WO2008048053A1

WO2008048053A1 - Pharmaceutical composition for prevention and treatment of parkinson's disease containing 1,2-di[2-methoxy-4-(2-carboxylvinyl)]phenoxyethane as an active ingredient

Info

Publication number: WO2008048053A1
Application number: PCT/KR2007/005102
Authority: WO
Inventors: Kye Jung Shin; Dong Jin Kim; Onyou Hwang
Original assignee: Korea Institute Of Science And Technology
Priority date: 2006-10-19
Filing date: 2007-10-18
Publication date: 2008-04-24
Also published as: KR20080035294A

Abstract

Having protective activity on dopaminergic neurons against toxic substances including l-methyl-4-phenylpyridinum (MPP+), tetrahydrobiopterin (BH4), hydrogen peroxide (H2O2) and l-methyl-4-phenyl-l, 2,4, β-tetrahyropyridine, 1, 2-di [2-methoxy- 4- (2-carboxylvinyl) ] phenoxyethane having the following chemical structure can be useful in the prevention and treatment of Parkinson's disease.

Description

[DESCRIPTION]

[invention Title]

PHARMACEUTICAL COMPOSITION FOR PREVENTION AND TREATMENT OF PARKINSON'S DISEASE CONTAINING 1,2-DI [2-METHOXΪ-4- (2- CARBOXYLVINYL) ] PHENOXYETHANE AS AN ACTIVE INGREDIENT [Technical Field]

The present invention relates to a pharmaceutical composition for the prevention and treatment of Parkinson' s disease, containing 1, 2-di [2-methoxy-4- (2- carboxylvinyl) ] phenoxyethane as an active ingredient.

[Background Art]

Parkinson' s disease is a degenerative disorder of the central nervous system, which is characterized by muscle rigidity, tremor, and a slowing of physical movement. This disease affects one of every 100 people after 65 years. Degenerative disorder as used herein is intended to refer to the cell death of neurons with age, like the symptom of senility. In 1817, Parkinson's disease was first reported by James Parkinson, a British physician, after whom the disease was named. However, Parkinson's disease has long remained unknown about the etiology thereof. It is at the time that 150 years had passed since the first report of Parkison' s disease that the etiology of Parkinson's disease was discovered so as to give a turning point for the treatment thereof. The patients suffering from Parkinson's disease in Korea approximates hundred thousands with a doubling increase in the number thereof for the last decade.

Part of the disease process develops as cells are destroyed in a degenerative manner in certain parts of the brain stem, particularly the crescent-shaped cell mass known as the substantia nigra. It is known that Parkinson's disease occurs when approximately 80% of the dopaminergic neurons, responsible for the production of the neurotransmitter dopamine, in the substantia nigra have been lost. Various suggestions have been raised for the pathology of Parkinson' s disease, including that the disease is associated with senility, or environmental factors, such as drinking water, a place of residence, etc. However, the exact pathology of Parkinson's disease has to be revealed. It is reasonable to accept that various factors account in combination for the onset of the disease. Sporadic Parkinson's disease for the onset of which a combination of environmental factors is responsible constitutes most (90% or greater) of the disorder, while the remaining portion of the cases is of familial origin, with which gene mutation is associated.

Oxidative stress is regarded as a main factor responsible for the onset of general Parkinson's diseases except for familial Parkinson's disease. Nerve cells in the central nervous system are at great risk of being damaged by oxygen radicals. Particularly, dopaminergic neurons in the substantia nigra are very likely to be exposed to oxidative stress because cytotoxic radicals are produced in situ during the oxidative metabolism of dopamine. In addition, when increased in in-vivo level due to, for example, a decreased level of an antioxidant glutathione (GSH) , hydrogen peroxide

(H₂O₂) reacts with iron ions (Fe²⁺) to hydroxide ions (0H^~), which mediate the oxidation-induced damage of neurons, resulting in promoting the death of neurons .

Recent study has disclosed that the activation of microglial cells around dopaminergic neurons is closely associated with the onset and progression of Parkinson' s disease (U. Wullner; T. Klockgether, J. Neurol 250, 35-38, 2003) . The exact mechanism of dopaminergic neuron cell death in the human substantia nigra has to be proven. In this regard, animal models using the neurotoxin l-methyl-4-phenyl- 1,2, 4, 6-tetrahydropyridine (hereinafter, referred to "MPTP") have been developed for study into the mechanism of dopaminergic neurons in the substantia nigra.

