WO2016083399A9 - Alpha-ketoglutarate in combination with glutamate dehydrogenase for treating hyperammonemia - Google Patents

Abstract

The invention relates to a pharmaceutical composition comprising alpha-ketoglutarate, optionally a glutamate dehydrogenase, and optionally a cofactor for glutamate dehydrogenase. The composition according to the invention is particularly useful for the treatment or prevention of a liver disease, wherein the liver disease is preferably hyperammonemia or a disease associated with hyperammonemia.

Description

Alpha-ketoglutarate in combination with glutamate dehydrogenase for treating hyperammonemia

[0001] The invention relates to a pharmaceutical composition comprising alpha-ketoglutarate, optionally a glutamate dehydrogenase, and optionally a cofactor for glutamate dehydrogenase. The composition according to the invention is particularly useful for the treatment or prevention of a liver disease, wherein the liver disease is preferably hyperammonemia or a disease associated with hyperammonemia.

[0002] Hyperammonemia is generally associated with severe complications, such as encephalopathy and coma. The most common causes of hyperammonemia are urea cycle defects in neonates, and liver cirrhosis or acute intoxications in adults. Therapy for hyperammonemia remains challenging.

[0003] Treatment of severe forms of hyperammonemia remains particularly difficult and often includes aggressive interventions. In emergency cases, hemodialysis is often considered to represent the most rapid and effective method of ammonia removal. However, hemodialysis remains associated with severe side effects, such as hemodynamic instability, hypertension, cramps, febrile reactions, arrhythmia, hemolysis and hypoxia. Nevertheless, up to now, hemodialysis remains the most efficient treatment for reducing severe hyperammonemia.

[0004] For milder forms of hyperammonemia pharmacologic management is possible. Efficient strategies for patients with urea cycle defects include infusion of phenyl acetate or benzoate. Phenyl acetate combines with glutamine to form a product which can be excreted by the kidneys, whereas benzoate combines with glycine to form hippurate which is also excreted in urine. Both compounds reduce the total body nitrogen content. However, the established pharmacotherapy with phenyl acetate and with benzoate can be a costly therapeutic option. Furthermore, this pharmacological treatment has also failed in a fraction of patients with hyperammonemic crisis who became refractory most probably due to the accumulation of nitrogen waste. This led to the concept that only blood ammonia concentrations below 500μΜ should be treated pharmacologically, whereas more severe hyperammonemia still requires the aggressive interventions with renal replacement therapies, such as hemodialysis.

[0005] Thus, there is a demand for methods of treating or preventing hyperammonemia and diseases or disorders that are associated with hyperammonemia, which methods overcome the drawbacks of the prior art. [0006] WO 2007/082914 relates to a method for diagnosing higher susceptibility for diseases and conditions associated with low levels of AKG in a human or animal comprising the following steps: a) obtaining a biological sample from said human or animal; b) measuring the alpha-ketoglutaric acid (AKG) level in the biological sample; and c) comparing said measured AKG level with normal average AKG levels, wherein a level of AKG in said sample lower than an average level is indicative of a higher susceptibility for various diseases. Further WO 2007/082914 relates to a use of a substance comprising at least one member selected from the group consisting of AKG and derivates, metabolites, analogues or salts thereof for the manufacture of a pharmaceutical preparation or a food or feed supplement for the treatment or prophylaxis of diseases and conditions associated with low levels of AKG in a human or animal with low levels of AKG compared with normal average AKG levels.

[0007] DE 199 29 993 relates to creatine alpha - ketoglutarates (1 :1) and creatine alpha -ketoglutarates (2:1) or other anion/creatine alpha -ketoglutarates (1 : 1 :1).

[0008] Szam I et al., Wiener Medizinische Wochenschrift, 1974, 319-325 relates to investigations concerning the efficacy of di-L(+)-ornithine-alpha-ketoglutarate in the treatment of hyperammonemia in rats.

[0009] US 3 441 650 relates to the therapy of ammonical intoxications by di-L-ornithine alpha- ketoglutarate.

[0010] WO 2007/122190 relates to a composition comprising alpha-ketoglutarate (AKG) for modulating muscle performance in a mammal including a human being. Also contemplated is a method for modulating muscle performance in a vertebrate, including mammal and bird, and the manufacture of a composition for the prevention, alleviation or treatment of muscle performance in said vertebrate.

[0011] WO 2014/046603 discloses a composition comprising alpha-ketoglutaric acid or a pharmaceutically acceptable salt thereof (AKG), and one or more enzymes selected from a group consisting of a lipase, a protease and an amylase, and medical uses thereof in, neurological and/or neurodegenerative disease, neurological trauma, depression or chronic fatigue syndrome.

[0012] US 3 929 581 discloses that blood ammonia is quantitatively determined by contacting a sample suspected of containing blood ammonia, with glutamate dehydrogenase, alpha -ketoglutarate, and, as a coenzyme in the reduced state, nicotinamide -adenine-dinucleotide phosphate in reduced form. [0013] CN 104 11 1 337 relates to a strong interference resistant homocysteine detection kit. The kit comprises a combination of 0.1 mM S-adenosyl-L-methionine, 0.3 mM NADH, 5mM tris(2-carboxyethyl) phosphine hydrochloric acid, 5.0 mM alpha-ketoglutarate, and 2.5 KU/I ascorbate oxidase (component 1 ) and a combination of 5 KU/I methyltransferase, 10 KU/I glutamate dehydrogenase, 2.5 KU/I S-adenosyl homocysteine hydrolase, 5.0 KU/I adenosine deaminase, and 20-30 KU/I cystathione beta-synthase (component 2) according to a volume ratio of 4: 1. The result obtained by using the kit is highly consistent with the result obtained through chemiluminescent detection, the accuracy and the sensitivity of detected clinic samples of the kit are better than that of routine reagents, and it is in favor of improving the clinic detection accuracy of homocysteine.

[0014] It is an object of the invention to provide a medicament that is useful for the treatment or prevention of hyperammonemia and of diseases or disorders that are associated with hyperammonemia which has advantages compared to the medicaments and to the alternative treatment options of the prior art.

[0015] This object has been achieved by the subject-matter of the patent claims.

[0016] The inventors have unexpectedly identified a so far unrecognized mechanism of ammonia detoxification. In particular, it has been surprisingly found that under conditions of hyperammonemia, glutamate dehydrogenase may change its enzymatic flow from ammonia production to ammonia consumption. This underlying pharmacological principle has been demonstrated in an animal model, namely by intravenous injection of glutamate dehydrogenase and its cofactors alpha-ketoglutarate and NADPH, together with aminooxy acetate, an inhibitor of competing metabolic pathways, into hyperammonemic mice. Therapy with a cocktail of these compounds at suitable doses and ratios reduced elevated blood ammonia concentrations close to normal levels within minutes after injection. Thus, especially in situations with either severe or with refractory hyperammonemia, the therapeutic strategy according to the invention is an alternative to hemodialysis.

[0017] It has been surprisingly found that the ammonia detoxification can take place in blood already, i.e. can be induced by intravenous administration. Its efficacy depends on the concentrations of substrate, product, and cofactors (ammonia, glutamate, alpha-ketoglutarate and NADPH).

[0018] A first aspect of the invention relates to a pharmaceutical composition comprising alpha- ketoglutarate (2-oxopentanedioic acid), optionally a glutamate dehydrogenase, and optionally a cofactor for glutamate dehydrogenase. [0019] Preferably, the pharmaceutical composition according to the invention comprises a glutamate dehydrogenase. However, the pharmaceutical composition according to the invention does not necessarily need to comprise a glutamate dehydrogenase, as depending upon the condition of the subject to be treated, the subject's own glutamate dehydrogenase may be sufficient in order to achieve the desired therapeutic effect when administering alpha-ketoglutarate. The serum concentration of glutamate dehydrogenase may be easily determined by a skilled person by means of routine tests that are well established and commercially available. Based upon the measured serum concentration it may then be decided whether glutamate dehydrogenase is to be co-administered or not. Therefore, the presence of the glutamate dehydrogenase in the pharmaceutical composition and its dose depends upon the individual needs of the subject to be treated. When for some reasons the concentration of glutamate dehydrogenase in the subject's serum is comparatively high, the pharmaceutical composition according to the invention does not need to comprise high doses of glutamate dehydrogenase or it may even contain no glutamate dehydrogenase at all.

[0020] Based on which cofactor is used, glutamate dehydrogenases are divided into the following three classes:

- only NAD⁺ is used as cofactor: EC 1.4.1.2;

- both NAD⁺ and NADP⁺ (i.e. NAD(P)⁺) are used as cofactors: EC 1.4.1.3; and

- only NADP⁺ is used as cofactor: EC 1.4.1.4.

[0021] Preferably, the glutamate dehydrogenase according to the invention belongs to class EC 1.4.1.3, i.e. is capable of using both, NAD⁺ and NADP⁺. In this regard it is to be noted that in accordance with the invention the relevant reaction that is catalyzed by the glutamate dehydrogenases is the reverse reaction such that the cofactor needs to be present in the pharmaceutical composition in its hydrogenated form, i.e., NADH and NADPH, respectively.

