Patent Application
20240293359
The present invention relates to a prophylactic or therapeutic agent for Parkinson's disease.
Parkinson's disease is a neurodegenerative disease that develops caused by dopaminergic neurons in the substantia nigra being selectively damaged, and that is diagnosed based on movement symptoms such as, primarily, bradykinesia, tremor, and muscle rigidity. The basis of treatment for Parkinson's disease is dopamine replacement therapy. However, there are therapeutic problems such that the amount of drugs administered required to obtain therapeutic effects gradually increases, or side effects due to dopamine replacement therapy appear, and therefore, development of a novel therapeutic agent has been expected.
In recent years, it has been reported that the deterioration of balance of the concentration of intracellular iron ions causes intracellular mitochondrial dysfunction and promoted production of reactive oxygen species (Non Patent Literature 1), leading to abnormal cell functions. In addition, when the intracellular redox balance is modulated, lipid oxidation reactions in which iron ions are involved are promoted, and eventually ferroptosis, which is a phenomenon that induces cell death from disruption of cell membrane function, receives attention.
On the other hand, a possibility that healthy and social activity impairment derived from other diseases may be aggravated caused by infectious diseases has attracted attention. For Parkinson's disease as well, a possibility that when a patient is infected with the new corona virus (SARS-CoV-2), effects of oxidative stress may enhance, and the damage to dopamine-expressing neurons may exacerbate has been reported (Non Patent Literature 2).
It has also been reported that oxidation reaction accelerated in the presence of iron ions is deeply involved also in pathogenesis and progression of Parkinson's disease. It has been pointed out that, while dopamine, neurotransmitter, has a catechol structure and originally has chemical properties which are susceptible to oxidation, the oxidation is further accelerated in the presence of iron ions. It has been pointed out that the oxidation of dopamine is a cause of loss of dopamine, and further, a dopamine oxidant produced in vivo polymerizes and forms insoluble neuromelanin, leading to a possibility of being involved in pathogenesis. In addition, it has been pointed out that the dopamine oxidant induces degeneration of α-synuclein and formation of aggregate thereof, which is involved in pathogenesis and aggravation of Parkinson's disease (Non Patent Literature 3).
Polysulfides such as glutathione trisulfide (GSSSG) are converted in vivo into reactive sulfur species such as glutathione persulfide (GSSH). It has been reported that the reactive sulfur species have potent antioxidant effect, and may have physiological function such as anti-aging (e.g., Non Patent Literatures 4 and 5). Further, as production methods of GSSSG, a method described in Patent Literature 1 is known.
Since it is desirable that drugs used for Parkinson's disease be highly efficiently delivered to nigrostriatal dopamine neurons in the brain, and nasal drop administration can avoid so-called first-pass metabolism, and directly deliver the drugs to the brain, the nasal drop administration attracts attention as an administration method for relatively increasing the concentration in the brain (Non Patent Literature 6). On the other hand, since the concentrations of the drugs that can be administered by nasal drop administration are limited, and in consideration of reducing burden on patients with medication intake, it is desirable that the drugs used for nasal trop administration can be administered as a solution and be without irritating. For example, a compound that is highly water-soluble, and free of ionizable functional group or ionically neutral is inferred as a suitable drug.
Any drug that suppresses dopamine oxidation accelerated in the presence of iron ions, and thereby suppresses a lack of dopamine has not been known. The present inventors have considered that by combining a compound having such effects with dopamine replacement therapy, there are a possibility to enhance therapeutic effects of the dopamine replacement therapy and a possibility to reduce the amount of a drug used for the dopamine replacement therapy to be administered, and thereby enabling the improvement of QOL of patients due to reduced side effects and the like. That is, an object of the present invention is to search for a compound having such effects and to provide a prophylactic or therapeutic agent for Parkinson's disease.
The present inventors have found that glutathione trisulfide, lipoic acid trisulfide, pantethine trisulfide, and N,N′-diacetyl-L-cysteine trisulfide suppress dopamine oxidation reaction accelerated in the presence of iron ions, and completed the present invention. The present invention provides the following [1] to [64].
wherein X represents —OR1 or —NR2R3, R1 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R2 and R3 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, the alkyl group optionally having one or more substituents selected from the group consisting of an amino group and a carboxy group (hereinafter, also described as Compound (1)), a pharmaceutically acceptable salt thereof or a cyclodextrin clathrate thereof, pantethine trisulfide or a pharmaceutically acceptable salt thereof, or N,N′-diacetyl-L-cysteine trisulfide or a pharmaceutically acceptable salt thereof.
According to the present invention, there are a possibility to enhance therapeutic effects of the dopamine replacement therapy and a possibility to reduce the amount of a drug used for the dopamine replacement therapy to be administered, and thereby enabling the improvement of QOL of patients due to reduced side effects and the like.
Hereinafter, the content of the present invention will be described in detail; however, the present invention is not limited to the following embodiments.
A prophylactic or therapeutic agent, and a prophylactic or therapeutic method of the present invention may be those administered or applied to a human.
