JPH0692302B2 - Neurological agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a neurological disease caused by a deficiency of tetrahydrobiopterin (hereinafter referred to as BH ₄ ) containing 6-hydroxymethyl-tetrahydropterin as an active ingredient. Regarding therapeutic agents.

[Problems to be Solved by Prior Art and Invention]

Examples of neurological diseases caused by BH ₄ deficiency referred to in the present specification include, specifically, Parkinson's disease, phenylketonuria,
Examples include intrinsic depression and childhood autism. Parkinson's disease is a neuropathy that occurs frequently in old age with movement disorder as the main symptom, and its cause is dopamine deficiency in the striatum of the brain associated with decreased degeneration of dopaminergic neurons in the substantia nigra-striatum It is said that there is. The deficiency of dopamine is believed to be due to the reduced activity of tyrosine hydroxylase for its precursor L-dopa biosynthesis and its coenzyme tetrahydrobiopterin. Phenylketonuria is also found in liver and brain neurons.
This is because BH ₄ cannot be biosynthesized, resulting in a lack of strength. Furthermore, BH ₄ is expected to have a therapeutic effect on autism in children with metabolic disorders.

Based on the above findings, L- was used to treat patients with Parkinson's disease.
Administration of dopa has been performed for the past 10 years, but it is known that administration of BH ₄ has a therapeutic effect recently (JP-A-59-25323). Furthermore, the development of a pterin derivative having a similar coenzyme activity of tyrosine hydroxylase is under way, for example, 1 ', 2'-diacetyl-.
It is known that tetrahydrobiopterin, 6-methyl-5,6,7,8-tetrahydropterin, L-septapterin and the like have a therapeutic effect (ibid.). These biopterin coenzyme-related compounds are natural coenzymes
The same effect as BH ₄ can be expected. However, a compound that can be expected to have an effect exceeding this has not been known.

For felketonuria, low-dose treatment that administers BH ₄ to the extent that phenylalanine hydroxylase in the liver acts, and large-dose method that administers a large amount and transfers it to the brain to restore the deficiency state in nerve cells Has been done. However, since BH ₄ does not easily cross the blood-brain barrier, it has not been sufficiently effective.

Thus, for the treatment of neurological diseases caused by BH ₄ deficiency, the emergence of therapeutic agents that have coenzyme activity of phenylalanine hydroxylase, tyrosine hydroxylase or tryptophan hydroxylase and that easily cross the blood-brain barrier. It was

[Object of the Invention]

An object of the present invention, must BH ₄ is a large amount administered BH ₄ to increase the concentration in the brain in treating neurological diseases caused by brain BH ₄ deficient by hardly cross the blood brain barrier An object of the present invention is to provide a therapeutic agent that solves the above-mentioned problems and that can easily cross the blood-brain barrier and that is effective even when administered in a small amount.

[Means for solving problems]

Formula of the invention: 6-Hydroxymethyl-tetrahydropterin (hereinafter referred to as 6HMPH ₄ ) is known to exhibit the coenzyme activity of tryptophan hydroxylase, which also has BH ₄ as a coenzyme (Kato et al .; Biosimika Ebio). Physica Acta (Biochimica et Biophysica Acta), 61
1 , 241-250 (1980), p. 246).

The present inventors show that 6HMPH ₄ has coenzyme activities of tyrosine hydroxylase and phenylalanine hydroxylase like BH ₄ and is far superior to BH _{4 in} that it easily crosses the blood-brain barrier. The present invention has been completed and the present invention has been completed.

That is, the present invention relates to a therapeutic agent for neurological diseases caused by BH ₄ deficiency such as Parkinson's disease and phenylketonuria, which contains 6HMPH ₄ as an active ingredient.

[Operation and Example]

6HMPH _4, which is an active ingredient of the therapeutic agent of the present invention, has stereoisomers with respect to the carbon atom at the 6-position, but the effective therapeutic agent of the present invention is the (6R) -isomer. However, (6
The (6S) form may coexist as long as the (R) form is included.

