CN108078989B - Pharmaceutical composition for treating malaria - Google Patents

Abstract

The invention belongs to the field of pharmacy, relates to a traditional Chinese medicine composition, and particularly relates to a pharmaceutical composition for treating malaria. The medicine has the characteristics of good curative effect, quick response, stable quality and capability of improving the symptoms of nerve injury. The pharmaceutical composition comprises artesunate and ligustrazine or medicinal salts thereof, and the weight ratio of the artesunate to the ligustrazine is 1-10: 10-1.

Description

Pharmaceutical composition for treating malaria

Technical Field

The invention belongs to the field of pharmacy, relates to a traditional Chinese medicine composition, and particularly relates to a pharmaceutical composition for treating malaria.

Background

Malaria is a significant infectious disease that seriously threatens human health and life safety, spreads epidemic in 108 countries and regions around the world, and is threatened by malaria in about half of the world, which is one of the three public health problems in the world. According to the world malaria report of WHO 2016, about 32 million people worldwide (accounting for nearly half of the total population worldwide) are at risk of malaria, with a total of 2.14 million new cases of malaria in 2015 and about 43.8 million people dying from malaria. Meanwhile, the drug resistance of plasmodium also makes malaria drug treatment a serious challenge. In order to improve clinical curative effect and delay drug resistance, WHO promoted A Combined Treatment Scheme (ACTs) based on Artemisinin antimalarials from 2001, and prohibited the use of Artemisinin antimalarials alone. 5 combinations of ACTs based on artemisinin drugs were specifically recommended in the 1 st version of malaria treatment guidelines issued by WHO in 2006. Combination therapy is the leading mode of malaria control today, with global ACT purchases reaching 3.11 parts per million in 2015, but the development of new drug combinations has progressed slowly in recent years, and 5 ACT drug combinations 10 years ago remain in the 3 rd version of the malaria treatment guideline published in 2015. Once plasmodium becomes resistant to current ACTs in a wide range, malaria faces the predicament of no drug cure, and the development of novel compound antimalarial drugs faces major challenges.

Cerebral Malaria (CM) is one of the most serious complications of plasmodium falciparum, with 74% of cases of cerebral Malaria occurring in african children under five years of age and a mortality rate of 15-20% (WHO, World Malaria Report 2015). Clinical manifestations of cerebral malaria include hemiparalysis, convulsion, ataxia, coma, disturbance of consciousness, and meningeal irritation. Most children's cerebral malaria patients die within one day after the onset of central nervous system symptoms, and 10-20% of surviving children develop nervous system sequelae such as persistent neurological impairment, cognitive dysfunction, behavioral dysfunction, and motor function impairment. It is considered that local microcirculation disturbance of brain tissue and secondary immunopathological damage caused by blood vessel blockage of cerebral vessels by plasmodium infection red blood cells are the most main pathogenesis of cerebral malaria.

Artesunate is considered to be the most effective drug for the treatment of severe malaria. Intravenous artesunate is the first choice therapeutic scheme recommended by WHO for cerebral malaria, and as the current first-line therapeutic drug, artesunate has anti-inflammatory effect in addition to antimalarial activity, but the reburning rate of artesunate after single drug therapy can reach 10% -100%.

Ligustrazine is the main active ingredient of ligusticum wallichii, and modern researches find that the ligustrazine mainly has the effects of treating ischemic stroke, headache, hypertension, coronary atherosclerotic heart disease (coronary heart disease), migraine, ischemic encephalopathy and the like.

After long-term research, an antimalarial drug with more obvious curative effect is obtained.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition for treating malaria. The medicine has the characteristics of good curative effect, quick response, stable quality and capability of improving the symptoms of nerve injury.

The pharmaceutical composition contains artesunate, ligustrazine or medicinal salts thereof.

Specifically, artesunate: the weight ratio of the ligustrazine or the medicinal salt thereof is 1-10: 10-1;

preferably, the weight ratio of artesunate: the weight ratio of the ligustrazine or the medicinal salt thereof is 1-2: 2-1.

