CN110152004B

CN110152004B - Application of glycine in preparation of drug delivery enhancer and cell transplantation reagent

Info

Publication number: CN110152004B
Application number: CN201910609203.3A
Authority: CN
Inventors: 尹海芳; 林曹瑞; 韩刚
Original assignee: Tianjin Medical University
Current assignee: Tianjin Medical University
Priority date: 2019-07-08
Filing date: 2019-07-08
Publication date: 2021-09-03
Anticipated expiration: 2039-07-08
Also published as: WO2021003864A1; CN110152004A

Abstract

The invention relates to application of glycine in preparing a drug delivery enhancer and a cell transplantation reagent, belonging to the field of biomedicine, and the application of the glycine in preparing the drug delivery enhancer is characterized in that the glycine enhances drug delivery by activating mTORC 1; meanwhile, based on the new application of the glycine, the invention also provides a novel medicine for treating muscle diseases, the application of the glycine in preparing an anti-aging medicine, the application of the glycine in preparing a muscle freezing medicine and a composition for enhancing medicine delivery. The invention discovers a new mechanism for enhancing drug delivery by glycine, can effectively enhance the drug delivery efficiency, improve the drug effect, reduce the drug dosage and improve the treatment effect.

Description

Application of glycine in preparation of drug delivery enhancer and cell transplantation reagent

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of glycine in preparation of a drug delivery enhancer and a cell transplantation reagent.

Background

The low efficacy of the drug system is the bottleneck of clinical transformation, particularly for nucleic acid drugs, and a safe and effective method and system for improving the systemic drug effect are lacked at present. Especially for muscular tissues, which occupy one third of the body composition, systemic therapy is the only effective method. Therefore, there is a need to develop new enhancers that can improve the efficiency of drug (including nucleic acid drugs) systems. In the prior art, a great deal of attempts have been made by different research groups to improve the transport efficiency of nucleic acid drugs in muscle tissues, including the utilization of polymers, nanomaterials, cell shuttle peptides, etc., but the systemic effect or safety of the nucleic acid drugs needs to be deeply studied. Also, there are studies showing that dantrolene, a muscle relaxant, can be used as an adjuvant to enhance the activity of nucleic acid drugs, but the results of systemic tests on muscle disease models show that the effect is very limited. The prior art discloses a novel transportation adjuvant-hexose, which can efficiently improve the transportation efficiency of nucleic acid drugs in muscle tissues, but has no obvious effect on old mice with muscular atrophy; and in some patients with muscular dystrophy, insulin resistance occurs in advanced stages of the patient. In addition, the biggest problem faced by cell therapy is low transplant rate. There is therefore a need in the art to develop new enhancers for nucleic acid and cell therapy that are safer and more efficient and that promote the efficiency of the drug system and the rate of cell transplantation and a wide range of applicable patients.

Disclosure of Invention

Based on the requirements, the invention screens different natural small molecule compounds, including ketonic acid and amino acid drugs, wherein in the screening of non-essential amino acids, the glycine has the most obvious enhancement effect on the nucleic acid drugs, and the glycine can obviously promote the proliferation of primary muscle cells and muscle stem cells, namely muscle satellite cells. The invention thus provides the use of glycine in the preparation of a pharmaceutical adjuvant.

The technical scheme of the invention is as follows:

use of glycine in the preparation of a drug delivery enhancer, characterized in that said glycine enhances drug delivery by activating mTORC 1.

The drug is selected from: nucleic acid drugs, and/or polypeptide drugs, and/or siRNA, and/or drugs with exosome as a transport carrier.

The medicament refers to a medicament for treating muscle-related diseases.

Glycine enhances muscle satellite cell proliferation and muscle regeneration by providing a one carbon unit.

The nucleic acid drug is selected from PMO, 2' Ome RNA, PNA, or peptide-PMO or plasmid; the polypeptide drug is selected from CP 05. The CP05 is a small peptide CP05 described in patent 201510520565.7.

The dosage form of the medicine is injection.

Glycine aqueous solution with the mass volume ratio of 2.5-10% enhances the delivery of nucleic acid drugs or polypeptide drugs; 0.8mM glycine enhances delivery of siRNA drugs, or drugs with exosomes as transport vehicles;

the use comprises the following steps: glycine and medicine are injected simultaneously, or glycine is injected first and then medicine is injected, or glycine is fed first and then medicine is injected; more specifically, glycine is injected first, and the drug is injected within 3 days.

Use of glycine in the preparation of a cell transplantation reagent.

The cell transplantation reagent is a glycine aqueous solution with the mass volume ratio of 2.5-10%;

the cell is selected from: a myosatellite cell, and/or, a primary myocyte;

preferably, the use comprises: glycine is injected or fed first and then cell transplantation is carried out, or glycine is injected at the same time of cell transplantation.

A novel medicament for treating muscle diseases is characterized by being a mixture of glycine and conventional medicaments for treating muscle diseases.

The conventional medicament for treating the muscle diseases is a nucleic acid medicament, the final concentration of the glycine in the mixture is 50mg/ml, and the administration concentration of the conventional medicament for treating the muscle diseases is 25 mg/kg;

the nucleic acid drug is selected from: PMO, 2' Ome RNA, PNA, or Peptide-PMO.

The dosage form is selected from oral preparation or injection; the concentration of glycine in the injection is 50 mg/ml.

Application of glycine in preparing antiaging medicine is provided.

The dosage form of the anti-aging drug is selected from oral preparation or injection.

Application of glycine in preparing muscle freezing medicine is provided.

The frostbite medicine is glycine water solution with the mass volume ratio of 2.5-10%.

