Reduction or prevention of malaria parasites or dengue virus using 1-methyl-9H-pyrido[3,4-b]indole

Abstract

This application relates to compositions comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for use in reducing or preventing the transmission of malaria and dengue fever, and corresponding methods. This application also relates to 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for use in inhibiting the development of protozoa into ookinetes, oocysts, or sporozoites, and to mosquito baits comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof and one or more sugar sources.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION This application relates to compounds and pharmaceutically acceptable salts thereof, compositions thereof, and their use in reducing or preventing the transmission of malaria by Anopheles mosquito vectors and dengue fever by Aedes mosquito vectors. [Background technology]

[0002] Background of the Invention Infectious diseases are responsible for a variety of medically and veterinarily important illnesses. Many of these diseases are transmitted by insect vectors. Vector-borne diseases are infections transmitted by the bites of infected arthropod species such as mosquitoes, ticks, assassin bugs, sand flies, black flies, and ectoparasites such as ticks and fleas.

[0003] Mosquitoes are vectors of various infectious diseases. Three medically relevant mosquito genera that transmit diseases in particular are Anopheles, Culex, and Aedes. Culex and Aedes belong to the subfamily Culicinae, while Anopheles belongs to the subfamily Anophelinae. Anopheles is a vector of malaria. Aedes is a vector of the dengue virus.

[0004] Malaria is a disease caused by parasitic protozoa of the genus Plasmodium, which infect and destroy red blood cells, causing fever, severe anemia, cerebral malaria, and death if left untreated. There are five species of Plasmodium: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. Plasmodium falciparum is the most virulent. In 2019, an estimated 229 million people were infected with malaria in 87 malaria-endemic countries, and an estimated 409,000 people died from malaria (WHO Malaria Report: https: / / www.who.int / teams / global-malaria-programme / reports / world-malaria-report-2021).

[0005] Dengue virus is a single-stranded, positive-stranded RNA virus in the Flaviviridae family that causes dengue fever. Dengue fever is transmitted by Aedes mosquitoes. Dengue fever is endemic in tropical and subtropical regions worldwide, with an estimated 100 million cases occurring annually. At least four serotypes of the virus have been identified, which cause approximately 400 million infections annually. Dengue virus infection can be asymptomatic or can cause a range of clinical manifestations, from mild fever to more life-threatening dengue hemorrhagic fever and often fatal dengue shock syndrome.

[0006] These diseases are of great medical importance. Numerous drugs are available for the treatment and / or prevention of some parasitic or vector-borne diseases. However, not all parasitic or vector-borne diseases can be effectively treated. For example, there are currently no chemotherapeutic drugs or vaccines available for dengue fever virus. Furthermore, with regard to antimalarial drugs, the increasing resistance of some Plasmodium strains has made currently available drug treatments less effective. Therefore, to prevent transmission, effective control of disease parasites and vectors is necessary. In this regard, mosquitoes can be targeted by various insecticides and repellents. Mosquitoes can be targeted by insecticides both in the larval stage and after they have matured into adults. However, mosquitoes have developed widespread resistance to currently used insecticides.

[0007] One approach to address this problem is to develop drugs that can reduce or prevent the transmission of vector-borne diseases without adversely affecting the insect vector, avoiding the development of resistance. In this regard, PCT / EP2020 / 069569 (published as WO 2021 / 009050) discloses bacteria of the genus Delftia and their use in reducing malaria transmission by mosquitoes. Summary of the Invention

[0008] Summary of the Invention According to a first aspect of the present invention, there is provided a composition for use in a method for reducing or preventing the transmission of malaria or dengue fever, the composition comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and the method comprising the step of contacting the vector mosquito with the composition.

[0009] According to a second aspect of the present invention, there is provided a composition for use in a method for reducing or preventing transmission of malaria parasites or dengue viruses, the composition comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and the method comprising the step of contacting the vector mosquito with the composition.

[0010] According to a third aspect of the present invention, there is provided a method of reducing or preventing the transmission of dengue fever or malaria, or malaria parasite or dengue virus infection, comprising the step of contacting at least one Anopheline or Namicinae mosquito vector of these vector-borne diseases with 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof.

[0011] In a further aspect of the invention there is provided the use of 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof in reducing or preventing malaria or dengue fever, or infection by a malaria parasite or dengue virus.

[0012] In a further aspect of the present invention, there is provided 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for use in inhibiting the formation of ookinetes, oocysts, or sporozoites of malaria parasites, or dengue virus infection.

[0013] In a further aspect of the present invention, there is provided a mosquito nectar bait comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof and glucose as a sugar source.

[0014] The present invention may be advantageous in many respects. In particular, the inventors have discovered that a compound called 1-methyl-9H-pyrido[3,4-b]indole (also known as harmane or 1-methyl-β-carboline) is produced by Delftia bacteria, which play a role in interfering with the transmission of malaria by Anopheles mosquitoes. When the compositions of the present invention are introduced into an environment where Anopheles vector mosquitoes are present, they prevent the development of the parasite in the mosquito, thereby blocking the transmission of the disease. For example, 1-methyl-9H-pyrido[3,4-b]indole inhibits the formation of ookinetes, an early stage of malaria parasite development, in the mosquito digestive tract. 1-methyl-9H-pyrido[3,4-b]indole can be used to combat the spread of malaria parasites. This compound can also be used to inhibit dengue virus infection.

[0015] The present invention will be further illustrated with reference to the accompanying drawings, which are not limiting. [Brief explanation of the drawings]

