What is the preparation method for the new antimalarial drug compound Artemether?

Drug-resistant malaria is increasing day by day around the world. More than 70% of Plasmodium falciparum parasites are resistant to existing antimalarial drugs such as chloroquine, chlorophenoquine, proguanil, pyrimethamine, mechloroquine and their combinations. All have developed varying degrees of drug resistance. In addition, the toxic and side effects of paraquine, mechloroquine and their compounds are also serious, directly affecting the treatment of 103 million malaria patients and 264 million malaria parasite carriers around the world every year.

Artemether (C18H28O5) and lumefantrine (C30H32CI3NO) are two new antimalarial drugs with different structures and effects that have been successfully developed in recent years. Both drugs are usually used as monotherapy. The characteristic of Artemether is that it kills Plasmodium quickly, but it does not kill parasites completely. After treatment, the protozoa remaining in the patient's blood have a high resurgence rate. Lumefantrine is just the opposite. It kills insects more thoroughly and has a high cure rate, but it works slowly. However, what they have in common is that they are highly effective against drug-resistant falciparum malaria.

The purpose of the present invention is to provide an antimalarial drug compound Artemether, which is based on the principle of synergistic and complementary effects between drugs, and through animal experimental research, to determine the type of interaction between drugs and to find the formula of the compound. The best composition, in order to achieve the purpose of maximizing strengths and avoiding weaknesses, not only takes advantage of Artemether's quick-acting effect, but also makes full use of lumefantrine's thorough insecticide and high cure rate. Up to now, there is no antimalarial compound product of the same kind at home and abroad.

The object of the present invention is achieved through the following technical solutions.

1. Determine the dose series ratio relationship between Artemether and lumefantrine and the synergistic effect under optimal ratio conditions through animal experiments. Using Plasmodium berghei-infected mice as the experimental model, an orthogonal design was adopted, and the "4-day inhibition test" method was used to conduct a parallel comparative test of the antimalarial potency of different dose series ratios of the two drugs. The linear regression equation calculation method is used to calculate ED50 or ED90 and its synergistic index.

Synergistic index = (ED5D or ED90 of a single drug)/(ED50 or ED90 of the corresponding drug in the compound)

According to this formula, the optimal ratio of the compound for anti-mouse malaria is calculated as 2:0.75 (the synergistic index of ED90 is >6).

Based on the mouse malaria experiment, a monkey Plasmodium (Plasmodium   Knowlesi)/rhesus monkey experiment was conducted. The results showed that the optimal ratio of the compound antimalarial compound is 1:3~6.

2. The evaluation of the type of interaction between drugs in this compound is based on the addition line diagram method of Peters (1969). Any coordinate point of the compound ED90 located near the addition line is judged to be an interaction between drugs. There is an additive effect. If the coordinate point is below the addition line and far away from the line, it is judged that there is a synergistic effect between the drugs. If the coordinate point is above the addition line and far away from the line, it is judged that there is an antagonism between the drugs. , this compound has a pharmacodynamic synergistic effect.

3. The insecticidal speed of the compound is determined by using a large dose (equivalent dose) of 20×ED90/ig when the density of protozoa in the blood of model animals artificially infected with malaria increases to a high density. According to the method, the decline rate of protozoa in the blood of animals was continuously observed after administration. Calculated based on the time for the protozoa to decline by 90%, the compound formula was 49.7 hours, and the single drug lumefantrine was 64.3 hours. Because Artemether failed to reduce the protozoa density to 90%, that is, Picked up. It can be seen that the insecticidal speed of the compound fully meets the design requirements.

IV. Clinical exploration of the optimal ratio of Artemether and lumefantrine. According to the animal test results, the optimal ratio of Western medicines is estimated to be 1:4 to 1:6 with reference to the clinical effective doses of Artemether and lumefantrine alone. Therefore, it is measured as 1:4. Each tablet contains 25 mg of Artemether and 100 mg of lumefantrine. According to the measurement of 1:5, each tablet contains Artemether 20mg and lumefantrine 100mg. According to the measurement of 1:6, each tablet contains Artemether 20mg and lumefantrine 120mg. There are three ratios. Choose 1:5 and 1:6 to set up two groups. , a clinical parallel comparative trial was conducted. Both groups were treated 4 times in 3 days, that is, 4 tablets were taken orally for the first time, and 4 tablets were taken at intervals of 8, 24, and 48 hours, for a total of 16 tablets for adults. 40 patients with falciparum malaria were selected and randomly divided into two groups. The test results showed that 4 hours after taking the drug, the protozoa reduction rates were 96.3% and 94.2% respectively; the average protozoa disappearance time was 34.8 hours and 36.0 hours, and the average fever reduction time was 23.2 hours and 22.4 hours. However, the relapse rate of protozoa on day 28 was 20% in the 1:5 group, while in the 1:6 group, no one relapsed and all were cured. It was proved in this experiment that the optimal ratio of Artemether and lumefantrine in the compound against human malaria was 1:6.

