Methylene blue for treatment of hepatitis b and / or hepatitis d infections

Abstract

The present invention relates to the use of methylene blue in the treatment of Hepatitis B (HBV) and / or Hepatitis D (HDV) infection in human patients and pharmaceutical compositions suitable for such treatment.

Description

TECHNICAL FIELD

[0001] The present invention relates to the use of methylene blue in the treatment of hepatitis B virus (HBV) and / or hepatitis D virus (HDV) infections in human patients and to pharmaceutical compositions suitable for such treatment. BACKGROUND OF THE INVENTION

[0002] Hepatitis B virus (HBV) and hepatitis D virus (HDV), a satellite virus of HBV, are two of the five hepatitis viruses that infect humans and have a significant impact on morbidity and mortality worldwide.

[0003] There are highly effective prophylactic HBV vaccines, but known antiviral drugs against HBV and HDV need significant improvement.

[0004] Methylene blue (MB) is the first synthetic molecule used in medicine, originally synthesized by Heinrich Caro in 1876. Since then, the molecule has had various applications in pharmaceuticals, particularly in the fields of antimalarial drugs, drugs for the treatment of methemoglobinemia, or as a drug for treating ifosfamide toxicity. MB drugs approved by the FDA and EMA have been widely used in clinical medicine for other applications, with few side effects and can be produced inexpensively. In the field of virology, photoactivated MB has been shown to clear various infectious virus particles from in vitro blood sources:

[0005] Wu W. et al., 2014, “Method of inactivating virus in circular blood and its applications in treating viral diseases”, U.S. Patent 8,808,977, describes an in vitro procedure of adding MB to blood. The blood is then recirculated under a light source with the help of a pump until the virus is completely neutralized. Before blood transfusion, a removal device is used to adsorb the photosensitizer.

[0006] Floyd R. et al., “Thiazine dyes used to inactivate HIV in biological fluids”, U.S. Patent 5,827,644, describes an in vitro method of using MB and light to treat biological fluids against the human immunodeficiency virus.

[0007] Zepp Ch. et al., "Method for inactivating non-enveloped viruses using aviricide-potentiating agent with a photoactivatable virucide", U.S. Patent 5,663,043, describes a method for inactivating non-enveloped viruses in blood with a photoactivatable virucide and a method for administering an aviricide-potentiating chemical agent such as a cationic lipid polyamine.

[0008] Swartz M.R., "Method for inactivating viruses, bacteria, etc. in vitro and production of vaccines", U.S. Patent 4,402,318. Discloses a method for inactivating infectious agents in vitro by applying an electric field and light simultaneously to activate MB.

[0009] Cerny E.H. et al., "Methylenblau und Riboflavin zur prophylaktischen und therapeutischen antiviralen Therapie", (MB and riboflavin for prophylactic and therapeutic antiviral therapy), Swiss Patent CH717522A2. Cerny, EH et al. have demonstrated that an extended incubation time corresponding to in vivo application and mammalian body temperature provide sufficient energy for the virucidal treatment of MB against coronavirus or influenza virus infection at physiological concentrations. The unusually long incubation time of in vivo application (i.e., days instead of minutes) compared to in vitro virus titration makes this possible. Strong antiviral efficacy against influenza N1H1 virus and SARS-CoV-2 was demonstrated in vitro in a completely dark environment (sealed box). This provides the possibility of using MB for in vivo treatment of influenza A virus and coronavirus infection.

[0010] The prior art does not show the antiviral efficacy of MB against HBV and HDV. The fact that HDV infection must be accompanied by HBV infection poses a challenge for the development of antiviral drugs because highly effective antiviral drugs against both viruses are needed.

[0011] There are drugs (nucleotide analogs, such as entecavir) that effectively inhibit HBV replication, but have no therapeutic effect on HDV replication. Another complication is that the two viruses are completely different: HBV belongs to the family Hepadnavirus, has an outer lipid envelope and a nucleocapsid, and its nucleocapsid contains viral DNA and a DNA polymerase with reverse transcriptase activity. On the other hand, HDV belongs to the order Ribozyviria and is an extremely small negative-sense single-stranded RNA virus with high polymorphism. The HDV virion is a spherical particle with a viral envelope containing host phospholipids and three proteins derived from HBV. The envelope contains an internal ribonucleoprotein (RNP) particle, and the genome is encapsulated by hepatitis delta antigen (HDAg). HDV has no druggable enzymes, but its ribozyme activity is a significant exception.

[0012] Therefore, there is a need for a novel therapeutic approach that can treat HBV and / or HDV infections in human patients. Summary of the Invention

[0013] The above problems are surprisingly solved by providing a methylene blue-based therapy for HBV and / or HDV infection as further defined hereinbelow.

[0014] This study of MB as an antiviral agent against HBV and HDV began with a speculative line of reasoning that the energy required for oxygen to transfer from its triplet ground state to its singlet excited state is relatively low (96 kJ / mol), and is sufficient to generate a significant antiviral level of singlet oxygen under in vivo treatment conditions (i.e., normal body temperature and extended treatment times).

[0015] In addition, in vitro testing of MB on HDV-infected cell cultures has been initiated, with the expectation of observing a further enhancement of antiviral activity against HDV. This is because HDV has an unusually high percentage of guanine nucleobases (70%) in its genome: the step that is very important or most important for the antiviral activity of MB is the oxidation of guanine to the 8-oxo-7,8-dihydroguanine form, which occurs due to the generation of singlet oxygen by MB in the presence of oxygen.

[0016] During in vitro testing of MB on cell cultures infected with HBV envelope proteins and HDV, the inventors surprisingly found that after treating the infected cells in the cell cultures with MB, not only was there antiviral activity against HDV, but also the secretion of hepatitis B surface antigen (HBsAg) decreased. The HDV RNA level measured by reverse transcription polymerase chain reaction (RT-PCR) decreased in a dose-dependent manner, thus demonstrating the efficacy of MB against HDV and even more surprisingly against HBV at physiological concentrations ( Figure 3 and 4 ).

[0017] Although there are effective prophylactic vaccines against HBV, the present invention for the first time teaches that MB is a broad-acting, substantially nucleic acid sequence-independent antiviral compound for therapeutic applications against HBV and / or HDV. This is an important advantage because both viruses, especially HDV, have significant mutational potential.

[0018] MB has been shown to have no known serious side effects even at very high doses and is a drug approved by the FDA and European medical institutions for applications outside virology. MB stains urine and, after long-term application, the sclera and skin in patients with white skin are also slightly blue, but the effect is reversible after treatment interruption.

[0019] In view of the above, MB will have a powerful non-viral sequence-specific broad antiviral activity against HBV and / or HDV as described in more detail below.

[0020] Depending on the concentration and reaction partner, MB can reduce or oxidize compounds. MB is capable of accepting electrons on its aromatic thiazine ring to be reduced to colorless methylene blue (MBH2) and transferring electrons to other compounds according to the redox state and concentration of MB. Singlet oxygen is in a quantum state in which all electrons are spin-paired and corresponds to the lowest excited state of the diatomic oxygen molecule. The combination of MB as a sensitizer with oxygen and an energy source results in the generation of singlet oxygen, which is a highly reactive reaction partner that destroys DNA, RNA, or protein moieties through mechanisms such as guanine oxidation, thus having broad non-sequence-specific antiviral activity. Known antiviral molecular modifications include but are not limited to a) 8-oxo-7,8-dihydroguanine (8-oxoguanine (8-oxoGua)) damage, b) modified carbonyl moieties on proteins, c) single-strand breaks (ssb) in the RNA genome, d) RNA-protein crosslinks. All of the cited damages have been shown in the literature to be well correlated with antiviral activity or activity against bacteriophages (Schneider, J.E., Jr., et al., Potential mechanisms of photodynamic inactivation of virus by methylene blue. I. RNA-protein crosslinks and other oxidative lesions in Q beta bacteriophage. Photochem Photobiol, 1998. 67(3): p. 350-7).

[0021] As described in more detail below Figure 2 、 3Figures 4 and 5 show significant dose-dependent antiviral activity in cell cultures infected with HBV and HDV after treatment with physiological concentrations of methylene blue (MB). MB induces the production of singlet oxygen, which acts as an antiviral compound by attacking RNA and DNA nucleotides as well as protein moieties such as carbonyl groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows Figure 3 and Figure 4 the timeline of the experimental setup.

