NL2031346B1 - Use of glucagon-like peptide-1 (glp-1) in preparation of drug for treating male hypogonadism syndrome - Google Patents
Use of glucagon-like peptide-1 (glp-1) in preparation of drug for treating male hypogonadism syndrome Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/26—Glucagons
Abstract
The present disclosure provides use of glucagon—like peptide—l (GLP—l) in preparation of a drug for treating male hypogonadism syndrome, belonging to the technical field of medicine. The GLP—l increases a serum testosterone (T) content and induces differentiation of Leydig cells (LCs), which is safe to use and has no obvious toxic and side effects.
Description
P1257 /NLpd
USE OF GLUCAGON-LIKE PEPTIDE-1 (GLP-1) IN PREPARATION OF DRUG FOR
TREATING MALE HYPOGONADISM SYNDROME
The present disclosure belongs to the technical field of med- icine, and in particular relates to use of glucagon-like peptide-1 (GLP-1) in preparation of a drug for treating male hypogonadism syndrome.
Hypogonadism syndrome, occurring frequently in males accompa- nied by testicular hypofunction, is associated with the decline of
Leydig cells (LCs). The LCs as cells that synthesize and secrete testosterone (T) are the main source of androgens in males. In se- rum, the T is produced by the LCs stimulated by luteinizing hor- mone secreted in the pituitary gland and regulated by a series of negative feedback mechanisms.
At present, the hypogonadism syndrome is mainly treated through a T supplementation therapy clinically; however, the ther- apy has significant safety concerns.
To solve the above problems, the present disclosure provides use of GLP-1 in preparation of a drug for treating male hypogonad- ism syndrome. The GLP-1 increases a T content, and induces differ- entiation of LCs with safety.
The present disclosure provides use of GLP-1 in preparation of a drug for treating male hypogonadism syndrome. The GLP-1 in- creases a T content in a serum or testis, and induces differentia- tion of LCs, which is safe to use and has no obvious toxic and side effects. Experiments show that: the GLP-1 significantly in- creases the T content in the serum of EDS model rats, and after continuous administration for 14 d, there is no significant change in a body weight and a testis wet weight of the rats, indicating a safe medication; meanwhile, the GLP-1 also induces differentiation of the LCs, up-regulates expression levels of key genes Scarbl,
Cypllal and Hsdllbl in androgen synthesis pathways, up-regulates expression levels of key proteins SCARB1, CYP11A1 and HSD11B1, and up-regulates expression levels of key proteins EPACl and pMEK1/2.
FIG. 1 shows differentiation results of LCs stimulated by
GLP-1 in Example 1; where A is a scheme for treating seminiferous tubules; B to D are staining of LCs on a surface of the seminifer- ous tubules by HSD3B1, where B is a BM treatment group, C is a DM treatment group, and D is a DM+30 nM GLP-1 treatment group; E is an effect of each treatment in Example 1 on the number of HSD3B1* cells; and F is an effect of each treatment in Example 1 on a T content;
FIG. 2 shows an effect of GLP-1 in Example 2 on expression of genes related to androgen synthesis pathways in LCs;
FIG. 3 shows an effect of the GLP-1 in Example 2 on expres- sion of proteins related to the androgen synthesis pathways in
LCS;
FIG. 4 shows an effect of GLP-1 in Example 3 on contents of the T, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in a serum of EDS model rats; where A is an operation plan of the EDS model; B is a T content in the serum of EDS model rats after administration; C is an LH content in the serum of EDS model rats after administration; and D is an FSH content in the serum of
EDS model rats after administration;
FIG. 5 shows an effect of the GLP-1 in Example 3 on the num- ber of LCsCYP11A1 positive cells and HSD11B1l positive cells in the
EDS model rats after administration; where A is expression of
HSD11B1 in cells of a control group (0 ng/testis GLP-1) after staining; B is expression of HSD11B1 in cells of a 100 ng/testis
GLP-1 group after staining; C is the number of cells expressing the HSD11B1 in groups of 0 ng/testis, 10 ng/testis and 100 ng/testis GLP-1 after staining; D is expression of CYP11Al in cells of the control group (0 ng/testis GLP-1) after staining; E is expression of CYP11A1 in cells of the 100 ng/testis GLP-1 group after staining; F is the number of cells expressing the CYP11A1 in the groups of 0 ng/testis, 10 ng/testis and 100 ng/testis GLP-1 after staining; and arrows indicate LCs expressing the HSD11B1, and triangles indicate LCs expressing the CYP11Al, with a scale bar = 50 um;