[0022] Preferably, the glutamate dehydrogenase according to the invention is human glutamate dehydrogenase (homo sapiens). While glutamate dehydrogenase in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two human isoenzymes (hGDHl and hGDH2), though highly homologous, differ markedly in their regulatory properties. For the purpose of the specification, human glutamate dehydrogenase encompasses both isoenzymes in any mixing ratio or either isoenzyme in substantially pure form. [0023] Besides the alpha-ketoglutarate and the optionally present glutamate dehydrogenase, the pharmaceutical composition according to the invention preferably comprises a cofactor for glutamate dehydrogenase. Preferably, the cofactor according to the invention is NADH or NADPH, particularly preferably NADPH. However, the pharmaceutical composition according to the invention does not necessarily need to comprise a cofactor for glutamate dehydrogenase, as depending upon the condition of the subject to be treated, the subject's own cofactor for glutamate dehydrogenase may be sufficient in order to achieve the desired therapeutic effect when administering alpha-ketoglutarate. The serum concentration of cofactor for glutamate dehydrogenase may be easily determined by a skilled person by means of routine tests that are well established and commercially available. Based upon the measured serum concentration it may then be decided whether cofactor for glutamate dehydrogenase is to be coadministered or not. Therefore, the presence of the cofactor in the pharmaceutical composition and its dose depends upon the individual needs of the subject to be treated. When for some reasons that concentrations of cofactor, particularly of NADH or NADPH in the subject's serum are comparatively high, the pharmaceutical composition according to the invention does not need to comprise high doses of cofactor or it may even contain no cofactor at all.

[0024] In preferred embodiments, the pharmaceutical composition according to the invention belongs to any of types (i), (ii), (iii) or (iv) and comprises

(i) alpha-ketoglutarate, but neither glutamate dehydrogenase nor cofactor; or

(ii) alpha-ketoglutarate and glutamate dehydrogenase, but no cofactor; or

(iii) alpha-ketoglutarate and cofactor, but no glutamate dehydrogenase; or

(iv) alpha-ketoglutarate and glutamate dehydrogenase and cofactor.

[0025] The majority of subjects suffering from hyperammonemia or a disease associated with hyperammonemia typically require administration of a pharmaceutical composition of type (iv), i.e. comprising all three components, alpha-ketoglutarate and glutamate dehydrogenase and cofactor. This is because ammonia may only be consumed by reaction with alpha-ketoglutarate if (1) sufficient glutamate dehydrogenase is present in blood and if (2) sufficient cofactor is present in blood. These requirements are usually not satisfied so that glutamate dehydrogenase and/or cofactor need to be coadministered with alpha-ketoglutarate.

[0026] In a minority of subjects, however, the above requirements are satisfied so that the sole administration of alpha-ketoglutarate (type (i)) or the administration of the combination of alpha- ketoglutarate either with only glutamate dehydrogenase (type (ii)) or with only cofactor (type (iii) may already be sufficient. In said minority of subjects both glutamate dehydrogenase and/or cofactor are already present in blood at relevant levels, e.g. because liver tissue has been damaged; in this case, glutamate dehydrogenase and/or cofactor are released from damaged liver cells. Nonetheless, levels of glutamate dehydrogenase and/or cofactor are increased only under rare circumstances, e.g. due to poisoning by acetaminophen or mushroom, e.g. amantia. Under such rare circumstances, the subjects typically merely require administration of a pharmaceutical composition of type (i), (ii) or (iii).

[0027] In contrast to such rare circumstances, in association of more frequent terminal stage chronic liver diseases, typically neither the level of glutamate dehydrogenase nor the level cofactor are significantly increased in blood so that both need to be substituted for therapy according to the invention (type (iv)).

[0028] In a preferred embodiment according to the invention, the threshold level for glutamate dehydrogenase, preferably for human glutamate dehydrogenase, in blood is 375 U/l, the threshold value for cofactor, preferably for NADPH, in blood is 0.2 μΜ, and the subjects to be treated in accordance with the invention can be divided into the following populations that are to be treated by administration of pharmaceutical compositions according to any of types (i), (ii), (iii) or (iv):

(i) level of glutamate dehydrogenase at threshold or above and level of cofactor at threshold or above: administration of pharmaceutical compositions of type (i);

(ii) level of glutamate dehydrogenase below threshold and level of cofactor at threshold or above: administration of pharmaceutical compositions of type (ii);

(iii) level of glutamate dehydrogenase at threshold or above and level of cofactor below threshold: administration of pharmaceutical compositions of type (iii); and

(iv) level of glutamate dehydrogenase below threshold and level of cofactor below threshold: administration of pharmaceutical compositions of type (iv).

[0029] In a preferred embodiment, besides the alpha-ketoglutarate, the glutamate dehydrogenase, and the optionally comprised cofactor for glutamate dehydrogenase, the pharmaceutical composition according to the invention additionally comprises a transaminase inhibitor. Suitable transaminase inhibitors are known to the skilled person and include but are not limited to aminooxy acetate.

[0030] The content of alpha-ketoglutarate within the pharmaceutical composition according to the invention is not particularly limited and may vary e.g. within the range of from 0.001 to 95 wt.-%, relative to the total weight of the pharmaceutical composition. Preferably, the content of alpha-ketoglutarate within the pharmaceutical composition according to the invention is within the range of from 30±25 wt.- %, 50±25 wt.-%, 70±25 wt.-%, 10±9 wt.-%, 15±9 wt.-%, 20±9 wt.-%, 25±9 wt.-%, 30±9 wt.-%, 35±9 wt.- %, 40±9 wt.-%, 45±9 wt.-%, 50±9 wt.-%, 55±9 wt.-%, 60±9 wt.-%, 65±9 wt.-%, 70±9 wt.-%, 75±9 wt- %, or 80±9 wt.-%, relative to the total weight of the pharmaceutical composition.

[0031] The content of the optionally present glutamate dehydrogenase within the pharmaceutical composition according to the invention is not particularly limited either and may vary e.g. within the range of from 0.0001 to 5 wt.-%, relative to the total weight of the pharmaceutical composition.

[0032] The content of the optionally present cofactor within the pharmaceutical composition according to the invention is not particularly limited either and may vary e.g. within the range of from 0.001 to 50 wt- %, relative to the total weight of the pharmaceutical composition.

[0033] The content of the optionally present transaminase inhibitor within the pharmaceutical composition according to the invention is not particularly limited either and may vary e.g. within the range of from 0.001 to 50 wt.-%, relative to the total weight of the pharmaceutical composition.

[0034] The pharmaceutical composition according to the invention may be solid, semisolid or liquid. Preferably, the pharmaceutical composition according to the invention is liquid.

[0035] Preferably, the pharmaceutical composition according to the invention comprises one or more physiologically acceptable pharmaceutical excipients such as carriers, solvents, fillers, binders and the like. The overall content of all pharmaceutical excipients within the pharmaceutical composition according to the invention is not particularly limited and may vary e.g. within the range of from 0.001 to 99.999 wt.-%, relative to the total weight of the pharmaceutical composition. Preferably, the overall content of all pharmaceutical excipients within the pharmaceutical composition according to the invention is within the range of from 30±25 wt.-%, 50±25 wt.-%, 70±25 wt.-%, 20±15 wt.-%, 30±15 wt.-%, 40±15 wt.-%, 50±15 wt.-%, 60±15 wt.-%, 70±15 wt.-%, 80±15 wt.-%, 10±9 wt.-%, 15±9 wt.-%, 20±9 wt.-%, 25±9 wt.-%, 30±9 wt.-%, 35±9 wt.-%, 40±9 wt.-%, 45±9 wt.-%, 50±9 wt.-%, 55±9 wt.-%, 60±9 wt.-%, 65±9 wt.-%, 70±9 wt.-%, 75±9 wt.-%, 80±9 wt.-%, 85±9 wt.-%, or 90±9 wt.-%, relative to the total weight of the pharmaceutical composition.

[0036] Another aspect of the invention relates to a pharmaceutical dosage form comprising the pharmaceutical composition according to the invention. Any preferred embodiment that has been defined in connection with the pharmaceutical composition according to the invention also analogously applies to the pharmaceutical dosage form according to the invention. [0037] For the purpose of the specification, the term "formulation" shall refer to both, to the pharmaceutical composition according to the invention as well as to the pharmaceutical dosage form according to the invention, respectively.

[0038] The formulation according to the invention may be administered by various means, depending on its intended use. For example, if the formulation according to the invention is to be administered orally, it may be provided in form of tablets, capsules, granules, powders or syrups. Alternatively, the formulation according to the invention may be administered parenterally as injections (intravenous, intramuscular or subcutaneous), drop infusion preparations or suppositories. These formulations may be prepared by conventional means, and, if desired, the formulations may be mixed with any conventional additive, such as an excipient, a binder, a carrier, a disintegrating agent, a buffer, an osmolality adjuster, a surfactant, a lubricant, a solubilizing agent, a suspension aid, an emulsifying agent, a coating agent and any mixtures thereof.

[0039] In formulations of the invention, wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may be present.

[0040] The formulation may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of composition that may be combined with a carrier material to produce a single dose varies depending upon the subject being treated, and the particular route of administration.

[0041] Formulations suitable for oral administration may be in the form of capsules, sachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of alpha-ketoglutarate, glutamate dehydrogenase, optionally present cofactor and optionally present transaminase as active ingredients. Formulations according to the invention may also be administered as a bolus, electuary, or paste.

