CN115487191B - Quisinostat, a novel high-efficiency antimalarial drug - Google Patents

Quisinostat, a novel high-efficiency antimalarial drug Download PDF

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CN115487191B
CN115487191B CN202211242735.6A CN202211242735A CN115487191B CN 115487191 B CN115487191 B CN 115487191B CN 202211242735 A CN202211242735 A CN 202211242735A CN 115487191 B CN115487191 B CN 115487191B
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active ingredient
acid
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plasmodium
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CN115487191A (en
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江陆斌
黄正辉
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Institut Pasteur of Shanghai of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention provides a novel high-efficiency antimalarial drug, in particular to application of Quisinostat, an analogue thereof or a pharmaceutically acceptable salt thereof in treating malaria. The invention also provides a pharmaceutical composition containing the Quisinostat. Through intensive researches, the quininostat has remarkable inhibition effect on plasmodium, and even shows better insecticidal effect than artemisinin in mice.

Description

Quisinostat, a novel high-efficiency antimalarial drug
The invention is based on the patent application of the application date of 2016, 2 and 22, the application number of 201610096388.9, the invention name of which is Quisinostat, and a novel efficient antimalarial drug.
Technical Field
The invention belongs to the field of medicines, and in particular relates to a novel high-efficiency 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 infected mosquitoes of the genus anopheles (Anophele). The disease is characterized by the onset of coldness, fever and night sweats that occur at intervals that depend on the time required for the development of a new generation of parasites in the body. After recovery from an acute episode, the disease tends to become chronic with occasional recurrence. There are four species of Plasmodium, plasmodium falciparum (Plasmodium falciparum/p.falciparum), plasmodium vivax (Plasmodium vivax/p.vivax), plasmodium malariae (Plasmodium malariae/p.malarial) and Plasmodium ovale (Plasmodium ovine/p.ovine).
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 hemoglobin of red blood cells, but also because it alters the adhesive properties of the inhabited cells, which causes the cells to adhere to the vessel wall. This becomes dangerous when the infected blood cells stick to the blood vessel and block the 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 world health organization 2011 world malaria report, 216,000,000 cases of malaria were present in 2010 and 81% of them were in WHO africa. There were estimated 655,000 malaria-related deaths in 2010, of which 86% of victims were children under 5 years of age.
Throughout the 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 treatment. WHO suggests artemisinin (artemisinin) in combination with other classes of antimalarial drugs (artemisinin-based combination therapy (ACT)) as a first line treatment for malaria caused by plasmodium falciparum. In 2009, the presence of garbodia-thailand border was confirmed and in the partial areas of burma and vietnam, the suspected presence of artemisinin-resistant plasmodium falciparum, and therefore, the presence of new highly potent antimalarials is urgently needed in the art to suppress 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
(1) A medicament for preventing and/or treating malaria; and/or
(2) Agents that inhibit and/or kill plasmodium.
In a second aspect of the invention there is provided an antimalarial parasite 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 analogue thereof, or a pharmaceutically acceptable salt thereof;
The second active ingredient is piperaquine, analogues thereof or pharmaceutically acceptable salts thereof, and/or
Lumefantrine, 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 from about 1 to about 20:20 to 1, preferably 1 to 10:10 to 1, more preferably 1 to 5:5 to 1.
In another preferred embodiment, a pharmaceutically acceptable carrier or excipient is also included in the composition.
In another preferred embodiment, the dosage form of the pharmaceutical composition comprises a tablet, granule, capsule, pill, injection, or oral liquid.
In another preferred embodiment, the composition is in unit dosage form, each unit dosage form having a content of the first active ingredient and the second active ingredient of about 0.1 to 1 (or 0.25-1, or 0.5-1) per 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 the present invention provides the use of an antimalarial composition according to the first aspect of the invention for the preparation of
(1) A medicament for preventing and/or treating malaria;
(2) Agents that inhibit and/or kill 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 to a subject in need thereof, quinnostat, an analog thereof, or a pharmaceutically acceptable salt thereof, or administering to a subject in need thereof, an antimalarial composition of claim 2, thereby preventing and/or treating malaria.
In another preferred embodiment, the subject includes humans and non-human mammals (e.g., rodents).
In another preferred embodiment, the amount of said administration is 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 understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the growth inhibitory effect of compound No.37 on in vitro culture of plasmodium (EC 503.91 nM), artemisinin as a positive control (EC 50.38 nM); the growth of plasmodium was determined by using a 3-day SYBR GREEN I cell growth assay.
