WO2019004825A1 - Tomatidine et analogues de celle-ci pour une utilisation en tant qu'agent antiviral - Google Patents

Tomatidine et analogues de celle-ci pour une utilisation en tant qu'agent antiviral Download PDF

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Publication number
WO2019004825A1
WO2019004825A1 PCT/NL2018/050413 NL2018050413W WO2019004825A1 WO 2019004825 A1 WO2019004825 A1 WO 2019004825A1 NL 2018050413 W NL2018050413 W NL 2018050413W WO 2019004825 A1 WO2019004825 A1 WO 2019004825A1
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tomatidine
virus
analog
denv
cells
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PCT/NL2018/050413
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English (en)
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Jolanda Mariske SMIT
Mayra Alejandra DIOSA TORO
Berit Helen TROOST
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Rijksuniversiteit Groningen
Academisch Ziekenhuis Groningen
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Publication of WO2019004825A1 publication Critical patent/WO2019004825A1/fr

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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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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

Definitions

  • the invention relates to the fields of medicine and virology. More in particular, it relates to means and methods for treating a viral disease caused by flaviviruses and alphaviruses, such as dengue and Chikungunya.
  • Dengue virus is a member of the family Flaviviridae which also includes West Nile virus, Zika virus, Yellow Fever virus, Japanese encephalitis (JE) virus, and the Tick-borne encephalitis (TBE) virus.
  • Flaviviruses are small enveloped viruses with a single positive strand RNA molecule. Flaviviruses are transmitted to humans and animals via bites with chronically infected mosquito or tick vectors. Flaviviruses contain three structural proteins capsid (C), membrane (M), and envelope (E). The E and M proteins are found on the surface of the virion where they are anchored in the membrane. Mature E is glycosylated, whereas M is not, although its precursor, prM, is a glycoprotein. The E glycoprotein, the largest structural protein, contains functional domains responsible for cell surface attachment, internalization and membrane fusion. The humoral response during infection is mainly directed towards the E and prM protein.
  • C capsid
  • M membrane
  • E envelope
  • Mature E is glycosylated
  • M is not, although its precursor, prM, is a glycoprotein.
  • the E glycoprotein the largest structural protein, contains functional domains responsible for cell surface attachment, internalization and membrane fusion. The humoral response during infection is mainly directed towards the E
  • Dengue virus is the causative agent of dengue fever and is transmitted to humans by Aedes mosquitoes, principally Aedes aegypti and Aedes albopictus. Annually, an estimated 390 million individuals are infected with dengue virus (DENV), of which 96 million individuals develop clinically apparent disease [1]. These staggering numbers make DENV the most common viral infection that is transmitted by arthropods worldwide. Clinical disease usually manifests as an acute self -limited illness with symptoms as high fever, severe headache, severe eye pain, muscle and/or bone pain and rash [2]. Approximately 0.5 to 1 million individuals, however, develop more extreme disease. Severe dengue is a potential fatal
  • Antiviral treatment is aimed at alleviating the viral titer , thereby decreasing the chance to develop severe disease [6]. Both direct- acting antivirals as well as host-directed antivirals have been pursued as potential candidates for dengue treatment.
  • WO2011/002635A1 discloses methods and pharmaceutical compositions for treating viral infections such as those caused by flavivirus e.g. Dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus, by administering certain 2-aryl- benzothiazole or 2-heteroaryl-benzothiazole derivative compounds.
  • flavivirus e.g. Dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus
  • ecdysones derived from Zoanthus spp. were found to inhibit DENV-2 replication in Huh7 cells and were predicted, by molecular docking studies, to associate with the NS5 polymerase of DENV [13].
  • carbenoxolone disodium was reported to reduce DENV infectivity due to direct virucidal activity of the compound [14].
  • coumarins were shown to be potent inhibitors of both DENV as well as Chikungunya virus (CHIKV) [15].
  • the compound tomatidine and analogs thereof have potent antiviral properties towards dengue virus (DENV) serotypes 1, 2, 3 and 4.