Further preferably, the weight ratio of artesunate: the weight ratio of the ligustrazine or the medicinal salt thereof is 2: 1.

Wherein, the medicinal salt of the ligustrazine is preferably ligustrazine hydrochloride.

Wherein, the artesunate and the ligustrazine or the medicinal salts thereof belong to the existing products, can be purchased in the market, and can also be prepared by the conventional method.

Another object of the present invention is to provide a process for the preparation of the pharmaceutical composition.

The preparation method comprises the following steps:

(1) artesunate powder is added with 5 percent sodium bicarbonate solution to shake, after the artesunate powder is completely dissolved, 0.9 percent sodium chloride injection is added to dilute the artesunate powder,

(2) adding ligustrazine or its medicinal salt into 0.9% sodium chloride injection, shaking to dissolve completely,

(3) and (3) uniformly mixing the solutions obtained in the two steps.

Preferably, the preparation method comprises the following steps:

(1) artesunate, adding 0.9% sodium chloride injection, dissolving completely, adding 0.9% sodium chloride injection for dilution,

(2) adding ligustrazine hydrochloride and 0.9% sodium chloride injection, shaking to dissolve completely,

(3) and (3) uniformly mixing the solutions obtained in the two steps.

It is another object of the present invention to provide a pharmaceutical formulation.

The pharmaceutical preparation comprises a pharmaceutical composition and a pharmaceutically acceptable carrier.

Wherein the pharmaceutically acceptable carrier may be 0.1-99.9% by weight of the total weight of the formulation.

The pharmaceutical preparation of the invention can be prepared into any pharmaceutical dosage form, and the pharmaceutical dosage forms comprise: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal agents, granules, pills, powders, ointments, pellets, suspensions, solutions, injections, suppositories, ointments, plasters, creams, sprays, patches, nasally administered formulations.

The preferred dosage form is nasal drops.

The administration mode of the invention comprises: injection administration, oral administration and nasal administration, preferably nasal administration.

Wherein the pharmaceutically acceptable carrier comprises: mannitol, sorbitol, sorbic acid or potassium salt, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin A, vitamin C, vitamin E, vitamin D, azone, disodium EDTA, calcium sodium EDTA, carbonates of monovalent alkali metals, acetates, phosphates or aqueous solutions thereof, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and derivatives thereof, alginate, gelatin, polyvinylpyrrolidone, glycerol, propylene glycol, ethanol, Tween 60-80, span-80, beeswax, lanolin, liquid paraffin, cetyl alcohol, gallic acid esters, agar, triethanolamine, basic amino acids, One or more of urea, allantoin, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, beta-cyclodextrin, phospholipid material, kaolin, pulvis Talci, calcium stearate, magnesium stearate, and microcrystalline; preferably, the carrier is one or more of microcrystalline cellulose, lactose, starch, sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose and talcum powder.

The invention also aims to provide application of the pharmaceutical composition.

The application of the pharmaceutical composition of the invention in preparing the medicines for treating malaria.

The invention also discloses application of the pharmaceutical composition in preparing a medicament for treating cerebral malaria.

The invention also discloses application of the pharmaceutical composition in preparing a medicament for treating severe malaria.

The pharmaceutical composition has the effects of improving the survival state, reducing the cerebrovascular blockage and inflammatory cell invasion, improving the nerve injury symptom and the like.

The invention also plays a role in improving a series of cerebral ischemia lesion and nerve function damage and other concurrent symptoms caused by accumulation of plasmodium in the brain in the cerebral malaria process.

The invention can obviously relieve the occurrence of cerebral malaria caused by infection of the plasmodium bovieri ANKA strain, prolongs the survival time and has more advantages than single use.

The invention can increase cerebral blood supply of cerebral malaria mice and alleviate ischemia and anoxic injury of cerebral tissues.

The invention has prominent effect on improving the blockage condition of the microvascular obstruction in the cerebellum.

