RU2826374C1 - Method of producing nanodispersions based on conjugate of alpha-lipoic acid with glutathione - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: disclosed is a method of producing a nanodispersion of a conjugate of alpha-lipoic acid with glutathione (LA-GSH), involving the following steps: (a) activation of the carboxyl group of lipoic acid with N-hydroxysuccinimide in presence of 1,3-dicyclohexylcarbodiimide in dry tetrahydrofuran to obtain an N-hydroxysuccinimide ester of lipoic acid; (b) blocking the mercapto group of glutathione by trityl protection to obtain S-tritylglutathione; (c) conjugating N-hydroxysuccinimide ester of lipoic acid obtained in step (a) with S-tritylglutathione obtained in step (b), in methylene chloride with the activating action of triethylamine to obtain a reaction product of N-lipoyl-S-trityl-glutathione; (d) acid hydrolysis of the reaction product obtained in step (c), with extraction of the LA-GSH conjugate; (e) dispersing the LA-GSH conjugate in an aqueous solution of Pluronic F68 with intense stirring to form a nanodispersion of the LA-GSH conjugate.

EFFECT: invention provides the development of a technology for producing nanodispersions of a conjugate of α-lipoic acid and glutathione, with output of all synthesis steps in range of 44–72% with increased colloidal stability, antioxidant and antiaggregation efficiency.

6 cl, 5 dwg, 1 tbl, 2 ex

Description

The invention relates to the pharmaceutical industry and can be used to produce medicines.

Stroke is a disorder of cerebral circulation that occurs due to a disruption of the blood supply to the brain. The ischemic process is aggravated by the formation of eicosanoids, which lead to activation and subsequently to the aggregation of formed elements of the blood [Gusev E.I. Cerebral Ischemia / E.I. Gusev, V.I. Skvortsova. - M .: Medicine, 2001. - 328 p.]. One of the approved schemes for the treatment of stroke is to restore blood flow to the brain, but most of the tissue damage occurs during subsequent reperfusion. The pathogenetic significance of oxidative stress in the development of ischemic brain damage determines the advisability of using neuroprotective drugs with antioxidant action [E.Yu. Solovieva, O.P. Mironova, O.A. Baranova, E.M. Bekman, A.V. Aseychev, A.I. Fedin, O.A. Azizova. Free radical processes and antioxidant therapy in cerebral ischemia // Journal of Neurology and Psychiatry named after S.S. Korsakov. - 2008. - Vol. 108, No. 45. 34-45]. Antioxidant therapy, which directly affects ischemia-reperfusion injury, has shown promising results in preclinical studies. Among low-molecular antioxidants, glutathione (GSH) and lipoic acid (LA) are of wide interest.

In various in vitro and in vivo studies, it has been shown that glutathione, LA, and dihydrolipoic acid (DGLA) suppress the formation of reactive oxygen species (ROS) and lipid peroxidation products (LPO), protecting cells from oxidative damage. LA enhances the activity of various antioxidants (vitamins C and E, ubiquinone), slows down the processes of neuronal damage by chelating metals and regulating the activity of enzymes and nuclear transcription factors (Nrf2 and NF-κB), and increases the concentration of reduced glutathione in cells. LA and DGLA are able to bind to divalent ions of heavy metals to form stable chelate complexes [Shchelkonogov V.A., Inshakova A.M., Shipelova A.V., Baranova O.A., Chekanov A.V., Shastina N.S., Solov'eva E.Yu., Fedin A.I. Nanoparticles of lipoic acid esters: preparationand antioxidant effect // Mendeleev Commun. - 2021. - V. 31. - P. 507-508].

