Patent Description:
Traumatic brain injury (TBI) is a form of non-degenerative acquired brain injury. TBI is a serious medical condition that may occur after the brain is subjected to a significant external physical impact. TBI is the leading cause of disability and death in people under <NUM> with approximately <NUM> million new cases each year worldwide.

According to the diagnostic criteria detailed in the "Diagnostic and Statistical Manual of Mental Disorders (DSM-<NUM>) TBI is associated with the unique combination of one or more of the following characteristics: changes in levels of consciousness; memory disturbances; confusion associated with deficits in orientation; neurological signs, such as brain injury observable on neuroimaging, new onset or worsening of seizure disorder, visual field deficits and hemiparesis. While some symptoms may appear immediately after the injury, others may evolve over time consistent with anatomical changes in the neural substrates following the injury.

The primary phase of TBI describes immediate brain tissue damage from contusions or oxygen deprivation caused by global mass effect. The primary injury in TBI can only be reduced through improved prevention. Secondary injury starts immediately after trauma and underlies the functional deficits associated with TBI. It occurs later via such mechanisms as reperfusion injury, delayed cortical edema, blood-brain barrier (BBB) breakdown, glutaminergic overexcitation and local electrolyte imbalance. These disturbances themselves result in reactive oxygen species (ROS)-mediated neurodegeneration through calcium release, glutamate toxicity, lipid peroxidation, and mitochondrial dysfunction. Such secondary injury may occur in brain adjacent to the site of initial supposed injury, yielding the potential for unexpected spread of the zone of damage over months post-injury. The damage resulting from this complex cascade of cellular, neurochemical, inflammatory and metabolic events may lead to neuronal loss, dendritic, axonal and synaptic changes, and persistent white matter abnormalities Presently, there are no treatments to counter such adverse outcomes. Thus, developing efficacious therapeutic interventions to protect the brain and promote repair after TBI is a particularly urgent pursuit.

<CIT> deals with improving neural survival following neurological injury induced by an external force, such as following traumatic brain injury.

An important problem induced by TBI is white matter loss or diffusivity leading to cognitive function disabilities. Reference is made to <NPL>. <CIT> describes the application of omega-<NUM> fatty acids in treatment of traumatic brain injury with an effect both on lesion size as well as white matter (by monitoring the fluorescence intensity of MBP), but still the effect is limited. There is thus still a need for improvement in the treatment of TBI in the art.

Not directed to TBI, <CIT> describes the use of one or more of uridine and cytidine, or salts, phosphates, acyl derivatives or esters thereof in the manufacture of a composition for treating, preventing or reducing the risk of occurrence of white matter lesions, white matter hyperintensities (WMH), Leukoaraiosis or periventricular white matter disease in elderly not suffering from a neurodegenerative disorder, preferably non-demented elderly and elderly not suffering from Alzheimer's Disease.

The inventors have observed that after administration of a product comprising (i) one or more of uridine and cytidine, or salts, phosphates, acyl derivatives or esters thereof, (ii) a lipid fraction comprising at least one of docosahexaenoic acid (<NUM>:<NUM>; DHA), eicosapentaenoic acid (<NUM>:<NUM>; EPA) and docosapentaenoic acid (<NUM>:<NUM>; DPA), or esters thereof, in which the lipid fraction comprises less than <NUM> weight% of α-linolenic acid (ALA), calculated on the weight of all fatty acids, (iii) choline, or salts or esters thereof, (iv) vitamins B6, B9 and B12, and (v) vitamin C, vitamin E and selenium, lesion size is reduced (<FIG>) and white matter is produced (<FIG>) after TBI. The effects of intervention in a TBI mouse model in terms of brain lesion size and white matter (by way of myelin production) were monitored. Intervention showed successful for each of these aspects which were found otherwise compromised in the context of TBI. The results are presented in the experimental part.

Taken from <FIG> herein, compared to the results obtained with DHA in <CIT>, the present intervention showed greater improvement in myelin production: the CCI-FC levels with the current intervention were almost at the same levels as observed for the craniotomy group (cranio-control), whereas theTBI intervention control (CCI control) group has a significantly lower myelin basic protein (MBP); In contrast, in <CIT> figures 6D and 6E the average MBP intensity is displayed for a craniotomy group (sham, white bar), a TBI intervention group (RD-CCI, grey bar) and a TBI intervention group treated with a fish oil diet comprising DHA (FOD-CCI, black bar). When the craniotomy group (cranio-control) results of the current invention are set at <NUM>, CCI-FC yielded about <NUM> (vs CCI ctrl <NUM>) whereas these values in Figure <NUM> in <CIT> are <NUM>, and about <NUM> and <NUM> respectively. Accordingly, the effect obtained with the present intervention shows an almost full restoration of myelin levels compared to control levels, whereas in the FOD-CCI group of <CIT> there is only slight improvement over the TBI invention group (RD-CCI) and no restoration towards the levels detected in the craniotomy group (sham).