Primarily aiming to reducing the symptoms of Parkinson' s disease, various studies and clinical trials have been developed on the basis of the research results described above. For instance, levodopa therapy, which is one of the most widely used therapies for Parkinson's disease, is applied to the recovery of the damaged motor function. When administered, levodopa is believed to activate the dopamine production of the dopaminergic neurons remaining alive. Like this, various pharmaceutical therapies have been studied to replenish the insufficient dopamine level attributed to the cell death of dopaminergic neurons, to inhibit the metabolism of dopamine, or to develop a dopamine-like compound (Johnston, Tom H; Brotchie, Jonathan M., Current Opinion in Investigational Drugs 25-32, 2005) .

In the same context, gene therapies for preventing the cell death of dopaminergic neurons, in which genes coding for tyrosine hydroxylase (hereinafter referred to as "TH") responsible for catalyzing the synthesis of dopamine or coding for neurotrophic factors (GDNF, BDNF) known to help the survival of neurons are introduced into cells (fibroblast cells, muscle cells, adrenal medulla cells, PC12 cells, etc.) or are administered directly using a viral vector such as adenovirus, adeno-associate virus, herpes virus, sindbis virus and the like, have been tried on animal models (Beverly L. Davidson and Xandra O. Breakefield, Nature Neuroscience 3, 353-364, 2003) .

Leading to the present invention, intensive and thorough research into the prevention and treatment of Parkinson' s disease, conducted by the present inventors, resulted in the finding that 1, 2-di [2-methoxy-4- (2- carboxylvinyl) ]phenoxyethane, one of the cinnamic acid dimmers which are patented for use in the treatment of dementia as disclosed in Korean Patent No. 0479405 issued to the present inventors, is highly protective of the dopaminergic neurons from the toxic substances including l-methyl-4-phenylpyridinium (hereinafter referred to as "MPP⁺"), tetrahydrobiopterin (hereinafter referred to as "BH₄") and H₂O₂.

[Disclosure] [Technical Problem]

It is an object of the present invention to provide a pharmaceutical composition useful in the prevention and treatment of Parkinson's disease.

[Technical Solution]

In order to accomplish the above object, there is provided a pharmaceutical composition containing l,2-di[2- methoxy-4- (2-carboxylvinyl) jphenoxyethane, represented by the following Chemical Formula 1, or a pharmaceutically acceptable salt thereof as an active ingredient for the prevention and treatment of Parkinson's disease. [Chemical Formula l]

[Advantageous Effects]

1,2-Di [2-methoxy-4- (2-carboxylvinyl) ] phenoxyethane has protective activity on dopaminergic neurons against toxic substances including l-methyl-4-phenylpyridinum (MPP⁺) , tetrahydrobiopterin (BH₄), hydrogen peroxide (H₂O₂) and 1- methyl-4-phenyl-1, 2, 4, 6-tetrahyropyridine, and thus can be useful in the prevention and treatment of Parkinson's disease.

[Description of Drawings] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a graph showing the protective effects of the compound of Chemical Formula 1 according to the present invention on dopaminergic neurons against toxic substances BH₄, H₂O₂ and MPP⁺ (*: p < 0.05);

FIG. 2 is a graph showing the protective effects of the compound of Chemical Formula 1 according to the present invention at various doses on dopaminergic neurons against BH₄ (*: p < 0.05);

FIG. 3 shows the protective effects of the compound of

Chemical Formula 1 according to the present invention on dopaminergic neurons against MPTP-induced cell death in mice in photographs (left panel) taken from live dopaminergic neurons after the iπimunostaining of tyrosin hydroxylase (TH) , a marker protein of dopaminergic neurons, and in a graph (right panel) in which the counts of the live cells are plotted (**: p < 0.01); and FIG. 4 shows the protective effects of the compound of Chemical Formula 1 according to the present invention on dopaminergic neurons against MPTP-induced cell death in mice as a result of staining with Fluoro-Jade C. [Best Mode] The compound of Chemical Formula 1 according to the present invention is derived from the compound, represented by the following Chemical Formula Ia, disclosed in Korean Patent No. 0479405, wherein R is hydrogen or an alkyl group of Ci ~ C₅; X is oxygen, -NH or -NCH₃-; Y is -OCH₃, -NHCH₃ or -N (CH₃) ₂; spacer is a carbon chain C₂ ~ Ce in length which may or may not contain one or more oxygen or nitrogen atoms; X and Y are in the ortho, meta or para position of the benzene ring of cinnamic acid.