[0042] In solid formulations for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the alpha-ketoglutarate, the glutamate dehydrogenase, the optionally present cofactor and the optionally present transaminase inhibitor are preferably mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, e.g., acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the formulations may also comprise buffering agents. Solid formulations of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0043] A tablet may be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art.

[0044] Liquid formulations for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition, the liquid formulations may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, cyclodextrins and mixtures thereof.

[0045] Suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. [0046] Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing the alpha-ketoglutarate, the glutamate dehydrogenase, the optionally present cofactor and the optionally present transaminase inhibitor with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent. Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0047] Formulations for parenteral administration preferably comprise the alpha-ketoglutarate, the glutamate dehydrogenase, the optionally present cofactor and the optionally present transaminase inhibitor in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0048] Examples of suitable aqueous and non-aqueous carriers which may be employed in the formulations according to the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate and cyclodextrins. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0049] The alpha-ketoglutarate, the glutamate dehydrogenase, the optionally present cofactor and the optionally present transaminase inhibitor can be formulated for parenteral administration, as for example, for subcutaneous, intramuscular or intravenous injection, e.g., the alpha-ketoglutarate, the glutamate dehydrogenase, the optionally present cofactor and the optionally present transaminase inhibitor can be provided in a sterile solution or suspension (injectable formulation).

[0050] The dosage of any formulation according to the invention will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the formulation. Any of the formulations may be administered in a single dose or in divided doses. Dosages for the formulations according to the invention may be readily determined by techniques known to those of skill in the art. Treatment may be initiated with smaller dosages which are less than the optimum dose of the alpha-ketoglutarate, the glutamate dehydrogenase, the optionally present cofactor and the optionally present transaminase inhibitor, respectively. Thereafter, the dosage may be increased by small increments until the optimum therapeutic effect is achieved.

[0051 ] For the treatment of hyperammonemia, alpha-ketoglutarate should only be administered in doses that return the decreased plasma concentrations back to normal levels.

[0052] Preferably, the administered dose of alpha-ketoglutarate is within the range of from 1 to 10,000 mg/kg body weight, more preferably from 10 to 1 ,000 mg/kg body weight, most preferably from 100 to 500 mg/kg body weight.

[0053] Preferably, the administered dose of glutamate dehydrogenase is within the range of from 1 to 10,000 U/kg body weight, more preferably from 10 to 5,000 U/kg body weight, most preferably from 100 to 2,000 U/kg body weight.

[0054] Preferably, the administered dose of cofactor, preferably of NADPH, is within the range of from 1 to 10,000 mg/kg body weight, more preferably from 10 to 1,000 mg/kg body weight, most preferably from 100 to 500 mg/kg body weight.

[0055] Preferably, the administered dose of transaminase inhibitor, preferably of aminooxy acetate, is within the range of from 0.1 to 1 ,000 mg/kg body weight, more preferably from 1 to 100 mg/kg body weight, most preferably from 5 to 50 mg/kg body weight.

[0056] Preferably, the formulation according to the invention is adapted for systemic administration, particularly for intravenous administration.

[0057] Preferably, the formulation according to the invention is for use in the treatment or prevention of a liver disease. In this regard, the invention also relates to a method of treating or preventing a liver disease comprising the administration of an effective amount of the formulation according to the invention to a subject in need thereof. Further, the invention also relates to the use of alpha-ketoglutarate, glutamate dehydrogenase and optionally a cofactor for the manufacture of a formulation according to the invention for the treatment or prevention of a liver disease.

[0058] Preferably, the liver disease is hyperammonemia or a disease associated with hyperammonemia. In a preferred embodiment, the liver disease is severe hyperammonemia or refractory hyperammonemia, primary hyperammonemia or secondary hyperammonemia, acquired hyperammonemia or congenital hyperammonemia, or the disease is associated with any of the forgoing hyperammonemia. Preferably, the liver disease is selected from the group consisting of urea cycle defects, liver cirrhosis and acute intoxication. Preferably, severe hyperammonemia is considered at serum ammonia levels of at least 500 μηιοΙ/L, more preferably at least 1000 μιηοΙ/L.

[0059] Primary hyperammonemia is caused by several inborn errors of metabolism that are characterized by reduced activity of any of the enzymes in the urea cycle. Secondary hyperammonemia is caused by inborn errors of intermediary metabolism characterized by reduced activity in enzymes that are not part of the urea cycle (e.g. propionic acidemia, methylmalonic acidemia) or dysfunction of cells that make major contributions to metabolism (e.g. hepatic failure). Acquired hyperammonemia is usually caused by liver diseases, such as viral hepatitis, or excessive alcohol consumption. Cirrhosis of the liver is formed, followed by a shunt of blood directly to the vena cava, resulting in decreased filtration of blood in the liver, which leads to hyperammonemia. Congential hyperammonemia is usually due to genetic defects in one of the enzymes of the urea cycle, which leads to lower production of urea from ammonia. The most common genetic defect is ornithine transcarbamylase deficiency. Specific types of hyperammonemia to be treated or prevented according to the invention include but are not limited to hyperammonemia due to ornithine transcarbamylase deficiency, hyperinsulinism-hyperammonemia syndrome (glutamate dehydrogenase 1), hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (ornithine translocase), hyperammonemia due to N-acetylglutamate synthetase deficiency, hyperammonemia due to carbamoyl phosphate synthetase I deficiency (carbamoyl phosphate synthetase I), hyperlysinuria with hyperammonemia (genetics unknown), methylmalonic acidemia, isovaleric acidemia, propionic acidemia, carnitine palmitoyltransferase II deficiency, and transient hyperammonemia of the newborn, specifically in the preterm.

[0060] Another aspect of the invention relates to a kit comprising a first pharmaceutical formulation which comprises alpha-ketoglutarate and a second pharmaceutical formulation which comprises a glutamate dehydrogenase. In a preferred embodiment, the first pharmaceutical formulation and the second pharmaceutical formulation are adapted for administration via the same route. In another preferred embodiment, the first pharmaceutical formulation and the second pharmaceutical formulation are adapted for administration via different routes. Optionally, the first pharmaceutical formulation and/or the second pharmaceutical formulation may independently of one another comprise the cofactor and optionally, the transaminase inhibitor. [0061] It has been surprisingly and unexpectedly found that infusion of a cocktail comprising alpha- ketoglutarate, glutamate dehydrogenase and NADPH reduces ammonia close to normal levels within minutes. Under these conditions, a glutamate dehydrogenase-catalyzed reaction takes place in blood where ammonia and alpha-ketoglutarate are consumed to form glutamate in an NADPH-dependent reaction. By releasing glutamate dehydrogenase from damaged hepatocytes into the blood, the intoxicated liver provides a mechanism which reduces blood ammonia levels. However, this protective mechanism is limited by the availability of the glutamate dehydrogenase substrate, alpha-ketoglutarate. The combined injection of alpha-ketoglutarate, glutamate dehydrogenase and optionally NADPH efficiently reduces blood ammonia concentrations. In the mouse experiments, the transaminase inhibitor aminooxy acetate was additionally injected to reduce the generation of glutamate from alpha-ketoglutarate. Reduction of ammonia per se does not depend on aminooxy acetate, but aminooxy acetate was shown to inhibit the increase in glutamate which is neurogenic at high concentrations.

[0062] The invention provides a form of therapy that allows the rapid correction of hyperammonemia by administration, e.g. infusion of alpha-ketoglutarate, glutamate dehydrogenase and NADPH. This pharmacotherapy is particularly relevant as an emergency therapy for episodes of hyperammonemia in urea cycle disease or liver cirrhosis. It may be used in combination with the established phenyl acetate therapy, because alpha-ketoglutarate/glutamate dehydrogenase/NADPH generate glutamate that is further metabolized to glutamine, which in turn binds to phenyl acetate and forms a product that is efficiently excreted in urine. Therefore, the alpha-ketoglutarate/glutamate dehydrogenase/NADPH cocktail according to the invention may serve as a short term emergency treatment for extreme hyperammonemia, while long term administration of phenyl acetate will reduce the overall nitrogen burden.