The main administration method of artesunate for severe malaria such as cerebral malaria in clinic is intravenous injection, but in high-incidence areas of cerebral malaria in Africa, extremely laggard medical conditions limit the clinical timely and effective application of artesunate to a great extent. Meanwhile, artesunate has the common characteristics of artemisinin drugs, namely the problems of fast metabolism in vivo, obvious first-pass effect, low bioavailability and the like exist, and the factors also limit the exertion of antimalarial effect to a certain extent and aggravate the problem of reburning after treatment. If the dosage of a single administration is increased or the administration frequency is increased, the drug resistance phenomenon is promoted and the toxic and side effects of the drug are possibly increased.

After the nasal administration is adopted, the invention effectively solves the problems in the prior art. The medicine can enter the brain through 3 channels of olfactory nerve channel, olfactory mucosal epithelial channel and blood circulation channel after being absorbed, and directly enters systemic circulation after being absorbed, so that the medicine can be prevented from being damaged by enzymes in gastrointestinal tracts and the first pass effect of the liver on the medicine, the average bioavailability is higher, and the nasal cavity absorption speed of the non-peptide medicine is close to intravenous injection; and because the nasal cavity and the cranial cavity are uniquely linked in anatomical physiology, the medicine can bypass the blood brain barrier and enter the brain, and the brain targeted therapy is realized; meanwhile, the medicine is convenient to take, has good patient compliance and is suitable for self-administration. Both artesunate and ligustrazine are suitable for nasal administration in terms of chemical properties, and are reported in documents, and the nasal administration has faster absorption. Therefore, the artesunate and the ligustrazine are combined in a compatible mode and are administrated through the nasal cavity, and the artesunate and the ligustrazine have great significance for treating cerebral malaria through the nasal cavity administration after the combination of the artesunate and the ligustrazine, regardless of the requirement of targeting administration in the cerebral malaria brain, the requirement of improving the bioavailability of the artesunate and the requirement of convenience in administration. Experimental results show that the artesunate and the ligustrazine are compatible and combined to be administrated through the nasal cavity, and the synergistic effect is achieved, the survival state of animals is improved, cerebral vessel blockage and inflammatory cell invasion of brain type malaria mice are reduced, and nerve injury symptoms are improved.

Drawings

FIG. 1 model mouse survival time

FIG. 2 weight gain in model mice

FIG. 3 model mouse blood insect ratio

FIG. 4 model mouse behavioural score

FIG. 5A control group cerebral microvasculature (HE staining, 20X)

FIG. 5B model group cerebral microvasculature (HE staining, 20x)

FIG. 6, growth curves of each group

FIG. 7 shows the weight growth curves of the respective groups

FIG. 8 infection rates of groups

FIG. 9A, Effect on serum inflammatory factor TNFa, FIG. 9B Effect on inflammatory factor IL-4

FIG. 10A, model group cerebral microvasculature (HE staining, 20X)

FIG. 10B, ARS (ip) cerebral microvasculature (HE staining, 20X)

FIG. 10C, ARS + CXQ (ip) cerebral microvasculature (HE staining, 20X)

FIG. 11, sets of growth curves

FIG. 12 shows the weight growth curves of the respective groups

FIG. 13 infection rates of the groups

FIG. 14, RMCBS scores of groups

FIG. 15A, Effect on serum inflammatory factor TNFa, FIG. 15B Effect on inflammatory factor IL-4

FIG. 16A, model group cerebral microvasculature (HE staining, 20x)

FIG. 16B, ARS + CXQ (ip) cerebral microvasculature (HE staining, 20X)

FIG. 16C, ARS + CXQ (IN) cerebral microvasculature (HE stain, 20X)

Detailed Description

The present invention is further illustrated by the following specific examples, which are not to be construed as limiting the invention thereto.

All components in the examples are commercially available.

The above-described embodiments are intended to be illustrative, but not limiting, of the present invention, and therefore any changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

The artesunate + ligustrazine used in the following test examples is artesunate in example 1 of the present invention: ligustrazine is 2: 1.