The therapeutic agent of the present invention may be administered by oral administration or injection. In the case of injection, conventional injection methods such as intravenous injection and intramuscular injection may be used. In the case of oral administration, it may be administered in a conventional dosage form such as tablets, pills, capsules, powders, solutions and suspensions. The therapeutic agent of the present invention contains a commonly used pharmaceutical carrier or excipient in addition to the active ingredient as a pharmaceutical composition, and may further contain other drugs that can be used in combination.

Oral preparations include, for example, 6HMPH ₄ ascorbic acid / 6HMPH ₄ =
It can be prepared by blending well with ascorbic acid in a ratio of 1/1 to 10/1 and encapsulating a predetermined amount in powder form or tableting by tableting. The injection can be prepared, for example, by dissolving a predetermined amount of 6HMPH ₄ powder in sterilized Ringer's water.

The effective dose of the therapeutic agent of the present invention can be appropriately determined depending on the time of administration, symptoms, etc., but is usually 0.1 to 500 mg / kg.
Body weight / day, preferably 1-100 mg / kg body weight / day.

6HMPH ₄ , which is an active ingredient of the therapeutic agent of the present invention, is, for example, Max Viscontini et al. [Helvetica CHimica Acta, 56 , 1710-1715].
(1973)].

(1) Measurement of the coenzyme activity of 6HMPH ₄ phenylalanine-4-hydroxylase Phenylalanine-4-hydroxylase was extracted from rat liver by R. SHIMAN et al.
Of Biological Chemistry (JBC) 254
Volume, 11300-11306 (1979), 255, 4793-4800 (1
980)] was used.

The following reaction mixture (total volume 40 μl): Phenylalanine-4-hydroxylase 6.7 units Dihydropterin reductase 7.8 units Catalase 500 units KH₂PO_FourBuffer solution (pH 6.8) 5.6 μM NADH (reduced nicotinamide adenine dinucleotide
D) 0.5M 4-tritium-phenylalanine 7.8mM (about 700000dpm) Coenzyme activity sample 0.1-1.0mM was incubated at 37 ° C for 45 minutes, and then PH5.5 acetic acid was added.
The reaction was terminated by adding 50 μl of sodium solution. reaction
Cool the vessel to 0 ° C. in an ice-water bath and use N-iodosuccinimi
25 μl of dimethyl sulfoxide containing 50 mg / ml of dimethyl sulfoxide
Of the newly formed tyrosine aromatic rings 3 and 5
Iodized the place. This makes tritium³As HOH
Liberated. After 5 minutes, add 50 μl of 30% trichloroacetic acid
To terminate the reaction. Tritiated water has the following three layers
By eluting from an ion exchange column consisting of
Separated from reaction system components. Column is from bottom to top
Toward Lewatit SP 1080 (60 to 150 mesh, H⁺Mold, 20 × 5mm) Activated carbon 1 × 5mm Lewatit MP It was composed of three layers of 5080 (60-160 mesh, acetate type, 5 × 5 mm). Tritiated water is a liquid
It was quantified with a body scintillation counter.

For 6HMPH ₄ and BH ₄ , as shown in FIG. 1, Lineweaver-Burk plot was performed to obtain Vmax and Km values. The results are shown in Table 1.

In Table 1, Vmax is 40 μl of reaction solution during 45 minutes of reaction time.
It represents the concentration of tyrosine generated in the product.

(2) 6HMPH_FourOf tyrosine-3-hydroxylase
Measurement of elementary activity Rat striatum was used as the enzyme source. Frozen filaments
Body tissue, 0.1% Triton X-100 Including 0.05M KH₂PO_Four
Homogenize in 5 volumes of buffer (pH 6.64). Homo
Centrifuge the ginate at 40,000 xg for 15 minutes at 4 ° C.
Then, the supernatant was collected. Enzyme activity measurement from the following composition
100 μl of the reaction solution from 7 ml of scintillation vial
Incubated for 15 minutes at 37 ° C.

