CN115487191A

CN115487191A - Quisinostat, a novel highly potent antimalarial drug

Info

Publication number: CN115487191A
Application number: CN202211242735.6A
Authority: CN
Inventors: 江陆斌; 黄正辉
Original assignee: Institut Pasteur of Shanghai of CAS
Current assignee: Institut Pasteur of Shanghai of CAS
Priority date: 2016-02-22
Filing date: 2016-02-22
Publication date: 2022-12-20
Anticipated expiration: 2036-02-22
Also published as: CN107095871A; CN115487191B; WO2017143964A1

Abstract

The invention provides a novel high-efficiency antimalarial drug, and particularly provides application of Quisinosistat, analogues thereof or pharmaceutically acceptable salts thereof in treating malaria. The invention also provides a pharmaceutical composition containing Quisinostat. Through deep research, quisinostat has a remarkable inhibition effect on plasmodium and even shows a better insecticidal effect than artemisinin in mice.

Description

Quisinostat, a novel highly potent antimalarial drug

The invention is a divisional application based on a patent application with the application date of 2016, 2, 22 and the application number of 201610096388.9, entitled "Quisinostat, a novel efficient antimalarial drug".

Technical Field

The invention belongs to the field of medicines, and particularly relates to a novel efficient antimalarial drug.

Background

Malaria is an infectious febrile disease caused by plasmodium protozoa that are parasitic in red blood cells and transmitted by the bite of malaria mosquitoes (anopheles). The disease is characterized by episodes of chills, fever and night sweats that occur at intervals that depend on the time required for the new generation of parasites to develop in vivo. After recovery from an acute episode, the disease tends to become chronic with occasional relapses. There are four types of Plasmodium, plasmodium falciparum/p.falciparum, plasmodium vivax/p.vivax, plasmodium malariae/p.malariae, and Plasmodium ovale/p.ovale.

Among the various forms of human malaria, malaria caused by plasmodium falciparum is characterized by severe systemic symptoms, sometimes even leading to death, which is responsible for most cases in human mortality. Plasmodium falciparum is dangerous not only because it digests the hemoglobin of the red blood cells, but also because it changes the adhesion properties of the inhabited cells, which causes them to stick to the vessel wall. This becomes dangerous when infected blood cells stick to blood vessels, thereby blocking blood flow. The disease is prevalent in tropical and subtropical regions of the world, including amazon, eastern and southern africa, and southeast asia of brazil. According to the world malaria report of the world health organization 2011, there were 216,000,000 cases of malaria in 2010, and 81% of them were in regions of WHO africa. An estimated 655,000 malaria-related deaths in 2010, with 86% of victims being children under the age of 5.

Throughout recent history, malaria has been treated with a variety of drugs, including combinations of drugs. However, the emergence of drug-resistant malaria insect strains has become a significant problem in malaria therapy. WHO has suggested artemisinin (artemisinin) in combination with other classes of antimalarial drugs (artemisinin-based combination therapy (ACT)) as a first line treatment of malaria caused by plasmodium falciparum. In 2009, it was confirmed in Cambodia-Thailand border that artemisinin resistant Plasmodium falciparum appears in Burma and parts of Vietnam, and therefore, the appearance of new highly effective antimalarial drugs is urgently needed in the art to inhibit malaria transmission.

Disclosure of Invention

The invention aims to provide a novel high-efficiency antimalarial drug.

In a first aspect of the invention, there is provided the use of Quisinostat, an analogue thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of cancer

(1) A drug for the prevention and/or treatment of malaria; and/or

(2) An agent that inhibits and/or kills plasmodium.

In a second aspect of the invention, there is provided an anti-plasmodium composition comprising Quisinostat, an analogue thereof, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the composition comprises a first active ingredient and a second active ingredient,

wherein the first active ingredient is Quisinostat, an analog thereof, or a pharmaceutically acceptable salt thereof;

the second active ingredient is piperaquine, an analogue thereof, or a pharmaceutically acceptable salt thereof, and/or

Benflumetol, an analog thereof, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the plasmodium is selected from the group consisting of: plasmodium falciparum, plasmodium vivax, plasmodium malariae, and plasmodium ovale.