It is known that nanoparticles can improve the penetration of drugs into cells due to their fusion with the plasma membrane during receptor-mediated endocytosis or through other mechanisms, including facilitating the transport of lipoic acid across the cell membrane [Rochette L. Alpha-lipoic acid: molecular mechanisms and therapeutic potential in diabetes / L. Rochette, S. Ghibu, A. Muresan, C. Vergely // Can. J. Physiol. Pharmacol. 1999. - V. 93, №12. P. 1021-1027; Duzgunes N. Mechanisms and kinetics of liposome-cell interactions / N. Duzgunes, S. Nir // Advanced Drug Delivery Reviews. - 1999. - V. 40. - P. 3-18]. It has been shown that nanodispersions containing GSH, which is used as an endogenous peptide for drug delivery to the brain, demonstrated enormous potential in both targeting and increased accumulation of drug components in brain cells [Brzica H., Abdullahi W., Ibbotson K., Ronaldson R.T. Role of Transporters in Central Nervous System Drug Delivery and Blood-Brain Barrier Protection: Relevance to Treatment of Stroke // J Cent Nerv Syst Dis. - 2017. - V. 9. - P. 1-12; Heba F.S., Sayed M.A., Ashraf E.H., Mahmoud M.O. Targeting brain cells with glutathione-modulated nanoliposomes: in vitro and in vivo study // Drug Des. Devel. Ther. 2015. V.9. - P. 3705-3727].

In publications [Nanoparticles with lipoic acid and glutathione, Inshakova A.M., Shchelkonogov V.A., Evstratova A.Yu., Al N.L., Shastina N.S., Baranova O.A., Chekanov A.V., Solovieva E.Yu., Fedin A.I. In the collection: collection of scientific papers of the VII Congress of Biophysicists of Russia. Collection of materials of the congress. In 2 volumes. Krasnodar, 2023. P. 176; Antioxidant and antiaggregation properties of nanoparticles with glutathione and lipoic acid. Shchelkonogov V.A., Inshakova A.M., Darnotuk E.S., Surikov N.A., Evstratova A.Yu., Baranova O.A., Chekanov A.V., Kazarinov K.D., Shastina N.S., Solovieva E.Yu., Fedin A.I. The book: Biotechnology: state and development prospects, materials of the international congress. Moscow, 2023. Pp. 46-49.] describes the production of nanoparticles based on phosphatidylcholine and pluronic F68, containing alpha-lipoic acid and glutathione (two different antioxidants are encapsulated in the nanoparticles). In addition, this nanodispersion was obtained using another technique: injection of a glutathione solution in a phosphate buffer, pH 7.4, and 1% aqueous solution of Pluronic F68 into a methanol solution of a mixture of alpha-lipoic acid and phosphatidylcholine with vigorous stirring, followed by removal of the solvent under reduced pressure and treatment of the resulting product in an ultrasonic bath. The resulting nanodispersions are characterized by heterogeneity (2 fractions of nanoparticles: 50-90 nm (15-25%) and 272-330 nm (75-85%), the efficiency of glutathione incorporation of 37±5% and LC 85±10% and a fairly low colloidal stability during storage at room temperature.

The closest to the patented method is the method for synthesizing derivatives (conjugates) of alpha-lipoic acid with glutathione (JIK-GSH) described in the publication [Inshakova A.M., Shchelkonogov V.A., Shastina N.S., Baranova O.A., Chekanov A.V., Solovieva E.Yu., Fedin A.I. Synthesis of glutathione conjugates with α-lipoic acid // VIII international scientific and practical conference of young scientists: biophysicists, biotechnologists, molecular biologists and virologists - 2021. Collection of abstracts of the conference, held within the framework of the open communications platform OpenBio-2021. Novosibirsk, 2021. - P. 108 - prototype.

A strategy plan for the synthesis of glutathione and alpha-lipoic acid derivatives via Steglich esterification of a monosubstituted alpha-lipoic acid ester (1-lipoyloxypropan-3-ol) at the carboxyl group, protected glutathione at the amino group and mercapto group is presented. Obtaining this substance was difficult due to the specific esterification conditions, low conversion of the initial protected glutathione, and difficulty in isolating the target substance. A possible variant of conjugation of alpha-lipoic acid at the amino group of glutathione was also described. However, this publication does not disclose the technology for obtaining the target substance, the conditions for the reactions, isolation and purification of compounds, physicochemical characteristics (NMR spectra, Rf values) of intermediate compounds and the target substance are not given, nanodispersions of the α-lipoic acid and glutathione conjugate have not been obtained and characterized, and studies on the biological activity of nanodispersions of the JIK-GSH conjugate have not been conducted (antioxidant and antiaggregation properties have not been studied).

