EP4110311A1

EP4110311A1 - Therapy

Info

Publication number: EP4110311A1
Application number: EP21708345.0A
Authority: EP
Inventors: Nicholas GREENE
Original assignee: UCL Business Ltd
Current assignee: UCL Business Ltd
Priority date: 2020-02-25
Filing date: 2021-02-22
Publication date: 2023-01-04
Also published as: GB202002639D0; WO2021170982A1

Abstract

Cinnamic acid and/or pharmaceutically acceptable salts thereof, and/or cinnamaldehyde, for use in the treatment and/or prevention of a disease or condition associated with hyperglycinemia and/or hyperammonemia.

Description

THERAPY

Field of the Invention

The present invention relates to therapies for the treatment and/or prevention of a disease or condition associated with hyperglycinemia and/or hyperammonemia using cinnamaldehyde, cinnamic acid and/or pharmaceutically acceptable salts thereof. For instance, the present invention is concerned with cinnamaldehyde, cinnamic acid and/or pharmaceutically acceptable salts thereof for the treatment and/or prevention of non-ketotic hyperglycinemia (NKH) and/or urea cycle disorders (UCDs; urea cycle defects).

Background of the Invention

Hyperglycinemia and hyperammonemia are conditions characterised by elevated levels of glycine and ammonia, respectively. These conditions arise in a number of inherited metabolic diseases, and typically in inherited childhood diseases. They can result in severe neurological conditions in those affected, including seizures, coma and death.

The glycine cleavage system (GCS), also known as the glycine decarboxylase complex (GDC), is an enzyme complex that breaks down the amino acid glycine. The GCS comprises four enzymes: aminomethyltransferase (AMT or GCS T-protein), glycine dehydrogenase (GLDC or GCS P-protein), dihydrolipoyl dehydrogenase (DLD or GCS L- protein) and ‘the GCS H protein’ (GCSH).

Non-ketotic hyperglycinemia (NKH), also known as ‘glycine encephalopathy’, is a rare genetic metabolic disorder caused by a defect in the GCS. Such defects result in an accumulation of glycine in the body’s tissues and fluids, and can result from mutations in GLDC, AMT or GCSH. Mutations in the GLDC gene account for 80% cases of NKH. ‘Classic NKH’ refers to NKH resulting from mutation in the GCS-encoding genes. Mutation in the accessory enzymes that are required for GCS function, particularly lipoylation of GCSH, can cause ‘atypical NKH’.

NKH is a life-limiting autosomal recessive neurometabolic disorder with a predicted incidence of around 1 in 50,000 worldwide. Without treatment, affected infants and children suffer severe neurological impairment and epilepsy. There are presently no curative treatments for NKH, though there are treatments that improve patient outcomes with respect to epilepsy (but not developmental progression).

One previously known method of treating NKH involves administering sodium benzoate. Anti-epileptic drugs are often co-administered to reduce seizures. Benzoate is converted in vivo to benzoyl-CoA (the conjugate of benzoic acid and enzyme CoA), which reacts with glycine to produce hippurate in glycine conjugation reactions. This reaction is catalysed by, at least, the enzyme Glycine -N-acyltransferase (GLYAT). Thus, administration of benzoate stimulates glycine conjugation reactions and decreases glycine levels. The resulting hippurate is excreted.

Urea cycle disorders (also known as urea cycle defects, and commonly abbreviated as UCDs) are a group of rare inherited genetic disorders associated with impaired function of the urea cycle, which reduces the metabolism of nitrogen and thus leads to hyperammonemia. In neonates, excess ammonia can cause hypotonia, seizures and lethal coma, while various neurological symptoms arise in mild and moderate UCDs which may present later in childhood. UCDs can be caused by mutations in a variety of enzymes, including carbamoyl phosphate synthase (CPS), ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL) and arginase (ARG1). The estimated incidence of UCDs is around 1 in 8,500. UCDs caused by mutation of OTC are referred to as ‘ornithine transcarbamylase deficiency’ (OTCD) and have an estimated incidence of between 1 in 56,500 and 1 in 77,000 live births. UCDs caused by mutation of the ASL gene are called argininosuccinic aciduria (ASA) and have an estimated incidence of 1 in 218,000 live births.

Current treatments for UCDs are complex and can be categorised as either emergency management or long term management. Emergency management may include a pharmacological reduction of levels of ammonia in the body by administration of sodium phenylacetate and sodium benzoate, followed by physical removal of the ammonia by dialysis. Long-term management focusses on reducing the number of trigger events by dietary modifications and, in some cases, through long-term use of pharmacological ammonia scavengers, for example benzoates. These work by reducing the amount of available glycine and thus lowering nitrogen levels and reducing the amount of ammonia that is generated.

One major disadvantage associated with current treatment methods for the above conditions is that therapeutically effective doses of benzoates can have severe gastrointestinal side-effects, cause vomiting, lead to carnitine deficiency, and cause toxicity. To counteract such symptoms, proton pump inhibitors are commonly co-administered as antacids. As a result of the administration of benzoate and long-term use of proton pump inhibitors, most NKH patients suffer gut motility and conditions associated with malabsorption. Still further, benzoate is widely considered to have a highly unpleasant taste. Consequently, patient compliance is an issue with benzoates. In animal models of UCDs, benzoates have also been found to deplete carnitine and disrupt metabolites associated with cerebral energy production, including pyruvate and acetyl CoA. Thus, benzoate may also lower levels of other important molecules required for brain function.