Test examples 1,

Experimental cerebral malaria model establishment:

6-8 week old female healthy C57BL6 XiaoIntraperitoneal inoculation of 1X 10 in mice⁶Individual p.burgdori strain ANKA-infected erythrocytes (pRBC) were scored as day 0 of infection. The observation is continued for 16 days, and cerebral malaria symptoms and death are observed.

Grouping experiments:

the C57BL6 mice were divided into 2 groups, i.e., a (control) control group, a (ANKA) model group, and each mouse was inoculated with 1X 106 pRBC in the abdominal cavity.

Evaluation indexes are as follows:

1. survival curve: the survival condition of the mice is observed day by day, and the survival curve and the weight change are drawn after 12 days. Survival rate-survival experimental mice/total experimental mice.

2. Protozoan rate: taking 1 droplet of infected mouse tail venous blood to prepare a thin blood sheet on a clean glass slide, fixing and air-drying the thin blood sheet by using methanol, dyeing the thin blood sheet for 15min by using Giemsa dye solution, washing and air-drying the thin blood sheet, and counting the number of protozoa under a 100X oil microscope, wherein the protozoa rate is pRBC/total red blood cell number;

3. evaluation of brain microvascular occlusion: brain tissue was taken 12 days after infection, and the tissue was cut into 4 μm sections fixed with 4% neutral formalin, stained by HE, and observed. Sites containing blood vessels were scored according to the severity of the obstruction and microhemorrhage using a semi-quantitative scale (0-5): 0, no obstruction; 1, partial obstruction; 2, the presence of leukocytes adhering to the endothelium; 3, local obstruction, coexistence of white blood cells and red blood cells; 4, complete occlusion, no bleeding; and 5, blocking the whole body. Data are presented as the average score for each brain.

4. Behavioral scoring mice were placed in a white box (bottom square) of 31.8 × 19.8 × 10.5cm long × wide × high, and 3mm diameter straight bars were prepared for touching the mice. The following criteria were used for scoring.

RMCBS score table:

and (3) testing seven indexes of the five aspects of coordination ability, exploration behavior, strength, skeleton, reflection, self-protection and sanitation behavior of the molded mouse every other day according to an RMCBS (national institutes laboratory) rating table, and giving a rating of 0-2. The sum of all the items is used as a total score, the seven items are divided into 14 points, and the lower the score is, the more serious the nerve function damage is. The experimental results are as follows:

1. survival time of model mice:

compared with the control group, the survival time of the mice in the ANKA group is obviously shortened, as shown in figure 1, the red curve is the control group, and the black curve is the ANKA.

2. Weight gain of model mice:

the mice in the ANKA group lost significantly compared to the control group, as shown in FIG. 2, with the red curve for the control group and the black curve for ANKA.

3. Model mouse blood rate:

compared with the control group, the blood protozoan rate of the mice in the ANKA group is obviously increased, as shown in figure 3, a red curve is the control group, and a black curve is the ANKA.

4. Evaluation of model brain microvascular occlusion:

in the ANKA group, mice had cerebral microvasculature clogged in the hippocampus and cerebellar tissues, as compared to the control group, as shown in FIG. 4.

Test examples 2,

Artesunate (ART), artesunate and ligustrazine (ART + CXQ) have protective effect on experimental cerebral malaria

Grouping experiments:

the C57BL6 mice were divided into 3 groups, which were (control) a model control group, (ART ip) an artesunate intraperitoneal injection group, (ART + CXQ ip) an artesunate + ligustrazine intraperitoneal injection group. Intraperitoneal inoculation of 1X 10 per mouse⁶After each pRBC had established cerebral malaria, the mice of the administration group were administered 4 times by intraperitoneal injection on the day before, the first day, the third day, and the fifth day after the inoculation.

Evaluation indexes are as follows:

1. observing the survival condition of the mice day by day, and drawing the survival curve and the weight change after 12 days

2. Blood smear is taken day by day and the infection rate is calculated by microscopic examination.

3. Blood was taken 12 days after infection, and the supernatant was centrifuged to detect inflammatory factors.

4. Brain tissue was taken 12 days after infection and fixed with 4% neutral formalin. The tissue is cut into 4 mu m sections, HE staining is carried out, and the condition of cerebral microvascular occlusion is observed.