FIG. 2 shows the cytotoxic effect of compound No.37 on human hepatoma cell line HepG2 and human renal epithelial cell line 293T; also shown is the selection index between the inhibitory effect of compound No.37 on plasmodium and toxicity on different cell lines, artemisinin 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 plasmodium was determined by the method of 3-day SYBR GREEN I.
FIG. 3 shows the drug interactions between compound No.37 and the usual antimalarial drugs, lumefantrine, artemisinin, piperaquine phosphate; drug interactions were performed in vitro cultured plasmodium, and the growth of plasmodium was determined by the 3-day SYBR GREEN I method; the FIC is the drug interaction index.
FIG. 4 shows the insecticidal effect of compound No.37 in mice; the compound No.37 is tested at two different concentrations of 10mg/kg and 20mg/kg, and the combined administration concentration is respectively that the compound No.37 (10 mg/kg) +benflumetol (10 mg/kg); no.37 compound (10 mg/kg) +piperaquine phosphate (10 mg/kg), artemisinin was used as positive control with 10mg/kg and 30mg/kg and DMSO as negative control.
FIG. 5 shows that the inventors prepared each compound solution at 400nM in advance, 100. Mu.l of complete medium was added to each of the B1-H12 wells in a 96-well plate, and two compound solutions of different volumes of interaction were added to each of the 200. Mu.l systems of the A1-A12 wells.
Detailed Description
Through extensive and intensive studies, the present inventors have unexpectedly found that quinnostat (i.e., compound No.37 in the present invention) has a remarkable inhibitory effect on plasmodium and exhibits even better insecticidal effect than artemisinin in mice. Further research shows that the quinnostat and Piperaquine and Lumefantrine respectively combined with each other are used for intravenous injection on mice, and show remarkable synergistic effect and better insecticidal effect than artemisinin. On this basis, the present invention has been completed.
Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods 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, as 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, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (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 described herein.
Quisinostat, an analog thereof, or a pharmaceutically acceptable salt thereof
In the present invention, the Quisinostat and No.37 compounds are the same compound. The compound No.37 as an active ingredient of the present invention is a novel second-generation Hdac inhibitor 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 compound No.37 is as follows:
Analogs of compound No.37 include, but are not limited to: a compound formed by substitution of one or more hydrogens, and/or hydroxy groups in the above formula, preferably the substituents are selected from the group consisting of: halogen (F, cl, br, I), C 1-8 alkyl, C 2-8 alkenyl, 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 tissue of a subject (e.g., a human) without undue adverse side effects. As used herein, pharmaceutically acceptable salts refer to salts of 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, and sulfuric acid; preferred organic acids include (but are not limited to): formic acid, acetic acid, propionic acid, succinic acid, naphthalene disulfonic acid (1, 5), asiatic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethyl acetic 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 compounds of No.37 of the present invention include salts of the compounds of the present invention having an acidic group (e.g., potassium, sodium, magnesium, calcium) or salts of the compounds of the present invention having a basic group (e.g., sulfate, hydrochloride, phosphate, nitrate, carbonate).
The compound No.37 showed a plasmodium growth inhibitory effect comparable to that of artemisinin in an in vitro test, and a better insecticidal effect than that of artemisinin in a mouse test. In the drug interaction experiment, no.37 and the most main antimalarial drugs at present are not antagonistic, and in the mouse combined drug administration experiment, no.37 and Piperaquine (piperaquine phosphate) and Lumefantrine (benflumetol) are respectively combined drug administration to carry out intravenous injection on mice, and the mice also show better insecticidal effect than artemisinin. This means that combination therapies can be performed in future patent drug development and clinical trials, which will greatly help delay the development of drug resistance, and at the same time will be better and more efficient for malaria treatment.
In an in vitro plasmodium growth inhibition experiment, artemisinin is used as a control, the No.37 is used as an experiment group to measure the plasmodium growth inhibition EC50, and the No.37 shows very good plasmodium growth inhibition function, and the EC50 is 3.91nM compared with artemisinin. The experiment is repeated for more than three times in biological sense, the result is stable, and the reliability is high.