In another preferred embodiment, the piperaquine is piperaquine phosphate.

In another preferred embodiment, the molar ratio of the first active ingredient to the second active ingredient is about 1 to 20:20 to 1, preferably 1 to 10:10 to 1, more preferably 1 to 5:5 to 1.

In another preferred embodiment, the composition further comprises a pharmaceutically acceptable carrier or excipient.

In another preferred embodiment, the dosage form of the pharmaceutical composition comprises tablets, granules, capsules, pills, injections, or oral liquids.

In another preferred embodiment, the composition is in unit dosage form, each unit dosage form containing the first active ingredient and the second active ingredient in an amount of about 0.1 to 1 (or 0.25-1, or 0.5-1) of the daily dose, wherein the daily dose is 20-100mg.

In another preferred embodiment, the daily dose is 25-70mg, such as 25mg, 40mg, 50mg.

In a third aspect of the invention there is provided the use of an anti-plasmodium composition according to the first aspect of the invention for the preparation of a medicament for the treatment of a disease or condition associated with a malaria parasite

(1) A drug for the prevention and/or treatment of malaria;

(2) An agent that inhibits and/or kills plasmodium.

In a fourth aspect of the invention, there is provided a method of preventing and/or treating malaria, the method comprising the steps of:

administering Quisinostat, an analog thereof, or a pharmaceutically acceptable salt thereof to a subject in need thereof, or administering the anti-malarial parasite composition of claim 2 to a subject in need thereof, thereby preventing and/or treating malaria.

In another preferred embodiment, the subject includes human and non-human mammals (e.g., rodents).

In another preferred embodiment, said administration is in an amount of 1-1000mg/kg body weight, preferably 15-700mg/kg body weight, more preferably 20-500mg/kg body weight, based on the weight of said first active ingredient.

In another preferred embodiment, the disease malaria.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the growth inhibitory effect of Compound No.37 on Plasmodium cultured in vitro (EC 503.91nM) with artemisinin as a positive control (EC 50.38 nM); the growth of Plasmodium was determined using a 3-day SYBR GREEN I cell growth assay.

FIG. 2 shows the cytotoxic effect of the No.37 compound on the human liver cancer cell line HepG2 and the human renal epithelial cell line 293T; simultaneously, the selective index between the inhibition effect of the No.37 compound on plasmodium and the toxicity on different cell lines is displayed, and artemisinin is used as a positive control; the growth of the cell line was determined by the method of 3-day cell titer Glo and the growth of the Plasmodium was determined by the method of 3-day SYBR GREEN I.

FIG. 3 shows the pharmacological interaction of the compound No.37 with the common antimalarial drugs benflumetol, artemisinin, piperaquine phosphate; the drug interaction is carried out in plasmodium cultured in vitro, and the growth condition of the plasmodium is measured by adopting a method of 3-day SYBR GREEN I; FIC is the drug interaction index.

FIG. 4 shows the pesticidal effect of the compound No.37 in mice; the compound No.37 was tested at two different concentrations, 10mg/kg and 20mg/kg, with the combined concentrations being compound No.37 (10 mg/kg) + lumefantrine (10 mg/kg); compound No.37 (10 mg/kg) + piperaquine phosphate (10 mg/kg), artemisinin 10mg/kg and 30mg/kg as positive controls, DMSO as negative controls.

FIG. 5 shows that the present inventors previously prepared each compound solution at a concentration of 400nM, added 100. Mu.l of complete medium to each of B1-H12 wells in a 96-well plate, and added two compound solutions interacting in different volumes to each of 200. Mu.l of a system in A1-A12 wells.