The freeze injury medicament is in an injection form.

A composition for enhancing drug delivery, comprising: aspartic acid, glycine, serine and tetrahydrofolic acid.

The composition comprises the following components in parts by weight: 0.5-5 parts of aspartic acid, 0.5-10 parts of glycine, 0.5-5 parts of serine and 0.001-0.04 part of tetrahydrofolic acid.

The composition comprises the following components in parts by weight: 3 parts of aspartic acid, 2.5 parts of glycine, 2.5 parts of serine and 0.002 part of tetrahydrofolic acid;

the composition is an aqueous solution containing 3% by mass and volume of aspartic acid, 2.5% by mass and volume of glycine, 2.5% by mass and volume of serine and 0.002% by mass and volume of tetrahydrofolic acid.

Experiments prove that the glycine alone can promote muscle regeneration but cannot improve muscle treatment and function. Glycine alone, used in the early stages in patients with progressive muscle wasting, also proved ineffective.

The invention discovers that: glycine enhances muscle satellite cell proliferation and muscle regeneration by providing a one carbon unit; glycine also enhances muscle stem cell proliferation and muscle regeneration by activating mTORC1, thereby promoting uptake and activity of PMO in differentiated regenerated muscle fibers.

Under the conditions allowed by some national or regional patent laws, the invention also claims the application of glycine in nucleic acid drug treatment;

the invention also claims the use of glycine to enhance drug delivery.

Specifically, the above uses include: glycine and medicine are injected simultaneously, or glycine is injected first and then medicine is injected, or glycine is fed first and then medicine is injected; more specifically, glycine is injected first, and the drug is injected within 3 days.

In another aspect, the invention also claims the use of glycine in cell transplantation;

the invention claims the application of glycine in treating muscle frostbite.

The invention also claims the use of glycine in the treatment of muscle disorders in the elderly.

The objects of the present invention include: 1) enhancing the drug effect of a drug system; 2) promoting proliferation of muscle stem cells; 3) improving the cell treatment efficiency; 4) can be applied to patients with wide age range. A safer and more efficient novel reinforcing agent is obtained, the drug effect is improved, the drug dosage is reduced, and the range of using people is enlarged; improving the cell treatment effect.

The invention 1) mixes different small molecule compounds with PMO respectively, and finally the concentration of each small molecule in the mixed solution is 5%, and the control group is the mixed solution of normal saline and PMO. The mixture is tested on mdx mice which are animal models of DMD, and finally, the mixture is compared with a physiological saline group mixture respectively to compare the level of produced dystrophin protein, so as to determine the influence of glycine on the improvement of PMO drug activity, and finally, the glycine group mixture can improve the expression level of the dystrophin protein in muscle to confirm the efficacy of the glycine. 2) After mixing glycine with PMO, testing is carried out on aged mdx mice, and finally, the glycine group can improve the expression level of dystrophin protein in the muscle of the aged mice so as to confirm that the glycine can act on the mdx mice of different ages. 3) Glycine was prepared as a 5% solution and tested alone in animal models of different muscle diseases and finally compared to saline group to compare the levels of myosatellite producing cells to compare the effect of glycine on increasing the number of myosatellite cells and finally to confirm the efficacy of glycine as a means of increasing the levels of myosatellite cells in muscle.

The invention has the advantages or positive effects that:

1) a new role for glycine as a novel enhancer;

2) the glycine has obvious enhancement effect on different medicines;

3) the glycine serving as a novel pharmaceutical adjuvant is suitable for different population ranges, and although experimental data of an aged mouse are only provided, the mouse model is a mouse model capable of simulating human diseases, and can be extended to human beings.

4) A novel mechanism of action of glycine on muscle stem cells: glycine enhances myocyte proliferation by activating mTORC1 and providing a one-carbon unit. Glycine was demonstrated to be a novel activator of the mTORC1 signaling pathway.

5) Enhancement of cell therapy by glycine, here cell therapy refers to cell transplantation.

Drawings

FIG. 1 is a graph of IHC results and quantitative statistics after 2 μ g PMO and different small molecule compounds tested topically intramuscularly in adult mdx mice at 6-8 weeks of age. IHC is immunofluorescent staining.

FIG. 2 shows the results of western-blot and quantitative statistical graphs showing that the expression level of the dystropin protein in the glycine group was higher than that in the other groups.

The meanings of the respective symbols in FIGS. 1-2 are listed below: c57-wild type mouse; mdx-untreated muscular dystrophy mice; saline-saline (placebo); AA-lithium acetoacetate; PA-sodium pyruvate; CA-sodium citrate; SA-sodium succinate; glu-glutamic acid; ala-alanine; gly-glycine; ser-serine; arg-arginine

FIG. 3 is the IHC results and quantitative statistics after topical testing of the tibialis anterior of adult mdx mice aged 6-8 weeks, following various concentrations of glycine mixed with PMO.

FIG. 4 is a western blot and quantitative statistical chart of different concentrations of glycine and PMO after local injection into the tibialis anterior of mdx mice.

FIG. 5 shows IHC results and quantitative statistics after local testing of tibialis anterior in 6-8 week old adult mdx mice after glycine was mixed with 2' OMe RNA, B-MSP-PMO, PNA and peptide-PMO, respectively.

FIG. 6 is a western blot result and a quantitative statistical chart, from which it can be seen that the expression level of the dystrophin protein in the glycine group is higher than that in the other groups.

