Substances for treatment of a retinal disease and / or retinal impairment

Abstract

The present disclosure generally relates to a composition for use in a therapeutic method of treatment or prevention of a retinal disease or retinal impairment, said composition comprising: A) carnitine, deoxycarnitine, acetyl-L-carnitine, gamma-butyrobetaine, 4- trimethylammoniobutanal, 3-hydroxy-N6,N6,N6-trimethyl-L-lysine, N6,N6,N6- trimethyl-L-lysine and / or lysine, preferably carnitine, such as L-carnitine; B) nicotinamide, nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D- ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, and / or nicotinate; C) optionally serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine, preferably serine or glycine; and D) optionally N-acetyl cysteine, cysteine and / or cystine.

Description

SUBSTANCES FOR TREATMENT OF A RETINAL DISEASE AND / OR RETINAL IMPAIRMENTTECHNICAL FIELD

[0001] The present disclosure relates the field prevention and / or treatment of a retinal disease and / or retinal impairment.BACKGROUND

[0002] Retinopathies are a group of eye diseases characterized by damage to the retina, the light-sensitive layer at the back of the eye that is crucial for vision. Retinopathies significantly impair vision and are among the leading causes of blindness.

[0003] Examples of retinopathies include diabetic retinopathy, hypertensive retinopathy, age-related macular degeneration (AMD), and retinopathy of prematurity (ROP).

[0004] In many retinopathies, retinal neovascularization is a common complication. Neovascularization is the process by which new blood vessels form from existing vessels. Neovascularization arises as the body attempts to compensate for oxygen-deprived retinal tissues. This is a natural response by the body to situations where tissues need more oxygen and nutrients. However, in the context of retinopathies, neovascularization is often abnormal and problematic. The new blood vessels that grow in the retina are usually fragile, leaky, and can lead to complications like bleeding, swelling, and even retinal detachment, all of which can impair vision.

[0005] Retinopathy of prematurity (ROP) primarily affects premature infants, particularly those born before 31 weeks of gestation or weighing less than 1250 grams at birth. The condition is caused by the incomplete development of blood vessels in the retina at birth and their abnormal growth thereafter, often exacerbated by the use of supplemental oxygen in neonatal care.

[0006] In full-term infants, retinal vascular development begins around the fourth month of gestation and is typically complete by the time of birth. This means that by birth, the blood vessels have fully formed throughout the retina, providing the necessary support and nourishment for the eye to function correctly. In contrast, premature infants are born before their retinal blood vessels have fully developed.The retinal blood vessels have not yet completed their growth towards the edges of the retina. This incomplete vascularization leaves parts of the retina without sufficient blood and nutrient supply.

[0007] When a preterm baby is exposed to high levels of oxygen in a neonatal intensive care unit (NICU), either from natural air or from medical intervention, the already formed blood vessels can constrict and cease growing. When normal oxygen levels are restored, areas of the retina that have not yet been vascularized become hypoxic (oxygen-starved), triggering the release of growth factors that stimulate the growth of new, abnormal blood vessels (neovascularization).

[0008] Retinopathy of prematurity (ROP) is the primary cause of preventable childhood blindness. ROP is also a robust biomarker for abnormal brain morphology / function [1] and long-term poor neurological outcomes [2].

[0009] Severe ROP causing visual impairment or blindness is increasing as developing countries improve survival of infants born at the limit of viability [3].

[0010] Current treatment of retinopathies, e.g. ROP is not ideal.

[0011] For example, laser photocoagulation may be used to treat the peripheral areas of the retina that have not been vascularized to reduce the drive for further abnormal blood vessel growth. Laser photocoagulation involves the use of a laser to burn areas of the peripheral retina. However, the destruction of retinal tissue is irreversible and can lead to permanent loss of peripheral or side vision.

[0012] Furthermore, local anti-VEGF eye injections have been implemented worldwide despite concern about long-term adverse systemic effects. More importantly, the long-term impact of anti-VEGF therapies in infants is not understood.

[0013] To date, no available preventative treatments for abnormal neurovascular development exist. Further exploration of molecular mechanisms underlying the disease and its progression could facilitate the development of effective and safe therapeutic options.

[0014] In view of this, there is a need for improving the prevention and treatment regimen associated with retinopathies, in particular retinopathy of prematurity (ROP).SUMMARY

[0015] An aim of the present disclosure is to provide for treatment or prevention of a retinal disease and / or retinal impairment.

[0016] In a first aspect, there is provided a composition for use in a therapeutic method of treatment or prevention of a retinal disease or retinal impairment, wherein the composition comprises:A) carnitine, deoxycarnitine, acetyl-L-carnitine, gamma-butyrobetaine, 4- trimethylammoniobutanal, 3-hydroxy-N 6,N6,N 6-trimethyl-L-lysine, N6,N6,N6- trimethyl-L-lysine and / or lysine, preferably carnitine, such as L-carnitine;B) nicotinamide, nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D- ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, and / or nicotinate;C) optionally serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine, preferably serine or glycine; andD) optionally N-acetyl cysteine, cysteine and / or cystine.

[0017] The substances defined hereinabove may stabilize retinal health, particularly in infants at risk of or suffering from ROP, and thereby reduce the severity or progression of the disease.

[0018] The inventors have utilized the mouse model of oxygen-induced retinopathy (OIR) and proteomics to investigate the underlying mechanisms of neovascularization and the effect that the substances mentioned above have on stabilizing the retina and reducing neovascularization.

[0019] OIR is a well-established animal model used to simulate human retinal diseases characterized by abnormal blood vessel growth, such as ROP. In this model, neonatal mice are exposed to alternating cycles of high and normal oxygen levels. This exposure mimics the conditions premature infants may experience, leading to similar pathological changes in the retina. A high oxygen exposure initially causes vaso-obliteration, where existing blood vessels in the retina constrict and degenerate due to the high oxygen environment, mimicking the loss of blood vessels observed in premature infants who receive high oxygen treatment. The shift of returning to normal oxygen creates hypoxic conditions in areas of the retina that lost their normal blood vessels. The hypoxia (lack of oxygen) triggers the body to release growth factorslike VEGF (Vascular Endothelial Growth Factor), leading to neovascularization, where new, but often abnormal and fragile, blood vessels begin to form.