FIG. 6 shows an effect of GLP-1 in Example 4 on expression levels of related genes in androgen synthesis pathways in the LCs after administration to the EDS model rats;
FIG. 7 shows an effect of the GLP-1 in Example 4 on expres- sion levels of related proteins in the androgen synthesis pathways in the LCs of EDS model rats; where A is a gel strip; B is results of Western blotting (WB) detection of expression levels of SCARBI,
CYP11A1, HSD3B1 and HSD11B1 in rat ILCs after administration of EDS and different concentrations of GLP-1 (0, 10 and 100 ng/testis) (where B-actin (ACTB) is an internal reference protein); C is a difference in expression levels of HSD11Bl and CYP11A1 proteins in testis detected by an immunohistochemical semi-quantitative method after administration of EDS and the different concentrations of
GLP-1 (0, 10 and 100 ng/testis); and arrows indicate LCs express- ing the HSD11B1 protein, and triangles indicate LCs expressing the
CYP11Al protein; and
FIG. 8 shows an effect of the GLP-1 in Example 4 on expres- sion levels of phosphorylated proteins and total proteins thereof in LCs of EDS model rats after administration; where A is the gel strip; B is a result of WB detection of EPAC1 expression level in rat LCs after administration of EDS and different concentrations of the GLP-1 (0, 10 and 100 ng/testis) (the ACTB is the internal reference protein); C is a result of WB detection of expression levels of MEK1/2 and its phosphorylated protein in rat LCs after administration of EDS and different concentrations of the GLP-1 (0, 10 and 100 ng/testis) (the ACTB is the internal reference pro- tein); and D is a result of WB detection of expression levels of
ERK1/2 and its phosphorylated protein in rat LCs after administra- tion of EDS and different concentrations of the GLP-1 (0, 10 and 100 ng/testis) (the ACTB is the internal reference protein).
The present disclosure provides use of GLP-1 in preparation of a drug for treating male hypogonadism syndrome. An active in- gredient of the drug is the GLP-1; the drug increases a serum T content, induces differentiation of LCs, and relieve or treat the hypogonadism syndrome; no obvious toxic and side effects are found after GLP-1 administration.
The GLP-1 in examples is a recombinant human GLP-1 of Pepro- tech (product number: 130-08).
The GLP-1 further includes aggregates formed by a plurality of GLP-1s being aggregated in an automatic manner (such as physi- cal adsorption); it should be understood as a spontaneous natural phenomenon of biology, or a form in which several GLP-1s are li- gated by genetic engineering.
The GLP-1 further includes a linker, that is, the GLP-1s are artificially ligated to each other using a biological or chemical ligation technology; it should be understood as a natural phenome- non that is different from living things, but achieved through hu- man intervention.
In the present disclosure, conventional dosage forms in the art can be used, not limited to aqueous injections, powder injec- tions, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols , sprays, dry powder inhalations, sustained-release agents and controlled-release agents.
In the present disclosure, conventional adjuvants in the art can be used, not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubri- cants; adjuvants to be compatible with the materials can also be used, such as emulsifiers, solubilizers, bacteriostatic agents, analgesics and antioxidants; such adjuvants can effectively im- prove the stability and solubility of a compound or change a re- lease rate and absorption rate of the compound, thereby metaboliz- ing the various compounds in the body to enhance a drug delivery effect. The adjuvants can also be used to achieve specific admin- istration purposes or methods, such as sustained-release admin- istration, controlled-release administration, and pulse admin- istration, including gelatin, albumin, chitosan, polyether and polyester polymer materials, polyethylene glycol, polyurethane,
polycarbonate and its copolymers.
When the drug is an aqueous injection, the adjuvants include isotonic agents and buffers, as well as necessary emulsifiers, solubilizers and bacteriostatic agents. In addition, the drug fur- 5 ther includes preferably other pharmaceutically acceptable pharma- ceutical adjuvants, such as antioxidants, pH adjusters and pain relievers.
