JPH0133116B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for increasing the intracellular concentration of glutathione.

Glutathione (L-γ-glutamyl-L-cysteinyl-glycine; GSH), a tripeptide thiol found in virtually all cells,
It is well known that it is involved in metabolism, transport, and cell protection. Glutathione plays a protective role against the effects of free radicals and reactive oxygen intermediates such as peroxides, which are formed during the reduction of disulfide bonds in proteins and other molecules, the synthesis of deoxyribonucleotide precursors of DNA, and metabolism. .

Modification of glutathione metabolism is accomplished by administering selective enzyme inhibitors to reduce intracellular glutathione concentrations or by providing compounds that increase glutathione synthesis. Such effects are useful in chemotherapy and radiotherapy or in protecting cells from the toxic effects of drugs, other external compounds, and oxygen. Indeed, the various functions of GSH make it relevant in many fields of biology, including not only oxygenology and migration, but also pharmacology, radiobiology, cancer therapy, toxicology, endocrinology, microbiology, agriculture, etc. It is. Enzymological and translocation phenomena of glutathione metabolism are described in Meister: “Selective Modification of Glutathione Metabolism” (Seience);
220, No. 4596, pp. 472-477 (April 1983)), which is hereby incorporated by reference.

Modification of GSH metabolism to decrease or increase intracellular GSH serves a variety of purposes. For example, thiols have long been known to protect cells from the effects of radiation. Since reducing intracellular GSH increases the sensitivity of the cell to irradiation, reducing glutathione is important for chemical reactions in which dying and viable cells have essentially different quantitative demands on GSH. It is useful in therapeutic situations. Also, reducing GSH by inhibiting its synthesis serves as a valuable adjunct in chemotherapy using drugs that are detoxified by reactions involving GSH.

Conversely, resistance to drugs and irradiation is caused by intracellular
It is associated with an increase in GSH. GSH works effectively in the detoxification of many pharmaceuticals, and it is already known that the detoxification process of acetaminophene involves binding with GSH.

Treatment with thiazolidines, such as L-2-oxothiazolidine-4-carboxylic acid, may be valuable in patients with liver disease and in premature infants with insufficient utilization of methionine sulfur for cysteine formation and defects in GSH synthesis. be. The effectiveness of thiazolidines as intracellular cysteine precursors depends on the presence of 5-oxoprolinase, an enzymatic activity found in almost all animal cells. This enzyme has also been found to occur in plants, including thiazolidine,
Therefore, it is suggested that glutathione is useful as an agricultural safety agent to protect grain plants from the toxic effects of herbicides.

Various methods are known for increasing cellular concentrations of glutathione. Glutathione is composed of three amino acids: glutamic acid, cysteine and glycine. Administration of amino acid precursors of glutathione to animals increases intracellular glutathione, but the effectiveness of this method has certain limitations. The concentration of GSH depends on the supply of cysteine, which is derived from dietary proteins and by sulfur transfer from methineon in the liver. Administration of cysteine itself is not an ideal way to increase GSH concentrations, as it is rapidly metabolized and is also highly toxic. Administration of compounds to animals that enter cells and convert to cysteine within the cells is often useful in increasing intracellular glutathione concentrations. For example, when the thiazolidine, L-2-oxothiazolidine-4-carboxylate, is transferred into cells, it is converted to L-cysteine by 5-oxoprolinase, and this L-cysteine is rapidly converted into L-cysteine. Used for GSH synthesis.

Other methods to increase tissue GSH concentration include:
There is a method of administering γ-glutamylcysteine or γ-glutamylcysteine. Administered γ-
The glutamyl amino acid is transferred intact and serves as a substrate for GSH synthetase. N-acetyl-
It is also known that administration of L-cysteine increases tissue concentrations of GSH.

It has also been thought that administration of glutathione itself may also increase glutathione concentrations. However, no literature has been published to date proving that glutathione enters cells. Rather, there are some reports on specific biological systems that show that glutathione itself does not move into cells. The increase in intracellular glutathione often observed after glutathione administration is due to (a) extracellular degradation of glutathione, (b) translocation into the cell of free amino acids or dipeptides derived from glutathione extracellularly, and (c)
This may be due to intracellular resynthesis of glutathione.