[0020] As demonstrated in the Examples, the inventors have found that carnitine may inhibit vaso-obliteration and retinal neovascularization.

[0021] As mentioned hereinbefore, neovascularization is the body’s response to lack of blood supply and is the process of forming new (abnormal) blood vessels which are prone to leaking.

[0022] Vaso-obliteration, i.e. loss or closure of blood vessels, may occur when the blood vessels in the retina begin to degenerate or cease to function properly. This may be due to exposure to high levels of oxygen, especially in premature infants who are often placed in high-oxygen environments to help them breathe. When these tiny blood vessels close up or obliterate, parts of the retina become deprived of blood, which carries essential oxygen and nutrients. This lack of blood supply creates areas of oxygen shortage (hypoxia) in the retina.

[0023] In diseases affecting the retina, such as ROP, vaso-obliteration may set off a chain reaction. The obliteration causes parts of the retina to become deprived of oxygen. In response, the body tries to compensate by triggering neovascularization to restore blood flow.

[0024] Accordingly, the dual action of carnitine provides a promising strategy in preventing the progression of retinal diseases like ROP where both vessel loss and abnormal vessel growth are central features.

[0025] The inventors have also found that nicotinamide may significantly reduce retinal neovascularization and improve revascularization.

[0026] Furthermore, as demonstrated in the Examples, serine has been shown to decrease retinal neovascularization.

[0027] In addition, the inventors have found that glycine may promote retinal revascularization. In other words, glycine may help restore the normal structure and function of blood vessels after damage or obliteration caused by high oxygen exposure. Accordingly, glycine may support the survival or health of existing retinal blood vessels or inhibit their initial degeneration in response to high oxygen levels. In this regard, the retina may be stabilized and the extent of hypoxic areas that trigger harmful neovascularization may be reduced.

[0028] These findings indicate that the substances may form a valuable treatment or prevention strategy for diseases characterized by similar vascular damage and maladaptive repair; i.e. retinopathies, such as ROP.

[0029] Preferably, the composition of the present disclosure comprises each of the components A-D mentioned hereinbefore. The inventors have found that a composition comprising each of the components in categories A-D significantly reduces neovascularization (NV) in mice.

[0030] Considering metabolic pathways, the following alternatives to the above- mentioned substances are conceivable:Substance Alternatives serine glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserineNAC Cysteine and / or cystineL-carnitine deoxycarnitine, acetyl-L-carnitine, gammabutyrobetaine, 4-trimethylammoniobutanal, 3- hydroxy-N 6,N6,N6-trimethyl-L-lysine, N6,N6,N6- trimethyl-L-lysine and / or lysineNR quinolinate, deamino-NAD+, nicotinate D- ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, nicotinamide and / or nicotinate

[0031] In exemplary embodiments, the retinal disease or retinal impairment is selected from the group consisting of diabetic retinopathy, retinopathy of prematurity (ROP), hypertensive retinopathy, central serous retinopathy, and age-related macular degeneration (AMD), preferably wherein the retinal disease or retinal impairment is retinopathy of prematurity (ROP).

[0032] In the context of the present disclosure, a retinal disease typically means a retinopathy. Retinopathies are a group of disorders affecting the retina, and whichshare common pathological features, such as vaso-obliteration and neovascularization, being critical factors in the progression of these diseases.

[0033] The composition of the present disclosure may advantageously be used in the prevention or treatment of each of the retinal diseases and / or impairments mentioned hereinabove.

[0034] In exemplary embodiments, the composition may further comprise pyruvate.

[0035] The inventors have found that pyruvate supplementation during the phase of neovessel formation significantly inhibits retinal neovascularization in mouse models of OIR.

[0036] In exemplary embodiments, the composition may further comprise MCT oil.

[0037] MCT oil (medium-chain triglyceride oil) is composed of fatty acids that have a length of 6 to 12 carbon atoms. MCT oil may be rapidly absorbed by the body as it may bypass certain processes in fat metabolism.

[0038] The inventors have found that MCT oil, administered during the early hyperoxic phase of development in OIR mice, may inhibit neovascularization. Accordingly, MCT oil may be a valuable component in preventing the progression of ROP in premature infants.

[0039] In exemplary embodiments, the composition may further comprise docosahexaenoic acid (DHA) and / or arachidonic acid (AA).

[0040] In exemplary embodiments, the composition may comprise docosahexaenoic acid (DHA) and arachidonic acid (AA), and wherein the molar ratio of arachidonic acid (AA) to docosahexaenoic acid (DHA) maybe from 1:1 to 1:7.

[0041] The inventors have evaluated the administration of DHA, and AA, as well as a combination of DHA and AA on retinal vascularization and neovascularization in an OIR model. A combined administration of AA and DHA (in lower concentrations) was shown to be more effective in improving retinal vascularization; i.e. in improving normal, healthy blood vessel growth in the retina compared to AA, and DHA alone (in higher concentration).

[0042] Furthermore, the inventors have found that the administration of DHA alone and a combination of DHA and AA suppressed retinal neovascularization in an OIR model. In other words, the administration of DHA alone and a combination of DHA and AA was effective at suppressing abnormal new blood vessel growth (neovascularization) in the retina compared to AA alone.

[0043] In exemplary embodiments, the composition may further comprise folic acid, 5-formyltetrahydrofolate, and / or 5-methyltetrahydrofolate (5-MTHF).

[0044] As demonstrated in the Example section, folic acid supplementation was shown to inhibit retinal neovascularization when administered during the hyperoxic phase, suggesting a preventative effect against ROP's progression by stabilizing the vascular development under oxidative stress conditions.

[0045] In exemplary embodiments, the method may comprise enteral or parental administration of the composition. Preferably, the method comprises enteral administration.