[0063] The following examples illustrate the invention but are not to be construed as limiting its scope. [0064] Example 1 - integrated spatial-temporal-metabolic model

[0065] To simulate the detoxification process in healthy, damaged and regenerating livers, a recently- established integrated metabolic spatial -temp oral model (IM) was used. However, this model did not yet consider possible changes in enzyme activities during the damage and regeneration process. To identify possible alterations, time resolved gene array analysis of mouse liver tissue was performed after CC1₄ intoxication (Fig. 1A-D; 9). Fuzzy clustering identified seven gene clusters which reflected time- dependent gene expression alterations. Clusters 4 and 6 contained genes whose expression was transiently repressed at early time points after CC1₄ intoxication (Fig. IB). Further bioinformatics analyses revealed an overrepresentation of nitrogen/ammonia metabolism KEGG and GO terms of genes in Cluster 4 (Tables 3 and 4). Genes relevant for ammonia metabolism were further studied by qRT-PCR, immunostaining and activity assays. Glutamine synthetase (GS) is the key enzyme of ammonia detoxification in the pericenfral compartment (Fig. 8). GS RNA levels started to decrease as early as at 6h after CC1₄ injection, went down to very low levels between days 1 and 4 and recovered between days 6 and 30 (Fig. 1C). A similar time-dependent curve was obtained for GS activity although the decrease occurred slightly later than that of RNA with very low levels between days 2 and 4 (Fig. 1C). The pattern and intensity of GS immunostaining was found to be comparable to GS activity (Fig. ID). The key enzymes of the periportal compartment- CPS1, ASS1, ASL and arginasel- were similarly analyzed in the same tissue (Fig. 10 and 11). CPS1 RNA levels decreased between 12h and day 3 (Fig. 10A). The typical periportal staining pattern for CPS1 was observed in the control liver samples. CC1₄ treatment led to a decreased immunostaining signal between day 1-3, followed by a period of overcompensation between days 4 and 6 where CPS1 was not only expressed in the periportal but also in the pericentral compartment of the liver lobule (Fig. 10B). ASS1 and ASL were also strongly decreased between 12h and day 3; whereas arginasel remained almost unaltered (Fig. 10A; 1 1 A and 1 1 B). Glutaminase showed a similar time course and spatial expression pattern as the urea cycle enzymes (Fig. 10A).

[0066] The influence of the CC -induced alterations on ammonia metabolism can be quite complex. The decrease in GS and urea cycle enzymes could lead to an increase in ammonia concentrations, whereas, the overcompensation of CPS1 (days 4-6) may decrease ammonia levels. Due to the complexity of the relationship between the CCl₄-induced gene alterations and their potential effect on ammonia concentration, the IM model was employed for blood ammonia concentrations (Fig. 2). To compare the simulated concentrations to the in vivo situation, an experiment was performed in which blood was collected from the portal vein (corresponding Lo the 'liver inflow'), the hepatic vein (corresponding to the 'liver outflow'), and mixed venous blood from the right heart chamber in a time resolved manner after CC1₄ injection (Fig. 2A; 9). The result shows that ammonia is detoxified during the liver passage as illustrated by the difference in ammonia concentrations between the portal vein and the hepatic vein in controls (Fig. 2B). This detoxification is compromised after liver damage, particularly on days 1 and 2. The inclusion of mixed venous blood demonstrates the contribution of the extrahepatic compartment to ammonia detoxification between days 1 and 4 after induction of liver damage. However, this extrahepatic contribution is small compared to detoxification by the liver (Fig. 15 and 16). Surprisingly, the ISTM model predicted much higher ammonia blood concentration than experimentally observed (Fig. 2D). The discrepancy was particularly high on day 1 after CC1₄ injection with measured liver vein ammonia concentrations of 380 and 179 μΜ, respectively (Fig. 2D). [0067] In addition to the time-resolved study, similar experiments were also performed in a dose dependent manner. For this purpose doses ranging between 10.9 and 1600 mg/kg CC1₄ were tested, resulting in a concentration dependent increase in the dead cell area which only at the highest dose destroys the entire CYP2E1 positive pericentral region of the liver lobule (Fig. 2E; 17A and B). Whereas the destruction of the GS positive area occurred in the dose range between 38.1 and 132.4 mg/kg; also CPSl showed a dose dependent decrease (Fig. 2E and F; 17C). Including these data into the IM model led to a dose dependent increase of the discrepancy between simulated and measured ammonia concentrations (Fig. 2G). The discrepancy could mean that our model lacks a relevant, but so far unrecognized mechanism of ammonia detoxification.

[0068] Example 2 - metabolic analysis

[0069] Further evidence that an unrecognized mechanism of ammonia detoxification exists arose from metabolic analysis performed using the plasma from mice after CC1₄ injection (Fig. 3).

[0070] Table 1 - Alteration of amino acids metabolism in mice liver tissue at different time intervals after intoxication with a single dose of CC (1.6 g/kg):

[nmol/mg Phenyl¬

Methionine Serine Threonine Aspartate Glutamate Ornithine protein] alanine

T M SD M SD M SD M SD M SD M SD M SD

Oh 0.68 0.12 2.10 0.31 1.62 0.14 0.80 0.16 1.52 0.65 12.44 3.19 0.91 0.19 lh 0.77 0.14 2.23 0.60 1.75 0.34 1.04 0.22 1.70 0.57 12.50 3.72 0.93 0.16

6h 0.77 0.20 2.63 0.57 1.97 0.45 0.94 0.21 1.78 0.69 14.79 0.75 1.00 0.22

12h 0.44 0.06 1.66 0.29 1.16 0.21 0.56 0.08 0.97 0.46 14.64 4.77 0.54 0.15

Id 0.49 0.02 2.15 0.19 1.85 0.17 0.71 0.07 3.97 0.47 16.29 2.93 0.99 0.19

2d 0.69 0.18 3.14 0.71 2.41 0.45 0.96 0.20 4.75 2.77 17.97 5.38 1.86 0.58

3d 0.81 0.15 3.20 0.77 2.60 0.40 1.06 0.11 2.77 1.25 21.59 9.00 2.19 0.91 4d 0.68 0.05 2.02 0.85 1.70 0.82 0.92 0.15 2.26 0.88 14.31 4.37 1.47 0.68

6d 0.73 0.06 1.90 0.26 1.81 0.33 0.95 0.04 1.83 0.68 18.11 3.32 1.24 0.18

12d 0.90 0.19 3.06 0.96 2.37 0.67 0.93 0.12 1.60 0.40 13.18 4.53 1.22 0.09

M = mean

SD = standard deviation

T = time after CC1₄ administration

Data are mean values and standard deviations of three mice analyzed per time point

Table 2 - Alteration of organic acids metabolism in mice liver tissue at different time intervals after intoxication with a single dose of CC (1.6 g/kg):

M = mean

SD = standard deviation

T = time after CC1₄ administration

Data are mean values and standard deviations of three mice analyzed per time point

[0071] Most of the analyzed factors in plasma (urea, glutamine, glucose, lactate, pyruvate, alanine, arginine and other amino acids: Fig. 1 1 ; 12; 13 and 14) were as expected except for alpha- ketoglutarate (alpha-ketoglutarate), which dramatically decreased between 12h and day 2 (Fig. 3A). This decrease was accompanied by a concurrent increase in glutamate levels, which persisted longer than the drop in alpha-ketoglutarate. One potential explanation is the delayed recovery of GS, which uses glutamate and ammonia to form glutamine Tables 3 and 4):

Table 3:

Table 4:

[0072] Table 3 shows changes in expression of genes associated to the KEGG terms ammonia/nitrogen metabolism (Gene Ontology ID 910) as revealed by KEGG pathways enrichment analysis in fuzzy cluster 4 (p=2.36xl0^~7). Table 4 shows changes in the expression of genes associated to the GO terms "urea cycle/urea metabolic process" (Gene Ontology ID 0000050 and 0019627 respectively) as revealed by GO enrichment analysis in fuzzy cluster 4 (p=3.83xl0 ). In both tables the values indicate fold of expression over healthy liver at each time point after CCU administration, and correspond to the average of 5 independent biological replicas. Time course of GS RNA levels, GS activity. [0073] The decrease in alpha-ketoglutarate (and the increase in glutamate) was also accompanied by increased glutamate dehydrogenase (glutamate dehydrogenase) activity in plasma, probably because glutamate dehydrogenase is released from damaged hepatocytes (Fig. 3A). The conventional reaction catalyzed by glutamate dehydrogenase in the periportal hepatocytes is the conversion of glutamate to alpha-ketoglutarate and ammonia. However, the present observations suggest that the released glutamate dehydrogenase catalyzes a reverse reaction that consumes ammonia to produce glutamate (Fig. 3C). To test this hypothesis, plasma from mice was taken on day 1 after CC injection. Addition of alpha- ketoglutarate led to a decrease in ammonia, but an increase in glutamate production (Fig. 3B), both of which were blocked by the glutamate dehydrogenase inhibitor, 2,6-pyridinedicarboxylic acid (PDAC). Under these conditions (l OmM PDAC) the glutamate dehydrogenase activity decreased to less than 15% of the control. Together, these experiments suggest that indeed a peripheral glutamate dehydrogenase reaction with 'switched' orientation (ammonia consumption instead of production) takes place when glutamate dehydrogenase is released from acutely damaged livers.

[0074] Example 3 - the 'glutamate dehydrogenase switch' in cultivated hepatocytes

[0075] The experiments described above suggest that high ammonia concentrations in plasma led to a 'reverse' glutamate dehydrogenase reaction, which consumes rather than produce ammonia. To test whether this 'glutamate dehydrogenase switch' occurs not only in plasma but also in cells, collagen sandwich cultures of mouse hepatocytes were used (Fig. 4). Without adding ammonia to the culture medium, glutamate dehydrogenase produces ammonia (Fig. 4A). This is illustrated by the small, but statistically significant decrease in ammonia levels in the culture medium upon glutamate dehydrogenase inhibition. With increasing ammonia concentrations in the culture medium glutamate dehydrogenase switches from ammonia production to ammonia consumption. Correspondingly, the glutamate dehydrogenase inhibitor decreases intracellular glutamate concentrations only if ammonia in the culture medium is high (Fig. 4B). The importance of glutamate dehydrogenase for ammonia detoxification is also illustrated in cytotoxicity experiments (Fig. 4D). Five mM NH₄C1 alone had no effect on the viability of cultivated hepatocytes; however, the addition of PDAC leads to massive cell killing. The results show that the catalytic direction of glutamate dehydrogenase can be changed both in plasma and also in hepatocytes, although un-physiologically high ammonia concentrations are required to induce the glutamate dehydrogenase switch in the latter.

