenoid, such as lutein and/or zeaxanthin and/or the AREDS cocktail (a component thereof) can be suggested or administered. The substance can be a xanthophyll (for example, a carotenoid possessing one or more oxygen atoms, such as an - OH or hydroxy group).
An effective amount of the carotenoid can be used. Preferably this is lutein, meso- zeaxanthin, and/or zeaxanthin and/or "the AREDS cocktail" (vitamin C, vitamin E, beta carotene, zinc and/or copper, AREDS Report No. 8, Arch. Ophthalmol. 2001;l 19:1417- 1436, referred to as "AREDS Cocktail", also at HKJ Ophthalmol. Vol. 4, Nr. 1, (2000), p. 31 -42) and/or one of the components of the AREDS cocktail. For the purposes of the present invention this can be e.g., within the range of from 0.001 mg per kg body weight to about 20 mg per kg body weight. More preferred is a daily dosage of about 0.01 to about 10 mg per kg body weight, and especially preferred is about 0.1 to 1.0 mg per kg body weight per day, especially for the carotenoid, e.g. lutein and/or zeaxanthin. Preferably the carotenoid, e.g. lutein and/or (optionally meso-)zeaxanthin are administered at a dosage of from 1 or 5 to 15, 30 or 50 mg/day, such as from 8 or 10 to 12, 15 or 20mg/day and may be present in compositions at that (daily) dosage. Preferred compositions can contain from 8 to 12mg lutein or zeaxanthin (and preferably both within this range).
Especially preferred for vitamin C is 1 to about 10 mg per kg body weight, for beta- carotene 0.1 to about 0.3 mg per kg body weight, for vitamin E HU to about 10 IU per kg body weight, for zinc 0.1 mg per kg body weight to about 1.5 mg kg body weight, and for copper 0.01 mg per kg body weight to about 0.05 mg per kg body weight. Zinc is preferably used as zinc oxide and copper as cupric oxide. Preferable daily dosages and/or amount in an oral (e.g. daily) formulation, such as a tablet, are as follows. The formulation may comprise an antioxidant. This may be vitamin C (such as at from 200 to 800mg, 400 to 600mg, such as 450 to 550mg). There may be 1, 2 or 3 antioxidants present. In addition or alternatively, another antioxidant is vitamin E. This may be present at a dosage of from 100 to 700 IU, such as from 200 to 600 IU, preferably from 300 to 500 IU. Another preferred antioxidant, instead of or in addition to vitamins C and E, is beta-carotene. Beta-carotene may be present at from 5 to 40mg, such as from 10 or 20 to 30 or 40mg, preferably from 13 to 18 mg. The zinc may be present as zinc oxide, and can be an amount of from 20 to 140mg, such as from 60 to lOOmg, preferably from 70 to 90mg. The copper may be present at from 1 to 2mg.
The duration of the treatment can be suitably life-long, and no shorter than the above- mentioned markers would indicate or suggest that the subject involved is no longer at risk for developing AMD or no longer suffers from AMD. Suitably, treatment is started with an initial dosage of 0.5 - l.Omg of carotenoid (eg. xanthophyll), such as lutein and/or zeaxanthin, per kg body weight per day for 1 -2 months whereupon the dosage may be lower to secure a macular pigment optical density of three times the threshold value, i.e. 0.6.
Preferably, two or more xanthophylls are present, such as a combination of lutein and zeaxanthin. In such combination these compounds are preferably used in a ratio of 0.1-1.0 : 1.0- 0.1 parts (by weight), such as from 0.5-1.0:1.0-0.5, especially 0.9-1.1 :0.9-1.1, to each other.
In accordance with the invention, the substance, such as lutein and/or zeaxanthin and/or the "AREDS Cocktail" or its individual components can be provided in any appropriate form, suitably for oral administration, e.g. as a pharmaceutical composition, or in food or beverage. The term "providing" as used herein is to be understood as denoting the act of collecting the desired active ingredients and processing them into a suitable administration form, as well as the direction for use and/or administration to the subject involved. Higher dosages and amounts can be provided to individuals who appear to be at greater risk, for example one or more polymorphisms associated or related to AMD, and so one can correlate higher dosages with greater risk (or more polymorphisms).
In still another aspect, the invention relates to the use of a (preferably macular) carotenoid, e.g. xanthophyll, such as lutein, meso-zeaxanthin and/or zeaxanthin and/or a vitamin C, beta-carotene, vitamin E, zinc and copper or a mixture thereof in the manufacture of a medicament for the treatment and/or prevention of age-related macular degeneration (AMD) in a subject which has been identified as being at risk of developing AMD, or as suffering from AMD, especially by one of the methods (of the invention) identified above. The nature of the substance (or its dosage for example), may be modified over time.
The substance and/or dosage may therefore change, depending on the individual's response to the treatment, to the subtance, or the nature or condition of AMD. The individual may be able to measure his/her response or reaction to the substance, for example the benefit of the substance administered, or of on going supplements. The individual, patient, physician, optician or other person may therefore change the substance, medicament or other composition, in view of variables such as increasing age, susceptibility to AMD and the progression (or otherwise) of AMD. The individual may therefore be able to self- monitor his/her response to the prevention or treatment that is ocurring.