When the drug is an oral liquid preparation, the adjuvants include solvents, flavoring agents, bacteriostatic agents, emulsi- fiers and colorants.
When the drug is a tablet, the adjuvants include fillers, binders, disintegrants and lubricants.
When the drug is an emulsion, the adjuvants include water, oil, emulsifiers, and necessary preservatives and flavoring agents.
When the drug is a granule, the adjuvants are similar to the tablets, with a different granulation process.
When the drug is a capsule, the adjuvants are similar to the granules and the granules and glidants are mixed into capsules.
The present disclosure provides use of GLP-1 in preparation of a drug for inducing LCs differentiation, increasing a T content in a serum and/or testis, up-regulating levels of key genes
Scarbl, Cypllal and Hsdilbl, or up-regulating contents of key pro- teins SCARB1, CYP11A1 and HSD11B1. The GLP-1 does not change the number of LCs, but can play a role through EPAC1 and pMEK1/2 path- ways in the LCs to significantly promote the differentiation of
LCs, thereby promoting the T secretion to significantly increase the T level in serum and testis.
In order to further illustrate the present disclosure, the technical solutions provided by the present disclosure are de- scribed in detail below in connection with examples, but these ex- amples should not be understood as limiting the claimed scope of the present disclosure.
Example 1
Materials included: a Corning 12-well plate, a T radioimmuno- assay kit, recombinant human GLP-1, ITS, LH, EDS, DMEM/F12, a Bio-
Rad cDNA synthesis kit and a Bio-Rad SYBR fluorescent dye.
Male Sprague-Dawley rats (90-day-old, 250420 g/rat) were in- traperitoneally injected with the EDS (75 mg/kg) 7 d before the experiment, sacrificed with CO:; testis was removed in a phosphate- buffered saline (PBS) at 4°C, and testicular capsule was cut off, seminiferous tubules were separated into single ones and divide into 4 groups in total in a 12-well plate: a BM group: the seminiferous tubules were cultured in a com- mon medium (BM) for 3 weeks, and the medium was changed every 3-4 d; a DM group: the seminiferous tubules were cultured in the BM for 7 d, and the medium was changed every 3-4 d; and the medium was changed to a differentiation-promoting medium (DM), and the medium was changed every 3-4 d, and GLP-1 was not added to the DM; a DM+3 nM GLP-1 group: the seminiferous tubules were cultured in the BM for 7 d, and the medium was changed every 3-4 d; and the medium was changed to the DM, and the medium was changed every 3-4 d, and 3 nM GLP-1 was added to the DM; and a DM+30 nM GLP-1 group: the seminiferous tubules were cul- tured in the BM for 7 d, and the medium was changed every 3-4 d; and the medium was changed to the DM, and the medium was changed every 3-4 d, and 30 nM GLP-1 was added to the DM.
The BM was prepared by: a M192 solution and a DMEM/F12 solu- tion were mixed at a volume ratio of 1:1, a double antibody (100
U/mL penicillin and 100 pg/mL streptomycin) was added, followed by adjusting pH to 7.2 and filtration through a 0.2 pM sterile fil- ter, and 1 g/L ITS was added. The M199 solution was: 500 mL of double distilled water + 1.0 g of BSA + 2.2 g of NaHCO; + 4.2 g of
HEPES + or 9.5 g of M199 powder were mixed and diluted to 990 mL; the DMEM/F12 solution was to replace the M199 powder in the M199 solution with the DMEM/F12.
DM was: LH (5 ng/ml) and lithium ion (LI, 5 mmol/L) were add- ed to the BM.