In the mouse experiments, mice were vaccinated with a murine malaria lethal strain Pb AnKa and were given different concentrations of artemisinin, compound No.37, and compound No.37 + Piperaquine, compound No.37 + Lumefantrine by intravenous injection 24 hours after the inoculation:
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 was used as replicates. DMSO was also injected as a negative control. The experimental results show that No.37, no.37+ piperaquine and No.37+ lumefantrine show better inhibitory effect on malaria of mice than artemisinin, and the experiment is repeated for a plurality of times in mice of different species, so that similar results are obtained, and the reliability is high.
Piperaquine (Piperaquine), analogues 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:
Analogues of piperaquine include, but are not limited to: a compound formed after substitution of one or more hydrogens, and/or one or more cls in the above formula, preferably the substituents are selected from the group consisting of: halogen (F, cl, br, I), C 1-8 alkyl, C 2-8 alkenyl, 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 tissue of a subject (e.g., a human) without undue adverse side effects. As used herein, pharmaceutically acceptable salts refer to salts of piperaquine or analogs 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, and sulfuric acid; preferred organic acids include (but are not limited to): formic acid, acetic acid, propionic acid, succinic acid, naphthalene disulfonic acid (1, 5), asiatic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethyl acetic 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 an acidic group (e.g., potassium, sodium, magnesium, calcium) or salts of compounds of the invention having a basic group (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 China and India for malaria prevention and treatment in the following 20 years until the drug-resistant insect strain of piperaquine phosphate appeared in the 80 s of the 20 th century; because of its long half-life, it is currently used in combination with Artemisinin (ACT) for the prevention and treatment of malaria.
Lumefantrine (Lumefantrine), analog thereof, or pharmaceutically acceptable salt thereof
Chemical name: (Z) -2, 7-dichloro-9- [ (4-chlorophenyl) methylene ] - α - [ (di-n-butylamino) methyl ] -9H-fluoren-4-methanol
The structural formula is as follows:
Analogues of lumefantrine include, but are not limited to: a compound formed by substitution of one or more hydrogen, chlorine and/or hydroxy groups in the above formula, preferably the substituents are selected from the group consisting of: halogen (F, cl, br, I), C 1-8 alkyl, C 2-8 alkenyl, 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 tissue of a subject (e.g., a human) without undue adverse 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, and sulfuric acid; preferred organic acids include (but are not limited to): formic acid, acetic acid, propionic acid, succinic acid, naphthalene disulfonic acid (1, 5), asiatic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethyl acetic 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 lumefantrine or analogs thereof of the present invention include salts of the compounds of the present invention having an acidic group (e.g., potassium, sodium, magnesium, calcium) or salts of the compounds of the present invention having a basic group (e.g., sulfate, hydrochloride, phosphate, nitrate, carbonate).
Lumefantrine has a long half-life and is currently used for preventing and treating malaria by combined Administration (ACT) with artemisinin.
Composition and method for producing the same
As used herein, the term "composition" includes pharmaceutical compositions and agents.
The composition of the invention comprises an anti-plasmodium active ingredient and a pharmaceutically acceptable carrier. The antimalarial active ingredients 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, analogues thereof, or pharmaceutically acceptable salts thereof, and/or
Lumefantrine, an analog thereof, or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition of the present invention may further comprise various pharmaceutical excipients compatible with the contained compounds or compositions and be formulated by conventional methods into dosage forms useful for administration, such as: but not limited to, aqueous injection, powder for injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray, powder fog, sustained release agent, controlled release agent, etc. The pharmaceutical excipients can be used conventionally in various preparations, such as: but are not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, lubricants, and the like; may also be selected for adaptation to the substance, such as: but not limited to emulsifying agent, solubilizer, bacteriostat, analgesic and antioxidant, etc., which can effectively improve stability and solubility of the compound contained in the composition or change release rate and absorption rate of the compound, etc., thereby improving metabolism of various compounds in living body and further enhancing administration effect of the composition. In addition, specific purposes or modes of administration may be achieved, such as: sustained release administration, controlled release administration, pulse administration, etc., and auxiliary materials used, such as: but are not limited to, gelatin, albumin, chitosan, polyethers and polyesters based polymeric materials (such as, but not limited to, polyethylene glycol, polyurethane, polycarbonate and copolymers thereof, etc.). The main manifestations of the beneficial drug administration are: but not limited to, improving therapeutic effect, improving bioavailability, reducing toxic side effects, improving patient compliance, and the like.
