use of alkylphospholipid compounds, preferably edelfosine, for the treatment of viral diseases, preferably hepatitis D virus infection

Abstract

A pharmaceutical preparation containing as an active ingredient a compound from the class of alkylphospholipids, and preferably edelfosine, for the treatment and / or prevention of infection by a hepatitis virus, and preferably the hepatitis D virus (HDV).

Description

Title of the invention: Use of alkylphospholipid compounds, preferably edelfosine, for the treatment of viral diseases, preferably hepatitis D virus infection

[0001] The present invention relates to the use of alkylphospholipid compounds, and in particular edelfosine, which is considered the reference molecule of this pharmacochemical class, for the treatment of viral diseases, in particular viral hepatitis D (HVD).

[0002] The pharmacochemical class of alkylphospholipids has been known for several decades for its pharmacological properties, which can be advantageously exploited for the treatment of various medical indications. The pharmacological properties most often described in scientific publications are the antitumor properties. A recent review of these antitumor properties and the mode of action of alkylphospholipids is given by V. Zaremberg et al. in Handb Exp Pharmacol. 2020;259:261-288.

[0003] The alkylphospholipid class also possesses antiparasitic properties, which enabled miltefosine to obtain international marketing authorization as a drug for the treatment of leishmaniasis and to be included on the World Health Organization's list of essential medicines. Currently, miltefosine is the only alkylphospholipid that has received regulatory approval as a drug.

[0004] The antiviral properties of alkylphospholipids are reported in only a few scientific publications, which only concern action against the human immunodeficiency virus HIV-1: i) Akt inhibitors as an HIV-1 infected macrophages-specific antiviral therapy, P Chugh et al., Retrovirology 2008;5:11-24; ii) Akt inhibitors as an HIV-1 infected macrophages-specific antiviral therapy, A Lucas et al., Plos One September 2010;5(9j:el3121). These publications suggest that the known inhibitory effect of alkylphospholipids on the PI3K / Akt cell survival pathway could be the mechanism of antiviral action.

[0005] The present invention, which describes an antiviral activity against the hepatitis D virus (HDV), is novel because such activity has never been reported for the alkylphospholipid class. Furthermore, this activity is surprising in light of the publication by K Xiang and B Wang (Role of the PI3K-Akt-mTOR pathway in hepatitis B virus infection and replication, Molecular Medicine Reports 2018;17:4713-4719), which shows that PI3K / Akt pathway inhibitors tend to increase hepatitis B virus (HBV) replication and transcription. This observation

[0006]

[0007] emphasizes the importance of testing and defining the antiviral effect of a compound against specific types of viruses. The present invention provides a method for treating viral hepatitis D by administering an alkylphospholipid compound, and in particular edelfosine. The chemical formulas of the principal alkylphospholipid compounds relevant to this invention are shown below. The compounds edelfosine and ilmofosine can consist of a single enantiomer (such as the R or S form) or a mixture of several enantiomers (such as the racemic R / S form). [Chem.l] cm, a / i P HXL MC ,.,-, .0' ' 0 Zx ...... ....... ....... . .. ... hsc v X O' o.. (BPQ) 9¾ Gy „O - xx. ​​x, xx xx. .--v ..-x xx .--x Yc '■■■' L Q- x-' 'x-'- x-" WO; ..-x x- . X, xx / x / X. Xx ...x, / xx, „,x M 0' y -g- xx v' v' x' -x Xx CH-j Çh3 G" .9 ,,x. xvx XX. XX.-, „.x, X-XX hsc ° 0' Y ■* v"z ■ ■■ Xz ■ Kz xz CY $CH;3 r X « > x Edelfome

[0008] Another objective of the present invention is to provide a method for treating hepatitis D by combining an alkylphospholipid compound, in particular edelfosine, or a pharmaceutically acceptable salt of this compound, with other drugs known for the treatment of viral hepatitis, such as nucleoside analogs that inhibit viral RNA polymerase or reverse transcriptase, inhibitors of HBV and / or HDV entry into hepatocytes, and analogs of interferon. More generally, an alkylphospholipid compound may be administered in combination with the following drugs: i) protease inhibitors (lopinavir, ritonavir, darunavir, danoprevir); ii) RNA polymerase inhibitors (remesdivir, fa-vipiravir, galidesivir, ribavirin, triazavirin, sofosbuvir); iii) reverse transcriptase inhibitors (emtricitabine, tenovir, elsulfavirin, azvudine, cleuvidine); iv) viral entry inhibitors (bulevirtide, umifenovir); v) viral protein replication inhibitors (ivermectin, nitazoxanide, lonafarnib); vi) interferons (interferon alpha-1b, interferon alpha-2b, Peg-interferon-alpha-2a, Peg-interferon-alpha-2b, interferon beta-la, interferon beta-lb).

