Medicine for treating trypanosomiasis

Abstract

The invention provides a medicine for treating trypanosomiasis, and belongs to the technical field of medicines. The medicine for treating trypanosomiasis provided by the invention comprises crizotinib and / or a salt of crizotinib. According to the present invention, the known anti-tumor drug crizotinib is used for treating trypanosomal infection through the drug relocation strategy, and the anti-kinase activity of the crizotinib is utilized to provide good trypanosomal resistance activity for trypanosoma buchneri so as to provide significant insecticidal effect. According to the technical scheme provided by the invention, clinical indications of crizotinib are expanded, so that crizotinib has a better application prospect. The result of the embodiment shows that crizotinib shows a relatively good in-vitro trypanosoma proliferation inhibition effect, and shows a remarkable insecticidal effect at a relatively low concentration.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, in particular to a medicine for treating trypanosomiasis. Background Art

[0002] Trypanosomiasis is a disease caused by Trypanosoma parasites, which mainly affects the health of humans and animals in tropical areas. Human trypanosomiasis, including African sleeping sickness and American trypanosomiasis, is caused by different species of trypanosomes and poses a major threat to public health. In particular, Human African trypanosomiasis (HAT) is a disease caused by infection with Trypanosoma parasites. It is also called sleeping sickness because of the symptoms of sleepiness it causes. The disease is transmitted by the bites of certain species of tsetse flies in the genus Glossina. It only occurs in Africa where tsetse flies survive. Patients in other regions are usually imported cases from endemic areas. After the tsetse flies infected with trypanosomes bite humans, local inflammatory reactions occur in the wound at the bite site, resulting in lumps and pain. The pathogen enters the blood circulation through the skin and multiplies in large numbers within two weeks, quickly reaching various tissues and organs of the body, followed by symptoms such as headache, irregular fever, swollen lymph nodes and rash. This stage is called the hemolymphatic stage or the first stage. In the second stage, the parasite can penetrate the blood-brain barrier and reach the brain, causing meningitis, whose symptoms include severe headache, drowsiness, slow reaction, difficulty walking, muscle tremors, etc., and sleep cycle disorders are also prominent features of the disease. Eventually, the patient falls into a coma or dies.

[0003] Sleeping sickness involves two types, depending on the type of infecting parasite, one is a chronic infection caused by Trypanosoma brucei gambiense, accounting for 92% of reported cases, and the other is an acute infection caused by Trypanosoma brucei rhodesiense. Patients with chronic infection may have no obvious symptoms and signs for a short period of time or even for several years. When obvious symptoms appear, they are often in the late stage of the disease and the patient's nervous system has been affected. Patients with acute infection usually do not last more than a few months. The initial symptoms can be observed within a few weeks of infection and the neurological course develops rapidly, with a high mortality rate.

[0004] At present, African trypanosomiasis is mainly treated with drugs. The choice of drugs depends on the type of pathogen and the stage of the disease. There are few types of drugs to choose from. Patients often need long-term injections of drugs and have serious side effects. In the first stage, trypanosomes are mainly present in the patient's subcutaneous, blood and lymph. At this time, intramuscular injection of pentamidine or oral fexinidazole is often used for infections caused by Gambian Trypanosoma, and suramin is often used for infections caused by Rhodesian Trypanosoma. In the second stage, drugs that can penetrate the blood-brain barrier are needed, and intravenous melarsoprol or eflornithine are often used. Among them, pentamidine is only effective for early Gambian trypanosomiasis and has limited application scenarios. Oral fexinidazole is more convenient than conventional injection treatment and has a good therapeutic effect, but recurrences may occur. Intravenous suramin may cause more severe toxic kidney damage and allergic reactions. Melarsoprol (melB) is an arsenic derivative that is effective against both types of trypanosomiasis. Due to its severe side effects, it is only used to treat patients in the late stages. It can cause very serious, life-threatening brain dysfunction, and reports show that 3 to 10% of patients die during treatment. Eflornithine (DFMO) is safer than melarsoprol and has become a first-line drug against human African trypanosomiasis as early as the 1990s, but it is only used for Gambian trypanosomiasis, so melarsoprol is still widely used in the late stages of the disease. In short, the current drugs for the treatment of human African trypanosomiasis still have many side effects, difficulty in using drugs, rapid development of drug resistance and high treatment failure rate.