The meanings of the respective symbols in FIGS. 3 to 4 are listed below: Saline-Saline group (blank control group); glycine group; PNA-S: PNA mixed group with physiological saline; PNA-Gly: mixed group of PNA and glycine; peptide-PMO-S (Pip 5e-PMO here): mixing peptide-PMO with normal saline; peptide-PMO-Gly: peptide-PMO mixed with glycine (referred to herein as Pip5 e-PMO); 2' OMe-S: 2' OMe and normal saline mixed group; 2' OMe-Gly: 2' OMe mixed with glycine; B-MSP-PMO-S: B-MSP-PMO and normal saline are mixed; B-MSP-PMO-Gly: B-MSP-PMO and glycine mixed group; 10% C57: 5 μ g total protein from C57 tibialis anterior; 5% C57: 2.5 μ g total protein from C57 tibialis anterior muscle; mdx: 50 μ g total protein from mdx tibialis anterior; α -actin: internal standard protein group (positive control group).

FIG. 7 is a graph of IHC results after tail vein system testing of 50mg/kg PMO and glycine in 13-month-old mdx mice.

FIG. 8 is a western blot result, from which it can be seen that the expression level of dystrophin protein in different muscles of the glycine group is higher than that of the control group.

FIG. 9 is a graph of IHC results after long-term systemic testing of 25mg/kg PMO with glycine on adult mdx mice.

FIG. 10 is a western blot result, from which it can be seen that the expression level of dystrophin protein in different muscles of glycine group is higher than that of control group.

FIG. 11 is a graph of grip results: as can be seen from the results, the level of grip in mdx mice was significantly increased after long-term treatment with glycine.

The meanings of the respective symbols in FIGS. 7 to 11 are listed below: PMO-S: PMO and normal saline mixed group; PMO-Gly (PMO-G): PMO and glycine mixed group; TA: the tibialis anterior muscle; quadrriceps: quadriceps femoris; gasrocnemius: gastrocnemius muscle; the Triceps: the triceps brachii muscle; diaphragm: the diaphragm muscle; abdominal: the abdominal muscles; 20% C57: 10 μ g total protein from C57 tibialis anterior; 10% C57: 5 μ g total protein from C57 tibialis anterior; 5% C57: 2.5 μ g total protein from C57 tibialis anterior muscle; 1% C57: 0.5 μ g total protein from C57 tibialis anterior muscle; mdx: 50 μ g total protein from mdx tibialis anterior; α -actin: internal standard protein group (positive control group); q-quadriceps femoris; g-gastrocnemius muscle; t-triceps brachii; d-diaphragm muscle; a-abdominal muscles.

FIG. 12 shows the IHC results, where the green parts are Brdu, eMyhc and pax7, respectively, indicating that the proliferation potency of microsatellite cells after glycine treatment was higher than that of the saline control group. The red part is the laminin staining result and the blue part is the DAPI staining result.

FIG. 13 shows the results of flow analysis, which shows that the expression level of myosatellite cells in glycine-treated group was higher than that in saline control group.

FIG. 14 shows the IHC results, in which eMyhc and pax7 are shown in the green parts, respectively, to demonstrate that the recovery of the frozen muscle tissue is better and the proliferation of the myosatellite cells is higher than that of the normal saline control group after the glycine treatment. The labeled meanings in FIG. 12 are listed below: saline-saline group (placebo); gly-glycine group; gly + Gly: glycine is pretreated for 1 week before frostbite, and is injected once every other day; BF-45: regenerating muscle fibers; DAPI: DAPI staining results; laminin: performing Laminin dyeing result; merge: and (4) displaying the dyeing results in a superposition manner.

Fig. 15 shows the results of cell transplantation assay IHC (immunohistochemistry): the red part is the dystrophin positive muscle fiber for restoring expression; blue is cell nucleus staining, which shows that the survival rate and the transplantation efficiency of transplanted muscle cells are obviously increased after the treatment of the glycine; (TA) is tibialis anterior; the anterior tibialis mass is significantly increased.

Fig. 16 shows the results of cell transplantation assay IHC (immunohistochemistry): the red part is the dystrophin positive muscle fiber for restoring expression; blue is cell nucleus staining; the survival rate and the transplanting efficiency of the transplanted muscle satellite meat cells are obviously improved after the glycine treatment. TA is tibialis anterior; the anterior tibialis mass is significantly increased. NC of the right panel of fig. 16 refers to muscle-wasting control mice that were not cell-transplanted.

FIG. 17 is a flow analysis and quantitative statistical plot of labeled exosomes in differentiated C2C12 cells, showing that glycine promotes Exosome uptake during C2C12 differentiation.

Fig. 18 is an IHC staining and quantitative statistical plot of labeled exosomes in differentiated C2C12 cells, demonstrating that glycine promotes Exosome uptake during C2C12 differentiation.

FIG. 19 is a graph of siRNA (targeting GAPDH gene) uptake in differentiated C2C12 cells, western blot to identify GAPDH protein knockdown and quantitative statistics, showing that: glycine facilitates the uptake of siRNA during C2C12 differentiation. Where NC refers to normal glycine concentration (0.4 mM); PC refers to GAPDH siRNA transiently transfected with transfection reagents.

The label meanings in FIGS. 17-19 are listed below: 0.4mM Gly (0.4 mM): glycine group at a concentration of 0.4 mM; 0.8mM Gly (0.8 mM): glycine group at a concentration of 0.8 mM; gly free: glycine is not added; NC: a normal cultured cell control to which unlabeled fluorescent exosomes are added; BF: indicating a field; EXO: refers to the observation of an exosome with a fluorescent label (PKH67 label) added under a fluorescent microscope field of view; MERGE: superposing the pictures; NC-C2C 12: group without siRNA; PC-C2C 12: transiently transferring the siRNA group using a transfection reagent; NC-dC2C 12: differentiating C2C12 in a normal differentiation culture medium and adding siRNA; Gly-dC2C 12: adding glycine and siRNA to normally differentiated C2C12 cells; tublin: internal control protein (positive control) group.

