Patent Application
20240207221
The present application relates to pharmaceutical compounds and compositions useful in the prevention or treatment of hyperammonemia, particularly drug-induced hyperammonemia.
Hyperammonemia is a metabolic disorder characterized by high levels of ammonia, a nitrogen-containing compound, in the blood. Ammonia is an important source of nitrogen and is required for amino acid synthesis. It is also necessary for normal acid-base balance. However, when present in high concentrations, ammonia is toxic.
Ammonia is normally produced in the colon and the small intestine, from where it is transported to the liver to be converted via the urea cycle into urea, a water-soluble compound which is then excreted by the kidneys.
The consequences of hyperammonemia are varied and can be serious. Ammonia is a potent neurotoxin, and its entry into the brain causes neurological disorders that can lead to convulsions, ataxia, stroke-like lesions, coma, psychosis, loss of vision, acute encephalopathy and cerebral edema. Other symptoms such as vomiting, respiratory alkalosis, hypothermia and liver damage leading to death are also observed in individuals with hyperammonemia.
Hyperammonemia must be detected early and treated immediately to avoid the development of potentially fatal complications such as cerebral edema or cerebral herniation.
Currently, the management of hyperammonemia involves a combination of measures to reduce ammonia levels in the blood. Typically, patients receive a low-protein diet combined with drugs intended to promote nitrogen elimination, such as sodium phenylbutyrate, sodium benzoate and glycerol phenylbutyrate (Enns et al., (2007) New Engl J Med., 356:2282-2292).
However, these drugs have to be administered several times a day and are associated with numerous side effects, in particular nausea, vomiting, irritability and anorexia. Moreover, because of the need to calculate dosages, overdosing can easily occur, leading to serious metabolic side effects and death.
Thus, there remains a need for effective alternative treatment and prevention of hyperammonemia.
The present invention stems from the inventors' unexpected finding that stiripentol reduces methionine sulfoximine (MSO)-induced hyperammonemia, as well as methionine sulfoximine (MSO)-induced hyperammonemia-related seizures.
Thus, the present invention relates to a compound of the following formula (I):
The present invention also relates to the compound of formula (I) or the pharmaceutically acceptable salt hydrate or solvate thereof for use as defined above in combination with at least one additional compound for the prevention or treatment of hyperammonemia or for the prevention or treatment of one or more symptoms associated with hyperammonemia.
The present invention also relates to a pharmaceutical composition comprising as active substance at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt, hydrate or solvate thereof, optionally in association with a pharmaceutically acceptable carrier, for use in the prevention or treatment of hyperammonemia.
The present invention also relates to a pharmaceutical composition comprising as active substance at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt, hydrate or solvate thereof, optionally in association with a pharmaceutically acceptable carrier, and optionally comprising at least one additional compound for the prevention or treatment of hyperammonemia or for the prevention or treatment of one or more symptoms associated with hyperammonemia.
The present invention also relates to a pharmaceutical composition comprising as active substance at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt, hydrate or solvate thereof, optionally in association with a pharmaceutically acceptable carrier, and optionally comprising at least one additional compound for the prevention or treatment of hyperammonemia or for the prevention or treatment of one or more symptoms associated with hyperammonemia selected from the group consisting of neuroprotective drugs, anti-inflammatory drugs, sodium benzoate, sodium phenylbutyrate, glycerol phenylbutyrate, N-carbamylglutamate, sodium benzoate, sodium phenylacetate, lactulose, arginine hydrochloride, L-arginine, carglumic acid, L-ornithine, L-aspartate, citrulline and mixtures thereof.
The present invention also relates to products containing:
The present invention also relates to a method of preventing or treating hyperammonemia in an individual comprising administering to the individual an effective amount of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt, hydrate or solvate thereof.
In an embodiment of the invention, the method as defined above also comprises the administration of at least one additional compound for the prevention or treatment of hyperammonemia or for the prevention or treatment of one or more symptoms associated with hyperammonemia.
The present invention also relates to the use of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for the preparation of a medicament intended for the prevention or treatment of hyperammonemia in an individual.
In one embodiment of the invention, the medicament as defined above comprises at least one additional compound for the prevention or treatment of hyperammonemia or for the prevention or treatment of one or more symptoms associated with hyperammonemia.
