USE OF MICRO- AND NANO-MgH2 COMPOUND PARTICLES IN INHIBITION OF LEISHMANIA INFECTION AND TREATMENT OF LEISHMANIASIS

Abstract

A use of micro- and nano-MgH.sub.2 compound particles in the inhibition of Leishmania infection and in the treatment of Leishmaniasis is provided. The micro- and nano-MgH.sub.2 compound particles can be used to prepare a pharmaceutical composition for inhibiting Leishmania or to prepare a pharmaceutical composition for treating a skin or mucosal ulcer or visceral damage caused by Leishmania infection. The present disclosure discovers for the first time that the micro- and nano-MgH.sub.2 compound particles can significantly reduce the number of Leishmania in macrophages and inhibit the proliferation of Leishmania, indicating a very prominent insecticidal inhibition effect. The micro- and nano-MgH.sub.2 compound particles of the present disclosure can quickly and effectively cure a skin ulcer and/or a mucosal ulcer caused by Leishmania and an impaired function of an internal organ (mainly liver and spleen) caused by Leishmania and have high biosafety and huge clinical application values.

Claims

1. A method of use of micro- and nano-MgH.sub.2 compound particles in a preparation of a pharmaceutical composition for inhibiting Leishmania, wherein the pharmaceutical composition comprises the micro- and nano-MgH.sub.2 compound particles and a pharmaceutically acceptable carrier and/or adjuvant.

2. A method of use of micro- and nano-MgH.sub.2 compound particles in a preparation of a pharmaceutical composition for treating a skin or mucosal ulcer caused by a Leishmania infection, wherein the pharmaceutical composition comprises the micro- and nano-MgH.sub.2 compound particles and a pharmaceutically acceptable carrier and/or adjuvant.

3. A method of use of micro- and nano-MgH.sub.2 compound particles in a preparation of a pharmaceutical composition for treating a visceral damage caused by a Leishmania infection, wherein the pharmaceutical composition comprises the micro- and nano-MgH.sub.2 compound particles and a pharmaceutically acceptable carrier and/or adjuvant.

4. The method of use according to claim 1, wherein the micro- and nano-MgH.sub.2 compound particles each have a particle diameter of 1 nm to 10 μm.

5. The method of use according to claim 4, wherein the micro- and nano-MgH.sub.2 compound particles each have the particle diameter of 100 nm to 1,000 nm.

6. The method of use according to claim 1, wherein in the pharmaceutical composition, an effective concentration of MgH.sub.2 is 1 mg/100 μL to 15 mg/100 μL.

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. The method of use according to claim 2, wherein the micro- and nano-MgH.sub.2 compound particles each have a particle diameter of 1 nm to 10 μm.

12. The method of use according to claim 3, wherein the micro- and nano-MgH.sub.2 compound particles each have a particle diameter of 1 nm to 10 μm.

13. The use according to claim 2, wherein in the pharmaceutical composition, an effective concentration of MgH.sub.2 is 1 mg/100 μL to 15 mg/100 μL.

14. The use according to claim 3, wherein in the pharmaceutical composition, an effective concentration of MgH.sub.2 is 1 mg/100 μL to 15 mg/100 μL.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 shows the influence of three types of micro- and nano-MgH.sub.2 compound particles on the hydrogen content, magnesium ion concentration, and pH value of a cell culture medium (RPMI1640), where FIG. 1A shows the hydrogen content; FIG. 1B shows the magnesium ion concentration; and FIG. 1C shows the pH value.

[0031] FIG. 2 shows the IC50 effect of micro- and nano-MgH.sub.2 compound particles on macrophages.

[0032] FIG. 3 is an image of Leishmaniadonovani-infected macrophages.

[0033] FIG. 4 shows the relationship between micro- and nano-MgH.sub.2 compound particle concentrations and Leishmania (Leishmaniadonovani)-infected cells.

[0034] FIG. 5 shows the assessment results of in vivo biosafety of micro- and nano-MgH.sub.2 compound particles, where FIG. 5A shows the body weight change; FIG. 5B shows the spleen weight; and FIG. 5C shows the liver and kidney function indexes.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The present disclosure is described in detail below with reference to specific examples. The following examples will help those skilled in the art to further understand the present disclosure but do not limit the present disclosure in any way. It should be noted that those of ordinary skill in the art can further make several variations and improvements without departing from the idea of the present disclosure. These all fall within the protection scope of the present disclosure.