  • DEV dengue virus
  • the effective tomatidine concentration in which a 50 and 90% reduction of infectious virus particle production is observed was 0.82 and 1.61 ⁇ following infection of Huh 7 cells with
  • the invention relates to the use of tomatidine or a functional derivative thereof as antiviral compound, in particular against flaviviruses and alphaviruses.
  • Tomatine is a steroidal alkaloid that can be extracted from the skin and leaves of tomatoes. Unripe green tomatoes contain up to 500 mg tomatine per kg, whereas ripe red tomatoes have less than 5 mg/kg [17]. In nature, tomatine functions as an important defense mechanism for pathogens [18]. Tomatidine is an aglycon metabolite of tomatine and was shown to exert a wide array of beneficial biological activities hke anticancer, anti -inflammatory and improvement of the muscle health span by stimulating muscle hypertrophy [19-21]. See also WO2014/022772 disclosing the ability of tomatidine to promote skeletal muscle hypertrophy, increase muscle strength, increase exercise capacity, and decrease adiposity.
  • tomatine When consumed by animals, tomatine is hydrolyzed by stomach acid and intestinal bacteria to tomatidine, which is absorbed by the gut.
  • Tomatidine appears to have a favorable safety profile based on several studies: 1) human consumption of indigenous variants of tomatoes with very high concentrations of a-tomatine (up to 0.05% (w/w) of dry tomato weight) appears to cause no adverse effects [22,23]); 2) tomatine content is twice as high in organically grown tomatoes compared to conventionally grown tomatoes [22]); and 3) in pregnant and non-pregnant mice, dietary
  • tomatidine possesses anti-hyperlipidemic and anti- atherosclerotic effects without evidence of toxicity [25]).
  • tomatidine was found to potently reduce replication of pathogenic S. aureus variants typically seen in cystic fibrosis [26].
  • the invention provides tomatidine or analog thereof, for use in a method of treating a viral infection caused by a flavivirus or an alphavirus, in particular Dengue Virus or Chikungunya virus.
  • the invention also provides a method of treating a viral disease (viral infection) caused by a flavivirus or an alphavirus in an animal, comprising administering to the animal an effective amount of tomatidine or an analog thereof.
  • Flaviviruses are small, enveloped, positive-strand RNA viruses that are of concern in many medical and veterinary settings throughout the world. Flavivirus proteins are produced by translation of a single, long open reading frame to generate a polyprotein, which undergoes a complex series of post-translational proteolytic cleavages by a combination of host and viral proteases to generate mature viral proteins (Amberg et al., J. Virol. 73:8083- 8094, 1999; Rice, "Flaviviridae,” In Virology, Fields (ed.), Raven-Lippincott, New York, 1995, Volume I, p. 937[30]).
  • the structural proteins are arranged in the polyprotein in the order C-prM-E, where "C” is capsid, “prM” (or “pre- membrane”) is a precursor of the viral envelope-bound M (membrane) protein, and ⁇ " is the envelope protein.
  • C capsid
  • prM or "pre- membrane”
  • ⁇ is the envelope protein.
  • These proteins are present in the N-terminal region of the polyprotein, while the non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are located in the C- terminal region of the polyprotein and these are required for replication of the viral genome.
  • said flavivirus is Dengue virus (serotype 1-4), Yellow Fever Virus (YFV), Zika virus or West-Nile virus.
  • the flavivirus is Dengue virus, more preferably dengue virus serotype 1, 2, 3 or 4.
  • the invention provides tomatidine or an analog thereof, for use in a method of treating a viral infection caused by dengue virus serotype 1, 2, 3 or 4.
  • the invention provides tomatidine or an analog thereof, for use in a method of treating a viral infection caused by an alphavirus.
  • Alphaviruses belong to the Togaviridae family viruses.
  • Alphaviruses like flaviviruses, have a positive sensed, single-stranded RNA genome.
  • the alphavirus is Chikungunya virus (CHIKV). It was first isolated in 1953 in Africa and is an RNA virus with a positive-sense single-stranded genome of about 11.6kb. It is a member of the Semliki Forest virus complex and is closely related to Ross River virus,
  • O'nyong'nyong virus and Semliki Forest virus. Because it is transmitted by arthropods, namely mosquitoes, it can also be referred to as an arbovirus (arthropod-borne virus). In the United States, it is classified as a category C priority pathogen, and work requires biosafety level III precautions.