Figure 20 is a glycine-facilitated uptake of short peptides in differentiated C2C12 muscle cells. After C2C12 cells are differentiated and cultured for 24 hours, glycine (the final concentration is 0.8mM) and 10 mu g of short peptide (green fluorescence labeling CP05) are added, and after differentiation and culture are induced for 72 hours, samples are collected to detect the short peptide uptake efficiency.

Fig. 21 is a statistical graph of muscle pathology, fibrosis and inflammation status identification and mouse grip, and the results show that glycine treatment effectively improves the muscle pathology and mouse grip function of aged mdx mice.

Figure 22 is a graph of pathological morphology in mice 3 weeks after treatment, HE staining results, showing that glycine treatment is effective in promoting improvement in muscle pathological morphology.

Figure 23 is a statistical analysis of muscle morphology and mass of mice 3 weeks after treatment, showing that glycine treatment improved muscle mass.

FIG. 24 shows the proliferation of myosatellite cells (IHC staining) and regeneration of myocytes (western blot and quantitative analysis) 3 weeks after treatment.

Gly in FIGS. 21-24 means glycine injection every other day after frostbite for 3 weeks; gly + Gly means glycine injection every other day for 1 week before cold injury; glycine was injected every 1 day after the cold injury for three weeks. Saline is the Saline group (blank).

FIG. 25 shows the IHC results. Beginning daily gavage of mdx mice 2 days before PMO injection (1%); at the same time, a 25mg/kg dose of PMO was injected via the tail vein system once a week in mdx mice for 3 weeks, and 2 weeks later, the samples were collected and tested. The test result shows that the PMO activity can be obviously promoted by feeding glycine.

FIG. 26 shows the results of IHC and western blot and quantitative analysis, which indicates that AGST is effective in promoting PMO drug activity. AG in the figure represents a mixture of aspartic acid and glycine; AGS represents a mixture of aspartic acid, glycine and serine; AGST denotes a mixture of aspartic, glycine and serine and tetrahydrofolic acid.

FIG. 27 shows the results of IHC and RT-PCR assays. The mdx mice were systemically injected with 5% glycine every other day, followed by a single injection of 50mg/kg of fluorescent labeled F-PMO one week after treatment, and harvested 48 hours later. The first and third rows of the leftmost column in the figure are eMyHC staining results, which are used to show regenerated muscle fibers; the second line and the fourth line in the leftmost column are FITC staining results, which refer to FITC fluorescently labeled PMO drugs; WGA indicates WGA staining results; DAPI is a DAPI staining result; merge is a superimposed result graph of the three staining results; PMO-S is a mixed group of PMO and normal saline; PMO-G is a mixed group of PMO and glycine; in the lower part of the electrophoretogram, -ve refers to the PCR reaction solution without template as negative control; c57 refers to wild type control; mdx refers to muscular dystrophy mouse control; g refers to gastrocnemius muscle; TA refers to the tibialis anterior.

Figure 28 is a validation test of upregulated expression of mTORC1 signaling pathway protein in skeletal muscle of glycine treated mdx mice. The results in the figure show that: in the glycine-treated group, the expression of phosphorylated mTOR (p-mTOR), S6K (p-S6K) and S6(p-S6) was significantly increased compared to the saline group, indicating that the mTORC1 pathway was further activated. p-mTOR/mTOR in the figure represents the ratio of phosphorylated mTOR to total mTOR protein; p-S6K/S6K represents the ratio of phosphorylated S6K to total S6K protein; p-S6/S6 represents the ratio of phosphorylated S6 to total S6 protein; p-mTOR means phosphorylated mTOR; mTOR means total mTOR protein; P-S6K represents phosphorylated S6K; S6K represents total S6K protein; P-S6 represents phosphorylated S6; s6 represents total S6 protein; alpha-actin refers to the internal reference protein actin (positive control).

FIG. 29 is a mixed local injection of mTORC1 inhibitor (5 μ g pp242) and 5% glycine into mdx mice to verify the modulation of the mTORC1 signaling pathway by glycine by detecting myosatellite cell proliferation and muscle regeneration; and further mixing with PMO for local injection to detect the expression of dystrophin protein. Compared with glycine-treated group (Gly), the number of PAX7 positive myosatellite cells and PAX7/Ki67 double-positive proliferating myosatellite cells is obviously reduced after co-injection of glycine and inhibitor pp242(Gly/pp242) in tibialis anterior muscles. The number of regenerated muscle fibers positive for BF-45 in the co-injected group of tibialis anterior (PMO-G/PP242) was significantly reduced for glycine, inhibitor PP242 and PMO compared to the glycine and PMO co-injected group (PMO-G); the number of dystrophin positive muscle fibers (red independent graph) and protein expression were significantly reduced, indicating inhibition of the mTORC1 pathway, affecting muscle regeneration and PMO activity. PMO-S is a group formed by mixing PMO and physiological saline.

Fig. 30 shows the results of local tests with different related substances in one carbon unit, which show that: co-injection of glycine and methotrexate (MTX-tetrahydrofolate reductase inhibitor) (MTX) in the tibialis significantly inhibited proliferation and number of regenerated muscle fibers (green) of PAX 7-positive and PAX7/Ki 67-double-positive proliferating muscle satellite cells compared to the glycine group (Gly). DAPI stained for nuclei. While the PAX7 positive myosatellite cells and PAX7/Ki67 double positive proliferating myosatellite cells and regenerating myofibers (green) in the formate (format) and Tetrahydrofolate (THF) treated groups were similar to those in the glycine treated group, but with slightly lower efficiency. Glycine metabolism can be inhibited by blocking the glycine decarboxylase (GLDC) key to glycine metabolism. The results show that: compared with the control group (SC), in the reduced GLDC group (shGLDC), the number (green) of PAX7 positive muscle satellite cells, PAX7/Ki67 double-positive proliferative muscle satellite cells and eMyHC positive regeneration muscle fibers is obviously reduced.