[0023] Figure 2 : Cytotoxicity of MB against HepG2-NTCP (A) and HepNB2.7 (B) as evaluated by MTT. Cells were treated with decreasing concentrations of MB maintained in the dark or photoactivated under visible light.

[0024] Figure 3 : Inhibitory effect of MB on HDV replication in HepG2-NTCP cells. Cells infected with 10 or 50 MOI were treated with MB (2.5 or 1.25 μg / ml), and HDV replication was evaluated by quantitative RT-PCR 3 days (A) or 6 days (B) post-infection.

[0025] Figure 4 : Inhibitory effect of MB on HDV replication in HepNB2.7 cells. Cells infected with 10 or 50 MOI were treated with MB (2.5 or 1.25 μg / ml), and HDV replication was evaluated by quantitative RC PCR 3 days (A) or 6 days (B) post-infection. (C) Secretion of hepatitis B surface antigen (HBsAg) was measured in the supernatants of cells infected with 10, 50, or 100 MOI and treated with MB (2.5 or 1.25 μg / ml). DETAILED DESCRIPTION

[0026] (1) Materials, Terms, Definitions

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of any conflict, the definitions provided in this application shall prevail. When a trade name appears herein, it refers to its corresponding commercial product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference.

[0028] Methylene blue:

[0029] “Methylene blue” (MB) refers to the FDA-approved drug (NDA 204630).

[0030] Compound CID: 6099, MF: C16H18ClN3S, MW: 319.9 g / mol, InChIKey: CXKWCBBOMKCUKX-UHFFFAOYSA-M, IUPAC name: [7-(dimethylamino)phenothiazin-3-ylidene]-dimethylammonium chloride. Absorption maxima: 668, 609 nm (PubChem database) or its salts or derivatives.

[0031] "Methylene blue cation":

[0032] 3,7-bis(dimethylamino)phenothiazine-5- Methylene blue Compound CID: 4139, MF: C16H18N3S+ MW: 284.4 g / mol; InChIKey: RBTBFTRPCNLSDE-UHFFFAOYSA-N, IUPAC name: [7-(dimethylamino)phenothiazin-3-ylidene]-dimethyl

[0033] MB can also exist in the form of its hydrates, which are also covered by the present invention. MB is a redox dye, which means that depending on the concentration and reaction partner, it can reduce or oxidize compounds. More precisely, it is able to accept electrons on its aromatic thiazine ring and be reduced to colorless methylene blue (MBH2), and transfer electrons to other compounds according to the redox state and concentration of MB.

[0034] "Singlet oxygen" is oxygen in a quantum state in which all electrons are spin-paired in the lowest excited state corresponding to the diatomic oxygen molecule. The combination of a sensitizer (such as methylene blue or riboflavin) with oxygen and an energy source results in the generation of singlet oxygen, a highly reactive reaction partner that disrupts DNA or RNA by mechanisms such as guanine oxidation, thus having broad non-sequence-specific antiviral activity: Chemical damage (induced by singlet oxygen) observed in the Qβ phage system includes: a) 8-oxo-7,8-dihydroguanine (8-oxoguanine) damage, b) modified carbonyl moieties on proteins, c) single-strand breaks (ssb) in the RNA genome, d) RNA-protein crosslinks. (Schneider, J.E., Jr., et al., Potential mechanisms of photodynamic inactivation of virus by methylene blue. I. RNA-protein crosslinks and other oxidative lesions in Q beta bacteriophage. Photochem Photobiol, 1998. 67(3): p. 350-7).

[0035] Other pharmacological effects of MB found in the literature may also contribute to its antiviral efficacy or have clinically beneficial effects:

[0036] - Distributive (hypovolemic) shock: MB produces vasoconstriction in distributive shock by inhibiting nitric oxide synthase and guanylate cyclase. This is an accompanying and unexpected beneficial effect of MB, as end-stage viral infections often present a clinical state of distributive shock. (Porizka, M., et al., Methylene blue administration in patients with refractory distributive shock - a retrospective study. Sci Rep, 2020. 10(1): p. 1828, Jang, D.H., L.S. Nelson, and R.S. Hoffman, Methylene blue for distributive shock: a potential new use of an old antidote. J Med Toxicol, 2013. 9(3): p. 242-9)

[0037] - Alzheimer's disease: MB oxidizes the cysteine thiol groups on tau protein to maintain tau monomers. A preclinical treatment study in a tauopathy mouse reported an anti-inflammatory neuroprotective effect mediated by Nrf2 / antioxidant response element (ARE); another reported a reduction in insoluble tau and learning and memory benefits when administered early.

[0038] - Methemoglobinemia: MB acts by reacting within red blood cells to form colorless methylene blue, which is a reducing agent for oxidized hemoglobin, converting the iron ion (Fe+++) back to its oxygen-carrying ferrous state (Fe++).

[0039] - As an antimalarial: MB, a specific inhibitor of Plasmodium falciparum glutathione reductase, has the potential to reverse CQ (chloroquine) resistance, and it resembles 4-amino-quinoline antimalarials in preventing the polymerization of heme into hemozoin.

[0040] - Ifosfamide-induced neurotoxicity: MB serves as an alternative electron acceptor. It acts to reverse the NADH inhibition caused by gluconeogenesis in the liver while blocking the conversion of chloroethylamine to chloroacetaldehyde. Additionally, it inhibits the activity of multiple amine oxidases, which also prevents the formation of chloroacetaldehyde.

[0041] Epidemic, pandemic:

[0042] The situation where a certain disease spreads rapidly to a large number of people in a specific population within a short period is an epidemic. Covering multiple countries or more is called a pandemic. HBV and HDV can occur in clusters and usually do not show an epidemic evolution.

[0043] Hepatitis B virus (HBV), Hepatitis D virus (HDV):

[0044] A liver-specific bile acid transporter called sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the cellular receptor for HBV and its satellite virus, Hepatitis D virus (HDV).

[0045] HBV is a partially double-stranded DNA virus belonging to the genus Orthohepadnavirus and is part of the family Hepadnaviridae. HDV contains different types of negative-sense single-stranded, closed-loop RNA viruses, which are collectively classified as the genus Deltavirus and belong to the realm Ribozyviria.

[0046] The classification of the serotypes and genotypes of HBV and HDV should not play a major role in the therapeutic response to MB because its mechanism of action is largely independent of the nucleic acid sequence.

[0047] Acute infection with HBV causes acute viral hepatitis, and the chronic form of HBV infection can lead to cirrhosis, hepatocellular carcinoma and increase the risk of related pathologies. Coinfection with HBV and HDV increases disease pathogenicity and reduces survival.

[0048] MOI (multiplicity of infection) describes the number of viral particles that can infect each cell in a tissue culture vessel.

[0049] Vaccines against the HBV surface antigen (HBsAg) are excellent prophylactic vaccines. Pegylated alpha interferon (PEG-IFN-α), entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are currently first-line agents used to treat hepatitis B disease.

[0050] Treatment of chronic hepatitis D includes conventional or pegylated alpha interferon therapy, and the benefits usually cease if the drug is discontinued. The antiviral Hepcludex (bulevirtide), which binds to and inhibits NTCP, has recently been approved for the treatment of hepatitis D and B.

[0051] The human hepatocellular carcinoma cell line HepG2 expressing NTCP provides a valuable tool for studying the basic biology of the virus and developing treatments for HBV and HDV infections.

[0052] Cell lines for studying HBV infection are described in R. Yan, Y. Zhang et al. “Spinoculation Enhances HBV Infection in NTCP-Reconstituted Hepatocytes”. PLOS ONE 2015 Vol.10 Issue 6. This publication reported the generation of the HepG2-NTCP cell line and the evaluation of spinoculation as a method to increase the HBV infection rate.

[0053] Youki Ueda showed that other cell lines such as A8.15.78.10 cells in addition to HepG2 can be used to generate cell lines with the same susceptibility to HBV infection as the HepG2-NTCB cell line (Ueda Y, Gu W et al., "A new hepatoma cell line exhibiting high susceptibility to hepatitis B virus infection". Biochem Biophys Res Commun. 2019 Jul 12;515(1):156-162).