Thus, according to one embodiment, the composition comprises:.

for use in producing myelin in a mammal suffering from or recovering from traumatic brain injury. The composition is preferably administered enterally (preferably orally).

While the individual active ingredients (i) - (v) are suitably administered enterally (preferably orally) in a single composition, they may also be administered in individual dosage units. Hence, the invention further relates to a kit of parts comprising (i) - (v) separately, for use together in producing myelin in a mammal suffering from or recovering from traumatic brain injury.

The ingredients (i), (ii), (iii), (iv) and (v) and further optional ingredients are detailed here below.

In one aspect, associated with the improved myelin production, the invention relates to the composition comprising (i), (ii), (iii), (iv) and (v) and further optional ingredients for use in improving the rate of recovery from TBI.

The inventors found that a composition according to the invention is effective in the treatment of TBI and improves the recovery from TBI. In a preferred aspect the composition according to the invention is administered shortly after diagnosis of TBI, preferably starting within <NUM> months, preferably within <NUM> months, more preferably within <NUM> month, more preferably within <NUM> weeks, most preferably within <NUM> week after the diagnosis of TBI. In a preferred aspect the administration or use of the composition is administered daily for at least <NUM> days, preferably at least <NUM> days, preferably at least <NUM> days, more preferably at least <NUM> month, more preferably at least <NUM> months.

The composition for use of the invention comprises administering the composition at outlined below to a mammal subject in need thereof, preferably a human person in need thereof, suffering from TBI. TBI refers to damage to the brain resulting from external mechanical force. In the context of the invention, TBI can be mild (level of unconsciousness of less than <NUM> minutes and/or a Glasgow Coma score of <NUM> - <NUM>) or moderate to severe TBI (level of unconsciousness of more than <NUM> minutes and/or a Glasgow Coma score of less than <NUM>). TBI can result from falls, firearm wounds, sports accidents, construction accidents and vehicle accidents, among other causes. As it appears, traumatic brain injury is the most prevalent injury of soldiers in combat (e.g. amongst the US troops in Iraq and Afghanistan). Victims of TBI can suffer from a number of physical, cognitive, social, emotional and/or behavioural disorders following injury. The primary impact results in direct neural cell loss predominantly exhibiting necrotic death, which is then generally followed by a wave of secondary injury cascades including excitotoxicity, oxidative stress, mitochondrial dysfunction, blood-brain barrier disruption, and inflammation. According to the diagnostic criteria detailed in the "Diagnostic and Statistical Manual of Mental Disorders (DSM-<NUM>) TBI is associated with the unique combination of one or more of the following characteristics: changes in levels of consciousness; memory disturbances; confusion associated with deficits in orientation; neurological signs, such as brain injury observable on neuroimaging, new onset or worsening of seizure disorder, visual field deficits and hemiparesis.

In one aspect the treatment involves improved myelinisation in a patient suffering or recovering from TBI. White matter loss in the context of TBI is different from white matter degeneration associated with neurodegenerative disorders such as Alzheimer's Disease. Treatment preferably involves improved myelin production. White matter disruption is observed in both mild as well as moderate and severe TBI. Intervention with the composition of the invention resulted in enhanced white matter production and improved myelin production. Myelin and white matter production can be monitored using imaging techniques available to skilled practitioners, such as there are Positron Emission Tomography (PET), Single Photon Emission Computerized Tomography (SPECT), Functional Magnetic Resonance Imaging (fMRI) and Diffuse Tensor Imaging (DTI).

In one aspect of the invention, the treatment does not relate to neurogenesis, neuronal survival and neuroinflammation. In another aspect of the invention, mammals suffering from PKU or hyperphenylalaninemia are excluded. In another aspect of the invention, the treatment of white matter disease or periventricular white matter disease or white matter degeneration associated with aging is also excluded.

In one aspect of the present invention, the composition according to the invention may be used as a pharmaceutical product comprising one or more pharmaceutically acceptable carrier materials.

In another aspect of the present invention, the composition according to the invention may be used as a nutritional product, for example as a nutritional supplement, e.g., as an additive to a normal diet, as a fortifier, to add to a normal diet, or as a complete nutrition.