[0016] [Figure 1] Liquid chromatography-high resolution accurate mass spectrometry trace for the active ingredient of Delftia tsuruhatensis. [Figure 2] Heteronuclear single quantum coherence spectroscopy of the active ingredient of Delftia tsuruhatensis is shown. [Figure 3] 1H NMR spectrum of the active ingredient of Delftia tsuruhatensis. [Figure 4] This shows the inhibitory effect of harman on the development of Plasmodium by feeding harman to Anopheles gambiae mosquitoes. [Figure 5-1] 1 shows the inhibitory effect of harman on the growth of Plasmodium in Anopheles gambiae mosquitoes. [Figure 5-2] 1 shows the inhibitory effect of harman on the growth of Plasmodium in Anopheles gambiae mosquitoes. [Figure 5-3]1 shows the inhibitory effect of harman on the growth of Plasmodium in Anopheles gambiae mosquitoes. [Figure 5-4] 1 shows the inhibitory effect of harman on the growth of Plasmodium in Anopheles gambiae mosquitoes. [Figure 5-5] 1 shows the inhibitory effect of harman on the growth of Plasmodium in Anopheles gambiae mosquitoes. [Figure 5-6] 1 shows the inhibitory effect of harman on the growth of Plasmodium in Anopheles gambiae mosquitoes. [Figure 6] 1 shows the duration of inhibitory action of harman in mosquitoes. [Figure 7] 1 shows the inhibition of ookinete formation by harman in vitro. [Figure 8] 1 shows the effect of proteinase K treatment of Delftia supernatant on the growth inhibition of Plasmodium falciparum in Anopheles gambiae mosquitoes. [Figure 9] 1 shows the effect of different concentrations of Delftia supernatant on the development of Plasmodium in Anopheles gambiae mosquitoes. [Figure 10] 1 shows the duration of inhibition of Plasmodium falciparum oocyst formation in Anopheles gambiae mosquitoes by Delftia supernatant. [Figure 11] Figure 1 shows the inhibition of Plasmodium growth in Anopheles gambiae mosquitoes by supernatants of different Delftia strains. [Figure 12-1] 1 shows the results of screening Delftia supernatant fractions for inhibition of Plasmodium falciparum oocyst development activity in Anopheles gambiae mosquitoes. [Figure 12-2] 1 shows the results of screening Delftia supernatant fractions for inhibition of Plasmodium falciparum oocyst development activity in Anopheles gambiae mosquitoes. [Figure 12-3] 1 shows the results of screening Delftia supernatant fractions for inhibition of Plasmodium falciparum oocyst development activity in Anopheles gambiae mosquitoes. [Figure 13] 1 shows the effect of harman on BHK21 cell proliferation. [Figure 14] 1 shows the effect of harman on dengue virus growth in cell culture. [Figure 15] Figure 1 shows the effect of harman on dengue virus replication in Aedes aegypti mosquitoes. [Figure 16] Figure 1 shows the effect of harman on mosquito mortality. [Figure 17] This shows the fitness cost of Delftia to Aedes aegypti mosquitoes. [Figure 18] 1 shows the effect of Delftia bacteria on dengue virus replication in Aedes aegypti mosquitoes. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Embodiments In one aspect, the invention provides a composition for use in a method for reducing or preventing the transmission of malaria or dengue fever, the composition comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and the method comprising the step of contacting at least one vector with the composition.

[0018] 1-Methyl-9H-pyrido[3,4-b]indole, also known as harman, is represented by the following structure: [ka]

[0019] The present compounds have been found to be active compounds secreted by Delftia bacteria and can inhibit the transmission of malaria parasites and / or dengue virus by various vectors. In particular, 1-methyl-9H-pyrido[3,4-b]indole has been shown to selectively inhibit the ookinetes and oocysts of Plasmodium in the mosquito midgut by inhibiting them. Thus, the compositions of the present invention can reduce or prevent mosquito-mediated malaria transmission and / or malaria parasite transmission. In other cases, the compositions of the present invention can reduce or prevent mosquito-mediated dengue fever transmission and / or dengue virus transmission. Furthermore, it will be understood that compounds of the present invention, such as compounds of Formula (I), can exist in different tautomeric forms. Tautomers refer to isomeric forms of a compound that are in equilibrium with each other. The concentration of isomeric forms depends on the environment in which the compound is present.

[0020] The compound may be protonated or deprotonated depending on the pH of the surrounding environment.The compound may be in the form of a pharmaceutically acceptable salt.Pharmaceutically acceptable salts include, but are not limited to, those described in Berge, J. Pharm. Sci., 1977, 66, 1-19, or those described in PH Stahl and CG Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Second Edition, John Wiley & Sons, March 2011.

[0021] When the functionality of the compound allows, suitable pharmaceutically acceptable salts of the compound of formula (I) can be formed, including acid addition salts or base addition salts. Acid addition salts can be formed by reacting with an appropriate acid, optionally in a suitable solvent, such as an organic solvent, to give a salt that can be isolated by crystallization and filtration. Base addition salts can be formed by reacting with an appropriate base, optionally in a suitable solvent, such as an organic solvent, to give a salt that can be isolated by crystallization and filtration.

[0022] Representative pharmaceutically acceptable acid addition salts include, but are not limited to, 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, Acid salts, dodecyl sulfate (estolate), edetate (ethylenediaminetetraacetate), estolate (lauryl sulfate), ethane-1,2-disulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate, galactarate (mucate), gentisate (2,5-dihydroxybenzoate), glucoheptonate (gluceptate), gluconate, glucuronate, glutamate, glutarate, glycerophosphate, glycolate, hexylresorcinate, hippurate , hydrabamine (N,N'-di(dehydroabietyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, mucate, naphthalene-1,5-disulfonate (napadisylate), naphthalene-2-sulfonate (napsylate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p-aminosalicylate Salicylate, pamoate (embonate), pantothenate, pectinate, persulfate, phenylacetate, phenylethylvalturate, phosphate, polygalacturonate, propionate, p-toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, teoclate (8-chlorotheophylline), thiocyanate, triethiodide, undecanoate, undecylenate, and valerate salts.

[0023] Representative pharmaceutically acceptable base addition salts include, but are not limited to, aluminum, 2-amino-2-(hydroxymethyl)-1,3-propanediol (tris, tromethamine), arginine, benethamine (N-benzylphenethylamine), benzathine (N,N'-dibenzylethylenediamine), bis-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole) (1-p chlorobenzyl- 2-pyrrolidin-1'-ylmethylbenzimidazole), cyclohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (N-methylglucamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, f-butylamine, and zinc.

[0024] The compounds are administered in an appropriate "effective amount," which will vary depending on numerous factors, including, for example, the size and weight of the subject, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and is ultimately at the discretion of one of ordinary skill in the art.

[0025] In a second aspect, the present invention provides a composition for use in a method for reducing or preventing transmission of a malaria parasite or a dengue virus, the composition comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and the method comprising the step of contacting at least one vector with the composition.

[0026] In one embodiment, the parasitic disease is malaria.

[0027] In one embodiment, the composition is used to reduce or prevent (i) malaria and / or (ii) malaria parasite transmission. In one embodiment, the composition is suitable for preventing malaria transmission by mosquitoes. In another embodiment, the composition is suitable for preventing malaria parasite transmission. In another embodiment, the composition is suitable for preventing malaria parasite transmission by mosquitoes.

[0028] As defined herein, "reducing or preventing malaria or malaria parasite transmission" refers to eliminating malaria by, for example, inhibiting the mosquito developmental stage of the malaria parasite (ookinete and subsequent oocyst formation). 1-methyl-9H-pyrido[3,4-b]indole, an active compound secreted by Delftia bacteria, specifically Delftia tsuruhatensis, has been shown to inhibit malaria parasites and thereby suppress mosquito-mediated malaria transmission.

