CN113491710A - β-As4S4Application of the same in preparing medicine for treating malignant tumor of blood system - Google Patents

Abstract

The present invention relates to beta-As₄S₄The application in preparing the medicine for treating the malignant tumor of the blood system. The hematologic malignancy is selected from leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome, refractory anemia with increased juvenile cells, and refractory anemia during the transition to leukemia. beta-As₄S₄Has stable and controllable quality, can be wetted by water, and has better wettability than natural realgar. Meanwhile, compared with natural realgar, beta-As₄S₄Has better dissolubility in pure water and human gastrointestinal fluid and better apoptosis inducing effect on malignant tumor cells in a blood system.

Description

β-As4S4Application of the same in preparing medicine for treating malignant tumor of blood system

The application is entitled 'beta-As' with application date of 2017, 2, 24₄S₄The divisional application of Chinese patent application No. 201710101939.0 for the preparation of a medicament for treating hematological malignancies.

Technical Field

The invention belongs to the field of medicine, and relates to beta-As₄S₄The application in preparing the medicine for treating the malignant tumor of the blood system.

Background

Realgar is a traditional mineral medicine, which was first recorded in Shen nong Ben Cao Jing, listed as a Chinese medicine. The traditional Chinese medicine is commonly used for treating symptoms such as carbuncle, furuncle, parasitic infestation, abdominal pain and the like in clinic, and the realgar and the compound preparation thereof have good effect on treating acute myelocytic leukemia in recent years.

Research finds that the crystal forms of realgar are four in total: alpha-As₄S₄、β-As₄S₄、χ-As₄S₄And realgar (paraalalgar). By adopting Raman spectrum and X-ray diffraction method, and combining with literature research and judgment, the realgar produced by Orpimento of Hunan province has alpha configuration, and the crystal structure of the medicinal realgar in China using the Orpimento As base source is alpha-As₄S₄(Zhang Shijie et al, Spectroscopy and Spectroscopy, 2.2011, vol.31, 2 nd p 291-. That is, the method is limited to natural arsenic sulfide (such As alpha-As) compounds₄S₄) The use of (1). As is well known, natural realgar is insoluble or even insoluble in water, insoluble in common dilute acid (only soluble in concentrated nitric acid) and insoluble in dilute alkali, so that the bioavailability of realgar in clinical application is extremely low; the natural realgar has different processing and using methods and great dosage difference, thereby bringing clinical risks.

Realgar nanoparticles (Ningning et al, Chinese traditional medicine journal, 1 month in 2005, vol. 30, 2 nd, 136 nd-140) were developed around 2000. However, the realgar nanoparticles are difficult to be clinically applied due to the great preparation difficulty. Other emerging preparation technologies are still in the exploration stage (Zhang Jinghong et al, J. Chinese medicine, 8.2010, 8 th vol., 25 th vol., 1321 st-. Therefore, it is necessary to research other crystal forms of realgar to overcome the problems of natural realgar.

The dissolution of the solid is a solid-liquid interface reaction, the dissolution process of the arsenic sulfide is a heterogeneous reaction, and the method comprises the following possible steps:

(1) wetting the arsenic sulfide particles by a solvent;

(2)As₄S₄solvation of (a);

(3)As₄S₄the solvated product diffuses into the solvent;

(4) further variations of the solvated products.

A schematic of the dissolution process of arsenic sulfide is shown in FIG. 1.

However, natural realgar has strong van der Waals forces between molecules, which cause (1) wetting of arsenic sulfide particles by solvent and (2) As₄S₄The solvation of the realgar is very difficult, and the natural realgar is particularly difficult to wet in water, so that the natural realgar presents strong powder agglomeration in water and most of aqueous solutions.

β-As₄S₄Having a structure with alpha-As₄S₄The same molecular structure, but different space accumulation mode, form C2/C space group. In a beta-type space structure, As₄S₄Intermolecular van der waals forces are much less than in the alpha form, which allows for wetting of the arsenic sulfide particles by (1) the solvent in the dissolution step described above; and (2) As₄S₄The difficulty of solvation is greatly reduced, thereby leading to beta-As₄S₄The dissolution rate and dissolution rate in water and aqueous solutions are greatly increased (E.J.Porter, and G.M.Sheldrick.J.chem.Soc., Dalton trans.,1972, 1347-1349).