[Chemical Formula Ia]

As can be seen in Chemical Formula Ia, the cinnamic acid dimers have a structure wherein two cinnamic acid monomers, each having an alkoxy group or alkyl amine group at the ortho, meta or para position of benzene ring, are connected via a carbon chain C₂ ~ C₈ in length which may or may not contain one or more oxygen or nitrogen atoms.

The compound of the present invention, 1, 2-di [2-methoxy- 4- (2-carboxylvinyl) ]phenoxyethane, corresponds to the compound of Chemical Formula Ia wherein X is an oxygen atom, spacer is ethyl and Y is a methoxy group located at the ortho position of the benzene ring. As mentioned above, 1, 2-di [2-methoxy-4- (2- carboxylvinyl) ] phenoxyethane, obtainable as a pale yellow solid, is useful as a therapeutic agent for dementia.

The compound of Chemical Formula 1 according to the present invention can be prepared as illustrated in the following Reaction Scheme 1. [Reaction Scheme l]

Maloπic acid

With reference to Reaction Scheme 1, a detailed description is given of the preparation of the compound according to the present invention.

First, two molecules of hydroxybenzaldehyde of Chemical Formula 2 are linked to each other by reaction with ethylene glycol ditosylate of Chemical Formula 3 in the presence of a base.

Then, the benzaldehyde dimmer of Chemical Formula 4 thus formed is reacted with malonic acid to give the object compound of Chemical Formula 1. This reaction is widely known as "Knoevenagel reaction" in the field of organic chemistry, and the reaction condition (solvents, reaction temperature and reaction time and so forth) may be suitably determined depending on reactants and products. For example, an alkaline organic solvent, such as piperidine, pyridine, lutidine, dimethylformamide and so on, may be employed. As for the reaction temperature, it is usually in the range from 80 to 100⁰C while the reaction time may be set within a period from 2 to 6 hours. It is preferred that the reaction be conducted at 80-90⁰C for 3-5 hours in the presence of piperidine and pyridine. By this method, the compound of Chemical Formula 1 according to the present invention can be produced at a yield of 98% or higher.

The compound of Chemical Formula 1 according to the present invention, 1, 2-di [2-methoxy-4- (2- carboxylvinyl) jphenoxyethane, can be applied to the treatment of Parkinson's diseases as follows.

In accordance with the present invention, 1, 2-di [2- methoxy-4- (2-carboxylvinyl) ] phenoxyethane or a pharmaceutically acceptable salt thereof may be used as an active ingredient of a pharmaceutical composition useful in the prevention and treatment of Parkinson's disease.

Parkinson's disease, as mentioned above, is a degenerative disorder caused by the progressive lack of dopaminergic neurons in the substantia nigra. Although the exact pathology of Parkinson's disease remains unknown to date, it is reported that the blockage of MPP⁺, BH₄, H₂O₂, and MPTP, known to be toxic to the dopaminergic neurons of the brain, leads to the protection of the dopaminergic neurons, thus resulting in the prevention and treatment of Parkinson' s disease (Miquel Vila; Serge Przedborski, Nature Neuroscience 3, 1-11, 2003) .

1,2-Di [2-methoxy-4- (2-carboxylvinyl) ] phenoxyethane was assayed for the protection of dopaminergic neurons from the toxic substances MPP⁺, BH₄ and H₂O₂. The compound of Chemical Formula 1 according to the present invention was found to almost completely suppress toxic substance-induced cell death when it was administered in an amount of 1 μM to dopaminergic neurons (see Table 1 and FIG. 1) .

Also, the compound of Chemical Formula 1 according to the present invention was examined as to the minimal concentration thereof required to suppress the toxic substance- induced cell death of dopaminergic neurons. Even as small as 100 nM of the compound was found to almost completely suppress the toxic substance-induced cell death of dopaminergic neurons. Thus, the compound of the present invention shows excellent neuroprotective effects on dompaniergic neurons even at a dosage of approximately 100 run (see Table 2 and FIG. 2) .

When administered to animal models, MPTP induced

Parkinson's disease therein to leave only 58% of the dopaminergic neurons . The administration of the compound of Chemical Formula 1 was found to recover 80% of the dopaminergic neurons (see Table 3 and FIG. 3) .