[0054] Sun Y et al. gave a detailed description of the construction of an HBV infection system based on NTCP. Sun Y, Qi Y, Peng B, Li W. “NTCP-Reconstituted In Vitro HBV Infection System.” Methods MolBiol. 2017;1540:1-14.

[0055] The NTCP-stable HepG2 cell line generated by infecting HepG2 cells with lentiviral NT-GFP (pLVX-IRES-ZsGreen) is commercially available from APM (Catalog number SKU T6190, Applied Biological Materials Inc., Richmond, Canada).

[0056] Another supplier of the NTCP-stable HepG2 cell line is BioCat (Catalog number T6190-GVO-ABM, BioCatGMBH, Heidelberg).

[0057] Cell lines and stem cells for studying HDV infection are discussed in “iPSC for Studying Infectious Diseases”, Volume 8, Advances in Stem Cell Biology 2021, Pages 149-213 (Academic Press), which is hereby incorporated by reference into this article.

[0058] Ni et al. established a Huh-7-derived cell line (Huh-7-END) for the continuous production of viral particles by integrating the HDV antigenome and HBsAg sequences (Ni, Y., Zhang et al. "Generation and characterization of a stable cell line persistently replicating and secreting the human hepatitis delta virus." Sci Rep 9, 10021, 2019).

[0059] HepaRG cells become sensitive to HDV infection upon differentiation or ectopic NTCP expression (Ni et al., "Hepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-Specific Entry into Hepatocytes", Gastroenterology, Volume 146, Issue 4, 2014, Pages 1070-1083).

[0060] Primary cultures of human hepatocytes (PHH) and primary cultures of tree shrew hepatocytes (PTH) (a tree shrew hepatocyte model that contributed to the identification of NTCP as a receptor for HBV and HDV) both permit HDV infection (Verrier et al., "Cell Culture Models for the Investigation of Hepatitis B and D Virus Infection." Viruses. 2016 Sep 20;8(9):261, and Walter, E., et al., (1996), "Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo". Hepatology, 24:1-5.).

[0061] Winer et al. (Winer et al., "Analysis of Host Responses to Hepatitis B and Delta Viral Infections in a Micro-scalable Hepatic Co-culture System." Hepatology. 2020 Jan;71(1):14-30) described a self-assembling co-culture of primary human hepatocytes (SACC-PHH) that, when co-cultured with stromal cells, supports long-term HBV / HDV infection.

[0062] Unzu et al. ("Pharmacological Induction of a Progenitor State for the Efficient Ex-pansion of Primary Human Hepatocytes." Hepatology. 2019 May;69(5):2214-2231) reported the infection and replication of HDV in PHH-derived hepatic progenitor cells differentiated into hepatocyte-like cells.

[0063] Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells induced pluripotent stem cells (iPSCs) are sensitive to HDV infection (F. Lange et al., "Hepatitis D virus infection of stem cell-derived hepatocytes triggers an IFN- and NFκB-based innate immune response unable to clear infection." bioRxiv 2022.08.11.502443).

[0064] Hepatitis B surface antigen (HBsAg) is a protein on the surface of HBV, which is released by infected cells and detected at high levels in serum during acute or chronic hepatitis B virus infection. There are three forms of HBsAg, and the maximum amount of HBsAg is required to produce infectious viral particles. The amount of HBsAg produced exceeds the amount required for the production of HBV viral particles, and the excess particles are secreted as HBsAg. HBsAg itself is used to prepare prophylactic hepatitis B vaccines.

[0065] HBV infection can be detected by detecting its nucleic acid, antigen, or antibody against one of its proteins. M. Krajden, G. McNabb et al., "The laboratory diagnosis of hepatitis B virus". Can J Infect Dis Med Microbiol. 2005 Mar-Apr;16(2):65–72. provides a comprehensive overview.

[0066] HDV infection is detected by anti-HDV immunoglobulin G (IgG) and immunoglobulin M (IgM), and confirmed by detecting HDV RNA in serum by RT-PCR (reverse transcription polymerase chain reaction).

[0067] Prophylactic and therapeutic applications of antiviral compounds:

[0068] The excellent efficacy of hepatitis B vaccine makes it unlikely that MB will be used as a prophylactic agent.

[0069] The prophylactic use of methylene blue (MB) may be applicable to rare cases in which, due to immunodeficiency, the patient may not be able to generate an effective antibody or cytotoxic T cell response. In the prophylactic situation, the subject has not been infected; while in the therapeutic situation, the subject has been infected by the virus. The population of subjects for prophylactic use involves those with a relatively high known risk of virus infection, such as healthcare workers treating the virus, intravenous drug users, those living with infected individuals, those with comorbidities (diabetes, leukemia, immunosuppression, etc.), the elderly, immunocompromised individuals, etc. The absence of side effects is particularly important for prophylactic applications because this is used in healthy populations in most cases. MB turns urine blue and also stains the skin and sclera after long-term prophylaxis. This effect is completely reversible. Considering the cost and side effects of antiviral compounds such as MB, when the virus exposure disappears, the treatment in the prophylactic mode is stopped, and when the diagnostic test shows that the virus is no longer detectable, the treatment in the therapeutic mode is stopped.

[0070] Antiviral activity, antiviral efficacy:

[0071] This describes the pharmacological effect caused by a compound that reduces the infectivity of the virus. Typical measurements in vitro are the counting of PFU (plaque-forming units) in a virus neutralization assay and the counting of LD50 or a clinical surrogate (such as elevated temperature) in in vivo tests. The counting of PFU is also possible in in vivo experiments: after infection, the organs can be homogenized and the PFU / weight ratio can be determined. In addition, those skilled in the art use generally accepted statistical methods to express LD50 in terms of PFU and vice versa. In alternative assays, quantitative measurements of viral nucleic acids or secreted viral antigens are commonly used for the quantification of the virus. For viruses that do not cause lysis of infected cells, such as HBV and HDV, quantitative RT-PCR specific for the viral nucleic acid sequence is used as an indication of the amount of virus present in the cells.

[0072] Active immunization (vaccination), passive immunization:

[0073] Historically, in passive immunization, convalescent serum (i.e., serum obtained from recovered patients of an infectious disease and containing antibodies against the infectious agent of the disease) or serum containing neutralizing antibodies generated by active immunization was used to prevent infection. Convalescent serum can be replaced by neutralizing monoclonal antibodies or genetically engineered antibody-like molecules against the neutralizing epitopes of the virus.

[0074] MB can be used in combination with active or passive immunization, any type of antiviral antibody, or any antiviral compound approved for anti-HBV or anti-HDV. In the case of maternal infection, passive immunization and / or MB treatment of the newborn can also be considered. MB is known to be teratogenic and should not be used during pregnancy (Tiboni GM, Lamonaca D. Transplacental exposure to methylene blue initiates teratogenesis in the mouse: preliminary evidence for a mechanistic implication of cyclic GMP pathway disruption. Teratology. 2001;64:213–220.).

[0075] Pegylated interferons and small molecule antiviral compounds for HBV and HDV:

[0076] Theoretically, the use of MB is compatible with that of alpha-2a interferon and pegylated alpha-2a interferon. No in vivo or in vitro data are available on the interaction of MB with other known antiviral compounds against HBV or HDV: lamivudine, adefovir, tenofovir disoproxil fumarate, tenofovir alafenamide, telbivudine, entecavir.

[0077] Dosage and toxicity of MB:

[0078] The present invention describes a compound containing MB for the prevention and / or treatment of HBV and / or HDV viral infections in humans, which acts by virtue of its antiviral efficacy after oral, intravenous, subcutaneous, intramuscular, intranasal, rectal or by nasal or oral application, wherein the daily dose is not less than 0.1 mg per application and not more than 20 mg per 24 hours and per kg of patient body weight.