Figure 31 is a graph of the effect of glycine alone on a mouse model of muscular dystrophy. The results show that: the number of regenerated muscle fibers (green) was significantly increased in the glycine-treated group compared to the saline group (saline), but there was no significant improvement in the body weight and grip strength of the mice.

Detailed Description

The invention is further described with reference to the drawings and the specific examples, but the scope of the invention is not limited thereto. Unless otherwise indicated, the reagents used in the examples and experimental examples of the present invention are commercially available, and the specific experimental procedures are all routine in the art.

The terms of the present invention:

glycine: glycine

PMO: phosphoric acid diamide morpholine Oligomer (phosphoamidite Morpholino Oligomer, PMO)

2' Ome RNA: dioxymethyl ribonucleotides

B-MSP-PMO: cell shuttle peptides and muscle targeting peptide modified PMO

PNA: peptide nucleic acids

Peptide-PMO: peptide modified PMO

brdu: 5-bromodeoxyuridine

eMyhc: embryonic myosin heavy chain

Pax 7: transcription factor paired protein 7

western blot: western blot

dysprophin protein: dystrophin protein

And (3) IHC: immunohistochemistry

mdx mice: dystrophin deficient mice

mTORC1 is a form of mTOR (mammalian target of rapamycin) that exists in two complexes on the organism, mTORC1 and mTORC 2.

All percentage concentrations herein are mass to volume ratios, for example, the "final concentration of glycine of 5%" of experimental example 2 means that the mass to volume ratio of glycine is 5%.

Group 1 examples glycine drug delivery uses

The present group of embodiments provides for the use of glycine in the preparation of a drug delivery enhancer, characterized in that said glycine enhances drug delivery by activating mTORC 1.

In specific embodiments, the drug is selected from: nucleic acid drugs, and/or polypeptide drugs, and/or siRNA drugs, and/or drugs with exosomes as transport carriers.

In a more specific embodiment, the drug refers to a drug for treating a muscle-related disorder.

In further embodiments, glycine inhibits or enhances muscle satellite cells and muscle regeneration by blocking or providing a one-carbon unit.

In some embodiments, the nucleic acid drug is selected from PMO, 2' Ome RNA, PNA, or peptide-PMO or a plasmid; the polypeptide drug is selected from CP 05.

In a specific embodiment, the pharmaceutical formulation is an injection.

In a preferred embodiment, the glycine refers to a 5-10% glycine aqueous solution by mass volume ratio.

Group 2 example cell transplantation use of Glycine

The present set of embodiments provides for the use of glycine in the preparation of a cell transplantation reagent.

In a preferred embodiment, the cell transplantation reagent is a glycine aqueous solution with a mass volume ratio of 5% -10%;

in specific embodiments, the cell is selected from the group consisting of: a myosatellite cell, and/or a primary myocyte, and/or a mesenchymal stem cell.

Group 3 example, novel remedy for muscular diseases

The group of embodiments provides a novel medicament for treating muscle diseases, which is characterized by being a mixture of glycine and conventional medicaments for treating muscle diseases.

In a specific embodiment, the conventional muscle disease treating drug is a nucleic acid drug, the glycine is present in the mixture at a final concentration of 50mg/ml, and the conventional muscle disease treating drug is administered at a concentration of 25 mg/kg;

in more specific embodiments, the nucleic acid drug is selected from the group consisting of: PMO, 2' Ome RNA, PNA, or Peptide-PMO.

In some embodiments, wherein the glycine dosage form is selected from the group consisting of an oral dosage form or an injectable dosage form; the concentration of glycine in the injection is 50 mg/ml.

Group 4 example Glycine anti-aging use

The present group of embodiments provides for the use of glycine in the preparation of an anti-aging medicament.

In a specific embodiment, the dosage form of the anti-aging drug is selected from oral agents or injections.

Group 5 examples glycine muscle frostbite uses

The present group of embodiments provides the use of glycine in the preparation of a muscle freezing medicament.

In a specific embodiment, the frostbite medicine is glycine aqueous solution with the mass volume ratio of 5% -10%.

In some embodiments, the freezing medicament is in the form of an injection.

Group 6 example compositions for enhanced drug delivery

The present group of embodiments provides a composition for enhancing drug delivery, comprising: aspartic acid, glycine, serine and tetrahydrofolic acid.

In a preferred embodiment, the composition comprises the following components in parts by weight: 0.5-5 parts of aspartic acid, 0.5-10 parts of glycine, 0.5-5 parts of serine and 0.001-0.04 part of tetrahydrofolic acid.

In a more preferred embodiment, the composition comprises the following components in parts by weight: 3 parts of aspartic acid, 2.5 parts of glycine, 2.5 parts of serine and 0.002 part of tetrahydrofolic acid;

in other preferred embodiments, the composition is an aqueous solution comprising 3% by volume aspartic acid, 2.5% by volume glycine, 2.5% by volume serine, and 0.04% by volume tetrahydrofolic acid.