Databases and foods/compositions
In the determining the susceptibility of an individual, one can obtain personal data, which may be obtained through automated data analysis, interview survey subjective analysis and/or laboratory testing. A database can be provided with information concerning available nutritional supplements, including contents, price and dosage form. A further database may include information, including risks and benefits, about constituency of nutritional supplements, for example information concerning the substance.
The invention can further include apparatus for formulating the substance, for example in a food or in a nutritional supplement, usually based on the determination of susceptibility. A specific formulation may then be provided or communicated to the individual, which may or may not be standard dosage form. The invention thus additionally contemplates a vending machine or point of sale dispensing machine which can formulate, or combine, pre-prepared dosage forms of nutritional supplements, based on the opposed nutritional supplementation or the substance to be taken by the individual. Where the point of sale dispensing machine is in a public location, an interface may be provided, for example a touch screen. Optionally, an individual may be interviewed, optionally in the presence of a trained professional, with the data inputted or accepted in an appropriate format. Thus, a trained professional, such as doctor, nurse, chiropractor, social worker or nutritionist may assist in the input in medical information, etc.
Detection of allelic variants or SNPs The detection of allelic variants according to the invention may comprise contacting a polynucleotide or protein of the individual with a specific binding agent for an AMD variant (related to or associated with AMD), and determining whether the agent binds to the polynucleotide or protein. The binding of the agent can indicate the presence of the age-related macular degeneration variant, and lack of binding of the agent may indicate the absence of the age-related macular degeneration variant.
The method is generally carried out in vitro on a sample from the individual. The sample typically comprises a body fluid and/or cells of the individual and may, for example, be obtained using a swab, such as a mouth or buccal swab. The sample may be a blood, saliva, skin, buccal mucosa (or cheek) cell or hair root sample. The sample is typically processed before the method is carried out, for example DNA extraction may be carried out. The polynucleotide or protein in the sample may be cleaved either physically or chemically, for example using a suitable enzyme. In one embodiment the part of polynucleotide in the sample is copied or amplified, for example by cloning or using a PCR based method prior to detecting the allelic variant(s) or SNP(s). In the present invention, any one or more methods may comprise determining the presence or absence of one or more age-related macular degeneration variants in the individual. The age-related macular degeneration variant is typically detected by directly determining the presence of the polymorphic sequence in a polynucleotide or protein of the individual. Such a polynucleotide is typically genomic DNA, mRNA or cDNA. The allelic variant may be detected by any suitable method such as those mentioned below.
A specific binding agent is an agent that binds with preferential or high affinity to the protein or polypeptide having the allelic variant but does not bind or binds with only low affinity to other polypeptides or proteins. The specific binding agent may be a probe or primer. The probe may be a protein (such as an antibody) or an oligonucleotide. The probe may be labelled or may be capable of being labelled indirectly. The binding of the probe to the polynucleotide or protein may be used to immobilise either the probe or the polynucleotide or protein. Generally in the method, determination of the binding of the agent to the age-related macular degeneration variant can be carried out by determining the binding of the agent to the polynucleotide or protein from the individual. However in one embodiment the agent is also able to bind the corresponding wild-type sequence, for example by binding the nucleotides or amino acids which flank the allelic variant position, although the manner of binding to the wild-type sequence will be detectably different to the binding of a polynucleotide or protein containing the allelic variant.
The method may be based on an oligonucleotide ligation assay in which two oligonucleotide probes are used. These probes can bind to adjacent areas on the polynucleotide which contains the allelic variant, allowing after binding the two probes to be ligated together by an appropriate ligase enzyme. However the presence of single mismatch within one of the probes may disrupt binding and ligation. Thus ligated probes will only occur with a polynucleotide that contains the allelic variant, and therefore the detection of the ligated product may be used to determine the presence of the allelic variant.
In one embodiment the probe is used in a heteroduplex analysis based system. In such a system when the probe is bound to polynucleotide sequence containing the allelic variant it forms a heteroduplex at the site where the allelic variant occurs and hence does not form a double strand structure. Such a heteroduplex structure can be detected by the use of single or double strand specific enzyme. Typically the probe is an RNA probe, the heteroduplex region is cleaved using RNAase H and the allelic variant is detected by detecting the cleavage products.
The method may be based on fluorescent chemical cleavage mismatch analysis which is described for example in PCR Methods and Applications 3, 268-71 (1994) and Proc. Natl. Acad. Sci. 85, 4397-4401 (1998).