[0079] MB may induce serotonin syndrome and should not be used concomitantly with serotonin reuptake inhibitors. It can cause hemolytic anemia in patients with glucose-6-phosphate dehydrogenase (G6PD) enzyme deficiency at high doses. Gastrointestinal symptoms may occur at higher oral doses due to the bitter taste, which can be masked by the dosage form. During World War I, soldiers took more than 400 mg of MB daily for weeks for malaria prophylaxis without serious side effects (Marshall DG. The "toxicity" of methylene-blue. Lancet. 1920; 195(5051): 1334). Brazilian children reportedly tolerated 20-50 mg / kg of MB daily well for a long period of time (Ferreira MC. Sur l'emploi du bleu de méthylène dans la malaria infantile. Ther Medico-Chirugicale. 1893; 124: 488–525). In general, orally administered MB appears to be largely well tolerated, and intravenous MB must be used with caution. For sheep, the LD50 of MB was found to be 42 mg / kg when applied intravenously (Burrows GE. Methylene blue: effects and disposition in sheep. J Vet Pharmacol Ther. 1984; 7(3): 225-231).

[0080] Virological methods:

[0081] Those skilled in the art are familiar with the widely used virology methods described herein. The book "Diagnostic Virology Protocols" was edited by John R. Stephenson and Alan Warnes in 2011 and published by Springer Verlag, and is one of the most comprehensive manuals on methods for studying, handling, and detecting viruses. This text book completes the concise description of the methods and procedures given here, and its contents are incorporated herein by reference.

[0082] (2) Embodiments of the present invention

[0083] (2.1) Specific implementation plans

[0084] The present invention relates to the following aspects and embodiments thereof:

[0085] According to a first aspect, the present invention relates to methylene blue (MB) compounds or active ingredients containing MB for the therapeutic or prophylactic treatment, particularly therapeutic treatment, of hepatitis B (HBV) and / or hepatitis D (HDV) infections in human patients.

[0086] According to one particular embodiment thereof, treat HDV infection.

[0087] According to another particular embodiment thereof, treat HBV infection.

[0088] According to another particular embodiment thereof, treat a combination (i.e., simultaneously or sequentially) infection of HBV and HDV.

[0089] In another particular embodiment of said first aspect, the virus is a genotype of HBV, HDV or HBV and HDV.

[0090] According to yet another particular embodiment, the treatment is a therapeutic treatment of such infection.

[0091] According to another particular embodiment, the treatment is a prophylactic treatment of such infection.

[0092] According to another particular embodiment, the compound or active ingredient containing MB consists essentially of MB as defined above, particularly any solid form of MB and / or its hydrate.

[0093] In particular, apply any anionic salt form of MB. Non-limiting examples of suitable and unique salts are selected from acetate, acetostearate, benzenesulfonate, bromide, chloride, citrate, fumarate, glucouronate, hydrobromide, hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate, pamoate, phosphate, succinate, sulfate, tartrate, toluenesulfonate and xinofoate. More particularly, the anionic salt is the chloride salt of MB.

[0094] According to another particular embodiment, the compound or active ingredient containing MB acts through its antiviral efficacy.

[0095] According to another particular embodiment, the treatment is for

[0096] According to another particular embodiment, the compound or active ingredient containing MB is applied to the patient for prophylactic treatment by the oral route, intranasal application, by intravenous, subcutaneous or intramuscular injection, by rectal or nebulization route or any combination thereof, particularly by the oral and intravenous routes.

[0097] According to another specific embodiment, the compound or the active ingredient containing MB is applied to the infected patient for therapeutic treatment by the oral route, intranasal application, by intravenous, subcutaneous or intramuscular injection, by rectal or nebulization route or any combination thereof, in particular by the oral and intravenous routes.

[0098] In a more specific embodiment of the first aspect, the MB is applied by the oral administration route.

[0099] In another more specific embodiment, the oral administration route is carried out by applying a solid preparation, which is encapsulated, in particular to mask the bitter taste of MB.

[0100] In another more specific embodiment, the formulation for oral administration enables slow release of MB.

[0101] According to another specific embodiment, the daily dose of the compound or the active ingredient containing MB administered for any of the above medical purposes is in the range of 0.1 mg to 20 mg per kilogram of human patient body weight.

[0102] According to a further specific embodiment, the compound or the active ingredient containing MB is administered.

[0103] a) Orally, at a daily dose of especially 0.1 to 20, more especially 0.5 to 7.5, even more especially 1 to 5, and most especially 2 to 4 mg per kilogram of patient body weight per day; or

[0104] b) By injection, at a daily dose of especially 0.1 to 20, more especially 0.5 to 7.5, even more especially 1 to 5, and most especially 2 to 4 mg per kilogram of patient body weight per day; or

[0105] c) Rectally, at a daily dose of especially 0.1 to 20, more especially 0.5 to 7.5, even more especially 1 to 5, and most especially 2 to 4 mg per kilogram of patient body weight per day.

[0106] According to a further specific embodiment, the compound or the active ingredient containing MB is administered.

[0107] d) By nebulizer, at a daily dose of especially 0.1 to 20 or 0.1 to 10, more especially 0.5 to 7.5, even more especially 1 to 5, and most especially 2 to 4 mg per kilogram of patient body weight; or

[0108] e) Intranasally, at a daily dose of especially 0.1 to 20 or 0.1 to 10, more especially 0.5 to 7.5, even more especially 1 to 5, and most especially 2 to 4 mg per kilogram of patient body weight.

[0109] Most particularly, the adult dose for antiviral treatment by the oral route is 3 mg / kg every 24 hours. If administered for a long period (such as more than 5 to 10 days), the maximum dose should not exceed 10 mg / kg per day.

[0110] In another specific embodiment of the first aspect, the compound or the active ingredient containing MB is administered by injection, particularly intravenously, more particularly by infusion, at a daily dose of 0.1 to 10, more particularly 0.5 to 7.5, even more particularly 1 to 5, and most particularly 2 to 4 mg per kilogram of patient body weight.

[0111] Most particularly, the adult dose for antiviral treatment by injection (particularly infusion) is 3 mg / kg every 24 hours. If administered as a bolus injection, it should be applied over a period of at least 5 minutes. If administered for a long period (such as more than 5 to 10 days), the maximum dose should not exceed 10 mg / kg per day.

[0112] In another specific embodiment of the first aspect, the compound or the active ingredient containing MB is administered by a combination of at least two administration routes selected from the group consisting of oral, intravenous, subcutaneous, intramuscular, intranasal, or nebulized routes, at a combined daily dose of particularly 0.1 to 20 or 0.1 to 10, more particularly 0.5 to 7.5, even more particularly 1 to 5, and most particularly 2 to 4 mg per kilogram of patient body weight per day.

[0113] In another specific embodiment of the first aspect, when the viral infection is caused by HBV; or when the viral infection is caused by simultaneous or sequential infection by HBV and HDV, the compound or the active ingredient containing MB is administered by any of the above administration routes.

[0114] In another specific embodiment of the first aspect, the compound or the active ingredient containing MB acts on the vasoconstriction of small blood vessels due to its action on nitric oxide and is administered by any of the above administration routes in distributive shock induced by viral infection.

[0115] In another specific embodiment of the first aspect, the compound or the active ingredient containing MB is administered simultaneously (i.e., concurrently or in any sequential order) with one or more other therapeutic agents by any of the above-mentioned administration routes, and the other therapeutic agents are particularly selected from interferon α-2a, pegylated interferon α-2a, interferon α-2b, pegylated interferon α-2b, 2b or any small molecule effective against antiviral drugs targeting HBV and / or HDV, such as lamivudine, adefovir, tenofovir disoproxil, tenofoviralafenamide, telbivudine, bulevirtide, and entecavir.

[0116] In another specific embodiment of the first aspect, the compound or the active ingredient containing MB is administered by any of the above-mentioned administration routes for therapeutic use, wherein the treatment is carried out for at least one day, particularly at least one month, more particularly 3 to 24 months, even more particularly 6 to 18 months, most particularly 12 months, or until the time when a negative virological test result for the presence of HBV and / or HDV in the patient is obtained.

[0117] In another specific embodiment of the first aspect, the compound or the active ingredient containing MB is administered by any of the above-mentioned administration routes, wherein the treatment is carried out in the absence of activation of methylene blue by an external (i.e., in vitro) high-energy light source.

[0118] In another specific embodiment of the first aspect, the compound or the active ingredient containing MB is administered by any of the above-mentioned administration routes in the form of a liquid pharmaceutical composition.