As intended herein, the term “comprising” is synonymous with “including”, “containing” or “encompassing”, i.e. when an object “comprises” one or more features, features other than those mentioned may also be comprised in the object. Conversely, the expression “consisting of” means “constituted by”, i.e. when an object “consists of” one or more features, the object cannot include features other than those mentioned.
Preferably, the compound of formula (I) as defined above is represented by the following formula (II):
in which n, A1, A2, A3 and R1 are as defined above.
More preferably, the compound of formula (I) or (II) defined above is represented by the following formula (III):
Preferred alkyl groups according to the invention include methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl and t-butyl.
Cl, I, Br or F atoms are preferred halogen atoms according to the invention.
French patent FR 2 173 691, incorporated herein by reference, describes the synthesis of stiripentol, in particular from methylenedioxy-3,4-phenyl-1-dimethyl-4,4-penten-1-on-3. It is well within the ordinary skills of the skilled person to synthesize the other compounds of formula (I) from this teaching.
As will be clear to those skilled in the art, the formulae (I), (II) and (III) defined above represent either the various stereoisomers encompassed by these formulae, or mixtures thereof, in particular racemic mixtures thereof.
Thus, the compound of formula (III) can be a compound of formula (IIIa), a compound of formula (IIIb), or a mixture of a compound of formula (IIIa) and a compound of formula (IIIb), in particular the racemic mixture thereof.
“Hyperammonemia” refers to increased ammonia concentrations in the body. Thus, hyperammonemia according to the invention can be caused, for example, by an increase in ammonia production or by a decrease in ammonia detoxification processes in the body.
Ammonia (NH3), which can coexist in the body with ammonium cation (NH4+) depending on pH, is a product of the catabolism of proteins and other nitrogenated compounds. It is converted by the enzymes of the urea cycle into the less toxic substance urea, before being excreted in the urine by the kidneys.
The urea cycle, which also functions as a source for the production of certain amino acids (arginine, citrulline and ornithine) in the body, comprises 6 enzymes: carbamoyl phosphate synthase I (CPS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), arginase and N-acetylglutamate synthase (NAGS) and two transport proteins: ornithine translocase (ORNT1) and citrin. A defect can occur in one or more of these 8 proteins, resulting in hyperammonemia, also known as primary hyperammonemia.
Examples of urea cycle disorder leading to primary hyperammonemia according to the invention include:
Hyperammonemia according to the invention may also be due to inhibition of the urea cycle as a result of errors of intermediary metabolism characterized by reduced activity of enzymes or proteins that are not part of the urea cycle, also known as secondary hyperammonemia.
By way of example of an anomaly causing inhibition of the urea cycle leading to secondary hyperammonemia according to the invention, it is possible to cite:
Hyperammonemia according to the invention can also be linked to or caused by other pathologies, in particular pathologies selected from the group consisting of liver diseases, such as acute or chronic hepatic insufficiency, viral hepatitis, cirrhosis, impaired liver function, and vascular bypass of the liver resulting in reduced filtration of blood in the liver. This is also known as acquired hyperammonemia.
Hyperammonemia according to the invention can also result from the intake of medication or be due to chemotherapy, it is referred to as drug-induced hyperammonemia. Examples of drugs liable to cause hyperammonemia include sodium valproate, barbiturates such as primidone, glycine gel, L-asparaginase, 5-fluorouracil, sunitinib, asparaginase chemotherapy and regorafenib.
Hyperammonemia according to the invention can also result from the complication of a solid organ transplantation, such as a lung transplant, and is referred to as post-operative hyperammonemia.
Hyperammonemia prevented or treated according to the invention can also be the transient hyperammonemia of the newborn.
In an embodiment, the present invention relates to the prevention or treatment of disorders associated with hyperammonemia preferably selected from the group consisting of hyperammonemia-related convulsions and hyperammonemia-related convulsive seizures.
The individual according to the invention is preferably a human being.
Preferably, the individual according to the invention suffers from hyperammonemia, in particular drug-induced hyperammonemia.
In one embodiment of the invention the individual is a full-term infant having a plasma ammonia concentration of at least 40 μM/L, at least 45 μM/L, at least 50 μM/L, at least 80 μM/L, or at least 90 μM/L.
