CN111821292A - Application method of nafamostat mesylate in treatment of chlamydia genital tract infection - Google Patents

Abstract

The invention relates to the technical field of biological medicines, in particular to an application method of nafamostat mesilate in treating chlamydia genital tract infection, which comprises the following steps: A. selecting an experimental material; B. making an experimental method; C. observing an experimental result; D. and (5) obtaining an experimental conclusion. The invention provides a new application of nafamostat mesilate in treating chlamydia genital tract infection, in particular to an application of nafamostat mesilate in preparing a medicament for treating chlamydia genital tract infection, and an application of nafamostat mesilate in preparing a medicament for preventing fallopian tube obstruction, hydrosalpinx and tubal infertility caused by chlamydia genital tract infection.

Description

Application method of nafamostat mesylate in treatment of chlamydia genital tract infection

Technical Field

The invention relates to the technical field of biomedicine, in particular to an application method of nafamostat mesilate in treating chlamydia genital tract infection.

Background

Chlamydia (Chlamydia) is a group of very small, non-motile, intracellularly growing microorganisms, and urogenital infections caused by them are the most common sexually transmitted diseases worldwide, with about 1.272 million new patients worldwide per year according to WHO statistics^[1]. Since the clinical manifestations of chlamydial infections are nonspecific, especially among women infected, asymptomatic individuals account for approximately 80% of the population, and are therefore often missed in clinical diagnosis. If the patient is not diagnosed and treated in time, the patient is easy to develop chronic and persistent infection, which causes the injury, adhesion and scar formation of the genital tract tissue, and then causes various complications such as tubal infertility, pelvic inflammation, ectopic pregnancy and the like^[2-4]And seriously affects the female reproductive health. In addition, genital tract chlamydia infection increases the risk of cervical cancer and AIDS^[5,6]It brings a heavy burden to the family and society.

Currently, according to the consensus of diagnosis and treatment of chlamydial genital tract infection recommended by WHO, azithromycin and doxycycline are first-line drugs for treating chlamydial genital tract infection, and although most chlamydial genital tract infections can be cured by antibiotic therapy, reports of antibiotic treatment failure have been increasing in recent years due to the emergence of multidrug-resistant chlamydia^[7,8]. Due to the broad spectrum of broad-spectrum antibioticsThe widespread use and the general use of empirical therapy during diagnosis and treatment may lead to the emergence of more multidrug-resistant chlamydiae in the future^[9]. Therefore, before the emergence of effective chlamydia vaccines, it is urgent to find other effective drugs capable of treating chlamydia genital tract infection and preventing the occurrence of pathological damage to the fallopian tube.

Nafamostat mesylate (CAS: 82956-11-4) is a synthetic serine protease inhibitor that has been approved by the FDA for: amelioration of acute symptoms of pancreatitis, treatment of patients with Disseminated Intravascular Coagulation (DIC), anticoagulation during extracorporeal circulation of blood in patients who are hemorrhagic or who have a tendency to bleed^[10,11]. Pharmacological studies show that nafamostat mesylate has strong selective inhibition effects on trypsin, thrombin, kallikrein, plasmin, fibrin protease, active blood coagulation factors (XIIa, Xa and VIIa) and trypsin-like serine protease of the classical pathway of the complement system. Recent studies have shown that the compounds are also useful in the treatment of diseases in other areas: the compound can relieve lung injury induced by inflammatory factor by its antiinflammatory effect^[12](ii) a Can be used for treating ischemia reperfusion injury of kidney and brain^[13](ii) a Pancreatic cancer inhibition by down-regulation of NF- κ B pathway^[14]Colorectal cancer^[15]Growing of (3); growth inhibition of triple negative breast cancer by downregulation of the Smad2-TGF β pathway^[16](ii) a Can be used for treating reactive arthritis induced by chlamydia^[17]。

At present, the application of nafamostat mesylate in treating chlamydia genital tract infection and preventing complications such as fallopian tube obstruction, hydrosalpinx, fallopian tube infertility and the like is not reported.