The present invention is aimed at developing a technology for obtaining nanodispersions of a conjugate of α-lipoic acid and glutathione, with a yield in all stages of synthesis in the range of 44-72% with an increase in colloidal stability, antioxidant and antiaggregation efficiency, which is the technical result of the invention.

The specified technical result is achieved in that the method for producing nanodispersions of the JIK-GSH conjugate comprises: (a) activating the carboxyl group of alpha-lipoic acid using N-hydroxysuccinimide in the presence of 1,3-dicyclohexylcarbodiimide in dry tetrahydrofuran to produce the N-hydroxysuccinimide ester of alpha-lipoic acid; (b) blocking the mercapto group of glutathione using trityl protection to produce the intermediate compound S-trityl glutathione; (c) conjugating the N-hydroxysuccinimide ester of lipoic acid with S-trityl glutathione in methylene chloride under the activating action of triethylamine; (d) acid hydrolysis of N-lipoyl-S-trityl glutathione to form the JIK-GSH conjugate; (d) dispersion of the LK-GSH conjugate in an aqueous solution of Pluronic F68 with vigorous stirring to form nanodispersions of the JIK-GSH conjugate.

The method may be characterized in that in step a) the activation of the carboxyl group of alpha-lipoic acid is carried out by adding N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide to alpha-lipoic acid in dry tetrahydrofuran.

The method can also be characterized by the fact that in stage b) the blocking of the mercapto group of glutathione is carried out by adding triphenylmethanol and boron trifluoride etherate to glutathione in glacial acetic acid.

The method can also be characterized by the fact that in step c) the conjugation of the products obtained in steps a) and b) is carried out by adding N-hydroxysuccinimide ester of alpha-lipoic acid and triethylamine to S-trityl glutathione in dry methylene chloride.

The method can also be characterized by the fact that in stage c), acid hydrolysis is carried out by adding trifluoroacetic acid and triisopropylsilane to N-lipoyl-S-trityl-glutathione obtained in stage c).

The method can also be characterized by the fact that at stage d) the production of a nanodispersion of the JIK-GSH conjugate is carried out by dispersing with a 1% aqueous solution of Pluronic F68.

Individual stages of the process were carried out taking into account known recommendations.

Activation of the carboxyl group of lipoic acid was carried out according to a known method by adding N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide to alpha-lipoic acid in dry tetrahydrofuran (US2013072533 (A1), NATIVI CRISTINA et al. - 21.03.2013). Blocking of the mercapto group of glutathione was carried out by adding triphenylmethanol and boron trifluoride etherate in glacial acetic acid [Photaki I., Taylor-Papadimitriou J., Sakarellos C., Mazarakis P., Zervas L. On cysteine and cystine peptides. V. S-trityl and S-diphenylmethyl-cysteine and - cysteine peptides // J Chem Soc Perkin 1. - 1970. - V. 19. - P. 2683-2687]. Acid hydrolysis of I-lipoid-D-trityl-glutathione was carried out by the action of trifluoroacetic acid [Pearson, D. A., Blanchette, M., Baker, M. L. and. Guindon, C. A. Trialkylsilanes as scavengers for the trifluoroacetic acid deblocking of protecting groups in peptide synthesis // Tetrahedron Letters. - 1989. - V. 30, No. 21. - P. 2739-2742]. To stabilize nanodispersions, various nonionic (uncharged) surfactants are used, including the most promising of them, Pluronic F68 [Wulff-Perez M., Torcello-G6mez A., Gdlvez-Ruiz M., Martin-Rodriguez, A. Stability of emulsions for parenteral feeding: Preparation and characterization of o/w nanoemulsions with natural oils and Pluronic f68 as surfactant // Food Hydrocolloids. - 2009 - V. 23. - P. 1096-1102].

The essence of the invention is explained in the drawings, where:

Fig. 1 shows the conventional designation of hydrogen protons of lipoic acid;

Fig. 2 shows a scheme for obtaining the JIK-GSH conjugate (6);

Fig. 3 shows electron micrographs of nanodispersions of the JIK-GSH conjugate (Clk-gsh = 1 mg/ml); A - TEM; B - SEM;

Fig. 4 shows the effect of nanodispersions with the JIK-GSH conjugate on the functional activity of neutrophils activated with PMA;

Fig. 5 shows the effect of nanodispersions with the JIK-GSH conjugate on the degree (A) and rate (B) of platelet aggregation induced by adenosine diphosphate (ADP).