EXAMPLE 1

[0036] MgH.sub.2 compound particles of different particle sizes were continuously monitored for hydrogen release, magnesium ion concentration, and pH. A specific process was as follows:

[0037] Three types of MgH.sub.2 particles respectively with particle diameters of 1 nm to 100 nm, 100 nm to 1,000 nm, and 1 μm to 10 μm each were added dropwise to a cell culture dish at a final concentration of 1 mg/mL and incubated for 48 h, and the hydrogen release, magnesium ion concentration, and pH were continuously monitored. Monitoring results were shown in FIG. 1. It can be seen from the results that the three types of MgH.sub.2 particles with different particle diameters can produce high-concentration hydrogen in an initial stage, and the hydrogen concentration continues to decrease over time, and the magnesium ion concentration continues to increase, and the pH value continues to rise and is gradually stabilized at about 8.5.

EXAMPLE 2

[0038] Micro- and nano-MgH.sub.2 compound particles were co-cultivated with Leishmania (Leishmaniadonovani)-infected macrophages as follows:

[0039] 1) The IC50 (half inhibition concentration, that is, a concentration at which half of the cell activity was inhibited) of MgH.sub.2 compound particles on macrophages was first determined. Magnesium hydride particles (100 nm to 1,000 nm) were co-cultivated with THP-1-derived macrophages for 72 h, and the cytotoxicity was tested by CCK-8 (FIG. 2). It was found that IC50 values at different cultivation time points were different:

[0040] 24 H: 0.261 mg/mL

[0041] 48 H: 0.290 mg/mL

[0042] 72 H: 0.306 mg/mL 2) The influence of micro- and nano-MgH.sub.2 compound particles on THP-1-derived macrophages infected by Leishmania (Leishmaniadonovani) was tested.

[0043] The optimal infection number and infection time were determined. Different Leishmania proliferation stages (5 d or 6 d), infection proportions (5 to 30 protozoa/cell), and infection times (3 h or 24 h) were set, and the number of infected cells was counted by Giemsa staining to determine the highest infection efficiency group and the lowest infection efficiency group. Results showed that the experimental group with 5 d Leishmania, 5 protozoa/cell, and 3 h infection had the lowest infection efficiency while the experimental group with 5 d Leishmania, 15 protozoa/cell, and 24 h infection had the highest infection efficiency (Table 1 and FIG. 3). Thus, the above two experimental groups were selected for follow-up investigation.

TABLE-US-00001 TABLE 1 Infection results of co-cultivation of Leishmaniadonovani and macrophages in different ratios Infection for 3 h Infection for 24 h Number of Total number Number of Total number infected cells of parasites infected cells of parasites  5 d Leishmania  5 parasites/cell 145 535 85 505 10 parasites/cell 26 1245 415 3545 15 parasites/cell 175 565 33 3565 20 parasites/cell 20 775 28 1975 30 parasites/cell 34 3755 45 5995  6 d Leishmania  5 parasites/cell 185 67 22 2355 10 parasites/cell 21 855 28 242 15 parasites/cell 225 130 31 537 20 parasites/cell 255 1145 325 461 30 parasites/cell 355 3085 40 521 Note: The number of infected cells and the number of parasites are expressed in a consistent unit.

[0044] Subsequently, micro- and nano-MgH.sub.2 compound particles (100 nm to 1,000 nm) were co-cultivated with infected macrophages (medium RPMI1640) for 48 h. The number of infected cells and the number of protozoa were observed through Giemsa staining. As shown in FIG. 4 and Table 2, with the increase of magnesium hydride concentration, the number of infected cells and the number of protozoa decreased in a concentration-dependent manner, and it was calculated that EC50=0.043 mg/mL (EC50 was a half effect concentration, namely, a dose causing 50% of subjects to produce a specific effect), which was significantly lower than IC50=0.290 mg/mL. It indicates that the micro- and nano-MgH.sub.2 compound particles can significantly inhibit the infection of macrophages without compromising biosafety.

TABLE-US-00002 TABLE 2 Therapeutic effects of micro- and nano-MgH.sub.2 compound particles at different concentrations on macrophages infected by Leishmaniadonovani 24 H 48 H 72 H Number of Number of Number of infected Number of infected Number of infected Number of mg/mL cells parasites cells parasites cells parasites 15-parasite group 0.6 22 66  7 15 ND ND 0.45 34 91 14 23 ND ND 0.3 27 119 24 85 61 254 0.15 27 88 23 65 60 281 Control group 59 195 79 196 71 608 5-parasite group 0.6 17 55 ND ND ND ND 0.45 28 59 13 31 ND ND 0.3 21 55 24 64 50 162 0.15 28 117 26 60 53 214 Control group 59 333 55 120 81 705 Notes: In the control group, a pure cell culture medium is added instead of the micro- and nano-MgH.sub.2 compound particles. The number of infected cells and the number of parasites are expressed in a consistent unit.