Disclosure of Invention

The invention aims to provide an application method of nafamostat mesilate in treating chlamydia genital tract infection, which has the advantage of providing a new application of nafamostat mesilate in treating chlamydia genital tract infection and solves the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: the application method of nafamostat mesylate in treating chlamydia genital tract infection comprises the following steps:

A. selecting an experimental material;

B. making an experimental method;

C. observing an experimental result;

D. and (5) obtaining an experimental conclusion.

Preferably, the experimental materials in the step A comprise any several of strains, animals, cells and main reagents.

Preferably, the experimental method in the step B is specifically divided into three,

the inhibition effect of nafamostat mesylate on chlamydia infection genital tract epithelial cells is achieved;

② the influence of nafamostat mesylate on the genital tract of mice infected with chlamydia;

③ the influence of the vaginal perfusion of nafamostat mesylate on the upper and lower genital tract cytokines of the mouse infected by the chlamydia.

Compared with the prior art, the invention has the following beneficial effects:

1. nafamostat mesylate was used for the first time to treat chlamydial genital tract infection;

2. because of the wide use of the traditional antibiotics, part of the germ-line chlamydiae generate mutation drug resistance, the traditional antibiotic therapy has the risk of treatment failure, but the technical scheme is not used clinically as the antibiotic, has low drug resistance and can replace the traditional antibiotic scheme to treat the acute stage chlamydia infection;

3. provides a new administration route for treating acute chlamydia infection, namely vaginal local administration, and compared with the traditional systemic antibiotic treatment, the administration route has the following advantages: the vaginal local drug application mode improves the local drug concentration of the focus, and the drug can more directly act on chlamydia and target cells infected by the chlamydia; secondly, the influence of the medicine on other organs is reduced by a local medicine application mode of the vagina, and the metabolic burden of organs such as the liver, the kidney and the like is reduced; and the mode of local vaginal administration also reduces the occurrence of flora imbalance after systemic administration of antibiotics.

Drawings

FIG. 1 is a graph showing the effect of nafamostat mesylate on the growth of chlamydia inclusion bodies according to the present invention;

(Note: the upper image is a fusion image of a bright field in which HeLa229 cells are present and red fluorescence in which chlamydia inclusion bodies are present)

FIG. 2 is a graph showing the effect of nafamostat mesylate on the number of chlamydia inclusions in accordance with the present invention;

(Note: P < 0.01X P <0.001, total 3 independent experiments, each time in different concentrations of the multiple holes)

FIG. 3 is a graph showing the effect of nafamostat mesylate on the diameter of chlamydia inclusion bodies according to the invention;

(Note: P <0.001, total 3 independent experiments, each experiment in different concentrations of the multiple holes)

FIG. 4 is a graph showing the change in vaginal bacterial load with time after infection with Chlamydia in different groups of mice according to the invention;

(Note: P < 0.05X P < 0.01X P <0.001, n ═ 5-7)

FIG. 5 is a graph showing the effect of nafamostat mesylate of the invention on cytokines in the upper and lower reproductive tracts of mice after infection;

(Note: P <0.05n ═ 5)

FIG. 6 is the structural formula of nafamostat mesylate of the invention;

FIG. 7 is a schematic flow chart of the method of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-7, a method of using nafamostat mesylate in the treatment of chlamydial genital tract infection comprising the steps of:

A. selecting an experimental material;

B. making an experimental method;

C. observing an experimental result;

D. and (5) obtaining an experimental conclusion.

The experimental materials in the step A comprise any several of strains, animals, cells and main reagents.

The experimental method in the step B is specifically divided into three types,

the inhibition effect of nafamostat mesylate on chlamydia infection genital tract epithelial cells is achieved;

② the influence of nafamostat mesylate on the genital tract of mice infected with chlamydia;

③ the influence of the vaginal perfusion of nafamostat mesylate on the upper and lower genital tract cytokines of the mouse infected by the chlamydia.