In one embodiment a PCR primer is used that primes a PCR reaction only if it binds a polynucleotide containing the allelic variant, for example a sequence- or allele-specific PCR system, and the presence of the allelic variant may be determined by the detecting the PCR product. Preferably the region of the primer which is complementary to the allelic variant is at or near the 3' end of the primer. The presence of the allelic variant may be determined using a fluorescent dye and quenching agent-based PCR assay such as the Taqman PCR detection system. In a preferred embodiment, one or more of the probes and/or primers are used in a Taqman assay to detect an allelic variant. The specific binding agent may be capable of specifically binding the amino acid sequence encoded by a variant sequence. For example, the agent may be an antibody or antibody fragment. The detection method may be based on an ELISA system.
The method may be an RFLP based system. This can be used if the presence of the allelic variant in the polynucleotide creates or destroys a restriction site that is recognised by a restriction enzyme.
The presence of the allelic variant may be determined based on the change it makes to the mobility of the polynucleotide or protein during gel electrophoresis. In the case of a polynucleotide, single-stranded conformation analysis (SSCP) or denaturing gradient gel electrophoresis (DDGE) analysis may be used. In another method of detecting the allelic variant a polynucleotide comprising the polymorphic region is sequenced across the region that contains the allelic variant to determine the presence of the allelic variant.
Detection kit The invention also provides a kit that comprises means for determining the presence or absence of one or more age-related macular degeneration allelic variant(s) in an individual, hi particular, such means may include a specific binding agent, probe, primer pair or combination of primers, or antibody, including an antibody fragment, as defined herein which is capable of detecting or aiding detection of an age-related macular degeneration allelic variant. The primer pair or combination of primers may be sequence specific primers which only cause PCR amplification of a polynucleotide sequence comprising the age-related macular degeneration variant(s) to be detected, as discussed herein. The kit may also comprise a specific binding agent, probe, primer pair or combination of primers, or antibody that is capable of detecting the absence of the allelic variant. The kit may further comprise buffers or aqueous solutions.
The kit may additionally comprise one or more other reagents or instruments that enable any of the embodiments of the method mentioned above to be carried out. Such reagents or instruments may include one or more of the following: a means to detect the binding of the agent to the allelic variant, a detectable label such as a fluorescent label, an enzyme able to act on a polynucleotide, typically a polymerase, restriction enzyme, ligase, RNAse H or an enzyme which can attach a label to a polynucleotide, suitable buffer(s) or aqueous solutions for enzyme reagents, PCR primers which bind to regions flanking the allelic variant, a positive and/or negative control, a gel electrophoresis apparatus, a means to isolate DNA from sample, a means to obtain a sample from the individual, such as swab or an instrument comprising a needle, or a support comprising wells on which detection reactions can be carried out. The kit may be, or include, an array such as a polynucleotide array comprising the specific binding agent, preferably a probe, of the invention. The kit may additionally comprise a substance (or composition) for administration to the individual, as discussed before. The kit typically includes a set of instructions for using the kit.
Screening for (therapeutic) substances In one embodiment the invention provides a method for identifying a substance useful for the treatment of age-related macular degeneration, which method comprises contacting a variant age-related macular degeneration polypeptide or a polynucleotide with a test agent and determining whether the agent is capable of binding to the polypeptide or modulating the activity or expression of the polypeptide or polynucleotide. Any suitable binding assay format can be used to determine whether the age-related macular degeneration variant binds the test agent, such as the formats discussed below.
The method may be carried out in vitro, either inside or outside a cell, or in vivo. In one embodiment the method is carried out on a cell, cell culture or cell extract that comprises a variant age-related macular degeneration protein or polynucleotide. The cell may be any suitable cell, and is typically a cell in which the product is naturally expressed. The term "modulate" includes any of the ways mentioned herein in which the agent is able to modulate activity of an age-related macular degeneration variant polypeptide or polynucleotide. This may be determined by contacting the polypeptide or polynucleotide with the test agent under conditions that permit activity of the polypeptide or polynucleotide, and then determining whether the test agent is able to modulate the activity of the polypeptide or polynucleotide.
In one aspect of the invention, the test agent is a food ingredient. Hence, the invention relates to a method of screening food ingredients to determine whether they contribute to or aggravate age-related macular degeneration in susceptible individuals, or if they prevent or alleviate age-related macular degeneration.
The present invention also provides an agent identified by a screening method of the invention. An agent identified in the screening method of the invention may be used in the therapeutic treatment of AMD. Such an agent may be formulated and administered in any means or amounts as discussed below.
Customised composition (eg. food) In one aspect, the invention relates to a customised diet for an individual that is susceptible to age-related macular degeneration (or an age-related macular degeneration- related disorder).
Accordingly, the present invention enables the preparation of a customised composition (or diet) suitable for an individual which is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), wherein the customised composition or diet comprises one or more ingredient(s) that can prevent or alleviate age-related macular degeneration (or age-related macular degeneration-related disorders) and/or does not comprise components that contribute to or aggravate age-related macular degeneration (or age-related macular degeneration-related disorders). Such ingredients may be any of those known in the art to prevent or alleviate age-related macular degeneration. Alternatively, screening methods as discussed herein may identify such ingredients. The preparation of customised food may be carried out using electronic means, for example by using a computer system.