[0076] Example 4 - therapy of hyperammonemia by the reverse glutamate dehydrogenase reaction [0077] In view of the above described switch of the glutamate dehydrogenase reaction (Fig. 3B and Fig. 4) and the aforementioned decrease in plasma alpha-ketoglutarate levels (Fig. 3A) it was tested whether supplementation of alpha-ketoglutarate in mice helps to detoxify ammonia. Therefore, mice received a hepatotoxic dose of CC1₄ (1.6 g/kg) and 24h later alpha-ketoglutarate (280 mg/kg) was injected into the tail vein. Blood was collected immediately before as well as 15, 30 and 60 min after injection of alpha- ketoglutarate. A decrease in ammonia plasma concentrations by 31, 40 and 43% was observed 15, 30 and 60 min after alpha-ketoglutarate injection, respectively (Fig. 5A). Glutamate increased after 15 min and decreased again after longer periods probably due to consumption by further metabolism. Alpha- ketoglutarate transiently increased in plasma after injection and then rapidly decreased. Analysis of glutamate dehydrogenase activity demonstrated that the experiment was performed under conditions of high plasma activity. In control mice, injection of alpha-ketoglutarate did not alter blood concentrations of ammonia or glutamate (Fig. 5B). In addition, plasma alpha-ketoglutarate levels were lower in CCI₄- treated mice compared to the control mice, suggesting increased consumption in mice with damaged livers.

[0078] In the aforementioned experiment, the amount of glutamate produced in the damaged liver after alpha-ketoglutarate injection was higher than ammonia consumption (Fig. 5 A). Therefore, the results can not only be explained by the reverse glutamate dehydrogenase reaction, but may be due to the consumption of alpha-ketoglutarate by transaminases which contribute to the generation of glutamate. Indeed, tail vein injection of the transaminases inhibitor aminooxy acetate prior to alpha-ketoglutarate injection reduced the production of glutamate (Fig. 5C) and improved ammonia detoxification. The efficiency of transaminases inhibition by aminooxy acetate in vivo has been confirmed in preliminary experiments (Fig. 19). Injection of 13 mg aminooxy acetate/kg reduced plasma AST and ALT activities by more than 65% for up to one hour. ALT and AST activities were also reduced in the mice shown in Fig. 5C (Fig. 20) while the activity of glutamate dehydrogenase remained unaltered.

[0079] The reverse glutamate dehydrogenase reaction requires reduced NADPH as a cofactor; however, NADPH concentrations are generally extremely low in blood. To determine how NADPH levels are altered in our model of liver damage, both NADPH and its oxidized form NADP⁺ were analyzed. An increase of blood NADPH and NADP⁺ was seen after induction of liver damage by CCI₄ (Fig. 21 A). In addition, an enhanced NADPVNADPH ratio was observed in both blood and liver tissue (Fig. 21B). This increase in NADPVNADPH ratio fits to a switch in the glutamate dehydrogenase reaction from NADPH generation to NADPH consumption. Despite the increased NADPH after induction of liver damage, the concentrations are still relatively low. To study if the influence of NADPH is concentration dependent, plasma from mice collected 24h after CC1₄ injection was incubated with varying concentrations of NADPH in the presence of NH₄C1 (ImM), alpha -ketoglutarate (3mM), aminooxy acetate (ImM) and glutamate dehydrogenase (12000U/1) for one hour. A concentration dependent decrease in plasma ammonia and an increase in glutamate were observed with increasing concentrations of NADPH (Fig. 6A). A similar trend for ammonia and glutamate was observed with increasing concentrations of alpha-ketoglutarate and glutamate dehydrogenase (Fig. 6 B and C). Moreover, addition of aminooxy acetate reduced both ammonia and glutamate concentrations (Fig. 22).

[0080] Based on these in vitro optimized concentrations, an in vivo study was designed to treat hyperammonemia in mice. After induction of liver damage by CC1₄ transaminases activities were inhibited by aminooxy acetate (13 mg/kg; tail vein injection; 24h after CC1₄ administration). Thirty minutes later a cocktail of alpha-ketoglutarate (280 mg/kg), glutamate dehydrogenase (720 U/kg) and NADPH (180 mg/kg) was intravenously injected. A dose of 280 mg/kg alpha-ketoglutarate was chosen because it transiently normalized alpha-ketoglutarate levels in mice 24h after CC . 720 U/kg glutamate dehydrogenase was used because it resulted in plasma levels to approximately 6000 U/l 15 min after injection (Fig. 23) - an activity level shown to allow maximal ammonia consumption in plasma in vitro (Fig. 6C). The dose of 180 mg/kg NADPH was also considered as adequate in a pharmacokinetic experiment (Fig. 24 as it transiently increased plasma NADPH of approximately 1.6 mM 2 min after injection. Injection of the alpha-ketoglutarate/glutamate dehydrogenase/NADPH cocktail (KGN-cocktail) reduced ammonia concentrations from 213 to 74 μΜ within 15 min after administration (Fig. 7). Simultaneously, glutamate increased from 131 to 369 μΜ. Analysis of alpha-ketoglutarate and glutamate dehydrogenase activity in the plasma showed that substitution was successful 15 min after injection of the KGN cocktail. Moreover, the activities of AST and ALT were successfully inhibited by aminooxy acetate. The mice were observed for three weeks after the experiment and did not show any complications.

[0081] Example 5 - influence of ammonia and glutamate concentrations on the orientation of the GDH reaction

[0082] To understand how the orientation of the GDH reaction is controlled by ammonia and glutamate concentrations, titration experiments were performed. For this purpose, plasma of untreated mice was used in presence of ammonium chloride (600 μΜ), GDH (6000 U/l), alpha-ketoglutarate (3 mM), NADPH (500 μΜ), aminooxy acetate (1 mM). These are conditions where GDH can work optimally but transaminases are inhibited. GDH switched to ammonia consumption already at ammonia concentrations of 150 μΜ and higher. This is of clinical relevance, since chronic liver diseases are often associated with even higher ammonia concentrations. In contrast, unphysiologically high concentrations of more than 10 niM glutamate were required to block the reaction (Fig. 25).

[0083] Example 6 - identification of the contribution of individual components of the cocktail

[0084] In order to study the contribution of each individual components of the cocktail (a-ketoglutarate, NADPH and GDH), plasma of mice 24h after CC1₄ intoxication was incubated with either a-KG alone or in combination with NADPH, GDH, AOA and the GDH inhibitor, PDAC. Already addition of a-KG alone slightly but statistically significantly decreased blood ammonia concentrations. This decrease was enhanced by further adding NADPH and particularly GDH, while the GDH inhibitor PDAC completely antagonized the effect (Fig. 26).

[0085] Example 7 - validation of the ' GDH -driven ammonia consumption' in hepatocytes

[0086] To test whether this 'GDH-driven ammonia consumption' occurs not only in plasma but also in cells, we used an in vitro system with primary mouse hepatocytes incubated with ammonia in suspension. PDAC was used to inhibit GDH and study its influence on ammonia metabolism. In hepatocytes isolated from control mice unphysiologically high ammonia concentrations (2 mM) were required until PDAC caused a significant increase of ammonia in the suspension buffer. However, using hepatocytes from mice 24h after CC1₄ intoxication PDAC caused increased ammonia concentrations in the suspension buffer already at 0.5 mM ammonia and even without ammonia addition hepatocytes secrete a small but statistically significant amount into the buffer. Similarly, glutamate production was reduced by PDAC and this effect was also stronger in hepatocytes from CC1₄ exposed mice. CC1₄ destroys the pericentral hepatocytes, which explains the reduced glutamine generation by GS and compromises urea cycle enzymes, which explains the reduced urea production. These results show that the catalytic direction of GDH can be changed to become markedly ammonia consuming also in hepatocytes to compensate compromised metabolism by urea cycle enzymes and GS after intoxication (Fig. 27).

[0087] Example 8 - extension of the integrated metabolic spatio-temporal model (IM) by including the GDH reaction

[0088] The previously established IM predicted higher ammonia output than experimental data upon liver damage. If a sink mechanism is included in the IM these discrepancies disappear. Here, the IM was extended by including the reversible GDH reaction. If a reversible GDH reaction was integrated into the hepatocyte compartment, the discrepancy between in vivo measured and simulated ammonia concentrations completely disappeared. The quantitative agreement was independent of the liver blood compartment suggesting that after CC1₄ damage, the ammonia consumption catalyzed by GDH in the hepatocytes represents the missing ammonia sink predicted by the IM (Fig. 28).

[0089] Description of the Figures:

[0090] Figure 1 : Spatio-temporal alterations of ammonia metabolizing enzymes after CC1₄ intoxication. A) Experimental design. B) Time dependent changes of gene expression in fuzzy cluster 4. The dots correspond to the average of the mean scaled values for all 310 genes, between their respective maximal and minimal expression levels at each time point, using healthy liver (i.e. time 0) as reference. Error bars indicate standard error. C) and immunostaining, Scale bars: 200μπι D). Similar analyses for further key factors of nitrogen metabolism are summarized in Fig. 10; 11 and 17. ***p<0.001, **p<0.01 & *p<0.05 when compared to the control group (Oh).