[0009] The present invention provides a method for treating and / or preventing disease caused by the hepatitis viruses HBV, HCV, HDV, or HEV, and in particular, chronic hepatitis induced by superinfection of hepatitis B with the HDV virus. This treatment method consists of administering a therapeutically active amount of an alkylphospholipid compound, and in particular edelfosine, in a pharmaceutical preparation to a subject in need of such treatment.

[0010] The compounds described in this document are administered in the form of pharmaceutical preparations known in the field of art. These are pharmaceutical forms for the oral route (tablet, granule, dragee, capsule, solution or syrup), for the parenteral route (solution or suspension for intravenous, intramuscular or subcutaneous injection) or for the inhalation route (powder or aerosol).

[0011] In the case of oral or parenteral treatment of an adult weighing approximately 80 kg, a daily dose of the alkylphospholipid compound of 20 mg to 900 mg should be administered, preferably between 100 mg and 400 mg (approximately 300 mg), although the upper limit may be exceeded when indicated. The daily oral dose may be administered as a single dose or in divided doses, or, for parenteral administration, it may be given by continuous infusion or by subcutaneous or intramuscular injection. Example

[0012] The following example describes tests performed to characterize the antiviral effect of edelfosine against hepatitis B (HBV) and D (HDV) viruses. The edelfosine used in these tests is a commercially available substance with a chemical purity > 95%, verified by HPLC analysis. The tests are designed to assess the effect of edelfosine on the following three essential steps in the development of viral hepatitis: i) HBV entry into hepatocytes; ii) HDV replication in hepatocytes; and iii) HDV spread from hepatocytes previously infected with HBV.

[0013] Evaluation of the effect of edelfosine on HBV entry into HepaRG cell cultures. HepaRG human hepatoma cells (commercially available) are cultivated as described in the publication Gripon et al., PNAS 2002;99(24):15655-60. They are infected with an inoculum prepared from HepAD38 cells, which replicate the HBV virus, according to Ladner et al. Antimicrob Agents Chemother 1997;41(8):1715-20. Simultaneously with infection, the HepaRG cells are treated with edelfosine or the reference substance (bulevirtide / Myrcludex®). One day after infection and treatment, the cells are washed and then incubated for six days in a culture medium without the addition of the substances to be tested. Each incubation condition is performed in triplicate. The amount of HBe antigen secreted into the medium is then measured by a commercially available method (AutoBio chemiluminescence immunoassay kit). The following [Fig.1] presents the combined results of two independent experiments, namely the mean and 95% confidence interval generated by six cell cultures for each experimental condition.

[0016] Figure 1 shows that edelfosine (from 0.039 pM to 10 pM) did not induce any inhibition of HBV entry into HepaRG hepatocytes because HBe antigen secretion by cells infected and simultaneously treated with edelfosine remained close to 100% of that of infected and untreated cells. In contrast, cells infected and treated with bulevirtide (100 nM), the reference substance known to inhibit HBV entry into hepatocytes, secreted only a trace amount of HBe antigen, which proves that HBV did not infect the bulevirtide-treated cells. Since HBV and HDV use the same mechanism of entry into hepatocytes (C. Sureau, Methods Mol Biol 2010;640:463-73), it can be concluded that treatment with edelfosine does not inhibit the entry of HBV and HDV viruses into he-

[0017] patocytes. Evaluation of the effect of edelfosine on HDV replication in HBV- and HDV-infected HepaRG cell cultures. Human hepatoma HepaRG cells were cultured as described in Gripon et al., PNAS 2002;99(24):15655-60. HepaRG cells were co-infected with an HBV inoculum prepared according to the previously described method and with an HDV inoculum prepared from Huh7 cells, which replicate HDV, according to C. Sureau, Methods Mol Biol 2010;640:463-73. Four days after infection, the cells were treated with either edelfosine or bulevirtide, and this treatment was repeated every two days until the eighth day of incubation. Each incubation condition was performed in triplicate.On day ten post-infection, HDV RNA was extracted from HepaRG cells (NucleoSpin RNA Plus, Mini kit for RNA purification with DNA removal – Macherey-Nagel) and quantified by RTqPCR (SuperScript III reverse transcriptase kit – Invitrogen, Thermo Fisher Scientific). Figure 2 below presents the combined results of two independent experiments, namely the mean and 95% confidence interval generated by six cell cultures for each experimental condition.