[0046] Enteral administration refers to the delivery of substances directly into the gastrointestinal (GI) tract. Enteral administration typically includes oral administration or administration through tube feeding.

[0047] Oral administration is preferred for adults, and may be used in the treatment or prevention regimen of e.g. diabetic retinopathy, hypertensive retinopathy, central serous retinopathy, and age-related macular degeneration (AMD).

[0048] Administration by tube feeding is generally preferred for infants and premature infants. Infants, especially premature infants often require administration by tube feeding (e.g., via nasogastric tubes) since they are not able to swallow safely or efficiently. Similarly, adults who are critically ill or have certain disabilities might also benefit from administration by tube feeding.

[0049] Administration by tube feeding may be used in the treatment or prevention regimen of retinopathy of prematurity (ROP).

[0050] Parenteral administration of the substances is also conceivable. Parenteral administration refers to the delivery of substances by injection or infusion.

[0051] In exemplary embodiments, the composition for use according to any one of the preceding items may be in the form of a powderous mixture.

[0052] The composition is preferably a solid, e.g. a solid powder. Such a powder can be mixed with water or breastmilk, e.g. by the patient / consumer, a nurse or a physician. It is also conceivable that the composition is an aqueous solution or suspension (“cocktail”), which facilitates convenient enteral administration.

[0053] The subject is preferably a human subject. The subject may be an adult or an infant, e.g. a premature infant.

[0054] In exemplary embodiments, the method may comprise administration of:A) in a dose of 0.100-2.50 mmol / kg / day, such as 0.200-2.00 mmol / kg / day, such as 0.230-1.00 mmol / kg / day, such as 0.300-0.800 mmol / kg / day;B) in a dose of 0.015-0.39 mmol / kg / day, such as 0.030-0.31 mmol / kg / day, such as 0.040-0.20 mmol / kg / day; optionally C) in a dose of 0.48-24 mmol / kg / day, such as 0.48-4.8 mmol / kg / day, such as 1.8-4.8 mmol / kg / day, such as 2.9-4. mmol / kg / day; and optionally D) in a dose of 0.31-3.05 mmol / kg / day, such as 0.31-1.84 mmol / kg / day, such as 0.40-1.23 mmol / kg / day.

[0055] In exemplary embodiments, the method may comprise administration of pyruvate in a dose of 0.08-9.9 mmol / kg / day.

[0056] In exemplary embodiments, the method may comprise administration of MCT oil in a dose of o.1-5.0 ml / kg / day.

[0057] In exemplary embodiments, the method may comprise administration of docosahexaenoic acid (DHA) in a dose of 0.152-1.52 mmol / kg / day.

[0058] In exemplary embodiments, the method may comprise administration of arachidonic acid (AA) in a dose of 0.164-16.4 mmol / kg / day.

[0059] In exemplary embodiments, the method may comprise administration of folic acid, 5-formyl-tetrahydrofolate, and / or 5-methyltetrahydrofolate (5-MTHF) in a dose of 0.045-1.0, e.g. 0.045-0.45 mmol / kg / day.

[0060] For the patient / consumer, it is not necessary to take the substances of the present disclosure simultaneously. A therapeutic effect can also be achieved if thesubstances are taken separately or sequentially, preferably within a day, more preferably within an hour.

[0061] Hence, according to another aspect, there is provided substances comprising:A) carnitine, deoxycarnitine, acetyl-L-carnitine, gamma-butyrobetaine, 4- trimethylammoniobutanal, 3-hydroxy-N 6,N6,N 6-trimethyl-L-lysine, N6,N6,N6- trimethyl-L-lysine and / or lysine, preferably carnitine, such as L-carnitine;B) nicotinamide, nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D- ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, and / or nicotinate;C) optionally serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine, preferably serine or glycine; andD) optionally N-acetyl cysteine, cysteine and / or cystine for simultaneous, separate or sequential use in a therapeutic method of treatment or prevention of a retinal disease and / or retinal impairment.

[0062] Preferably, each of the components A-D is used for simultaneous, separate or sequential use in a therapeutic method of treatment or prevention of a retinal disease and / or retinal impairment.

[0063] Further features of, and advantages with, the present disclosure will become apparent when studying the appended claims and the following description. The skilled addressee realizes that different features of the present disclosure may be combined to create embodiments other than those described in the following, without departing from the scope of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 illustrates A) flat mounted retinas with less neovascularisation (NV) in carnitine treated animals than in controls and similar amount of vaso- obliteration (VO), which indicates that carnitine does not affect normal vessel growth. In Fig 1B), the data is illustrated in bar graphs with individual values and p-values for NV and VO respectively. Bodyweight was similar in the groups.

[0065] Figure 2 illustrates A) flat-mounted retinas with significantly less neovascularization (NV) in Nicotinamide-treated animals compared to control animals and non-significant difference in vaso-obliteration (VO) compared tocontrols. In Fig 2B), the data is illustrated in bar graphs with individual values and p- values for NV and VO, respectively. Bodyweight was similar between the two groups.

[0066] Figure 3 illustrates A) flat mounted retinas with less neovascularisation (NV) in serine treated animals than in controls and similar amount of vaso- obliteration (VO), which indicates that serine does not affect normal vessel growth. In Fig 3B), the data is illustrated in bar graphs with individual values and p-values for NV and VO respectively. Bodyweight was similar in the groups.

[0067] Figure 4 illustrates A) flat mounted retinas with less vaso-obliteration (VO) in glycine treated animals than in controls, indicating improved normal vessel growth and similar amount of neovascularisation (NV), which indicates that glycine does not affect abnormal vessel growth. In Fig 4B), the data is illustrated in bar graphs with individual values and p-values for NV and VO respectively. Bodyweight was similar in the groups.