The pharmaceutical product, preferably for enteral application, may be a solid or liquid galenical formulation. Examples of solid galenical formulations are tablets, capsules (e.g. hard or soft shell gelatine capsules), pills, sachets, powders, granules and the like which contain the active ingredient together with conventional galenical carriers. Any conventional carrier material can be utilized. The carrier material can be organic or inorganic inert carrier material suitable for oral administration. Suitable carriers include water, gelatine, gum Arabic, lactose, starch, magnesium stearate, talc, vegetable oils, and the like. Additionally, additives such as flavouring agents, preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of pharmaceutical compounding.

If the composition is a pharmaceutical product, such product may contain the daily dosage in one or more dosage units. The dosage unit may be in a liquid form or in a solid form, wherein in the latter case the daily dosage may be provided by one or more solid dosage units, e.g. in one or more capsules or tablets.

In another aspect of the present invention, the composition according to the invention may be used in a nutritional product comprising fats, proteins, and carbohydrates. It is understood that a nutritional product differs from a pharmaceutical product by the presence of nutrients which provide nutrition to the subject to which the composition is administered, in particular the presence of protein, fat, digestible carbohydrates and dietary fibres. It may further contain ingredients such as minerals, vitamins, organic acids, and flavouring agents. Although the term "nutraceutical product" is often used in literature, it denotes a nutritional product with a pharmaceutical component or pharmaceutical purpose. Hence, the nutritional composition according to the invention may also be used in a nutraceutical product.

The product of the invention is an enteral composition, intended for oral administration. It is preferably administered in liquid form. In one embodiment, the product comprises a lipid fraction and at least one of carbohydrates and proteins, wherein the lipid composition provides between <NUM> and <NUM> energy % of the food product. This is based on the assumption that lipid, carbohydrates and protein generate approximately <NUM>, <NUM> and <NUM> kcal/g, respectively, the <NUM> together making up for all caloric contributions of the composition. In one embodiment, the food product is a liquid composition containing between <NUM> and <NUM> kcal per ml.

The method, product, composition or kit of the invention comprises therapeutically effective amounts of at least one omega-<NUM> long-chain polyunsaturated fatty acid (LC-PUFA; having a chain length of <NUM> and more carbon atoms) selected from the group consisting of docosahexaenoic acid (<NUM>:<NUM>, ω-<NUM>; DHA), eicosapentaenoic acid (<NUM>:<NUM>, ω-<NUM>; EPA) and docosapentaenoic acid (<NUM>:<NUM>ω-<NUM>; DPA), preferably at least DHA and/or EPA, more preferably at least DHA, most preferably DHA and EPA. EPA is converted to DPA (ω-<NUM>), increasing subsequent conversion of DPA to DHA in the brain. Hence, the present composition preferably contains a significant amount of EPA, so to further stimulate in vivo DHA formation.

The DHA, EPA and/or DPA are preferably provided as triglycerides, diglycerides, monoglycerides, free fatty acids or their salts or esters, phospholipids, lysophospholipids, glycerol ethers, lipoproteins, ceramides, glycolipids or combinations thereof. Preferably, the present composition comprises at least DHA in triglyceride form. Suitable ω-<NUM> LCPUFA and/or DHA sources include tuna oil, (other) fish oils, DHA-rich alkyl esters, algae oil, egg yolk, or phospholipids enriched with ω-<NUM> LCPUFA e.g. phosphatidylserine-DHA. Preferably, a product, composition or kit according to the invention comprises fish oil providing the omega-<NUM> LCPUFA(s). Another particularly suitable source for the omega-<NUM> LCPUFA(s) is algae oil.

In terms of daily dosage, the present composition for use preferably comprises the administration of <NUM> to <NUM> DHA+EPA+DPA per day. In a preferred embodiment, in terms of daily dosage, the present composition for use preferably comprises the administration of <NUM> - <NUM> DHA+EPA per day, more preferably <NUM> - <NUM> per day. DHA is preferably administered in an amount of <NUM> - <NUM> per day, more preferably <NUM> - <NUM> per day. In addition, EPA is preferably administered in an amount of <NUM> - <NUM> per day, more preferably <NUM> - <NUM> per day.

In terms of unit dosage, the proportion of DHA+EPA+DPA (preferably DHA+EPA) of the total fatty acids is preferably <NUM> to <NUM> weight%, more preferably <NUM> to <NUM> weight%, most preferably <NUM> to <NUM> weight%. The present composition preferably comprises <NUM> to <NUM> weight% DHA based on total fatty acids, preferably <NUM> to <NUM> weight% DHA based on total fatty acids, more preferably <NUM> to <NUM> weight% DHA based on total fatty acids. The present composition preferably comprises <NUM> to <NUM> weight% EPA based on total fatty acids, preferably <NUM> to <NUM> weight% EPA, most preferably <NUM> to <NUM> weight%, based on total fatty acids.