In one embodiment, the composition may be formulated to alter the profile of food proteins in order to minimise the potential for secondary dietary sensitivity. The customised food may be hypoallergenic and/or may exclude ingredients that are poorly tolerated or cause allergies, for example gluten-containing grains such as wheat, particular protein sources such as animal proteins, milk (lactose), eggs, soy, peanuts, shellfish, fruits or tree nuts. In another embodiment, the (customised) composition may be formulated to include functional or active ingredients that help prevent or alleviate age-related macular degeneration (or an age-related macular degeneration-related disorder).
The present invention also relates to a method of providing a composition (eg. food) suitable for an individual who has been determined to be susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder) such as by a method of the invention.
The customised composition can be made to an inventory and supplied from inventory, i.e. is pre-manufactured rather than being made to order. Therefore the composition may not be specifically designed for one particular individual but may be suitable for a relative of the individual that may also be susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder). Alternatively, the composition may be suitable for a group of individuals that are susceptible to an age- related macular degeneration-related disorder, such as members of a family. In preferred embodiment, the composition is personalised or customised to meet the nutritional requirements of a specific individual.
Bioinformatics The sequences of the age-related macular degeneration variants or SNPs may be stored in an electronic format, for example in a computer database. Accordingly, the invention provides a database comprising information relating to age-related macular degeneration allelic variant sequences, which may include further information about the allelic variant, for example the level of association of the allelic variant with an age-related macular degeneration-related disorder or the frequency of the allelic variant in the population. The database can comprise information regarding the substance(s), which are suitable and/or not suitable for individuals (e.g. who may possess a particular allelic variant of age-related macular degeneration).
A database may be used to determine the susceptibility of an individual to age-related macular degeneration (or an age-related macular degeneration-related disorder). Such a determination may be carried out by electronic means, for example by using a computer system (such as a PC). Typically, the determination will be carried out by inputting genetic data from the individual to a computer system; comparing the genetic data to a database comprising information relating to age-related macular degeneration allelic variants; and on the basis of this comparison, determining the susceptibility of the individual to an age-related macular degeneration-related disorder.
The invention also provides a computer program comprising program code means for performing all the steps of a method of the invention when said program is run on a computer. Also provided is a computer program product comprising program code means stored on a computer readable medium for performing a method of the invention when said program is run on a computer. A computer program product comprising program code means on a carrier wave that, when executed on a computer system, instruct the computer system to perform a method of the invention is additionally provided. The invention also provides an apparatus arranged to perform a method according to the invention. The apparatus typically comprises a computer system, such as a PC. In one embodiment, the computer system comprises: means for receiving genetic data from the individual; a module for comparing the data with a database comprising information relating to age-related macular degeneration allelic variants; and means for determining on the basis of said comparison the susceptibility of the individual to an age-related macular degeneration-related disorder.
Composition/food manufacturing In one embodiment of the invention, the manufacture of a customised composition may be controlled electronically. Typically, information relating to the age-related macular degeneration allelic variant(s) present in an individual may be processed electronically to generate a customised composition. The customised composition may then be used to generate electronic manufacturing instructions to control the operation of composition manufacturing apparatus.
The apparatus used to carry out these steps will typically comprise a computer system, such as a PC, which comprises means for processing the nutritional information to generate a customised composition; means for generating electronic manufacturing instructions to control the operation of composition manufacturing apparatus; and a composition manufacturing apparatus.
The composition manufacturing apparatus may comprise a packaging apparatus. The packaging apparatus typically packages the composition into a container (such as a plastic or paper bag or box). The apparatus may also comprise means for labelling the composition, typically after packaging. The label may provide information such as: ingredient list; nutritional information; date of manufacture; best before date; weight; and types of individual(s) for which the composition is suitable.
Preferred features and/or characteristics of one aspect of the invention are applicable to another aspect mutatis mutandis.
The invention is illustrated further by the Examples given below which should not be construed as being limiting. Example 1
From an adult individual a buccal (cheek) swab using a fibre brush or a Q-tip is taken. The buccal (cheek) swab is stored at 4°C until analysis. The buccal mucosa cells derived from this swab are used for DNA analysis and determination of the genotypes. DNA extraction is performed according to commercial suppliers (e.g. Qiagen Ltd, 8634 Hombrechtikon, Switzerland) using standardized protocols e.g. "Isolation of DNA from buccal cells using the EZl DNA Tissue Kit (Qiagen Ltd, 8634 Hombrechtikon, Switzerland)". This protocol is designed for the isolation of total genomic and mitochondrial DNA from buccal cells. The genotype analysis can be performed involving diverse technologies which are known to a skilled person and which are available through commercial services.
The following genotypes as a risk factor is included in the analysis: a) The SNP rs 11200638 ! , located 626bp upstream of the translational start site of the HTRAl gene (also known as PRSSl 1, NM 002775); b) The SNP rsl04909242, residing between two genes on chromosome 10q26;
PLKHAl, encoding a pleckstrin homology domain-containing protein (GenBank ID
59338) and HTRAl encoding a heat shock serine protease also known as PRSSl 1
(GenBank ID 5654).