[0005] After invading the human body, trypanosomes will cause a large increase in immunoglobulins, mainly IgM, and specific antibodies to aggregate, which will be engulfed by phagocytes or dissolved under the action of complement. However, because trypanosomes can continuously change antigens to escape the host's immune system, the number of trypanosomes in the blood fluctuates and is difficult to kill. Even after receiving drug treatment for several months, they continue to survive and cause disease again. At the same time, the continuous production of immune complexes will also cause extensive tissue damage. Patients can recover quickly after receiving treatment in the early stage of the disease, but in the late stage of the disease with neurological damage, some patients have permanent neurological sequelae after achieving clinical cure, and there is a possibility of recurrence, especially patients who enter the stage of lethargy and cardiovascular and cerebrovascular damage have a poor prognosis. At present, there is no detection method to confirm whether the patient has recovered. In this context, it is still very necessary to develop anti-trypanosome drugs that can cope with different types and different stages of the disease and are safer and can be taken orally.

[0006] In recent years, drug repositioning strategies have been widely used to find new treatments for diseases. Drug repositioning, also known as new uses for old drugs, is a strategy used to discover old drugs or drugs under development that go beyond their originally approved indications and expand their scope of application and use. New drug development is a high failure rate, high cost and slow process, while drug function repositioning has the advantages of low R&D costs and short development time, so using old drugs to treat common and rare diseases has become increasingly attractive.

[0007] Therefore, how to use drug repositioning strategy to provide a new drug for the treatment of trypanosomiasis has become a technical problem that needs to be solved urgently in this field. Summary of the invention

[0008] The object of the present invention is to provide a drug for treating trypanosomiasis. The drug provided by the present invention not only has good antitrypanosomal activity against Trypanosoma brucei and exhibits significant insecticidal effect, but also expands the clinical indications of crizotinib, so that crizotinib has better application prospects.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a drug for treating trypanosomiasis, wherein the drug for treating trypanosomiasis comprises crizotinib and / or a salt of crizotinib.

[0011] Preferably, the structural formula of crizotinib is as shown in Formula I:

[0012]

[0013] Preferably, the salt of crizotinib is an organic salt of crizotinib or an inorganic salt of crizotinib.

[0014] Preferably, the salt of crizotinib is a pharmaceutically acceptable salt of crizotinib.

[0015] Preferably, the drug for treating trypanosomiasis further comprises a carrier and / or an excipient.

[0016] Preferably, the carrier and / or excipient is a pharmaceutically acceptable carrier and / or excipient.

[0017] Preferably, the trypanosomiasis preferably includes Trypanosoma brucei and trypanosomiasis caused by it.

[0018] The present invention provides a drug for treating trypanosomiasis, wherein the drug for treating trypanosomiasis comprises crizotinib and / or a salt of crizotinib. The present invention uses a drug repositioning strategy to use the known anti-tumor drug crizotinib for treating trypanosomiasis infection, and utilizes its anti-kinase activity to have good anti-trypanosomiasis activity against Trypanosoma brucei, showing a significant insecticidal effect. The present invention provides a technical solution to expand the clinical indications of crizotinib, so that crizotinib has a better application prospect. The results of the embodiment show that the drug provided by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the inhibition curve of crizotinib on trypanosome growth;

[0020] Figure 2 is the parasitemia curve in mice;

[0021] Figure 3 This is the curve of mouse weight change over time;

[0022] Figure 4 This is the mouse survival rate curve. DETAILED DESCRIPTION

[0023] The present invention provides a drug for treating trypanosomiasis, wherein the drug for treating trypanosomiasis comprises crizotinib and / or a salt of crizotinib.

[0024] In the present invention, the structural formula of crizotinib is as shown in Formula I:

[0025]

[0026] In the present invention, the salt of crizotinib is preferably a pharmaceutically acceptable salt of crizotinib; the salt of crizotinib is preferably an organic salt of crizotinib or an inorganic salt of crizotinib. In the present invention, the pharmaceutically acceptable salt of crizotinib is a salt form used in a pharmaceutical preparation and is harmless to the human body.

[0027] In the present invention, when the drug for treating trypanosomiasis includes crizotinib and a salt of crizotinib, the present invention has no particular limitation on the specific dosage relationship of the crizotinib and the salt of crizotinib, and any ratio can be used for compounding.