A trisulfide compound used for the prophylactic or therapeutic agent of the present invention is glutathione trisulfide or a pharmaceutically acceptable salt thereof, Compound (1), a pharmaceutically acceptable salt thereof or a cyclodextrin clathrate thereof, pantethine trisulfide or a pharmaceutically acceptable salt thereof, or N,N′-diacetyl-L-cysteine trisulfide or a pharmaceutically acceptable salt thereof. The glutathione trisulfide is represented by Formula (2). The pantethine trisulfide is represented by Formula (2A). The N,N′-diacetyl-L-cysteine trisulfide is represented by Formula (2B).
In the present invention, examples of the pharmaceutically acceptable salt can include salts with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; salts with organic acids, such as acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, and p-toluenesulfonic acid; salts with alkali metals, such as sodium and potassium; salts with alkali earth metals, such as calcium and magnesium; ammonium salts; and salts with amino acids, such as arginine.
It is preferable for the pharmaceutically acceptable salt of glutathione trisulfide and pantethine trisulfide to be an amino acid salt or an alkali metal salt, and more preferable to be an arginine salt or a sodium salt. It is preferable for the pharmaceutically acceptable salt of Compound (1) to be a salt with an alkali metal, and more preferable to be a sodium salt.
The glutathione trisulfide or a pharmaceutically acceptable salt thereof, Compound (1) or a pharmaceutically acceptable salt thereof, pantethine trisulfide or a pharmaceutically acceptable salt thereof, or N,N′-diacetyl-L-cysteine trisulfide or a pharmaceutically acceptable salt thereof may have a crystal polymorph, but not limited to any crystal form, and may be a single product or a mixture of any crystal form. An amorphous body is also included in the glutathione trisulfide or a pharmaceutically acceptable salt thereof, Compound (1) or a pharmaceutically acceptable salt thereof, pantethine trisulfide or a pharmaceutically acceptable salt thereof, or N,N′-diacetyl-L-cysteine trisulfide or a pharmaceutically acceptable salt thereof. An anhydride and a solvate (in particular, hydrate) are included in the glutathione trisulfide or a pharmaceutically acceptable salt thereof, Compound (1) or a pharmaceutically acceptable salt thereof, pantethine trisulfide or a pharmaceutically acceptable salt thereof, or N,N′-diacetyl-L-cysteine trisulfide or a pharmaceutically acceptable salt thereof. The glutathione trisulfide or a pharmaceutically acceptable salt thereof, Compound (1) or a pharmaceutically acceptable salt thereof, pantethine trisulfide or a pharmaceutically acceptable salt thereof, or N,N′-diacetyl-L-cysteine trisulfide or a pharmaceutically acceptable salt thereof may be a cocrystal with an amino acid (e.g., arginine).
In one embodiment, Compound (1) is a compound represented by Formula (3):
wherein R1 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R1 may be, for example, a hydrogen atom, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, or a hexyl group. Preferably, R1 is a hydrogen atom, wherein the compound represented by Formula (3) is lipoic acid trisulfide represented by Formula (4).
In another embodiment, Compound (1) is a compound represented by Formula (5):
wherein R2 and R3 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, the alkyl group optionally having one or more substituents selected from the group consisting of an amino group and a carboxy group. R1 and R2 may be a hydrogen atom, or an alkyl group such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, or a hexyl group. These alkyl groups optionally have substituents of either or both of the amino group and the carboxy group. R1 and R2 may be, for example, a group represented by Formula (6), wherein * represents a bond. Specific examples of the compound represented by Formula (5) include a compound in which R2 and R3 are both hydrogen atoms, and a compound in which R2 is a hydrogen atom and R3 is the group represented by Formula (6).
The compound represented by Formula (5) can be produced by a step of oxidizing a compound represented by Formula (5a) with an oxidant to obtain a sulfoxide compound (Step 1), and a step of causing the obtained sulfoxide compound to be reacted to a sulfur source (Step 2).
wherein R1 and R2 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, the alkyl group optionally having one or more substituents selected from the group consisting of the amino group and the carboxy group.
The above production method may be conducted by performing the reactions of Step 1 and Step 2 in one pot without isolating the sulfoxide compound.
A solvent to be used in Step 1 is not particularly limited, as long as it dissolves the compound represented by Formula (5a) and the oxidant, and does not inhibit oxidation reaction. Examples of such a solvent include water, sulfuric acid aqueous solution, ethanol aqueous solution and acetonitrile aqueous solution, and it is preferably water. The amount of the solvent to be used in Step 1 can be 1 mL to 500 mL and is preferably 10 mL to 20 mL per 1 g of the compound represented by Formula (5a).
Examples of the oxidant to be used in Step 1 include potassium peroxymonosulfate (sold under the commercial name, e.g., Oxone (R)), peracetic acid, hydrogen peroxide and sodium periodate. Hydrogen peroxide may be used with the catalytic amount of methyltrioxorhenium. From the viewpoints of safety and cost, potassium peroxymonosulfate is a preferable oxidant. The amount of the oxidant to be used can be 0.8 equivalents to 2.0 equivalents and is preferably 1.0 equivalent to 1.3 equivalents per 1 equivalent of the compound represented by Formula (5a).
The reaction temperature in Step 1 can be −20° C. to 30° C. and is preferably −5° C. to 5° C.
The reaction time in Step 1 can be 5 minutes to 24 hours and is preferably 0.5 hours to 2 hours.