[0091] Figure 2: Evidence for a so far unrecognized mechanism of ammonia detoxification. A) Experimental design. Blood was taken from the 'liver inflow' (portal vein), 'liver outflow' (hepatic vein) and mixed venous blood from the right heart chamber. B) Ammonia concentrations in the portal vein, hepatic vein and heart. ***p<0.001, **p<0.01 & *p<0.05 compared to the corresponding control (Oh). Data of all further analyzed parameters are in Fig. 1 1 ; 12; 1 and 18. C) Integrated metabolic spatio- temporal model. The model simulates concentrations of ammonia and metabolites in the "liver inflow" for a given concentration in the "liver outflow" and for a given extent of tissue destruction. D) Model simulations after consideration of the altered expression/ activities of ammonia metabolizing factors described in Fig. 1. The simulated ammonia concentrations are higher than the experimental data, particularly on day one. E) Dose dependent experiment (10.9 to 1600 mg/kg CC1₄ 24h after administration) showing macroscopic alterations with a spotted pattern at 132.4 mg/kg and higher doses, corresponding to the central necrotic lesion in hematoxylin/eosin staining, scale bars: 100 μιη. Destruction of the pericentral CYP2E1 positive region which begins at 132.4 mg/kg with central necrosis still surrounded by CYP2E1 positive surviving hepatocytes; the entire CYP2E1 positive region was destroyed at the highest dose of 1600 mg/kg. The GS positive region was destroyed only at 132.4 mg/kg and higher doses, which correspond to the decrease in GS activity (F), scale bars: 200 μιη. **p<0.01 & *p<0.05 when compared to the control group (0). G) Comparison of analyzed and simulated ammonia concentrations in the liver vein, meas, in: analyzed concentrations in the portal vein; meas. out: analyzed concentrations in the liver vein; sim. out: simulated concentrations in the liver vein. Data for ammonia and further metabolites relevant for ammonia metabolism in the portal vein, liver vein and heart blood are in Fig. 18 and simulations in Fig. 19. Data are mean values and SD of three mice per time point and dose of CC1₄.

[0092] Figure 3 : Detoxification of ammonia by a reverse glutamate dehydrogenase reaction. A) After induction of liver damage by CC1₄ plasma activity of glutamate dehydrogenase transiently increases. This is accompanied by a decrease in the glutamate dehydrogenase substrate alpha-ketoglutarate and an increase in the glutamate dehydrogenase product, glutamate. ***p<0.001, **p<0.01 & *p<0.05 when compared to the corresponding control (Oh). Similar results were observed in liver tissue (Table 1 and Table 2). B) Validation of the reverse glutamate dehydrogenase reaction using an inhibitor of glutamate dehydrogenase (PDAC). Plasma from mice 24h after CC injection, a time period when glutamate dehydrogenase activity in blood reaches its highest values, was analyzed. Ammonia was added at a concentration typical for severe hyperammonaemia. alpha-ketoglutarate decreases ammonia and increases glutamate concentrations which can be blocked by 10m PDAC. ***p<0.001, **p<0.01 & *p<0.05 when compared to NH₄CI group (Oh). D) Concept of the reverse glutamate dehydrogenase reaction. Glutamate dehydrogenase released from damaged hepatocytes provides systemic protection against hyperammonaemia. This detoxifying reaction is limited by the availability of alpha-ketoglutarate and NADPH. Data are mean values and SD of three mice. C) Concept of the reverse glutamate dehydrogenase reaction (glutamate dehydrogenase). In normal liver, glutamate dehydrogenase generates ammonia which is detoxified by the urea cycle in the periportal hepatocytes. In the pericentral hepatocytes glutamate dehydrogenase generates glutamate which is required as a substrate for the glutamine synthetase (GS) reaction to form glutamine (Gin). After induction of liver damage the expression of urea cycle enzymes decreased and the pericentral region with GS is completely destroyed. This leads to increased blood ammonia concentrations. However, also glutamate dehydrogenase is released from damaged hepatocytes and catalyzes a reaction in blood consuming ammonia and alpha-ketoglutarate (alpha-ketoglutarate) to generate glutamate (Glu). This reaction can go until alpha-ketoglutarate in blood is consumed. In this situation alpha-ketoglutarate and NADPH should be therapeutically substituted.

[0093] Figure 4: Confirmation of the reversibility of the glutamate dehydrogenase reaction in cultivated primary mouse hepatocytes. Ammonia (A) and glutamate (B) concentrations in the culture medium in the presence and absence of the glutamate dehydrogenase inhibitor PDAC (5mM). C) Efficiency of glutamate dehydrogenase inhibition by PDAC (5mM). ***p<0.001, **p<0.01 & *p<0.05 when compared to the corresponding control. D) Cytotoxicity of ammonia in cultivated mouse hepatocytes. PDAC enhances morphological alterations, such as cell fragmentation and condensation, at 5 and 10 mM ammonia. Data are mean values and SD of three biological replicates. [0094] Figure 5: Reduction of blood ammonia concentrations by alpha-ketoglutarate. A) Tail vein injection of 280 mg/kg alpha-ketoglutarate into mice 24h after induction of liver damage by CCU (1.6 g/kg). B) Control experiment with alpha-ketoglutarate (280 mg/kg) injected into the tail vein of untreated mice. ***p<0.001, **p<0.01 & *p<0.05 when compared to the control group (0). C) Influence of the transaminase inhibitor aminooxy acetate (13 mg/kg; tail vein injection) on ammonia detoxification by alpha-ketoglutarate. The experimental design was identical to A with the difference that aminooxy acetate was injected 30 min prior to alpha-ketoglutarate. **p<0.01 & *p<0.05 when compared to the corresponding control. Data are mean values and SD of three mice treated at different experimental days with individually prepared alpha-ketoglutarate.

[0095] Figure 6: Optimization of cofactor concentrations for the glutamate dehydrogenase reaction and identification of maximal glutamate dehydrogenase activity. A) NADPH: mice plasma collected 24h after CCU injection was incubated with varying concentrations of NADPH in the presence of NH₄C1 (ImM) and other cofactors; aminooxy acetate (aminooxy acetate) (ImM), alpha-ketoglutarate (alpha- ketoglutarate) (3mM) and glutamate dehydrogenase (12000U/1) for one hour. B) Alpha-ketoglutarate (alpha-ketoglutarate): plasma was collected from mice at day one after CCU administration and incubated with varying concentrations of alpha-ketoglutarate in the presence of NH₄C1 (ImM) and other cofactors; aminooxy acetate (ImM), NADPH (ImM) and glutamate dehydrogenase (12000U/1). C) Glutamate dehydrogenase (glutamate dehydrogenase): plasma from mice collected 24h after CCU injection was incubated with varying concentrations of glutamate dehydrogenase in the presence of NH₄C1 (ImM) and other cofactors; aminooxy acetate (ImM), alpha-ketoglutarate (3mM), and NADPH (ImM) for one hour. Data are mean values and SD of three biological replicates. ***p<0.001, **p<0.01 & *p<0.05 compared to the situation where the plasma was incubated with only NH₄C1.

[0096] Figure 7: Treatment of Hyperammonaemia by injection of a cocktail of glutamate dehydrogenase and optimized cofactor doses. A) A cocktail of glutamate dehydrogenase (720 U/kg), alpha-ketoglutarate (280 mg/kg) and NADPH (180 mg/kg) (KGN) was injected into mice 24h after induction of liver damage using CCU (1.6 g/kg). Thirty minutes prior to treatment with the cocktail mice received a single dose of 13 mg/kg aminooxy acetate to block transaminases. Injection of the KGN cocktail reduced ammonia and increased glutamate concentrations in the blood of mice. Alpha-ketoglutarate (alpha-ketoglutarate) and glutamate dehydrogenase (glutamate dehydrogenase) activity increased while AST and ALT activities decreased. Data are mean values and SD of four mice treated at different experimental days with individually prepared therapeutic cocktails. ***p<0.001, **p<0.01 & *p<0.05 when compared to the control group (-30). [0097] Figure 8: Acinar compartmentation of ammonia detoxification in intact liver. The liver can be considered to consist of two compartments (periportal and pericentral). Ammonia (NH4⁺), glutamine (Gin) and other metabolites contained in blood enter periportal hepatocytes. Gin is broken down into additional NH4+ and glutamate (Glu) by the ammonia-activated glutaminase (GLNase). NH4⁺ is detoxified by carbamoyl phosphate synthetase (CPS) to carbamoyl phosphate (Cbm-P) which is entering the urea cycle (Citcitrulline, ArgS: arginino succinate, Arg: arginine, Orn: ornithine) to form urea. N-acetylglutamate (N-Acetylglu) as a glutamate-derived compound activates CPS. Since CPS has low affinity to NH4⁺, the remaining NH4⁺ is further detoxified by the high-affinity pericentral glutamine synthetase (GS) forming Gin. In both compartments glutamate dehydrogenases (GDH) are active which either deaminate Glu (periportal) or form Glu (pericentral) resulting in respective changes of NH4⁺.