Experimental example 1

Mixing different small molecule compounds with PMO, wherein the total volume is 40 μ l, the final concentration of small molecule compound is 5% (mass/volume ratio), and the dosage of PMO is 2 μ g. Control group was a mixture of physiological saline and PMO by tibialis anterior intramuscular injection, administered in a single dose in adult mdx mice at 6-8 weeks, and sampled two weeks later for testing. Immunohistochemistry (IHC) and western-blot were used to detect the expression level of dystrophin protein. FIG. 1 shows the IHC result, the red part is the dystrophin protein, and from the IHC result, it can be seen that the dystrophin protein positive region of the glycine group is more than that of the other small molecule compound group. FIG. 2 shows the results of western-blot and quantitative statistical graphs, from which it can be seen that the expression level of the dystrophin protein is higher in the glycine group than in the other groups.

Experimental example 2

2.5%, 5% and 10% glycine, respectively, were mixed with 2 μ g PMO in a total volume of 40 μ l, with a final concentration of 2.5%, 5% or 10% glycine, respectively, and injected locally once in the anterior tibialis of mdx mice, two weeks later with sampling. Immunohistochemistry (IHC) was used to detect the expression level of the dystropin protein. FIG. 3 shows the IHC staining results and quantitative statistics that the red part is the dystrophin protein, and from the IHC results, it can be seen that the 5% glycine group is more than 2.5% of the area in the nucleic acid drug mixture that is positive for the dystrophin protein; similar to the 10% glycine group. FIG. 4 shows the results of western blot and the results of quantitative statistics, the results of quantitative analysis also showing: the glycine improves the PMO drug activity, and the 5% glycine group is superior to the 2.5% glycine group, and the effect is close to that of the 10% glycine group.

Experimental example 3

Glycine was mixed with 2 'Ome RNA, B-MSP-PMO, PNA and Pip5e-PMO, respectively, in a total volume of 40. mu.l, wherein the final concentration of glycine was 5% and 2' Ome 5. mu.g or B-MSP-PMO 1. mu.g or PNA 5. mu.g or Pip5e-PMO 1. mu.g was injected locally in the tibialis anterior. The control group was prepared by mixing physiological saline with each nucleic acid drug, and the others were identical to the glycine group except that only glycine was changed to physiological saline. One local injection was performed on each of the tibialis anterior of mdx mice, and two weeks later, samples were taken. Immunohistochemistry (IHC) was used to detect the expression level of the dystropin protein. FIG. 5 shows the IHC staining results and quantitative statistics, the red part is the dystrophin protein, and from the IHC results, it can be seen that the glycine group has more dystrophin protein positive regions in different nucleic acid drug mixtures than the control group. FIG. 6 shows the results of western blot and the results of quantitative statistics, the results of quantitative analysis also showing: the glycine group can improve the activity of the 4-structure antisense oligonucleotide drugs.

Experimental example 4

Glycine was mixed with PMO in a total volume of 120. mu.l, at a final glycine concentration of 5% and at a PMO concentration of 50mg drug/kg mouse body weight, and injected via the tail vein system. The control group was a mixture of physiological saline and PMO, and the others were the same as the glycine group except that only glycine was changed to physiological saline. The administration is repeated for three weeks in 13-month-old mdx mice once a week, during which glycine or physiological saline is administered separately every other day, and two weeks after the last administration, the mice are sampled and tested. Immunohistochemistry (IHC) and western blot were used to detect the expression level of dystrophin protein. FIG. 7 shows the IHC results, where the red part is the dystrophin protein, and it can be seen from the IHC results that the glycine group had more positive areas for the dystrophin protein in different muscles than the control group. FIG. 8 is a western blot result, from which it can be seen that the expression level of dystrophin protein in different muscles of the glycine group is higher than that of the control group.

Experimental example 5

Glycine and PMO were mixed in a total volume of 120. mu.l, a final concentration of glycine of 5% and a concentration of PMO of 25mg drug/kg mouse body weight, and injected via the tail vein system for long-term systemic treatment. The control group was a mixture of physiological saline and PMO, and the others were the same as the glycine group except that only glycine was changed to physiological saline. The preparation method comprises the following steps of administering the mdx mice in 6-8 weeks once a week, repeatedly administering for three weeks, independently administering glycine or physiological saline every other day, injecting PMO-glycine mixed solution or PMO-physiological saline mixed solution every month after three weeks, continuously injecting for 5 months, independently administering glycine or physiological saline every other week, and sampling and detecting after two weeks of the last administration. Immunohistochemistry (IHC) and western-blot were used to detect the expression level of dystrophin protein. FIG. 9 shows the IHC results, where the red part is the dystrophin protein, and it can be seen from the IHC results that the glycine group had more positive areas for the dystrophin protein in different muscles than the control group. FIG. 10 shows the results of western-blot, from which it can be seen that the expression level of dystrophin protein in different muscles is higher in the glycine group than in the control group. FIG. 11 is a graph showing the results of the grip test (procedures of the grip test can be referred to as "Han et al. Nature Communications (2016)7: 10981"), from which it can be seen that the grip level of mdx mice is significantly improved after the glycine treatment for a long period of time.

All Immunohistochemistry (IHC) and western-blot experimental procedures herein are experimental procedures well known to those skilled in the art (Han et al. Nature Communications (2016)7: 10981).