These conventional methods of increasing intracellular glutathione concentrations suffer from disadvantages such as efficiency, toxicity, and limitations on the effective concentrations achieved, as discussed above. In addition, known methods for synthesizing GSH by increasing the supply of substrates to two synthetases include:
One of them relies on the presence of a synthetase that follows the feedback blocking action of GSH.

Therefore, one of the objects of the present invention is to achieve an increase in the intracellular concentration of glutathione by transmitting glutathione itself to cells rather than amino acid substrates.

Another object of the present invention is to provide pure derivatives of glutathione and to use them in glutathione delivery systems.

Another object of the present invention is to efficiently increase the intracellular concentration of glutathione without depending on the presence of synthetase.

Yet another object of the present invention is to achieve an increase in the intracellular concentration of glutathione without the toxic effects observed with other known methods.

Yet another object of the present invention is to detoxify medicines,
oxygen and its metabolites (e.g. peroxides),
The goal is to efficiently and rapidly achieve the intracellular glutathione concentration necessary to achieve a specific purpose, such as the protection of cells from free radicals, external compounds, etc.

The present invention overcomes the difficulties, such as toxicity, limited effectiveness, and dependence on intracellular synthetases, encountered with prior art techniques for increasing intracellular glutathione concentrations. More specifically, lower alkyl esters of glutathione, preferably monomethyl esters and/or monoethyl esters, in which esterification has occurred at the glycine carboxyl group, are administered to increase the intracellular glutathione concentration, thereby achieving the above-mentioned or other purposes. The goal is to achieve the following. The above-mentioned esters move into cells of the liver or kidney, are deesterified within the cells, and contribute to an increase in the intracellular concentration of glutathione.

According to the present invention, which involves administration of a GSH ester,
Increased concentration of GSH is achieved efficiently and quickly.

In addition, the present invention provides pure lower alkyl esters, preferably monomethyl and/or monoethyl esters, and processes for their preparation. Diesters, which are byproducts of monoester synthesis, are extremely toxic.

Figures 1a and 1b show glutathione concentrations in the liver and kidneys of fasted mice administered glutathione esters or glutathione compared to controls.

Figures 2a and 2b are graphs showing changes in glutathione concentration in mice pretreated with buthionine sulfoximide.

FIG. 3 is a graph showing changes in glutathione concentration in the liver of mice pretreated with acetaminophene.

According to the present invention, esterified glutathione is directly transferred into cells and deesterified by the action of hydrolase within the cells, resulting in an increase in the intracellular concentration of glutathione. Glutathione has two carboxyl groups, one in the glutamic acid residue and the other in the glycibone residue. The compounds used in the present invention are lower alkyl esters of glutathione, preferably monomethyl or monoethyl esters, in which esterification occurs only at the glycine carboxyl group.

The source of glutathione used in the invention is not critical. That is, glutathione may be synthesized and separated by conventional methods, or may be purchased.

Esterification can be carried out using conventional methods.
The resulting ester is sufficiently purified to achieve a monoester purity of at least 98% (by weight), preferably 90% (by weight).
%. The reason for this is that in the conventional methods described in the prior literature, such as the method of treatment with ethanolic hydrochloric acid, monoesters containing diesters in a proportion of about 5 to 20% are obtained.
Experiments using mice have revealed that diesters are extremely toxic. A typical example of a method for producing a pure monoester is shown in Synthesis Example 1 below. Monoesters are highly water soluble at basic PH, and diesters are organic solvent soluble at basic PH. Therefore, by dissolving the impure product obtained by conventional synthetic methods in water,
pH around 8.0 using common bases such as amines
9.2 and then treatment with a water-immiscible organic solvent such as chloroform or ethyl acetate, the pure target product can be obtained.