The ratio of the weights of DHA to EPA is preferably larger than <NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>. The above-mentioned ratios and amounts take into account and optimise several aspects, including taste (too high LCP levels reduce taste, resulting in a reduced compliance), balance between DHA and precursors thereof to ensure optimal effectiveness while maintaining low-volume formulations.

It is preferred that the alpha-linolenic acid [ALA] content of the composition is maintained at low levels. The inventors believe that due to the inflammatory nature of traumatic brain injury, excess supply of highly unsaturated fatty acids increases the risk of further damage to injury tissue due to the effect of peroxidized PUFAs, even though it has been observed that in vivo supply of α-linolenic acid is neuroprotective in neurotrauma (<NPL>). It is discovered by the inventors that the ALA concentration is maintained at levels less than <NUM> weight%, more preferably below <NUM> weight%, particularly below <NUM> weight%, calculated on the weight of all fatty acids. In the animal studies attached levels were about <NUM> per <NUM> fatty acids.

Linoleic acid [LA] concentrations can be maintained at normal levels, i.e. between <NUM> to <NUM> weight%, although in one embodiment the LA concentration is also significantly reduced to an amount of less than <NUM>/<NUM> fatty acids and preferably even less than <NUM> weight%. The LA concentrations are preferably at least <NUM> weight% of the fatty acids. The LA:ALA ratio is preferably in the range of <NUM>: <NUM> to <NUM>:<NUM>.

In one embodiment, the weight ratio ω-<NUM>/ ω-<NUM> in the composition of the invention is preferably in the range <NUM> to <NUM>, preferably in the range <NUM>:<NUM> to <NUM>:<NUM>, more preferably in the range <NUM>:<NUM> to <NUM>:<NUM>, most preferably <NUM>:<NUM> to <NUM>:<NUM>, in particular less than <NUM>:<NUM>. The amount of ω-<NUM> LCPUFAs is preferably less than <NUM>, preferably <NUM> to <NUM>, more preferably <NUM> to <NUM> weight% of the fatty acids in the formula.

In one embodiment, the composition, product or kit contains less than <NUM> weight%, preferably less than <NUM> weight% of fatty acids of less than <NUM> carbon atoms. Medium chain fatty acids [MCT] are defined to be linear or branched saturated carboxylic acids having six (C6:<NUM>), seven (C7:<NUM>), eight (C8:<NUM>), nine (C9:<NUM>) or ten (C10:<NUM>) carbon atoms. The amount of MCTs is preferably lower than <NUM> weight%, more preferably lower than <NUM> weight%, most preferably lower than <NUM> weight% of the total fatty acids. In one embodiment, the sum of the medium chain fatty acids C6:<NUM> + C7:<NUM> + C8:<NUM> over the sum of C9:<NUM> and C10:<NUM> is less than <NUM>:<NUM>, more preferably less than <NUM>:<NUM>, most preferably less than <NUM>:<NUM>, particularly less than <NUM>:<NUM>.

The present composition, productor kit involves therapeutically effective amounts of uridine, cytidine and/or an equivalent thereof, including salts, phosphates, acyl derivatives and/or esters. In terms of uridine, the composition preferably comprises at least one uridine or an equivalent thereof selected from the group consisting of uridine (i.e. ribosyl uracil), deoxyuridine (deoxyribosyl uracil), uridine phosphates (UMP, dUMP, UDP, UTP), nucleobase uracil and acylated uridine derivatives. In one embodiment, cytidine, CMP, citicoline (CDP-choline) may also be applied. Preferably, the present composition comprises an uridine phosphate selected from the group consisting of uridine monophosphate (UMP), uridine diphosphate (UDP) and uridine triphosphate (UTP); and/or a cytidine phosphate (CMP, CDP, CTP, preferably CMP). Most preferably the present composition comprises UMP, as UMP is most efficiently being taken up by the body. Preferably at least <NUM> weight% of the uridine in the present composition is provided by UMP, more preferably at least <NUM> weight%, most preferably at least <NUM> weight%. Doses that must be administered are given as UMP. The amount of uracil sources can be calculated taking the molar equivalent to the UMP amount.