Existing methods of treatment for hyperglycinemia and hyperammonemia are therefore problematic and there is a significant need for improved therapies. For instance, it would be desirable to provide treatments that have improved therapeutic efficacy, reduced toxicity, a better therapeutic window, improved symptom management, improved tolerance at high dosages, and/or improved long-term tolerability. Further objects of the present invention include to provide a simple, cost-effective treatment for hyperglycinemia and/or hyperammonemia that has high patient compliance. Furthermore, objects of the present invention include providing a treatment for hyperglycinemia and/or hyperammonemia that has a reduced tendency to cause gastrointestinal distress and/or carnitine deficiency and does not require co-administration with antacids.

The present invention achieves these goals by providing cinnamic acid and/or pharmaceutically acceptable salts thereof for use in treating and/or preventing for hyperglycinemia and/or hyperammonemia.

Summary of the Invention

The applicant has generated a mouse model of hyperglycinemia and screened compounds for their ability to activate glycine conjugation and alleviate accumulation of glycine. Cinnamate has been found to achieve excellent reduction of levels of excess glycine and related compounds and to be well-tolerated at high dosages. In particular, cinnamate has been found to be at least as effective in lowering excess glycine levels in blood as an equal amount of benzoate. Furthermore, cinnamate is better tolerated than benzoate by oral administration and can likely be used at higher doses without toxicity and thus may be able to achieve superior glycine reduction.

Cinnamate has been found to lower the abundance of glycine in liver and brain tissues, lower the levels of a series of glycine derivatives in the liver and brain, and increase the abundance of hippurate. Notably, cinnamate has been found to lower the abundance of guanidinoacetate, a metabolite of glycine that has been implicated in disorders of the nervous system.

Additionally, there is evidence that oxidative stress plays a role in hyperglycinemia and hyperammonemia. Cinnamate may attenuate such oxidative stress and thus provide an improved treatment of hyperglycinemia and hyperammonemia by this additional mechanism. Cinnamaldehyde, a component of certain foods and a common food additive, which is metabolised in vivo to form cinnamate, may also be capable of achieving analogous advantageous results in treating the disease states discussed herein ( via its metabolism in vivo to yield cinnamate).

The present invention relates to a compound that is cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde, for use in the treatment or prevention of a disease or condition associated with hyperglycinemia or hyperammonemia.

The present invention also relates to a pharmaceutical composition comprising: cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde; and at least one further therapeutic agent selected from carnitine, benzoic acid and a pharmaceutically acceptable salt of benzoic acid.

The present invention also relates to a kit comprising: cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde; and, in admixture or in one or more separate containers, at least one further therapeutic agent selected from carnitine, benzoic acid and a pharmaceutically acceptable salt of benzoic acid.

The present invention also relates to a product containing: (a) cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde; and (b) at least one further therapeutic agent selected from carnitine, benzoic acid and a pharmaceutically acceptable salt of benzoic acid; for simultaneous, separate or sequential use in the treatment or prevention of a disease or condition associated with hyperglycinemia or hyperammonemia.

The present invention also relates to a method of treating or preventing a disease or condition associated with hyperglycinemia or hyperammonemia, in a patient a need thereof, which method comprises administering to said patient an effective amount of cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde.

The present invention also relates to use of cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde, in the manufacture of a medicament for use in the treatment or prevention of a disease or condition associated with hyperglycinemia or hyperammonemia. Brief Description of the Drawings

Figure 1 Diagrams for the metabolic pathway of the GCS (A) and key metabolic pathways in glycine and N¾ regulation (B).

A: The Glycine Cleavage System. GLDC, AMT and GCSH are specific to the GCS, while DLD is a house-keeping enzyme. The key functions of GCS are to decarboxylate glycine and to transfer a one-carbon to tetrahydrofolate (THF). The majority (80%) of NKH patients carry mutations in GLDC, with remaining patients carrying mutations in AMT.

B: The GCS regulates glycine levels and produces NH₃. The urea cycle (UC) removes NH₃. Mutations in the GLDC, OTC and ASL proteins are known to result in elevated levels of glycine or NH₃. Glycine conjugation is stimulated by exogenous benzoate, which results in a reduction in glycine and NH₃ levels. Pilot data shows that exogenous cinnamate is converted to benzoyl-CoA by beta-oxidation and also stimulates glycine conjugation.

Figure 2 Graphs showing the suitability of G/ c-deficient mice as a model for NKH.

A: Gldc- deficient mice ( Gldc^GT1/GT1 ) exhibit diminished Gldc rnRNA.

B: G/ c-deficient mice exhibit loss of GCS enzymatic activity.

C: G/i/c-deficient mice exhibit an elevated abundance of glycine in plasma and urine.

Figure 3 Altered levels of glycine and glycine derivatives in liver tissue of Gldc- deficient mouse.

A: Gldc rnRNA is restored by liver-specific genetic rescue.

B: This normalises the concentration of glycine in plasma.

C-F: Concentration of (C) glycine and glycine derivatives including (D) guanidinoacetate (formed from glycine combined with arginine), and (E, F) acyl glycines, are increased in liver tissue of Gldc-deficient ( Gldc^GT1/GT1 ) mice and normalised by liver-specific genetic rescue (** different to wild-type, +/+ mice; * different to Gldc^GT1/GT1).