In an embodiment of the invention, the individual is a premature infant with a plasma ammonia concentration of at least 70 μM/L, at least 80 μM/L or at least 90 μM/L.
In an embodiment of the invention the individual is a child over one month old having a plasma ammonia concentration of at least 40 μM/L, at least 45 μM/L, at least 50 M/L, at least 80 μM/L, or at least 90 μM/L.
In an embodiment of the invention, the individual is an adult with a plasma ammonia concentration of at least 30 μM/L, at least 40 μM/L at least 50 μM/L, at least 80 M/L, at least 150 μM/L, at least 180 μM/L, at least 200 μM/L, at least 300 μM/L or at least 400 μM/L.
In an embodiment of the invention, the individual according to the invention has a urea cycle disorder.
In an embodiment of the invention, the individual according to the invention presents a disturbance of cerebral functions. Preferably, the individual according to the invention presents at least one disorder selected from the group consisting of convulsions, convulsive seizures, confusion, agitation, disorders of consciousness, lethargy, ataxia, stroke-like lesions, coma, psychosis, loss of vision, acute encephalopathy, cerebral edema, and cerebral herniation. More preferably, the individual according to the invention has convulsions or convulsive seizures.
Preferably, the compound of formula (I) as defined above, or its pharmaceutically acceptable salt, hydrate or solvate, is administered or administrable at a unit dose, or is packaged in a unit dose, of from about 50 mg to about 1500 mg, particularly of from about 150 mg to 300 mg. Also preferably, the compound of formula (I) as defined above or its pharmaceutically acceptable salt, hydrate or solvate is administered or administrable with a dosage regimen of from about 5 g/day to about 45 g/day. Preferably also the compound of formula (I) as defined above or its pharmaceutically acceptable salt, hydrate or solvate is administered or administrable with a dosage regimen of from 50 mg/kg/day to 500 mg/kg/day, preferably of from 250 mg/kg/d to 350 mg/kg/d, more preferably of about 300 mg/kg/d.
Preferably, the compound of formula (I) as defined above, or its pharmaceutically acceptable salt, hydrate or solvate, the pharmaceutical composition as defined above or the medicament as defined above is in a form acceptable for oral or rectal administration. Preferably, the compound of formula (I) as defined above, or its pharmaceutically acceptable salt, hydrate or solvate, the pharmaceutical composition as defined above or the medicament as defined above is administered or administrable in the form of a powder, tablets, capsules, sachets or suppositories.
In an embodiment, the compound of formula (I) as defined above, or its pharmaceutically acceptable salt, hydrate or solvate, the pharmaceutical composition as defined above or the medicament as defined above is not administered with another anticonvulsant or antiepileptic substance, such as sodium valproate and clobazam.
In an embodiment, the compound of formula (I) as defined above, or its pharmaceutically acceptable salt, hydrate or solvate, the pharmaceutical composition as defined above or the medicament as defined above is not administered with a carnitine treatment.
Preferably, the additional compound for the prevention or treatment of hyperammonemia or for the prevention or treatment of one or more symptoms associated with hyperammonemia according to the invention is intended to prevent or treat hyperammonemia, in particular by decreasing the production of ammonia in the blood or by increasing the mechanisms for eliminating ammonia from the blood.
Preferably also, the additional compound for the prevention or treatment of hyperammonemia or for the prevention or treatment of one or more symptoms associated with hyperammonemia can be any compounds intended to alleviate or treat one or more symptoms associated with hyperammonemia, such as neuroprotective or anti-inflammatory drugs.
Examples of symptoms associated with hyperammonemia include vomiting, loss of appetite, respiratory alkalosis, hypothermia, liver damage, neurological disorders such as convulsions, convulsive seizures, confusion, agitation, disturbed consciousness, lethargy, ataxia, stroke-like lesions, coma, psychosis, loss of vision, acute encephalopathy, cerebral edema, cerebral herniation.
Preferably, the additional compound useful for the prevention or treatment of hyperammonemia is selected from the group consisting of neuroprotective drugs such as N-acetylcysteine, indomethacin, propofol, minocycline and mannitol anti-inflammatory drugs, sodium benzoate, sodium phenylbutyrate, glycerol phenylbutyrate, N-carbamylglutamate, sodium benzoate/sodium phenylacetate, lactulose, arginine hydrochloride, L-arginine, carglumic acid, a mixture of L-ornithine and L-aspartate, citrulline, and mixtures thereof.