  • Chikungunya is a physically debilitating disease of humans mainly in Africa, Asia and the Americas.
  • the disease is caused by CHIKV, and is spread by Aedes spp. mosquitoes, principally Aedes aegypti and Aedes albopictus.
  • the symptoms include abrupt onset of high fever, rash or hemorrhages, arthralgia and occasional involvement of the nervous system, heart and liver.
  • the incapacitation is due to arthralgia, which can persist for years. Treatment is only palliative and there is no commercially available vaccine.
  • the presently disclosed antiviral activity of tomatidine or a tomatidine analog against CHIKV is therefore a major breakthrough in the management of Chikungunya.
  • the invention also provides a method of treating a disease caused by a flavivirus or an alphavirus in an animal, comprising administering to the animal an effective amount of tomatidine or an analog thereof.
  • a method of treating a disease caused by Dengue virus preferably dengue virus serotype 1, 2, 3 or 4 in an animal, comprising administering to the animal an effective amount of tomatidine or analog thereof.
  • a method of treating a disease caused by an alphavirus, preferably Chikungunya virus, in an animal comprising administering to the animal an effective amount of tomatidine or a analog thereof.
  • the invention also provides a method for inhibiting replication of Dengue virus or Chikungunya virus in a cell comprising administering tomatidine or analog thereof.
  • treating refers to obtaining a desired pharmacological and physiological effect.
  • the effect may be prophylactic in terms of preventing or partially preventing a viral disease, symptom, or condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom, or adverse effect attributed to the disease.
  • treatment covers any treatment of a viral disease caused by a flavivirus or alphavirus in mammal, such as a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it, i.e., causing the clinical symptoms of the disease not to develop in a subject that may be predisposed to the viral disease but does not yet experience or display symptoms of the disease; (b) inhibiting the disease, i.e., arresting or reducing the
  • the subject is a mammal such as a primate, and, in a preferred aspect, the subject is a human.
  • administering and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not hmited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, subhngual
  • administration including injectable such as intravenous administration, intra- arterial administration, intramuscular administration, and subcutaneous
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a viral disease or condition.
  • the tomatidine or tomatidine analog may be administered in any order
  • compositions may include any of a variety of standard pharmaceutically accepted carriers employed by those of ordinary skill in the art. Examples include saline, phosphate buffered saline (PBS), water, aqueous ethanol, emulsions, such as oil/water emulsions or triglyceride emulsions, tablets and capsules.
  • PBS phosphate buffered saline
  • emulsions such as oil/water emulsions or triglyceride emulsions, tablets and capsules.
  • suitable physiologically acceptable carrier will vary dependent upon the chosen mode of administration.
  • the method of the present invention may also comprise co-administration of: a) other antivirals such as Ribavirin or cidofovir; b) vaccines; and/or c) interferons or pegylated interferons.
  • a pharmaceutical composition comprising tomatidine or analog thereof and at least one further antiviral agent.
  • the pharmaceutical composition comprises tomatidine, solasodine or sarsasapogenin. and comprising at least one further antiviral agent.
  • the at least one further antiviral agent is suitably selected from the group consisting of coumarins, ribavirin, cidofovir, anti-flavi virus vaccines, anti- alphavirus vaccines and (pegylated) interferons.
  • derivatives refers to a compound having a structure derived from the structure of the tomatidine parent compound and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds.
  • exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound as disclosed e.g. WO2014/022772.
  • the tomatidine derivative or analog has a structure
  • R-> is selected from H. C1-C6 alkyl, COR 53 , C1-C6 alkylamino, C 1-C6 dialkylamino, C6-C 10 aryl. C3-C 10 cvcloalkyl. C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cvcloalkyl, C5-C heteroaryl.