The experimental results are as follows:

1. effect on survival time of mice:

compared with a model control group, the artesunate intraperitoneal injection group, the artesunate + ligustrazine intraperitoneal injection group can obviously prolong the survival time of the mice, the artesunate + ligustrazine intraperitoneal injection group is superior to the artesunate single-use group, as shown in figure 6, a black curve is the control group, a green curve is the artesunate intraperitoneal injection group, and a blue curve is the artesunate + ligustrazine intraperitoneal injection group.

2. Effect on weight gain in mice:

compared with the model control group, the artesunate intraperitoneal injection group, the artesunate + ligustrazine intraperitoneal injection group can obviously increase the weight of the mice, and as shown in fig. 7, the artesunate + ligustrazine intraperitoneal injection group at the end of 12 days has obvious weight increase.

3. Effect on mouse blood rate:

compared with the model control group, the artesunate intraperitoneal injection group and the artesunate + ligustrazine intraperitoneal injection group can reduce the blood protozoan rate of mice, and as shown in figure 8, 2 groups have equivalent effects.

4. Effects on mouse serum inflammatory factors:

compared with the model control group, the artesunate and ligustrazine intraperitoneal injection group can reduce the serum inflammatory factor TNFa of mice and has an increasing trend of anti-inflammatory factor IL-4. As shown in fig. 9.

5. Influence on brain microvascular obstruction

Compared with the model control group, the artesunate intraperitoneal injection group, the artesunate + ligustrazine intraperitoneal injection group can remarkably reduce the brain microvascular blockage in the hippocampus and cerebellum tissues of the mice, and as shown in figure 10, the artesunate + ligustrazine intraperitoneal injection group is superior to the artesunate intraperitoneal injection group.

Test examples 3,

Comparison of protective effects of different administration routes of artesunate and ligustrazine (ART + CXQ) on experimental cerebral malaria

Grouping experiments:

the C57BL6 mice were divided into 3 groups, which were (control) a model control group, (ART + CXQ ip) an artesunate + ligustrazine intraperitoneal injection group, (ART + CXQ IN) an artesunate + ligustrazine nasal administration group. Intraperitoneal inoculation of 1X 10 per mouse⁶After each pRBC had established cerebral malaria, the mice of the administration group were administered 4 times by intraperitoneal injection or nasal administration on the day before, the first day, the third day, and the fifth day of inoculation, respectively.

Evaluation indexes are as follows:

1. observing the survival condition of the mice day by day, and drawing the survival curve and the weight change after 12 days

2. Blood smear is taken day by day and the infection rate is calculated by microscopic examination.

3. Mice were scored for behavioural behaviour 4 days later.

4. Blood was taken 12 days after infection, and the supernatant was centrifuged to detect inflammatory factors.

5. Brain tissue was taken 12 days after infection and fixed with 4% neutral formalin. The tissue is cut into 4 mu m sections, HE staining is carried out, and the condition of cerebral microvascular occlusion is observed.

The experimental results are as follows:

1. effect on survival time of mice:

compared with the model control group, the artesunate and ligustrazine nasal administration group can obviously prolong the survival time of the mice, as shown in fig. 11, the black curve is the control group, the blue curve is the artesunate and ligustrazine intraperitoneal injection group, and the red curve is the artesunate and ligustrazine nasal administration group.

2. Effect on weight gain in mice:

compared with the model control group, the artesunate + ligustrazine nasal administration group showed a significant weight gain at 12 days of exposure to the virus, as shown in fig. 12.

3. Effect on mouse blood rate:

compared with the model control group, the artesunate + ligustrazine intraperitoneal injection group and the artesunate + ligustrazine nasal administration group can reduce the blood rate of the mouse protozoa, and as shown in figure 13, 2 groups have equivalent effects.