β-As₄S₄The method comprises 3 sources, namely, mixing pure arsenic and pure sulfur according to the mass ratio of 1: 1, heating, and controlling the temperature to be 252-306 ℃, so that elemental arsenic, elemental sulfur and a combined product are obtained; ② realgar (alpha-As) of raw ore₄S₄) Heating at 295 deg.C for 24 hr, and cooling to room temperature to obtain product; ③ heating the realgar (realgar) and controlling the temperatureThe product is prepared by heating at 220-230 ℃ for about 5 minutes (Guanjun et al, university of Beijing Chinese medicine, 9.2010, No. 9, No. 33, No. 9, No. 623-.

However, in the prior art, beta-As was not used₄S₄The report of treating malignant tumor of blood system.

Disclosure of Invention

Through a series of researches, the inventor discovers that the beta-As serving As a high-temperature crystal form₄S₄The dissolution rate and solubility in pure water, artificial gastric juice and artificial intestinal juice are all obviously higher than those of natural realgar. Further research shows that compared with natural realgar, the beta-As₄S₄Has better effect of inducing apoptosis to malignant tumor cells in the blood system.

Accordingly, the object of the present invention is beta-As₄S₄The application in preparing the medicine for treating the malignant tumor of the blood system.

β-As₄S₄The crystal structure of (a): monoclinic system; a cyclic molecular structure; space group C2/C (15); a is₀＝9.95700nm，b₀＝9.33500nm，c₀＝8.88900nm，β＝102.380。

Herein, the hematologic malignancy is selected from leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome, refractory anemia with increased juvenile cells, and refractory anemia during transformation to leukemia.

The leukemia is selected from acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute non-lymphocytic leukemia, acute promyelocytic leukemia, acute monocytic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia and chronic myelomonocytic leukemia. Preferably, the leukemia is selected from acute promyelocytic leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia.

The lymphoma is selected from the group consisting of severe lymphoma, moderate lymphoma, and mild lymphoma. Preferably, the lymphoma is selected from non-hodgkin's disease lymphoma.

β-As₄S₄The effective dosage of (a) varies according to a number of parameters, such as the chosen route of administration, the weight, age, sex and sensitivity of the subject to be treated. Thus, the optimal dosage should be determined by the relevant specialist in view of the relevant parameters he considers. Although effective dosages may vary in large proportions, the daily dosage of one or more doses may vary from 0.1 to 2000mg, and preferably from 1 to 500mg, per 24 hours for an average adult human body weight of 70 kg.

In the present invention, the term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce any adverse or allergic or any other unwanted reaction when administered to a human. As used herein, the term "pharmaceutically acceptable carrier or excipient" includes any diluent, adjuvant, such as preservatives, fillers, disintegrants, wetting agents, emulsifiers, dispersants, antibacterial or antifungal agents, or agents that allow delayed absorption and intestinal and digestive resorption. The use of such media or carriers is well known to those skilled in the art.

β-As₄S₄Can be administered with one or more other active agents, such as an anti-cancer agent, or in combination with radiation therapy or surgical therapy, or in combination with radiation therapy and surgical therapy. Administration may be simultaneous, separate or staggered from other treatments. The anticancer agent is selected from cisplatin, taxol, chunhorine, vincristine, vinorelbine, camptothecin, epipodophyllotoxin, colchicine and 5-fluorouracil.

β-As₄S₄Can be administered to a human. The beta-As containing compositions may be administered by oral, sublingual, subcutaneous, intramuscular, intravenous, topical, rectal or intranasal routes₄S₄The pharmaceutical composition of (1). In such cases, the active ingredient may be administered to a human in a single dosage form (in admixture with conventional pharmaceutical carriers). Suitable single dosage forms include oral forms such as tablets, capsules, powdersPowders, granules and oral solutions or suspensions, sublingual and buccal dosage forms, subcutaneous or transdermal, external, intramuscular, intravenous, intranasal or intraocular dosage forms, rectal dosage forms.

When preparing solid compositions in the form of tablets, the principal active ingredient is mixed with a pharmaceutical carrier, for example, gelatin, starch, lactose, magnesium stearate, talc, acacia, silicon dioxide or equivalents. The tablets may be coated with sucrose or other suitable material, or may be treated to provide sustained or delayed activity and sustained release of a predetermined amount of the active ingredient.

The capsule preparation is obtained by mixing the active ingredient with a diluent and pouring the obtained mixture into a soft or hard capsule.

Formulations in the form of syrups or elixirs may contain the active ingredient together with sweetening agents, preservatives and agents to impart a flavour and suitable colouring agents.