The SNP rs 12006381 is located 626bp upstream of the HTRAl putative translational start site and 6096bp downstream of SNP rs 10490924.
SEQ. ID No. 1 shows a sequence that includes the SNP rs 10490924, which is shown at position 26 (GfT). The total length of the sequence is 51 bp, so the sequence includes 25bp flanking either side of the SNP.
In a similar fashion, SEQ ID NO:2 shows the SNP rsl 1200638 at position 26, the total length of the sequence being 51bp. For this second SNP, therefore, the sequence also gives 25bp flanking either side of the SNP.
The risk evaluation according to step (a) of the claimed method thus involved one or more of the individual methods discussed above, i.e., by genome or genetic analysis and/or determining the macular pigment optical density and/or xanthophyll and carotenoid plasma or cellular level. Example 2
A buccal (cheek) swab is taken from an individual (adult man, aged 60, different from the individual in Example 1) and DNA extracted as described in Example 1. The DNA is analysed for the SNP rsl 1200638, (SEQ ID NO:2) located 626 base pairs (bp) upstream of the translational start site of the HTRAl gene1. The adult having this polymorphism, and is recommended a course of zeaxanthin at a dosage of 12mg/day, reducing to 6mg/day after one month.
Example 3 A buccal (cheek) swab was taken from an individual (adult man, aged 56) and DNA extracted as described in Example 1. The DNA is analysed for the SNP rsl 0490924 (SEQ ID NO:1), located 6722 base pairs (bp) upstream of the translational start site of the HTRAl gene2. The adult having this polymorphism, is recommended a course of zeaxanthin at a dosage of 12mg/day, reducing to 6mg/day after one month.
Examples 4 and 5
Soft gelatin capsules to be administered to an individual determined to be susceptible to AMD are described in the following Examples. These capsules were prepared comprising the following ingredients: Ingredient Amount per Capsule
Lutein 8 and 10 mg
Lecithin 50 mg
Soy bean oil 200 mg
One or more capsules may be taken, suitably with breakfast.
Examples 6 and 7
Soft gelatin capsules were prepared comprising the following ingredients: Ingredient Amount per Capsule
Lutein 8 and 10 mg Zeaxanthin 8 and 10 mg
Lecithin 50 mg
Soy bean oil 200 mg Examples 8 and 9
Soft gelatin capsules were prepared comprising the following ingredients: Ingredient Amount per Capsule
Lutein 6 mg Zeaxanthin (or meso-zeaxanthin) 6 mg
Lecithin 50 mg
Soy bean oil 200 mg
Example 10 Soft gelatin capsules were prepared comprising the following ingredients:
Ingredient Amount per Capsule
Lutein 12 mg
Lecithin 50 mg
Soy bean oil 200 mg
Example 11
Soft gelatin capsules was prepared comprising the following ingredients:
Ingredient Amount per Capsule
Zeaxanthin 12 mg Lecithin 50 mg
Soy bean oil 200 mg
Example 12
Soft gelatin capsules were prepared comprising the following ingredients: Ingredient Amount per Capsule
Lutein 12 mg
Zeaxanthin 12 mg
Lecithin 50 mg
Soy bean oil 200 mg Examples 13 to 19
Eight soft gelatin capsules were prepared comprising the following ingredients:
Figure imgf000038_0001
References
1. Yang et al, Science, Vol. 314 (5801), pages 992-993, 10 November 2006.
2. DeWan et al, Science, Vol. 314 (5801), pages 989-992, 10 November 2006.

Claims

1. A method for treatment and/or prevention of age-related macular degeneration (AMD), which method comprises: a) detecting one or more polymorphism(s) in a sample taken from an individual, or typing the individual for one or more polymorphism(s), wherein the polymorphism is an SNP shown in Table 1, such as rsl 1200638 or rs 10490924 or is related to or associated with a (e.g. high temperature requirement) serine protease; and b) providing (an effective amount of) a substance to the individual, wherein the substance is able to prevent or treat age-related macular degeneration, or mitigate or alleviate symptoms of age-related macular degeneration (AMD).
2. A method according to claim 1 wherein the substance comprises (an effective amount of) a carotenoid and/or vitamin C, vitamin E; beta carotene, zinc and/or copper, and/or or a mixture thereof (the AREDS Cocktail) to said subject.
3. A method of claim 1 or 2 wherein: a) the substance is a carotenoid such as lutein, zeaxanthin and/or meso-zeaxanthin; or b) the substance is a carotenoid, such as a xanthophyll;
4. A method according to any preceding claim wherein the serine protease has a (trypsin like) catalytic or serine protease domain and/or at least one PDZ domain.
5. A method according to any preceding claim wherein the serine protease has a IGF BP binding domain and/or a serpin inhibitor motif.
6. A method according to any preceding claim wherein the serine protease is a high temperature requirement serine protease.