[0029] Specifically, this specification demonstrates that harman, when introduced into an environment where mosquitoes are present, can prevent malaria parasite transmission by inhibiting Plasmodium ookinetes and oocysts in the mosquito midgut. In some embodiments, harman is introduced to mosquitoes via contact with sugar bait, nectar bait, blood bait, and / or other feeding bait, in which case 1-methyl-9H-pyrido[3,4-b]indole can be transmitted to and / or within mosquitoes through cuticular absorption and / or oral ingestion. Thus, the compositions of the present invention can reduce or prevent malaria transmission and / or malaria parasite transmission by mosquitoes. The mosquitoes can be any mosquito capable of transmitting malaria, such as Anopheles mosquitoes. It is contemplated that the compositions and methods of the present invention are applicable to all Anopheles mosquito species. In one embodiment, the mosquito is Anopheles gambiae, Anopheles stephensi, Anopheles culicifacies, or Anopheles coluzzi. In one embodiment of the invention, the mosquito is Anopheles gambiae or Anopheles stephensi. In one embodiment, the mosquito is Anopheles stephensi. In another embodiment, the mosquito is Anopheles gambiae.

[0030] The malaria parasite can be any malaria parasite. In some embodiments, the malaria parasite is a malaria parasite of the genus Plasmodium. In some embodiments, the malaria parasite is Plasmodium falciparum, Plasmodium berghei, Plasmodium vivax, or a combination thereof. In some embodiments, the malaria parasite is Plasmodium falciparum. In other embodiments, the malaria parasite is Plasmodium berghei. In yet other embodiments, the malaria parasite is Plasmodium vivax.

[0031] In one embodiment, the composition is used to reduce or prevent (i) dengue fever and / or (ii) dengue virus transmission. In one embodiment, the composition is used to reduce or prevent (i) dengue fever and / or (ii) dengue virus transmission by mosquitoes. In one embodiment, the composition is used to reduce or prevent dengue fever. In another embodiment, the composition is used to reduce or prevent dengue virus transmission (e.g., by mosquitoes). The mosquito can be any mosquito. In one embodiment, the mosquito is an Aedes mosquito. In one embodiment, the mosquito is an Aedes albopictus or an Aedes aegypti mosquito.

[0032] As defined herein, "reducing or preventing dengue virus transmission" refers to eliminating dengue fever by, for example, inhibiting the mosquito developmental stage of dengue virus load. 1-Methyl-9H-pyrido[3,4-b]indole, an active compound secreted by Delftia bacteria, specifically Delftia tsuruhatensis, has been shown to inhibit dengue virus and thereby suppress mosquito-mediated dengue fever transmission.

[0033] In another aspect, the present invention provides a method for reducing or preventing the transmission of malaria or dengue fever, the method comprising the step of contacting at least one vector, or parasite, of the disease with 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof.

[0034] The step of contacting the vector, parasite, or virus with the compound can be carried out by any suitable method. For example, it is not necessary to physically contact the vector, parasite, or virus with 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof. The compound can be left in place where it will come into contact with the vector. The compound can be in the form of a composition described above or below.

[0035] In certain embodiments of the invention, the contacting can be achieved by treating an area with a composition of the invention using a spray formulation, such as, for example, an aerosol or pump spray. In certain embodiments of the invention, an area can be treated using, for example, air delivery, truck-mounted equipment, etc. In some embodiments, the composite is sprayed, for example, by backpack spraying, aerial spraying, spraying / dusting, etc. The parasitic or viral vector can be any parasitic or viral vector capable of transmitting disease. In one embodiment, the vector is a mosquito.

[0036] In the case of malaria, the mosquito can be any mosquito capable of transmitting malaria, such as an Anopheles mosquito. It is contemplated that the compositions and methods of the present invention are applicable to any species of mosquito. In one embodiment, the mosquito is an Anopheles mosquito. In one embodiment, the mosquito is an Anopheles gambiae, Anopheles stephensis, Anopheles chrysifacies, or Anopheles coluzzi. In one embodiment, the mosquito is an Anopheles stephensis or Anopheles gambiae. In one embodiment, the mosquito is an Anopheles stephensis. In another embodiment, the mosquito is an Anopheles gambiae. The malaria parasite can be any malaria parasite. In one embodiment, the malaria parasite is a Plasmodium genus. In one embodiment, the malaria parasite is Plasmodium falciparum. In another embodiment, the malaria parasite is Plasmodium berghei. In yet another embodiment, the malaria parasite is Plasmodium vivax. In one embodiment, the malaria parasite is selected from Plasmodium vivax, Plasmodium ovale curtisi, P. ovale wallikeri, and Plasmodium knowlesisel.

[0037] In the case of dengue virus, the method comprises contacting a mosquito with 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof. The mosquito may be any mosquito capable of transmitting dengue virus. It is contemplated that the compositions and methods of the present invention are applicable to any species of mosquito. In one embodiment, the mosquito is of the Aedes genus. In one embodiment, the mosquito is Aedes albopictus or Aedes aegypti.

[0038] In another aspect, the present invention provides the use of 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof in reducing or preventing malaria or dengue fever, or infection by a malaria parasite or dengue virus.

[0039] In another aspect, the present invention provides 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for use in inhibiting the development of malaria parasites into ookinetes, oocysts, or sporozoites in mosquito vectors, or the replication of dengue virus.

[0040] composition The compositions of the present invention may be in any suitable form and may include any suitable carrier. The compositions may be feed compositions. That is, the compositions may be in a form that can be provided to vectors or parasites for oral ingestion. In some embodiments, the feed composition is an attractive sugar bait, a sugar source, or nectar. In one embodiment, the feed composition is a sugar source. The sugar source may be an attractive sugar bait or may be included in an attractive sugar bait. Attractive sugar baits generally comprise a sugar and a toxic component. It is contemplated that an attractive sugar bait of the present invention may comprise a composition of the present invention instead of a toxic component, i.e., the attractive sugar bait may comprise a sugar and a composition of the present invention. In some embodiments, the attractant is a sugar source comprising a mixture of fruit juice and / or syrup. In one embodiment, the composition is in the form of a bait. The bait is designed to attract vector mosquitoes to come into contact with the composition. In one embodiment, the composition is taken up by the vector (e.g., mosquito) upon contact, for example, by oral ingestion and / or cuticular absorption. Attractants can also be used. The attractant can be a sugar or sugar mixture, fruit juice or pulp, or a pheromone, for example, a male or female pheromone. The attractant serves to attract the vector or parasite (e.g., mosquito) to the bait. The bait can be in a suitable form, for example, a solid, paste, pellet, or powder.

[0041] The bait can be provided in a suitable "housing" or "trap." Such housings and traps are commercially available, and existing traps can be adapted to contain the composition of the present invention. The housing or trap can be, for example, box-shaped, provided in a preformed state, or made of, for example, foldable cardboard. Suitable materials for the housing or trap include plastic and cardboard, particularly corrugated cardboard. The inner surface of the trap can be coated with an adhesive substance to restrict the movement of vectors or parasites (e.g., mosquitoes) that have entered the trap. The housing or trap can be equipped with a suitable trough inside which the bait can be fixed. Traps differ from housings in that mosquitoes cannot easily escape from the trap after entry, whereas housings function as "feeding stations" that provide vectors with a favorable environment in which they can feed and feel safe from predators.