5. Determination of the effect of the compound in delaying the development of drug resistance in protozoa. A drug-resistant strain of protozoa was cultivated through a small-dose ascending continuous blood transmission method, and a parallel comparative test was conducted on the rate of drug resistance of the drug-resistant strain to the compound Artemether and the single drug lumefantrine. It lasted 560 days and was continuously cultivated for 80 generations. The results proved that The combination of the two drugs can delay the development of drug resistance in protozoa and reduce the degree of resistance in protozoa. For example, when calculating the drug tolerance dose of Plasmodium bred for 20 generations, the tolerance dose to lumefantrine alone is 100 times higher than the original dose, and to Artemether it is 20 times higher, but to this compound it is only 2.9 times higher. . Calculated according to the 30th generation resistance index (Ⅰ90), lumefantrine is >410 and the compound is >19.3, indicating that this test method can more accurately assess the effect of this compound on delaying the development of drug resistance in protozoa and is less likely to produce drug resistance in actual use. .

6. Determine the clinical side effects of the compound according to pharmacological methods. Mice, rats and cats were used as test models. The two compound drugs were prepared at a ratio of 1:6. The total dosage was 112 mg/kg (equivalent to 10 times the human dose). The mice and rats were administered the same volume by gavage. Both are 10ml/kg, cats are 2ml/kg. During the observation period, the animals’ nervous system, cardiovascular system and respiratory system were examined. However, the results showed no changes in pharmacological activity.

7. Conduct a safety evaluation of the compound according to toxicological methods, in which the ratio of Artemether and lumefantrine is 1:6. The LD50 for acute toxicity tests in mice was 4455 mg/kg by intragastric administration and 1554 mg/kg by intraperitoneal injection. According to chemical toxicity classification standards, this compound is of low toxicity level. A 14-day toxicity test on rats and Bea-gle dogs, each with three dose groups: large, medium and small, administered orally once a day for 14 consecutive days. The animals' diet, weight, hematology, biochemistry and other indicators as well as major organs were observed and examined. and pathological examination of drug target organs. The results show that the basic safe dose for rats is 448MKD, which is equivalent to 40 times the clinical dose; the safe dose for dogs is 556MKD, which is equivalent to 50 times the human dose. In the high-dose group, although there were abnormal changes in the liver and kidneys, the target organs, all examinations returned to normal 28 days after stopping the drug. This shows that the compound has low toxicity, a wide safety range, and no irreversible toxic reactions.

8. Comparison of the efficacy of compound and single drugs against human falciparum malaria. Two test groups were set up, oral administration, 4 times of therapy in 3 days, 20 patients with falciparum malaria in each group, and the efficacy of the compound and its corresponding doses of Artemether and lumefantrine alone were observed.

The decline rate of protozoa 24 hours after administration: 97% for the compound, 95.1% for Artemether, and 74.5% for lumefantrine; the average disappearance time of protozoa: 35.6 hours for the compound, 38.7 hours for Artemether, and 74.5% for this drug. Fluorenol is 68.4 hours; the average antipyretic time of the compound is 23.8 hours, Artemether is 19.7 hours, lumefantrine is 40 hours, and the 28-day cure rate is 95%, 45% and 65% respectively. This experimental treatment plan can be compared Clearly explain the difference in efficacy between a compound formula and a single drug. The compound formula is better than a single drug.

9. Clinical expansion trial of the compound and comparative trial of the efficacy of oxyquine. The compound Artemether was administered orally, 4 times in 3 days, and a total of 400 cases of falciparum malaria were treated. The main indicators of clinical observation: (1) The average time for protozoa to disappear (the test result is 23.2 to 41.0 hours); (2) The average antipyretic time (the result is 20.4 to 25.7 hours); (3) The cure rate 28 days after administration ( The average results are 95-100%), indicating that the efficacy of the compound is more than 1.3 times higher than that of Artemether alone (45%).

Comparative trial on the efficacy of compound and chloroquine. The compound is administered four times in three days. Chloroquine is administered according to international standards, that is, 4 tablets for the first time, 2 tablets each at 8, 24 and 49 hours, for a total of 10 tablets for adults. There were 35 patients in the compound group and 22 patients in the chloroquine group. Observation content: (1) Average protozoal disappearance time (results were 37.8 and 87.3 hours respectively); (2) Average fever defervescence time (results were 24.2 and 58.5 hours respectively); (3) 28-day cure rate (results were 97.1%) and 40.9%). It is proved that the compound formula has significant effect on anti-chloroquine malaria, which is significantly more than 1.4 times better than chloroquine.

No abnormal findings were found in the patient's blood, urine and electrocardiogram examinations.

The examples given according to the technical solution proposed by the compound Artemether of the present invention are as follows: Example 1: Through animal experimental research, the dosage series relationship between Artemether and lumefantrine in the compound is determined, and the dosage after mixing the two drugs is determined. synergistic effect. Using white mice infected with Plasmodium berghei as the experimental model, an orthogonal design and "4-day inhibition test" method were used to conduct a parallel comparative test of the antimalarial potency of different dose series ratios of the two drugs. , use the linear regression equation calculation method to calculate ED or ED and its synergy index.

Synergistic index = (ED50 or ED90 of a single drug)/(ED50 or ED90 of the corresponding drug in the compound). This formula determines the optimal ratio of the compound against mouse malaria.

Based on the mouse malaria test, the test was conducted using Plasmodium Knowlesi/rhesus monkey as the model. Each monkey was intravenously inoculated with 5 × 10 red blood cells harboring Plasmodium parasites. When the protozoal parasite rate in the animal's blood reaches 3 to 5%, drug administration (gavage) is started according to the designed plan. Smear microscopy will be performed every 12 hours after the first dose, once a day after the protozoa turns negative, and once every other day after 15 days. Continuous observation will be conducted for 105 days, and the test results will be determined according to pharmacodynamic standards.