7. A method according to any preceding claim wherein the polymorphism is in, or related to, HTRAl, 2, 3 or 4.
8. A method according to any preceding claim wherein the polymorphism is near or associated with the HTRAl gene.
9. A method according to any preceding claim wherein the polymorphism is shown in SEQ. ID No. 1 or 2 or any of SEQ ID NOs:3 to 70.
10. A method according to any preceding claim wherein the polymorphism is: a) associated with ABCAl, e.g. rs2230806, rs28587567, rs35819696, rs9282543, or rs2066718; b) associated with APOB; c) associated with APOE, e.g. rs429358 or rs7412; d) associated with CD36; e) associated with CYPlAl, or CYP1A2 e.g. rs2470893, rs2606345, rsl799814, or rs4646903; f) associated with CYP2D6, e.g. rs2854740 or rs2743456; g) associated with ELOVL4, e.g. rs3812153; h) associated with LDLR; i) associated with LRP6, e.g. rs7294695; j) associated with PONl, e.g. rs2237583, rs854560, rs662, or rs854552; k) associated with PPARA, e.g. rsl 800206; 1) associated with PPARG, e.g. rsl 801282; and/or m) associated with VLDLR.
11. A method of any preceding claim wherein, in (b) the substance is provided at from 0.001 mg to 20 mg, preferably 0.1 mg to 1.0 mg of carotenoid (lutein and/or zeaxanthin) per kg body weight per day.
12. A method of any preceding claim wherein, in (b) the substance is provided at from 0.001 mg to 20 mg, preferably 0.1 mg to 1.0 mg per kg body weight of lutein and/or zeaxanthin plus vitamin C (1 to about 10 mg per kg body weight), beta-carotene (0.1 to about 0.3 mg per kg body weight), vitamin E (HU to about 10 IU per kg body weight), zinc (0.1 mg per kg body weight to about 1.5 mg kg body weight), copper (0.01 mg per kg body weight to about 0.05 mg per kg body weight) per day.
13. (The use of) a substance that is able to prevent or treat AMD or mitigate or alleviate symptoms of AMD, optionally in the manufacture of a medicament, for the treatment and/or prevention of age-related macular degeneration (AMD) in a subject who, on the basis of a sample taken therefrom, has been tested for the presence of a polymorphism, wherein the polymorphism is rsl 1200638 or rsl 0490924 or an SNP shown in Table 1 or is related to or associated with a (e.g. high temperature requirement) serine protease .
14. The use according to claim 13 wherein the polymorphism is as defined in any one of claims 4 to 9 and/or the substance comprises a carotenoid, such as lutein, meso-zeaxanthin and/or zeaxanthin, and/or a mixture of vitamin C, beta-carotene, vitamin E, zinc and copper.
15. The use according to claim 13 or 14 wherein the medicament contains an amount of carotenoid, such as lutein/and or zeaxanthin, which is sufficient to administer 0.001 mg to 20 mg, preferably 0.1 mg to 1.0 mg of lutein and/or zeaxanthin per kg body weight per day.
16. A method of determining a substance to be administered to an individual, the method comprising: a) detecting one or more polymorphism(s) in a sample taken from an individual, or typing an individual for one or more polymorphism(s), wherein the polymorphism is rsl 1200638 or rs 10490924 or an SNP shown in Table 1, or is related to or associated with a (e.g. high temperature requirement) serine protease; and b) on the basis of (a), identifying a substance capable of treating and/or preventing age-related macular degeneration in that individual.
17. A method according to claim 16 additionally comprising: c) providing (such as administering or communicating) the substance (or its identity) to the individual.
18. A method according to claim 16 or 17 when the determination comprises additionally: a) obtaining relevant information from the individual, such as personal and/or clinical information; b) preparing a pharmacogenomic, nutrigenomic, nutrigenetic, transcriptomic, epigenetic or proteomic and/or metabolomic profile and/or identity based on personal and/or clinical information from or about the individual.
19. A method according to claim 18 when the information comprises personal information on the lifestyle, health, nutritional status, ethnic background, diet, age, sex and/or weight of the individual.
20. A method according to claim 18 when the information comprises clinical or medical information such as current or past vitamin and/or drug regime or treatments, medical conditions and/or any allergies.
21. A method according to any of claims 16 to 20 when the determination comprises taking a biological sample of the individual, such as a body fluid, and/or a sample comprising cells and/or analysing a sample for a biomarker as an indicator of age-related macular degeneration.
22. A method according to claim 21 wherein the body fluid is urine, saliva and/or blood and/or the biological sample comprises buccal cells.
23. A method according to any of claims 16 to 22 wherein in (b), a proposal, suggestion or recommendation is made to the individual concerning the substance to be administered.
24. A method according to any of claims 16 to 23 wherein the substance is a compound (such as drug, pharmaceutical, or nutraceutical), a foodstuff, feed or dietary supplement.