[0042] In another embodiment, the present invention provides a mosquito bait comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof and one or more sugar sources.

[0043] combination It is envisioned that the present invention will be deployed in conjunction with other malaria or dengue eradication efforts. For example, the compositions, methods, and compounds for use of the present invention may be used in conjunction with known anti-vector or anti-parasitic agents (e.g., antimalarials). In one embodiment, the compositions or compounds for use of the present invention may be used in combination with one, two, or three additional anti-parasitic or antimalarial agents. Integrated Vector Management (IVM) suggests making the best use of available tools.

[0044] The at least one other antimalarial agent may be selected from ferroquine, KAF156, cipargamin, DSM265, artemisone, artemisinone, artefenomel, MMV048, SJ733, P218, MMV253, PA92, DDD498, AN13762, DSM421, UCT947, ACT 451840, OZ609, OZ277 and SAR97276. For the treatment of Plasmodium falciparum infection, the at least one, two, or three additional antimalarials may be selected from the following list, where at least one antimalarial is an artemisinin: artemether and lumefantrine, artesunate and amodiaquine, artesunate and mefloquine, dihydroartemisinin and piperaquine, or artesunate and sulfadoxine-pyrimethamine (SP). These combination therapies are known as artemisinin-based combination therapies (ACTs). The selection of an ACT is typically based on the results of therapeutic efficacy studies against endemic strains of Plasmodium falciparum malaria. For the treatment of Plasmodium vivax infection, an ACT may be used, as described above. Alternatively, the at least one other antimalarial may be chloroquine, particularly in areas where chloroquine-resistant Plasmodium vivax is not present. In areas where resistant P. vivax strains have been identified, the infection may be treated with ACT as described above. The therapeutic combination is conveniently presented for use in the form of a pharmaceutical composition or formulation and may be administered together or separately, and if administered separately may be administered separately or sequentially in any order (by the same or different routes of administration).

[0045] The compositions or bacteria for use in the present invention may be used in combination with insecticide-treated nets (ITNs), including long-acting insecticidal nets (LLINs) and / or IRSs (indoor residual sprays). Attractive toxic sugar baits (ATSBs) use sugars containing toxic compounds that attract and kill mosquitoes. To increase efficiency, ATSBs can also contain the aforementioned harman compounds instead of toxic compounds. [Example]

[0046] The present invention will be described below by way of non-limiting examples. Specific embodiments of the present invention are described below, but those skilled in the art will understand that various changes and modifications are possible. References to other similar preparation methods or preparations carried out by other common preparation methods may include variations in normal parameters, such as slight changes in time, temperature, post-treatment conditions, and reagent amounts.

[0047] material microbial strains Delftia tsuruhatensis GSK TC1 Delftia tsuruhatensis Carb * , and Mut1 ** * Carb-USA: Delftia tsuruhatensis (ATCC collection), obtained from Dr. Ramesh Goel (University of Utah, USA). ** Mut1-USA; Delftia tsuruhatensis, obtained from Dr. Goel (2). Delftia acidovorans: Ingrid * * Ingrid: Delftia acidovorans, obtained from Dr. Ingrid Faye (Stockholm University, Sweden). Pseudomonas putida GSK TC2 Pantoea agglomerans mosquito Anopheles gambiae -Anopheles Stephens Parasites: Plasmodium falciparum NF54 Plasmodium falciparum ANKA 234 Culture medium: M9 minimal medium:

[0048] [Table 1]

[0049] Filter sterilize and store at 4°C. Autoclave and store at room temperature.

[0050] BHK21 medium: DMEM + 10% heat-inactivated FBS + 1% L-glutamine + 1% penicillin-strep solution (Pen-strep) + 5 μg plasmocin.

[0051] Layer medium: DMEM + 2% heat-inactivated FBS + 1% L-glutamine + 1% penicillin-streptomycin solution + 5 μg plasmocin + 0.8% methycellulose Drugs: Harman (CHEM-IMPEX INT'L INC, Catalog No. 21682) DMSO (Amresco Life Science, Catalog No. 0231-500ML)

[0052] Example 1: Bioassay-guided purification of active natural products from Delftia tsuruhatensis supernatant For flash fractionation, the fermentation supernatant (10 L) was passed through a C-18 reverse-phase silica gel column (160 x 30 mm; Sepra™ C-18-E (50 μm, 65 Å)).

[0053] Material not retained on the column (follow-through) was collected for activity testing. The column was subjected to isocratic elution (HO / CHCN 95:5), followed by a 40-minute gradient of 5% to 100% acetonitrile in water (CHCN), and an isocratic step of 100% CHCN in 20 minutes at 10 mL / min. 18 mL fractions were collected. UV detection at 210 nm and 280 nm was used.

[0054] Additionally, 100 mL of unfermented medium was passed through a 60 x 15 mm C-18 cartridge and eluted with 100 mL of 100% CH3CN, which was dried (12.5 mg) and dissolved in 1 mL of MeOH.

[0055] For shipping and activity assessment, 500 μL of the supernatant, 500 μL aliquots of each fraction, 500 μL of follow-through obtained when 10 L of supernatant was loaded onto a C-18 column, and 100 μL of blank medium extract were transferred to an AB-Gene 0765 800 μL 96-well storage plate and dried in an HT-8 Genevac vacuum centrifuge.

[0056] LC-HRMS dereplication method Active fractions were analyzed using an Agilent 1200 Rapid Resolution HPLC interfaced to a Bruker maXis mass spectrometer. Sample injection volume was 2 μL. Separation was performed using a Zorbax SB-C8 column (2.1 × 30 mm, 3.5 μm particle size). Two solvents were used as mobile phases: Solvent A 90:10 H2O:CH3CN, Solvent B 10:90 water:CH3CN, both containing 13 mM ammonium formate and 0.01% TFA. The gradient composition was as follows: [Table 2]

[0057] The mass spectrometer was operated in positive ESI mode. The instrument parameters were: capillary voltage 4 kV, drying gas flow rate 11 L / min at 200 °C, and nebulizer pressure 2.8 bar. TFA-Na cluster ions were used for mass calibration of the instrument before sample injection. Each sample run was recalibrated by injecting the same TFA-NA calibrant before starting the chromatography.

[0058] NMR dereplication method For NMR analysis, samples were dissolved in CD3OD. After dissolution, each sample was transferred to a 1.7 mm tube. Spectra were acquired on a Bruker AVANCE III 500 MHz spectrometer equipped with a 1.7 mm TCI micro-cryoprobe (1D 1 H spectrum and 2D HSQC spectrum). All spectra were recorded at 24 °C.