The present composition for use preferably comprises the administration of uridine (the cumulative amount of uridine, deoxyuridine, uridine phosphates, nucleobase uracil and acylated uridine derivatives) in an amount of (i) <NUM> to <NUM> per day, preferably <NUM> to <NUM> per day, more preferably <NUM> to <NUM> per day, and/or (ii) <NUM> to <NUM> per <NUM> (liquid) composition, preferably <NUM> to <NUM> per <NUM> (liquid) composition, more preferably <NUM> to <NUM> per <NUM> (liquid) composition. The above amounts also account for any amounts of cytidine, cytidine phosphates and citicoline incorporated in the composition.

Preferably, the present composition comprises uridine phosphate, preferably uridine monophosphate (UMP). The UMP is very efficiently taken up by the body. Hence, inclusion of UMP in the present composition enables a high effectivity at the lowest dosage and/or the administration of a low volume to the subject.

The present composition, product or kit involves therapeutically effective amounts of choline, a choline salt and/or choline ester. The choline salt is preferably selected from choline chloride, choline bitartrate, or choline stearate. The choline ester is preferably selected from a phosphatidylcholine and lyso-phosphatidyl choline. The present composition for use preferably comprises the administration of more than <NUM> choline per day, preferably <NUM> to <NUM> choline per day, more preferably <NUM> to <NUM> choline per day, most preferably <NUM> to <NUM> choline per day. The present composition preferably comprises <NUM> to <NUM> gram choline per <NUM> of the liquid composition, preferably <NUM> to <NUM> choline per <NUM>, preferably <NUM> to <NUM> choline per <NUM> composition, most preferably <NUM> to <NUM> choline per <NUM>. The above numbers are based on choline, the amounts of choline equivalents or sources can be calculated taking the molar equivalent to choline into account.

The present composition, product or kit involves therapeutically effective amounts of vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride), vitamin B9 (folic acid or folate), and vitamin B12 (cobalamins). Functional equivalents are encompassed within these terms. Good results have been achieved with a combination comprising therapeutically effective amounts of all of vitamin B6, vitamin B12 and vitamin B9.

The vitamins B are to be administered in an effective dose, which dose depends on the type of vitamin B used. As a rule of thumb, a suitable minimum or a maximum dose may be chosen based on known dietary recommendations, for instance as recommended by Institute of Medicine (IOM) of the U. National Academy of Sciences or by Scientific Committee on Food (a scientific committee of the EU), the information disclosed herein and optionally a limited amount of routine testing. A minimum dose may be based on the estimated average requirement (EAR), although a lower dose may already be effective. A maximum dose usually does not exceed the tolerable upper intake levels (UL), as recommended by IOM.

The vitamin B6 is preferably present in an amount to provide a daily dosage in the range of <NUM> to <NUM>, in particular in the range of <NUM> to <NUM>, more in particular in the range of <NUM> to <NUM>. The present composition preferably comprises <NUM> - <NUM> vitamin B6 per <NUM> (liquid) product, more preferably <NUM> - <NUM> vitamin B6 per <NUM> (liquid) product, more preferably <NUM> - <NUM> vitamin B6 per <NUM> (liquid) product.

The vitamin B12 is preferably present in an amount to provide a daily dosage in the range of <NUM> to <NUM>µg, in particular in the range of <NUM> to <NUM>µg, more in particular in the range of <NUM> to <NUM>µg. The present composition preferably comprises <NUM>-<NUM>µg vitamin B12 per <NUM> (liquid) product, more preferably <NUM>-<NUM>µg vitamin B12 per <NUM> (liquid) product, more preferably <NUM>-<NUM>µg vitamin B12 per <NUM> (liquid) product. The term 'vitamin B12' incorporates all cobalbumin equivalents known in the art.

The vitamin B9 (folic acid) is preferably present in an amount to provide a daily dosage in the range of <NUM> to <NUM>µg, in particular in the range of <NUM> to <NUM>µg, more in particular in the range of <NUM> to <NUM>µg. The present composition preferably comprises <NUM>-<NUM>µg folic acid per <NUM> (liquid) product, more preferably <NUM>-<NUM>µg folic acid per <NUM> (liquid) product, more preferably <NUM> - <NUM>µg folic acid per <NUM> (liquid) product. Folates include folic acid, folinic acid, methylated, methenylated and formylated forms of folates, their salts or esters, as well as their derivatives with one or more glutamic acid, and all in either reduced or oxidized form.

It is preferred to incorporate at least one phospholipid in the composition. The term "phospholipid" excludes PC that is already accounted for in the choline fraction. The present composition preferably comprises at least one phospholipid in an amount of <NUM> to <NUM> gram per <NUM>, more preferably between <NUM> and <NUM> gram per <NUM>, most preferably <NUM> to <NUM> per <NUM>. The at least one phospholipid is preferably provided for using lecithin, more preferaly soy lecithin.