The method is carried out as follows.

Below are examples that disclose the technology for obtaining nanodispersions of the JIK-GSH conjugate. Fig. 1 shows the conventional designation of the protons of α-lipoic acid. The preparation of the amide derivative of lipoic acid and glutathione (6) consists of several stages and consists of adding lipoic acid with an activated carboxy group (2) to the amino group of glutathione (3) (see Fig. 2).

In the first stage, the carboxyl group of alpha-lipoic acid (1) was activated with N-hydroxysuccinimide in the presence of 1,3-dicyclohexylcarbodiimide to dicyclohexylcarbodiimide in dry tetrahydrofuran.

At the second stage, the mercapto group of glutathione (3) was blocked using trityl protection. The choice of the protective group was due to the ease of introduction. The reaction was carried out in glacial acetic acid with triphenylmethanol under the activating action of boron trifluoride etherate, followed by precipitation of the reaction products with ice water.

In the third step, the reaction of protected glutathione (4) with activated lipoic acid (2) was carried out in the presence of triethylamine in dry methylene chloride with heating for 9 h. N-Lipoyl-S-trityl-glutathione (5) was purified by column chromatography on silica gel in the chloroform:methanol system (20% methanol) and subjected to acid hydrolysis with trifluoroacetic acid in methylene chloride in the presence of triisopropylsilane. The target compound (6) of the JIK-GSH conjugate was purified by column chromatography on silica gel in the chloroform:methanol system (30% methanol).

At the fourth stage, nanodispersions containing the LK-GSH conjugate were obtained. For this, the conjugate was dispersed with an aqueous solution of Pluronic F68 (1%, pH 6.2) for 10 min with vigorous stirring.

The effect of the obtained nanoparticles of the LK-GSH conjugate (6) on the functional activity of neutrophils and platelets was assessed. Nanodispersions without antioxidants and a glutathione solution in PBS (pH 7.4) were used as controls. The studies were conducted in vitro on blood samples obtained from conditionally healthy donors. The kinetics of platelet aggregation was recorded for 10 min.

Example 1. Synthesis of LA-GSH conjugate

The compound was obtained in 4 stages. The synthesis scheme is shown in Fig. 2.

In the first step, 240 mg (1.16 mmol) of 1,3-dicyclohexylcarbodiimide were added to a solution of 200 mg (0.97 mmol) of α-lipoic acid (1) and 134 mg (1.16 mmol) of N-hydroxysuccinimide in tetrahydrofuran THF (30 ml) at 4°C with stirring and the mixture was stirred for 5.5 h at room temperature, filtered, evaporated and purified by flash chromatography, eluting the target compound (2) with chloroform.

Yield 206 mg (72%, oil), R _f 0.72 (chloroform:methanol 9.9:0.1).

¹ H-NMR spectrum: (CDCl ₃ , δ, ppm): 1.52-1.71 (m, 2H, Na), 1.73-1.85 (m, 4H, Hb), 1.87-2.01 (m, 1H, Hc ), 2.41-2.55 (m, 1H, He), 2.69 (t, J=7.3 Hz, 2H, Hd), 2.81 (s, 4H, 2CH ₂ succinimide), 3.11-3.26 (m, 2H, Hf, Hg) , 3.52-3.63 (m, 1H, Hh).

In the second stage, a mixture of 500 mg (1.63 mmol) of glutathione (3), 424 mg (1.63 mmol) of triphenylmethanol and 255 mg (1.79 mmol) of boron trifluoride etherate in glacial acetic acid (85 ml) was stirred at 85°C for 30 min. Then it was stirred for 60 min at room temperature, cooled water (100 ml), sodium acetate (1 g) and water again (50 ml) were added to the reaction mixture until a milky-white suspension appeared, the precipitated white crystals were filtered, washed with water, acetone, diethyl ether and purified by crystallization from acetone.

The yield of compound (4) was 611 mg (71.1%, amorphous), Rf 0.39 (chloroform:methanol:water 6:4:0.8).