[0059] result As shown in Figure 1, LC-HRMS revealed that the molecular formula for the active ingredient was C 12 H 10 It was determined to be N2.

[0060] HSQC of active ingredients and 1 The 1 H NMR spectrum was consistent with that of harman, as shown in Figures 2 and 3, respectively.

[0061] Biological data Mosquito breeding and protozoan culture Anopheles gambiae (Keele strain) and Anopheles stephensis (Nijmegen strain) were reared as described and known in the art. For fitness assessment, mosquitoes were fed on Swiss Webster mice.

[0062] Using the membrane feeding method, female Anopheles gambiae mosquitoes were infected with Plasmodium falciparum gametocyte cultures to generate P. falciparum NF54 gametocytes. Briefly, parasites were maintained in RPMI 1640 medium supplemented with 25 mM HEPES, 50 mg / L hypoxanthine, 25 mM NaHCO3, and 10% (v / v) heat-inactivated O2+ human serum (Interstate Blood Bank, Inc.) at 37°C in O2+ human red blood cells using a gas mixture of 5% O2, 5% CO2, and balanced N2. For feeding, 14- to 17-day-old mature gametocytes were pelleted by centrifugation (5 min, 2,500 g) and resuspended in O2+ human red blood cells to a gametocyte concentration of 0.15% to 0.2% and diluted to 40% hematocrit with human serum. All manipulations were performed while maintaining the cultures, tubes, and feeders at 37°C.

[0063] Supernatant preparation Delftia and Pantoea agglomerans (control) were grown overnight in LB liquid medium (200 rpm, 28 °C). Bacteria were washed and resuspended in M9 medium (10 9 The supernatant was filtered through a 0.22 μm filter to prepare Delftia supernatant (D-8h) and Pantoea agglomerans supernatant (P-8h). An aliquot of the Delftia supernatant was passed through a 3 kDa centrifugal filter (Amicon Ultra-3K, REF: UFC500396) (D-8h<3KD). Another aliquot of the supernatant was boiled for 10 min (boiled D-8h). An aliquot of the Delftia cell suspension in M9 medium (10 9 / ml) was heated at 70°C for 15 minutes to prepare dead bacteria (Dead-B).

[0064] Proteinase K treatment of Delftia supernatant M9 medium (control) and D-8h were incubated with 1.25 μg / ml proteinase K in 30 mM Tris HCl, pH 8.0, at 50°C for 1 hour (M9+K and D-8h+K). Mosquitoes were fed with P. falciparum gametocytes (150 μl infected red blood cells + 150 μl normal human serum) supplemented with 50 μl of M9, M9+K, D-8h, or D-8h+K. After 7 days of feeding, the number of oocysts per midgut was determined. Parasite counts between the control and experimental groups were compared using the nonparametric Mann-Whitney test (GraphPad, Prism).

[0065] Inhibition of Plasmodium falciparum oocyst development by Delftia supernatants Two-day-old mosquitoes were fed with or without Delftia supernatant on days 0, 1, 2, and 4. On day 4, mosquitoes were fed with P. falciparum gametocytes. Seven days after blood feeding, the number of oocysts per midgut was determined. Data were pooled from two independent experiments. Parasite counts between control and experimental groups were compared using the nonparametric Mann-Whitney test (GraphPad, Prism).

[0066] Inhibition of oocyst formation by Delftia supernatant Plasmodium falciparum gametocytes (150 μl infected red blood cells + 150 μl normal human serum, final 0.02% gametocyte blood) were mixed with different concentrations of Delftia supernatant in M9 medium and fed to Anopheles gambiae mosquitoes. Seven days after feeding, oocyst numbers were determined. Parasite counts between control and experimental groups were compared using the nonparametric Mann-Whitney test (GraphPad, Prism).

[0067] Inhibition of ookinete formation by Delftia supernatants Anopheles gambiae mosquitoes were fed with Plasmodium falciparum-infected blood meal (150 μl red blood cells + 150 μl normal human serum) supplemented with 50 μl of M9 medium, D-8h, boiled D-8h, Dead-B, and P-8h. 22 h after feeding, the number of ookinetes in the midguts was determined. Each midgut was placed in 20 μl of PBS, homogenized by pipetting, and placed in an 8-well slide (1 midgut / well). After drying at room temperature, the samples were fixed with 80% methanol for 15 s and allowed to dry at room temperature. The samples were blocked with 5% BSA for 1 h at room temperature and then incubated with the Pfs25 antibody (Mab4b7) (38) in blocking buffer (1:250–1:500 dilution) for 1 h at room temperature. After washing three times with PBS for 5 min, the samples were incubated with Alexa fluor 488 goat anti-mouse IgG (Life Technologies, Catalog No.: A11001) in blocking buffer (1:250–1:500 dilution) for 1 h at room temperature. After washing three times with PBS for 5 min, the slides were dried and covered with a coverslip. Ookinetes were counted using a fluorescence microscope. Parasite counts between control and experimental groups were compared using the nonparametric Mann-Whitney test (GraphPad, Prism).

[0068] Inhibitory activity of harman against asexual blood stages of Plasmodium falciparum This assay is based on the uptake of labeled hypoxanthine by P. falciparum parasites, which is proportional to their proliferation. Briefly, cultures of 3D7A and Dd2 parasitized red blood cells (RBCs) (0.5% parasitemia and 2% hematocrit in RPMI-1640 supplemented with 25 ml Albumax and 5 μM hypoxanthine, final volume 500 ml) were exposed to two-fold serial dilutions, starting at 10 μM, for a total of 10 concentrations. Plates were incubated at 37°C with [5% CO2, 5% O2, 90% N2]. After 24 h of incubation, 3H-hypoxanthine (0.025 μCi / μl in RMPI-1640) was added and incubated for an additional 24 hours, after which the plates were frozen and stored at -80°C. Parasites were harvested onto glass fiber filters using a TOMTEC Cell Harvester 96. The filters were dried and radioactivity was measured in a MicroBeta counter using melt-on scintillator sheets. 3 H-hypoxanthine uptake was determined.

[0069] Flagellar release assay A total of 1 ml of P. falciparum NF54 gametocyte culture was centrifuged at 700 x g for 1 minute. The supernatant was discarded, and the pellet was resuspended in an equal volume of normal human serum and incubated at room temperature for 10-15 minutes. Ten microliters of this suspension was placed on a glass slide, covered with a coverslip, and the average number of flagellar extrusion centers per field was determined using a light microscope at 400x magnification. At least 10-20 fields were randomly counted. The number of extrusion centers between the control and experimental groups was compared using a Student's test.