The first embodiment is as follows:

inhibition effect of nafamostat mesylate on chlamydia infection of genital tract epithelial cells

1. Experimental Material

Strains and cells: the mouse Chlamydia (Chlamydia muridarum, Cm) Cm-mCherry fluorescent strain is constructed by the laboratory, is subjected to conventional passage, purification and storage, the fluorescent strain introduces a red fluorescent protein mCherry gene into a wild type Cm plasmid, the strain has fluorescence, fixation and antibody incubation are not needed, and fluorescent observation can be carried out in vivo after the cell is infected. HeLa229 cells were purchased from American culture Collection (ATCC) and routinely passaged and stored in the laboratory;

the main reagents are as follows: nafamostat mesylate was purchased from Abcam, high-glucose DMEM medium, pancreatin was purchased from Hyclone, usa; fetal bovine serum was purchased from Gibco, usa; cycloheximide, gentamicin, DEAE-dextran, purchased from Sigma-Aldrich, USA; sucrose, KH₂PO₄、K₂HPO₄L-glutamic acid was purchased from chemical reagents of the national drug group, Inc., China. Sucrose-phosphate-glutamate buffer (SPG buffer) configuration: mixing sucrose 37.31g and KH₂PO₄0.2558g，K₂HPO₄Dissolving 0.6185g of L-glutamic acid 0.3605g in ultrapure water, diluting to a constant volume of 500mL, adjusting pH value to 7.2-7.4, filtering with 0.2 μm filter tip, packaging, and sealingMouth, 4 ℃ for storage.

2. Experimental methods

When the HeLa229 cells were passaged well, they were seeded in a 96-well plate and placed at 37 ℃ in 5% CO₂The culture box is used for culturing for 12 hours; when the cell fusion degree in the 96-well plate reaches about 90%, the culture medium is sucked away, the cells are washed once by PBS buffer, 100 mu l of DMEM culture medium containing DEAE-dextran with the final concentration of 30 mu g/ml is added into each well to pretreat the cells, the cells are placed at 37 ℃ and 5% CO₂The incubator is 20 min; inoculating Chlamydia murinus infection solution to HeLa229 cells according to the multiplicity of infection (MOI) of 0.8, centrifuging at room temperature at 500g/min for 60 min; removing the infection liquid, adding high-sugar DMEM medium (containing 10% fetal calf serum, 10. mu.g/ml gentamicin, 2. mu.g/ml cycloheximide and different concentrations of nafamostat mesylate), placing at 37 deg.C and 5% CO₂After 24h in the incubator, the cells were washed once with PBS buffer, and placed under an inverted fluorescence microscope for in vivo fluorescence observation, 3 fields were counted at random with a half-mirror (20 ×) per well, the number of inclusion bodies in each well was counted, and after storage by photographing, the size of the inclusion bodies was measured using ImageJ software. Data processing, statistical analysis and mapping were performed using GraphPad Prism 8 software.

3. Results of the experiment

As shown in figure 1, in the control group without nafamostat mesylate, HeLa229 cells can see abundant chlamydia inclusion bodies in cells, the growth of the chlamydia inclusion bodies is inhibited along with the increase of the concentration of nafamostat mesylate in the culture medium, the number and the diameter of the inclusion bodies are gradually reduced, the morphology of the inclusion bodies is gradually changed from a full circle to an irregular shape, and the inclusion bodies are completely eliminated when the drug concentration is more than 50 mug/ml. As shown in fig. 2, when the drug concentration is greater than 5 μ g/ml, the number of inclusion bodies is significantly reduced compared to the control group, and the difference is statistically significant (P < 0.01); as shown in FIG. 3, when the drug concentration is higher than 2.5. mu.g/ml, the diameter of the inclusion bodies is significantly reduced compared with the control group, and the difference is statistically significant (P < 0.001).