[0119] In particular, the pharmaceutical composition is in solid or liquid form and contains a virucidal effective amount of the compound or the active ingredient containing MB in a pharmaceutically acceptable carrier or diluent.

[0120] More particularly, the pharmaceutical composition contains the compound or the active ingredient containing MB in a pharmaceutically acceptable liquid carrier in a proportion ranging from 0.1 to 2 wt.-% based on the total weight of the composition, particularly 0.5 to 1.5 wt.-%, more particularly 0.8 to 1.2 wt.-%, especially about 1 wt.-%.

[0121] More particularly, the pharmaceutical composition comprises the compound or the active ingredient containing MB in a pharmaceutically acceptable solid carrier, in a proportion ranging from 0.1 to 2 wt.-%, particularly from 0.5 to 1.5 wt.-%, more particularly from 0.8 to 1.2 wt.-%, especially about 1 wt.-%, based on the total weight of the composition.

[0122] According to a second aspect, the present invention relates to a solid or liquid pharmaceutical composition as defined above.

[0123] According to a third aspect, the present invention relates to a method for the therapeutic treatment of a human patient infected with hepatitis B (HBV) and / or hepatitis D (HDV), the method comprising administering to the patient a virucidal effective amount of a methylene blue compound as further defined in the first aspect of the present invention.

[0124] More particularly, the use, composition or method of any of the foregoing aspects or embodiments is applied with MB in a substantially pure form, particularly a pharmaceutically acceptable salt or hydrate thereof.

[0125] More specifically, during the use or method of any of the foregoing aspects or embodiments, the MB treatment is applied in combination with active immunization or passive immunization effective against HBV and / or HDV, any type of antiviral antibody and / or any type of antiviral compound, such as lamivudine, adefovir, tenofovir disoproxil fumarate, tenofovir alafenamide, telbivudine, brexpiprazole and entecavir.

[0126] (2.2) Further embodiments

[0127] One or more of the compounds or "active agents" disclosed herein can be administered to a patient alone or in the form of a pharmaceutical composition, in which they are mixed with a biologically suitable carrier or excipient in a dose effective to prevent, treat, alleviate or improve a disease or disorder as described herein. Mixtures of these compounds can also be administered to a patient as a simple mixture or in a suitable formulated pharmaceutical composition.

[0128] As used herein, "patient" refers to human or non-human, particularly human, animal.

[0129] In the context of the present invention, an "active agent" or "compound" refers to any compound, element or mixture that directly or indirectly produces a physiological effect on a patient when administered to the patient alone or in combination with another agent. When the active agent is a compound, it includes salts, free compounds or solvates (including hydrates) of salts, crystalline and non-crystalline forms, and various polymorphs of the compound. The compound may contain one or more asymmetric elements, such as a stereocenter, a stereaxis, etc., such as an asymmetric carbon atom, so that the compound can exist in different stereoisomeric forms. These compounds can be, for example, racemates or optically active forms. All stereoisomers, diastereoisomers, Z and E forms, in purified and mixed forms are included. Thus, when a compound is referred to by a specific name or a class of compounds is mentioned, all these forms are intended to be included.

[0130] A "dosage form" is any administration unit ("unit dose") of one or more active agents as described herein.

[0131] The terms "treating" or "treatment" mean: (i) preventing a disease, disorder and / or condition from occurring in a patient who may be exposed to or is susceptible to a disease, disorder and / or condition but has not been diagnosed as having the disease, disorder and / or condition; (ii) inhibiting a disease, disorder or condition, i.e., preventing its development; and (iii) alleviating a disease, disorder or condition, i.e., causing regression of the disease, disorder and / or condition.

[0132] The compounds or active ingredients of the present invention are generally administered as pharmaceutical compositions, which contain a prophylactically or therapeutically effective amount of at least one such compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, and may contain conventional excipients.

[0133] The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of reasonable medical judgment, suitable for contact with the tissues of a patient without excessive toxicity, irritation, allergic response or other problems or complications commensurate with a reasonable risk / benefit ratio.

[0134] The present invention includes all "pharmaceutically acceptable salt forms" of the compounds. Pharmaceutically acceptable salts are those salts in which the counterion does not significantly contribute to the physiological activity or toxicity of the compound and thus acts as a pharmacological equivalent. These salts can be prepared according to conventional organic techniques using commercially available reagents. Some anionic salt forms include acetate, acistrate, benzenesulfonate, bromide, chloride, citrate, fumarate, glucuronate, hydrobromide, hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate, pamoate, phosphate, succinate, sulfate, tartrate, tosylate, and xinofoate. Some cationic salt forms include ammonium, aluminum, benzathine, bismuth, calcium, choline, diethylamine, diethanolamine, lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine, potassium, sodium, tromethamine, and zinc.

[0135] "Therapeutically effective amount" means an amount that, when administered to a human or non-human patient, is effective in providing any therapeutic benefit. More specifically, a "therapeutically effective amount" is an amount of a compound disclosed herein or a combination of two or more such compounds that completely or partially inhibits the progression of a disorder or at least partially alleviates one or more symptoms of the disorder.

[0136] The therapeutic benefit can be improvement of the symptoms of the afflicted patient, such as an amount effective in reducing the symptoms of the patient or the laboratory indicators of liver function. The ultimate goal is to eradicate HBV and / or HDV.

[0137] The "frequency" of dosing can vary depending on the compound used and the particular type of infection being treated. A dosing regimen of once a day or even over a longer time period is possible. A dosing regimen in which the active agent is administered several times a day (e.g., 2 to 10 times, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 times) may sometimes be more helpful.

[0138] However, it should be understood that the specific dose level and frequency for any particular patient will depend on a variety of factors, including the activity of the particular compound employed, age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease being treated in the patient undergoing therapy. The therapeutic effectiveness of a patient can generally be monitored using assays suitable for the disorder being treated that are familiar to those of ordinary skill in the art.

[0139] Solid compositions are generally formulated in dosage units, and compositions providing about 0.1 to 2000 mg of active ingredient per dose are of interest. Some examples of doses are 1 mg, 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, and 1000 mg.

[0140] Liquid compositions are generally within a dosage unit range. Typically, the unit dosage range of the liquid composition is 1 - 100 mg / ml. Some examples of dosages are 1 mg / ml, 10 mg / ml, 25 mg / ml, 50 mg / ml, and 100 mg / ml.

[0141] The present invention also includes methods of administering the compounds in combination therapy. That is, the compounds can be used in combination with other agents for treating infections, but are administered separately therefrom. In these combination methods, the compounds are generally administered in the daily dosage as described above, together with the other agents. The other agents are generally administered in amounts used for therapeutic use. However, the specific dosing regimen will be determined by a physician using reasonable medical judgment.

[0142] For any compound or combination thereof used in accordance with the present invention, the therapeutically effective dose can initially be estimated from cell assays or animal models. For example, doses can be formulated in cell and animal models to achieve a range of circulating concentrations that includes the IC50 determined in cell assays (i.e., the concentration of the test compound that produces half-maximal inhibition of a given activity). In some cases, it is appropriate to determine the IC50 in the presence of 3 to 5% serum albumin, since this assay approximates the binding effect of plasma proteins on the compound. Such information can be used to more accurately determine useful doses in humans.

[0143] The toxicity and therapeutic efficacy of such compounds or combinations thereof can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as for determining the maximum tolerated dose (MTD) and ED 50 (effective dose at 50% maximum response). The dose ratio between toxicity and therapeutic effect is the therapeutic index, and it can be expressed as the ratio between MTD and ED 50 The ratio between. Compounds or combinations thereof that preferably exhibit a high therapeutic index are preferred. Data obtained from these cell culture assays and animal studies can be used to formulate a dosage range for humans. The dose of such compounds is preferably within a range of circulating concentrations that includes the ED50, with little or no toxicity. The dose can vary within this range, depending on the dosage form used and the route of administration employed. The exact formulation, route of administration, and dose can be selected by the individual physician according to the condition of the patient (see, for example, Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1).