EXAMPLE 3

Verification by Animal Experiments

[0045] 1) Biosafety verification: C57BL/6 mice were selected and intragastrically administered with micro- and nano-MgH.sub.2 compound particles (100 nm to 1,000 nm) at different concentrations (0 mg/kg, 60 mg/kg, 120 mg/kg, 250 mg/kg, and 500 mg/kg) every day, and various physiological indexes were observed on day 8. As shown in FIG. 5, the survival rate of mice was 100%, and the body weight, spleen weight, and liver and kidney indexes were normal.

[0046] 2) Research on the treatment with micro- and nano-MgH.sub.2 compound particles:

[0047] 2.1 MgH.sub.2 compound particles with a particle diameter of 100 nm to 1,000 nm were first dissolved in a phosphoric acid buffer to prepare 5 concentration groups (1 mg/100 uL, 3 mg/100 uL, 5 mg/100 uL, 10 mg/100 uL, and 15 mg/100 uL).

[0048] 2.2 A Leishmaniabraziliensis-infected mouse model was established, which was specified as follows:

[0049] Various hamsters (Mesocricetus auratus) were selected, the lower dorsal margin was shaved, and Leishmania (1 million/100 uL/mouse) was injected subcutaneously at 2 cm of an upper margin of a tail. A skin change at an injection site was observed every day, and after the hamsters were raised for 4 to 8 weeks, it was observed that a skin tissue at the injection site was bright red, tended to bleed, and underwent fresh granulation tissue hyperplasia and hair loss that could not be cured in a short time, indicating that a skin ulcer was formed and the model was successfully established.

[0050] 2.3 35 hamster models infected by Leishmania were divided into 7 groups, with 5 hamster models per group. MgH.sub.2 compound particles (with a particle size of 100 nm to 1,000 nm) at different concentrations (100 uL) were smeared to a skin lesion of an infected hamster once every 24 h continuously for 14 d. An untreated group (namely, a blank control group, infected mice were not treated) and an antimony preparation subcutaneous injection group (sodium antimony gluconate (5-valent antimony preparation, 200 μg/mL) was injected subcutaneously at an ulcer site 100 μL/mouse/d) were set. After treatment in each experimental group, a healing rate of a skin ulceration wound was shown in Table 3 below.

TABLE-US-00003 TABLE 3 Efficacy of micro- and nano-MgH.sub.2 compound particles at different doses on skin ulceration of Leishmania-infected hamsters Healing rate of Healing rate of Healing rate of Healing rate of MgH.sub.2 particle a skin ulceration a skin ulceration a skin ulceration a skin ulceration diameter Concentration wound, %/day 0 wound, %/day 3 wound, %/day 7 wound, %/day 14 100 nm-1000 nm 1 mg/100 ul 0  3.3 ± 2.1 18.4 ± 3.2 42.5 ± 6.5 3 mg/100 ul 0  5.2 ± 1.5 38.7 ± 5.5 71.3 ± 6.1 5 mg/100 ul 0  7.4 ± 1.8 53.5 ± 4.4 91.2 ± 5.4 10 mg/100 ul  0 13.1 ± 2.3 75.3 ± 4.6 100 15 mg/100 ul  0 14.4 ± 2.5 80.2 ± 4.9 100 Untreated group 0 0 0  0 Antimony preparation 0 15.3 ± 2.0 75.2 ± 4.8 88.9 ± 5.1 subcutaneous injection group

[0051] The above-mentioned animal studies have shown that, compared with the untreated group, the MgH.sub.2 compound particle treatment group can improve the clinical symptoms of a skin lesion in a concentration-dependent manner, inhibit the number of Leishmania in macrophages, reduce the infiltration of inflammatory cells and inflammatory factors at a skin lesion, significantly enhance the tissue repair, significantly reduce the area of a skin lesion, make hair follicles grow vigorously, and accelerate the healing of a wound in a skin lesion area. In addition, studies have shown that, in the magnesium hydride particle oral administration group, there is no significant damage to the liver and kidney functions of mice (FIG. 5C), the body weight (FIG. 5A), spleen (FIG. 5B), cardiopulmonary and brain tissues, and blood routine are normal, and there is no organ and tissue damage. Existing studies have shown that the antimony preparation can cause serious liver and kidney function damage and even cause side effects such as death.

[0052] There are many ways to specifically apply the present disclosure, and the above are merely preferred implementations of the present disclosure. It should be noted that the above examples are provided only for illustrating the present disclosure and are not intended to limit the protection scope of the present disclosure. For a person of ordinary skill in the art, several improvements may further be made without departing from the principle of the present disclosure, and such improvements should also be considered as falling within the protection scope of the present disclosure.