After the test, the T to be tested was collected in the medi- um; the seminiferous tubules were collected for HSD3B1 staining (marking differentiated LCs): the seminiferous tubules were placed on a glass slide, air-dried naturally with a hairdryer, an appro- priate amount of an HSD3B1l enzyme cytochemical staining solution was quickly added dropwise, and the staining solution was circled using an immunohistochemical pen; the glass slide was placed in a wet box, followed by staining at 34°C in the dark for 45 min to 120 min (the staining solution included: a solution A: 1 mg of
NBT+0.6 mg of DHEA+0.6 ml of DMSO; and a solution B: 10 mg of $-
NAD" +9.5 ml of D-PBS; before staining, the A and B solutions were mixed, shook well, and dropped on a cell smear); after staining, the samples were rinsed with distilled water, and placed in a fix- ative (10% Formalin in D-PBS with 5% Sucrose, pH 7.4) for 10 min, the fixative was washed off with the D-PBS, followed by mounting with 50% glycerol (Glycerol: PBS=1: 1, V/V); observation and pho- tographing were conducted under a microscope, the number of blue- purple cells (differentiated cells) were recorded around the semi- niferous tubules, and a differentiation percentage of the LCs was calculated as a ratio of cell number/tubule area (cm?) . The detec- tion results are shown in FIG. 1.
As can be seen from FIG. 1, after 3 weeks of BM culture, there are no HSD3Bl-positive mesenchymal cells (FIG. 1-B); in the
DM group (FIG. 1-C}) and the DM+30 nM GLP-1 group (FIG. 1-D), in- duction differentiation results in the HSD3Bl-positive mesenchymal cells. Compared with the DM (FIG. 1-C} and BM (FIG. 1-B) groups, the GLP-1 group (FIG. 1-D) significantly increases the number of
HSD3B1-positive mesenchymal cells (FIG. 1-E).
After culturing the seminiferous tubules in BM for 3 weeks, the T level is almost 0 (FIG. 1-F). After 3 weeks of culture in
DM, T can be produced; the 3 nM GLP-1 and the 30 nM GLP-1 further increase the T production compared to the DM group (FIG. 1F). This indicates that the GLP-1 stimulates the differentiation of stem
Leydig cells (SLCs) into the ILCs line, thereby inducing the pro- duction of T. A same letter indicates no difference between the groups (p > 0.05), and different letters indicate significant dif- ference between the groups (p < 0.05).
Example 2
According to the treatment method in Example 1, a single sem- iniferous tubule of a male rat was isolated and evenly divided in- to a 12-well plate, and BM was added. Incubation was conducted for 3 weeks at 37°C, 5% CO,. In the first week, culture was conducted with only BM free of GLP-1; on the 2nd to 3rd week of culture, the medium was replaced with DM, 3 nM and 30 nM GLP-1s were added, the medium was changed every 3-4 d, seminiferous tubules were collect- ed to extract RNA, 1 ug of gRNA was reverse transcribed at 42 °C for 30 min using a reverse transcription kit (Promega), followed by treating at 85°C for 5 min to terminate the reaction, to obtain a 40S ribosomal protein S16 gene (Rpslé and Rpsl6 were used as an internal reference); the expression of related genes Lhcgr,
Scarbl, Star, Cypllal, Hsd3bl, Cypl7al, Hsd17b3, Srdsal and
Hsdilbl as well as proteins LHCGR, SCARB1, STAR, CYP11A1, HSD3B1,
CYP17A1, HSD17B3, SRD5A1 and HSD11B1l in the T synthesis pathway was detected by QPCR and WB. The detection results are shown in
FIG. 2 and FIG. 3.
As can be seen from FIG. 2, the GLP-1 significantly up- regulates the expression levels of genes Scarbl, Cypllal, Hsd3bl and Hsdllbl, but does not affect the expression levels of other androgen synthesis-related genes Lhcgr, Star, Cypl7al, Hsd17b3 and
Srd5al. It shows that the GLP-1 selectively up-regulates the ex- pression levels of key genes in the androgen synthesis pathway of
LCs to promote the differentiation of LCs.
As can be seen from FIG. 3, compared with the control group, the GLP-1 significantly up-regulates the expression levels of pro- teins SCARB1, CYP11A1, HSD3Bl and HSD11B1, which is consistent with changes in their corresponding mRNA levels.
Example 3 18 90-day-old male Sprague-Dawley rats were injected intra- peritoneally with 75 mg/kg of EDS (prepared for immediate use, dissolved in a solution of DMSO: saline = 1:3) to kill LCs to cre- ate a T-free serum model; the rats were randomly divided into 3 groups with 6 in each group: 1) a group of GLP-1-0 ng/testis/day (as a 0.9% saline control group); 2) a group of GLP-1-10 ng/testis/day (GLP-1 dissolved in 0.9% saline); and 3) a group of
GLP-1-100 ng/testis/day (GLP-1 dissolved in 0.9% saline).