A solvent to be used in Step 2 is not particularly limited, as long as it dissolves the sulfoxide compound and the sulfur source, and does not inhibit subsequent reaction. Examples of such a solvent include water, sulfuric acid aqueous solution, ethanol aqueous solution and acetonitrile aqueous solution, and it is preferably water. The amount of the solvent to be used in Step 2 can be 1 mL to 500 mL and is preferably 10 mL to 20 mL per 1 g of the sulfoxide compound.
Examples of the sulfur source to be used in Step 2 include sodium sulfide, potassium sulfide, sodium hydrosulfide, potassium hydrosulfide and hydrogen sulfide. The amount of the sulfur source to be used can be 0.5 equivalents to 4.0 equivalents and is preferably 0.9 equivalents to 1.2 equivalents per 1 equivalent of the sulfoxide compound.
The reaction temperature in Step 2 can be −20° C. to 30° C. and is preferably −5° C. to 25° C.
The reaction time in Step 2 can be 10 minutes to 2 days and is preferably 0.5 hours to 2 hours.
When Step 1 and Step 2 are conducted in one pot, examples of a reaction solvent include water, sulfuric acid aqueous solution, ethanol aqueous solution and acetonitrile aqueous solution, and it is preferably water; and the amount of the solvent can be 1 mL to 500 mL and is preferably 10 mL to 20 mL per 1 g of the compound represented by Formula (5a). Examples of the oxidant to be used include potassium peroxymonosulfate, peracetic acid, hydrogen peroxide (may be used with the catalytic amount of methyltrioxorhenium) and sodium periodate, and it is preferably potassium peroxymonosulfate; and the amount of the oxidant to be used can be 0.8 equivalents to 2.0 equivalents and is preferably 1.0 equivalent to 1.3 equivalents per 1 equivalent of the compound represented by Formula (5a). Examples of the sulfur source to be used include sodium sulfide, potassium sulfide, sodium hydrosulfide, potassium hydrosulfide and hydrogen sulfide; and the amount of the sulfur source to be used can be 0.5 equivalents to 4.0 equivalents and is preferably 0.9 equivalents to 1.2 equivalents per 1 equivalent of the compound represented by Formula (5a). The reaction temperature can be −20° C. to 30° C. and is preferably −5° C. to 25° C. The reaction time can be 15 minutes to 2 days and is preferably 1 hour to 4 hours.
In addition to Step 1 and Step 2, the method may include a step of protecting functional groups such as a hydroxy group, a carbonyl group, an amino group, and a carboxy group and a step of deprotecting the protected functional groups, as needed. Protective groups, protection/deprotection reactions of these functional groups are well-known to those skilled in the art, and suitable protective groups, protection/deprotection reactions can be chosen in reference to “Greene's Protective Groups in Organic Synthesis” or the like.
The compound represented by Formula (5a) can be produced by condensing the lipoic acid and NHR2R3. Examples of solvent for condensation reaction include dichloromethane, chloroform, and tetrahydrofuran, and it is preferably tetrahydrofuran. The amount of the solvent can be 1 mL to 200 mL and is preferably 3 mL to 35 mL per 1 g of the compound represented by Formula (5a). Examples of condensing agent to be used include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and a salt thereof, N,N′-dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC) (N-hydroxysuccinimide (NHS), or 1-hydroxybenzotriazole (HOBt) may be used as an additive). The amount of the condensing agent to be used can be 0.8 equivalents to 2.0 equivalents and is preferably 1.0 equivalent to 1.5 equivalents per 1 equivalent of the compound represented by Formula (5a). The reaction temperature can be −10° C. to 40° C. and is preferably 15° C. to 25° C. The reaction time can be 1 hour to 3 days and is preferably 1 hour to 24 hours.
The compound represented by Formula (5a) can also be produced by condensing the lipoic acid trisulfide and NHR2R3. The condensation conditions are the same as above.
The compound represented by Formula (3) in which R1 is an alkyl group having 1 to 6 carbon atoms can be produced by a step of oxidizing a compound represented by Formula (3a) with an oxidant to obtain a sulfoxide compound (Step 1), and a step of causing the obtained sulfoxide compound to be reacted to a sulfur source (Step 2). The reaction conditions are the same as above.
wherein R1 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
The compound represented by Formula (3a) can be produced by condensing the lipoic acid and R1OH. The same applies as above.
The compound represented by Formula (3) in which R1 is an alkyl group having 1 to 6 carbon atoms can also be produced by condensing the lipoic acid trisulfide and R1OH. The condensation conditions are the same as above.
Cyclodextrin may be α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin or derivatives thereof. Here, the term “derivative” means that a hydrogen atom of at least one hydroxyl group that each cyclodextrin carries is substituted with an alkyl group optionally having a substituent, or sugar. For cyclodextrin derivatives, for example, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, dimethyl-α-cyclodextrin, dimethyl-β-cyclodextrin, dimethyl-γ-cyclodextrin, hydroxyethyl-α-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxyethyl-γ-cyclodextrin, 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 2-hydroxypropyl-7-cyclodextrin, glucosyl-α-cyclodextrin, glucosyl-β-cyclodextrin, glucosyl-γ-cyclodextrin, maltosyl-α-cyclodextrin, maltosyl-β-cyclodextrin, maltosyl-γ-cyclodextrin, sulfobutylether-β-cyclodextrin, or the like can be used.