The present composition, product or kit involves therapeutically effective amounts of the antioxidants vitamin C, vitamin E and selenium.

Vitamin C, or a functional equivalent thereof, may be present in an amount to provide a daily dosage in the range of <NUM> to <NUM>, in particular in the range of <NUM> to <NUM>, more in particular in the range of <NUM> to150 mg. In one embodiment, vitamin C , or a functional equivalent thereof, is present in an amount in the range of <NUM> to <NUM>, in particular in the range of <NUM> to <NUM>, more in particular in the range of <NUM> to150 mg per <NUM> of the composition.

Tocopherol and/or an equivalent thereof (i.e. a compound having vitamin E activity) may be present in an amount to provide a daily dosage in the range of <NUM> to <NUM>, in particular in the range of <NUM> to <NUM>, more in particular in the range of <NUM> to100 mg, to prevent oxidative damage to the injury site resulting from dietary PUFA. In one embodiment, tocopherol and/or equivalent is present in an amount in the range of <NUM> to <NUM>, in particular in the range of <NUM> to <NUM>, more in particular in the range of <NUM> to100 mg per <NUM> of the composition. The term "tocopherol and/or an equivalent there of", as used in this description, comprises tocopherols, tocotrienols, pharmaceutical and/or nutritional acceptable derivatives thereof and any combination thereof. The above numbers are based on tocopherol equivalents, recognized in the art.

The present composition also contains selenium. The antioxidant activity of selenium advantageously prevents and/or inhibits damages to the brain areas. Preferably the present composition for use provides the administration of a composition comprising <NUM> and <NUM> selenium per <NUM> liquid product, preferably <NUM> and <NUM> selenium per <NUM> liquid product. The amount of selenium administered per day is preferably more than <NUM>, more preferably <NUM> to <NUM>.

The composition, product or kit may further comprise proteinaceous material. Should a protein fraction be included, the protein fraction comprises intact proteins, peptides as may be obtained by hydrolyses of intact proteins and by syntheses, derivatives of peptides comprising more than <NUM> weight% amino acids. In the context of the invention, nitrogen from nucleosides material and choline will not be calculated as being protein.

It is preferred that the amount of taurine (including taurine salts) is less than <NUM>, preferably less than <NUM> per daily dose. Additionally or alternatively, it is preferred that the amount of taurine (including taurine salts) is less than <NUM>, more preferably less than <NUM> per <NUM> composition. In one embodiment, the composition comprises less than <NUM>, more preferably less than <NUM>, most preferably less than <NUM> cysteine and taurine per <NUM> of the (liquid) composition. In one embodiment, the composition comprises less than <NUM>, more preferably less than <NUM>, most preferably less than <NUM> cysteine per <NUM> of the (liquid) composition. It is preferred that the protein fraction comprises more than <NUM> weight% of casein or caseinates, or hydolysates thereof, and more preferably <NUM> weight% or more, because caseins comprise relatively low amounts of cysteine compared to other protein sources. It is further preferred to heat the liquid composition in order to oxidize the cysteine molecules present in the protein. This impairs biological availability of any residual cysteine as present in the formula. A preferred heat treatment involves sterilization. It is preferred to maintain the temperature remains below <NUM>, preferably less than <NUM> combined with a sufficient long time to have the cysteine oxidized, i.e. more than <NUM> seconds, preferably more than <NUM> seconds.

In one embodiment, it is preferred that the composition has a protein content of less than <NUM> en%, more preferably less than <NUM> en%, most preferably less than <NUM> en% of the total energy content of the composition. The energy percentages of the components are calculated using the calculation factors <NUM> kcal per g lipid, <NUM> kcal per g protein or g digestible carbohydrates, <NUM> kcal per g dietary fibers and zero kcal for the other components in the composition. In one embodiment, it is preferred that the composition comprises less than <NUM> to <NUM> protein per <NUM>, more preferably less than <NUM> to <NUM> gram protein per <NUM>, most preferably <NUM> to <NUM> gram protein/<NUM>.

It has been found that enhanced levels of manganese and molybdenum are not necessary in the composition for use according to the invention to achieve a beneficial effect on TBI. The amount of manganese consumed/administered in the composition for use of the invention is preferably less than <NUM>µg per <NUM>, preferably less than <NUM>µg per <NUM>, more preferably less than <NUM>µg per <NUM>, in particular less than <NUM>µg per <NUM>. In one embodiment, the amount of manganese administered per day is preferably less than <NUM>µg, more preferably less than <NUM>µg. In one embodiment, <NUM> liquid composition according to the invention comprises less than <NUM> molybdenum, preferably less than <NUM> molybdenum.