¹ H-NMR spectrum (DMSO-d ₆ , δ, ppm): 1.78-1.95 (m, 2H, Glu _β ), 2.26-2.35 (m, 2H, Cys _β ), 2.37-2.41 (m, 2H, Glu _γ ), 3.51-3.66 (m, 3H, Glu _α , Gly _α ), 4.19-4.38 (m, 1H, Cys _α ), 7.18-7.35 (m, 15H, Tr), 8.33-8.50 (m, 2H, Glv- NH , Cys- NH ).

In the third step, a solution of 300 mg (0.55 mmol) of S-trityl glutathione (4), 331 mg (1.09 mmol) of activated lipoic acid (2) and 386 mg (3.82 mmol) of triethylamine in anhydrous methylene chloride (40 ml) was stirred with heating at 40° for 9 h. Purification was carried out by column chromatography, eluting compound (5) with the solvent system chloroform: methanol (20% methanol).

Yield 254 mg (56.7%, amorphous), Rf 0.71 (chloroform:methanol:water 6:4:0.8).

¹ H-NMR spectrum: (DMSO-d ₆ , δ, ppm): 1.21-1.39 (m, 2H, Na), 1.41-1.59 (m, 4H, Hb), 1.74-1.95 (m, 3H , Hc, Glu _β ), 2.01-2.16 (m, 4H, Cysp, Glu _γ ), 2.18-2.29 (m, 1H, He), 2.35 (t, J=12.6 Hz, 2H, Hd), 3.02-3.33 ( m, 2H, Hf, Hg), 3.49-3.61 (m, 3H, Glu _α , Gly _α ), 3.93-4.02 (m, 1H, Hh), 4.11-4.24 (m, 1H, Cys _α ), 7.18-7.35 (m, 15H, Tr), 8.33-8.50 (m, 2H, Gly -NH , Cys -NH ).

In the fourth step, a solution of 200 mg (0.27 mmol) of the protected alpha-lipoic acid-glutathione conjugate (5), 214 mg (1.35 mmol) of triisopropylsilane, and 154 mg (1.35 mmol) of trifluoroacetic acid in methylene chloride (30 ml) was stirred for 2 h at room temperature, concentrated under vacuum, and reevaporated with toluene, ethanol, and methylene chloride. The residue was purified by column chromatography on silica gel in the chloroform:methanol system (30% methanol).

The yield of compound (6) - the LK-GSH conjugate - was 60.1 mg (44.6%, amorphous), Rf 0.67 (chloroform:methanol:water:aqueous:ammonia 6:4:0.8:0.2).

¹ H-NMR spectrum: (D ₂ O, δ, ppm): 1.28-1.46 (m, 2H, Na), 1.48-1.71 (m, 4H, Hb), 1.78-1.97 (m, 2H, Glu _β ), 1.99-2.13 (m, 1H, He), 2.17-2.28 (m, 2H, Glu _γ ), 2.29-2.46 (m, 2H, Cys _β ), 2.59-2.63 (m, 1H, He), 2.86 (t, J=9.6 Hz, 2H, Hd), 3.05-3.22 (m, 2H, Hf, Hg), 3.52-3.74 (m, 3H, Glu _α , Gly _α ), 4.07-4.18 (m, 1H, Hh), 4.44-4.53 (m, 1H, Cys _α ).

Example 2. Method for preparing nanodispersion with LK-GSH conjugate based on nonionic surfactant - Pluronic F68. To obtain nanodispersion of LK-GSH conjugate (6) in the amount of 6 mg was dispersed with an aqueous solution of Pluronic F68 (1%, pH 6.2, 2 ml) for 10 minutes with vigorous stirring. Thus, homogeneous nanodispersions with LK-GSH conjugate based on Pluronic F68 with particle size of 243±24 nm were obtained, possessing high dispersion stability during storage at room temperature (Table 1).

Using transmission and scanning electron microscopy, it was shown that the obtained nanodispersions with the LK-GSH conjugate consist mainly of spherical nanoparticles with sizes from 100 to 270 nm (Fig. 3: A - TEM, B - SEM).

Below are experimental data showing the effect of nanodispersions of the LK-GSH conjugate on free radical processes in neutrophils and on platelet aggregation.