Detailed Description

The present inventors have unexpectedly found, through extensive and intensive studies, that Quisinostat (i.e., the compound No.37 in the present invention) has a significant inhibitory effect on plasmodium and even exhibits a better insecticidal effect than artemisinin in mice. Further research finds that intravenous injection of the combination drug of Quisinosistat, piperaquine and Lumefantrine to mice shows remarkable synergistic effect and also shows better insecticidal effect than artemisinin. On the basis of this, the present invention has been completed.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

Quisinosistat, analogue thereof, or pharmaceutically acceptable salt thereof

In the present invention, quisinostat and the No.37 compound are the same compound. The compound No.37, which is the active ingredient of the present invention, is a novel second-generation inhibitor of Hdac, which acts mainly on Hdac1 in Hdac family Class I. The chemical name of the No.37 compound is: n-hydroxy-2- [4- { [ (1-methylindol 1-3-ylmethyl) amino ] methyl } -1-piperidinyl ] -5-pyrimidinecarboxamide

The structure of the compound No.37 is as follows:

analogs of the No.37 compounds include, but are not limited to: compounds formed by substituting one or more hydrogens, and/or hydroxyl groups in the above formula, preferably the substituents are selected from the group consisting of: halogen (F, cl, br, I), C _1-8 Alkyl radical, C _2-8 Alkenyl radical, C _2-8 Alkynyl, C _3-8 Cycloalkyl, 3-to 12-membered heterocyclyl, aryl, heteroaryl, CN, NO ₂ 。

The term "pharmaceutically acceptable salt" refers to salts that are suitable for contact with the tissues of a subject (e.g., a human) without undue side effects. As used herein, a pharmaceutically acceptable salt refers to a salt of the compound No.37 or an analog thereof with pharmaceutically acceptable inorganic and organic acids, wherein preferred inorganic acids include (but are not limited to): hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid; preferred organic acids include (but are not limited to): formic acid, acetic acid, propionic acid, succinic acid, naphthalenedisulfonic acid (1, 5), sulfinic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, and amino acids. In some embodiments, pharmaceutically acceptable salts of the No.37 compound of the present invention include salts of the compounds of the present invention having acidic groups (e.g., potassium, sodium, magnesium, calcium salts) or salts of the compounds of the present invention having basic groups (e.g., sulfate, hydrochloride, phosphate, nitrate, carbonate).

The compound No.37 showed a growth inhibitory effect of plasmodium comparable to that of artemisinin in vitro test, and showed a better insecticidal effect than artemisinin in mouse test. In drug interaction experiments, no.37 was found to have no mutual antagonism with the current most important classes of antimalarials, and in mouse combination experiments, it was found that intravenous injection of No.37 in combination with Piperazine (Piperaquine phosphate) and Lumefantrine (Lumefantrine) respectively in mice also showed better insecticidal effect than artemisinin. This means that combination therapy can be used in future patent drug development and clinical trials, which will greatly help to delay the development of drug resistance, while malaria treatment will be better and more efficient.

In vitro plasmodium growth inhibition experiments, artemisinin is used as a control, no.37 is used as an experimental group to determine the plasmodium growth inhibition EC50, and the No.37 shows very good plasmodium growth inhibition function, and is equivalent to artemisinin, and the EC50 is 3.91nM. The experiment is repeated for more than three times in the biological sense, the result is stable, and the reliability is high.

In mouse experiments, mice were inoculated with the murine malarial lethal strain Pb AnKa and 24 hours after inoculation were administered artemisinin, the No.37 compound and the No.37 compound + pipraquinone, the No.37 compound + lumefarntrine by intravenous injection at different concentrations, respectively:

artemisinin 10mg/kg,30mg/kg;

No.37 10mg/kg,20mg/kg；

No.37(10mg/kg)+Piperaquine(10mg/kg)；

No.37(10mg/kg)+Lumefantrine(10mg/kg)；

three of each group were used as replicates. DMSO was also injected as a negative control. The experimental results show that the No.37 and the No.37+ piperaquine and the No.37+ lumefarnine show better inhibition effect on the rat malaria than the artemisinin, and the experiments are repeated for many times in rats of different species, so that similar results are obtained, and the reliability is high.