In one embodiment, the composition according to the invention comprises per daily dosage or per <NUM> of liquid (preferably water) :.

The compositions as described above can be used as a nutritional therapy, nutritional support, as a medical food, as a food for special medical purposes or as a nutritional supplement. Such product can be consumed at one, two or three servings of <NUM> per day during recovery and/or rehabilitation from TBI.

For a more complete understanding of the present disclosure, reference is now made to the following examples taken in conjunction with the accompanying drawings.

The effects of the composition comprising a diet according to the invention comprising and comprising ingredient as set out in table <NUM> was assessed in an experimental model of traumatic brain injury. Table <NUM> sets out the ingredients of the control and FC diet.

Adult <NUM>-<NUM> week-old male C57BL/<NUM> mice, weighing <NUM>-<NUM> (Charles River Laboratories, Harlow, UK), were used. Mice were housed in groups of four in standard cages provided with enrichment objects, in a <NUM> light/dark cycle, and given diet and water ad libitum. Food intake and body weight were monitored daily. All animal procedures were approved by the Animal Welfare and Ethical Review Body, at Queen Mary University of London and the UK Home Office, in accordance with the EU Directive <NUM>/<NUM>/EU.

A controlled cortical impact (CCI) TBI model and a craniotomy only (control) model were used in this study. Briefly, after a <NUM>-week acclimatisation period, mice were anaesthetized using a mixture of ketamine (<NUM>/Kg) and medetomidine (<NUM>/Kg) in sterile saline, administered intraperitoneally (i. Mice were placed in a stereotaxic frame and a midline longitudinal incision was performed to expose the skull. A right lateral craniotomy was carried out using a pneumatic drill, <NUM> behind Bregma and <NUM> lateral to the midline. CCI injury was induced using the following settings: a <NUM> impactor tip with a speed of <NUM>/s, a depth of <NUM> and a dwell time of <NUM>, applied using the PCI3000 Precision Cortical Impactor™ (Hatteras Instruments, Inc. A control group underwent craniotomy only. After injury, the skull flap was placed back and the skin was sutured. Mice were allowed to recover in an incubator (<NUM>) until they were fully awake and active. Buprenorphine (<NUM>/kg) administered subcutaneously (s. ) was used preoperatively for pre-emptive analgesia and post-operatively every <NUM> for <NUM> days post-TBI.

Following the injury, the CCI mice were randomized into two groups and fed daily with either a fresh control diet ('CCI-Control'; n=<NUM>), or with a multi-nutrient intervention diet ('CCI-FC'; n=<NUM>) for <NUM> days. The craniotomy group of mice were daily fed with control diet (Craniotomy; n=<NUM>). The diets were formulated by Nutricia Research, Nutricia Advanced Medical Nutrition (Utrecht, The Netherlands) and manufactured and pelleted by Ssniff (Soest, Germany). Diets were stored at - <NUM> until use, to prevent lipid oxidation, and fresh diet was given to the animals daily. No significant differences were observed in the mean daily food intake and body weight gain (between groups), throughout the experiment.

At day <NUM> post-TBI, <NUM> animals from each group were deeply anaesthetized with sodium pentobarbital (<NUM>/kg, i. ; Sagatal, Rhone Merieux, Harlow, UK), and received a transcardiac perfusion with phosphate-buffered saline (PBS; <NUM>, pH <NUM>), followed by <NUM>% paraformaldehyde (PFA) in phosphate buffer (<NUM>, pH <NUM>, <NUM>). The brains were dissected out and brain tissue blocks were paraffin-fixed for histology and immunohistochemistry (IHC).

All tissue staining was performed between bregma - <NUM> and bregma -<NUM>, where the lesion was located. Briefly, <NUM> sections were deparaffinised and hydrated through a series of xylene and ethanol baths. Sections were subjected to antigen retrieval with <NUM> citrate buffer (pH <NUM>) for <NUM> at <NUM> and allowed to cool at room temperature for <NUM>. Then, the tissue was blocked with <NUM>% normal donkey serum in <NUM>% Triton X-<NUM> in PBS for an hour, followed by three PBS washes and overnight staining with a primary antibody. The secondary antibodies were Alexa <NUM> or Alexa <NUM> (Molecular Probes, Leiden, The Netherlands; <NUM> :<NUM>), and Hoechst <NUM> stain (Sigma, UK; <NUM>µg/ml PBS) was used to visualize nuclei. Slides were mounted and cover-slipped using Vectashield fluorescent mounting medium (H-<NUM>; Vector Laboratories, Burlingame, CA).