After 14 d of EDS treatment (T was zero at this time), each rat was injected into the testis for administration every day, with an administration volume of 50 pL; during the period of 14 d to 28 d after the EDS treatment, continuous daily administration was conducted.
After the experiment, serum and testes were collected. The serum was used to detect the levels of T, LH and FSH; one testis was stored at -80°C for RNA extraction to conduct QPCR and WB; the other testis was fixated in a Bouin's fixative for immunohistolog- ical examination in paraffin section. The detection results were shown in FIG. 4 to FIG. 8. The Bouin's fixative was prepared by: 2 clean and dry 100 mL glass bottles were prepared; 100 mL of dou- ble-distilled water was added to 1-2 g of picric acid and mix well to obtain a saturated picric acid solution; 75 mL of the saturated picric acid solution, 25 mL of formaldehyde, and 5 mL of glacial acetic acid were mixed well, and stored at room temperature for later use.
It can be seen from FIG. 4 that the GLP-1 significantly in- creases the T content in the serum of EDS model rats, but has no significant effect on the LH and FSH in serum. It shows that the
GLP-1 can increase the T level in the serum of EDS model rats, which can be used for the treatment of low-T syndrome caused by the testis, and has no effect on the functions of the pituitary and hypothalamus.
As can be seen from FIG. 5, the GLP-1 does not increase or decrease the number of CYP11A1-positive cells and HSD11B1-positive cells. This shows from the side that the GLP-1 increases the level of androgen (T) by not simply increasing the number of cells, but improving the function of cells.
As can be seen from FIG. 6, the GLP-1 significantly up- regulates the expression levels of genes Scarbl, Cypllal, Hsd3bl and Hsd11b1, but does not affect the expression levels of other androgen synthesis-related genes Lhcgr, Star, Cypl7al, Hsdl7b3 and
Srdbal. It shows that the GLP-1 selectively up-regulates the ex- pression levels of key genes in the T synthesis pathway of LCs to promote the differentiation of LCs. The results are consistent with those of the in vitro test of Example 2.
As can be seen from FIG. 7, compared with the control group, the GLP-1 significantly up-regulates the expression levels of pro- teins SCARB1, CYP11A1, HSD3B1 and HSD11Bl, which is consistent with changes in their corresponding mRNA levels.
It can be seen from FIG. 8 that GLP-1 can up-regulate the ex- pression level of EPAC1l protein; meanwhile, the GLP-1 up-regulates the expression level of MEK1/2 phosphorylated protein (pMEK1/2), thereby up-regulating a ratio of pMEK1l/2 and MEK1/2; the GLP-1 al- so up-regulates the expression level of total ERK1/2 protein, in- dicating that the GLP-1 can act through EPACl, MEK1/2 and ERK1/2.
Example 4
An in vivo administration schedule was the same as in Example 3. At the end of the test, the rats were weighed and dissected, and bilateral testis and epididymis were taken to weigh and record a wet weight, and the test results were shown in Table 1.
Table 1 Effects of GLP-1 on body weight and weights of testis and epididymis of rats
Parameter Dosage (ngftestis) 0100 “Body weight (8) ~~ 313.7#40.11 3246£13.9 293.8¢7.514 “Testisweight (g) ~~ 1.164$0.1103 1.09+0.08344 094:0.07613
Epididymis weight (g) ~~ 0.354:0.0275 0.241:0.0324 0.2430.03521
Mean + SEM, n = 6-12
It can be seen from Table 1 that: after continuous admin- istration for 14 d, there is no statistical significance in the comparison of body weight and testis wet weight of rats in each group (P > 0.05), indicating that the GLP-1 does not affect the body weight and the weights of testis and epididymis of rats, such that the GLP-1 is safe for administration with no adverse effects on the rats.
Although the present disclosure has been described in detail through the above examples, the examples are only a part rather than all of the examples of the present disclosure. All other ex- amples obtained by persons based on these examples without crea- tive efforts shall fall within a protection scope of the present disclosure.
Claims (1)
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