[0070] Inhibition of Plasmodium growth in Anopheles gambiae mosquitoes by harman Feeding assay: Plasmodium falciparum NF54 gametocytes (150 μl red blood cells + 150 μl normal human serum) were fed to 2-day-old mosquitoes after adding 50 μl of M9 medium containing or without harman at 1 nM, 10 nM, 25 nM, 50 nM, or 150 nM. Oocyst counts were determined 7 days after feeding. Oocyst counts between control and experimental groups were compared using a nonparametric Mann-Whitney test (GraphPad, Prism).

[0071] Contact assay: Harman was dried onto a 9 cm glass Petri dish at a final coverage of 1 nmol / m 2 , 10 nmol / m 2 , 100 nmol / m 2 , 1 μmol / m 2 and 10 μmol / m 2 Atovaquone (ATQ) was dried on a 9 cm glass Petri dish to a final coverage of 100 nmol / m2 , 1 μmol / m 2 , 10 μmol / m 2 and 100 μmol / m 2 A total of 50 μl of D-8h (positive control), and M9 medium and / or P-8h (Pantoea supernatant as a negative control) were also dried on a 9 cm glass Petri dish. Two-day-old mosquitoes were sugar-starved overnight (provided only with water), knocked down with CO2 for 2–3 min, and transferred to a coated Petri dish for 1 h at 27°C. The mosquitoes were knocked down, transferred to a mosquito cup, and fed with a Plasmodium falciparum-infected blood meal 3 h later. Oocyst counts were determined 7 days after feeding. Oocyst counts between the control and experimental groups were compared using a nonparametric Mann-Whitney test (GraphPad, Prism).

[0072] Inhibitory activity of harman on asexual growth of Plasmodium falciparum This assay is based on the uptake of labeled hypoxanthine by P. falciparum parasites, which is proportional to their growth. Briefly, 3D7A- and Dd2-parasitized red blood cell (RBC) cultures (500 ml of RPMI-1640 supplemented with 25 ml Albumax and 5 μM hypoxanthine at 0.5% parasitemia and 2% hematocrit) were exposed to two-fold serial dilutions of compounds. Starting at 10 μM, a total of 10 concentrations were used. Plates were incubated at 37°C in 5% CO2, 5% O2, and 90% N2. After 24 hours of incubation, 3 H-hypoxanthine (0.025 μCi / μl in RMPI-1640) was added and incubated for another 24 hours, after which the plates were frozen at -80°C. Parasites were then harvested onto glass fiber filters using a TOMTEC Cell Harvester 96. The filters were dried and radioactivity was measured in a Microbeta counter using melt-on scintillator sheets. 3 H-hypoxanthine uptake was determined.

[0073] Inhibitory activity of harman on male and female gametogenesis in Plasmodium falciparum Double gametogenesis assay or DGFA. Mature stage 5 gametocytes were exposed to two-fold serial dilutions of compounds. A total of 10 concentrations were used, ranging from 10 μM to 0.01 μM. Compounds were incubated for 48 h and then activated in ookinete medium using the DGFA protocol described by Ruecker (39).

[0074] In vitro ookinete production assay Mice were treated with phenylhydrazine 3 days before infection by intraperitoneal inoculation with Plasmodium berghei (ANKA) strain parasites obtained from cryopreserved stocks from donor mice between passages 2 and 6. From day 3 postinfection onward, parasitemia was confirmed by microscopic examination of thin blood smears to determine the presence of flagellated gametocytes (more than 15 flagella per 20x field). Gametocyte blood was collected by cardiac puncture into a heparinized syringe. This was achieved by passing the blood through an equilibrated 5 ml sterile column containing 1 ml glass wool and 3 ml Whatmann CF11 cellulose powder (Beckton & Dickenson). The blood was washed with RPMI, and 1 ml of blood was added to 19 ml of fresh complete ookinete medium. The blood solution was transferred to a 24-well plate (0.5 ml per well), mixed with DMSO (control) or harman, and the plate was shaken at 19°C for 24 hours.

[0075] Cytotoxicity assay BHK21 cells were harvested at 80% confluence and the cell suspension was diluted 50-fold. The cell suspension was transferred to a 24-well plate, and harman was added to each well to final concentrations of 0 (control), 1 nM, 10 nM, 100 nM, 1 uM, and 10 uM. The cells were cultured at 37°C and 5% CO2 for 2 days, and the number of cells in each well was counted. Inhibition rate = 100 x [cell number (control) - cell number (harman-treated wells)] / cell number (control).

[0076] Dengue infection inhibition assay BHK21 cells were cultured in 24-well plates and used for dengue infection when they reached 80% confluence. The cells were diluted 10-fold and transferred to 24-well plates. Harman was diluted in complete DMEM medium to final concentrations of 1 nM, 10 nM, 100 nM, 1 uM, and 10 uM and added to the cells. The cells were then incubated on a rocking platform at room temperature for 15 minutes, followed by 1 hour in an incubator at 37°C and 5% CO2. Approximately 1 hour after the addition of the test compound, dengue virus was added to each well and incubated with gentle rocking for 15 minutes, followed by 45 minutes in an incubator at 37°C and 5% CO2. 1 ml of overlay medium was added to each well, and the cells were incubated at 37°C and 5% CO2 for 5 days. The plates were then fixed and visualized with a mixture of methanol / acetone (1:1 volume) and 1% crystal violet at room temperature for 30 minutes, and the plaque-forming units were counted. The results of drug-treated cells were compared with those of the DMSO control. Experiments were performed in triplicate.

[0077] Figure Discussion See Figure 4: Harman fed to Anopheles gambiae mosquitoes inhibits the development of Plasmodium spp. (A) Mosquitoes were fed Plasmodium falciparum gametocytes with or without the indicated concentrations of harman. M9: M9 medium control; D-8h: Delftia supernatant cultured in M9 medium for 8 h. Red bars indicate median values. Numbers: number of mosquitoes analyzed; Prevalence: percentage of mosquitoes carrying one or more oocysts. Data are pooled from three independent experiments. (B) Dose-response curve fit for oral feeding of Harman {nonlinear regression, n = 13, df = 12, sum of squares = 1054, R 2 = 0.9082. The IC50 for HA feeding was calculated by interpolation and is shown below the graph. The mean inhibitory potency relative to control oocysts is shown. Error bars represent the 95% confidence interval (CI). 50% inhibition was observed at 45.9 nM. Related to data in panel A. Statistical analysis by Mann-Whitney U test. ***: P value < 0.001; ** : P value < 0.001; * : P value < 0.05; NS, not significant difference.