Water-dispersible powders or granules may contain the active ingredient in admixture with dispersing or wetting agents, or suspensions, and flavouring or sweetening agents.

For rectal administration, suppositories are used which are prepared with binders which melt at the rectal temperature, for example cocoa butter or polyethylene glycols.

For parenteral (intravenous, intramuscular, intradermal, subcutaneous), intranasal or intraocular administration, aqueous suspensions, isotonic saline solutions or sterile and injectable solutions containing pharmaceutically compatible dispersing and/or wetting agents are used.

The active ingredient may also be formulated in microencapsulated form, optionally with one or more additional carriers.

Advantageously, the pharmaceutical composition according to the invention is intended for oral or topical administration.

The invention will be further elucidated by the following figures and examples. These examples and drawings, however, should not be construed as limiting the scope of the invention in any way.

Drawings

FIG. 1 is a schematic diagram of the dissolution process of arsenic sulfide.

FIG. 2 shows beta-As₄S₄X-ray diffraction powder diffraction pattern of (a).

Figure 3 shows the contact angle of natural realgar and water.

FIG. 4 shows β -As₄S₄Contact angle with water.

Fig. 5 shows the time-concentration curve of natural realgar dissolved in artificial gastric juice, artificial intestinal juice and pure water.

FIG. 6 shows β -As₄S₄Time-concentration curve of dissolution in artificial gastric juice, artificial intestinal juice and pure water.

Detailed Description

The source of the natural realgar used in the examples was purchased from zhengzhou ruilong pharmaceutical industry gmbh; beta-As₄S₄Prepared according to the method disclosed in E.J.Porter, and G.M.Sheldrick.J.chem.Soc., Dalton trains, 1972, 1347-1349.

beta-As used in the present invention₄S₄The crystal structure is as follows: monoclinic system; a cyclic molecular structure; space group C2/C (15); a is₀＝9.95700nm，b₀＝9.33500nm，c₀＝8.88900nm，β＝102.380。

Example 1: beta-As₄S₄Identification of Crystal samples

1. A detection instrument: bruker powder X-ray diffractometer, model: d8 Advance.

2. The main working parameters of the instrument are as follows:

cu target, Ka radiation, 40kV,40mA, RS slit: 3mm, scanning range: 5-80 degrees, scanning mode: continuous scan, data interval: 0.02 °, count time: 0.1 sec/step.

3. And (3) detection results: see fig. 2.

The measured X-ray diffraction spectrum of the sample is shown above, and the beta-As in JCPDS spectrogram library (PDF2) is shown below₄S₄The standard spectrum (card number: 01-088-1657), the main diffraction peak on the actually measured X-ray diffraction spectrum of the sample and the beta-As in PDF2₄S₄The standard spectrogram (card number: 01-088-1657) is well matched, and the main crystal phase component in the sample is proved to be As₄S₄(arsenic sulfide) (note: 17.002 degrees, 20.60 degreesImpurity peaks at 0 °).

Example 2: wettability

Wettability refers to the tendency of a solid to readily contact a fluid rather than another fluid. Can be generally defined as the phenomenon of transition of a solid interface from a solid-gas interface to a solid-liquid interface. Wettability is determined by the structure of the outermost layer of the solid and is one of its inherent characteristics.

1. A detection instrument: high-speed video optical contact angle measuring apparatu: OCAH 200, Dataphysics, germany.

2. The test method comprises the following steps: pressing realgar fine powder (200 meshes) into a wafer with a smooth surface and a diameter of 1cm, placing the wafer on an objective table, slowly extruding liquid through a needle head to drip on the wafer, shooting the contact process of the liquid drops and the wafer through a high-speed camera, and recording and measuring the contact angle of the liquid and the wafer. The shooting speed is 200 frames/min.

3. And (3) testing results: the contact angle of the natural realgar powder and water is 133 degrees, the natural realgar powder belongs to a non-wetting sample (the contact angle theta is more than 90 degrees), and the strongly hydrophobic realgar particles are not easy to wet, so that the method is a rate-limiting reaction step for dissolving out the arsenic sulfide. The results are shown in FIG. 3.

β-As₄S₄The contact angle with water was 87 deg., which is a wettable sample (contact angle theta < 90 deg.), and the results are shown in fig. 4.

Thus, it can be seen that beta-As₄S₄Can be used for moistening water, and has better wettability than natural Realgar.

Example 3: dissolution property

1. ICP-AES: american VARIN corporation, model: the VISTA-MPX (temperature induced stress) reactor,

2. analysis conditions were as follows: RF, power: 1.15kw, cooling air flow: 15L/min, atomizer gas flow: 0.75L/min, height observed: 12mm, auxiliary gas flow rate: 1.5L/min.