In aqueous injection solutions, the excipients generally include isotonic and buffering agents, as well as, if necessary, emulsifiers (e.g., tweeen-80, pluronic, and Poloxamer, etc.), solubilizers, bacteriostats, and the like. In addition, the composition also comprises other pharmaceutically acceptable pharmaceutical excipients, such as: antioxidants, pH adjusters, analgesics, and the like.
The auxiliary materials for preparing the oral liquid preparation generally comprise solvents, necessary flavoring agents, bacteriostats, emulsifying agents, coloring agents and the like.
The auxiliary materials for preparing the tablet generally comprise a filler (such as starch, sugar powder, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, calcium hydrophosphate, mannitol and the like), a binder (such as ethanol, starch slurry, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatin solution, sucrose solution, aqueous or alcoholic solution of polyvinylpyrrolidone and the like), a disintegrating agent (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, crosslinked polyvinylpyrrolidone and crosslinked sodium carboxymethyl cellulose) and a lubricant (such as magnesium stearate, micro-powder silica gel, talcum powder, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000, magnesium lauryl sulfate and the like) and the like.
The auxiliary materials for preparing the emulsion are water, oil (such as fatty acid), emulsifying agent, necessary preservative, flavoring agent and the like.
The auxiliary materials used for preparing the granules are similar to tablets, but the granulation process is different. Mixing the 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). Wild animals are animals which have not been artificially domesticated in a natural state. Livestock are animals that are raised artificially to provide a source of food, such as: but are not limited to, dogs, cats, mice, rats, hamsters, pigs, rabbits, cows, buffalo, bulls, sheep, goats, geese, chickens, and the like. The administration of the treatment to a "patient" or "organism" preferably selects a mammal, particularly a human.
As used herein, the term "prevention" refers to various means or measures for preventing the occurrence or progression of a disease, including medical, physical or chemical methods, to prevent and reduce the occurrence or progression of various symptoms of the disease, prior to the disease not being recognized by clinical standards.
As used herein, the term "treatment" refers to the inhibition, containment, alleviation, amelioration, slowing, stopping, delaying or reversing of the progression or exacerbation of a disease in order to prevent or reduce the occurrence or progression of the disease, and the various indicators of the disease, disorder or pathological state described as maintaining and/or administering include alleviation or reducing symptoms or complications, or curing or eliminating the 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 comprising a single compound as the primary active ingredient, and also refers to a composition or formulation comprising multiple compounds as the active ingredient, which may be used to prevent or treat a disease. "pharmaceutical" is understood to mean not only the products approved and approved for production by the authorities established in accordance with the legal regulations of the country, but also the forms of the various substances formed in order to obtain the products approved and approved for production, which contain the single compound as active ingredient. "formed" is understood to mean obtained by chemical synthesis, bioconversion or purchase, among other means.
The present invention provides routes of administration as pharmaceutical compositions, including, but not limited to, oral (Oral), nasal (Nasal), (facial) buccal (Buccal), transdermal (TRANSDERMAL), pulmonary (Pulmonal), vaginal (Vaginal), subcutaneous (Subcutaneous), or intravenous (Intravenous) administration to a living organism.
The invention has the main advantages that:
(1) The Quisinostat (namely, the compound No.37 in the invention) has obvious inhibition effect on plasmodium for the first time;
(2) The first finding shows that the combined use of Quisinostat, piperaquine and Lumefantrine shows remarkable synergistic inhibition effect on plasmodium.
(3) Provides a new antimalarial drug with remarkable effect.
(4) Provides a more effective preventive and therapeutic scheme for malaria parasite strains, and simultaneously the scheme can effectively slow down the occurrence of drug resistance of malaria parasites.
The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratories, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
Materials and methods
1. Material
Artemsinin (artemisinin) SIGMA ALDRICH cat.no.361593-100MG.
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 (Hypoxanthine, 25 g) SIGMA Cat.No. H9377
2. Method of
1. Plasmodium culture: plasmodium cultures were grown in 37℃incubator (5% CO 2,5％02) using RPMI (NaHCO 3, HEPES, albumax I, hypoxanthine, genaotamicin) Complete Medium.
2. Mammalian cell culture: mammalian cell lines 293T and HepG2 were cultured in an incubator (5% CO 2) at 37℃using DMEM (10% FBS,1 XP/S).