G: A heat-map (black = low, white = high) showing the relative abundance, determined by mass spectrometry, of glycine and various glycine metabolites in the liver tissues of wild-type mice (“+/+”), Gldc- deficient mice (“Gdlc^GT1/GT1 ”) and Gldc- deficient mice that have been treated by liver-specific genetic resue (“Gdlc^GT1/GT1 Liver-rescue”). Each column represents a sample from an individual mouse. A series of glycine conjugates are significantly elevated in the G/ c-deficient mice and normalised in the genetic rescue mice. Figure 4 Gldc-deficiency leads to increased concentration of (A) glycine and (B) guanidinoaceate in plasma and these can be lowered by oral sodium benzoate or sodium cinnamate.

Administration of sodium benzoate in drinking water (for 7 days) significantly lowers (A) plasma glycine and (B) plasma guanidinoacetate in Gldc^GT1/GT1 mice compared with untreated control mutants (*p<0.01), confirming that circulating glycine is amenable to lowering by conjugation (as in humans). Higher doses of benzoate are poorly tolerated (as in NKH patients).

A: Oral treatment with cinnamate (7 days) significantly lowers plasma glycine levels in Gldc^GT1/GT1 mice compared with untreated mutants (both doses; p<0.01).

B: Cinnamate also lowers plasma guanidinoacetate.

Cinnamate doses: Cinn is equimolar with benzoate, Cinn x2 is double the concentration (** increased in Gldc^GT1/GT1 vs wild-type, p<0.01).

Figure 5 Graph showing relative abundance of glycine and glycine derivatives in liver of treated Gldc-deficient mice.

Cinnamate treatment achieves lowering of abundance. The graph (left side) shows the relative abundance of each glycine derivative in liver of treated Gldc-deficient mice (Gldc^GT1/GT1 ; n = 4-6 per group) compared with untreated Gldc-deficient mice (treated Gldc^GT1/GT1 ÷ Gldc^GT1/GT1). Bars below line indicate reduction in abundance with significant reduction indicated (*p<0.05; #p<0.1; ANOVA). The graph (right side) shows corresponding results for hippurate abundance.

Figure 6 Graphs showing abundance of glycine and glycine derivatives in brain tissue of wild-type mice (n=5), untreated G/ c-deficient mice (n=5), and G/ c-deficient mice treated with sodium benzoate (“+ benzoate” n=5), an equal quantity of sodium cinnamate (“+ cinnamate”, n=4), or a double quantity of sodium cinnamate (“+ 2x cinnamate”, n=4). This shows that benzoate and cinnamate can lower the abundance of glycine and glycine derivatives in the brain tissues of G/ c-deficient mice and that 2x cinnamate can lower concentration of guanidinoacetate and glycylleucine that are not achieved by benzoate. Increased levels of hippurate are found with each treatment. Detailed Description of the Invention

Definitions

As used herein, the term "patient" typically refers to a human patient. Patients may, however, be other vertebrate animals, such as mammals. The terms “subject” and “patient” are used interchangeably herein.

As used herein, the words "treatment" and "treating" are to be understood as embracing treatment and/or amelioration and/or prevention of or reduction in aggravation/worsening of symptoms of a disease or condition as well as treatment of the cause of the disease or condition, and may include reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilise a subject's condition.

Reference to "prevention" and "preventing" a disease or condition embraces prophylaxis and/or inhibition of the disease or condition. The term "preventing" is art- recognized, and when used in relation to a condition, such as hyperglycinemia, hyperamminemia, or their associated symptoms, is well understood in the art, and includes administration of a drug and/or composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the drug or composition.

As used herein, the term "pharmaceutically acceptable" refers to a material that does not interfere with the effectiveness of the compound of the invention and is compatible with a biological system such as a cell, cell culture, tissue, or organism. Preferably, the biological system is a living organism, such as a vertebrate.

As used herein, the phrase “therapeutically effective amount” refers to an amount of a compound, material or composition that is effective for producing some desired therapeutic effect, such as treating, preventing or ameliorating hyperglycinemia or hyperammonemia or reducing levels of glycine, at a reasonable benefit/risk ratio applicable to any medical treatment. In one embodiment, the therapeutically effective amount is sufficient to reduce or eliminate at least one symptom. A therapeutically effective amount may partially improve a disease or symptom without fully eradicating the disease or symptom.

Compounds

The compound for use in the present invention is cinnamic acid or a pharmaceutically acceptable salt thereof, or cinnamaldehyde. Cinnamic acid and pharmaceutically acceptable salts thereof may be produced using known methods. In particular, cinnamic acid and cinnamate salts are known compounds and can be produced, for example, using the Perkin reaction with benzaldehyde and acetic anhydride, as discussed in Johnson et al, Org. React., 1942, 1, 210. Cinnamaldehyde can also be produced using known methods, including from steam distillation of oil from cinnamon bark and by aldol condensation of benzaldehyde and acetaldehyde.

Cinnamic acid, its pharmaceutically acceptable salts, and cinnamaldehyde all contain an alkene motif and thus may be present as the trans- or the cis- isomer. In the present invention, the compound may be used as the cis-isomer, as the trans-isomer, or in the form of a mixture of configurational isomers.