Piperaquine (Piperaquine), analogs thereof, or pharmaceutically acceptable salts thereof

Chemical name: 1, 3-bis [4- (7-hydroxyquinolin-4-yl) piperazin-1-yl ] propane

The structural formula is as follows:

analogs of piperaquine include, but are not limited to: compounds formed after substitution of one or more hydrogens, and/or one or more Cl in the above formula, preferably the substituents are selected from the group consisting of: halogen (F, cl, br, I), C _1-8 Alkyl radical, C _2-8 Alkenyl radical, C _2-8 Alkynyl, C _3-8 Cycloalkyl, 3-to 12-membered heterocyclyl, aryl, heteroaryl, CN, NO ₂ 。

The term "pharmaceutically acceptable salt" refers to a salt that is suitable for contact with the tissues of a subject (e.g., a human) without undue side effects. As used herein, pharmaceutically acceptable salts refer to salts of piperaquine or an analog thereof with pharmaceutically acceptable inorganic and organic acids, wherein preferred inorganic acids include (but are not limited to): hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid; preferred organic acids include (but are not limited to): formic acid, acetic acid, propionic acid, succinic acid, naphthalenedisulfonic acid (1, 5), sulfinic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, and amino acids. In some embodiments, pharmaceutically acceptable salts of piperaquine or analogs thereof of the invention include salts of compounds of the invention having acidic groups (e.g., potassium, sodium, magnesium, calcium salts) or salts of compounds of the invention having basic groups (e.g., sulfate, hydrochloride, phosphate, nitrate, carbonate).

Piperaquine phosphate was synthesized in the 60 s of the 20 th century, and was widely used in malaria prevention and treatment in china and india for the following 20 years, until the 80 s of the 20 th century, drug-resistant strains of piperaquine phosphate emerged; because of its long half-life, it is now used in combination with Artemisinin (ACT) for the prevention and treatment of malaria.

Benflumetol (Lumefantrine), analogue thereof, or pharmaceutically acceptable salt thereof

Chemical name: (Z) -2, 7-dichloro-9- [ (4-chlorophenyl) methylene ] - α - [ (di-n-butylamino) methyl ] -9H-fluorene-4-methanol

The structural formula is as follows:

analogs of lumefantrine include, but are not limited to: compounds formed by substituting one or more hydrogen, chlorine and/or hydroxyl groups in the above formula, preferably the substituents are selected from the group consisting of: halogen (F, cl, br, I), C _1-8 Alkyl radical, C _2-8 Alkenyl radical, C _2-8 Alkynyl, C _3-8 Cycloalkyl, 3-to 12-membered heterocyclyl, aryl, heteroaryl, CN, NO ₂ 。

The term "pharmaceutically acceptable salt" refers to salts that are suitable for contact with the tissues of a subject (e.g., a human) without undue side effects. As used herein, pharmaceutically acceptable salts refer to salts of lumefantrine or an analog thereof with pharmaceutically acceptable inorganic and organic acids, wherein preferred inorganic acids include (but are not limited to): hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid; preferred organic acids include (but are not limited to): formic acid, acetic acid, propionic acid, succinic acid, naphthalenedisulfonic acid (1, 5), sulfinic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, and amino acids. In some embodiments, the pharmaceutically acceptable salts of the lumefantrine or the analog thereof of the present invention include salts of the compounds of the present invention having an acidic group (e.g., potassium salt, sodium salt, magnesium salt, calcium salt) or salts of the compounds of the present invention having a basic group (e.g., sulfate, hydrochloride, phosphate, nitrate, carbonate).

Benflumetol has a very long half-life and is now used mainly in the prevention and treatment of malaria in combination with Artemisinin (ACT).

Composition comprising a metal oxide and a metal oxide

As used herein, the term "composition" includes pharmaceutical compositions and agents.