4. Conclusion of the experiment

The experiment uses a female genital epithelial cell line, and simulates the chlamydia infection process through in vitro experiments. After infection with chlamydia, cells were intervened with different concentrations of nafamostat mesylate. The experimental result shows that the nafamostat mesylate can inhibit the growth of chlamydia in cells, the inhibition effect is positively correlated with the dosage, when the drug concentration is more than 50 mu g/ml, chlamydia inclusion bodies are completely eliminated, and the cell state is good, which indicates that the nafamostat mesylate can be used for treating female genital tract infection of chlamydia.

Example two:

effect of nafamostat mesylate on Chlamydia infection of mouse reproductive tract

1. Experimental Material

Animals: c57BL/6 mice, SPF grade, 6 weeks old, female, purchased from south of the lake slaikatia laboratory animals ltd, license number: SCXK (Xiang) 2019-;

the cells and main reagents were the same as in example 1.

2. Experimental methods

Animal grouping: 5 abdominal cavity control groups, 5 vaginal control groups, 6 abdominal cavity administration groups and 7 vaginal administration groups.

Establishing an infection model: 5 days before chlamydia infection (-5d), 2.5mg of progesterone is pre-injected subcutaneously into each mouse, so that the oestrus cycle of the mice is synchronous, and chlamydia infection is facilitated. On the day of infection (0d), each mouse was vaginally inoculated with an infection of 5X 10⁵Cm of chlamydia Inclusion body forming units (IFUs) according to 5X 10⁵Diluting and re-suspending the Cm mother solution in precooled SPG buffer solution, adjusting a sample application gun to sample 10 mu L of mice, slowly and gently extending the tip into the vagina of the mice, continuously pressurizing after a button of the sample application gun is pressed down for a first file and encountering a blocking feeling until a second file so as to completely eject the infection solution, keeping the gear of the sample application gun unchanged and slightly stagnating, and gently withdrawing the tip from the vagina of the mice after the infection solution is brought in.

The administration method comprises the following steps: at 4 hours post-infection, dosing groups administered nafamostat mesylate at a dose of 10mg/kg per mouse for intervention once daily for 21 days in total. The intraperitoneal administration group carries out intraperitoneal injection of the medicine, and the intraperitoneal control group replaces nafamostat mesylate with normal saline; vaginal administration group was administered with vaginal douche, and vaginal control group was prepared by replacing nafamostat mesylate with physiological saline.

Lower genital tract bacterial load test: the amount of chlamydial load of the secretions of the lower genital tract of each mouse was measured on days 3, 7, 10, 14, 21 and 28 after infection, respectively. Taking genital tract (vagina and cervical body) secretion by using a sterile cotton swab, re-suspending the genital tract (vagina and cervical body) secretion in 500 mu L of SPG buffer solution of prepared glass beads, and oscillating by using a vortex oscillator to release chlamydia in the secretion into the mixed solution; infecting HeLa229 cells in a monolayer in CO with a mixture of appropriate dilutions₂After culturing for 24h in an incubator, placing the mice under an inverted fluorescence microscope for living body observation, counting inclusion bodies, randomly counting 3 visual fields in each hole by a medium-magnification lens (20 x), and calculating the chlamydia carrying amount of the genital secretion of each mouse according to the parameters of the inclusion body counting, the dilution multiple of an infection solution, the area of the visual fields under the lens, the bottom area of a 96-pore plate and the like.

3. Results of the experiment

In the experiment, the influence of nafamostat mesylate on the genital tract of a mouse infected with chlamydia is researched by two modes of intraperitoneal injection administration and vaginal perfusion administration. As shown in fig. 4, there was no significant difference in vaginal bacterial load at different time periods in the mice of the abdominal cavity administration group compared to the abdominal cavity control group; compared with a vaginal control group, the vaginal bacterial load of mice in the vaginal administration group is remarkably reduced at 3, 7 and 10 days after infection (P < 0.01); compared with the two administration modes, the inhibition effect of the vaginal administration group on the vaginal bacterial load is obviously better than that of the abdominal administration group at 3, 10 and 14 days after infection (P < 0.05). On day 21 post-infection, vaginal bacterial load was not detectable in the mice of the vaginal administration group earlier than in the other 3 groups.