The compound of the invention may be used in any tautomeric form.

The cinnamic acid can be used in the form of a pharmaceutically acceptable salt (i.e. as a cinnamate). As used herein, a pharmaceutically acceptable salt of cinnamic acid is a salt with a pharmaceutically acceptable base. Representative and non-limiting pharmaceutically acceptable bases include alkali metals (e.g. lithium, sodium and potassium), alkali earth metals (e.g. calcium and magnesium), hydroxides and organic bases such as alkyl amines, aralkyl amines and heterocyclic amines, lysine, guanidine, diethanolamine and choline. Preferably, the pharmaceutically acceptable salt is an alkali metal salt. More preferably, the pharmaceutically acceptable salt is sodium cinnamate.

The pharmaceutically acceptable salts may be obtained as the direct products of compound synthesis. Alternatively, the free acid may be dissolved in a suitable solvent containing the appropriate base, and the salt isolated by evaporating the solvent or otherwise separating the salt and the solvent.

Cinnamates occur naturally in plants and are used in a large range of synthetic pathways, including the production of lignin. Cinnamate is approved for human consumption as a food additive. Current industrial uses include application as a flavouring and food additive and as a precursor in manufacturing a range of dyes, perfumes and flavourings, including the sweetener aspartame. Cinnamic acid is also well known to have antimicrobial activities, and has recently been reported to have antioxidant properties and free radical scavenging properties that may provide a range of health benefits.

Animals are not known to naturally produce cinnamates, but may ingest dietary cinnamate from plant matter. The metabolism of cinnamates in several mammals, including rats, mice and humans, has previously been investigated. Hippurate is the major metabolite, while other metabolites include 3 -hydroxy-3 -phenylpropionic acid, acetophenone, benzoic acid, benzoyl glucuronide, cinnamoyl glucuronide and cinnamoyl glycine.

Without being limited to theory, it is believed that the cinnamic acid or salt thereof, and similarly cinnamate, exerts its therapeutic effect in the context of the pathological conditions discussed herein, at least in part, by being metabolised to generate benzoyl-CoA in vivo, which then reacts with glycine to reduce glycine content (see Figure 1, illustrating key metabolic pathways in glycine and NH₃ regulation, for further details; in Figure IB, the glycine cleavage system (GCS) regulates glycine levels and produces NH₃, the urea cycle (UC) removes NH₃ and enzymes whose mutation results in elevated glycine or NH₃ are highlighted).

The previous metabolic studies concerning cinnamate metabolism, the pathway shown in Figure IB, and present experimental observations as described in the Examples section herein, suggest that a proportion of ingested cinnamate compounds may ultimately exert a glycine reducing effect by a similar mechanism to that giving rise to sodium benzoate’s utility in this context. However, cinnamaldehyde, cinnamic acid and salts thereof have not previously been investigated for their suitability for treating hyperglycinemia and/or hyperammonemia, or for use in therapeutically reducing glycine levels. Prior to the present studies it was not evident whether cinnamaldehyde, cinnamic acid or salts thereof could be administered, in place of benzoate, in order ultimately to reduce glycine levels to a therapeutically acceptable extent, e.g. bearing in mind the complexity of the total human metabolism, possible alternative metabolic fates of administered cinnamaldehyde and/or cinnamate, bioavailability considerations, and the like. Still further, it was not apparent that cinnamaldehyde and/or cinnamate might be susceptible to administration to achieve analogous therapeutic effects at similar or even lower doses than benzoate, or that it might be administered at higher concentrations without the deleterious side effects associated with benzoate administration. Still further, and as discussed elsewhere herein, the additional anti oxidant properties of cinnamate may provide further unexpected advantages in the context of hyperglycinemia and/or hyperammonemia therapy.

Treatment

The compound of the present invention is able to reduce glycine and/or ammonia levels in a subject. Thus, the compound of the invention may be used in a method of treating a subject suffering from or susceptible to hyperglycinemia and/or hyperammonemia, which method comprises administering to said subject an effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof. The compound may be used in combination with additional therapeutic agent(s), as desired.

The compound of the invention may be used in a method of treating or preventing a disease or condition associated with hyperglycinemia and/or hyperammonemia.

In one embodiment, the disease or condition associated with hyperglycinemia and/or hyperammonemia is a disease or condition associated with hyperglycinemia. In a further embodiment, the disease or condition associated with hyperglycinemia and/or hyperammonemia is a disease or condition associated with hyperammonemia.

In one embodiment, the condition associated with hyperglycinemia and/or hyperammonemia is a condition in which hyperglycinemia and/or hyperammonemia is a secondary characteristic of the condition, such as methylmalonic acidemia or hepatic encephalopathy.

In one embodiment the compound of the invention may be for use in treating and/or preventing hyperglycinemia and/or hyperammonemia. In a further embodiment, the hyperglycinemia and/or hyperammonemia may be a symptom of, and/or associated with, another condition.

In a preferred embodiment, the hyperglycinemia is ketotic glycinemia (also known as propionic acidemia) or non-ketotic glycinemia (NKH). Preferably, the hyperglycinemia is non-ketotic glycinemia (NKH). Thus, in a preferred embodiment, the compound of the invention is for use in treating or preventing NKH.