The composition of the present invention comprises an active ingredient against plasmodium and a pharmaceutically acceptable carrier. The active ingredients of the anti-malarial parasite comprise a first active ingredient and a second active ingredient, wherein the first active ingredient comprises Quisinostat, an analogue thereof, or a pharmaceutically acceptable salt thereof; and the second active ingredient is piperaquine phosphate, an analog thereof, or a pharmaceutically acceptable salt thereof, and/or

Benflumetol, an analog thereof, or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition of the present invention may further comprise various pharmaceutical excipients suitable for the contained compounds or compositions, and may be prepared into dosage forms advantageous for administration by conventional methods, such as: but not limited to aqueous solution injection, powder injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray, powder spray, sustained release agent, controlled release agent, etc. The pharmaceutic adjuvant can be conventionally used in various preparations, such as: but are not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrating agents, lubricants, and the like; it may also be selected for use in accordance with the substance, such as: but not limited to emulsifier, solubilizer, bacteriostatic agent, analgesic, antioxidant and the like, and the auxiliary materials can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate, absorption rate and the like of the compounds, thereby improving the metabolism of various compounds in organisms and further enhancing the administration effect of the composition. In addition, specific administration purposes or modes may be achieved, such as: sustained release administration, controlled release administration, pulse administration, and the like, and used auxiliary materials such as: but are not limited to, gelatin, albumin, chitosan, polyethers, and polyester-based polymeric materials (e.g., but not limited to, polyethylene glycol, polyurethane, polycarbonate, copolymers thereof, etc.). The main indications for facilitating administration are: but not only improving the treatment effect, improving the bioavailability, reducing the toxic and side effects, improving the compliance of patients and the like.

In aqueous injection, the adjuvant generally comprises isotonic agent, buffer, necessary emulsifier (such as Tween-80, pluronic and Poloxamer), solubilizer, bacteriostatic agent, etc. In addition, the pharmaceutical composition also comprises other pharmaceutically acceptable pharmaceutic adjuvants, such as: antioxidants, pH modifiers, analgesics, and the like.

The adjuvants used for preparing oral liquid preparation generally include solvent, and necessary correctant, bacteriostat, emulsifier and colorant, etc.

The excipients used for preparing tablets generally include fillers (e.g., starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, mannitol, etc.), binders (e.g., ethanol, starch slurry, sodium carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, gelatin solution, sucrose solution, and an aqueous or alcoholic solution of polyvinylpyrrolidone, etc.), disintegrants (e.g., dry starch, sodium carboxymethylcellulose, low-substituted hydroxypropylcellulose, crospovidone, and croscarmellose sodium), lubricants (e.g., magnesium stearate, aerosil, talc, hydrogenated vegetable oils, polyethylene glycol 4,000, polyethylene glycol 6,000, magnesium lauryl sulfate, etc.), and the like.

The adjuvants used for preparing emulsion are generally water, oil (such as fatty acid), emulsifier, and necessary antiseptic and correctant.

The excipients used to make granules are similar to tablets, but the granulation process is different. Mixing the obtained granule with glidant, and encapsulating to obtain capsule.

As used herein, the terms "subject", "organism", "animal" or "patient" include humans, wild animals and Livestock (Livestock). The wild animal is an animal which is not artificially domesticated in a natural state. Livestock are animals that are artificially raised to provide a food source, such as: but are not limited to, dogs, cats, mice, rats, hamsters, pigs, rabbits, cows, buffalos, bulls, sheep, goats, geese, chickens, and the like. The "patient" or "organism" to which treatment is administered is preferably a mammal, particularly a human.

As used herein, the term "prevention" refers to various means or measures for preventing the onset or progression of a disease, including medical, physical, or chemical means, to prevent or reduce the onset or progression of various symptoms of the disease, prior to the disease being recognized by clinical standards.

As used herein, the term "treating" or "treatment" refers to inhibiting, suppressing, reducing, ameliorating, slowing, halting, delaying or reversing the progression or exacerbation of a disease process in order to arrest or reduce the occurrence or progression of a disease, and describes maintaining and/or reducing the various indicators of a disease, disorder or pathological condition as applied, including reducing or alleviating symptoms or complications, or curing or eliminating a disease, disorder or condition.