The cyclodextrin clathrate can be produced by a step of dissolving cyclodextrin in a solvent (Step a), a step of adding Compound (1) or a pharmaceutically acceptable salt thereof to the resulting dissolved solution and stirring the mixed solution (Step b), and a step of filtering the stirred solution, washing the resulting filtrate with the same solvent as that used in Step a, freezing the filtrate to freeze-dry (Step c). Note that filtering and washing operations in Step c can be omitted.
The solvent to be used in Step a is preferably water.
The amount of the solvent to be used in Step a can be 1 to 350 ml and is preferably 1 to 80 ml per 1 g of the cyclodextrin.
In Step b, the mass ratio of cyclodextrin to Compound (1) or a pharmaceutically acceptable salt thereof can be 2 to 20, and is preferably 5 to 16.5.
The stirring temperature in Step b can be 20 to 50° C. and may be room temperature.
The stirring time in Step b can be 0.25 to 40 hours and is preferably 2 to 35 hours.
In Step b, after adding Compound (1) or a pharmaceutically acceptable salt thereof to the solution and before stirring the mixed solution, the same solvent as that used in Step a can be added thereto. Here, the amount of the solvent can be 0 to 30 ml and is preferably 0 to 20 ml per 1 g of the cyclodextrin.
The amount of the solvent to be used in Step c can be 0 to 150 ml and is preferably 0 to 20 ml per 1 g of the cyclodextrin.
The freezing temperature in Step c can be −30 to −20° C. and is preferably −20° C.
The freezing time in Step c can be 10 to 50 hours.
The freeze-drying in Step c can be performed at an absolute pressure of 20 to 100 Pa, and an external temperature of 10 to 40° C., preferably 20° C.
The freeze-drying time in Step c can be 1 to 5 days.
The pantethine trisulfide or a pharmaceutically acceptable salt thereof can be produced by the method descried in Patent Literature 3.
The N,N′-diacetyl-L-cysteine trisulfide or a pharmaceutically acceptable salt thereof can be produced by the method descried in Patent Literature 4.
Regarding the drug used for dopamine replacement therapy, for example, “Clinical Practice Guideline for Parkinson's Disease 2018” (Igaku-Shoin Ltd.) supervised by the Japanese Society of Neurology can be used as reference. In one embodiment, the drug used for dopamine replacement therapy include at least one selected from the group consisting of a dopamine precursor, a dopa-decarboxylase inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, a monoamine oxidase (MAOB) inhibitor, a dopamine release accelerator, and a dopamine agonist. Examples of the dopamine precursor include levodopa (L-dopa). Examples of the dopa-decarboxylase inhibitor include carbidopa and benserazide. Examples of the COMT inhibitor include entacapone. Examples of the MAOB inhibitor include safinamide, selegiline, and rasagiline. Examples of the dopamine release accelerator include amantadine. Examples of the dopamine agonist include cabergoline, bromocriptine, pergolide, talipexole, pramipexole, ropinirole, rotigotine, and apomorphine. These drugs may be in a form of a pharmaceutically acceptable salt; and these drugs or pharmaceutically acceptable salts thereof may be crystalline or amorphous body, and anhydride or solvate (in particular, hydrate).
Specific examples of the combination of two or more drugs used for dopamine replacement therapy include concurrent use of a dopamine precursor and a dopa-decarboxylase inhibitor (e.g., concurrent use of levodopa and carbidopa, or concurrent use of levodopa and benserazide), concurrent use of a dopamine precursor, a dopa-decarboxylase inhibitor and a COMT inhibitor (e.g., concurrent use of levodopa, carbidopa and entacapone), concurrent use of a dopamine precursor and a dopamine agonist (e.g., concurrent use of levodopa and talipexole, concurrent use of levodopa and cabergoline, or concurrent use of levodopa and ropinirole), and concurrent use of a dopamine precursor and a MAOB inhibitor (e.g., concurrent use of levodopa and selegiline).
In the present invention, the phrase “administration in combination” or “concurrent use” refers to using active ingredients in combination and includes: (1) a mode of administering a single formulation comprising the drug used for dopamine replacement therapy and the trisulfide compound; and (2) a mode of separately administering the drug used for dopamine replacement therapy and the trisulfide compound as individual formulations simultaneously or at time intervals. In the case of (2), the drug used for dopamine replacement therapy may be administered first, or the trisulfide compound may be administered first. Further, in the case of (2), either of the following can be performed: (i) a mode in which the drug used for dopamine replacement therapy and the trisulfide compound are separately prepared and administered simultaneously through the same administration route; (ii) a mode in which the drug used for dopamine replacement therapy and the trisulfide compound are separately prepared and administered separately at time intervals through the same administration route; (iii) a mode in which the drug used for dopamine replacement therapy and the trisulfide compound are separately prepared and administered simultaneously through different administration routes (administered from different sites on the same patient); and (iv) a mode in which the drug used for dopamine replacement therapy and the trisulfide compound are separately prepared and administered separately at time intervals through different administration routes. Note that, in the case of (i), both of the preparations may be mixed immediately before administration.