[0028] In the present invention, the drug for treating trypanosomiasis preferably also includes a carrier and / or an excipient. The present invention has no particular limitation on the specific type and source of the carrier and excipient, and the carrier and excipient well known to those skilled in the art can be used. As an embodiment of the present invention, the carrier and / or excipient are preferably a pharmaceutically acceptable carrier and / or excipient. The present invention can play a role in fixing and adsorbing crizotinib and the salt of crizotinib by adding a carrier or excipient, thereby helping to further improve its therapeutic effect.

[0029] In the present invention, the trypanosomiasis preferably includes Trypanosoma brucei and trypanosomiasis caused by it.

[0030] The present invention uses the known anti-tumor drug crizotinib to treat trypanosome infection through a drug repositioning strategy, and utilizes its anti-kinase activity to have good anti-trypanosome activity against Trypanosoma brucei, showing a significant insecticidal effect. The present invention provides a technical solution to expand the clinical indications of crizotinib, giving crizotinib a better application prospect.

[0031] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] A drug used to treat trypanosomiasis, crizotinib.

[0034] The inhibitory effect of crizotinib on the growth activity of trypanosomes was tested by:

[0035] (1) The blood type of Trypanosoma brucei brucei AnTat1.1 strain was used, and the culture medium was HMI-9 (Hirumi's Modified Iscove's Medium-9), which contained 1.512 g / L sodium bicarbonate, 8.83 g / L IMDM (Iscove's Modified Dulbecco's Medium) powder, 0.068 g / L xanthine, 0.055 g / L sodium pyruvate, 0.0195 g / L thymidine, 0.014 g / L bathocuproine sulfate, 0.091 g / L L-cysteine ​​and 7 μL / L β-mercaptoethanol, and fetal bovine serum (fetal bovine serum) was added. serum, FBS) to obtain 10% FBS / HMI-9 medium, put Trypanosoma brucei into 10% FBS / HMI-9 medium, and then culture the strain in a 37°C incubator containing 5% carbon dioxide;

[0036] (2) dissolving crizotinib in DMSO to obtain a drug stock solution with a concentration of 10 mM, diluting the drug stock solution with a culture medium based on the concentration of the drug stock solution, setting the concentration gradient of the drug solution to 1 μM, 0.5 μM, 0.1 μM, 0.05 μM and 0.01 μM, respectively, to obtain experimental groups with concentration gradients of 1 μM, 0.5 μM, 0.1 μM, 0.05 μM and 0.01 μM, respectively, and setting a DMSO-containing culture medium without adding crizotinib and a culture medium without adding crizotinib and DMSO as control groups. For the experimental group and the control group containing DMSO, the DMSO content was controlled to be equal and the volume content was 0.2%, and then the experimental group and the control group were subjected to an in vitro activity test experiment;

[0037] (3) Count and dilute the Trypanosoma brucei being cultured in step (1) to prepare a concentration of 1×10 4 The experimental group and the control group obtained in step (2) were mixed with the culture solution at a volume ratio of 1:1. At this time, the volume content of DMSO was 0.1%, and the trypanosome density was 5×10 3 The volume of the experimental group and the control group was 6 mL. Then, a 12-well cell culture plate was used for the insect resistance experiment. Three duplicate wells were set for each group, and the sample volume was 2 mL. After 48 hours of culture, the counts were made and the growth rate was calculated. The calculation formula of the growth rate is shown in Formula II:

[0038]

[0039] The obtained data were entered into GraphPad Prism software for plotting and analysis to obtain IC50 The numerical value, the result is Figure 1 described.

[0040] Figure 1 The inhibition curve of crizotinib on trypanosome growth is shown in Figure 2. Figure 1 It can be seen that with the increase of crizotinib concentration in the drug solution, the growth rate of trypanosomes showed a significant downward trend, and the IC50 value was 0.124±0.019 μM, indicating that crizotinib showed a good inhibitory effect on trypanosome proliferation in vitro.

[0041] The inhibitory effect of crizotinib on parasitemia in mice was tested by the following method:

[0042] (1) 25 healthy, mature, nulliparous female Kunming mice were obtained and routinely raised with pelleted food and tap water at room temperature of 20-22°C and a light-dark cycle of 12 h each. The mice weighed 15-25 g and were purchased from the Experimental Animal Center of Sun Yat-sen University.