  • C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, Cl- C6 alkylamino, and C1-C6 dialkylamino;
  • R 53 ⁇ 4 is selected from C 1-C6 alkyl, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, CI- C6 alkylamino, and C 1-C6 dialkylamino;
  • ⁇ ⁇ 1 is selected from O, S, and NR 54 ; preferably Z 51 is O or wherein R 3 ⁇ 4 is selected from H, C 1-C6 alkyl, COR ⁇ s, C 1-C6 alkylamino, C 1-C6 dialkylamino, C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, Cl- C6 alkylamino, and C1-C6 dialkylamino;
  • R 55 is selected from C 1-C6 alkyl, C 1-C6 monohaloalkyl,
  • C 1-C6 polyhaloalkyl, C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl wherein C6-C 10 aryl, C3-C 10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, CI- C6 alkylamino, and C 1-C6 dialkylamino; or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.
  • R 51 is selected from H, C 1-C6 alkyl and COR 53 , wherein R 53 is CI- C6 alkyl.
  • R 51 is H.
  • Z 51 is NR 5 ' 1 .
  • Z 51 is NR 54 , wherein R 54 is selected from H, C 1-C6 alkyl, and COR ⁇ , wherein R35 i s C 1-C6 alkyl.
  • R 51 is selected from H, C1-C6 alkyl and COR 53 , wherein R r !
  • R 51 and R 54 are identical.
  • the structure is represented by the formula
  • R 51 is selected from H. C1-C6 alkyl, COR 53 , C1-C6 alkylamino, Cl- C6 dialkylamino.
  • C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2- C9 heterocyclyl wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl.
  • C1-C6 polyhaloalkyl C1-C6
  • alkylamino and C1-C6 dialkylamino
  • R 53 is selected from C1-C6 alkyl, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl.
  • C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy.
  • R 54 is selected from H, C1-C6 alkyl, COR 55 , C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl. C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, C l- C6 alkylamino, and C1-C6 dialkylamino;
  • R 55 is selected from C 1-C6 alkyl, C 1-C6 monohaloalkyl.
  • C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C 1-C6 alkyl, C 1-C6 alkoxy, C 1-C6 monohaloalkyl, C 1-C6 polyhaloalkyl, C 1-C6 alkylamino, and C 1-C6 dialkylamino; or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.
  • the structure is represented by the formula:
  • the formula has the structure:
  • tomatidine 25 ⁇ S -226N-5a-spirolane.
  • solasodine ((25i?)-22aN-spiroal-5-ene).
  • Solasodine is a main active component isolated from Solatium incanum L. that has been reported to perform a wide range of functions containing anti-oxidant, anti -infection, and neurogenesis promotion. Furthermore, Zhuang et al. (Cancer Sci. 2017 Nov; 108(11): 2248-2264) showed that solasodine prohibited human colorectal cancer cell proliferation dose- and time-dependently, suggesting that solasodine may be a therapeutic drug for CRC treatment.
  • the tomatidine analog has a structure represented by the formula
  • the invention provides a method for treating a viral infection caused by Dengue Virus or Chikungunya virus in a subject, comprising administering to the subject an effective amount of tomatidine, solasodine or sarsasapogenin.
  • the invention provides a method for treating a viral infection caused by Dengue Virus, comprising
  • the invention provides a method for treating a viral infection caused by Chikungunya Virus, comprising administering to the subject an effective amount of tomatidine, solasodine or sarsasapogenin.
  • the terms "effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition.
  • a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The dose will be adjusted to the individual requirements in each particular case.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration.
  • compositions can contain such amounts or submultiples thereof to make up the daily dose.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • a preparation can be administered in a "prophylactically effective amount"; that is, an amount effective for prevention of a disease or condition.
  • a daily dosage of between about 0.01 and about 1000 mg/kg body weight per day should be appropriate in
  • a preferred daily dosage is between about 0.1 and about 500 mg/kg body weight, more preferred 0.1 and about 100 mg/kg body weight and most preferred 1.0 and about 10 mg/kg body weight per day. Thus, for administration to a 70 kg person, the dosage range would be about 7 mg to 0.7 g per day.
  • the daily dosage can be administered as a single dosage or in divided dosages, typically between 1 and 5 dosages per day. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect for the individual patient is reached.
  • One of ordinary skill in treating diseases described herein will be able, without undue experimentation and in reliance on personal knowledge, experience and the disclosures of this application, to ascertain a therapeutically effective amount of the
  • Tomatidine might act directly on the viral proteins or indirectly by controlling the expression of a cellular factor that is important in the late stages of viral replication.