In other words, the phrase “administration in combination” or “concurrent use” in the present invention can be said to be a usage mode of administration of either one of drugs in a state where the actions and effects of the other drug express in the patient body. That is, in the present invention, it is preferable to be a mode in which the drug used for dopamine replacement therapy and the trisulfide compound be administered so as to be present in the patient body, for example, in the blood, at the same time; and it is preferable to be a mode in which within 24 hours after one drug has been administered to the patient, the other drug is administered.
It is preferable for the amount of the drug used for dopamine replacement therapy to be administered (the amount of each drug to be administered in a case of the concurrent use of two or more drugs) to be 0.1 to 50 mg per 1 kg of body weight a day (0.1 to 50 mg/kg/day), more preferable to be 0.5 to 20 mg/kg/day, and further more preferable to be 1 to 10 mg/kg/day.
It is preferable for the amount of the trisulfide compound to be administered to be 0.5 to 800 mg per 1 kg of body weight a day (0.5 to 800 mg/kg/day), more preferable to be 1 to 400 mg/kg/day, and further more preferable to be 2 to 200 mg/kg/day.
It is preferable that the amount of the drug used for dopamine replacement therapy to be administered be 0.1 to 50 mg/kg/day and the amount of the trisulfide compound to be administered be 0.5 to 800 mg/kg/day, more preferable that the amount of the drug used for dopamine replacement therapy to be administered be 0.5 to 20 mg/kg/day and the amount of the trisulfide compound to be administered be 1 to 400 mg/kg/day, and further more preferable that the amount of the drug used for dopamine replacement therapy to be administered be 1 to 10 mg/kg/day and the amount of the trisulfide compound to be administered be 2 to 200 mg/kg/day. When the amounts of the drug used for dopamine replacement therapy and the trisulfide compound to be administered fall within this range, it is considered that higher prophylactic effects on Parkinson's disease are obtained, and further, higher therapeutic effects are exhibited. In addition, effects of further reducing side effects are also expected.
The number of administration of the drug used for dopamine replacement therapy and the trisulfide compound can be 1 to 8 times a day or 1 to 4 times a day. With the number of administration like this, the drug is considered to reduce patient's burden of taking medication and to improve medication compliance. As a result, it is expected that prophylactic or therapeutic effects of the present invention and effects of reducing side effects will further improve. When the administration of the drug is discontinued, the dose of the drugs may be gradually reduced. Examples of such a way include a method in which, while the administration of one drug is suspended, the other drug is administered.
The prophylactic or therapeutic agent for Parkinson's disease of the present invention can be administered orally as tablets, capsules, powders, granules, liquids or syrups, or parenterally as nasal drops, injections, infusions, or suppositories. The drug can be formulated in these dosage form by a known formulation technique. In a case of solids, when being formulated, a pharmacologically acceptable excipient such as starch, lactose, refined white sugar, glucose, crystalline cellulose, carboxycellulose, carboxymethylcellulose, carboxyethylcellulose, calcium phosphate, magnesium stearate, or gum arabic can be blended thereto, and if necessary, a lubricant, a binder, a disintegrant, a coating agent, a colorant or the like can be blended. In addition, in a case of liquids, a stabilizer, a dissolving aid, a suspending agent, an emulsifier, buffer, a preservative or the like can be blended.
The present invention will be described in more detail using Examples described below; however, the present invention is not limited to these.
For a test, dopamine hydrochloride (DA·HCl), iron(III) nitrate 9-hydrate (Fe(NO3)3·9H2O) as Fe3+ source, hydrogen peroxide solution as oxidant, glutathione trisulfide 2-hydrate, lipoic acid trisulfide, pantethine trisulfide, or N,N′-diacetyl-L-cysteine trisulfide as trisulfide compound were used.
A method of preparing each substance is as follows.
The pre-prepared preparation solution of each substance (mentioned above) and 0.2 M phosphate buffer solution (pH=6.5) were mixed in a 50 mL or 5 mL test tube to prepare 20 mL or 2 mL reaction solution so that each substance has the following concentration in the reaction solution.
The reaction solution was stirred at 25° C., 500 rpm for 6 to 9 hours. The dopamine concentration in the reaction solution was quantified with high performance liquid chromatography (HPLC).
The conditions for HPLC is as follows.
As shown in
Glutathione trisulfide suppressed the dopamine oxidation in the presence of Fe3+. Likewise, as shown in
Since glutathione trisulfide, lipoic acid trisulfide, pantethine trisulfide and N,N′-diacetyl-L-cysteine trisulfide were effectively able to suppress dopamine oxidation reaction accelerated in the presence of Fe3+, it is expected that the concurrent use of the drug with the conventional dopamine replacement therapy may effectively address the problems of conventional dopamine replacement therapy, the problems in which symptoms are not improved (wearing off), the effects of the drug suddenly run out (on-off phenomenon), side effects of the drug occur (dyskinesia, etc.) and the like.