[0043] (2) The mice in step (1) were randomly divided into 5 groups, each with 5 mice. The 5 groups of mice were infected with an equal amount of Trypanosoma brucei brucei AnTat1.1 strain. Four of the groups were administered with 150 mg / kg, 100 mg / kg, 50 mg / kg and 10 mg / kg of crizotinib, respectively. The remaining group was used as a control group and was administered with DMSO instead of crizotinib. The administration was performed by intraperitoneal injection. The parasitemia of the mice was observed and counted by blood sampling from the tail vein under a 20x microscope. The data were entered into GraphPad Prism. The experimental results are shown in FIG. Figures 2 to 4 shown.

[0044] Figure 2 This is the parasitemia curve in mice, where the “-” below the X-axis represents that the mice were not given medication on that day, and the “+” represents that the mice were given crizotinib on that day. The value is mean±SD. Figure 3 This is the curve of mouse body weight change over time, and the values ​​are mean±SD. Figure 4 is the mouse survival rate curve, and the values ​​are mean±SD. Figure 2It can be seen that after the mice were infected with Trypanosoma brucei, the number of Trypanosoma brucei showed a clear increasing trend in the first 4 days. After the drug was administered on the 3rd to 6th days, it can be clearly seen that the number of Trypanosoma brucei in the 4 groups of mice injected with crizotinib was significantly lower than that in the control group, indicating that crizotinib exhibited significant insecticidal effects at lower concentrations. At the same time, when the dosage was 150 mg / kg and 100 mg / kg, the number of Trypanosoma brucei decreased significantly, indicating that by controlling the dosage of crizotinib, it can exhibit excellent insecticidal effects. After stopping the drug administration within 7 to 13 days, the number of Trypanosoma brucei in the mice began to increase significantly again. After the drug was administered every other day within 14 to 20 days, the number of Trypanosoma brucei decreased again, showing a significant insecticidal effect. Figure 3 and Figure 4 It can be seen that when the dosage of crizotinib is too high (150 mg / kg and 100 mg / kg), although it has a good inhibitory effect on Trypanosoma brucei, the weight of mice will drop rapidly and the survival rate will be greatly reduced. When the dosage of crizotinib is 50 mg / kg and 10 mg / kg, the weight of mice increases normally and no mice die. However, when the dosage is 10 mg / kg, the content of Trypanosoma brucei in mice is higher. Although the weight of mice that have not been injected with crizotinib is normal, they begin to die after 8 days. This shows that crizotinib can inhibit the growth of Trypanosoma brucei, thereby improving the survival rate.

[0045] Depend on Figures 2 to 4 It can be seen that crizotinib shows obvious insecticidal effect in vivo at around 50 mg / kg, but if the dose is too high, it will have a greater impact on the health of mice and even cause death, while if the dose is too low, the insecticidal effect in vivo is weak. After comprehensive consideration, it is recommended to control the dose of crizotinib within the range of 30-70 mg / kg.

[0046] From the above analysis, it can be seen that crizotinib exhibits significant insecticidal effects at relatively low concentrations; in vivo experiments confirm the effectiveness of crizotinib in inhibiting trypanosomiasis, which shows that the present invention provides a scientific basis for expanding the clinical indications of crizotinib and provides an effective new solution for the treatment of trypanosome infections.

[0047] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A drug for treating trypanosomiasis, characterized in that The drug for treating trypanosomiasis includes crizotinib and / or a salt of crizotinib.

2. The drug for treating trypanosomiasis according to claim 1, characterized in that The structural formula of crizotinib is shown in Formula I:

3. The drug for treating trypanosomiasis according to claim 1, characterized in that The salt of crizotinib is an organic salt of crizotinib or an inorganic salt of crizotinib.

4. The drug for treating trypanosomiasis according to claim 1 or 3, characterized in that: The salt of crizotinib is a pharmaceutically acceptable salt of crizotinib.

5. The drug for treating trypanosomiasis according to claim 1, characterized in that The medicament for treating trypanosomiasis further comprises a carrier and / or an excipient.

6. The drug for treating trypanosomiasis according to claim 1, characterized in that The carrier and / or excipient is a pharmaceutically acceptable carrier and / or excipient.

7. The drug for treating trypanosomiasis according to claim 1, characterized in that The trypanosomiasis preferably includes Trypanosoma brucei and trypanosomiasis caused by it.

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