阅读说明：本技术 一种线粒体靶向谷胱甘肽衍生物、制备方法及应用 (Mitochondrion targeted glutathione derivative, preparation method and application ) 是由 林森 李青 黄宝珊 金佳惠 李岳亭 潘露 于 2021-06-17 设计创作，主要内容包括：本发明属于药学技术领域,具体涉及一种线粒体靶向谷胱甘肽衍生物、制备方法及应用,其为单三苯基膦化合物修饰的谷胱甘肽TPP-GSH。本发明提供一种新的谷胱甘肽衍生物,其具有线粒体靶向作用,相比未修饰的谷胱甘肽,具有更好的细胞保护效果以及更好的抑制眼压升高的效果。(The invention belongs to the technical field of pharmacy, and particularly relates to a mitochondrion targeted glutathione derivative, a preparation method and application thereof, wherein the mitochondrion targeted glutathione derivative is glutathione TPP-GSH modified by a single triphenylphosphine compound. The present invention provides a novel glutathione derivative which has a mitochondrial targeting effect, a better cytoprotective effect and a better intraocular pressure increase-suppressing effect than unmodified glutathione.)

1. A mitochondrially targeted glutathione derivative, characterized by: the glutathione TPP-GSH is modified by a mono triphenylphosphine compound, and the molecular formula is shown as the formula (I):

2. the method for producing a mitochondrially targeted glutathione derivative according to claim 1, wherein: the oxidized glutathione GSSH reacts with excessive triphenylphosphine compound TPP with a carboxyl end to form glutathione TPP-GSSH-TPP modified by a ditriphenylphosphine compound, then the glutathione TPP-GSSH-TPP modified by a single triphenylphosphine compound is decomposed under the action of dithiothreitol to form glutathione TPP-GSH modified by a single triphenylphosphine compound, and the mitochondrion targeting glutathione derivative is obtained after separation and purification.

3. The method of claim 2, wherein: the oxidized glutathione is carried out with excessive triphenylphosphine compound with carboxyl terminal under the catalyst system of EDC/NHS, EDC/DMAP or DIPEA/HATU.

4. The method of claim 2, wherein: the separation and purification comprises the steps of firstly obtaining an initial product through dialysis, then further separating and purifying through a sephadex column, and finally carrying out preparation liquid phase gradient elution to obtain a pure product; the sephadex column adopts methanol as a mobile phase, and the liquid phase gradient elution adopts methanol and water as the mobile phase.

5. The use of the mitochondrion-targeted glutathione derivative of claim 1 in the preparation of an antioxidant medicament.

6. Use of the mitochondrion-targeted glutathione derivative of claim 1 in the preparation of a medicament for inhibiting ocular tension elevation.

7. Use of a mitochondrially-targeted glutathione derivative according to claim 1 in the manufacture of a medicament for the treatment of glaucoma.

8. A medicament for treating a disease caused by oxidative stress, characterized by: comprising the mitochondrially-targeted glutathione derivative of claim 1.

9. The medicament of claim 8, wherein: for use in the treatment of ocular surface or intraocular diseases associated with oxidative stress.

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to a mitochondrion targeted glutathione derivative, a preparation method and application thereof.

Background

Glutathione (GSH) is a sulfhydryl-containing tripeptide composed of glutamic acid, cysteine and glycine and widely exists in various animal and plant cells. As a natural sulfhydryl-containing molecule, glutathione can be used as a reducing agent, participates in the regulation of oxidation and reduction states in organisms, and is a key component for maintaining the balance of oxidation and reduction in cells and promoting the repair of oxidative damage of biological macromolecules (lipid, protein, DNA and the like). Meanwhile, GSH is also an important regulation metabolite in cells, and participates in the tricarboxylic acid cycle and carbohydrate, fat and protein metabolism in vivo. The shortage of intracellular glutathione is one of the key factors for oxidative damage of cells and organelles and further induction of programmed cell death (such as iron death, apoptosis, etc.). Clinically, glutathione can be used as an auxiliary medicament for treating hepatitis, keratitis, cataract, retinal diseases and the like. At the same time, she can also be used as a food additive to delay aging and enhance immunity.