Synthesis Example 1 Production of glutathione monoethyl ester (L-γ-glutamyl-L-cysteinyl-glycylethyl ester) In a glass stoppered container, glutathione (20 g) was treated with ethanol (100 ml) containing hydrogen chloride (4.8 g). , and left at 0°C for 6 hours. After adding cold ethanol (200ml), triethylamine was added to bring the pH to 6. The mixture was kept at 0°C for 18 hours and the precipitate was collected. Add this precipitate to water (100ml)
and cooled to 0°C. The pH of this solution was adjusted to 8.8 with triethylamine and rapidly extracted three times with chloroform (250 ml each). The aqueous layer was quickly adjusted to pH 5 with 6M hydrochloric acid, and the solvent was evaporated and removed under reduced pressure. The dried specimens were treated with dry diethyl ether and ethanol. The obtained crystals were collected and washed with diethyl ether.
This was dried under reduced pressure over phosphorus pentate and calcium chloride and recrystallized from aqueous ethanol. Yield 80-90%.

Glutathione monoethyl ester infrared absorption spectrum (KBr, cm ^-1 ): 1735,
1635, 1520, 1400, 1200 Specific rotation ([α] ²¹ _D ): −27.2 (c = 1.0, water) Nuclear magnetic resonance spectrum (DMSO-d ₆ , δ): 1.16
(3H, t, J=7); 1.70-2.04 (2H, m);
2.16-2.40 (2H, m); 2.60-2.82 (2H, m);
3.16-3.40 (1H, m); 3.80 (2H, s); 4.04
(2H, q, J=7); 4.20-4.48 (1H, m) Glutathione monoethyl ester hydrochloride Melting point 148°C (decomposition) Synthesis example 2 Glutathione monomethyl ester (L-γ-glutamyl-L-cysteinyl-glycylmethyl Production of ester) The target product was obtained in the same manner as in Synthesis Example 1, using methanol instead of ethanol.

Glutathione monomethyl ester infrared absorption spectrum (KBr, cm ^-1 ): 1740,
1635, 1520, 1400, 1210 Specific rotation ([α] ²⁵ _D ): -32.0 (c = 1.0, water) Nuclear magnetic resonance spectrum (DMSO-d ₆ , +CD ₃ OD,
δ): 1.72-2.04 (2H, m); 2.20-2.40 (2H,
m); 2.64-2.88 (2H, m); 3.16-3.40 (1H,
m); 3.60 (3H, s); 3.80 (2H, s); 4.20~
4.60 (1H, m) The lower alkyl ester of glutathione thus obtained, preferably monomethyl ester or monoethyl ester, is usually dissolved in water for injection. However, these esters may also be administered orally. Further, these esters may be mixed with a suitable pharmaceutically acceptable carrier to form a formulation. For example, it can be dissolved in water to make a liquid preparation, or mixed with lactose or the like to make a powder preparation.

Therapeutically effective amounts are appropriately determined by routine experimentation and based on routine techniques for glutathione and the experimental examples below, especially taking into account that nearly stoichiometric intracellular hydrolysis is believed to proceed. Good. In either case, glutathione levels within the patient can be monitored based on the nature of the toxicity being treated, and other parameters of efficacy can be used. The amount of esters that can be proposed at this time is approximately
0.5-1.0 mmol, preferably about 2-5 esters
Millimoles, administered 1 to 6 times a day.

Administration is usually about 0.5 to 3 hours after administration.
This is done to obtain millimolar intracellular concentrations of glutathione.

Although the detailed mechanism of the reaction is still not clear, the increase in intracellular glutathione concentration achieved by the present invention is due to the fact that the administered glutathione ester moves into at least the cells of the liver or kidney, where it is hydrolyzed. This can be interpreted as indicating that glutathione is converted into glutathione. Such hydrolysis was demonstrated by in vitro experiments in which glutathione monoester was incubated with liver and kidney homogenates.

Incidentally, the LD ₅₀ (mouse, intraperitoneal injection) shows values far exceeding 10 mmol/Kg for both glutathione methyl ester and glutathione ethyl ester.

The specification of Japanese Patent Publication No. 43-20166 discloses the fact that glutathione diesters such as glutathione diethyl ester and glutathione di-n-butyl ester have good biomembrane permeability. However, there is no disclosure as to how these diesters behave after permeating through biological membranes.