3. As a result:

natural realgar: see table 1 and fig. 5.

Table 1: time-concentration data of natural Realgar dissolved in artificial gastric juice, artificial intestinal juice, and pure water

β-As₄S₄Sample preparation: see table 2 and fig. 6.

Table 2: beta-As₄S₄Time-concentration data of dissolution in artificial gastric juice, artificial intestinal juice and pure water

Thus, it can be seen that beta-As₄S₄The dissolution rate in pure water and artificial gastrointestinal fluid is higher than that of natural realgar, and the dissolution amount is larger than that of artificial realgar. Taking the dissolution in the artificial gastric juice As an example, the total dissolution rate of the beta-As 4S4 in each liter of artificial gastric juice is 0.014mg/min, and the total dissolution rate of the natural realgar in each liter of artificial gastric juice is 0.0085 mg/min.

Example 4: quality of

Determination of beta-As₄S₄Total arsenic and soluble arsenic for three samples.

The detection method comprises the following steps:

1. a detection instrument: ICP-AES: american VARIN corporation, model: the VISTA-MPX (temperature induced stress) reactor,

2. detection conditions are as follows: RF, power: 1.15kw, cooling air flow: 15L/min, atomizer gas flow: 0.75L/min, height observed: 12mm, auxiliary gas flow rate: 1.5L/min.

3. As a result: actually measured data: the total arsenic content of samples No. 1, No. 2, and No. 3 was 69.36%, 69.92%, and 69.12%, respectively, with an average value of 69.47%.

The measured data of the soluble arsenic content in the three samples are respectively as follows: 0.029%, 0.036%, 0.039%, with an average value of 0.035%, i.e. 0.35 mg/g.

And (4) conclusion: relative atomic weight of arsenic 74.92159, relative atomic weight of sulfur 32.066, Compound As₄S₄The theoretical content of arsenic element in the alloy is 70.028%. beta-As₄S₄Measured average of Total arsenic and As₄S₄The ratio of the theoretical total arsenic values can then be converted into purity, i.e. beta-As₄S₄The sample purity was 99.20% based on arsenic.

Thus, it can be seen that beta-As₄S₄The quality is stable and controllable, the content of arsenic sulfide is more than or equal to 99 percent, and the content of soluble arsenic is less than or equal to 2 mg/g.

Example 5: antitumor activity

Respectively adopting NB4 cell line and HL-60 cell line to treat natural realgar and beta-As₄S₄The sample No. 1 is subjected to an activity verification experiment of flow cytometry, the condition of tumor cell apoptosis induction of different samples is detected, and the dose-response curves of different samples are determined.

1. Laboratory apparatus

Flow cytometry analysis by Becton Dickinson, model BD Calibur, PI Monostain, excitation wavelength 488nm, emission wavelength 635nm, and 20000 cells analyzed.

2. Samples and Experimental methods

Cell culture: HL-60 cells (provided by basic research institute of Chinese medical science), NB4 cell line (provided by American Type Culture Collection (ATCC)), 1640 medium (containing 10% fetal calf serum) 36.5 + -0.5 deg.C, 5% CO₂And carrying out conventional subculture in a saturated humidity incubator.

Sample preparation: natural realgar fine powder (200 meshes) and beta-As₄S₄Sample No. 1 (200 mesh), stored in a cool and dark place.

Preparing a sample solution: respectively and precisely weighing natural realgar fine powder and beta-As₄S₄The sample No. 1 was prepared by adding sterilized medium precisely to prepare a stock solution having a concentration of 10mM, and adding 0, 20. mu.L, 40. mu.L, 80. mu.L and 160. mu.L of 1mL of the stock solution → 10mL (1mM) of the 1mM stock solution to the culture well, respectively, to give a final volume of 2mL of the culture system, and to give final concentrations of 0, 10. mu.M, 20. mu.M, 40. mu.M and 80. mu.M, respectively, to the two samples.

Flow cytometry to determine apoptosis rate: respectively taking logarithmic growthThe HL-60 cells and NB4 cells were centrifuged at 500r/m for 5m at 2X 10⁵The cell concentration of (3) is plated, the sample solution is respectively added, the cell concentration is repeated in 5 holes, the culture is carried out for 48 hours, the sample is centrifuged for 5 minutes at 800 revolutions, the supernatant and the cells are separated, the cells are fixed by cold ethanol, the temperature is kept overnight at 4 ℃, PBS is washed, RNA enzyme digestion is carried out, PI staining is carried out, and the cell cycle and the apoptosis condition are measured by flow cytometry.