4. Effect on mouse behavioural score

Compared with the model control group, the artesunate + ligustrazine intraperitoneal injection group and the artesunate + ligustrazine nasal administration group can remarkably increase the behavioral score of the mice, wherein the artesunate + ligustrazine nasal administration group is superior to the artesunate + ligustrazine intraperitoneal injection group after 10 days of administration, as shown in fig. 14.

5. Effects on mouse serum inflammatory factors:

compared with a model control group, the artesunate + ligustrazine intraperitoneal injection group and the artesunate + ligustrazine nasal administration group can reduce the serum inflammatory factor TNFa of mice and increase the anti-inflammatory factor IL-4. As shown in fig. 15.

6. Effect on microvascular occlusion

As shown in fig. 16, artesunate + ligustrazine nasal administration group showed prominent effect of improving occlusion of microvasculature and hemorrhage in cerebellum, compared to the model control group.

The above-mentioned experiments only illustrate the efficacy of some of the drugs of the present invention, and actually, other drugs of the present invention have the same or similar efficacy, which are not illustrated herein.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Example 1 pharmaceutical compositions of the invention

Artesunate 40mg, ligustrazine hydrochloride 20mg

(1) Taking 40mg artesunate, adding 1ml 0.9% sodium chloride injection, dissolving completely, adding 3ml 0.9% sodium chloride injection for dilution,

(2) 20mg of ligustrazine hydrochloride is added with 4ml of 0.9 percent sodium chloride injection and shaken till being completely dissolved,

(3) and (3) uniformly mixing the solutions obtained in the two steps.

Example 2 pharmaceutical compositions of the invention

Artesunate 20mg, ligustrazine hydrochloride 20mg

(1) Taking 20mg artesunate, adding 1ml 0.9% sodium chloride injection, dissolving completely, adding 1ml 0.9% sodium chloride injection for dilution,

(2) 20mg of ligustrazine hydrochloride is added with 4ml of 0.9 percent sodium chloride injection and shaken till being completely dissolved,

(3) and (3) uniformly mixing the solutions obtained in the two steps.

Example 3 pharmaceutical compositions of the invention

Artesunate 20mg, ligustrazine hydrochloride 40mg

(1) Taking 20mg artesunate, adding 1ml 0.9% sodium chloride injection, dissolving completely, adding 1ml 0.9% sodium chloride injection for dilution,

(2) adding ligustrazine hydrochloride 40mg into sodium chloride injection 8ml 0.9%, shaking to dissolve completely,

(3) and (3) uniformly mixing the solutions obtained in the two steps.

Example 4 pharmaceutical compositions of the invention

Artesunate 20mg, ligustrazine hydrochloride 200mg

(1) Taking 20mg artesunate, adding 1ml 0.9% sodium chloride injection, dissolving completely, adding 19ml 0.9% sodium chloride injection for dilution,

(2) adding ligustrazine hydrochloride 200mg into sodium chloride injection 20ml 0.9%, shaking to dissolve completely,

(3) and (3) uniformly mixing the solutions obtained in the two steps.

Example 5 pharmaceutical compositions of the invention

Artesunate 200mg, ligustrazine hydrochloride 20mg

(1) Taking 200mg artesunate, adding 10ml 0.9% sodium chloride injection, dissolving completely, adding 10ml 0.9% sodium chloride injection for dilution,

(2) 20mg of ligustrazine hydrochloride is taken, 20ml of 0.9 percent sodium chloride injection is added and shaken till being completely dissolved,

(3) and (3) uniformly mixing the solutions obtained in the two steps.

Claims (1)

1. The pharmaceutical composition for treating severe malaria is characterized in that the pharmaceutical composition is prepared into nasal drops and consists of 40mg of artesunate and 20mg of ligustrazine hydrochloride, and the preparation process comprises the following steps:

(1) taking 40mg artesunate, adding 1ml 0.9% sodium chloride injection, dissolving completely, adding 3ml 0.9% sodium chloride injection for dilution,

(2) 20mg of ligustrazine hydrochloride is added with 4ml of 0.9 percent sodium chloride injection and shaken till being completely dissolved,

(3) and (3) uniformly mixing the solutions obtained in the two steps.

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