24.38 g (118.17 mmol) of (R)-α-lipoic acid and 488 mL (20.0 v/w) of a 75% ethanol aqueous solution were added to a 200 mL four-neck flask. After confirming that the contents of the flask had dissolved, the internal temperature was cooled to 0° C. Oxone (registered trademark) (41.40 g, 124.20 mmol, 1.05 equivalents) was added thereto in two portions to cause a reaction for about 50 minutes. An insoluble substance in the reaction solution was removed by filtration and then washed with 65 mL (2.67 v/w) of ethanol. 400 mL (206.93 mmol, 1.75 equivalents) of a Na2S aqueous solution (70.70 g of Na2S·9H2O dissolved in 569 mL of water) was added dropwise to the filtrate and wash liquid at an internal temperature of 2° C. to 6° C. over about 2.5 hours (during the dropwise addition and the reaction, a 3 mol/L sulfuric acid aqueous solution was used to control the pH at 6 to 7, and the total amount thereof used was 14 mL). After causing a reaction at an internal temperature of 3° C. at a pH 7 for about 50 minutes, 41 mL (1.7 v/w) of a 3 mol/L sulfuric acid aqueous solution was added dropwise thereto to adjust the pH to 1.3. Next, 320 mL (13.1 v/w) of water and 320 mL (13.1 v/w) of ethyl acetate were added thereto to perform extraction with ethyl acetate. The aqueous layer was extracted four times with 160 mL (6.6 v/w) of ethyl acetate, and organic layers were combined and concentrated under reduced pressure at an external temperature of 30° C. Ethanol was added to the concentrate to dissolve it, followed by column purification with ODS. The fraction was concentrated under reduced pressure at an external temperature of 30° C. and then dried with an oil pump to obtain 10.69 g of (R)-lipoic acid trisulfide (44.84 mmol, yield 38%, HPLC purity of 99.7%, white solid).
2.00 g (8.39 mmol) of (R)-lipoic acid trisulfide and 65 mL (32.5 v/w) of methylene chloride were added to a 200 mL four-neck flask. After confirming that the contents of the flask had dissolved, 2.07 g (10.77 mmol, 1.28 equivalents) of 1-(3-diinethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl) and 1.42 g (12.33 mmol, 1.47 equivalents) of N-hydroxysuccinimide (NHS) were added thereto. After replacing the air in the flask with nitrogen, a reaction was carried out at room temperature for about 8 hours. Next, 2.28 mL (33.74 mmol, 4.02 equivalents) of 28% aqueous ammonia was added dropwise at room temperature over 5 minutes and allowed to react overnight. Thereafter, at room temperature, 60 mL of water (30.0 v/w) was added thereto and the mixture was subjected to liquid separation. Then, the organic layer was washed three times with 60 mL (30.0 v/w) of a 2.5% sodium hydrogen carbonate aqueous solution and additionally washed four times with 60 mL of water (30.0 v/w). Thereafter, the organic layer after washing was concentrated under reduced pressure at an external temperature of 25° C., and then dried with an oil pump to obtain 1.93 g of (R)-lipoamide trisulfide (8.13 mmol, yield 97%, HPLC purity of 99.6%, white solid).
1H-NMR: (CDCl3, 400 MHz) δ (ppm)=5.36 (bs, 2H), 3.33 (m, 1H), 3.13 (m, 2H), 2.22 (m, 3H), 1.89 (m, 1H), 1.74-1.42 (m, 6H).
HR-ESI-TOF-MS: m/z 236.0238 ([M-H]−), calcd for [C8H14NOS3]−236.0243.
1.00 g (4.87 mmol) of lipoamide (racemate) and 182 mL (182.0 v/w) of an 85% dimethylformamide aqueous solution were added to a 500 mL four-neck flask. After confirming that the contents of the flask had dissolved, the internal temperature was cooled to 4° C. 1.63 g (4.89 mmol, 1.00 equivalent) of Oxone (registered trademark) was added to the flask in three portions every 10 minutes to cause a reaction for about 1 hour. 1.24 g (5.16 mmol, 1.06 equivalents) of sodium sulfide nonahydrate was added in portions at an internal temperature of 5° C. while controlling the pH of the reaction solution at 5 to 11 using a 3 mol/L sulfuric acid aqueous solution to cause a reaction for about 1.5 hours. 180 mL (180.0 v/w) of water and 50 mL (50.0 v/w) of methylene chloride were added thereto to perform extraction with methylene chloride. Then, the aqueous layer was extracted twice with 50 mL (50.0 v/w) of methylene chloride, and organic layers were combined and concentrated under reduced pressure at an external temperature of 30° C. or lower. 80 mL (80.0 v/w) of water was added dropwise to the concentrated residue over 30 minutes at room temperature for crystallization, and the slurry was filtered and then washed with 50 mL (50.0 v/w) of water. Wet crystals were dried under reduced pressure at 25° C. to obtain 510 mg of lipoamide trisulfide (racemate) (2.15 mmol, yield 44%, HPLC purity of 92%, white solid).
Hereinafter, “HP”, “Me”, and “Mal” are respectively abbreviations for “hydroxypropyl”, “methyl”, and “maltosyl.”