In one embodiment, the NKH is associated with defects in the GCS. Preferably, the NKH is associated with defects in a protein selected from GLDC, GCSH, DLD and AMT. More preferably, the NKH is associated with defects in GLDC.

In one embodiment, the NKH is variant NKH, transient NKH or classic NKH. Preferably, the NKH is classic NKH.

In one embodiment, the NKH is severe NKH, attenuated poor NKH, attenuated intermediate NKH or attenuated mild NKH.

In one embodiment, the compound of the invention may be for use in treating and/or preventing hyperammonemia.

In one preferred embodiment, the hyperammonemia is associated with a UCD.

In another preferred embodiment, the compound of the invention is for use in treating or preventing a UCD. Preferably, the UCD is associated with defects in CPS, OTC, ASS, ASL or ARG1. More preferably, the UCD is associated with defects in OTC or ASL. Further preferably, the UCD is associated with defects in ASL.

Preferably, the UCD is N-acetylglutamate synthetase deficiency (NAGS), carbamoylphosphate synthetase I deficiency (CPSI deficiency), ornithine transcarbamylase deficiency (OTCD), argininosuccinate synthetase seficiency (ASSD), citrin deficiency, argininosuccinic aciduria (ASA), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH Syndrome) or argininemia (ARG). More preferably, the UCD is OTCD or ASA. Further preferably, the UCD is ASA.

Many clinical features of NKH are thought to result from excess glycine in the brain. Unlike the liver, acyl-glycine compounds do not accumulate in the brains of G/ c-deficient mice, confirming that glycine conjugation pathways are not active in the brain, which lacks expression of GLYAT. Thus, benzoate’s ability to lower glycine levels in brain tissue is a secondary effect of lowering the concentration of circulating glycine by activating conjugation reactions elsewhere, such as the liver and kidneys.

RNA-sequence based transcrip tomic analyses of bulk brain samples from Gldc- deficient and wild- type mice identified gene expression changes. This analysis revealed down-regulation of several ion channels implicated in epilepsy and up-regulation of specific genes associated with gliosis and microglial activation. This gene regulation provides a potential measure of hyperglycinemia treatment success.

Thus, the compound of the invention may be for use in treating or preventing neurological conditions associated with hyperglycinemia and/or hyperammonemia. Preferably, the neurological condition is associated with hyperglycinemia.

Additionally, there is evidence of increased oxidative stress in mouse models of hyperglycinemia and hyperammonemia as part of the pathophysiology, and in particular in mouse models of NKH and ASA. Cinnamate attenuates oxidative stress, possibly mediated via activation of the Nrf2 pathway. Thus, an additional benefit of cinnamate (or its precursor cinnamaldehyde) may be amelioration of oxidative stress.

Thus, the compound of the invention may be for use in treating or preventing oxidative stress associated with hyperglycinemia and/or hyperammonemia. In one embodiment, the oxidative stress is associated with NKH. In another embodiment, the oxidative stress is associated with a UCD. Preferably, the UCD is NAGS, CPSI deficiency, OTCD, ASSD, ASA, HHH syndrome or ARG, and more preferably OTCD or ASA. More preferably still, the UCD is ASA. In a preferred embodiment, the compound of the present invention is for use in the treatment of a patient that has been diagnosed with hyperglycinemia and/or hyperammonemia.

In another preferred embodiment, the compound is for use in the treatment of a patient which is at risk of developing hyperglycinemia and/or hyperammonemia. Preferably, the patient has a genetic defect associated with hyperglycinemia and/or hyperammonemia and/or has a family history of hyperglycinemia and/or hyperammonemia. More preferably, the patient has a defect in the Gldc gene, the Otc gene or the Aal gene. Further preferably, the patient has a defect in the Gldc gene.

The present invention additionally provides a method of treating and/or preventing a disease or condition associated with hyperglycinemia and/or hyperammonemia as described above in a patient which comprises administering to said patient an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof. Preferred features of the compound for use as defined herein are also preferred features of the method of the invention.

The present invention further provides the use of the compound of the present invention as described above or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment and/or prevention of a disease or condition associated with hyperglycinemia and/or hyperammonemia as described above. Preferred features of the compound for use as defined herein are also preferred features of the use of the invention.

Pharmaceutical Compositions and Administration

The present invention also provides a pharmaceutical composition comprising the compound of the invention or a pharmaceutically acceptable salt thereof for use in treating and/or preventing hyperglycinemia and/or hyperammonemia. In one embodiment, this composition further comprises one or more pharmaceutically acceptable carriers diluents, excipients and/or additives.

Preferably, the composition is a solution of the compound of the invention in a liquid carrier. Preferred pharmaceutical compositions are sterile.

The concentration of the compound of the invention in a pharmaceutical composition will vary depending on several factors, including the dosage of the compound to be administered. In one embodiment, the compound of the invention is administered as a monotherapy. In another embodiment, the compound of the invention is administered in combination with one or more additional therapeutic agent(s). The present invention also provides a pharmaceutical combination of the compound of the invention or a pharmaceutically acceptable salt thereof, with one or more additional therapeutic agent(s). Thus, the compound of the invention may be present in the combinations, compositions and products of the invention with one or more additional therapeutic agent(s).