1. The effect of nanodispersions of the LK-GSH conjugate on free radical processes in neutrophils.

The effect of nanodispersions of the LK-GSH conjugate on the functional activity of neutrophils was studied using luminol-dependent chemiluminescence (LCL). Phorbol-12-myristate-13-acetate (PMA) was used as an activator of free-radical processes in NF, which leads to an increase in ROS formation due to the regulation of the activity of various cellular enzymes (NADPH oxidase, superoxide dismutase, peroxidase and other enzymes), as well as the signaling pathways of Nrf2 and NF-κB factors. Experiments were carried out in vitro on neutrophils isolated from blood obtained from conditionally healthy donors aged 20 to 30 years.

The studies showed that the addition of PMA to native neutrophils resulted in an increase in the intensity of luminol-dependent chemiluminescence of NF compared to intact cells. In turn, nanoparticles containing the LK-GSH conjugate significantly (by 3-15 times) reduced the concentration of active forms of oxygen and nitrogen in activated neutrophils. The most significant antioxidant effect was demonstrated by nanodispersions with the maximum studied concentration of the LK-GSH conjugate (Fig. 4).

2. Effect of nanodispersions of the LK-GSH conjugate on platelet aggregation. The effect of the obtained nanoparticles with the LK-GSH conjugate on the degree and rate of platelet aggregation activated by adenosine diphosphate (ADP) was assessed. Nanodispersions without antioxidants and a glutathione solution in PBS (pH 7.4) were used as controls. The studies were conducted in vitro on blood samples obtained from conditionally healthy donors. The kinetics of platelet aggregation was recorded for 10 min.

As a result of the experiments, it was found that nanoparticles containing the LK-GSH conjugate (0.75-1.5 mM) reduced the degree (by 20-40%) and rate of platelet aggregation activated by ADP. The most significant antiaggregatory effect was demonstrated by nanodispersions with the maximum studied concentration of the LK-GSH conjugate. In addition, it was found that the GSH solution in PBS had virtually no effect on platelet aggregation caused by this inducer (Fig. 5 A, B). Note: * - p<0.05 relative to the control (Tc+ADP).

Thus, the presented data indicate the promise of the technology for producing nanodispersions of the LK-GSH conjugate, which ensures an increase in colloidal stability, antioxidant and antiaggregation efficiency, which is the technical result of the invention.

Claims (11)

1. A method for producing a nanodispersion of alpha-lipoic acid conjugate with glutathione (LA-GSH), comprising the following stages: (a) activation of the carboxyl group of lipoic acid with N-hydroxysuccinimide in the presence of 1,3-dicyclohexylcarbodiimide in dry tetrahydrofuran to obtain N-hydroxysuccinimide ester of lipoic acid; (b) blocking the mercapto group of glutathione using trityl protection to obtain S-trityl glutathione; (c) conjugating the N-hydroxysuccinimide ester of lipoic acid obtained in step (a) with S-trityl glutathione obtained in step (b) in methylene chloride under the activating action of triethylamine to obtain the reaction product N-lipoyl-S-trityl glutathione; (d) acid hydrolysis of N-lipoyl-S-trityl-glutathione obtained in step (c) to isolate the LK-GSH conjugate; (d) dispersing the LK-GSH conjugate in an aqueous solution of Pluronic F68 with vigorous stirring to form a nanodispersion of the LK-GSH conjugate. 2. The method according to claim 1, wherein in step (a) the activation of the carboxyl group of alpha-lipoic acid is carried out by adding N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide to alpha-lipoic acid in dry tetrahydrofuran. 3. The method according to claim 1, wherein in step (b) the blocking of the mercapto group of glutathione is carried out by adding triphenylmethanol and boron trifluoride etherate to glutathione in glacial acetic acid. 4. The method according to claim 1, wherein in step (c) the conjugation of the products obtained in steps (a) and b) is carried out by adding N-hydroxysuccinimide ester of alpha-lipoic acid and triethylamine to S-trityl glutathione in dry methylene chloride. 5. The method according to claim 1, wherein in step (d) the acid hydrolysis is carried out by adding trifluoroacetic acid and triisopropylsilane to the N-lipoyl-S-trityl-glutathione obtained in step (c). 6. The method according to claim 1, wherein in step (d) obtaining a nanodispersion of the LK-GSH conjugate is carried out by dispersing with a 1% aqueous solution of Pluronic F68.