The compound of formula (I) as defined above, or its pharmaceutically acceptable salt, hydrate or solvate, the pharmaceutical composition as defined above or the medicament as defined above may also be associated with therapies intended to alleviate or reduce one or more symptoms associated with hyperammonemia or to prevent or treat hyperammonemia such as hemofiltration, intermittent hemodialysis, peritoneal dialysis, induced hypothermia, renal replacement therapy.
The compound of formula (I) as defined above, or its pharmaceutically acceptable salt, hydrate or solvate, the pharmaceutical composition as defined above or the medicament as defined above may also be combined with a low-protein diet and/or a diet in which caloric intakes are replaced by sugar and lipids.
As used herein, the expression “combined” or “in combination” or “combination product” means that the compound of formula (I) as defined above, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in particular stiripentol, and the additional compound for the prevention or treatment of hyperammonemia or for the prevention or treatment of one or more symptoms associated with hyperammonemia as defined above, may be combined within the same pharmaceutical composition or medicament, and thus administered together, or be administered separately, i.e. by separate routes of administration and/or separate administration regimens, provided that when administered separately the period of time during which the compound of formula (I) as defined above or its pharmaceutically acceptable salt, hydrate or solvate exerts its pharmacological effects on the individual and the period of time during which the additional compound as defined above exerts its pharmacological effects on the individual overlap in whole or in part.
The invention will be further explained with the following non-limiting Examples and Figure.
*p<0.05: ANOVA statistical test of treatment versus control groups.
#p<0.05: ANOVA statistical test of groups receiving MSO and stiripentol versus group receiving MSO only.
The inventors studied the efficacy of stiripentol on hyperammonemia and on hyperammonemia-associated convulsions in a model of methionine sulfoximine (MSO)-induced convulsions according to Hevor et al. (1985) Neuropathol. Appl. Neurobiol. 11: 129-139, as follows.
Male CBA mice (Janvier) weighing between 23 and 27 grams are used after acclimatization for at least 4 days in the animal house (t°=22±2° C.; housing conditions are as follows: SAFE “A04” food; drinking water: tap water; nycthemeral cycle: 12 h/12 h (light: 7 h/19 h-dark: 19 h/7 h)).
On reception, mice are grouped in batches of 8 to 10 in 1500 U cages (floor area 1500 cm2) with bedding and sizzle dry (SDS) enrichment, in line with the ethical recommendations of European Parliament Directive 2010/63/EU (Sep. 22, 2010).
On test day, mice are weighed, marked and placed in individual cages. At t ═O minutes, MSO (0.1 mL/10 g) is administered intraperitoneally, and the mice are carefully observed for 8 hours. Animals are normal for the first four hours. Tonic and clonic seizures appear for a few hours, then gradually disappear.
The parameters evaluated are the general condition of the animals, according to the Irwin grid (Irwin 1968), the number of convulsions, their onset times and mortality. Animals are euthanized as soon as limit points are reached, or at the end of the 8-hour observation period, according to the ANI07 procedure approved by the Ethics Committee.
Blood and whole brain with cerebellum samples will be taken in some studies to determine ammonia levels at sacrifice or at death for animals that do not complete the study.
Blood is collected under isoflurane anesthesia by cardiac puncture (0.5 to 1.0 mL per mouse) to determine ammonia levels. In the case of brain and blood sampling, the mouse is rapidly sacrificed by decapitation: the brain is removed and frozen in liquid nitrogen. The liver can then be removed and rapidly frozen in liquid nitrogen. Samples are stored at −80° C.
Stiripentol (0.1 mL/10 g) is administered intraperitoneally at different times with respect to MSO.
3. Ammonemia Measurement with the ABCAM Kit
Blood is collected after decapitation or by cardiac puncture, under isoflurane anesthesia (flow rate 1.5 l/min, evaporator at 4.3% for induction and maintenance) using an orange 25 G needle (Terumo, NN2516R) and a 1.3 ml S-Monovette tube containing EDTA (purple cap, ref 41.1395.105, Sarstedt). The tube is centrifuged at 2000 g for 10 minutes in a refrigerated 4° C. Beckman Allegra centrifuge. Plasma is collected in a 1 ml microtube (Brand, ref 780500). Microtubes are placed in a freezer at −80° C. before analysis, otherwise on crushed ice.