2.0 g (9.02 mmol) of lipoic acid and 40 mL of a 75% ethanol aqueous solution were added to a reaction container, and the mixture was cooled to an internal temperature of 0° C. 3.4 g (10.20 mmol) of Oxone (registered trademark) was added thereto to cause a reaction for about 2 hours. Inorganic salts in the reaction solution were filtered and then washed in 7 mL of ethanol. 5.8 g (24.1 mmol) of sodium sulfide nonahydrate was added to the filtrate to cause a reaction for about 1 hour. After 7 mL of a 3 mol/L sulfuric acid aqueous solution was added dropwise to this reaction solution, 20 mL of water and 45 mL of ethyl acetate (AcOEt) were subsequently added thereto to perform extraction with AcOEt. The aqueous layer was extracted twice with 20 mL of AcOEt, and organic layers were combined and concentrated under reduced pressure. After 3 mL of ethanol was added to the concentrate to dissolve it, the solution was purified with an ODS column (YMC Dispo PackAT, mobile phase: acetonitrile aqueous solution) to obtain 0.7 g (2.39 mmol, HPLC purity: 100%) of lipoic acid trisulfide.
1,020.0 mg (0.899 mmol) of β-CD and 80 mL of water were added to a 100 mL eggplant flask. After confirming that the contents of the flask had dissolved, 99.8 mg (0.419 mmol) of lipoic acid trisulfide was added thereto, and the inside of the flask was washed with 20 mL of water. The washed mixture was stirred at 45° C. for 15 minutes and then filtered, and the inside of the flask and the crystals were washed with 10 mL of water. The obtained filtrate was frozen in a −20° C. freezer for 23 hours. The frozen filtrate was freeze-dried at an external temperature of 20° C. for about 4.5 days to obtain 980.0 mg of a clathrate (white solid).
1291.0 mg of HP-β-CD and 16 mL of water were added to a 50 mL eggplant flask. After confirming that the contents of the flask had dissolved, 100.0 mg (0.419 mmol) of lipoic acid trisulfide was added thereto. The mixture was stirred at room temperature for about 28 hours and then filtered, and the inside of the flask and the crystals were washed with 10 mL of water. The obtained filtrate was frozen in a −20° C. freezer for about 2 days. The frozen filtrate was freeze-dried at an external temperature of 20° C. for about 2 days to obtain 1330.0 mg of a clathrate (white solid).
969.9 mg of HP-β-CD and 10 mL of water were added to a 50 mL eggplant flask. After confirming that the contents of the flask had dissolved, 100.3 mg (0.421 mmol) of (R)-lipoic acid trisulfide was added thereto, and the inside of the flask was washed with 4 mL of water. The washed mixture was stirred at room temperature for about 25 hours and then filtered, and the inside of the flask and the crystals were washed with 12 mL of water. The obtained filtrate was frozen in a −20° C. freezer for 15 hours. The frozen filtrate was freeze-dried at an external temperature of 20° C. for about 2 days to obtain 1040.0 mg of a clathrate (white solid).
1,616.0 mg of Me-β-CD (mixture of several methylated) and 12 mL of water were added to a 50 mL eggplant flask. After confirming that the contents of the flask had dissolved, 101.0 mg (0.424 mmol) of lipoic acid trisulfide was added thereto, and the inside of the flask was washed with 4 mL of water. The washed mixture was stirred for 21 hours and then filtered, and the inside of the flask and the crystals were washed with 12 mL of water. The obtained filtrate was frozen in a −20° C. freezer for 20 hours. The frozen filtrate was freeze-dried at an external temperature of 20° C. for about 4 days to obtain 1665.2 mg of a clathrate (white solid).
1,616.0 mg of Me-β-CD (mixture of several methylated) and 16 mL of water were added to a 50 mL eggplant flask. After confirming that the contents of the flask had dissolved, 99.9 mg (0.420 mmol) of (R)-lipoic acid trisulfide was added thereto, and the inside of the flask was washed with 4 mL of water. The washed mixture was stirred at room temperature for about 6 hours and then filtered, and the inside of the flask and the crystals were washed with 13 mL of water. The obtained filtrate was frozen in a −20° C. freezer for 28 hours. The frozen filtrate was freeze-dried at an external temperature of 20° C. for about 3 days to obtain 1610.9 mg of a clathrate (white solid).
1,224.2 mg (0.839 mmol) of Mal-β-CD and 14 mL of water were added to a 50 mL eggplant flask. After confirming that the contents of the flask had dissolved, 100.4 mg (0.421 mmol) of lipoic acid trisulfide was added thereto, and the inside of the flask was washed with 2 mL of water. The washed mixture was stirred at room temperature for about 31 hours and then filtered, and the inside of the flask and the crystals were washed with 10 mL of water. The obtained filtrate was frozen in a −20° C. freezer for 22 hours. The frozen filtrate was freeze-dried at an external temperature of 20° C. for about 46 hours to obtain 1180.0 mg of a clathrate (white solid).
1,224.2 mg (0.839 mmol) of Mal-β-CD and 10 mL of water were added to a 50 mL eggplant flask. After confirming that the contents of the flask had dissolved, 100.1 mg (0.420 mmol) of (R)-lipoic acid trisulfide was added thereto, and the inside of the flask was washed with 5 mL of water. The washed mixture was stirred at room temperature for about 4.5 hours and then filtered, and the inside of the flask and the crystals were washed with 11 mL of water. The obtained filtrate was frozen in a −20° C. freezer for 24 hours. The frozen filtrate was freeze-dried at an external temperature of 20° C. for about 41 hours to obtain 1319.6 mg of a clathrate (white solid).