3. Compound plasmodium in vitro growth inhibition EC50 assay: 100ul of complete medium was added to 96-well plates, an appropriate amount of 1uM of compound was added to the first well and the complete medium was sized to 200ul to give a final concentration of 400nM, then gradient diluted (11 concentration gradients) with a robot at a 1/2 ratio, ARTEMSININ as positive control, no compound as negative control, no plasmodium and compound as experimental background. 100ul of plasmodium culture (1%ring parasitemia,4%HCT) was then added per well using a robot arm to give final HCT and parasitemia of 2% and 1% and final concentration gradients of compounds (200 nm, 100nm, 50nm, 25nm, 12.5nm, 6.25nm, 3.125nm, 1.5625nm, 0.78125nm, 0.390625nm, 0.195313 nm). After the completion of the loading, the 96-well plate was placed in an incubator (5% CO 2, 5% O2) at 37℃and cultured for 72 hours. After 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 to each well after the completion of the culture, the mixture was incubated at room temperature for 2 hours in the dark. After incubation was completed, the values were read with an orifice plate fluorescence reader (maximum excitation light/maximum acceptance light: 485nm/535 nm). Inhibition was calculated from fluorescence readings, inhibition = (negative control group-experimental group)/(negative control group-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, hepG were cultured to the p5 generation, digested with pancreatin-EDTA, resuspended in DMEM (10% fbs,1x p/S), counted with a hemocytometer plate and diluted to 10 5 cells/ml. 100ul of the cell suspension was added to each well of a 96-well plate by a robot, and cultured at 37℃for 24 hours with 5% CO 2. 100ulDMEM (10% FBS, no. p/s) were added to each well of the new 96-well plate, 200ul of 1uM compound (dissolved in DMEM) was added to the first well of the 96-well plate and diluted in a 1/2 ratio (11 concentration gradients) with a robotic arm, without any compound as a negative control and with DMEM as background. The culture solution in the previous 96-well plate is then aspirated, and the culture solution after gradient dilution is then added to the 96-wellk-well plate. The cells were incubated at 37℃for 72 hours with 5% CO 2. After the completion of the incubation, the 96-well plate was taken out and equilibrated at room temperature for 30min or more, then 50ul of cell title-Glo reagent (equilibrated at room temperature in advance for 1h or more in the dark) was added to each well, and incubated at room temperature for 10min with shaking, followed by fluorescence detection by a fluorescence detection instrument. Inhibition was calculated from fluorescence readings, inhibition = (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 with commonly used antimalarials: in this experiment, the initial concentration of the compound and the common antimalarial was set to 200nM, and thus the inventors prepared each compound solution at 400nM in advance, 100ul of complete medium was added to each of the B1-H12 wells in 96-well plates, and two compound solutions of different volumes of interactions were added to each of the 200ul systems of the A1-A12 wells, respectively, according to the initial concentration of the compound and the antimalarial, which was optimal to ensure that the concentration value of the EC50 was located in the middle of the concentration gradient.
Then, the solution was diluted in a gradient from A to B at a ratio of 1/2, and finally, a culture with a volume of 100ulRBC and a protozoa rate of 4% and a protozoa rate of 1% was added to each well so that the final volume of RBC was 2% and the protozoa rate was 1%, the concentration gradient of the compound was 200nM,100, 50, 25, 12.5,6.25,3.125,1.5625nM, and after the completion of the addition, the 96-well plate was placed in a three-gas incubator (5% CO 2,5％O2) and incubated for 72 hours, and after the completion of the incubation, 100ul Lysis 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 mixed uniformly, and incubated at room temperature in a dark place for 2 hours. After incubation was completed, the values were read with an orifice plate fluorescence reader (maximum excitation light/maximum acceptance light: 485nm/535 nm). Inhibition was calculated from fluorescence readings, inhibition = (negative control group-experimental group)/(negative control group-experimental background), growth inhibition curves for different groups were plotted according to inhibition at different concentrations and EC50 for each group was calculated by Graph Pad 6. Finally, the correlation coefficient of the two compounds is calculated according to the EC50 of each group, and the interaction relation graph of the two compounds is drawn.