[0098] Figure 9: Liver damage and regeneration after intoxication with a single dose of CCU (1.6 g/kg). A) Macroscopic appearance of mouse liver at different time intervals after CCI₄ injection, showing characteristic spotted pattern. The white spots at days one, two and three correspond to the centrilobular necrotic lesion which is visualized by hematoxylin/eosin staining. B), Scale bars: ΙΟΟμιη. The area of the necrotic lesion was quantified using image analysis software and it was evident at days one to four: while it was not detectable at all other tested time points. C) Activities of alanine transaminase (ALT) and aspartate transaminase (AST) were measured in blood plasma collected from the heart of mice at different time intervals alter CC1₄ injection as markers of liver damage. The activity of both ALT and AST was elevated after CCI₄ administration , reached the maximal level on day one and returned to the basal level on day four. Data are mean values and standard error of three mice analyzed per time point.

[0099] Figure 10: Alterations of urea cycle enzymes and glutaminase after CCI₄ intoxication. A) NA expression of carbamoyl phosphate synthetase 1 (CPS1), argininosuccinate synthetase 1 (ASS1), argininosuccinate lyase (ASL) and glutaminase (Glnase) at different time intervals after CCI₄ administration showing down regulation between 12h and day 3. During the regeneration phase (days 4-12) slight upregulation was observed and became similar to the control an day 30. Data are mean values and standard errors of three mice analyzed per time point. B) CPSl immuno staining. The control liver shows the typical periportal zonation. The Signal decreased between days 1 -3 after CCI₄ administration. On days 4 and 6, CPSl was overexpressed and extended also to the pericentral compartment of the liver lobule and returned to its normal periportal zonation on day 12 after CCI₄ injection. Scale bars: 200μιη.

[0100] Figure 11 : Arginasel expression and its influence on blood levels of arginine and urea. A) RNA expression of arginasel showing modest alterations after CCI₄ intoxication. B) arginasel immuno staining. The control liver shows the typical periportal zonation of arginasel. The signal was decreased slightly at day one after CC1₄ administration while it remained almost unaltered at all other time points. Scale bars: 200μηι. C) Concentrations of arginine and urea measured in plasma collected from the portal vein, hepatic vein and heart at different time intervals after CC1₄ injection. Concentrations of arginine decreased to undetectable levels between 12h and day 2 after CC1₄ administration. In contrast, plasma urea concentrations remained unaltered, except at day one where a slight decrease was recorded, despite the down regulation of urea cycle enzymes. The loss of arginine might result in production of urea in the blood. Data are mean values and standard errors of three mice analyzed per time point.

[0101] Figure 12: Concentrations of glutamine, alanine, serine, glucose, lactate and pyruvate in the portal vein, hepatic vein and heart of mice after intoxication with a single dose of CC1₄ (1.6 g/kg). The concentrations of glutamine were markedly elevated in the heart blood after CCI₄ administration suggesting the extrahepatic contribution by other body organs which express glutamine synthetase. The levels of alanine, serine, lactate and pyruvate were elevated at all analyzed positions of the vascular system of the mouse after CCI₄ injection particularly by day one and returned to the basal levels by day 6. In contrast, analysis of glucose revealed systemic decrease especially by day one after intoxication and recovered until day 6. Data are mean values and standard errors of three mice analyzed per time point.

[0102] Figure 13: Concentrations of amino acids in the portal vein, hepatic vein, and heart of mice before as well as two days after intoxication with a single dose of CCI₄ (1.6 g/kg).

[0103] Figure 14: Simulations of ammonia, glutamine and urea concentrations after CCl₄-intoxication using the integrated model. Experimentally determined liver input concentrations, composed of arterial (30%) and portal (70%) blood (meas, in) of ammonia, glutamine and urea were used to simulate the respective output concentrations in the hepatic vein (sim, out) and compared with the experimentally measured concentrations (meas, out). A) Simulations in a time-dependent manner after CCI₄ administration. All enzymes concentrations were considered constant over time. Parameter values are as determined in Schliess et al. (2014). B) Measured (white) and simulated (black) steady-state outflow concentrations of glutamine, ammonia and urea in the case of liver ex -vivo perfusions in antegrade and retrograde direction. Same simulations as in Schliess et al. All enzymes concentrations are assumed not to change after CC^-intoxication. Parameter values are as determined in Schliess et al. (2014). Errors bars represent standard deviation. The inflow concentrations for glutamine (Glnin) and ammonia (NH₄ in) in the perfusion medium are given on the top of each plot. C) Simulations in a dose-dependent manner at day 1 after CC1₄ administration. All enzymes concentrations were considered constant over time. D) Simulations in a time-dependent manner after CCI₄ administration. Measured alteration of enzyme concentrations due to liver damage were included in the model. E) Measured (white) and simulated (black) steady-state outflow concentrations of glutamine, ammonia and urea in the case of liver ex- vivo perfusions in antegrade and retrograde direction. Measured alteration of enzyme concentrations due to liver damage were included in the model. F) Simulations in a dose-dependent manner at day 1 after CC1₄ administration. Measured alteration of enzyme concentrations due to liver damage were included in the model. G) Simulations in a time-dependent manner after CC1₄ administration. Measured alteration of enzyme concentrations due to liver damage were included in the model. The model parameters were fitted with the perfusion data and subsequently the time course data was predicted by the model. H) steady-state outflow concentrations of glutamine, ammonia and urea in the case of liver ex -vivo perfusions in antegrade and retrograde direction. The model parameters were fitted with the perfusion data. I) Simulations in a dose-dependent manner at day 1 after CCI₄ administration. Measured alteration of enzyme concentrations due to liver damage were included in the model. The model parameters were fitted with the perfusion data and subsequently the dose dependent data was predicted by the model. J) Simulations in a time-dependent manner after CCI₄ administration. Measured alteration of enzyme concentrations due to liver damage were included in the model. The model parameters were fitted with the perfusion data with a 5 fold higher weight on glutamine compared to ammonia and urea. Subsequently the time course data was predicted by the model. K) steady-state outflow concentrations of glutamine, ammonia and urea in the case of liver ex- vivo perfusions in antegrade and retrograde direction. The model parameters were fitted with the perfusion data with a 5 fold higher weight on glutamine compared to ammonia and urea. L) Simulations in a dose-dependent manner at day 1 after CCI₄ administration. Measured alteration of enzyme concentrations due to liver damage were included in the model. The model parameters were fitted with the perfusion data with a 5 fold higher weight on glutamine compared to ammonia and urea. Subsequently the dose dependent data was predicted by the model. M) C alculation of the enzyme quantities for (A) glutamine synthetase, (B) carbamoyl phosphate synthetase and (C) glutaminase from the mRNA measurement. The translation model described in the supplemental methods is calibrated using the experimentally obtained mRNA (black line) and activity data for GS (dashed red line). The enzyme quantities of CPS and GLNase (red line) are then simulated with the same model from the respective mRNA quantities.

[0104] Figure 15: Structural model of blood flow for calculating organic contribution to metabolic conversion. Blood is distributed between three compartments representing liver, a first extrahepatic compartment (muscle, kidneys, brain,...) and a second (gastrointestinal) compartment. In each compartment the rate v depicts the metabolic contribution. The rates can be calculated from concentration values c measured at three sites (heart, portal vein, hepatic vein) and proportions of blood flow F described by respective factors like \epsilon. [0105] Figure 16: A): Extrahepatic contribution of ammonia, glutamate, alpha-KG and glutamine. Measured (single data points) and calculated (lines) consumption in two extrahepatic compartments (organ groups) is compared at days of regenerating liver. Extrahepatic 1 : muscle, kidneys, brain, etc. Extrahepatic 2: gastrointestinal tract. B): Extrahepatic contribution of glucose, pyruvate and lactate. Measured (single data points) and calculated (lines) consumption in two extrahepatic compartments (organ groups) is compared at days of regenerating liver. Extrahepatic 1 : muscle, kidneys, brain, etc. Extrahepatic 2: gastrointestinal tract. C): Extrahepatic contribution of alanine, arginine, histidine and serine. Measured (single data points) and calculated (lines) consumption in two extrahepatic compartments (organ groups) is compared at days of regenerating liver. Extrahepatic 1 : muscle, kidneys, brain, etc. Extrahepatic 2: gastrointestinal tract. D): Change of extrahepatic contribution of various amino acids depending on liver damage. Extrahepatic consumption is compared between healthy liver (black) and day 2 of liver damage (white) for two extrahepatic compartments (organ groups). Extrahepatic 1 : muscle, kidneys, brain, etc. (left). Extrahepatic 2: gastrointestinal tract (right). Amino acids: asparagine, aspartic acid, citruilline, glycine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, tryptophane, and valine.

[0106] Figure 17: Dose dependent experiment. A) Experimental design: mice were intoxicated with various doses of CC1₄ ranging between 10.9 and 1600 mg/kg. 24h later blood samples were collected from the portal vein, hepatic vein and heart to be used for the metabolic analysis. In addition, liver tissue samples were formalin fixed and paraffin embedded to be used for immuno staining and the histopathological examination. Moreover, snap frozen liver tissue samples were collected for RNA isolation and qRT-PCR analysis. B) whole slide scan of hematoxylin/eosin stained livers showing a dose dependent increase in the diameter of necrotic lesions, which was quantified by image analysis software. C) RNA expression of key nitrogen metabolizing enzymes. The expression of glutamine synthetase (GS), glutaminase (Glnase) and carbamoyl phosphate synthetase 1 (CPSl) was downregulated in a dose dependent manner after CC1₄ injection. Data are mean values and standard errors of three mice analyzed per time point.