3. The experimental results are as follows: see tables 3 and 4.

TABLE 3 Natural Realgar and beta-As₄S₄Effect on M phase in HL-60 cell cycle: (

n＝5，(％))

	0μM	10μM	20μM	40Μm	80μM
						Control group	1.87±0.21
Natural realgar		11.72±1.38	18.52±0.82	35.83±2.53	53.61±3.60
						β-As4S4		56.25±1.99	85.48±1.72	/	/

P values among the three experimental groups are less than 0.01.

TABLE 4 Natural Realgar and beta-As₄S₄Comparison of Induction of apoptosis of NB4 (

n＝5，(％))

	0μM	10μM	20μM	40μM	80μM
						Control group	3.83±0.10
Natural realgar		13.32±3.21	19.36±2.17	33.32±3.85	56.68±5.10
						β-As4S4		54.26±4.0	68.27±3.01	76.10±5.01	83.55±5.47

The P values between groups were less than 0.01.

Realgar inhibits tumor growth by retarding tumor cell cycle and inducing tumor cell apoptosis. Through blank control experiment, different concentrations and beta-As of natural realgar samples are detected₄S₄The number of M1 cells of samples with different concentrations can be known As natural realgar and beta-As₄S₄The sample can obviously interfere the mitosis of HL-60 cells, thereby leading the HL-60 cells to be blocked in the mitosis phase. beta-As₄S₄The activity of the sample is stronger than that of natural realgar, and the beta-As is 10 mu M₄S₄The activity of blocking HL-60 cell cycle is equivalent to the activity of 80 mu M natural realgar; natural realgar and beta-As₄S₄The sample can induce NB4 cell apoptosis, beta-As₄S₄The activity of the sample is stronger than that of natural realgar, and the beta-As is 20 mu M₄S₄The activity of inducing NB4 apoptosis is equivalent to that of 80 μ M natural realgar.

In conclusion, compared to natural realgar, beta-As₄S₄Stable and controllable quality, can be wetted by water, has remarkably improved dissolution rate and solubility in pure water, artificial gastric juice and artificial intestinal juice, and has beta-As₄S₄Has better effect of inducing apoptosis to malignant tumor cells in the blood system. Thus, beta-As₄S₄Can be developed into a novel medicine for treating hematological malignant tumors.

Claims (10)

1.β-As₄S₄The use in the manufacture of a medicament for the treatment of leukaemia.

2. Use according to claim 1, wherein β -As₄S₄The crystal structure of (A) is: monoclinic system; a cyclic molecular structure; space group C2/C (15); a is₀＝9.95700nm，b₀＝9.33500nm，c₀8.88900nm, 102.380; preferably, beta-As₄S₄The contact angle theta is less than 90 degrees; more preferably, the overall dissolution rate in each liter of simulated gastric fluid is 0.014 mg/min; also preferably, beta-As₄S₄The content of the arsenic sulfide is more than or equal to 99 percent and the content of the soluble arsenic is less than or equal to 2 mg/g.

3. The use of claim 1, wherein the leukemia is selected from acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute non-lymphocytic leukemia, acute promyelocytic leukemia, acute monocytic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and chronic myelomonocytic leukemia; preferably, the leukemia is acute promyelocytic leukemia.

4. The use according to claim 1, wherein the medicament is between 0.1 and 2000mg β -As₄S₄The daily dosage unit form of (a); preferably, the drug is between 1 and 500mg beta-As₄S₄In the form of daily dosage units.

5. The use according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.

6. The use of claim 1, wherein the medicament further comprises another anticancer agent selected from the group consisting of cisplatin, taxol, paclitaxel, chamomiline, vincristine, vinorelbine, camptothecin, epipodophyllotoxin, colchicine, and 5-fluorouracil.

7. The use of claim 1, wherein the medicament is administered to a human.

8. The use according to claim 1, wherein the medicament is administered by oral, sublingual, subcutaneous, intramuscular, intravenous, topical, rectal or intranasal route.

9. Use according to any one of claims 1 to 8, wherein the cell line associated with the leukaemia is the HL-60 cell line and/or the NB4 cell line.

10.β-As₄S₄In preparation of the extract for blocking HL-60 cell line in mitosis and/or inducing NB4 fineThe application of the cell strain apoptosis medicine.

Images