As described above, the compound of Chemical Formula 1 according to the present invention can be used for the prevention and treatment of Parkinson' s disease as it almost completely protects dopaminergic neurons from toxic substances even at a low dosage (100 nM or lower) .

The pharmaceutical composition comprising the compound of Chemical Formula 1 according to the present invention may be provided in general medicine forms . The compound of Chemical Formula 1 in accordance with the present invention can be administered in various oral or non-oral dosage forms. Preferable are oral dosage forms. The compound is usually formulated in combination with a diluent or excipient, such as a filler, a thickening agent, a binder, a wetting agent, a disintegrant, a surfactant, etc.

Solid agents intended for oral administration of the compound of the present invention may be in the form of tablets, pills, powders, granules, capsules, and the like. These solid agents are formulated in combination with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatine. Besides, a lubricant, such as magnesium stearate, talc and the like, may also be added.

Liquid agents intended for oral administration include suspensions, internal use solutions, emulsion, syrups, and the like. In addition to a simple diluent such as water or liquid paraffin, various excipients, such as wetting agents, sweetening agents, aromatics, preservatives, and the like may be contained in the liquid agents for the oral administration of the compound of the present invention. Also, non-oral dosage forms of the compound of the present invention include injections, emulsions, inhalations, and suppositories. For injections, non-aqueous solvents and suspensions made from propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and esters such as ethyl oleate may be used.

The pharmaceutical composition comprising the compound of Chemical Formula 1 according to the present invention may be administered in a single dose or in several doses per day, each dose ranging from 1 ~ 250 mg/kg of the active ingredient, depending on various factors including the patient' s age, weight, gender, state of health, diet, administration route, number of administration, excretion rate, severity of illness, and the like. [Mode for Invention]

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

EXAMPLE 1: Assay of Compound of Chemical Formula 1 for Protection of Dopaminergic Neurons from Toxic Substance

In order to examine the neuroprotective effect of 1,2- di [2-methoxy-4- (2-carboxylvinyl) ] phenoxyethane on dopaminergic CATH. a cells, an assay was conduced as follows. BH₄, H₂O₂ and MPP⁺, known to be toxic to dopaminergic cells, were administered in doses of 200 μM, 50 nM and 1 itiM, respectively, to the dopaminergic cells, after which the cells were measured for the degree of cell death using an LDH (lactate dehydrogenase) release assay.

In addition, dopaminergic cells were co-administered with 1 μM of 1, 2-di [2-methoxy-4- (2-carboxylvinyl) ] phenoxyethane and the same concentration of BH₄, H₂O₂ or MPP⁺ and analyzed for cell death using an LDH release assay so as to determine the neuroprotective effect of the compound of Chemical Formula 1. The results, expressed as percentages of the control, are given in Table 1 and FIG. 1.

TABLE 1

When administered, as is apparent from the data of Table 1 and FIG. 1, 1 μM of 1, 2-di [2-methoxy-4- (2- carboxylvinyl) ] phenoxyethane was found to completely suppress the toxic substance-induced cell death. EXAMPLE 2: Protective Effect of Compound of Chemical Formula 1 on Dopaminergic Cells by Doses

The minimum concentration of 1,2-di [2-raethoxy-4- (2- carboxylvinyl) ] phenoxyethane required to protect dopaminergic cells from BH₄ was determined as follows.

To dopaminergic cells treated with 200μM of BH₄, the compound of Chemical Formula 1 according to the present invention was administered in doses of 0, 0.1, 0.5, 1, 5 and 25 μM. The protective effects of the compound on the dopaminergic cells were determined as the degree of cell death was measured using an LDH release assay. The results, expressed as percentage of control, are given in Table 2 and FIG. 2.

TABLE 2

Doses of Cpd. of Formula KμM) LDH Activity (% of control) *

Control (no BH₄) 100 + 8

0 177 + 30

0.1 92 + 4

0.5 109 + 5

93 ± 14

¹

5 73 ± 11

25 75 + 25

LDH activity of the dopaminergic cells which were treated with BH₄, but not with the compound of Chemical Formula 1 was as twice as that ofthe control which was not administered with BH₄. However, the groups treated with 0.5 μM or higher of compound of Chemical Formula 1 were measured to show LDH activity similar to or less than that of the control. Particularly, the compound of Chemical Formula 1 according to the present invention was found to have potent protective activity on dopaminergic neurons even in a dose of 100 nM or less .