Mitochondria are the major organelles in cells that produce free radicals, and are the most vulnerable organelles to oxidative damage. It has been shown that intracellular biomacromolecules such as mitochondrial dna (mtdna), etc. are more susceptible to oxidative damage. In pathological conditions, mitochondrial free Radicals (ROS) burst, causing oxidative damage to the biomacromolecules within the mitochondria. The mitochondrion apoptosis inducing agent is mainly characterized in that the whole structure and function of mitochondria are abnormal, the ridge structure formed by an inner membrane disappears, the potential difference between the inner membrane and the outer membrane is reduced or disappears, the integrity of the membrane structure is lost, and the permeability is increased, so that macromolecules such as cytochrome C, mtDNA in the mitochondria are released from the mitochondria firstly, a series of biological manifestations such as high expression of NLRP3 inflammatory corpuscles are triggered, and programmed cell death is induced. Therefore, the antioxidant substances are delivered into the mitochondria in a targeted way, so that the imbalance of ROS in the mitochondria can be restrained from the source, and the normal structure and function of the mitochondria are protected, thereby achieving the treatment effect.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a mitochondrion targeting glutathione derivative, a preparation method and application.

The technical scheme adopted by the invention is as follows: a mitochondrion targeting glutathione derivative is glutathione TPP-GSH modified by a mono triphenylphosphine compound, and the molecular formula of the derivative is shown as a formula (I):

according to the preparation method of the mitochondrion targeting glutathione derivative, oxidized glutathione GSSH reacts with excessive triphenylphosphine compound TPP with a carboxyl terminal to form glutathione TPP-GSSH-TPP modified by a ditriphenylphosphine compound, then the glutathione TPP-GSSH-TPP modified by a single triphenylphosphine compound is decomposed under the action of dithiothreitol to form glutathione TPP-GSH modified by a single triphenylphosphine compound, and the mitochondrion targeting glutathione derivative is obtained after separation and purification.

The oxidized glutathione is carried out with excessive triphenylphosphine compound with carboxyl terminal under the catalyst system of EDC/NHS, EDC/DMAP or DIPEA/HATU.

The separation and purification comprises the steps of firstly obtaining an initial product through dialysis, then further separating and purifying through a sephadex column, and finally carrying out preparation liquid phase gradient elution to obtain a pure product; the sephadex column adopts methanol as a mobile phase, and the liquid phase gradient elution adopts methanol and water as the mobile phase.

The application of the mitochondrion targeting glutathione derivative in preparing antioxidant drugs.

The application of the mitochondrion targeting glutathione derivative in preparing the medicine for inhibiting the increase of intraocular pressure.

Use of a mitochondrially targeted glutathione derivative as described above for the preparation of a medicament for the treatment of glaucoma.

A medicament for the treatment of a disease caused by oxidative stress comprising a mitochondrially targeted glutathione derivative as described above.

The medicament is used for the treatment of ocular surface or intraocular diseases associated with oxidative stress.

The invention has the following beneficial effects: the present invention provides a novel glutathione derivative which has a mitochondrial targeting effect, a better cytoprotective effect and a better intraocular pressure increase-suppressing effect than unmodified glutathione.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.

FIG. 1 is a preparation scheme for TPP-GSH;

FIG. 2 is a mass spectrum of the prepared TPP-GSH;

FIG. 3 shows the preparation of TPP-GSH¹An H-NMR spectrum;

FIG. 4 is a comparison of the effect of protecting mitochondria in cells under oxidative stress;

FIG. 5 shows the comparison of the cytoprotective effect in the low-nutrition state;

FIG. 6 is a comparison of the cell protection effect in the oxidative stress model;

FIG. 7 shows the comparative results of the efficacy studies of animal ocular hypertension models.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

Example 1:

as shown in figure 1, oxidized GSH (GSSG) and triphenylphosphine compound with carboxyl terminal react (TPP excess) under catalyst (EDC/NHS, EDC/DMAP, or DIPEA/HATU, etc., in this example EDC/NHS is specifically adopted) to form TPP modified GSSG (TPP-GSSG-TPP), dialysis is carried out to remove small molecular substances, and then TPP-GSSG-TPP is opened under the action of Dithiothreitol (DTT) to form 2 molecules of TPP-GSH (Mito-GSH). Performing dialysis primary treatment to obtain a primary product, further separating and purifying by a Sephadex LH-20 column (methanol is a mobile phase), and further preparing a liquid phase and performing gradient elution (methanol and water are mobile phases) on the product to obtain a Mito-GSH pure product.