In one embodiment the present invention provides a pharmaceutical composition comprising (i) a compound of the invention or a pharmaceutically acceptable salt thereof, (ii) one or more additional therapeutic agent(s), which additional therapeutic agent(s) may be as defined herein and (iii) one or more pharmaceutically acceptable carriers and/or excipients.

Typically, the combination is a combination in which the compound of the invention or a pharmaceutically acceptable salt thereof, and the additional therapeutic agent(s) are formulated for separate, simultaneous or successive administration. The combination may optionally also comprise a pharmaceutically acceptable carrier or diluent.

When, for example, the compound of the invention is part of a combination (such as a pharmaceutical combination) as defined herein, formulated for separate, simultaneous or successive administration, (a) the pharmaceutical compound of the invention, and (b) the additional therapeutic agent(s) may be administered by the same mode of administration or by different modes of administration.

For simultaneous administration, the compound of the invention or a pharmaceutically acceptable salt thereof, and the additional therapeutic agent(s) may for example be provided in a single composition. Thus, the composition may, for example, comprise the compound of the invention or a pharmaceutically acceptable salt thereof, and the additional therapeutic agent(s), and optionally a pharmaceutically acceptable carrier or diluent. For separate or successive administration, the compound of the invention or a pharmaceutically acceptable salt thereof, and the additional therapeutic agent(s) may, for example, be provided as a kit.

The additional therapeutic agent(s) used in the invention can be any suitable therapeutic agent that the skilled person would judge to be useful in the circumstances. Particularly suitable classes of therapeutic agents include agents that are suitable for the treatment and/or prevention of hyperglycinemia and/or hyperammonemia. In one embodiment, the additional therapeutic agent(s) include benzoates. Preferably, the benzoate is sodium benzoate. In another embodiment, the additional therapeutic agent(s) include nutrients. Specific nutrients may overcome undesirable side-effects associated with treatments for hyperglycinemia and hyperammonemia. In one embodiment, the additional therapeutic agent(s) include agents that are suitable for the treatment and/or prevention of a carnitine deficiency. In one embodiment, the additional therapeutic agent(s) include carnitine.

In another embodiment, the additional therapeutic agent(s) includes a gene therapy agent. For instance, the gene therapy agent may be an agent for inserting, into cells of the subject, a functional (e.g., non-mutated) version of the mutated gene that is responsible for the specific pathological condition to be treated. For example, where the condition is classic NKH caused by mutations in the GLDC gene, the gene therapy agent may be an agent for inserting a functional GLDC gene. Methods for gene therapy, and for creating gene therapy agents for delivering a given gene of interest, are known in the art.

The compound, combinations, compositions and products of the invention may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. The compound, combinations, compositions and products of the invention may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrastemally, transdermally or by infusion techniques. Depending on the vehicle and concentration used, the drugs can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agent can be dissolved in the vehicle. The compound, combinations, compositions and products may also be administered as suppositories. The compounds, combinations, compositions and products may be administered by inhalation in the form of an aerosol via an inhaler or nebuliser. The pharmaceutical compound of the invention, pharmaceutical combinations and pharmaceutical compositions may be administered topically, for example, as a cream, foam, gel, lotion, or ointment.

A compound of the invention, and optionally additional therapeutic agent(s), is typically formulated for administration with a pharmaceutically acceptable carrier or diluent. For example, solid oral forms may contain, together with the active compound, solubilising agents, e.g. cyclodextrins or modified cyclodextrins; diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.

Liquid dispersions for oral administration may be solutions, syrups, emulsions and suspensions. The solutions may contain solubilising agents e.g. cyclodextrins or modified cyclodextrins. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may include pharmaceutically active compounds in which the average particle size has undergone particle size reduction by micronisation or nanonisation technologies. For instance, the average particle size of the compound of the invention may have undergone particle size reduction by micronisation or nanonisation technologies.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol; solubilising agents, e.g. cyclodextrins or modified cyclodextrins, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous or infusions may contain as carrier, for example, sterile water and solubilising agents, e.g. cyclodextrins or modified cyclodextrins or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

For topical application to the skin, the compound may, for example, be made up into a cream, lotion or ointment. Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.

For topical application by inhalation, the compound may be formulated for aerosol delivery for example, by pressure-driven jet atomizers or ultrasonic atomizers, or preferably by propellant-driven metered aerosols or propellant-free administration of m i cron i zed powders, for example, inhalation capsules or other “dry powder” delivery systems. Excipients, such as, for example, propellants (e.g. Frigen in the case of metered aerosols), surface-active substances, emulsifiers, stabilizers, preservatives, flavorings, and fillers (e.g. lactose in the case of powder inhalers) may be present in such inhaled formulations. For the purposes of inhalation, a large number of apparata are available with which aerosols of optimum particle size can be generated and administered, using an inhalation technique which is appropriate for the patient. In addition to the use of adaptors (spacers, expanders) and pear-shaped containers (e.g. Nebulator®, Volumatic®), and automatic devices emitting a puffer spray (Autohaler®), for metered aerosols, in particular in the case of powder inhalers, a number of technical solutions are available (e.g. Diskhaler®, Rotadisk®, Turbohaler® or the inhalers for example as described in European Patent Application EP 0 505 321).