  • a use or method of the invention advantageously allows to treat subjects that have acquired or are suspected of having acquired the viral disease.
  • the disclosed compounds i.e. tomatidine or analog thereof
  • the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 5 mg per day in a human.
  • the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 10 mg per day in a human.
  • the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 25 mg per day in a human.
  • the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 50 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 75 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 100 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 150 mg per day in a human. In a further aspect, the disclosed have antiviral activity when administered at an oral dose of greater than about 200 mg per day in a human.
  • the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 250 mg per day in a human. In a yet further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 300 mg per day in a human. In a still further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 400 mg per day in a human. In an even further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 500 mg per day in a human. In a further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 750 mg per day in a human.
  • the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 1000 mg per day in a human. In a still further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 1500 mg per day in a human. In an even further aspect, the disclosed compounds have antiviral activity when administered at an oral dose of greater than about 2000 mg per day in a human.
  • FIG. 1 Tomatidine reduces the production of infectious DENV particles.
  • Huh7 cells were infected with DENV serotype 2 at MOI 1 and 10. Simultaneously with the infection, cells were treated with 10 ⁇ of tomatidine, the equivalent volume of EtOH or left un-treated (NT).
  • NT left un-treated
  • Dose-response curve showing the inhibition of DENV infection at increasing concentrations of tomatidine in relation to the equivalent EtOH-treated control. EC50 values were calculated with GraphPacl Prism software.
  • C 1 ⁇ 10 ⁇ PFU of DENV was incubated for 2 h at room temperature RT or 37 °C with 10 ⁇ of tomatidine. The infectivity was determined by plaque assay on BHK-15 cells. Data is presented as mean ⁇ SEM from three independent experiments.
  • FIG. 1 Tomatidine decreases the percentage of DENV-infected Huh7 cells.
  • Huh7 cells were infected with DENV serotype 2 at MOI 1 and 10 in the presence of 1 or 10 ⁇ tomatidine as indicated. At 2 hpi, DENV inoculum was removed and incubation was continued in the presence of the compound until harvesting of cells at 24 hpi. As control, cells were infected with DENV in presence of an equal volume of EtOH.
  • A Huh7 cells were infected with DENV serotype 2 at MOI 1 and 10 in the presence of 1 or 10 ⁇ tomatidine as indicated. At 2 hpi, DENV inoculum was removed and incubation was continued in the presence of the compound until harvesting of cells at 24 hpi. As control, cells were infected with DENV in presence of an equal volume of EtOH.
  • FIG. 3 Tomatidine reduces DENV infectivity when added up to 12 hpi.
  • A Outhne of the experimental set-up.
  • B Infectious virus particle production following the conditions presented in (A). The EtOH control was added to all experimental conditions and the average titer is depicted. For the tomatidine samples, data is presented as mean ⁇ SEM from three independent experiments.
  • FIG. 4 Tomatidine reduces the number of secreted DENV genome-equivalent copies.
  • Huh7 cells were infected with DENV serotype 2 at MOI 1 and treated with 10 ⁇ tomatidine at 12 hpi.
  • the number of DENV genome-equivalent copies per ml was determined in the cell culture supernatant at 24 hpi. Data is presented as mean ⁇ SEM from three independent experiments.
  • FIG. 6 Dose-response curve showing the inhibition of DENV-1, DENV-3 and DENV-4 infection at increasing concentrations of tomatidine in relation to the equivalent EtOH-treated control.
  • Huh7 cells were infected with DENV serotype 1 (MOI 1), serotype 3 (MOI 0.5) and serotype 4 (MOI 1).
  • the supernatants were harvested at 24 hpi for DENV-1 and DENV -3 and at 30 hpi for DENV-4.
  • the number of infectious virus particles was determined by an immunofocus assay on HK-15 cells.
  • EC50 values were calculated with GraphPad Prism software. Data is presented as mean ⁇ SEM from three independent experiments.
  • FIG. 7 Tomatidine reduces the number of secreted genome- equivalent copies of Chikungunya (CHIKV) virus.