Tissue from <NUM> animals from each group (CCI or craniotomy) was used for western blot analysis. After decapitation on day <NUM>, brains were quickly removed and a cube of the right hemisphere around the lesion was dissected using a sagittal brain slicer matrix. The tissue was snap frozen and stored at -<NUM>°C until analysis. Brain samples were prepared in RIPA lysis buffer (Sigma-Aldrich) complete with Protease Inhibitor Cocktail (Sigma-Aldrich) and sonicated. Samples were then centrifuged at <NUM>,<NUM> for <NUM> at <NUM> and the supernatant was taken. Protein concentrations were determined using the Bradford protein assay. Equal amounts of protein (<NUM>µg) from each sample were mixed with NuPAGE® LDS sample buffer (Thermo Fisher Scientific) and dithiothreitol (DTT) and boiled at <NUM> for <NUM> before loading. Proteins were separated using Mini-Protean TGX Gels, <NUM>% (Biorad, UK) and electro-transferred onto polyvinylidene difluoride membranes (Biotrace). Membranes were blocked in <NUM>% non-fat dry milk in Tris-buffered saline (pH <NUM>), with <NUM>% Tween-<NUM> (Tris-buffered saline-Tween) for <NUM> at room temperature. The primary antibodies used was: mouse anti-MBP (<NUM>:<NUM>,<NUM>; Abeam), diluted in <NUM>% bovine serum albumin solution, and membranes were incubated overnight at <NUM>°C. The primary antibody was removed and the blots were washed in Tris-buffered saline-Tween and incubated for <NUM> at room temperature in horseradish peroxidase-conjugated secondary antibodies (<NUM>:<NUM>,<NUM>; Jackson Immunoresearch). Reactive proteins were visualized using an enhanced chemiluminescence reagent (VWR International). Optical density was determined using Imaged software (National Institutes of Health). All membranes were also incubated with a mouse polyclonal antibody for β-actin (<NUM>:<NUM>,<NUM>; Merck Millipore). Protein level was expressed as relative optical density, representing the optical density of the band revealed by the primary antibody, divided by the optical density of β-actin within the same lane.

For calculation of the lesion size, sections of <NUM>, <NUM> apart and spanning the entire rostro-caudal extent of the injured cortex were stained with haematoxylin and eosin. The size of the lesion was measured with imaged software (National Institutes of Health, Bethesda, MD, USA). The lesion size was calculated using the following equation: the contralateral (non-lesioned) hemisphere size minus the injured hemisphere size and divided by the contralateral hemisphere size,. The results are expressed as a percentage of hemispheric tissue.

Analysis of the lesion size in the CCI model showed that at <NUM> dpi there was a significant loss of brain tissue, with almost total loss of the ipsilateral hippocampus. FC supplementation led to a significantly decreased lesion size (<FIG>).

A subset of representative randomly selected sections across the whole lesion was used for Luxol Fast Blue (Sigma, UK) myelin staining. Luxol Fast Blue staining (LFB) of the brain sections from animals following injury showed myelin disruption after CCI, particularly apparent within the caudate-putamen and internal capsule. Qualitative observations indicated preserved patterns of myelin in these regions in CCI-FC and craniotomy-control mice (<FIG>) with myelin-stained tracts that look continuous, whereas CCI-control diet animals showed severely disrupted patterns.

Tissue myelin basic protein (MBP) measurements using Western Blotting showed a statistically significant decrease in expression levels in CCI mice fed with the control diet, with the levels being fully restored after FC-treatment (<FIG>). Overall, these results indicate that the FC diet restores the TBI associated demyelinisation process and protects the white matter post-TBI.

Claim 1:
A composition for use in producing myelin in a mammal suffering from or recovering from traumatic brain injury, comprising enterally administering to the subject a composition comprising therapeutically effective amounts of:
(i) one or more of uridine, cytidine, or salts, phosphates, acyl derivatives or esters thereof;
(ii) a lipid fraction comprising at least one of docosahexaenoic acid (<NUM>:<NUM>; DHA), eicosapentaenoic acid (<NUM>:<NUM>; EPA) and docosapentaenoic acid (<NUM>:<NUM>; DPA), preferably at least DHA, more preferably DHA and EPA,
wherein the lipid fraction comprises less than <NUM> weight% of α-linolenic acid (ALA), calculated on the weight of all fatty acids;
(iii) choline, or salts or esters thereof;
(iv) vitamins B6, B9 and B12; and
(v) vitamin C, vitamin E and selenium.