EXAMPLE 3: Assay of Compound of Chemical Formula 1 for Neuroprotective Effect on Dopaminergic Neurons in MPTP-Induced Parkinson' s Disease Mice

The compound of Chemical Formula 1 in accordance with the present invention was assayed for neuroprotective activity on dopaminergic neurons in the substantia nigra of MPTP-induced Parkinson's disease mice, as follows.

C57B1/6 male mice weighing 23-25 g were divided into groups of 8. The compound of Chemical Formula 1 in accordance with the present invention was orally administered to the mice in a dose of 30 mg/kg at regular intervals of 24 hours for three days, with four intraperitoneal injections of MPTP in a dose of 20 mg/kg at regular intervals of 2 hours. The first injection of MPTP followed 30 min after the first administration of the compound of Chemical Formula 1. The mice were sacrificed 7 days after the first injection of MPTP, followed by histochemical analysis.

Tyrosine hydroxylase (TH) , a marker protein of dopaminergic neurons, was immunostained to visualize and count dopaminergic neurons. Fluoro-Jade staining was performed to observe the protective effect of the compound of Chemical Formula 1 on the dopaminergic neurons of the substantia nigra against MPTP

The results are given in Table 3 and FIGS. 3 and 4.

The data of Table 3 and FIG. 3 show that the dopaminergic neurons of the substantia nigra survived approximately 58% upon the administration of MPTP, but approximately 80% upon the administration of the compound of Chemical Formula 1 according to the present invention.

When treated with MPTP, the dopaminergic neurons were stained with Floro-Jade C, which stains specifically dying cells, to high extent (predominantly green) while co^¬ administration with the compound of Chemical Formula 1 significantly decreased the Fluoro-Jade staining (predominantly red) .

Taken together, the data obtained in Examples demonstrate that the compound of Chemical Formula 1 in accordance with the present invention effectively suppresses the MPTP-induced cell death of dopaminergic neurons in vivo, as proven in animal models, leading to the treatment of Parkinson's disease.

The composition of the present invention can be formulated as follows.

FOPMUIATION EXAMPLE: Preparation of Oral Dosage Forms

1. Preparation of Powder

Compound of Chemical Formula 1 2g Lactose Ig

The above ingredients were mixed and loaded into an airtight sac to produce powder.

2. Preparation of Tablet Compound of Chemical Formula 1 lOOmg Corn Starch lOOmg

Lactose lOOmg

Mg Stearate 2mg

These ingredients were mixed and prepared into tablets using a typical tabletting method.

3. Preparation of Capsule

Compound of Chemical Formula 1 lOOmg

Corn Starch lOOmg Lactose lOOmg

Mg Stearate 2mg

These ingredients were mixed and loaded into gelatin capsules according to a typical method to produce capsules .

4. Preparation of Injection

Compound of Chemical Formula 1 10 μg/ml Diluted HCl BP added to form pH 3.5

NaCl BP injection up to 1 ml

The compound of Chemical Formula 1 was dissolved in a suitable volume of an NaCl BP injection and the solution was adjusted to pH 3.5 with diluted HCl BP and to a desired volume with NaCl BP injection, followed by sufficiently mixing. The solution was loaded into transparent 5 ml type I ampules which were hermetically sealed by melting, followed by autoclaving at 120⁰C for 5 min to prepare injections. [industrial Applicability]

Having protective activity on dopaminergic neurons against toxic substances including l-methyl-4-phenylpyridinum (MPP⁺) , tetrahydrobiopterin (BH₄) , hydrogen peroxide (H₂O₂) and l-methyl-4-phenyl-l,2, 4, 6-tetrahyropyridine, as described hitherto, the compound of Chemical Formula 1 in accordance with the present invention can be useful in the prevention and treatment of Parkinson's disease.

Although the preferred embodiment (s ) of the present invention have (has) been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims .

Claims

[CLAIMS]

[Claim l]

A pharmaceutical composition for the prevention and treatment of Parkinson' s disease, comprising as an active ingredient 1,2-di [2-methoxy-4- (2-carboxylvinyl) Jphenoxyethane, represented by the following Chemical Formula 1, or a pharmaceutically acceptable salt thereof.