4. Conclusion of the experiment

The nafamostat mesylate is infused into the vagina, so that the growth and the reproduction of chlamydia in the genital tract can be inhibited, and the removal of the chlamydia by the body is accelerated. Suggesting that the vaginal perfusion of nafamostat mesylate can be used for treating the acute chlamydia infection of the genital tract.

Example three:

effect of vaginal infusion of nafamostat mesylate on upper and lower genital tract cytokines in mice infected with chlamydia

1. Experimental Material

Animals, cells, and main reagents were the same as in example 2.

2. Experimental methods

Animal grouping: 10 vagina control groups and 10 vagina administration groups;

the infection model was established and the administration was performed in the same manner as in example 2.

Upper and lower genital tract cytokine assay: 5 mice in each group were sacrificed randomly at 7, 14 days post infection, respectively, and the upper and lower reproductive tracts were carefully isolated. The upper and lower genital tract tissues and 500. mu.l of SPG buffer were added to the homogenizer, ground on ice, collected for homogenization, centrifuged at 500g for 5 minutes at 4 ℃ and the supernatant was collected. Using BD^TMThe Cytomeric Bead Array kit (BD CBA mouse in-migration kit, cat #552364) was used to detect the levels of IL-6, IL-10, MCP-1, IFN-. gamma., TNF-. alpha., IL-12p70 factor in the supernatant of the homogenate of the upper and lower genital tract tissues of each mouse, respectively.

3. Results of the experiment

As shown in fig. 5, the levels of TNF- α were significantly reduced in the lower genital tract of mice in the vaginal dose group at day 14 post infection (P <0.05), with no statistical difference in the remaining factor levels between the groups.

4. Conclusion of the experiment

TNF-alpha is mainly produced by monocyte-macrophage and is an important inflammatory mediator, according to previous research reports, chlamydia infection can obviously increase TNF-alpha level, and high level of TNF-alpha is reflected by inflammatory reaction after chlamydia infection. On day 14 post-infection, the treatment regimen of vaginal infusion of nafamostat mesylate effectively reduced the TNF- α level in the lower genital tract, reduced the inflammatory response following lower genital tract infection, indirectly reflecting that the lower genital tract chlamydia infection has been substantially controlled in the treatment group. The experimental result indicates that the nafamostat mesylate can be used for treating the acute chlamydia infection of the genital tract by vaginal infusion.

Reference to the literature

[1]Giffard P M,Lilliebridge R A,Wilson J,et al.Contaminated fingers:apotential cause of Chlamydia trachomatis-positive urine specimens[J].SexTransm Infect,2018,94(1):32-36.

[2]Rowley J,Vander Hoorn S,Korenromp E,et al.Chlamydia,gonorrhoea,trichomoniasis and syphilis:global prevalence and incidence estimates,2016[J].Bull World Health Organ,2019,97(8):548-562p.

[3]Schuchardt L,Rupp J.Chlamydia trachomatis as the Cause ofInfectious Infertility:Acute,Repetitive or Persistent Long-Term Infection？[J].Curr Top Microbiol Immunol,2018,412:159-182.

[4]Reekie J,Donovan B,Guy R,et al.Risk of Pelvic Inflammatory Diseasein Relation to Chlamydia and Gonorrhea Testing,Repeat Testing,and Positivity:A Population-Based Cohort Study[J].Clin Infect Dis,2018,66(3):437-443.

[5]Karim S,Souho T,Benlemlih M,et al.Cervical Cancer InductionEnhancement Potential of Chlamydia Trachomatis:A Systematic Review[J].CurrMicrobiol,2018,75(12):1667-1674.