[0078] See Figure 5: Contact inhibition of Plasmodium development in Anopheles gambiae mosquitoes. (A) Delftia supernatant contains compounds that penetrate the mosquito cuticle and inhibit the development of Plasmodium falciparum. Female mosquitoes were exposed to D-8h, atovaquone (ATQ), or P-8h (Pantoea supernatant) dried on glass plates for 60 minutes before infection with P. falciparum gametocytes. D-8h feeding: As a positive control, the supernatant was mixed with infected blood and fed to mosquitoes. Data are pooled from two independent experiments. (B) and (F) harmane (HA) or (C) and (G) atovaquone (ATQ) were dried onto glass plates at the indicated final concentrations. Mosquitoes were exposed to these plates for 60 minutes and then fed on P. falciparum gametocytes. M9: 3 ml of M9 medium dried onto the plate. D-8h: 3 ml of Delftia supernatant dried onto the plate. Data were pooled from two independent experiments. Statistical analysis was performed using the Mann-Whitney U test. *** : P value < 0.001; ** : P value < 0.01; NS, not significant. Number: number of mosquitoes analyzed; Prevalence: percentage of mosquitoes carrying one or more oocysts. (D) Dose-response curve fit for harmane (HA) exposure {nonlinear regression, n = 17, degrees of freedom df = 16, sum of squares = 6698, R 2 = 0.6122. The IC50 for HA challenge was calculated by interpolation and is shown below the graph. The mean inhibitory potency relative to control oocysts is shown. Error bars are 95% confidence intervals (CI). Corresponds to panel B. 102.2 nmol / m 2 A 50% HA inhibition was observed. (E) Dose-response curve fit for atovaquone (ATQ) exposure {nonlinear regression, n = 13, degrees of freedom df = 12, sum of squares = 4120, R2 = 0.8085. The IC50 for ATQ exposure was calculated by interpolation and is shown below the graph. The mean inhibitory potency relative to control oocysts is shown. Error bars are 95% confidence intervals (CI)}. 3,634 nmol / m 2 50% ATQ inhibition was observed. Corresponds to panel C.

[0079] See Figure 6: Duration of harmane inhibitory action in mosquitoes. (A) Schematic of the experiment. Harman was administered to female mosquitoes either by feeding (B) or contact (C) on days 0, 1, 2, and 4 before feeding with P. falciparum-infected blood. Seven days later, the number of oocysts per midgut was determined. Number: number of mosquitoes analyzed; prevalence: percentage of mosquitoes carrying one or more oocysts. Data are pooled from two independent experiments. Statistical analysis by Mann-Whitney U test. *** : P value < 0.001; NS, not significant difference.

[0080] See Figure 7: Inhibition of ookinete formation by harman in vitro. (A) Schematic of the experiment. (B) Blood containing Plasmodium gametocytes was incubated with DMSO (control) or 1 μM harman at 0 or 4 h after culture setup, and ookinete counts were measured 24 h later. Data are pooled from two independent experiments. Statistical analysis by Student's test. ** : P value < 0.01.

[0081] See Figure 8: Treatment of Delftia supernatant with proteinase K does not alter the growth inhibition of Plasmodium falciparum in Anopheles gambiae mosquitoes.

[0082] M9 medium and D-8h (supernatant of Delftia cultured in M9 medium for 8 hours) were treated with 1.25 μg / ml proteinase K at 50°C for 1 hour and designated as M9+K and D-8h+K. Mosquitoes were fed with P. falciparum gametocytes treated with M9, M9+K, D-8h, or D-8h+K and monitored for oocyst formation. Horizontal lines represent median values. Values: number of mosquitoes analyzed; prevalence: percentage of mosquitoes carrying one or more oocysts. Statistical analysis by Mann-Whitney U test. *** : P value < 0.001; NS, not significant. Data are from one experiment.

[0083] See Figure 9: Effect of different concentrations of Delftia supernatant on the development of Plasmodium parasites in Anopheles gambiae mosquitoes. A total of 50 μl of M9 medium or Delftia supernatant, undiluted (100%) or diluted (1% or 10%), was added to 300 μl of P. falciparum-carrying blood (150 μl infected red blood cells + 150 μl normal human serum, final 0.02% gametocyte blood) and fed to mosquitoes. Values: number of mosquitoes analyzed; prevalence: percentage of mosquitoes carrying one or more oocysts. Data are from a single experiment. Statistical analysis was performed using the Mann-Whitney U test (GraphPad, Prism). *** : P value < 0.0001.

[0084] See Figure 10: Duration of inhibition of Plasmodium falciparum oocyst formation in Anopheles gambiae mosquitoes by Delftia supernatant.

[0085] Mosquitoes were fed with P. falciparum gametocytes on days 0, 1, 2, and 4 after feeding with Delftia supernatant cultured in M9 medium for 8 hours, and oocyst formation was measured. Numerical values: number of mosquitoes analyzed; prevalence: percentage of mosquitoes carrying one or more oocysts. Statistical analysis was performed using the Mann-Whitney U test. *** : P value < 0.001; NS, not significant. Data are pooled from two independent experiments.

[0086] See Figure 11: Inhibition of Plasmodium growth in Anopheles gambiae mosquitoes by supernatants from different Delftia strains. Plasmodium falciparum gametocytes were mixed with supernatants from different strains cultured in M9 medium for 8 h and fed to mosquitoes. Pantoea: Pantoea agglomerans control; Dtsu1: Delftia tsuruhatensis strain from GSK used in this study; Dtsu2: Delftia tsuruhatensis strain from ATCC; Dtsu3: Delftia tsuruhatensis strain from Gilcrease et al. (57); Dacido, Delftia acidovorans strain (obtained from Dr. Ingrid Faye, Stockholm University, Sweden). Numbers: number of mosquitoes analyzed; Prevalence: percentage of mosquitoes carrying one or more oocysts. Data are pooled from two experiments. Statistical analysis by Mann-Whitney U test. *** : P value < 0.0001; NS: no significant difference.

[0087] See Figure 12: Screening of Delftia supernatant fractions for inhibition of Plasmodium falciparum oocyst development activity in Anopheles gambiae mosquitoes. (A) A total of 10 L of Delftia supernatant was fractionated on a C18 column, and 1 / 36 of each fraction (equivalent to 278 mL of supernatant) was dried in a multiwell plate. "PL_ROW" and "PL_COLMN" indicate the position of the fraction within the plate. The seven fractions pooled for testing are color-coded and labeled on the right. Crude: 500 μL of dried Delftia supernatant; FL: 500 μL of dried flow-through from the C18 column; M9 FL: As a control, only M9 medium was passed through the column, and 100 mL of flow-through was collected and dried. (B) Fractions resuspended in DMSO and tested for inhibition of Plasmodium falciparum oocyst formation in Anopheles gambiae mosquitoes. C: DMSO control; D-8h: fresh Delftia 8-h culture supernatant in M9 medium positive control. (C) Separate assay of P4 and P5 fractions. (D) Separate assays of P6 and P7 fractions. Data are from one experiment. Statistical analysis by Mann-Whitney U test. *** : P value < 0.001; **: P value < 0.01; * : P value < 0.05; NS, not significant difference.

