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 infection by the hepatitis D virus

[0001] The present invention relates to the use of alkylphospholipid compounds, and in particular of edelfosine which is considered to be 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 anti-tumor properties. A recent review of these anti-tumor 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 has anti-parasitic properties, which has allowed miltefosine to obtain international marketing authorization as a drug for the treatment of leishmaniasis and its inclusion in the World Health Organization's List of Essential Medicines. Currently, miltefosine is the only alkylphospholipid that has obtained regulatory approval as a drug.

[0004] The anti-viral properties of alkylphospholipids are reported only in a few scientific publications, which only concern an action against the human immunodeficiency virus HIV-1: i) Akt inhibitors as an HIV-1 infected macrophages-specific anti-viral therapy, P Chugh at al., Retrovirology 2008;5:11-24; ii) Akt inhibitors as an HIV-1 infected macrophages-specific anti-viral 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 the anti-viral action.

[0005] The present invention, which describes an anti-viral activity against the hepatitis D virus (HDV), is novel because such activity has never been reported for the alkylphospholipid class. Moreover, this activity is surprising given the publication of 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 inhibitors of the PI3K / Akt pathway tend to increase the replication and transcription of the hepatitis B virus (HBV). This observation

[0006]

[0007] highlights 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 main alkylphospholipid compounds covered by this invention are shown below. The edelfosine and ilmofosine compounds may consist of a separate enantiomer (such as the R-form or the 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 object 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 thereof, with other drugs known for the treatment of viral hepatitis, such as nucleoside analogues which are inhibitors of RNA polymerase or reverse transcriptase of viruses, inhibitors of the entry of HBV and / or HDV into hepatocytes and analogues 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 of treating and / or preventing a disease induced by the hepatitis viruses HBV, HCV, HDV or HEV, and in particular, chronic hepatitis induced by superinfection of hepatitis B by the HDV virus. This method of treatment 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 oral administration (tablet, granule, dragee, capsule, solution or syrup), for parenteral administration (solution or suspension for intravenous, intramuscular or subcutaneous injection) or for inhalation administration (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 (i.e. approximately 300 mg), although the upper limit may be exceeded when indicated. The oral daily dose may be administered as a single or divided dose or, for parenteral administration, it may be given as a continuous infusion or by subcutaneous or intramuscular injection. Example

[0012] The following example describes the tests carried out 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% controlled by HPLC analysis. The tests are intended to test the effect of edelfosine on the following three steps essential for the development of viral hepatitis: i) entry of HBV into hepatocytes; ii) replication of HDV in hepatocytes; iii) propagation of HDV from hepatocytes previously infected with HBV.

[0013] Evaluation of the effect of edelfosine on HBV entry into HepaRG cell cultures. Human hepatoma cells HepaRG (commercially available) are cultured 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 the substance 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 test substances. Each incubation condition is carried out in triplicate. The amount of HBe antigen secreted into the medium is then measured by a commercially available method (Chemiluminescence immunoassay kit AutoBio). The following [Fig.l] presents the combined results of two independent experiments, i.e., the mean and 95% confidence interval generated by six cell cultures for each experimental condition.

[0016] [Fig.l] shows that edelfosine (from 0.039 pM to 10 pM) did not induce any inhibition of HBV entry into HepaRG hepatocytes because the secretion of HBe antigen by infected cells treated simultaneously with edelfosine remained close to 100% of that of infected and untreated cells. On the other hand, infected cells treated with bulevirtide (100 nM), the reference substance known to inhibit HBV entry into hepatocytes, secreted only a tiny amount of HBe antigen, which proves that HBV did not infect the cells treated with bulevirtide. 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 hepatocytes.

[0017] patocytes. Evaluation of the effect of edelfosine on HDV replication in HepaRG cell cultures infected with HBV and HDV. Human hepatoma HepaRG cells were cultured as described in Gripon et al., PNAS 2002;99(24):15655-60. HepaRG cells were coinfected with an HBV inoculum prepared according to the method described previously 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, the cells were treated with edelfosine or bulevirtide and this treatment was repeated every other day until the eighth day of incubation. Each incubation condition was performed in triplicate.On day 10 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). The following [Fig.2] presents the combined results of two independent experiments, i.e., the mean and 95% confidence interval generated by six cell cultures for each experimental condition.

[0018]

[0019] [Fig.2]

[0020] [Fig. 2] shows that edelfosine between 0.039 pM and 2.5 pM did not reduce HDV replication. The decrease in the amount of HDV RNA measured in cultures treated with 10 pM edelfosine is probably related to cytotoxicity of edelfosine at this concentration as revealed by microscopic observation of the treated cells. In accordance with what is known about the 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 infected with HBV and HDV to Huh7.5-hNTCP cells. Human hepatoma HepaRG cells were cultured as described in Gripon et al., PNAS 2002;99(24):15655-60. Huh7.5-hNTCP cells resulted from hNTCP gene transfection of human hepatoma Huh7.5 cells cultured according to Blight et al., J Viral. 2003;77(5):3181-90. HepaRG cells were coinfected with an HBV inoculum prepared according to the method described previously 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 were treated with edelfosine or the reference substance lonafarnib and this treatment was repeated every other day until the eighth day of incubation. Each incubation condition was performed in triplicate. On the tenth day after infection, the HepaRG culture media supernatants were transferred to Huh7 cells.5-hNTCP cells and six days later HDV RNA was 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). The following [Fig.3] presents the combined results of three independent experiments, i.e. the mean and the 95% confidence interval generated by nine cell cultures for each experimental condition.

[0022] [Fig.3]

[0024] [Fig.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 decrease in HDV spread

[0025]

[0026]

[0027]

[0028] measured with 10 μM edelfosine-treated cultures is probably related to cytotoxicity of edelfosine at this concentration as revealed by microscopic observation of the treated cells. Edelfosine at 2.5 μM appears as effective as the reference substance lonafarnib at 10 μM in inhibiting the spread of HDV. Lo-nafarnib is a farnesyltransferase 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. In order to ensure that the inhibitory effect of edelfosine on the spread of HDV is not the consequence of a toxic effect of the substance on HepaRG cells, the cytotoxicity of edelfosine at active concentrations on HDV was evaluated on uninfected HepaRG cells. Cytotoxicity was measured with the Promega CellTiter-Glo® assay. The following [Fig. 4] presents the combined results of two independent experiments, i.e., the mean and the 95% confidence interval generated by sixteen cell cultures for each experimental condition. [Fig.4] [Fig. 4] shows that edelfosine induces significant cytotoxicity at concentrations above 5 pM on HepaRG cells. It can be deduced 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

Claims

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

Images