  • Huh7 cells were infected with CHIKV (MOI 1) and treated with 10 ⁇ tomatidine.
  • the number of CHIKV genome-equivalent copies per ml (GEC/ml) was determined in the cell culture supernatant at 24 hpi. Data is presented as mean ⁇ SEM from three independent experiments. "NT" denotes non- treated and EtOH served as an ethanol solvent control.
  • FIG. 8 Anti-viral activity of tomatidine analogs.
  • Huh7 cells were infected with (panel A) DENV serotype 2 or (panel B) CHIKV at MOI 1.
  • Test compound was added to the cells at the time-point of infection at the highest non-toxic dose.
  • the equivalent concentration of the solvent (96% w/v ethanol; end concentration ⁇ 0.05%) was added to the cells at the time point of infection.
  • the supernatants were harvested at (panel A) 24 hpi or (panel B) 9 hpi.
  • the number of produced DENV genome- equivalent copies were determined by Q-RT-PCR.
  • panel B the number of produced infectious CHIKV particles was determined by plaque assay on BHK -15 cells.
  • Baby hamster kidney-21 cells clone 15 was a kind gift from Richard Kuhn (Purdue University). BHK-15 cells were grown in Dulbecco's minimal essential medium (DMEM) (Gibco, the Netherlands) supplemented with 10% fetal bovine serum (FBS) (Lonza, Basel,
  • DMEM Dulbecco's minimal essential medium
  • FBS fetal bovine serum
  • Human hepatocarcinoma (Huh 7) cells (JCRB0403) were a kind gift from Tonya Colpitts (University of South Carolina) and cultured in DMEM/Glutamax supplemented with 10% FBS, lOOU/mL penicillin and lOOmg/mL streptomycin.
  • Vero WHO cells (WHO Reference Cell Bank 10-87) were grown in DMEM supplemented with 10% FBS, lOOU/mL penicillin and lOOmg/mL streptomycin.
  • Aedes albopictus C6/36 cells (ATCC: CRL-1660) were maintained in minimal essential medium (Invitrogen, Carlsbad, California, USA) supplemented with 10%> FBS, 25 mM HEPES, 7.5% sodium bicarbonate, lOOU/mL penicillin and lOOmg/mL streptomycin, 200 mM glutamine, and 100 ⁇ nonessential amino acids. All mammalian cells were cultured at 37°C and 5% CO2 and C6/36 cells were cultured at 28°C and 5% CO 2 . Virus stocks and titration.
  • DENV serotype 2 strain 16681, DENV serotype 1 strain 16007, DENV serotype 3 strain H87 and DENV serotype 4 strain 1036 were propagated on C6/36 cells as described before [28].
  • CHIKV strain OPY LR2006 was propagated on Vero cells. The number of infectious particles was determined by plaque assay on BHK-15 cells[31]. For plaque assays, BHK-15 cells were seeded in 12-well plates at a cell density of 9.0xl0 cells per well. At 24 h post-seeding, cells were infected with 10-fold serial dilutions of the sample.
  • hpi At 2 h post-infection (hpi), an overlay of 1% seaplaque agarose (Lonza, Swiss) prepared in MEM was added and plaques were counted 2 (for CHIKV) and 5 (for DENV) days post-infection. Titers are reported as plaque forming units (PFU) per ml. The number of genome equivalent copies (GEC) in a solution was determined by Q-RT-PCR as described previously [31]. Briefly, viral RNA was extracted using a QIAamp viral RNA mini kit (QIAGEN, Venlo, The Netherlands) following
  • cDNA was synthesized from viral RNA using Omniscript (QIAGEN) and the primers and probes (Eurogentec, Maastricht, The Netherlands) are listed in Table 1.
  • DENV-1 pcDNA3 encoding the M protein sequence of DENV- 1 strain 16007
  • DENV-2 pSINDENCprME encoding the CprME sequence of DENV- 2 strain 16681
  • DENV-4 pcDNA3 encoding the E protein sequence of DENV-4 strain 1036.