[0144] The dosage and interval can be adjusted individually to provide a plasma level or minimum effective concentration (MEC) of the active moiety sufficient to maintain the desired effect. The MEC will vary with each compound but can be estimated from in vitro data; for example, the concentration required to achieve 50 - 90% inhibition of a protein kinase using the assays described herein. The dosage required to achieve the MEC will depend on individual characteristics and the route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

[0145] The dosage interval can also be determined using the MEC value. The compound should be administered using a regimen that maintains the plasma level above the MEC for 10 - 90%, preferably 30 - 90%, most preferably 50 - 90% of the time until the desired symptomatic improvement is achieved.

[0146] Of course, the amount of the composition administered will depend on the subject being treated, the subject's weight, the severity of the affliction, the mode of administration, and the judgment of the prescribing physician.

[0147] The term "pharmaceutical composition" refers to a composition comprising a combination of at least one pharmaceutically active compound as described herein with at least one additional pharmaceutical carrier (i.e., adjuvant, excipient, or vehicle, such as diluent, preservative, filler, stabilizer, extender, binder, humectant, flow regulator, disintegrant, wetting agent, emulsifier, suspending agent, sweetening agent, flavoring agent, fragrance, antibacterial agent, antifungal agent, lubricant, and dispersant), depending on the mode of administration and the nature of the dosage form. For example, the ingredients listed in Remington’s Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA (1999) can be used.

[0148] The pharmaceutical compositions used herein can exist in the form of a "dosage form" or "unit dose" and can contain one or more active agents. Thus, a pharmaceutical composition as used herein can, for example, provide two active agents mixed together in a unit dose or provide two active agents in a dosage form combination, where the active agents are physically separated and / or have different release rates.

[0149] As used herein, a "combination pharmaceutical product" is a combination of two or more doses of two or more different active agents combined in separate, un-mixed dosage forms.

[0150] Pharmaceutical compositions include any suitable "formulations", including, for example, capsules, tablets, coated tablets, injections, and liquid preparations, and can be administered by any suitable route.

[0151] Suitable administration routes may include, for example, oral, rectal, transmucosal or enteral administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injection and intrathecal, intravenous or intraperitoneal application.

[0152] In addition, the drug can be administered in a targeted drug delivery system, for example, in liposomes coated with endothelial cell-specific antibodies or peptides.

[0153] The pharmaceutical compositions of the present invention can be manufactured in a manner known per se, for example, by conventional mixing, dissolving, granulating, sugar coating preparation, grinding, emulsifying, encapsulating, entrapping or lyophilization processes. The appropriate formulation depends on the selected route of administration.

[0154] For injection and administration in liquid form, the reagents of the present invention can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks solution, Ringer's solution or physiological saline buffer.

[0155] For transmucosal administration, permeants suitable for the barrier to be penetrated are used in the formulation. Such permeants are generally known in the art.

[0156] For oral administration, the compound can be easily formulated by combining the active compound with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds disclosed herein to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by the patient to be treated. Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding the resulting mixture, and processing the granule mixture to obtain tablets or dragee cores after adding suitable auxiliaries (if necessary). Suitable excipients are especially fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If necessary, disintegrants can be added, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or its salts, such as sodium alginate.

[0157] The dragee cores have suitable coatings. Considering bitterness, coating is particularly important for MB, as bitterness may cause gastrointestinal symptoms and reduce the patient's compliance with the treatment schedule. For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the tablet or dragee coating for the identification or characterization of different combinations of active compound doses.

[0158] Pharmaceutical preparations for oral use include push-fit capsules made of gelatin, and sealed soft capsules made of gelatin and a plasticizer (such as glycerol or sorbitol). The push-fit capsules may contain the active ingredient mixed with a filler (such as lactose), a binder (such as starch), and / or a lubricant (such as talc or magnesium stearate), and optionally a stabilizer. In the soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, a stabilizer may be added. All preparations for oral administration should be in a dosage suitable for such administration.

[0159] The compounds can be formulated for parenteral administration by injection (such as bolus injection or continuous infusion). Preparations for injection may be in unit dosage forms, such as in ampoules or multi-dose containers, and contain a preservative. The composition may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulating agents, such as suspending agents, stabilizers, and / or dispersing agents.

[0160] Pharmaceutical preparations for parenteral administration include aqueous solutions of the active compound in water-soluble form. Additionally, suspensions of the active compound may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of highly concentrated solutions.

[0161] Alternatively, the active ingredient may be in powder form for formulation with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0162] The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, for example containing conventional suppository bases such as cocoa butter or other glycerides. Rectal application is attractive because it avoids problems due to the bitter taste of MB.

[0163] In addition to the previously described preparations, the compounds can be formulated as long-acting preparations. Such long-acting preparations can be administered by injection or implantation (such as subcutaneously or intramuscularly). Thus, for example, the compounds can be formulated with a suitable polymer or a hydrophobic material (such as an emulsion in an acceptable oil), or an ion-exchange resin, or as a slightly soluble derivative, such as a slightly soluble salt.

[0164] Examples of pharmaceutical carriers for the hydrophobic compounds disclosed herein are cosolvent systems comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system can be a VPD cosolvent system. VPD is a solution of 3% w / v benzyl alcohol, 8% w / v nonpolar surfactant polysorbate 80, and 65% w / v polyethylene glycol 300, made up to the desired volume with absolute ethanol. The VPD cosolvent system (VPD:5W) consists of VPD diluted 1:1 with 5% aqueous glucose solution. This cosolvent system dissolves hydrophobic compounds well and is itself less toxic when administered systemically. Of course, the proportions of the cosolvent system can vary significantly without disrupting its solubility and toxicity characteristics. In addition, the properties of the cosolvent components can be altered: for example, other low-toxicity nonpolar surfactants can be used in place of polysorbate 80; the molecular weight of polyethylene glycol can be changed; other biocompatible polymers can replace polyethylene glycol, such as polyvinylpyrrolidone; and other sugars or polysaccharides can replace glucose.

[0165] Alternatively, other delivery systems for hydrophobic pharmaceutical compounds can be employed. Liposomes and emulsions are well-known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethyl sulfoxide can also be used, although usually at the cost of greater toxicity. Additionally, the compound can be delivered using sustained-release systems, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. A variety of sustained-release materials have been developed and are well-known to those skilled in the art. Sustained-release capsules can release the compound for several hours up to over several days depending on their chemical nature.

[0166] The pharmaceutical composition can also include a suitable solid or gel-phase carrier or excipient. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

[0167] If desired, the composition can be present in a package or dispenser device, which can contain one or more unit dosage forms containing the active ingredient. The package can include, for example, a metal or plastic foil, such as a blister pack. The package or dispenser device can be accompanied by instructions for administration. Compositions comprising the compounds disclosed herein formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for the treatment of the designated condition. Commercial products approved and sold in Switzerland for the treatment of HBV and HDV include "ProveblueTM", which contains 50 mg of MB diluted in a 10 ml injection solution, approved for the treatment of methemoglobinemia toxicity, and "LumeblueTM", Cosmo Pharmaceuticals, a 25 mg coated tablet for diagnostic staining of the intestinal mucosa during colonoscopy.

[0168] In some formulations, it may be beneficial to use the compounds disclosed herein in the form of very small-sized particles obtained, for example, by air jet milling.

[0169] The use of the compounds disclosed herein in the preparation of pharmaceutical compositions is illustrated by the following description. In this specification, the term "active compound" means any compound of the invention, but in particular any compound that is the end product of one of the following non-limiting examples.

[0170] a) Capsules

[0171] In the preparation of capsules, 100 parts by weight of the active compound and 150 parts by weight of lactose can be depolymerized and mixed. The mixture can be filled into hard gelatin capsules, each capsule containing a unit dose or a partial unit dose of the active compound.

[0172] b) Tablets

[0173] Tablets can be prepared, for example, from the following ingredients.

[0174] Parts by weight: 100 parts of active compound, 150 parts of lactose, 22 parts of corn starch, 10 parts of polyvinylpyrrolidone, 3 parts of magnesium stearate. The active compound, lactose and some of the starch can be depolymerized and blended, and the resulting mixture can be granulated with an ethanolic solution of polyvinylpyrrolidone. The dry granules can be mixed with magnesium stearate and the remaining starch. The mixture is then compressed in a tableting machine to obtain tablets, each tablet containing a unit dose or a partial unit dose of the active compound.