[0068] Figure 5 illustrates A) flat mounted retinas with less neovascularisation (NV) in folic acid treated animals than in controls and similar amount of vaso- obliteration (VO), which indicates that folic acid does not affect normal vessel growth. In Fig 5B), the data is illustrated in bar graphs with individual values and p-values for NV and VO respectively. Bodyweight was similar in the groups.

[0069] Figure 6 illustrates A) flat-mounted retinas with less neovascularization (NV) in pyruvate-treated animals than in controls and a trend of improved vaso- obliteration (VO), which indicates that pyruvate might improve normal vessel growth. In Fig 6B), the data is illustrated in bar graphs with individual values and p- values for NV and VO respectively.

[0070] Figure 7 illustrates A) flat mounted retinas with less neovascularisation (NV) in MCT-oil treated animals than in controls and less vaso-obliteration (VO), which indicates that MCT-oil also improves normal vessel growth. In Fig 7B), the data is illustrated in bar graphs with individual values and p-values for NV and VO respectively. Bodyweight was similar in both groups.

[0071] Figure 8 illustrates A) flat-mounted retinas with less neovascularization (NV) in DHA and AA / DHA- treated animals compared to animals treated with AA only and less vaso-obliteration (VO) in DHA and AA / DHA- treated compared to AA- treated mice, wherein the most profound effect on the normalization of vessel growthwas observed in the AA / DHA treated mice. In Fig 8B), the data is illustrated in bar graphs with individual values and p-values for NV and VO respectively. Bodyweight was similar in all three groups.

[0072] Figure 9 illustrates A) flat-mounted retinas with less neovascularization (NV) and similar amount of vaso-obliteration (VO) observed in mice treated with a “fuel mixture”, i.e. a composition comprising L-serine, L-carnitine, nicotinamide and N-acetyl cysteine compared to vehicle-treated animals. In Figure 9B), the data is illustrated in bar graphs with individual values and p-values for NV and VO respectively. Bodyweight was similar in both groups.

[0073] Figure 10 illustrates the serum levels of carnitine in extremely preterm infants during the first four weeks of life with ROP (lower, dark grey line) and without ROP (upper, grey line).DETAILED DESCRIPTION

[0074] As a first aspect of the present disclosure, there is provided a composition for use in a therapeutic method of treatment or prevention of a retinal disease or retinal impairment.

[0075] The retinopathy may be diabetic retinopathy, retinopathy of prematurity (ROP), hypertensive retinopathy, or central serous retinopathy. Preferably, the composition is for use in a therapeutic method of treatment or prevention of retinopathy of prematurity (ROP).

[0076] The composition comprises:A) carnitine, deoxycarnitine, acetyl-L-carnitine, gamma-butyrobetaine, 4- trimethylammoniobutanal, 3-hydroxy-N 6,N6,N 6-trimethyl-L-lysine, N6,N6,N6- trimethyl-L-lysine and / or lysine, preferably carnitine, such as L-carnitine;B) nicotinamide, nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D- ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, and / or nicotinate;C) optionally serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine, preferably serine or glycine; andD) optionally N-acetyl cysteine, cysteine and / or cystine.

[0077] The substance of group A) is preferably carnitine, optionally in the form of a carnitine salt, such as carnitine tartrate. Most preferably, the substance of group A) is L-carnitine, optionally in the form of a L-carnitine salt, such as L-carnitine tartrate.

[0078] The substance of group B) is preferably nicotinamide, nicotinamide riboside (NR), nicotinamide D-ribonucleotide or nicotinate. In the art, nicotinamide D-ribonucleotide is also referred to as nicotinamide mononucleotide (NMN). NR is typically provided as a salt, preferably NR chloride. Nicotinate maybe provided as inositol hexanicotinate, which is also referred to as flush free niacin since it allows slower absorption.

[0079] In group C), serine and glycine are preferred. The most preferred substance in group C) is serine, typically L-serine.

[0080] In group D), N-acetyl cysteine (NAC) and cysteine are preferred. The most preferred substance in group D) is NAC.

[0081] In embodiments including the substance(s) of group C), the substance(s) of group C) may be included in a higher molar amount than the substance(s) of group B). With efficacy and toxicity also considered, the molar ratio of C) to B) maybe between 250:1 and 1.5:1 and typically between 150:1 and 3:1. Preferably, the molar ratio is between 90:1 and 10:1, more preferably between 50:1 and 20:1.

[0082] The molar ratio of C) to D), considering efficacy and toxicity, is typically between 20:1 and 1:4, such as between 16:1 and 1:4, such as between 16:1 and 1.5:1, preferably between 12:1 and 1.5:1 and more preferably between 10:1 and 3:1.

[0083] The molar ratio of C) to A), considering efficacy and toxicity, is normally between 150:1 and 1:1, typically between 100:1 and 2:1, preferably between 30:1 and 3:1, more preferably between 15:1 and 4:1.

[0084] The above ratios entail that a patient consuming the composition can obtain appropriate doses of the respective substances.

[0085] The mode of administration may be parenteral or enteral administration. Typically, the mode of administration is enteral administration. The method may comprise administration of:A) in a dose of 0.100-2.50 mmol / kg / day, such as 0.200-2.00 mmol / kg / day, such as 0.230-1.00 mmol / kg / day, such as 0.300-0.800 mmol / kg / day;B) in a dose of 0.015-0.39 mmol / kg / day, such as 0.030-0.31 mmol / kg / day, such as 0.040-0.20 mmol / kg / day; optionally C) in a dose of 0.48-24 mmol / kg / day, such as 0.48-4.8 mmol / kg / day, such as 1.8-4.8 mmol / kg / day, such as 2.9-4. mmol / kg / day; and optionally D) in a dose of 0.31-3.05 mmol / kg / day, such as 0.31-1.84 mmol / kg / day, such as 0.40-1.23 mmol / kg / day.

[0086] The daily dose may be reached by administering one or more doses per day to the patient. The number of daily doses may e.g. be one, two or three.

[0087] The method of treatment may be carried out for a period of at least one week, e.g. 1-52 weeks, such as 3-46 weeks, such as 6-30 weeks.