  • GGCGAACA-3' AGAGG AC AT- 3' GGCCCCACA-TAMRA-3 '
  • CHIKV La Reunion 2006 OPY-strain was propagated on Vero -WHO cells, as described before (Richter et al. 2015. J. Gen. Virol., 96: 2122-2132 [32]). Plaque assay was performed to determine the infectious titer and Q-RT-PCR was performed to determine the number of genome equivalent copies (GEC).
  • Q-RT-PCR viral cDNA was synthesized by reverse transcriptase (RT) PCR using the forward primer 5'-AGCTCCGCGTCCTTTACCA-3' and the reverse primer 5'-GCCAAATTGTCCTGGTCTTCCT-3'.
  • RT reverse transcriptase
  • TaqMan probe 5'-FAM-CAC TGTAACTGCCTATGCAAACGGCGAC-
  • TAMRA-3' was added.
  • a CHIKV plasmid containing the E l sequences pCHIKV-LS3 IB was used.
  • tomatidine Cytotoxicity of tomatidine was assessed in vitro by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazohum bromide (MTT) assay.
  • Huh7 cells were seeded in 96-well plates at a density of 1.0x10 4 and 7.0xl0 3 cells per well, respectively. At 24 h post-seeding, cells were treated with
  • MTT was added at a final concentration of 0.45mg/ml and incubated for 3 h.
  • Huh7 cells were infected with the indicated viruses
  • multiplicity of infection (MOI) of 1 or 10.
  • Tomatidine tomatidine analogor the equivalent volume of EtOH, was added at different stages of infection. In most experiments, increasing concentrations of tomatidine (analog) was added together with the virus to the cells. At 2 hpi, the virus inoculum was removed, cells were washed three times and drug-containing medium was added for the duration of the experiment. In case of pre-treatment experiments, tomatidine (analog) was added 1 or 2 h prior to infection. At the time of infection, cells were washed three times before the virus inoculum was added. The condition "during” relates to the presence tomatidine during the infection for 2 h. Also, tomatidine was added 2, 4, 6, 12, 16, 20 hpi.
  • virus inoculum was removed at 2 hpi, cells were washed three times and incubation was continued. At 24 hpi, cell supernatants were harvested and the titer was determined by plaque assay or Q-RT-PCR.
  • DENV serotype 2 (lxlO 5 PFU) was incubated for 2 h at room temperature or 37°C in the absence or presence of 10 ⁇ tomatidine in a final volume of 250 ⁇ . Upon incubation, the infectious titer was determined by plaque assay.
  • Huh7 cells were trypsinized using IX Trypsin/EDTA (Gibco). Cells were fixed with 2% paraformaldehyde and permeabilized with 0.5% saponin. Staining was performed with 4G2 antibody and a rabbit anti- mouse IgG coupled to AF647 (Molecular probes, Eugene, Oregon, USA). Flow cytometry was carried out in a FACSCalibur cytometer (BD
  • the tomatidine concentration at which 50 and 90% reduction in virus particle production is seen is referred to as EC50 and EC90,
  • EC50 and EC90 concentration of tomatidine that caused 50 and 90% cellular cytotoxicity
  • concentration of tomatidine that caused 50 and 90% cellular cytotoxicity is referred to as EC50 and EC90, respectively.
  • Dose- response curves were fitted by non-linear regression analysis employing a sigmoidal model.
  • the selectivity index (SI) was determined by the ratio of CC50 to EC50. All data were analyzed in GraphPad Prism software (La Jolla, CA, USA). Data is presented as mean ⁇ SEM. Student T test was used to evaluate statistical differences and a p value ⁇ 0.05 was considered significant with *p ⁇ 0.05 , **p ⁇ 0.01 and ***p ⁇ 0.001.
  • EXAMPLE 1 Tomatidine inhibits the production of progeny infectious virus particles.
  • hepatocytes are important target cells during DENV infection. Furthermore, this cell line is permissive to DENV infection. Indeed, at 24 hpi, on average 8.3xl0 4 progeny infectious particles per ml are produced following infection at MOI 1.