[0107] Figure 18: Metabolic analysis in plasma collected from the portal vein, hepatic vein and heart of mice 24h after intoxication with various doses of CCI₄ (10.9-1600 mg/kg). Blood ammonia concentrations were elevated in a dose dependent manner after CCI₄ injection. The levels of ammonia were lower in the heart blood compared to the hepatic vein. Blood urea concentrations remained unaltered at all tested doses of CC1₄. Analysis of glutamine revealed a dose dependent increase in heart blood. The concentrations of glutamate increased in a dose dependent manner after CCI₄ administration with lower values in the heart compared to the hepatic and portal veins. The results suggest extrahepatic consumption of ammonia plus glutamate and generation of glutamine. Glucose concentrations decreased in a dose dependent manner after CC1₄ injection at all measured positions of the vascular system of the mouse. In contrast, the levels of lactate, pyruvate and alanine increased after CC1₄ administration. Data are mean values and standard errors of three mice analyzed per time point.

[0108] Figure 19: Inhibition of transaminases activities by aminooxy acetate (AOA). A) Experimental design: mice received a single hepatotoxic dose of CC1₄ (1.6 g/kg); 24h later various doses of AOA were intravenously injected via the tail vein. Transaminases activities were measured in blood plasma collected from the eye plexus of the same mouse before (0) as well as 15, 30 and 60 min after AOA injection. B) Dose dependent inhibition of aspartate transaminase (AST) and alanine transaminase (ALT) activities by AOA. Injection of 13 mg/kg AOA reduced the plasma activities of both AST and ALT by more than 65%. The data of 0.86 and 4.31 mg/kg AOA were obtained from one mouse only as a range finding experiment to reduce the number of required animals, while the results with 13 mg/kg AOA were reproduced in three different mice and expressed as mean values with standard errors.

[0109] Figure 20: Experimental design of the results shown in main Fig. 5C. A) Mice were intoxicated with a single dose of CC1₄ (1.6 g/kg): 24h later blood samples were collected from the eye plexus for metabolic analysis. Subsequently, the mice received a single dose of aminooxy acetate (AOA) (13 mg/kg) or saline via the tail vein and 30 minutes later all mice were intravenously injected with 280 mg/kg alpha-ketoglutarate. The metabolic analysis was performed in blood samples collected from the eye plexus before as well as 15 minutes after a-KG injection in the same mouse. B) Successful inhibition of transaminases activities by AOA. The activity of both aspartate transaminase (AST) and alanine transaminase (ALT) was decreased by more than 65% after AOA injection. Data are mean values end standard errors of three mice.

[0110] Figure 21 : Analysis of NADP⁺ and NADPH in blood plasma and liver tissue of control and CC1₄ treated mice. A) Concentrations of NADP⁺ and NADPH in blood plasma collected from the portal vein, hepatic vein and heart of control mice and at day one after intoxication with 1.6 g/kg CC1₄. Both NADP⁺ and NADPH were not detectable in control mice. In CC1 treated mice both NADP⁺ and NADPH were detected in plasma with an increased NADPVNADPH ratio. B) Analysis of NADP⁺ and NADPH in liver tissue showing an increased NADPVNADPH ratio after CC1 intoxication.

[0111] Figure 22: Influence of transaminases inhibition an ammonia consumption and glutamate generation by the reversed glutamate dehydrogenase (GDH) reaction. A) Experimental design: mice received a single dose of CC1₄ (1.6 g/kg); 24h later plasma was separated from heart blood and incubated with either ammonium chloride (NH₄C1, ImM) or NH₄C1 (ImM) plus GDH (12000 U/I) and its cofactors alpha-ketoglutarate (a-KG. 3mM) and NADPH (1 mM) in the presence or absence of the transaminases inhibitor aminooxy acetate (AOA, 1 mM). After 1 h incubation at 37 °C the concentrations of ammonia and glutamate were analyzed. B) Inhibition of transaminases slightly improved the detoxification of ammonia by the reverse GDH reaction, while the production of glutamate was strongly decreased after inhibition of transaminase. Data are mean values and standard errors of three replicates.

[0112] Figure 23: Increase of blood glutamate dehydrogenase (GDH) activity. Mice received various doses of GDH intravenously via the tail vein. The activity of GDH was measured in blood plasma collected from the eye plexus before (0) as well as 2, 10 and 15 minutes after GDH injection. Analysis of GDH shows that injection of external GDH successfully enhanced the blood activity of GDH, which remained increased for at least 15 minutes after injection.

[0113] Figure 24: NADPH and NADP⁺ concentrations in mouse blood alter injection of various doses of NADPH into the tail vein. A) Experimental design: various doses of NADPH (dissolved in NaCl) were administered in the tail vein of male C57BI6/N mice. Blood samples were collected from the eye plexus before (0) as well as 2 and 10 minutes following NADPH injection. B) Transient elevation of NADPH concentration in the blood after intravenous administration of 150 mg/kg NADPH.

[0114] Figure 25: A) Ammonia detoxification by the cocktail. Different ammonia concentrations were added to plasma of untreated mice and a cocktail of a-KG (3 mM), NADPH (0.5 mM), GDH (6000 U/l) and AOA (1 mM) was used to analyze ammonia and glutamate lh later. B) Ammonia (600 μΜ) detoxification by the cocktail was only blocked by unphysiologically high glutamate concentrations.

[0115] Figure 26: Validation of the reverse GDH reaction using an inhibitor of GDH (PDAC). Plasma of mice 24h after CCI₄ injection was analyzed. a-KG was added alone or in combination with aminooxy acetate (AOA), NADPH, GDH and pyridine dicarboxylic acid (PDAC).

[0116] Figure 27: Reversibility of the GDH reaction in primary mouse hepatocytes. Hepatocytes were isolated from CC1₄ (1.6 g/kg) intoxicated (day 1) and untreated mice and suspended at a concentration of 2 million hepatocytes/ml for lh with different concentrations of ammonia. A) Inhibition of GDH activity by PDAC. B) Compromised ammonia detoxification after GDH inhibition. C) Reduced glutamate production by GDH inhibition. ***p<0.001, **p<0.01 & *p<0.05 compared to -PDAC. ⁰⁰ p<0.01 & °p<0.05 compared to hepatocytes from untreated mice. D& E) compromised urea and glutamine production by hepatocytes of CC1₄ intoxicated mice. **p<0.01 & *p<0.05 compared to hepatocytes from untreated mice. Data are mean values and SD of three in dependent experiments.

[0117] Figure 28: Integration of the GDH reaction into the metabolic model. A) Simulation of ammonia metabolism by the IM without including the reverse GDH reaction. Predicted ammonia concentrations in the liver outflow are higher compared to the experimental data. B)Scheme of the metabolic reactions and zones of the extended model including GDH in the blood of the liver and hepatocytes. B) The model extension leads to a better fit between simulated and experimental data.

Claims

Patent claims:

1. A pharmaceutical composition comprising alpha-ketoglutarate, optionally a glutamate dehydrogenase, and optionally a cofactor for glutamate dehydrogenase.

2. The pharmaceutical composition according to claim 1, which comprises alpha-ketoglutarate and a glutamate dehydrogenase and a cofactor for glutamate dehydrogenase.

3. The pharmaceutical composition according to claim 1 or 2, for use in the treatment or prevention of a liver disease.

4. The pharmaceutical composition for use according to claim 3, wherein the liver disease is hyperammonemia or a disease associated with hyperammonemia.

5. The pharmaceutical composition for use according to claim 3 or 4, wherein the liver disease is selected from the group consisting of urea cycle defects, liver cirrhosis and acute intoxication.

6. The pharmaceutical composition according to any of the preceding claims, wherein the glutamate dehydrogenase is human glutamate dehydrogenase.

7. The pharmaceutical composition according to any of the preceding claims, wherein the cofactor is NADPH.

8. The pharmaceutical composition according to any of the preceding claims, which is liquid.

9. The pharmaceutical composition according to any of the preceding claims, which is adapted for systemic administration, preferably for intravenous administration.

10. The pharmaceutical composition according to any of the preceding claims, wherein the content of alpha-ketoglutarate is within the range of from 0.001 to 95 wt.-%, relative to the total weight of the pharmaceutical composition.

11. The pharmaceutical composition according to any of the preceding claims, which additionally comprises a transaminase inhibitor.

12. The pharmaceutical composition according to claim 11, wherein the transaminase inhibitor is aminooxy acetate.

13. A pharmaceutical dosage form comprising the pharmaceutical composition according to any of the preceding claims.

14. A kit comprising a first pharmaceutical formulation which comprises alpha-ketoglutarate and a second pharmaceutical formulation which comprises a glutamate dehydrogenase.

15. The kit according to claim 14, wherein the first pharmaceutical formulation and the second pharmaceutical formulation are adapted for administration via the same route or via different routes.