[0018]

[0019] [Fig.2]

[0020] Figure 2 shows that edelfosine at concentrations between 0.039 pM and 2.5 pM did not reduce HDV replication. The decrease in HDV RNA levels measured in cultures treated with 10 pM edelfosine is likely due to cytotoxicity of edelfosine at this concentration, as revealed by microscopic observation of the treated cells. Consistent with the known pharmacological properties of bulevirtide, no inhibition of HDV replication was observed with this treatment.

[0021] Evaluation of the effect of edelfosine on the propagation of HDV from cells HepaRG cells infected with HBV and HDV are transfused to Huh7.5-hNTCP cells. HepaRG human hepatoma cells are cultured as described in Gripon et al., PNAS 2002;99(24):15655-60. Huh7.5-hNTCP cells result from transfection with the hNTCP gene of cultured Huh7.5 human hepatoma cells according to Blight et al., J Viral. 2003;77(5):3181-90. HepaRG cells are co-infected with an HBV inoculum prepared according to the method described above and with an HDV inoculum prepared from Huh7 cells, which replicate the HDV virus, according to C. Sureau, Methods Mol Biol 2010;640:463-73. Four days after infection, cells are treated with edelfosine or the reference substance lonafarnib, and this treatment is repeated every two days until the eighth day of incubation. Each incubation condition is performed in triplicate. On the tenth day after infection, supernatants from HepaRG culture media are transferred to Huh7 cells.Fresh 5-hNTCPs and, six days later, HDV RNA is extracted from Huh7.5-hNTCP cells (NucleoSpin RNA Plus, Mini kit for RNA purification with DNA removal column - Macherey-Nagel) and quantified by RTqPCR (SuperScript III reverse transcriptase kit - Invitrogen - Thermo Fisher Scientific). Figure 3 below presents the combined results of three independent experiments, namely the mean and 95% confidence interval generated by nine cell cultures for each experimental condition.

[0022] [Fig.3]

[0024] Figure 3 shows that edelfosine at concentrations above 0.156 pM significantly reduced the spread of HDV from infected HepaRG cells to Huh7.5-hNTCP cells. The strong reduction in HDV spread

[0025]

[0026]

[0027]

[0028] The measured effect of edelfosine 10 pM on cultures is likely related to cytotoxicity of edelfosine at this concentration, as revealed by microscopic observation of the treated cells. Edelfosine at 2.5 pM appears to be as effective as the reference substance lonafarnib at 10 pM in inhibiting HDV propagation. Lo-nafarnib is a famesyltransferase inhibitor that has demonstrated its potential to interfere with the formation of HDV viral particles (Glenn et al. Science 1992;256:1331-3). Evaluation of the cytotoxicity of edelfosine on HepaRG cells. To ensure that the inhibitory effect of edelfosine on HDV propagation is not due to toxicity of the substance to HepaRG cells, the cytotoxicity of edelfosine at HDV-active concentrations was evaluated in uninfected HepaRG cells. Cytotoxicity was measured using the Promega CellTiter-Glo® assay. Figure 4 below presents the combined results of two independent experiments, namely the mean and 95% confidence interval generated by sixteen cell cultures for each experimental condition. [Fig.4] Figure 4 shows that edelfosine induces significant cytotoxicity at concentrations above 5 pM in HepaRG cells. It can therefore be inferred that the inhibitory effect of edelfosine on HDV propagation observed at concentrations between 0.156 pM and 2.5 pM cannot be explained by a toxic effect of edelfosine on HepaRG cells.

Claims

Demands

1. A pharmaceutical preparation containing as an active ingredient a compound from the class of alkylphospholipids, and preferably edelfosine, for the treatment and / or prevention of infection by a hepatitis virus, and preferably the hepatitis D virus (HDV).

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