Experimental example 6

Glycine and water were mixed to make a 5% strength solution, the total volume being 120. mu.l. Injections were given via the tail vein system. The control group was a physiological saline solution group. The drug is administered once a week to adult mdx mice of 6-8 weeks of age, and the drug is repeated for 5 weeks, and the sample is taken two days after the last drug administration. The proliferation level of the myosatellite cells was determined by measuring the expression level of pax7 protein using Immunohistochemistry (IHC) and flow cytometry. FIG. 12 shows the IHC results, where the green signal portions are brdu, eMyhc, and pax7 from top to bottom; brdu is a cell proliferation index, and the higher the expression is, the stronger the proliferation capacity is shown; the eMyhc is an index of new muscle fibers, and the higher the expression is, the more the number of new muscle fibers is; pax7 is a muscle satellite cell marker, and higher expression indicates that the number of muscle satellite cells is higher, so that the IHC result shows that the proliferation level of muscle satellite cells of the glycine group is higher than that of the control group. FIG. 13 shows the results of flow analysis, which shows that the expression level of myosatellite cells in glycine-treated group was higher than that in saline control group.

Experimental example 7

Glycine and water were mixed to make a 5% strength solution, the total volume being 120. mu.l. Injections were given via the tail vein system. The control group was a physiological saline solution group. Two control groups were set, the first: glycine is injected into the upper tail vein of an adult wild type mouse after the frostbite once a week for 1 week, and sampling and detection are carried out two days after the last injection; second group: glycine was administered to wild type mice in advance every other day for one week, and after continuous injection, frostbite modeling was performed. Glycine was injected into the tail vein of a frostbitten adult wild-type mouse once a week for 1 week continuously, and the samples were collected and examined two days after the last injection. The proliferation level of the myosatellite cells was determined by measuring the expression level of pax7 protein using immunohistochemical technique (IHC). FIG. 14 shows the IHC results, where the green components are eMyhc and pax7, eMyhc is the index for neogenic muscle fibers, and higher expression indicates a greater number of neogenic muscle fibers; pax7 is a marker for myosatellite cells, and higher expression indicates a higher number of myosatellite cells; from the IHC results, the proliferation level of the muscle satellite cells was higher in the glycine group than in the control group, in which the proliferation of the muscle satellite cells was more promoted by the pre-injection of glycine.

Experimental example 8

Glycine and water are prepared into a solution with the concentration of 5-10% (mass-volume ratio), and the total volume is 120 mu l. Injections were given via the tail vein system. The control group was a physiological saline solution group. The experimental design was as follows: pre-systematically injecting 5% glycine or normal saline for 1 time every other day, continuously injecting for one week, performing CTX induction treatment, then performing myoblast transplantation experiment of in vitro separation on the injured muscle, continuously systematically injecting glycine or normal saline after transplantation is completed, continuously injecting for 1 week or 3 weeks, sampling and detecting. The cell transplantation efficiency was examined by measuring the expression level of the dystrophin protein using Immunohistochemical (IHC) technique. FIG. 15 shows the immunohistochemical results of cell transplantation test, with red part representing restored expression of dystrophin positive muscle fibers and blue part representing nuclear staining, indicating that the transplantation efficiency is significantly enhanced after glycine treatment; (TA) is tibialis anterior; the anterior tibialis mass is significantly increased.

Experimental example 9

FIGS. 16-19 show that 0.8mM glycine facilitates transport of exosomes and siRNAs (targeting the GAPDH gene) in differentiated C2C12 cells (dC2C12), respectively: after the C2C12 cells were cultured for 24 hours in normal differentiation, glycine (final concentration: 0.8mM) and siRNA were added, and after 48 hours in induced differentiation culture, samples were collected to examine the knockdown efficiency of GAPDH protein.

Experimental example 10

The effect of glycine on anti-aging is as follows: FIG. 20 is a systematic test of 5% glycine on aged mdx mice. The injection is systemically injected every other day for 5 weeks, and the sample is taken two days after the last injection.

Experimental example 11

Data for glycine in terms of frostbite: results after 3 weeks of glycine treatment: systematically injecting 5% glycine every other day after the frostbite, continuously injecting for 3 weeks, and sampling and detecting two days after the last injection; gly + Gly group was glycine induction treatment for 1 week before frostbite (injection every other day), and continuous injection for 3 weeks after frostbite, as above. As shown in fig. 21-24, the results indicate that glycine treatment is effective in promoting improvement in muscle pathological morphology, and that glycine treatment promotes improvement in muscle mass.

Experimental example 12

Data for glycine fed to promote nucleic acid drug activity: beginning daily gavage of mdx mice 2 days before PMO injection (1%); at the same time, a 25mg/kg dose of PMO was injected via the tail vein system once a week in mdx mice for 3 weeks, and 2 weeks later, the samples were collected and tested. As shown in fig. 25, the results indicate that feeding glycine also significantly promoted PMO activity.

Experimental example 13

Glycine acts as an aid to drug delivery as one of the AGST complexes: AGST refers to: 3% aspartic acid + 2.5% glycine + 2.5% serine + 0.04% tetrahydrofolic acid. The PMO dose was 2. mu.g. Control group was a mixture of physiological saline and PMO by intratibialis injection, administered in a single dose in adult mdx mice 6-8 weeks old, and sampled two weeks later for testing. As shown in fig. 26, the results indicate that AGST can effectively promote the pharmaceutical activity of PMO, and AGST can more efficiently transport more PMO to the target cell, thus effectively promoting the pharmaceutical activity of PMO.

Experimental example 14

To demonstrate that glycine promotes the transport and uptake and activity of fluorescently labeled PMO in regenerating muscle fibers by promoting muscle regeneration and fusion, the following experiments were performed herein: the mdx mice were systemically injected with 5% glycine every other day, treated for one week and then subjected to a single injection of 50mg/kg fluorescence labeled FITC-PMO, followed by a sample collection test after 48 hours, and their muscle regeneration and expression of FITC-PMO in the nascent fiber were examined, as well as their exon skipping efficiency. Results as shown in fig. 27, IHC results indicate that glycine is able to significantly promote muscle regeneration and has significant fluorescently labeled PMO co-localized expression in the nascent fibers. RT-PCR detection results show that the activity of PMO is remarkably improved by glycine.