According to experiments conducted by the inventors using mouse liver and kidney homogenates, it was confirmed that although glutathione diester is converted to glutathione to some extent, the conversion rate is extremely slow compared to glutathione monoester. Ta. Furthermore, according to an intraperitoneal administration experiment to mice, it was not possible to detect an increase in glutathione concentration in the liver or kidneys with glutathione diester even after 8 hours, whereas with glutathione monoester, there was a significant increase in glutathione concentration after 2 hours. An increase in concentration could be confirmed. Furthermore, intraperitoneal administration of glutathione diester at 5 mmol/Kg or more causes mouse death, but as described above, glutathione monoester does not cause mouse death even when administered at 10 mmol/Kg.

In this way, both glutathione monoester and glutathione diester have excellent biomembrane permeability, but the former is quickly converted to glutathione after permeation and exerts glutathione action, and is less toxic. While it is suitable for use as a medicine, the latter is difficult to convert into glutathione after permeation and has relatively high toxicity, making it essentially unsuitable for use as a medicine. .

Although the present invention has been generally described above, the present invention will be described in more detail with reference to Examples below.

Example 1 Glutathione monomethyl ester was used in this example (purity 99%). A solution of glutathione ester was prepared using sterile water. Takonik・
Mice (TAC; (SWFBR)) weighing 20-25 g obtained from Farms Inc.
After 24 hours fasting, 10 glutathione esters
It was administered intraperitoneally at a rate of mmol/Kg. As shown in Figures 1a and 1b, mice were dissected every 2 hours, and the liver and kidneys were removed and analyzed for glutathione. Three mice were used per group, and values are expressed as mean ± SD.

As shown in FIG. 1a (liver), in the livers of mice administered with glutathione ester (GSH-ESTER), a substantial increase in glutathione concentration was observed 2 hours after administration, and the concentration gradually decreased thereafter. In addition, as shown in Figure 1b (kidney), glutathione concentration in the kidney also increased substantially 2 hours after administration, and then decreased. Control group (CONTROL)
When administered an equal volume of 0.15M sodium chloride solution, no increase in glutathione concentration was observed in either the liver or kidneys.

Example 2 In the same manner as in Example 1, glutathione monomethyl ester was administered to mice. However, mice were pretreated with 2 mmol/Kg of buthionine sulfoximine, an inhibitor of glutathione synthesis, and glutathione ester was administered 4 hours after administration of the inhibitor. In this example, glutathione concentrations in the liver and kidneys initially decreased to significantly lower values because the enzyme required for glutathione synthesis (γ-glutamylcysteine synthetase) was significantly inhibited by buthionine sulfoximine. was. When glutathione ester (GSH-ESTER) was administered to mice pretreated as described above, a substantial increase in glutathione concentration was observed in the liver and kidneys. See Figure 2a (liver) and Figure 2b (kidney). Control group (CONTROL)
were given saline solution and at the above mentioned time intervals.
The tissue concentration of GSH was investigated. Three mice were used in each group, and the values are expressed as the mean ± SD.

Comparative Example 1 The same procedure as in Example 1 was carried out except that the same amount of glutathione was used in place of glutathione monomethyl ester. As shown in FIG. 1a, no effect was observed on the glutathione concentration in the liver of the treated group (GSH). In addition, compared to the control group (CONTROL) in which there was virtually no change in the kidney shown in Figure 1b,
A slight effect was observed in the treated group (GSH).

Note that the effects observed for glutathione monomethyl ester are approximately equivalent to those observed for glutathione monoethyl ester.

Example 3 A significant decrease in hepatic glutathione concentration was observed in mice treated with sublethal doses of acetaminophene (Figure 3; control group (CONTROL)).
The treatment group received acetaminophen treatment followed by injection of glutathione monomethyl ester (GSH-
ESTER), no such decrease in glutathione concentration was observed. In contrast, hepatic glutathione concentrations were significantly elevated. Under the same conditions, administration of other thiols, namely L-cysteine (CYSTEINE) and L-cysteine methyl ester (CYSTEINE-ESTER),
The liver concentration of glutathione was significantly low (third
figure).