[0088] As mentioned hereinbefore, the composition may be in the form of a powderous mixture. Alternatively, the composition is in the form of an aqueous solution.

[0089] In embodiments where the composition is in the form of an aqueous solution, the concentration of A) maybe 0.009-0.38 mmol / ml, typically 0.009- 0.19 mmol / ml, preferably 0.016-0.16 mmol / ml and more preferably 0.028- 0.12 mmol / ml.

[0090] The concentration of B) maybe 0.006-0.12 mmol / ml, preferably 0.012- 0.08 mmol / ml and more preferably 0.018-0.07 mmol / ml.

[0091] The concentration of C) maybe 0.20-2.4 mmol / ml, preferably 0.40-2.4 mmol / ml and more preferably 0.60-2.4 mmol / ml.

[0092] The concentration of D) maybe 0.09-0.90 mmol / ml, typically 0.09-0.54 mmol / ml, preferably 0.11-0.40 mmol / ml and more preferably 0.013-0.30 mmol / ml.

[0093] The composition may further comprise pyruvate.

[0094] The dose of pyruvate maybe 0.08-9.9 mmol / kg / day, e.g. o.1-5.0 mmol / kg / day.

[0095] The composition may further comprise MCT oil.

[0096] The MCT oil may be composed of caproic acid, caprylic acid, capric acid and / or lauric acid.

[0097] The MCT oil may be derived from coconut oil and / or palm kernel oil.

[0098] The dose of MCT oil maybe 0.1-5.0 ml / kg / day.

[0099] The composition may further comprise docosahexaenoic acid (DHA) and / or arachidonic acid (AA).

[0100] The dose of docosahexaenoic acid DHA may be 0.152-1.52 mmol / kg / day.

[0101] The dose of arachidonic acid (AA) maybe 0.164-16.4 mmol / kg / day.

[0102] The composition may comprise docosahexaenoic acid (DHA) and arachidonic acid (AA). The molar ratio of arachidonic acid (AA) to docosahexaenoic acid (DHA) maybe from 1:1 to 1:7.

[0103] The composition may further comprise folic acid, 5- formyltetrahydrofolate, and / or 5-methyltetrahydrofolate (5-MTHF), preferably folic acid.

[0104] The dose of folic acid, 5-formyltetrahydrofolate, and / or 5- methyltetrahydrofolate (5-MTHF) maybe 0.045-0.1, e.g. 0.045-0.45 mmol / kg / day.

[0105] The substances of the present disclosure are preferably a significant part of the composition. For example, the substances included in groups A)-D) may amount to at least 10 %, such as at least 25 %, such as at least 50 % of the dry weight of the composition of the first aspect. In one embodiment, the weight of serine is at least 10 %, such as at least 25 %, such as at least 40 % of the dry weight of the composition.

[0106] The composition may comprise one or more tasting agent(s), such as one or more sweetener(s) (e.g. sucralose) and / or one or more flavor agent(s). It may also comprise a lubricant, such as a polyethylene glycol lubricant (e.g. Polyglykol 8000 PF (Clariant)).

[0107] For the patient / consumer, it is not necessary to take the substances of the present disclosure simultaneously. A therapeutic effect can also be achieved if the substances are taken separately or sequentially, preferably within a day and more preferably within an hour.

[0108] Accordingly, as a second aspect of the present disclosure, there is provided substances comprisingA) carnitine, deoxycarnitine, acetyl-L-carnitine, gamma-butyrobetaine, 4- trimethylammoniobutanal, 3-hydroxy-N 6,N6,N 6-trimethyl-L-lysine, N6,N6,N6- trimethyl-L-lysine and / or lysine, preferably carnitine, such as L-carnitine;B) nicotinamide, nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D- ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, and / or nicotinate;C) optionally serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine, preferably serine or glycine; andD) optionally N-acetyl cysteine, cysteine and / or cystine for simultaneous, separate or sequential use in a therapeutic method of treatment or prevention of a retinal disease and / or retinal impairment.

[0109] The embodiments and examples of the first aspect apply to the second aspect mutatis mutandis.

[0110] As a third aspect of the present disclosure, there is provided a method of treatment or prevention of a retinal disease or retinal impairment, comprising administration of:A) carnitine, deoxycarnitine, acetyl-L-carnitine, gamma-butyrobetaine, 4- trimethylammoniobutanal, 3-hydroxy-N 6,N6,N 6-trimethyl-L-lysine, N6,N6,N6- trimethyl-L-lysine and / or lysine, preferably carnitine, such as L-carnitine;B) nicotinamide, nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D- ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, and / or nicotinate;C) optionally serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine, preferably serine or glycine; andD) optionally N-acetyl cysteine, cysteine and / or cystine.

[0111] The administration of A), B), C) and D) maybe simultaneous, separate or sequential.

[0112] In exemplary embodiments, A), B), C) and D) are contained in a composition that is administered to the subject.

[0113] As a fourth aspect, there is provided substances for use in a therapeutic method of treatment or prevention of a retinal disease or retinal impairment comprising:A) carnitine, deoxycarnitine, acetyl-L-carnitine, gamma-butyrobetaine, 4- trimethylammoniobutanal, 3-hydroxy-N 6,N6,N 6-trimethyl-L-lysine, N6,N6,N6- trimethyl-L-lysine and / or lysine, preferably carnitine, such as L-carnitine;B) nicotinamide, nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D- ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, and / or nicotinate;C) optionally serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine, preferably serine or glycine; andD) optionally N-acetyl cysteine, cysteine and / or cystine.

[0114] Components A), B), optionally C) and D) maybe administered simultaneously, separately or sequentially.

[0115] The embodiments and examples of the first, second and third aspect apply to the fourth aspect mutatis mutandis.