[0175] c) Enteric-coated tablets

[0176] Tablets can be prepared by the method described in (b) above. The tablets can be enteric-coated in a conventional manner using a solution of 20% cellulose acetate phthalate and 3% diethyl phthalate in ethanol:dichloromethane (1:1).

[0177] d) Suppositories

[0178] In the preparation of suppositories, for example, 100 parts by weight of the active compound can be incorporated into 1300 parts by weight of a triglyceride suppository base, and the mixture is formed into suppositories, each suppository containing a therapeutically effective amount of the active ingredient.

[0179] The invention will now be described in more detail with reference to the following non-limiting illustrative examples.

[0180] Embodiment

[0181] Materials

[0182] a) Chemicals

[0183] The compound methylene blue (methylene blue chloride The solution was purchased from ProVepharm in Marseille, France.

[0184] All other chemicals used in this study were of analytical grade and were commercially available products.

[0185] The cell culture media and supplements used in this study were commercially available products.

[0186] b) Viral particles

[0187] HDV particles were generated and titrated as previously described (Alfaiate D, Lucifora J et al; "HDV RNA replication is associ - ated with HBV repression and interferon - stimulated genes induction in super - infected hepatocytes." Antiviral Res 2016;136:19 - 31.).

[0188] c) Cell lines and cell culture

[0189] HepG2 - NTCP is a hepatocellular carcinoma - derived cell line that expresses NTCP (the cellular receptor for HBV and HDV). When infected with HDV, there is replication of HDV but no production of new virus (Ni, Y. et al. Hepatitis B and D viruses exploit sodium taurocholate co - transporting polypeptide for species - specific entry into hepatocytes. Gastroenterology 146, 1070–1083 (2014)).

[0190] HepNB2.7 is a hepatocellular carcinoma - derived cell line that has constitutive expression of the HBV envelope protein (HBsAg) but does not express other proteins required for the production of HBV particles. When infected with HDV, there is replication, production, and new HDV particle formation. The production of new HDV particles requires the cell to express HBsAg.

[0191] Cell lines have been specifically developed for the detection and evaluation of antiviral compounds against HBV and HDV (Lempp FA et al., "Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation". Nat Commun 2019; 10:2265).

[0192] HepG2-NTCP cells and HepNB2.7 cells (each provided by Prof. Stephan Urban, University Hospital Heidelberg, Germany) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin-streptomycin (all from Gibco, Thermo Fisher Scientific) and 5 μg / ml puromycin (ant-pr-1, InvivoGen).

[0193] Examples of alternative cell test systems suitable for evaluating the efficacy of MB in treating or preventing HBV and HDV infections are provided in the general part of the specification above.

[0194] Methods

[0195] a) Cell infection and cell analysis

[0196] HepG2-NTCP or HepNB2.7 cells were infected with different titers of HDV (10, 50 MOI) and treated with 2.5 or 1.25 μg / ml of MB for 5 hours.

[0197] Cells were analyzed on day 3 or day 6 post-infection according to the MTT assay protocol (see below).

[0198] b) MTT cell viability assay

[0199] The cultures were maintained in a humidified atmosphere at 37 °C and 5% CO2. Cell viability was assessed using the MTT assay (Sigma-Aldrich, In Vitro Toxicology Assay Kit, TOX1-1KT). HepG2-NTCP and HepNB2.7 cells were seeded at 10x103 cells / well in 96-well plates 16 hours before treatment and treated with different doses of MB for 6 days. The cells were incubated for different time periods (as shown in the respective experiments below; 1 hour / 3 days / 6 days) at room temperature in the dark or in visible light (for photoactivated methylene blue). The assay used MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to determine mammalian cell viability.

[0200] c) Viral infection and MB HDV inhibition assay

[0201] As described previously (Alfaiate D, Lucifora J et al.; see above), HepG2-NTCP and HepNB2.7 cells (seeded at 10x103 cells / well) were infected with HDV at 10 or 50 MOI using 4% PEG (polyethylene glycol) overnight. MB (2.5 or 1.25 μg / ml) was added 16 hours after HDV infection for 5 hours and the cells were incubated at room temperature in the dark. At three and six days post-infection, RNA was extracted and HDV RNA levels were monitored by RT-qPCR to determine HDV replication as described below. For HepNB2.7, the level of HBsAg in the supernatant was measured as described below.

[0202] d) HBsAg measurement

[0203] For HepNB2.7, the level of HBsAg in the supernatant was measured at two time points by ELISA (Enzyme-Linked Immosorbent Assay, ABBOTT HBsAg).

[0204] e) HDV replication quantification:

[0205] Total intracellular RNA was extracted using the Nucleospin RNA kit (Macherey-Nagel AG). cDNA was synthesized from 100 ng of total RNA using Superscript II and random hexamer primers (Roche Diagnosis). Quantification of HDV was performed by qRT-PCR as described (Alfaiate D, Lucifora J et al.) using the primers described by Scholtes & colleagues (Scholtes C, Icard V, Amiri M et al; "Standardized one-step real-time reverse transcription-PCR assay for universal detection and quantification of hepatitis delta virus from clinical samples in the presence of a heterologous internal-control RNA." J Clin Microbiol 2012;50:2126-2128.) and EEF1A1 as a housekeeping gene.

[0206] Using the above materials and methods, the following experiments were conducted:

[0207] Example 1: Study of the cytotoxicity of MB on the hepatoma cell lines HepG2-NTCP and HepNB2.7

[0208] The cytotoxicity of MB on HepG2-NTCP and HepNB2.7 was evaluated by MTT. Cells were treated with MB at decreasing concentrations maintained in the dark or photoactivated under visible light.

[0209] MTT cell viability assay The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is used to determine mammalian cell viability. The application of this assay in infected and non-infected cells is crucial to demonstrate that the measured cytotoxic effect is due to the antiviral activity of the drug and not due to the direct cytotoxic effect of the drug being evaluated on the cultured cells. The redox potential in active mammalian cells reduces MTT to a strongly colored formazan product. After solubilization, the absorbance of the formazan product can be measured using a microplate absorbance reader.

[0210] Figure 2Show the cytotoxicity of MB against HepG2-NTCP (A) and HepNB2.7 (B) evaluated by MTT. Cells were treated with MB at concentrations that decreased upon being kept in the dark or photoactivated under visible light. The figure shows the percentage of viable HepG2-NTCP (A) and HepNB2.7 (B) cells after incubation with different concentrations of MB for 3 days. Compared to the dark, the cytotoxic effect of MB was not significantly enhanced in the presence of light, but the long-term effect (3 days) showed significant toxicity.

[0211] Example 2: Study the inhibitory effect of MB on HBV / HDV infection

[0212] HepG2-NTCP or HepNB2.7 cells were infected with different titers of HDV (10, 50, 100 MOI) and treated with 2.5 or 1.25 μg / ml of MB for 5 hours. Cells were analyzed on day 3 or day 6 after infection, and HDV replication was evaluated by RT-PCR. For HepNB2.7, the HBsAg level in the supernatant was measured at both time points.

[0213] Figure 1 Show the general timeline of the experimental setup for these experiments.

[0214] The following experiments were performed using infected HepG2-NTCP or HepNB2.7 cells:

[0215] a) The inhibitory effect of MB on HDV replication in HepG2-NTCP cells.

[0216] Study the inhibitory effect of MB on HDV replication in HepG2-NTCP cells. Cells infected with 10 or 50 MOI virus were treated with MB (2.5 or 1.25 μg / ml), and HDV replication was evaluated 3 days (A) or 6 days (B) after infection.

[0217] Figure 3 Show a significant antiviral effect of MB against HDV-infected HepG2-NTCP cells at physiological drug concentrations. The discovery of the NTCP receptor as a common entry portal for HBV and HDV infection of hepatocytes enabled the cloning of HepG2 cells overexpressing NTCP that are susceptible to HBV and HDV infection. This provided a cell line of HDV-susceptible cancer origin suitable for high-throughput studies. Cells were infected with 10 or 50 MOI and treated with MB (2.5 or 1.25 μg / mL) for 3 to 6 days. The measured HDV RNA concentrations showed an obvious dose-response relationship 3 days and 6 days after infection at 50 MOI. The HDV RNA concentration at 10 MOI was too low to distinguish the antiviral effect of MB.