As used herein, the term "drug" refers to a single compound, a composition of multiple compounds, or a composition or formulation (formulation) having a single compound as a main active ingredient, and also refers to a composition or formulation having multiple compounds as active ingredients, which can be used for preventing or treating a certain disease. "medicament" is understood to mean not only the product approved and approved for production by the regulatory agency established in accordance with the state of law, but also the forms of the various substances formed in order to obtain the approved and approved production, which contain the single compound as active ingredient. "Forming" is understood to mean obtaining it by chemical synthesis, biotransformation or by purchase.

Routes of administration provided by the present invention as pharmaceutical compositions include, but are not limited to, oral (Oral), nasal (Nasal), (Buccal), transdermal (Transdermal), pulmonary (Pulmonal), vaginal (Vaginal), subcutaneous (Subcutaneous), or Intravenous (Intravenous) administration to an organism.

The main advantages of the invention are:

(1) It was revealed for the first time that Quisinostat (i.e., the No.37 compound in the present invention) has a significant inhibitory effect on plasmodium;

(2) The first discovery shows that the combination of Quisinosistat, piperazine and Lumefantrine respectively shows remarkable synergistic inhibition effect on plasmodium.

(3) Provides a new antimalarial drug with remarkable effect.

(4) Provides a more effective prevention and treatment scheme for plasmodium strains, and simultaneously, the scheme can effectively slow down the occurrence of plasmodium drug resistance.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Materials and methods

1. Material

Artemisinin (artemisinin) Sigma Aldrich cat.no.361593-100MG.

2.no.37 compound: selleckchem Cat. No. S1096

3. Plasmodium falciparum (3D 7C 8): purchased from ATCC.

4.293T cell line, purchased from ATCC.

HepG2 cell line, purchased from ATCC.

6.SYBR Green I:Invitrogen Cat.No.S-7585

7.Cell titer Glo:Promega Cat.No.G7572

8.96-well plate Corning

9. pancreatin-EDTA Gbico Cat.No.25200-072

10.DMEM:Gbico Cat.No.11965-092

11.FBS:Gbico Cat.No.10082-147

HEPES, ULTROL grade (100 g) Calbiochem Cat.No.391338

14. Hypoxanthine (25 g) SIGMA Cat.No. H9377

2. Method of producing a composite material

1. Culturing plasmodium: culture of Plasmodium Using RPMI (containing NaHCO) ₃ HEPES, albumax I, hypoxanthine, genaotamicin) Complete Medium (Complete Medium), incubator at 37 deg.C (5% CO) ₂ ，5％0 ₂ ) Culturing in medium.

2. Mammalian cell culture: mammalian cell lines 293T and HepG2 use DMEM (containing 10% FBS,1X P/S) at 37 ℃ incubator (5% CO) ₂ ) And (4) medium culture.

3. Compound plasmodium in vitro growth inhibition EC50 assay: 100ul of complete medium was added to a 96 well plate, an appropriate amount of 1uM of the compound was added to the first well and made up to 200ul with complete medium to give a final concentration of 400nM, followed by dilution graduations (11 concentration graduations) at a ratio of 1/2 using a robot, artemisin as a positive control, no compound as a negative control, no Plasmodium and no compound as experimental background. 100ul of Plasmodium culture (1% ring parasitemia,4% HCT) was then added per well with robotic arm to give final HCT and parasitemia of 2% and 1%, and final concentration gradients of compounds (200 nm,100 nm, 50nm, 25nm, 12.5nm, 6.25nm, 3.125nm, 1.5625nm, 0.78125nm, 0.390625nm, 0.195313 nm). After completion of the sample addition, the 96-well plate was placed in a 37 ℃ incubator (5% CO) ₂ 5% O2) for 72h. The culture is completedThen 100ul Lysis Buffer (10x SYBR Green I,0.5% v/v Triton X-100,0.5mg/ml saponin,0.75% EDTA in Tris-Cl Buffer) was added per well, after mixing, incubation for 2h at room temperature in the dark. After the incubation was complete, the values were read using a well plate fluorescence reader (maximum excitation/maximum acceptance: 485nm/535 nm). Inhibition was calculated from fluorescence readings, = (negative control-experimental)/(negative control-experimental background), growth inhibition curves were plotted from inhibition at different concentrations and EC50 was calculated by Graph Pad 6.