Experimental example 15

This experimental example demonstrates a new mechanism of action of glycine on muscle stem cells: glycine enhances myocyte proliferation by activating mTORC1 and providing a one-carbon unit. Glycine was demonstrated to be a novel activator of the mTORC1 signaling pathway. The specific experimental procedures of this experimental example are as follows: mdx mice are subjected to mixed local injection of mTORC1 inhibitor (5 mug pp242) and 5% glycine, the regulation of a mTORC1 signal channel by glycine is verified by detecting the muscle satellite cell expression condition and the muscle regeneration condition of the mdx mice, the mdxC mice are further subjected to mixed local injection with PMO, and the PMO activity is improved by regulating the mTORC1 signal channel by glycine by detecting the expression condition of dystrophin protein. Results are shown in fig. 28 and 29: the systemic injection of 5% glycine can remarkably promote the activation of mTORC1 signal channel, and the added local detection of mTOR inhibitor remarkably inhibits the effect of glycine on promoting the proliferation of muscle satellite cells and the muscle regeneration, thereby inhibiting the effect of glycine on PMO activity.

Meanwhile, in the experimental example, different carbon unit related substances are selected for local testing: respectively co-injecting glycine, 0.2 mu g of tetrahydrofolic acid and 0.2 mu g of methotrexate on tibialis anterior muscles, and collecting samples after 3 days for detection; separately, 0.4. mu.g of tetrahydrofolic acid and 5% sodium formate were injected into the tibialis anterior and examined 3 days later. The influence of the one-carbon unit on the action mechanism of the glycine is determined by detecting the proliferation and muscle regeneration conditions of the muscle satellite cells, and meanwhile, GLDC (GLDC), which is a key enzyme of glycine metabolism, is knocked down in a body, so that the important influence of the one-carbon unit channel on the action of the glycine is further verified. As shown in fig. 30, the results show that: by blocking and providing other one-carbon unit carriers, the proliferation of muscle satellite cells and muscle regeneration can be inhibited or enhanced; and the inhibition of the glycine metabolism can obviously inhibit the proliferation of muscle satellite cells and the muscle regeneration.

Experimental example 16

The mdx mice were systemically injected with 5% glycine every other day, and after one week of treatment, the mice were subjected to a sample collection test to detect the systemic regeneration of each muscle and the muscle function, i.e., the recovery of grip strength. As shown in fig. 31, the results show that: after one week of treatment, glycine significantly promotes muscle regeneration of each muscle tissue, but has no significant effect on improving muscle quality and function, i.e., grip strength.

Claims

1. Use of glycine in the preparation of a drug delivery enhancer, wherein said glycine enhances drug delivery by activating mTORC 1; the drug is selected from: nucleic acid drugs, polypeptide drugs or drugs with exosomes as transport carriers.

2. Use of glycine according to claim 1 for the preparation of a drug delivery enhancing agent, wherein said drug is a drug for the treatment of muscle related disorders.

3. Use of glycine according to claim 2 for the preparation of a drug delivery enhancer, wherein glycine enhances muscle satellite cell proliferation and muscle regeneration by providing one carbon unit.

4. Use of glycine for the preparation of a drug delivery enhancing agent according to claim 1 wherein said nucleic acid based drug is selected from PMO, 2' Ome RNA, PNA, siRNA, peptide-PMO or plasmid; the polypeptide drug is selected from CP 05.

5. Use of glycine according to any one of claims 1 to 4 in the preparation of a drug delivery enhancer wherein the drug is in the form of an injection.

6. Use of glycine in the preparation of a drug delivery enhancer according to any one of claims 1 to 4 wherein the delivery of nucleic acid drugs or polypeptide drugs is enhanced by an aqueous glycine solution having a mass to volume ratio of 2.5% to 10%.

7. Use of glycine according to any one of claims 1 to 4 for the preparation of a drug delivery enhancer, characterized in that 0.8mM glycine enhances the delivery of siRNA drugs, or drugs with exosomes as transport vehicles;

the use comprises the following steps: glycine is injected simultaneously with the drug, or glycine is injected first and then the drug is injected, or glycine is fed first and then the drug is injected.

8. Use of glycine for the preparation of a cell transplantation reagent, wherein glycine promotes cell transplantation regeneration by promoting restoration of expression of a dystrophin protein by transplanted cells; the cell is selected from: a myosatellite cell, and/or a primary myocyte.

9. The use according to claim 8, wherein the cell transplantation reagent is 2.5-10% by mass/volume glycine aqueous solution.

10. Use according to claim 8 or 9, characterized in that it comprises: glycine is injected or fed first and then cell transplantation is carried out, or glycine is injected at the same time of cell transplantation.

11. A composition for enhancing drug delivery, comprising: aspartic acid, glycine, serine and tetrahydrofolic acid.

12. The composition of claim 11, comprising the following components in parts by weight: 0.5-5 parts of aspartic acid, 0.5-10 parts of glycine, 0.5-5 parts of serine and 0.001-0.04 parts of tetrahydrofolic acid.

13. The composition of claim 12, comprising the following components in parts by weight: 3 parts of aspartic acid, 2.5 parts of glycine, 2.5 parts of serine and 0.002 part of tetrahydrofolic acid.

14. The composition of claim 13, wherein the composition is an aqueous solution comprising 3% by mass/volume aspartic acid, 2.5% by mass/volume glycine, 2.5% by mass/volume serine, and 0.002% by mass/volume tetrahydrofolic acid.