[0088] Referring to Figures 13 and 14, harman has no effect on BHK21 cell growth in culture (except at the highest concentration tested: 10 μM), but significantly inhibits dengue virus infection at 1 μM.

[0089] Referring to FIG. 15, mosquitoes aged 3 to 5 days were infected with approximately 10 6~7 They were fed a blood meal containing PFU / ml of dengue virus. (A) Blood meals contained harman at the indicated concentrations. (B) Before blood feeding, mosquitoes were exposed to DMSO (control) or the indicated concentrations of harmane (HA) dried onto glass plates for 60 min.

[0090] After 7 days, mosquitoes were dissected and their midguts were homogenized and analyzed. Plaque-forming unit (PFU) counts were determined as described in "Dengue infection inhibition assay" and are shown in Figure 15. Figure 15A shows that harman concentrations of 100 nM or less did not significantly inhibit dengue virus growth in mosquitoes, whereas 1 μM and 10 μM strongly inhibited it. Figure 15B shows that harman concentrations of 100 nM or less significantly inhibited dengue virus growth in mosquitoes, whereas 1 μM and 10 μM strongly inhibited it. -1 mmol / m 2 (100 μmol / m 2 ) shows that exposure to harman can inhibit dengue virus infection. (Data are pooled from three independent experiments.)

[0091] Figure 16 shows approximately 10 6~7 Survival of 3-5 day-old Aedes mosquitoes fed blood meals containing PFU / ml dengue virus and different concentrations of harman: 0, 10nM, 100nM, 1µM, and 10µM. Mosquito survival after 7 days was not affected by feeding 10µM harman. (Data pooled from three independent experiments.)

[0092] Figure 17 shows the fitness cost of Delftia to mosquitoes.

[0093] 17A and 17B, two-day-old female Aedes aegypti mosquitoes were fed Delftia or Pantoea bacteria. A "control" group was not fed any bacteria. (A) Bloodfeeding rate and (B) blood meal volume were determined by measuring the hemoglobin content of individual guts 2 days after bloodfeeding. Error bars indicate the standard deviation of the mean. Data are pooled from three independent experiments (30 mosquitoes per sample). Statistical analysis was performed using a t-test.

[0094] 17C and 17D, two-day-old female mosquitoes were fed Delftia or Pantoea bacteria. A "control" group received no bacteria. Five days later, the mosquitoes were blood-fed. (C) Fecundity (number of eggs laid) and (D) fertility (percentage of eggs that hatched) were measured. Horizontal lines indicate medians. No significant differences were found using the Mann-Whitney U test. Three biological replicates were combined (total of 96 mosquitoes). NS: not significant; * : P value < 0.05.

[0095] 17E and 17F, two-day-old female Aedes mosquitoes were fed Delftia or Pantoea bacteria. A "control" group received no bacteria. The survival rates of (E) male and (F) female mosquitoes were measured. No significant difference was observed in the male group. In the female group, slight differences were observed between the survival rates of Delftia-fed and control mosquitoes. Survival rates were calculated using Kaplan-Meier survival curves, and multiple comparisons were performed using the log-rank test. Three biological replicates were combined (a total of approximately 330 mosquitoes). NS: not significant; * : P value < 0.05.

[0096] The data in Figure 17 show that Delftia does not impose a fitness cost on mosquitoes.

[0097] See Figure 18: Inhibition of dengue infection in Aedes mosquitoes by Delftia. (A) and (B) Aedes mosquitoes were fed Delftia bacteria and subsequently infected with dengue virus 2 days later. (C) Aedes mosquitoes were fed Delftia bacteria and subsequently infected with dengue virus 16 days later.

[0098] Figures 18A and 18B show that Aedes mosquitoes fed Delftia bacteria had reduced PFUs in the midgut and salivary glands. Figure 18C shows that this effect persists for a long period of time, indicating that Delftia can inhibit dengue virus infection at the midgut and salivary gland stages, and that this effect persists for at least 16 days. Plaque-forming units (PFU) were determined as described in "Dengue Infection Inhibition Assay." Delftia bacteria inhibited dengue virus growth in Aedes aegypti by 78.4%. (Data pooled from three independent experiments.)

[0099] Magazine Feature Further biological data was published after the priority date of this application in Huang, Science, 381, 533-540 (2023).

Claims

1. 1. A composition for use in a method for reducing or preventing the transmission of malaria or dengue viruses, comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, the method comprising the step of contacting at least one species of vector mosquito of the genus Anopheles or subfamily Anophelinae with the composition.

2. 1. A composition for use in a method for reducing or preventing transmission of malaria parasites or dengue viruses, the composition comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, the method comprising contacting at least one vector mosquito species with the composition.

3. 10. The composition for use of claim 1, further comprising at least one carrier, delivery vehicle, adjuvant, solvent, stabilizer, or preservative.

4. A composition for use according to any one of claims 1 to 3, comprising an attractant.

5. The composition for use according to claim 4, wherein the attractant is a sugar source or a pheromone.

6. 2. The composition for use according to claim 1, wherein the vector-borne disease is selected from dengue fever and malaria.

7. 3. The composition for use according to claim 2, wherein the protozoal disease is malaria.

8. 8. A composition for use according to any one of claims 1 to 7 for use in reducing or preventing (i) malaria, and / or (ii) malaria parasite transmission.

9. 9. The composition for use according to claim 8, wherein the malaria parasite is Plasmodium falciparum.

10. 10. A composition for use according to claim 9 for use in reducing or preventing (i) malaria, and / or (ii) mosquito transmission of the malaria parasite.

11. 11. The composition for use according to claim 10, wherein the mosquito is an Anopheles mosquito.

12. 12. The composition for use of claim 11, wherein the mosquito is Anopheles gambiae, Anopheles stephensis, Anopheles chrysifacies, Anopheles gambiae, or Anopheles stephensis.

13. 8. A composition for use according to any one of claims 1 to 7 for use in reducing or preventing (i) dengue fever, and / or (ii) dengue virus transmission.

14. 14. A composition for use according to any one of claims 13 for use in reducing or preventing (i) dengue fever, and / or (ii) mosquito transmission of dengue virus.

15. 15. A composition for use according to claim 13 or claim 14, wherein the mosquito is an Aedes mosquito.

16. 16. The composition for use according to claim 15, wherein the mosquito is Aedes albopictus or Aedes aegypti.

17. 1. A method for reducing or preventing the transmission of dengue fever or malaria, comprising contacting mosquito vectors of at least one of these diseases with 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof.

18. Use of 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof in reducing or preventing malaria or dengue fever.

19. 1-Methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for use in inhibiting the development of a parasite into an ookinete, oocyst, or sporozoite.

20. A mosquito bait comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof and one or more sugar sources.

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