A therapeutically effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof is administered to a patient. A typical daily dose is, for example, from 10 mg to 3000 mg per kg of body weight, preferably 50 mg to 1500 mg per kg of body weight (e.g. from 100 to 500 mg per kg of body weight), according to the activity of the compound or combination of specific therapeutic agents used, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. In one embodiment the daily dosage level is about 250 mg per kg of body weight. Where a combination is administered, the compound of the invention or a pharmaceutically acceptable salt thereof is typically administered in an amount of at least 250 mg per kg of body weight. A preferred upper limit on the amount of compound of the invention or a pharmaceutically acceptable salt thereof administered is typically 3000 mg per kg body weight. Any additional therapeutic agent(s) are typically administered at or below the standard dose used for that drug. The compound, combination or composition of the invention is typically administered to the patient in a non-toxic amount.

It is possible for the compound of the invention to be administered in at least two phases, corresponding respectively to an initial loading phase and then a subsequent maintenance phase. The daily dose in the maintenance phase may, for instance, correspond to the daily doses described above. The dose in the loading phase may correspond to the dose described as a typical daily dose above, but contracted into a shorter “loading” time frame such a period of 1 to 5 hours (e.g., 1 to 3 hours).

In an embodiment of the invention, the compound or composition of the invention is delivered in vivo in a mammal. In another embodiment the mammal is a human.

The present invention also provides a kit comprising the compound of the invention, or a pharmaceutically acceptable salt thereof, or a composition of the invention, for use in the treatment and/or prevention of the pathological conditions described herein. The kit optionally further comprises, in admixture or in separate containers, an additional pharmaceutically active agent(s) as described above. Preferred features of the compound or composition for use as defined herein are also preferred features of the kit of the invention.

Examples

Example 1 - A mouse model for NKH

A mouse model for NKH was developed. As discussed above, 80% of human NKH patients carry a mutation in the Gldc gene, which is responsible for the coding of glycine decarboxylase (GLDC). A GLDC-deficient mouse model was developed by introducing loss of function mutations to the ortholog of the human Gldc gene. Thus, the Gldc^GT1/GT1 mouse was developed, which is homozygous for a loss of function allele in its Gldc gene. This mouse model recapitulates the key features of NKH, including significant elevation of plasma, urine and tissue glycine, abnormal electroencephalogram and premature lethality. Gldc^GT1/GT1 mice also altered one-carbon metabolism.

As shown in Figure 2, the Gldc^GT1/GT1 mice exhibit diminished Gldc mRNA (Fig. 2A), loss of GCS enzymatic activity (Fig. 2B) and elevated abundance of glycine in body fluids (Fig. 2C).

Guanidinoacetate is formed from glycine and arginine. Quantification of the guanidinoacetate levels in Gldc^GT1/GT1 mice shows increased levels in the brain and liver tissue and plasma, as shown in Figure 3D, Figure 4 and Figure 6. Figure 3G shows a heat- map of the relative abundance of metabolites in the liver as determined by mass spectrometry. It can be seen that a series of glycine conjugates are significantly elevated in the liver tissue of Gldc^GT1/GT1 mice, and that these elevated levels may be normalised in liver tissue by conditional reinstatement of hepatic Gldc expression.

Thus, a series of metabolic abnormalities in the livers and brains of the Gldc^GT1/GT1 mice were identified, which provide metrics for the effectiveness of treatments of their hyperglycinemia.

Example 2 - The effect of oral sodium benzoate and sodium cinnamate on plasma glycine levels

Sodium benzoate and sodium cinnamate were administered to Gldc^GT1/GT1 mice in their drinking water for 7 days. Sodium benzoate was tolerated at doses of up to 0.5% in drinking water. Two different dosage levels of sodium cinnamate were used; ‘dosage 1 ’ matches the levels of sodium benzoate administered, while ‘dosage 2’ is double that of ‘dosage 1 ’ (n=4 for each group). The results are shown in Figure 4.

Sodium benzoate was found to significantly lower plasma glycine in Gldc^GT1/GT1 mice in comparison to untreated control mutants (p<0.001). This confirms that circulating glycine in Gldc^GT1/GT1 mice is amenable to lowering by conjugation, as is known to be the case in humans. As in human NKH patients, higher doses of benzoate were poorly tolerated by the Gldc^GT1/GT1 mice.

Sodium cinnamate was also found to significantly lower plasma glycine levels in Gldc^GT1/GT1 mice in comparison to untreated mutants at both dosage levels (p<0.001). In particular, dosage 1 of sodium cinnamate provided similar levels of glycine reduction to sodium benzoate, while dosage 2 of sodium cinnamate was well-tolerated by the mice and achieved a markedly improved reduction in lower plasma glycine levels. Similarly, both benzoate and cinnamate (tested at cinnamate dosage 2) achieved lowering of plasma guanidinoacetate (Figure 4B).

Example 3 - Levels of glycine and glycine derivatives in mouse liver tissue

Sodium benzoate and sodium cinnamate were administered to adult Gldc^GT1/GT1 mice and the levels of glycine and various glycine derivatives in liver tissue were measured.

As can be seen from Figure 5, sodium benzoate and sodium cinnamate both lowered the abundance of glycine and its derivatives in liver tissue, indicating activation of glycine conjugation reactions, as confirmed by an observed increased abundance of hippurate in each experiment. Note that 3-phenylpropionate is hydrocinnamate and is therefore more abundant in cinnamate-treated mice. Hydrocinnamate may act as an intermediate in glycine conjugation.