[0116] The subject of the first, second, third, and fourth aspect is preferably a human subject. The subject maybe an adult or an infant, e.g. a premature infant.EXAMPLES:Materials and methodMouse model of oxygen-induced retinopathy (OIR)

[0117] OIR is a well-established animal model used to simulate human retinal diseases characterized by abnormal blood vessel growth, such as ROP. In this model, neonatal mice are exposed to alternating cycles of high and normal oxygen levels. This exposure mimics the conditions premature infants may experience, leading to similar pathological changes in the retina. Oxygen consumption rates (OCR) of OIR vs. control retinas were assessed using BaroFuse ex vivo.

[0118] Oxygen-induced retinopathy (OIR) modeling retinal neovascularization was induced in C57BL / 6 J mice. Global proteomics of OIR vs. control mouse retinas was conducted, and pathways were analyzed. The top findings were validated with immunostaining of synaptic markers.

[0119] Carnitine, nicotinamide, pyruvate, serine, glycine, folic acid, MCT-oil, docosahexaenoic acid (DHA), arachidonic acid (AA), and a combination of DHA and AA vs. vehicle control were supplemented in OIR mice, and retinal vasculature was examined.Mice

[0120] C57BL / 6J mouse pups and their nursing dam were exposed to 75% 02(ProOx Model no, Biospherix) from postnatal day (P) 7 to P12, and returned to room air (21% 02) at P12, resulting in hyperoxia-induced central retinal vaso-obliteration and relative hypoxia-induced midperipheral retinal neovascularization which peaked at P17. Healthy control mice were maintained in the room air.

[0121] For in vivo testing, the respective substance or vehicle control (phosphate buffered saline, PBS) was administered to littermate mouse pups daily from P12 to P16. Retinas were collected at P17 and stained with isolectin GS-IB4 (vessel marker, Invitrogen, I21413). Images were taken using a Zeiss confocal microscope. Retinal vascular pathology was quantified with Image J plugin SWIFT_NV [4,5].Immunohistochemistry (IHC)

[0122] Mouse eyes were vertically sectioned using a Leica CM3050 S Cryostat (Leica). Sections with retina and the optic nerve were treated with ice-cold methanol and 0.1% Triton PBS subsequently. Retinal sections were blocked with 3% BSA for i hr at RT and stained with primary antibodies against synaptophysin (1:200, MAB368, Millipore) and postsynaptic density protein 95 (PSD95) (1:200, 75-028, UC Davis / NIH NeuroMab Facility) overnight at 4°C. Corresponding fluorescent secondary antibodies were then incubated for 1 hr at RT, and sections were covered in mounting medium with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI for cell nuclei, H-1200, Vector Laboratories). Images were taken using a Zeiss confocal microscope at 200X magnification.Statistics

[0123] Data were further analysed using the statistical software, Qlucore (version 3.5). A two-group comparison (OIR vs. normal control) using the log-transformed protein group means was performed, the student’s t-test for each protein’s comparison, and the Benjamini-Hochberg (p) procedure to calculate the FDR (q).

[0124] Statistics are described in the figure legends. In general, normality (quantile-quantile (QQ) plot) and F-test (for variance) were conducted first, followed by the according parametric unpaired t-test (or Welch’s t-test) or the non-parametric Mann-Whitney test to compare groups (Prism V9.0; GraphPad Software, Inc.). P<o.O5 was considered as statistically significant.ResultsEffect of carnitine

[0125] C57BL / 6J mice at postnatal day 7 (P7) were exposed to 75% oxygen with their nursing dam until P12. Mouse neonates were delivered L-carnitine (300 ug / g ip injection) or vehicle (10% water in PBS) from P7 to P11 (hyperoxia, phase I) or P12 to P16 (hypoxia, phase II). At P17, the neonatal mice were euthanized. Retinal vessels were visualized with isolectin (white). Retinal vaso-obliteration (VO) and neovascularization (NV) was quantified, and ratio of change was calculated compared with littermate vehicle groups. Unpaired t test. Each dot represented one retina (VO, NV), or one mouse (body weight plot, BW). P value was labelled. In preterm infants Carnitine was given 25mg / kg by IV route in the first 6 hours of life with total parenteral nutrition (TPN).

[0126] As illustrated in figure 1, L-carnitine from P7-11 inhibits vaso-obliteration at P12 and neovascularization at P17 in OIR. Hence, L-carnitine exhibits a dual action, and provides a promising strategy in preventing the progression of retinopathies like ROP where both vessel loss and abnormal vessel growth are central features.Effect of nicotinamide

[0127] C57BL / 6J mice at postnatal day 7 (P7) were exposed to 75% oxygen with their nursing dam until P12. Mouse neonates were delivered nicotinamide (NAM, 500ug / g, i.p.) or vehicle (PBS) from P12 to P16 (hypoxia, phase II). At P17, the neonatal mice were euthanized. Retinal vessels were visualized with isolectin (white). Retinal vaso-obliteration (VO) and neovascularization (NV) was quantified, and ratio of change was calculated compared with littermate vehicle groups. Unpaired t test. Each dot represented one retina (VO, NV), or one mouse (body weight plot, BW). P value was labelled.

[0128] As illustrated in figure 2, nicotinamide from P12-16 (phase 2) significantly inhibits retinal neovascularization at P17 in OIR.Effect of serine

[0129] Serine- C57BL / 6J mice at postnatal day 7 (P7) were exposed to 75% oxygen with their nursing dam until P12. Mouse neonates were delivered L-serine (o.6ug / g ip injection) or vehicle (PBS) from P12 to P16. At P17, the neonatal micewere euthanized. Retinal vessels were visualized with isolectin (white). Retinal vaso- obliteration (VO) and neovascularization (NV) was quantified, and ratio of change was calculated compared with littermate vehicle groups. Unpaired t test. Each dot represented one retina (VO, NV), or one mouse (body weight plot, BW). P value was labelled.