1. And (3) analyzing the structure of the product:

mass spectrum of the product obtained and¹as shown in FIGS. 2 and 3, the product had a molecular ion peak 652.2 according to the molecular structure of the compound to be synthesized, as seen from the mass spectrum results. From¹The structure of the prepared product can be analyzed on an H-NMR spectrum to be consistent with the molecular structure of the compound to be synthesized.

2. Evaluation of cell and mitochondrial protective effects:

cells were seeded at the same density in 96-well plates. The cells were pre-cultured at 37 ℃ in a 5% CO2 incubator for 24 h. When the cells grow to about 70% -80% of the area of the pore plate, H2O2 with different concentrations is added respectively, and the cell activity is measured by a CCK8 method. H2O2 semilethal concentration is obtained, and a corneal epithelial cell oxidative damage model is prepared. HCECs cells were seeded at the same density in 35mm confocal dishes, adding 2.5mL of media per well. To validate the cytoprotective effect of the drugs, we comparatively analyzed the protective effect of GSH and Mito-GSH on starvation and hydrogen peroxide treatment of 2 stress-induced cell damage.

Cytoprotective effect under starvation model: co-culturing GSH and Mito-GSH with different concentrations with cells for a certain time (2-8h), changing the culture medium into a serum-free culture medium, continuing to culture for 24-72h, and determining the cell viability by a CCK-8 method.

Cell protection effect under hydrogen peroxide oxidation damage model: after GSH with different concentrations, Mito-GSH and cells are co-cultured for a certain time (2-8h), the culture medium is changed into a culture medium containing hydrogen peroxide (semilethal concentration), the activity of the cells is measured after the cells are continuously cultured for 24h (CCK8 method), and the protective effect on mitochondria is measured by JC-1 kit method.

After the damage of the mitochondria of the cells, the potential difference between the inner and outer membranes of the mitochondria is usually disappeared. JC-1 kit is a method for maturing mitochondrial membrane potential difference. JC-1 has two existing states of monomer and polymer, where green fluorescence can be detected in the presence of monomer at low concentration and red fluorescence can be detected in the presence of polymer at high concentration. In normal cells, when the mitochondrial membrane potential is normal, JC-1 enters mitochondria through the polarity of the mitochondrial membrane and forms multimers emitting red fluorescence due to the increase of concentration, while in cells with abnormal mitochondria, the mitochondrial transmembrane potential depolarizes, JC-1 is released from mitochondria, the concentration is reduced, and the JC-1 is reversed to be a monomer form emitting green fluorescence. Mitochondrial protective effects can be assessed by detecting green and red fluorescence. As can be seen from fig. 4, only a small amount of green fluorescence is visible in normal cells, while after hydrogen peroxide treatment, the green fluorescence is significantly increased, indicating that mitochondria are destroyed, and the Mito-GSH treatment can significantly reduce the intensity of the green fluorescence, indicating that Mito-GSH has an effect of protecting mitochondria.

As shown in fig. 5, the mitochondria-targeted modified glutathione (Mito-GSH) can better protect the apoptosis induced by adversity (low nutrition state) (CCK-8 method) than the natural GSH, and the protective effect on the cells is improved by more than 100 times (the protective effect of 153 μ M Mito-GSH on the cells is equivalent to that of 15300 μ M GSH).

As shown in FIG. 6, in the concentration range of 75-750. mu.M, mitochondrially targeted modified glutathione (Mito-GSH) is better able to protect hydrogen peroxide-induced (oxidative damage) apoptosis than native GSH (CCK-8 method).

3. Evaluation of ocular hypotensive effect:

selecting 180-200g of healthy SD rats which are female, have no ocular infection and inflammation and have no old corneal leukoplakia. An animal model of ocular hypertension was prepared by intracameral injection of 15 microliters of alpha-chymotrypsin (7.5 mg/ml).

The experiment is divided into: 1) healthy animals were dosed with PBS group (normal control); 2) the animals with high intraocular pressure are added with PBS (blank control), and 3) the animals with high intraocular pressure are added with Mito-GSH (1 time per day, 15 uL); 4) the animals with high intraocular pressure are added with the Mito-GSH treatment group (1 time per day, 15 uL);

as can be seen from FIG. 7, the pharmacodynamic study of animal ocular hypertension model found that Mito-GSH has better effect of inhibiting the increase of intraocular pressure than natural GSH.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.