These results show that cinnamate treatment (dosage 2) can bring levels of glycine and its derivatives closer to wild-type levels than benzoate.

Example 4 - Levels of glycine and associated molecules in mouse liver and brain tissue

Sodium benzoate and sodium cinnamate were administered to Gldc^GT1/GT1 adult mice (6 weeks) and the levels of glycine, glycine derivatives and downstream metabolites in their brain were measured. Acyl-glycines such as isovalerylglycine and butyrylglycine accumulated in the liver but not the brain, while the brain showed accumulation of glycine dipeptides such as gamma-glutamylglycine and glycylleucine.

The results are set out in Figure 3 and Figure 6. It can be seen that the Gldc^GT1/GT1 mice have significantly increased abundance of glycine and associated compounds in both liver and brain tissues when compared with wild- type mice. Sodium benzoate provides a significant lowering of the levels of glycine and associated compounds in these tissues in Gldc^GT1/GT1 mice when compared to untreated Gldc^GT1/GT1 mice as shown in Figure 5 and Figure 6.

An equal dosage of sodium cinnamate also provides a significant lowering of the levels of glycine and associated compounds in both liver and brain tissues in Gldc^GT1/GT1 mice when compared to untreated Gldc^GT1/GT1 mice. A double dosage of sodium cinnamate is known to be well-tolerated and provides a particularly excellent decrease in the levels of glycine and associated compounds in both liver and brain tissues of Gldc^GT1/GT1 mice in comparison to both untreated Gldc^GT1/GT1 mice and Gldc^GT1/GT1 mice treated with sodium benzoate. Sodium benzoate was not well-tolerated at such ‘double dosage’ levels.

As noted above, acyl-glycine compounds did not accumulate in brains of the Gldc- deficient mice. This evidences that the ability of benzoate to lower glycine levels in brain tissue is a secondary effect of reducing circulating glycine concentrations via conjugation reactions in the liver and kidneys.

Claims

1. A compound that is cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde, for use in the treatment or prevention of a disease or condition associated with hyperglycinemia or hyperammonemia.

2. The compound for use according to claim 1, wherein the compound is a pharmaceutically acceptable salt of cinnamic acid.

3. The compound for use according to claim 2, wherein the pharmaceutically acceptable salt of cinnamic acid is sodium cinnamate.

4. The compound for use according to any one of claims 1 to 3, wherein the treatment or prevention of a disease or condition associated with hyperglycinemia or hyperammonemia is the treatment of a disease or condition associated with hyperglycinemia or hyperammonemia.

5. The compound for use according to any one of claims 1 to 4, wherein the disease or condition associated with hyperglycinemia or hyperammonemia is a disease or condition associated with hyperglycinemia.

6. The compound for use according to claim 5, wherein the hyperglycinemia is non ketotic glycinemia.

7. The compound for use according to claim 6, wherein the non-ketotic glycinemia is classic non-ketotic glycinemia.

8. The compound for use according to any one of claims 5 to 7, wherein the hyperglycinemia is associated with defects in the glycine cleavage system.

9. The compound use according to any one of claims 5 to 8, wherein the disease or condition associated with hyperglycinemia is a neurological condition.

10. The compound for use according to any one of claims 1 to 4, wherein the disease or condition associated with hyperglycinemia or hyperammonemia is a disease or condition associated with hyperammonemia.

11. The compound for use according to claim 10, wherein the hyperammonemia is associated with a urea cycle disorder (UCD).

12. The compound for use according to claim 11, wherein the UCD is associated with defects in ornithine transcarbamylase or argininosuccinate lyase.

13. The compound for use according to claim 12, wherein the UCD is ornithine transcarbamylase deficiency or argininosuccinic aciduria.

14. The compound for use according to claim 13, wherein the UCD is argininosuccinic aciduria.

15. A pharmaceutical composition comprising: cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde, as defined in any one of claims 1 to 3; and at least one further therapeutic agent selected from carnitine, benzoic acid and a pharmaceutically acceptable salt of benzoic acid, preferably sodium benzoate.

16. A kit comprising: cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde, as defined in any one of claims 1 to 3; and, in admixture or in one or more separate containers, at least one further therapeutic agent selected from carnitine, benzoic acid and a pharmaceutically acceptable salt of benzoic acid, preferably sodium benzoate.

17. A product containing: (a) cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde, as defined in any one of claims 1 to 3; and (b) at least one further therapeutic agent selected from carnitine, benzoic acid and a pharmaceutically acceptable salt of benzoic acid, preferably sodium benzoate; for simultaneous, separate or sequential use in the treatment or prevention of a disease or condition associated with hyperglycinemia or hyperammonemia as defined in any one of claims 5 to 14.

18. A method of treating or preventing a disease or condition associated with hyperglycinemia or hyperammonemia, as defined in any one of claims 5 to 14, in a patient a need thereof, which method comprises administering to said patient an effective amount of cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde, as defined in any one of claims 1 to 3.

19. Use of cinnamic acid or a pharmaceutically acceptable salt thereof or cinnamaldehyde, as defined in any one of claims 1 to 3, in the manufacture of a medicament for use in the treatment or prevention of a disease or condition associated with hyperglycinemia or hyperammonemia, as defined in any one of claims 5 to 14.