WO2025014360A1

WO2025014360A1 - Guanosine nucleotide analogs for use in preventing and/or treating hepatitis e virus infection

Info

Publication number: WO2025014360A1
Application number: PCT/NL2024/050364
Authority: WO
Inventors: Qiuwei Pan; Pengfei Li; Kuan LIU
Original assignee: Erasmus University Medical Center
Current assignee: Erasmus University Medical Center
Priority date: 2023-07-07
Filing date: 2024-07-05
Publication date: 2025-01-16
Anticipated expiration: 2026-01-07

Abstract

The present invention solves the problem of providing novel and improved therapeutic agents for preventing and/or treating Hepatitis E virus (HEV) infections. The present invention provides guanosine nucleotide analogs or a pharmaceutical composition comprising said analogs for preventing and/or treating HEV infections. The guanosine nucleotide analogs or a pharmaceutical composition comprising guanosine nucleotide analogs for preventing and/or treating HEV infections are for example administered in combination with one or more additional antivirals, such as IFN-α and/or ribavirin. Further, the present invention also provides a method of preventing and/or treating a HEV infection in a subject, comprising the step of administering a therapeutically effective amount of the guanosine nucleotide analogs to said subject.

Description

Title: Guanosine nucleotide analogs for use in preventing and/or treating Hepatitis E virus infection

FIELD OF THE INVENTION

The present invention relates to the field of antiviral therapy, in particular to the treatment of Hepatitis E virus (HEV) infection in humans. Particularly, the present invention relates to the use of the guanosine nucleotide analog a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof, in a method of preventing and/or treating a HEV infection.

BACKGROUND OF THE INVENTION

Hepatitis E virus (HEV) is a single-stranded positive-sense RNA virus. As a leading cause of acute viral hepatitis, approximately 939 million global populations have been ever experienced HEV infection according to a large-scale meta-analysis. Generally, HEV infections are asymptomatic or self -limiting in healthy individuals, but infections in specific populations are prone to develop severe complications and even death. In particular, infections with genotype 3 (GT3) HEV in immunocompromised patients can progress to fulminant or chronic hepatitis E, and these patients are mainly from European countries. Acute infection with genotype 1 (GT1) HEV in pregnant women can cause severe maternal and infant complications, resulting in up to 30% mortality in mother and child. These patients are mainly located in resource-limited regions. Considering the complicated health conditions in these specific populations and the lack of approved treatment, developing HEV therapeutics with favorable safety is of paramount importance.

Currently, mono- or combined administration of ribavirin and pegylated interferon-alpha (IFN-a) is explored as off-label therapeutic against chronic HEV infection in the clinic. Generally, ribavirin treatment can achieve sustained clearance of the viral RNA in most of the solid organ transplant patients who are eligible for the treatment. However, the newly emerged clinical mutant appears to have low sensitivity to ribavirin, leading to growing failure rates of ribavirin therapy against HEV infection. Moreover, ribavirin can cause substantial side effects and is contraindicative in pregnant women due to its teratogenicity. IFN-a administration in a small cohort of chronic hepatitis E patients has been shown to induce HEV viral clearance, but the considerable adverse effects and the risk of triggering acute rejection in organ transplant patients caution its application.

Therefore, there is an urgent need for further developing new therapeutics against HEV infections. Considering the substantial costs and slow pace of developing a new drug, re-purposing existing drugs for treating diseases such as hepatitis E is becoming increasingly an attractive option.

SUMMARY OF THE INVENTION

Unexpectedly, the inventors of the present invention have discovered that a guanosine nucleotide analog according to formula 3, preferably the guanosine nucleotide analog AT-527, or AT-752 or a combination thereof can be successfully employed to treat a HEV infection in a range of liver cells and organoids based models. Without wishing to be bound by theory, it is believed that the anti-HEV activity of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof function through impairing the nucleotide biosynthesis pathway of HEV. The Examples and Figures herein below show, inter alia, that a guanosine nucleotide analog according to formula 3, preferably AT- 527, AT-752, or a combination thereof are potent compounds to treat and/or prevent a HEV infection alone. Further, it is shown that the combination of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof with additional antivirals, for example IFN-a and/or ribavirin, provides advantageous synergistic effects for preventing and/or treating HEV infections.

Therefore, the present invention provides in a first aspect a guanosine nucleotide analog according to formula 3, preferably AT-527 or a salt, AT-752 or a salt, or a combination of AT-527 and AT-752, for use in a method of preventing and/or treating a HEV infection in a subject, preferably a human.

In preferred embodiments of the invention, the HEV infection is a GT1, GT2, GT3 and/or GT4 HEV infection, more preferably the HEV infection is a GT1 and/or GT3 infection. The HEV infection may be an acute or chronic HEV infection, preferably chronic.

In one embodiment of the invention, the guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof is administered as monotherapy or as sole antiviral compound of a pharmaceutical composition of the invention.