The yield and solubility of the clathrates obtained in Reference Examples 3 to 9 are shown in Table 2.
1)represents solubility in water at 20° C.
2)Percent yield (%) = (yield × content)/theoretical yield × 100
3)represents solubility of lipoic acid trisulfide in a clathrate in water at 20° C. A statement such as “≥49 g/L” indicates dissolution at 49 g/L.
After charging 18.75 g (27.04 mmol, 15.00 g as pantethine) of a 80% pantethine aqueous solution and 180 ml (12.0 v/w) of water to a 1 L four-neck flask, the internal temperature was cooled to 1° C. 10.34 g of Oxone (registered trademark) (31.02 mmol, 1.15 equivalents, calculated as available oxygen of 4.8%) was added thereto in 4 portions every 10 minutes, and then the flask was washed with 8 mL of water and a reaction was carried out for about 3 hours. 45 mL of a 0.67 mol/L sodium sulfide aqueous solution (29.98 mmol, 1.11 equivalents) was added dropwise thereto over 45 minutes at an internal temperature of 0° C. to 1° C., and the pH was controlled at 7 or lower during dropwise addition using 1.6 mL of a 3 mol/L sulfuric acid aqueous solution. After the mixture was allowed to react at an internal temperature of 1° C. and at pH 4 for 40 minutes, 500 mL (33.3 v/w) of ethanol was added thereto at an internal temperature of 1° C. to 5° C. to precipitate inorganic salts, and then the resultant was stirred at an internal temperature of 1° C. to 5° C. for 30 minutes. The inorganic salts were filtered and then washed with 50 mL (3.3 v/w) of ethanol, and the filtrate and wash liquid were concentrated under reduced pressure at an external temperature of 23° C. to obtain 34 g of a crude body of pantethine trisulfide. Then, the crude body was dissolved by charging 6 mL of water thereto, and 40 g (2.7 w/w) of a stock solution for column purification was prepared. Purification was performed using an ODS column, and fractions with an LC purity of 95% or higher were collected. The fractions were concentrated under reduced pressure at an external temperature of 30° C. and then dried with an oil pump to obtain 9.57 g of pantethine trisulfide (16.31 mmol, yield 60%, white solid).
1H NMR: (D20, 400 MHz) δ (ppm)=3.97 (s, 2H), 3.44-3.58 (m, 10H), 3.37 (d, J=11.4 Hz, 2H), 3.04 (t, J=6.2 Hz, 4H), 2.50 (t, J=6.2 Hz, 4H), 0.91 (s, 6H), 0.87 (s, 6H).
HR-ESF-TOF-MS: m/z 585.2086 ([M-H]−), calcd for [C22H41N4O8S3]−585.2092.
Pantethine trisulfide (10% by mass), benzalkonium (0.01% by mass), carboxyvinyl polymer (0.5% by mass), L-arginine (1% by mass), and physiological saline (88.49% by mass) were mixed and stirred in a vacuum stirrer under shade, filtered and sterilized under sterile conditions, and aseptically filled in a sterilized container to produce a nasal drop formulation comprising pantethine trisulfide.
1.0 g (3.08 mmol) of N,N′-diacetyl-L-cystine and 10 mL of water were added to a reaction container, and the mixture was cooled to an internal temperature of 1° C. 1.25 g (3.72 mmol) of Oxone (registered trademark) was added thereto to cause a reaction for about 3 hours. Subsequently, 8.5 mL (3.71 mmol) of a 0.44 mol/L sodium sulfide aqueous solution was added dropwise thereto to cause a reaction for about 3 hours. After adding 33 mL of acetonitrile to the reaction mixture, inorganic salts were filtered and washed with 5 mL of acetonitrile. This filtrate was concentrated under reduced pressure with an evaporator, and the concentrate was purified with an ODS column (mobile phase: acetonitrile aqueous solution) to give 0.2 g (0.56 mmol) of N,N′-diacetyl-L-cysteine trisulfide.
The HPLC conditions are as follows.
1.0 g (6.13 mmol) of N-acetyl-L-cystine and 40 mL of a 20% acetonitrile aqueous solution were added to a reaction container, and the mixture was cooled to an internal temperature of 5° C. 3.4 g (10.14 mmol) of Oxone (registered trademark) was added thereto to cause a reaction for about 2.5 hours. Subsequently, 1.5 g (6.12 mmol) of sodium sulfide nonahydrate was added thereto to cause a reaction for about 1 hour. After adding 33 mL of acetonitrile thereto, inorganic salts were filtered and washed with 3 mL of acetonitrile. This filtrate was concentrated under reduced pressure with an evaporator, and the concentrate was purified with an ODS column (mobile phase: acetonitrile aqueous solution) to give 0.1 g (0.28 mmol) of N,N′-diacetyl-L-cysteine trisulfide.
HPLC conditions are the same as those described in Reference Example 12.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-113378 | Jul 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/026755 | 7/5/2022 | WO |