25. A method of preparing a customised composition for an individual, the method comprising: a) determining whether the individual has a polymorphism, or typing the individual for the presence of a polymorphism, wherein the polymorphism is rs 11200638 or rs 10490924 or an SNP shown in Table 1 or is related to or associated with a (e.g. high temperature requirement) serine protease; and b) preparing a composition suitable for, or tailored to, the individual.
26. A method according to claim 25, wherein the customised composition comprises ingredients which prevent or alleviate age-related macular degeneration (or an age-related macular degeneration -related disorder) and/or does not comprise ingredients which contribute to or aggravate age-related macular degeneration (or an age-related macular degeneration-related disorder).
27. A method according to claim 25 wherein the customised composition comprises a therapeutic substance.
28. A method of providing a customised composition, the method comprising providing a composition suitable for an individual who has been tested for the presence of a polymorphism, wherein the polymorphism is rsl 1200638 or rs 10490924 or an SNP shown in Table 1 or is related to or associated with a (e.g. high temperature requirement) serine protease.
29. A method for identifying a substance for the prevention or treatment of age-related macular degeneration (or an age-related macular degeneration-related disorder), the method comprising: a) contacting an age-related macular degeneration haplotype and/or allelic variant polypeptide or a polynucleotide which is related to or associated with an age-related macular degeneration allelic variant or polymorphism, wherein the polymorphism is rsl 1200638 or rsl 0490924 or an SNP shown in Table 1 or is related to or associated with a (e.g. high temperature requirement) serine protease, with a test agent; and b) determining whether the agent is capable of binding to the polypeptide or modulating the activity or expression of the polypeptide or polynucleotide; and c) optionally, providing (such as administering or communicating) the substance (or its identity) to an individual.
30. Use of a compound which is therapeutic for age-related macular degeneration, or an AMD-related disorder (optionally, in the manufacture of a medicament) for the prevention or treatment of age-related macular degeneration (or an age-related macular degeneration- related disorder) in an individual that has been tested for the presence of a polymorphism, wherein the polymorphism is rsl 1200638 or rs 10490924 or an SNP shown in Table 1 or is related to or associated with a (e.g. high temperature requirement) serine protease.
31. A method of treating an individual for or preventing, age-related macular degeneration (or an age-related macular degeneration-related disorder), the method comprising administering to the individual (an effective amount of) a therapeutic compound which prevents or treats AMD or the related disorder, wherein the individual has been tested for the presence of a polymorphism, wherein the polymorphism is rsl 1200638 or rsl 0490924 or an SNP shown in Table 1 or is related to or associated with a (e.g. high temperature requirement) serine protease.
32. A database comprising information relating to age-related macular degeneration haplotype(s) and/or polymorphisms, wherein the polymorphism is rsl 1200638 or rsl 0490924 or an SNP shown in Table 1 or is related to or associated with a (e.g. high temperature requirement) serine protease, and optionally their association with age-related macular degeneration, or age-related macular degeneration-related disorder(s) and/or substances capable of preventing or treating age-related macular degeneration.
33. A method for determining whether an individual is susceptible to age-related macular degeneration (AMD), or an age-related macular degeneration-related disorder, the method comprising: a) inputting data of one or more age-related macular degeneration haplotype(s), allelic polymorphism(s) and/or SNPs (or a combination thereof) wherein the polymorphism is rsl 1200638 or rsl 0490924 or an SNP shown in Table 1 or is related to or associated with a (e.g. high temperature requirement) serine protease present in the individual to a computer system; b) comparing the data to a computer database, which database comprises information relating to haplotypes and/or allelic variants related or associated with AMD and the age-related macular degeneration susceptibility associated with the haplotype(s) and/or polymorphism(s); and c) determining on the basis of the comparison whether the individual is susceptible to age-related macular degeneration (or an age-related macular degeneration- related disorder).
34. A computer program comprising program code means for performing all the steps of claim 33 when said program is run on a computer.
35. A computer program product comprising program code means stored on a computer readable medium for performing the method of claim 16 or 33 when said program product is run on a computer.
36. A computer program product comprising program code means on a carrier wave, which program code means, when executed on a computer system, instruct the computer system to perform a method according to claim 35.
37. A computer system arranged to perform a method according to claim 33 comprising: a) means for receiving data of the one or more haplotype(s), allelic variant(s) and/or SNPs (or combination thereof) related to or associated with AMD present in the individual; b) a module for comparing the data with a database comprising information relating to age-related macular degeneration haplotype(s), allelic variants and/or SNPs (or combination thereof) related to or associated with AMD and the age-related macular degeneration susceptibility associated with the haplotype(s) and/or variants; and c) means for determining on the basis of said comparison whether the individual is susceptible to age-related macular degeneration, or an age-related macular degeneration- related disorder.
38. A method of preparing a customised composition for an individual, the method comprising: a) determining whether the individual possesses a polymorphism, wherein the polymorphism is rsl 1200638 or rs 10490924 or an SNP shown in Table 1 or is related to or associated with a (e.g. high temperature requirement) serine protease; b) (e.g. electronically) generating a customised composition suitable for the individual; c) optionally, generating electronic manufacturing instructions to control the operation of composition manufacturing apparatus in accordance with the customised composition; and d) manufacturing the customised composition (according to the electronic manufacturing instructions).