[0218] b) Inhibitory effect of MB on HDV replication in HepNB2.7 cells.

[0219] The inhibitory effect of MB on HDV replication in HepNB2.7 cells was investigated. In the first experiment, cells infected with 10 or 50 MOI were treated with MB (2.5 or 1.25 μg / ml), and HDV replication was evaluated 3 or 6 days after infection.

[0220] In the second experiment, the secretion of hepatitis B surface antigen (HBsAg) was measured in the supernatants of cells infected with 10, 50, or 100 MOI and treated with MB (2.5 or 1.25 μg / ml).

[0221] The dose-dependent effect of MB on virus proliferation of HDV at 50 MOI on day 6 after infection was confirmed in HepNB2.7 cells under physiological conditions.

[0222] The results are as Figure 4 shown. The weak signals on day 3 and day 6 using 10 MOI were due to the low-infectivity challenge (Figures A and B). Figure C shows a clear dose / response relationship of the antiviral activity of MB against virus replication under all tested conditions - using HBsAg as a surrogate marker for HBV virus and also as a surrogate marker for HBV-dependent HDV virus. The antiviral effect of MB accumulates over time, as shown by the results on day 3 and day 6. Extrapolating this data, it seems reasonable that under conditions of long-term treatment (weeks instead of days), virus markers (except components of HBV) completely disappear from the cell line (data not shown).

[0223] Example 3: Methylene blue formulations for different application routes:

[0224] In the following sections, non-limiting examples of suitable MB formulations for the treatment of HDV / HBV infections are described.

[0225] a) Injection formulations (for intravenous, subcutaneous, or intramuscular application):

[0226] Methylene blue chloride (MB, chloride salt) was diluted in distilled water at a concentration of 5 mg / ml as a solution for intravenous injection. The ionic strength of pure water was adjusted with KCl because chloride ions reduce solubility. The pH was adjusted to pH = 4.5 because long-term stability is impaired at higher pH. The solution was stored in dark glass ampoules in volumes of 5, 10, and 20 mL. Dark glass was used due to the photosensitivity of MB. This solution is compatible with 5% glucose or 5% dextran solutions but is incompatible with 0.9% saline solution due to the risk of precipitation.

[0227] The recommended dose for antiviral treatment is 1 to 10 mg / kg every 24 hours for adults. If administered as a bolus injection, it should be applied over at least 5 minutes. In the case of bolus application, the maximum dose should not exceed 5 mg / kg. Caution is recommended in case of impaired renal function. Sensitivity to thiazine dyes and G-6-PD deficiency are further contraindications. Pulse oximeters cannot be used.

[0228] b) Formulations for oral administration:

[0229] Formulations for injection can also be administered by the oral route or as nasal drops. The bioavailability of MB after oral administration is 72%, with a peak plasma concentration after two hours and an elimination half-life of 18 hours. The half-life of MB in the human circulation is 5 to 10 hours. The recommended dose per kilogram every 24 hours is the same as that recommended for injection. The excellent reabsorption of MB after oral ingestion makes oral ingestion an attractive option.

[0230] MB can of course also be formulated in dry form, in combination with fillers as tablets or capsules. Given the bitter taste, coating of the tablets can be used to minimize gastrointestinal symptoms and maximize patient compliance.

[0231] c) Sustained-release formulations for oral administration:

[0232] For example, MB tablets containing doses of 25, 50, 100, 300 mg are formulated into sustained-release tablets in combination with 2.1 grams of a mixture consisting of a pharmaceutical coating material, rice bran, hydroxypropyl methylcellulose, dicalcium phosphate, stearic acid, magnesium stearate, lecithin such as soy lecithin, and silica. The gastric-resistant coating prepared according to a formulation well-known to those skilled in the art protects the patient from the bitter taste and reduces gastrointestinal irritation. Due to sustained release and reabsorption, the peak plasma concentration of MB is significantly prolonged.

Claims

1. A methylene blue (MB) compound for the therapeutic or prophylactic treatment, particularly the therapeutic treatment, of hepatitis B (HBV) and / or hepatitis D (HDV) infection in human patients.

2. The compound according to claim 1, wherein the compound acts through its antiviral efficacy.

3. The compound according to claim 1 or 2, wherein the compound is administered to the patient, particularly the infected patient, by the oral route, intranasal application, by intravenous, subcutaneous or intramuscular injection, by the rectal or nebulization route or any combination thereof, particularly by the oral and intravenous routes.

4. The compound according to any one of claims 1 to 3, wherein the daily dose of the compound ranges from 0.1 mg to 20 mg per kilogram of human patient body weight.

5. The compound according to any one of claims 1 to 4, wherein the compound is administered: a) orally, at a daily dose of particularly 0.1 to 20, more particularly 0.5 to 7.5, even more particularly 1 to 5, and most particularly 2 to 4 mg per kilogram of patient body weight; or b) by injection, at a daily dose of particularly 0.1 to 20, more particularly 0.5 to 7.5, even more particularly 1 to 5, and most particularly 2 to 4 mg per kilogram of patient body weight; or c) rectally, at a daily dose of particularly 0.1 to 20, more particularly 0.5 to 7.5, even more particularly 1 to 5, and most particularly 2 to 4 mg per kilogram of patient body weight.

6. The compound according to any one of claims 1 to 5, wherein the compound is administered by a combination of at least two administration routes selected from the oral, intravenous, subcutaneous, intramuscular, intranasal or nebulization routes, at a combined daily dose of particularly 0.1 to 20, more particularly 0.5 to 7.5, even more particularly 1 to 5, and most particularly 2 to 4 mg per kilogram of patient body weight.

7. The compound according to any one of claims 3 to 6, wherein the oral formulation is encapsulated.

8. The compound according to any one of claims 3 to 7, wherein the oral formulation allows for slow release of methylene blue.

9. The compound according to any one of claims 1 to 8, wherein the viral infection is caused by the hepatitis B virus; or wherein the viral infection is caused by simultaneous or sequential infection by the hepatitis B virus and the hepatitis D virus.

10. The compound according to any one of claims 1 to 9, which is applied to distributive shock induced by viral infection by acting on the vasoconstriction of small blood vessels due to its action on nitric oxide.

11. The compound according to any one of claims 1 to 10, which is administered simultaneously, particularly simultaneously, or sequentially in any order with one or more additional therapeutic agents, particularly selected from α-2a interferon, pegylated α-2a interferon, lamivudine, adefovir, tenofovir disoproxil fumarate, tenofovir alafenamide, telbivudine, brexpiprazole and entecavir.

12. A compound for therapeutic use according to any one of claims 1 to 11, wherein the treatment is carried out for at least one day, in particular for at least one month, more particularly for 3 to 24 months, even more particularly for 6 to 18 months, most particularly for 12 months, or until the time when a negative virological test result for HBV and / or HDV present in the patient is obtained.

13. A compound used according to any one of claims 1 to 12, wherein the treatment is carried out in the absence of activation of methylene blue by an external, in particular in vitro, high-energy light source.

14. A compound used according to any one of claims 1 to 13, wherein the compound is applied in the form of a liquid pharmaceutical composition.

15. A compound used according to claim 14, wherein the pharmaceutical composition is in liquid form and contains a virucidal effective amount of the compound in a pharmaceutically acceptable carrier or diluent.

16. A compound used according to claim 14 or 15, wherein the pharmaceutical composition contains the compound in a pharmaceutically acceptable liquid carrier in a proportion ranging from 0.1 to 2 wt.-%, in particular 0.5 to 1.5 wt.-%, more particularly 0.8 to 1.2 wt.-%, especially approximately 1 wt.-%, based on the total weight of the composition.

17. A pharmaceutical composition as defined in any one of claims 14 to 16.

18. A method for the therapeutic treatment of hepatitis B (HBV) and / or hepatitis D (HDV) infection in a human patient, the method comprising administering to the patient a virucidal effective amount of a methylene blue compound further defined in any one of claims 1 to 16.

19. Use, composition or method according to any one of the preceding claims, wherein MB or a pharmaceutically acceptable salt or hydrate thereof is applied.

20. Use or method according to any one of the preceding claims, wherein the MB treatment is applied in combination with active or passive immunization effective against HBV and / or HDV, any type of antiviral antibody or any type of antiviral compound.

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