[0130] As illustrated in figure 3, exogenous L-serine (i.p., P12-16) decreases retinal neovascularization, and does not change vaso-obliteration in C57BL / 6J OIR mice.Effect of glycine

[0131] C57BL / 6J mice at postnatal day 7 (P7) were exposed to 75% oxygen with their nursing dam until P12. Mouse neonates were delivered glycine (o.8ug / g ip injection) or vehicle (PBS) from P7 to P11 (hyperoxia, phase I) or P12 to P16 (hypoxia, phase II). At P17, the neonatal mice were euthanized. Retinal vessels were visualized with isolectin (white). Retinal vaso-obliteration (VO) and neovascularization (NV) was quantified, and ratio of change was calculated compared with littermate vehicle groups. Unpaired t test. Each dot represented one retina (VO, NV), or one mouse (body weight plot, BW). P value was labelled.

[0132] As illustrated in figure 4, exogenous glycine treatment promoted retinal revascularization (reflected by decreased vaso-obliteration) in OIR mice. Accordingly, glycine may help restore the normal structure and function of blood vessels after damage or obliteration caused by high oxygen exposure.Effect of folic acid

[0133] C57BL / 6J mice at postnatal day 7 (P7) were exposed to 75% oxygen with their nursing dam until P12. Mouse neonates were delivered folic acid (0.5 ug / g ip injection) or vehicle (DMSO) from P7 to P11 (hyperoxia, phase I). At P17, the neonatal mice were euthanized. Retinal vessels were visualized with isolectin (white). Retinal vaso-obliteration (VO) and neovascularization (NV) was quantified, and ratio of change was calculated compared with littermate vehicle groups. Unpaired t test. Each dot represented one retina (VO, NV), or one mouse (body weight plot, BW). P value was labelled.

[0134] As illustrated in figure 5, early direct supplementation of folic acid during hyperoxic phase inhibits retinal neovascularization in OIR mice.Effect of pyruvate

[0135] C57BL / 6J mice at postnatal day 7 (P7) were exposed to 75% oxygen with their nursing dam until P12. Mouse neonates were delivered pyruvate (50 ug / g, ip injection) or vehicle (PBS) from P12 to P14 or P14 to P16. At P17, the neonatal mice were euthanized. Retinal vessels were visualized with isolectin (white). Retinal vaso- obliteration (VO) and neovascularization (NV) was quantified, and ratio of change was calculated compared with littermate vehicle groups. Unpaired t test. Each dot represented one retina (VO, NV), or one mouse (body weight plot, BW). P value was labelled.

[0136] As illustrated in figure 6, pyruvate supplementation during the phase of neovessel formation inhibits retinal neovascularization in mouse OIR. Furthermore, an improved vaso-obliteration (VO) is observed.Effect of MCT oil

[0137] MCT-oil - C57BL / 6J mice at postnatal day 7 (P7) were exposed to 75% oxygen with their nursing dam until P12. Mouse neonates were delivered MCT oil (4011I, oral) or vehicle (PBS) from P7 to P11 (hyperoxia, phase I). The MCT oil used was supplied by Neste (https: / / mvw.nestlemedicalhub.com / products / rnct-oil). At P17, the neonatal mice were euthanized. Retinal vessels were visualized with isolectin (white). Retinal vaso-obliteration (VO) and neovascularization (NV) was quantified, and ratio of change was calculated compared with littermate vehicle groups.Unpaired t test. Each dot represented one retina (VO, NV), or one mouse (body weight plot, BW). P value was labelled, n.s., not significant.

[0138] As illustrated in figure 7, MCT oil from P7-11 (phase I) inhibits retinal neovascularization at P17 in OIR. Furthermore, less vaso-obliteration (VO) is observed, which indicates that MCT-oil also improves normal vessel growth.Effect of AA and DHA

[0139] C57BL / 6J mice at postnatal day 7 (P7) were exposed to 75% oxygen with their nursing dam until P12. The nursing dam were fed 1%DHA2%AA, or 3%DHA, or 3% AA enriched customized diet after giving birth (Pi). At P17, the neonatal mice were euthanized. Retinal vessels were visualized with isolectin (white). Retinal vaso- obliteration (VO) and neovascularization (NV) was quantified, and percentage was calculated to the total retinal area. Ratio of change was calculated compared to 3%AAgroup. AN OVA. Each dot represented one retina (VO, NV), or one mouse (body weight plot, BW). P value was labelled, n.s., not significant.

[0140] The doses used were calculated with the basis that a mouse eats about 3 grams of food per day. 3g*ig / kg=o.oo3g DHA, 3g*2g / kg=o.oo6g AA consumed per day for the nursing dam.

[0141] As illustrated in figure 8, less neovascularization (NV) is observed in DNA and AA / DHA- treated animals compared to animals treated with AA only. Furthermore, less vaso-obliteration (VO) is observed in in DHA and AA / DHA- treated compared to AA-treated mice. The most profound effect on the normalization of vessel growth was observed in the AA / DHA treated mice.Effect of composition comprising serine, carnitine, nicotinamide, and N- acetyl cysteine

[0142] C57BL / 6J mice at postnatal day 7 (P7) were exposed to 75% oxygen with their nursing dam until P12. Mouse neonates were intraperitoneally delivered a “Fuel mixture” containing; L- serine o.6ug / g, L-carnitine 300 ug / g, Nicotinamide 500 ug / g, N-acetyl-L-cysteine 200 ug / g or vehicle (PBS) from P12 to P16. At P17, the neonatal mice were euthanized. Retinal vaso-obliteration (VO) and neovascularization (NV, highlighted in white in figure 9A) were quantified, and the ratio of change was calculated compared with littermate vehicle groups. n=i2-i4 retinas per group. Birth weights were similar between groups. Unpaired t test. Each dot represents one retina (VO, NV). The p-value was labelled.

[0143] As illustrated in figure 9, significantly less neovascularization (NV) was observed in mice treated with the fuel mixture compared to vehicle-treated mice.Serum levels of carnitine in preterm infants

[0144] The inventors have also investigated the serum level of carnitine in extremely preterm infants (mean [IQR] gestational age at birth = 25+5 [24+3 to 26+6] weeks+days) during the first four weeks of life, stratified by retinopathy of prematurity (ROP) status: No Severe ROP (stage 0-2, n = 127) or Severe ROP (stage 3 and / or type I, n = 50).