4. Compound cytotoxicity IC50 assay: mammalian cells 293T, hepG2 were cultured to p5 passages, digested with pancreatin-EDTA, resuspended in DMEM (10% FBS,1X p/S), counted in a haemocytometer and diluted to 10 ⁵ cell/ml. Adding 100ul of cell suspension per well in a 96-well plate by a robot, 37 ℃ and 5% CO ₂ And culturing for 24h. 100ul of DMEM (10% FBS, no p/s) was added per well to a new 96-well plate, and 200ul of 1uM of compound (in DMEM) was added to the first well of the 96-well plate and diluted in a gradient of 1/2 by the robot (11 concentration gradients), without any compound as a negative control and with DMEM alone as a background. Then, the culture medium in the previous 96-well plate is sucked up, and the culture medium after the gradient dilution is correspondingly added into the 96-well k-well plate. 37 ℃ 5% of CO ₂ And culturing for 72h. After the culture is completed, the 96-well plate is taken out and balanced for more than 30min at room temperature, 50ul of cell title-Glo reagent (pre-balanced for more than 1h at room temperature in a dark place) is added into each well, the incubation is carried out for 10 min at room temperature by shaking, and then the fluorescence detection is carried out by using a fluorescence detector. Inhibition was calculated from fluorescence readings, = (negative control-experimental group)/(negative control-background), growth inhibition curves were plotted from inhibition at different drug concentrations and IC50 was calculated by Graph Pad 6.

Interaction assay with commonly used antimalarial drugs: an optimal starting concentration was chosen based on the individual EC50 of the compound and the antimalarial to ensure that the concentration of the EC50 was in the middle of the concentration gradient, which was 200nM for both the compound and the conventional antimalarial in this experiment, so the inventors previously prepared 400nM concentrations of each compound, added 100ul of complete medium to each of the B1-H12 wells in a 96-well plate, and added different volumes of interacting solutions of the two compounds to each of the 200ul system of the A1-a12 wells, as shown in fig. 5.

Then, the wells were subjected to 1/2 dilution from A-B, and finally, 100ul of a culture having a 4% protozoan rate of 1% RBC packed volume was added to each well so that the final RBC packed volume was 1% protozoan rate of 2%, and the concentration gradient of the compound was (200nM, 100, 50, 25, 12.5,6.25,3.125, 1.5625nM), and after completion of the addition, the 96-well plates were placed in a three-gas culture tank (5% CO) ₂ ,5％O ₂ ) After culturing for 72 hours, 100ul of lysine Buffer (10X SYBR Green I,0.5% v/vTriton X-100,0.5mg/ml saponin,0.75% EDTA in Tris-Cl Buffer) was added to each well, and after mixing, the wells were incubated at room temperature for 2 hours in the dark. After the incubation was completed, the values were read using a well plate fluorescence reader (maximum excitation/maximum acceptance: 485nm/535 nm). Calculating the inhibition rate according to the fluorescence reading value, wherein the inhibition rate = (negative control group-experimental group)/(negative control group-experimental background), drawing growth inhibition curves of different groups according to the inhibition rates of different concentrations, and calculating the EC50 of each group through Graph Pad 6. And finally, calculating the correlation coefficient of the two compounds according to the EC50 of each group, and drawing an interaction relation graph of the two compounds.

Insecticidal efficiency analysis of compounds in mice: two different strains of mice were selected (ICR and BALB/c were used for this experiment, respectively, and on day 0, all mice were inoculated with the murine Plasmodium lethal strain Pb ANKA, 10 per mouse ⁶ 24 hours later, performing intravenous injection administration of artemisinin, no.37+ piperaqioine, no.37+ lumefantinine with different concentrations to mice, wherein the concentration of artemisinin is 10mg/kg, and the concentration of artemisinin is 30mg/kg; no.3710mg/kg,20mg/kg; no.37+ pipraquinone (10mg + 10mg)/kg; no.37+ lumefarntrine (10mg + 10mg)/kg, three mice per group as replicates. DMSO was also injected as a negative control. The administration is carried out continuously for four days in the same way, all mice are subjected to tail blood sampling every day to prepare blood smears, the protozoan rate in the mice is calculated, the mice are continuously observed for more than 10 days, and the graph is drawn according to the results.