3,5-Ditritium tyrosine (100M) (approx. 600,000dpm) Ferrous ammonium sulfate 10μM Catalase 7500 units Ascorbic acid 1.0mM KH ₂ PO ₄ buffer (pH6.64) 50mM Coenzyme ^* active sample enzyme 1.0 to 10mM * The (6R, S) body was used for both BH ₄ and 6HMPH ₄ .

The reaction was terminated by adding 50 μl of 3.0 M sodium carbonate (pH 11.69), further adding 5 ml of toluene and swirling vigorously for 10 seconds. The mixture was allowed to stand for 15 minutes to separate the organic solvent layer, and the tritium content of the vial was measured with a scintillation counter.

For 6HMPH ₄ and BH ₄ , as shown in FIG. 1, Lineweaver-Burk plot was performed to obtain Vmax and Km values.
The results are shown in Table 2.

In Table 2, Vmax is 100μ of reaction solution during 15 minutes of reaction time.
It represents the concentration of dopa produced in 1 l.

(3) Transfer test of 6HMPH ₄ into rat brain 6HMPH ₄ and BH ₄ were injected into the abdominal cavity of rats at 50 mg / kg body weight, respectively, and the rats were sacrificed 2 hours later. The striatum, hippocampus and cerebellum of the brain were taken out, and the contents of 6HMPH ₄ and BH ₄ in their tissues were examined by the following method.

Using 0.1 N HCl containing 5 mg of dithioerythritol in 100 ml, the linear bodies were homogenized in 200 μl, the cerebellum in 1200 μl, and the hippocampus in 400 μl.
The homogenate was stored frozen in dry ice. Thaw if necessary, centrifuge at 39000 xg for 20 minutes, and add supernatant to H
It was analyzed by PLC. The yield of the homogenate was corrected by adding neopterin as an internal standard.

HPLC: S5 ODS column eluate H ₂ O in _{2l Na 2 HPO 4 · 2H 2} O 2.35g Citric acid 5.6g octanoic acid 650 mg EDTA 45 mg elution rate 1 ml / min 6HMPH ₄ containing dithioerythritol 50 mg 2-propanol 140ml Table 3 shows the results of comparing the transferability of BH ₄ and BH ₄ to the tissue in the brain.

From Table 3, it can be seen that 6HMPH ₄ has BH
You can see that it is superior to ₄ . Tokui 6 HMPH ₄ is B
3 times in striatum and 6-7 in hippocampus and cerebellum compared to H _4.
It can be seen that the concentration has doubled.

〔The invention's effect〕

The therapeutic agent of the present invention does not need to be administered in a large amount like BH ₄ because 6HMPH _4, which is its active ingredient, easily crosses the blood-brain barrier, and is deficient in BH _{4 in} the brain such as Parkinson's disease and phenylketonuria. It can be effectively used for the treatment of neurological diseases caused by.

[Brief description of drawings]

FIGS. 1 and 2 show 6HMPH in (1) 6HMPH ₄ phenylalanine-4-hydroxylase coenzyme activity measurement, and (2) 6HMPH ₄ tyrosine-3-hydroxylase coenzyme activity measurement. Lineweaver-Burk plots for ₄ and BH ₄ , respectively.

Claims (5)

[Claims] 1. A therapeutic agent for neurological diseases caused by tetrahydrobiopterin deficiency, which comprises 6-hydroxymethyl-tetrahydropterin as an active ingredient. 2. The therapeutic agent according to claim 1, wherein the neurological disease is Parkinson's disease. 3. The therapeutic agent according to claim 1, wherein the neurological disease is phenylketonuria. 4. The therapeutic agent according to claim 1, wherein the neurological disease is intrinsic depression. 5. The therapeutic agent according to claim 1, wherein the nerve disease is childhood autism.