Ammonia is determined colorimetrically using a microplate reader (Dynex, model MRX), and an ABCAM kit (ref AB83360, ammonia assay kit) containing several reagents. This kit enables quantitative enzymatic determination of ammonium present in the samples. During the assay, ammonium is converted to a product that reacts with the OxiRed probe for quantification at 570 nm using a microplate reader. This test can detect 1 nmol (#20 μM) of ammonium. The kit, stored at −20° C., includes the following components:
Standard at 1 mM, i.e. 1 mmol in 1 L or 1 nmol in 1 μL, so 6 nmol in 6 μl. Taking 50 μl out of 150 μL (6+144), we have 2 nmol/well.
Sa=(corrected OD−y)/slope
Methionine sulfoximine (Sigma, ref M5379, batches SLBN0115V, SLBP0971V) was solubilized in 0.9% NaCl.
Stiripentol (batch 176) was suspended in 5% (v/v) Tween 80 and 0.9% NaCl.
The ABCAM assay kit (ref AB83360, ammonia assay) and NH4Cl (ammonium chloride, Sigma, ref 254134, molar mass 53.49 g) are used for ammonemia determinations.
Results are expressed as the mean value±MSE. The statistical tests used are:
Methionine sulfoximine (MSO) was administered intraperitoneally at different concentrations. Mouse behavior and mortality were observed up to 8 hours after administration:
Based on these results, a dose of 50 mg/kg methionine sulfoximine was chosen to study the protective effects of stiripentol on MSO-induced disorders.
Stiripentol was administered intraperitoneally at 300 mg/kg without methionine sulfoximine.
It was observed that stiripentol did not alter ammonemia (176±14 μM) compared with the control group (179±11 μM).
3. Effects of Stiripentol after Administration of 50 mg/kg MSO
Stiripentol is administered just before methionine sulfoximine according to Hevor et al. (1985) Neuropathol. Appl. Neurobiol. 11: 129-139.
Administration of 50 mg/kg methionine sulfoximine resulted in behavioral signs of toxicity, with a convulsion rate of 90% and a mortality rate of 75%.
Administration of 300 mg/kg stiripentol, just prior to MSO, significantly reduced the convulsion rate to 50% and the mortality rate to 25%, suggesting a protective effect.
In this study, ammonemia determinations gave the following results:
Stiripentol is administered intraperitoneally 30 minutes before methionine sulfoximine according to Cloix et al. (2010). Epilepsia, 51: 118-128.
Administration of 50 mg/kg methionine sulfoximine resulted in behavioral signs of toxicity, with a convulsion rate of 100% and a mortality rate of 80%.
The administration of 300 mg/kg stiripentol, 30 minutes prior to MSO, significantly reduced the convulsion rate to 45% and the mortality rate to 25%, suggesting a protective effect.
During this study, ammonemia determinations gave the following results (
In the methionine sulfoximine (MSO)-induced convulsion model, the inventors have shown that a single intraperitoneal dose of 50 mg/kg MSO results in hyperammonemia, convulsions and mortality in 80-100% of mice.
The inventors have shown that intraperitoneal administration of stiripentol at 300 mg/kg significantly reduces methionine sulfoximine-induced convulsions and mortality, suggesting a protective effect.
MSO, which main mechanism of action is the irreversible inhibition of glutamine synthetase, an enzyme involved in ammonia detoxification, notably in the brain, leads to hyperammonemia (Ratnakumari, et al. (1985) J. Neurosci. Res. 14: 449-59). In astrocytes, glutamine synthetase uses ammonium and glutamate to manufacture glutamine, so inhibition of its activity by MSO leads to an increase in ammonia.
In terms of ammonemia, the inventors have shown that intraperitoneal administration of stiripentol at 300 mg/kg significantly reduces MSO-induced hyperammonemia.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR2104119 | Apr 2021 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP22/60445 | 4/20/2022 | WO |