In this example, acetaminophene (2.5 mmol/Kg) was intraperitoneally injected into fasted mice, and 30 minutes later (arrow in Figure 3), GSH monomethyl ester,
GSH, L-cysteine, L-cysteine methyl ester (10 mmol/Kg) or equivalent volume
0.15M saline (control) was administered. organization
GSH concentrations were determined for each group 3 at the indicated time intervals.
determined for one mouse. Values are mean ± S.
D.

Example 4 A lethal dose of acetaminophene (5 mmol/Kg) was administered to mice, and 1 hour later, glutathione monomethyl ester (10 mmol/Kg) was administered, but no mice were found to be lethal. In this experiment, 15 mice were treated as described above; all survived and showed obvious recovery after 7 days. In a group of 40 mice that received an equal amount of acetaminophen but no glutathione monomethyl ester, all died within 7 days.

The disclosure herein shows that administered glutathione esters migrate into liver and kidney cells where they are hydrolyzed to glutathione. Experiments in which mice were pretreated with buthionine sulfoximine provide strong evidence for the transfer of glutathione esters; under these conditions glutathione synthesis from constituent amino acids is severely inhibited.

Since glutathione synthesis is significantly inhibited by buthionine sulfoximine, it is thought that glutathione is transmitted into cells. That is,
The present invention makes it possible to increase the intracellular glutathione concentration even when the necessary synthetase for glutathione is deficient.

The technical scope of the present invention is not limited to the examples disclosed in detail herein, but is intended to include modifications obvious to those skilled in the art. For example, according to the invention it is possible to form lower alkyl esters, preferably methyl or ethyl esters, for other thiols that do not easily cross the cell membrane, in order to allow their translocation into the cell. The esters of the present invention can also be administered to cereal plants or to their seeds before they develop in order to reduce the effects of herbicides that may be applied to them by administering them in the form of an absorbable liquid formulation. It can also be used as a safety agent to prevent

The term "lower alkyl" used herein has its usual meaning and includes propyl in addition to methyl and ethyl.

[Brief explanation of drawings]

Figures 1a and 1b are graphs showing a comparison of glutathione ester or glutathione concentration in the liver and kidneys between the glutathione ester or glutathione administration group and the control group, and Figures 2a and 2b are graphs showing buthionine sulfoximide concentrations in the liver and kidneys, respectively. Graph showing changes in glutathione concentration in pretreated mice. FIG. 3 is a graph showing changes in glutathione concentration in the liver of mice pretreated with acetaminophen.

Claims (1)

[Claims] 1. A mono-lower alkyl ester of glutathione related to the glycine carboxyl group. 2. The ester according to claim 1, wherein the lower alkyl ester is a methyl ester. 3. The ester according to claim 1, wherein the lower alkyl ester is an ethyl ester. 4. Dissolving the mono-lower alkyl ester related to the glycine carboxyl group of glutathione in which the glutathione di-lower alkyl ester is mixed in an aqueous medium, and adjusting the pH of this solution to 8.0 to 9.2,
A method for purifying a mono-lower alkyl ester of glycine carboxyl group of glutathione, which comprises extracting and removing the di-lower alkyl ester of glutathione using an organic solvent that is immiscible with the aqueous medium. 5. A glutathione-like metabolic pharmaceutical composition containing a mono-lower alkyl ester related to the glycine carboxyl group of glutathione as an active ingredient. 6. The composition according to claim 5, wherein the lower alkyl ester is a methyl ester. 7. The composition according to claim 5, wherein the lower alkyl ester is an ethyl ester. 8. The composition according to claim 5, which has improved cell permeability for animal cells. 9 Lower alkyl ester is 0.5 to kg body weight
9. A composition according to claim 8 in a dosage form such that 10 mmol is administered. 10. The composition according to claim 9, wherein the dosage form is an injection dosage form. 11. The composition according to claim 5, which is used as a pharmaceutical antidote. 12. The composition according to claim 5, which is used as an irradiation resistance enhancer. 13. The composition according to claim 11, wherein the medicament is acetaminophenone. 14. A composition according to claim 5 for use as a resistance enhancer against the effects of reactive oxygen intermediates.