[6]Bernstein K T,Marcus J L,Nieri G,et al.Rectal gonorrhea andchlamydia reinfection is associated with increased risk of HIV seroconversion[J].J Acquir Immune Defic Syndr,2010,53(4):537-43.

[7]Mohammadzadeh F,Dolatian M,Jorjani M,et al.Urogenital chlamydiatrachomatis treatment failure with azithromycin:Ameta-analysis[J].Int JReprod Biomed(Yazd),2019,17(9):603-620.

[8]Mestrovic T,Ljubin-Sternak S.Molecular mechanisms ofChlamydiatrachomatis resistance to antimicrobial drugs[J].Front Biosci(Landmark Ed),2018,23:656-670.

[9]Tavares E,Ouellette L,Law J,et al.Effect of provider type and sexonempiric treatment of STD infections in adolescent patients[J].Am J EmergMed,2018,36(10):1918-1919.

[10]Nakase H,Itani T,Mimura J,et al.Successful treatment of severeacute pancreatitis by the combination therapy of continuous arterial infusionof a protease inhibitor and continuous hemofiltration[J].J GastroenterolHepatol,2001,16(8):944-5.

[11]Makino S,Egi M,Kita H,et al.Comparison of nafamostat mesilate andunfractionated heparin as anticoagulants during continuous renal replacementtherapy[J].Int J Artif Organs,2016,39(1):16-21.

[12]Miyaso H,Morimoto Y,Ozaki M,et al.Protective effects ofnafamostat mesilate on liver injury induced by lipopolysaccharide in rats:possible involvement of CD14 and TLR-4 downregulation on Kupffer cells[J].DigDis Sci,2006,51(11):2007-12.

[13]Na K R,Choi H,Jeong J Y,et al.Nafamostat Mesilate AttenuatesIschemia-Reperfusion-Induced Renal Injury[J].Transplant Proc,2016,48(6):2192-9.

[14]Fujiwara Y,Furukawa K,Haruki K,et al.Nafamostat mesilate canprevent adhesion,invasion and peritoneal dissemination of pancreatic cancerthorough nuclear factor kappa-B inhibition[J].J Hepatobiliary Pancreat Sci,2011,18(5):731-9.

[15]Lu Y X,Ju H Q,Wang F,et al.Inhibition of the NF-kappaB pathway bynafamostat mesilate suppresses colorectal cancer growth and metastasis[J].Cancer Lett,2016,380(1):87-97.

[16]Mander S,You D J,Park S,et al.Nafamostat mesilate negativelyregulates the metastasis of triple-negative breast cancer cells[J].Arch PharmRes,2018,41(2):229-242.

[17]Inman R D,Chiu B.Nafamostat mesylate,a serine protease inhibitor,demonstrates novel antimicrobial properties and effectiveness in Chlamydia-induced arthritis[J].Arthritis Res Ther,2012,14(3):R150.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (3)

1. An application method of nafamostat mesilate in treating chlamydia genital tract infection is characterized in that: the method comprises the following steps:

A. selecting an experimental material;

B. making an experimental method;

C. observing an experimental result;

D. and (5) obtaining an experimental conclusion.

2. The method of claim 1 for the treatment of chlamydial genital tract infections with nafamostat mesylate, wherein the method comprises: the experimental materials in the step A comprise any several of strains, animals, cells and main reagents.

3. The method of claim 1 for the treatment of chlamydial genital tract infections with nafamostat mesylate, wherein the method comprises: the experimental method in the step B is specifically divided into three types,

the inhibition effect of nafamostat mesylate on chlamydia infection genital tract epithelial cells is achieved;

② the influence of nafamostat mesylate on the genital tract of mice infected with chlamydia;

③ the influence of the vaginal perfusion of nafamostat mesylate on the upper and lower genital tract cytokines of the mouse infected by the chlamydia.

Images