[0145] In this study, blood was collected in serum-separating tubes, allowed to clot at room temperature for 0.5 to 2 hours, and centrifuged at 1500 x g for 10 minutes. Serum aliquots were stored at -20 °C for up to one week before long-term storage at -80 °C. Twenty-five microliters of serum were subjected to protein precipitation in methanol; the resulting supernatant was filtered and analyzed by untargeted metabolomics using UHPLC-HRMS with reversed-phase chromatography in positive ionization mode.

[0146] As illustrated in figure 10, the serum level of carnitine in preterm infants with severe ROP, was significantly lower (see lower, dark line in figure 10)References[1] Sveinsdottir, et al. Neonatology 20185114: 46-52.[2] Huang CC et al. Neonatology 2O22;ii9(6):727-734.[3] Blencowe H, Indian Pediatr 2016; 53 Suppl 2: S89-S92.[4] Pubmed id: 19816419[5] Pubmed id: 19757106

Claims

CLAIMS1. A composition for use in a therapeutic method of treatment or prevention of a retinal disease or retinal impairment, wherein said composition comprises:A) carnitine, deoxycarnitine, acetyl-L-carnitine, gamma-butyrobetaine, 4- trimethylammoniobutanal, 3-hydroxy-N 6,N6,N 6-trimethyl-L-lysine, N6,N6,N6- trimethyl-L-lysine and / or lysine, preferably carnitine, such as L-carnitine;B) nicotinamide, nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D- ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, and / or nicotinate;C) optionally serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine, preferably serine or glycine; andD) optionally N-acetyl cysteine, cysteine and / or cystine.

2. The composition for use according to claim 1, wherein said retinal disease or retinal impairment is selected from the group consisting of diabetic retinopathy, retinopathy of prematurity (ROP), hypertensive retinopathy, central serous retinopathy, and age-related macular degeneration (AMD), preferably wherein said retinal disease or retinal impairment is retinopathy of prematurity (ROP).

3. The composition for use according to claim 1 or claim 2, wherein said composition further comprises pyruvate.

4. The composition for use according to any one of the preceding claims, wherein said composition further comprises MCT oil.

5. The composition for use according to any one of the preceding claims, wherein said composition further comprises docosahexaenoic acid (DHA) and / or arachidonic acid (AA).

6. The composition for use according to claim 5, wherein said composition comprises docosahexaenoic acid (DHA) and arachidonic acid (AA), and wherein the molar ratio of arachidonic acid (AA) to docosahexaenoic acid (DHA) is from 1:1 to 1:7.

7. The composition for use according to any one of the preceding claims, wherein said composition further comprises folic acid, 5-formyltetrahydrofolate, and / or 5-methyltetrahydrofolate (5-MTHF).

8. The composition for use according to any one of the preceding claims, wherein said method comprises enteral administration of the composition.

9. The composition for use according to any one of the preceding claims, in the form of a powderous mixture.

10. The composition for use according to any one of the preceding claims, wherein said method is carried out on a human subject.

11. The composition for use according to any one of the preceding claims, wherein said method comprises administration of:A) in a dose of 0.100-2.50 mmol / kg / day, such as 0.200-2.00 mmol / kg / day, such as 0.230-1.00 mmol / kg / day, such as 0.300-0.800 mmol / kg / day;B) in a dose of 0.015-0.39 mmol / kg / day, such as 0.030-0.31 mmol / kg / day, such as 0.040-0.20 mmol / kg / day; optionally C) in a dose of 0.48-24 mmol / kg / day, such as 0.48-4.8 mmol / kg / day, such as 1.8-4.8 mmol / kg / day, such as 2.9-4. mmol / kg / day; and optionally D) in a dose of 0.31-3.05 mmol / kg / day, such as 0.31-1.84 mmol / kg / day, such as 0.40-1.23 mmol / kg / day.

12. The composition for use according to any one of the preceding claims when dependent on claim 3, wherein said method comprises administration of pyruvate in a dose of 0.08-9.9 mmol / kg / day.

13. The composition for use according to any one of the preceding claims when dependent on claim 4, wherein said method comprises administration of MCT oil in a dose of o.1-5.0 ml / kg / day.

14. The composition for use according to any one of the preceding claims when dependent on claim 5, wherein said method comprises administration of docosahexaenoic acid (DHA) in a dose of 0.152-1.52 mmol / kg / day.

15. The composition for use according to any one of the preceding claims when dependent on claim 5, wherein said method comprises administration of arachidonic acid (AA) in a dose of 0.164-16.4 mmol / kg / day.

16. The composition for use according to any one of the preceding claims when dependent on claim 7, wherein said method comprises administration of folic acid, 5-formyltetrahydrofolate, and / or 5-methyltetrahydrofolate (5-MTHF) in a dose of 0.045-1.0, e.g. 0.045-0.45 mmol / kg / day.

17. Substances for use in a therapeutic method of treatment or prevention of a retinal disease or retinal impairment comprising:A) carnitine, deoxycarnitine, acetyl-L-carnitine, gamma-butyrobetaine, 4- trimethylammoniobutanal, 3-hydroxy-N 6,N6,N 6-trimethyl-L-lysine, N6,N6,N6- trimethyl-L-lysine and / or lysine, preferably carnitine, such as L-carnitine;B) nicotinamide, nicotinamide riboside, quinolinate, deamino-NAD+, nicotinate D- ribonucleotide, nicotinamide D-ribonucleotide, nicotinate D-ribonucleoside, and / or nicotinate;C) optionally serine, glycine, betaine, N-acetylglycine, N-acetylserine, dimethylglycine, sarcosine and / or phosphoserine, preferably serine or glycine; andD) optionally N-acetyl cysteine, cysteine and / or cystine.

18. Substances for use according to claim 17, wherein A), B), optionally C) and D) are administered simultaneously, separately or sequentially.

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