Insecticidal efficacy analysis of compounds in mice: two different strains of mice were selected (ICR and BALB/c were each run, on day 0, all mice were vaccinated with the malaria lethal strain Pb ANKA, 10 6 malaria parasites were vaccinated per mouse, after 24 hours, mice were given different concentrations of artemisinin, no.37 and No.37+ piperaqiuoine, no.37+ lumefantrine by intravenous injection, respectively, artemisinin 10mg/kg,30mg/kg;No.3710mg/kg,20mg/kg;No.37+piperaquine(10mg+10mg)/kg;No.37+lumefantrine(10mg+10mg)/kg, each group of three mice was repeated, and DMSO was injected simultaneously as a negative control, four days following the same dosing, tail blood sampling was performed on all mice daily to make blood smears to calculate the internal protozoa rate of the mice, and continuous observation was performed for more than 10 days, and plotted according to the results.
Example 1 compound plasmodium in vitro growth inhibition EC50 assay:
the inventors performed an EC50 assay on the No.37 compound, performed a gradient dilution (11 gradients) with Artemisinin (ARTEMSININ) as a positive control and 200nm as an initial concentration in a 1/2 ratio, performed a culture and calculated inhibition rates as described above, and finally calculated an EC50 of the compound against plasmodium growth inhibition using Graph Pad. As a result of the present inventors (FIG. 1), compound No.37 showed a potent inhibitory effect on the growth of plasmodium falciparum (3D 7C 8)) as compared with artemisinin.
Example 2 compound cytotoxicity EC50 assay:
the inventors used human kidney epithelial cells 293T and human liver cancer cell line HepG2 for cytotoxicity assay, compound 1um as initial concentration and gradient dilution (11 gradients) at 1/2 ratio, artemisinin as control, culture and inhibition calculation as described above. EC50 calculations were finally performed using Graph Pad. As shown in the figure (FIG. 2), compound No.37 showed very little toxicity to cells, with an approximately 1000-fold higher cytotoxic EC50 compared to the EC50 for inhibition of plasmodium growth (FIG. 2).
Example 3 interaction detection with commonly used antimalarials:
The present inventors conducted a drug interaction test of the compound No.37 with several common antimalarials, artemisinin (ARTEMISININ), piperaquine phosphate (Piperaqiuoine), lumefantrine (Lumefantrine), and as shown in FIG. 3, the antagonism of the compound No.37 with none of the three common antimalarials was observed.
Insecticidal efficacy analysis of the compound of example 4 in mice:
In the mouse test, the present inventors used mice (BALB/c, ICR) of two different strains to perform the drug test, respectively, and inoculated 10 6 of plasmodium to each mouse before the administration, and administered 24 hours later, continuously administered for 4 days, DMSO as a negative control, artemisinin as a positive control, and continuously recorded the change of the mouse protozoal rate, as shown in fig. 4, the present inventors could see that the control group became very high in protozoal rate immediately after the inoculation, while the experimental group hardly seen plasmodium, or the occurrence time thereof was much delayed from the control group, and the present inventors could see that the effect of the compound No.37 group and its combination group was significantly superior to that of the control group of artemisinin.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (9)

1. An antimalarial composition comprising a first active ingredient and a second active ingredient, wherein the first active ingredient is Quisinostat or a pharmaceutically acceptable salt thereof; the second active ingredient is piperaquine or pharmaceutically acceptable salt thereof, or lumefantrine or pharmaceutically acceptable salt thereof,
Wherein the molar ratio of the first active ingredient to the second active ingredient is 1-20:20-1;
The formulation of the composition comprises tablets, granules, capsules, pills, injections and oral liquids;
the composition is in unit dosage form, and the content of the first active ingredient and the second active ingredient in each unit dosage form is 0.1-1 of daily dose, wherein the daily dose is 20-100mg.
2. The composition of claim 1, wherein the first active ingredient and the second active ingredient are present in a molar ratio of 1-10:10 "1.
3. The composition of claim 1, wherein the first active ingredient and the second active ingredient are present in a molar ratio of 1-5:5-1.
4. 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 from 0.25 to 1 on a daily basis.
5. 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 from 0.5 to 1 on a daily basis.
6. The composition of claim 1, wherein the plasmodium is selected from the group consisting of plasmodium falciparum, plasmodium vivax, plasmodium malariae, and plasmodium ovale.
7. The composition of claim 1, wherein the piperaquine is piperaquine phosphate.
8. The composition of claim 1, further comprising a pharmaceutically acceptable carrier or excipient.
9. Use of an antimalarial parasite composition according to claim 1 for the manufacture of a medicament for the prevention and/or treatment of malaria.
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