Example 1 compound plasmodium in vitro growth inhibition EC50 assay:

the inventors measured the EC50 of the compound No.37, cultured and calculated the inhibition rate according to the above-described method using artemisinin (artemisin) as a positive control and 200nm as an initial concentration at a rate of 1/2 (11 gradients), and finally calculated the EC50 of the compound against growth inhibition of plasmodium using Graph Pad. The present inventors obtained the results (fig. 1), that compound No.37 is equivalent to artemisinin and shows a strong inhibitory effect on the growth of plasmodium falciparum (3D 7C 8).

Example 2 compound cytotoxicity EC50 assay:

the present inventors carried out cytotoxicity experimental determination using human kidney epithelial cell 293T and human liver cancer cell line HepG2, compound 1um as an initial concentration and dilution gradient (11 gradients) at a ratio of 1/2, artemisinin as a control, culture and inhibition calculation as described above. Finally EC50 calculations were performed using Graph Pad. As shown (fig. 2), compound No.37 showed very weak toxicity to cells, with approximately 1000-fold higher cytotoxic EC50 compared to the plasmodium growth inhibitory EC50 (fig. 2).

Example 3 detection of interaction with commonly used antimalarial drugs:

the inventor conducts drug interaction test on the compound No.37 and several common antimalarials, namely Artemisinin (Artemisinin), piperaquine phosphate (Piperaqiuoine) and Lumefantrine (Lumefantrine), and the experimental result is shown in figure 3, and the compound No.37 and the three common antimalarials have no antagonism.

Insecticidal efficacy analysis of the compound of example 4 in mice:

in the mouse test, the present inventors used two different strains of mice (BALB/c, ICR) to perform drug experiments separately, and inoculated 10 times per mouse before administration ⁶ 24 hours later, 4 days after continuous administration, DMSO as a negative control, artemisinin as a positive control, and continuously recording the change of the protozoan rate of mice, as shown in fig. 4, the inventors can see that the protozoan rate of the control group becomes very high immediately after inoculation, while the experimental group hardly sees malaria parasites or the appearance time thereof is much delayed with respect to the control group, andthe inventors have seen that the No.37 compound group and the combination group thereof are significantly superior in effect to the control group of artemisinin.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. An anti-malarial protozoan composition comprising a first active ingredient which is Quisinostat or a pharmaceutically acceptable salt thereof; the second active ingredient is piperaquine or pharmaceutically acceptable salts thereof, or lumefantrine or pharmaceutically acceptable salts thereof,

wherein the molar ratio of the first active ingredient to the second active ingredient is from about 1 to about 20:20-1, preferably 1-10:10-1, more preferably 1-5:5-1.

2. The composition of claim 1, wherein said plasmodium is selected from the group consisting of: plasmodium falciparum, plasmodium vivax, plasmodium malariae, and plasmodium ovale.

3. The composition of claim 1, wherein said piperaquine is piperaquine phosphate.

4. The composition of claim 1, further comprising a pharmaceutically acceptable carrier or excipient.

5. The composition of claim 1, wherein the pharmaceutical composition is in a dosage form comprising a tablet, a granule, a capsule, a pill, an injection, or an oral liquid.

6. The composition of claim 1, wherein the composition is in unit dosage form, and wherein the first active ingredient and the second active ingredient are present in each unit dosage form in an amount of about 0.1 to about 1 (or 0.25 to about 1, or 0.5 to about 1) of the daily dose, wherein the daily dose is about 20 to about 100mg.

7. Use of the anti-plasmodium composition according to claim 1 for the preparation of a medicament for the prevention and/or treatment of malaria.