  • Fig. 1A shows that tomatidine has potent antiviral activity towards DENV serotype 2. At a concentration of 10 ⁇ tomatidine, infectious virus particle production was reduced 2.02 log when compared to DENV-infected cells treated with equivalent volumes of EtOH. The final concentration of EtOH was below 0.01% and had no effect on virus particle production when compared to non-treated cells. The EC50 and EC90 values are 0.82 and 1.61 ⁇ for MOI 1, respectively.
  • EXAMPLE 2 Tomatidine decreases the number of infected cells.
  • EXAMPLE 3 Tomatidine has a potent antiviral effect when added post-infection.
  • Fig. 4 shows that the number of GEC is reduced by 1 Log (90.2%) when compared to EtOH-treated control cells.
  • the number of secreted infectious particles (Fig. 3B) and GEC (Fig.4) is correspondingly reduced, thereby confirming that tomatidine acts at a step prior to virion secretion.
  • EXAMPLE 4 Antiviral activity of tomatidine is independent of the DENV serotype.
  • hepatocarcinoma human hepatocarcinoma (Huh 7) cells. This cell line was chosen since hepatocytes are important target cells during DENV infection. Furthermore, this cell line is permissive to DENV infection. Tomatidine was added at a concentration of 10 ⁇ and remained present for the duration of the experiment (24 hr). For DENV serotype 1, cells were infected at MOI 1 and for DENV serotype 4 MOI 0.1 was used. Fig. 5 shows that tomatidine exerts antiviral activity towards DENV serotype 1 and 4. At a concentration of 10 ⁇ tomatidine more than 75% reduction in GEC production is seen.
  • Figure 6 shows a dose-response curve showing the inhibition of DENV- 1, DENV-3 and DENV-4 infection at increasing concentrations of tomatidine.
  • EXAMPLE 5 Tomatidine also has antiviral activity against alphavirus.
  • tomatidine The effect of tomatidine on CHIKV was determined in human hepatocarcinoma (Huh 7) cells. Tomatidine was added at a concentration of 10 ⁇ and remained present for the duration of the experiment (16 hr). Fig. 7 shows that tomatidine exerts potent antiviral activity towards CHIKV- LR. At a concentration of 10 ⁇ tomatidine, more than 99% reduction in GEC production is seen.
  • EXAMPLE 6 Anti-viral activity of tomatidine analogs.
  • Solasodine and Sarsasapogenin are two exemplary tomatidine analogs showing a high structural similarity to tomatidine.
  • solasodine and sarsasapogenin were tested for their antiviral activity against different viruses.
  • Huh7 cells were infected with DENV serotype 2 strain 16681 at MOI 1. Test compound was added to the cells at the time-point of infection at the highest non-toxic dose. Tomatidine 10 ⁇ ; Solasodine 5 ⁇ ; Sarsasapogenin 20 ⁇ . In control conditions, the equivalent concentration of the solvent (96% w/v ethanol; end concentration ⁇ 0.05%) was added to the cells at the time point of infection.
  • the equivalent concentration of the solvent (96% w/v ethanol; end concentration ⁇ 0.05%) was added to the cells at the time point of infection.
  • Huh7 cells were infected with Chikungunya virus OPY LR2006 at MOI 1.
  • Test compound was added to the cells at the time-point of infection at the highest non-toxic dose.
  • the supernatants were harvested at 9 hpi and the number of produced infectious particles was determined by plaque assay on BHK -15 cells. Q-RT-PCR.
  • solasodine showed a strong antiviral activity to dengue virus, whereas for sarsasapogenin a negative trend in virus particle production was observed.

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Abstract

L'invention concerne les domaines de la médecine et de la virologie, plus particulièrement des moyens et des méthodes de traitement d'une maladie virale provoquée par des flavivirus ou des alphavirus. L'invention concerne la tomatidine ou un analogue de celle-ci pour une utilisation dans une méthode de traitement d'une infection virale provoquée par un flavivirus ou un alphavirus, tel que le virus de la dengue ou le virus du Chikungunya. L'invention concerne également une composition pharmaceutique comprenant de la tomatidine ou un analogue de celle-ci, et au moins un autre agent antiviral.
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CN114712370A (zh) * 2022-05-07 2022-07-08 中国农业科学院麻类研究所 一种番茄碱在制备抗以色列急性麻痹病毒药物中的应用

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