39. A computer system according to claim 38, further comprising: e) means for electronically generating a customised composition formulation suitable for the individual; f) means for generating electronic manufacturing instructions to control the operation of composition manufacturing apparatus in accordance with the customised composition; and g) a composition product manufacturing apparatus.
40. Use of a computer system as defined in claim 39 to make a customised composition.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011006161A2 (en) 2009-07-10 2011-01-13 The Regents Of The University Of Michigan Compositions and methods for diagnosing and treating macular degeneration
US7972787B2 (en) 2007-02-16 2011-07-05 Massachusetts Eye And Ear Infirmary Methods for detecting age-related macular degeneration
ES2399897A1 (en) * 2011-09-20 2013-04-04 Segugen, S.L. Method for predicting the risk of developing age-related macular degeneration disease in the spanish population (Machine-translation by Google Translate, not legally binding)
WO2015004266A1 (en) * 2013-07-12 2015-01-15 Novogenia Gmbh Personalized nutrient compositions and methods for producing these
CN105331682A (en) * 2015-09-24 2016-02-17 郑州市职业病防治院 Human PON1 gene rs854560 site polymorphism detection method and kit
CN109055526A (en) * 2018-08-17 2018-12-21 潍坊德诺泰克生物科技有限公司 For detecting the primed probe group and its application of rs662

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006088950A2 (en) * 2005-02-14 2006-08-24 University Of Iowa Research Foundation Methods and reagents for treatment and diagnosis of age-related macular degeneration

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006088950A2 (en) * 2005-02-14 2006-08-24 University Of Iowa Research Foundation Methods and reagents for treatment and diagnosis of age-related macular degeneration

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AGE-RELATED EYE DISEASE STUDY RESEARCH GROUP: "A randomized, placedo-controlled, clinical trial of high-dose supplementation with Vitamins C and E, beta carotene and zinc for age-related macular degeneration and vision loss", ARCHIVES OF OPHTHALMOLOGY, XX, XX, vol. 119, no. 10, 1 October 2001 (2001-10-01), pages 1417 - 1436, XP001098778, ISSN: 0003-9950 *
ALLIKMETS R ET AL: "Mutation of the Stargardt Disease gene (ABCR) in Age-related Macular Degeneration", SCIENCE, WASHINGTON, DC, vol. 277, 19 September 1997 (1997-09-19), pages 1805 - 1807, XP002099899, ISSN: 0036-8075 *
DEWAN ANDREW ET AL: "HTRA1 promoter polymorphism in wet age-related macular degeneration.", SCIENCE (NEW YORK, N.Y.) 10 NOV 2006, vol. 314, no. 5801, 10 November 2006 (2006-11-10), pages 989 - 992, XP002486674, ISSN: 1095-9203 *
HAGEMAN GREGORY S ET AL: "A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, vol. 102, no. 20, 17 May 2005 (2005-05-17), pages 7227 - 7232, XP002391909, ISSN: 0027-8424 *
YANG ZHENGLIN ET AL: "A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration.", SCIENCE (NEW YORK, N.Y.) 10 NOV 2006, vol. 314, no. 5801, 10 November 2006 (2006-11-10), pages 992 - 993, XP002486673, ISSN: 1095-9203 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7972787B2 (en) 2007-02-16 2011-07-05 Massachusetts Eye And Ear Infirmary Methods for detecting age-related macular degeneration
US8232056B2 (en) 2007-02-16 2012-07-31 Massachusetts Eye And Ear Infirmary Methods for detecting neovascular age-related macular degeneration
WO2011006161A2 (en) 2009-07-10 2011-01-13 The Regents Of The University Of Michigan Compositions and methods for diagnosing and treating macular degeneration
EP2451983A2 (en) * 2009-07-10 2012-05-16 The Regents of the University of Michigan Compositions and methods for diagnosing and treating macular degeneration
EP2451983A4 (en) * 2009-07-10 2012-12-05 Univ Michigan Compositions and methods for diagnosing and treating macular degeneration
EP2963128A1 (en) * 2009-07-10 2016-01-06 The Regents of the University of Michigan Compositions and methods for diagnosing and treating macular degeneration
ES2399897A1 (en) * 2011-09-20 2013-04-04 Segugen, S.L. Method for predicting the risk of developing age-related macular degeneration disease in the spanish population (Machine-translation by Google Translate, not legally binding)
WO2015004266A1 (en) * 2013-07-12 2015-01-15 Novogenia Gmbh Personalized nutrient compositions and methods for producing these
CN105331682A (en) * 2015-09-24 2016-02-17 郑州市职业病防治院 Human PON1 gene rs854560 site polymorphism detection method and kit
CN109055526A (en) * 2018-08-17 2018-12-21 潍坊德诺泰克生物科技有限公司 For detecting the primed probe group and its application of rs662

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