In another embodiment of the invention, the guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof is administered in combination with one or more additional antivirals. In preferred such embodiments, the additional antiviral is selected from the group consisting of protease inhibitors, interferon alpha (IFN-a), non-substrate based inhibitors, helicase inhibitors, direct-acting antivirals, antisense oligodeoxynucleotides (S-ODN), aptamers, nuclease resistant ribozymes, iRNA, inducing microRNA, siRNA, viral antigens, nucleoside analogues, antibodies and a combination thereof. Preferably, the additional antiviral is IFN-a and/or ribavirin.

In another embodiment of the invention, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof, optionally in combination with one or more additional antivirals, is administered for at least 3 days. In another embodiment of the invention, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof optionally in combination with one or more additional antivirals, is administered three times a day, twice daily, once daily, every two days, once weekly, or once every two weeks, preferably once daily, every two days.

In embodiments of the invention, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof is administered in an amount ranging from 5 mg to 2000 mg, preferably from 50 mg to 1000 mg, more preferably from 100 mg to 800 mg. In another aspect, the present invention provides a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof, for use in a method of preventing and/or treating a Hepatitis E virus (HEV) infection in a subject, preferably a human.

The invention also provides a method of preventing and/or treating a HEV infection in a subject, comprising the step of administering a therapeutically effective amount of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof to said subject.

All embodiments and/or aspects described in relation to a medical use of the invention also apply in relation to a pharmaceutical composition for said use and to methods of treatment of the invention (which amounts to a medical use of the invention).

The invention also provides a use of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for the manufacture of a medicament for preventing and/or treating a HEV infection in a subject. DESCRIPTION OF THE DRAWINGS

Figure 1. AT-527 inhibited HEV replication in Huh7-based cell models.

(A) The effects of AT-527 treatment for 24, 48 or 72 h on viral replication related luciferase activity in Huh7-p6-Luc cells of genotype 3 model. The untreated group serves as control (CTR) (set as 1) (n = 9). (B) The 50% inhibitory concentration (IC50) and 50% cytotoxic concentration (CC50) of AT-527 in Huh7-p6-Luc cell model and Huh7 cell line were calculated using GraphPad Prism software (n = 5-6). (C) The infectious Huh7-p6 cell model was treated with indicated concentrations of AT-527 (n = 6-8) or ribavirin (n=8) for 48 h. The effects on viral RNA were quantified by qRT-PCR. (D) Western blot analysis of HEV capsid protein level in Huh7- p6 cells treated with AT-527 for 48 h. The uninfected group (mock) serves as the negative control, whereas the infected but untreated group set as 1 (n = 4). (E) Immunofluorescence analysis of viral ORF2-encoded capsid protein (red) in Huh7 cells treated with 10 pM of AT-527 for 48 h. Huh7 cells incubated with the anti-HEV capsid protein antibody serves as Mock. HEV infected Huh7 cells untreated and incubated with the anti-HEV capsid protein antibody serves as the positive control. DAPI (blue) was applied to visualize nuclei. (Scale bar, 100 pm. 40x oil immersion objective). (F) The viral replication-related luciferase activity of a Huh7 cell-based genotype 1 HEV replicon (Sar55 clone) was assessed after treatment with AT-527 for 24, 48, or 72 hours, the untreated group serves as control (CTR) (set as 1) (n = 8). (G) Naive Huh7 cells were inoculated with HEV particles produced from HEV infected Huh7 cells 48 h post-treatment with different concentrations of AT-527(untreated set as control group). After the inoculation for 48 h, the Huh7 cells were lysed to qRT-PCR analysis of HEV RNA (n = 4) and (H) Immunofluorescence analysis of HEV ORF2-encoded capsid protein. (Scale bar, 100 pm. 40x oil immersion objective) RLU: relative luciferase unit. The results are shown as means ± SEM. (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 2. Long-term effects of AT-527 on different Huh7-based HEV models.

(A) The effects of 7 days treatment with AT-527 on GT3 Huh7-p6- Luc model were quantified by luciferase units. (B) The effects of 21 days treatment with AT 527 on GT3 Huh7-p6 model were measured by qRT-PCR, the untreated (CTR) group serve as control (set as 1) (n = 4). (C) Western blot analysis of HEV ORF2-encoded capsid protein was performed on 10 days and 21 days of treatment. (D) The effects of 7 days with AT-527 on GT1 HEV were tested by luciferase units, the untreated (CTR) group serve as control (set as 1) (n=4).

Figure 3. AT-527 suppressed HEV replication in human liver organoids.

(A) Schematic representation of the HEV infection in human liver organoids. (B) The effects of AT-527 treatment for 24, 48 or 72 hours on viral replication related luciferase activity in GT3 HEV subgenomic replicon model in Ever organoids. The untreated group serves as control (CTR) (set as 1) (n=4) (C) The human liver organoids harboring full length genomic RNA was treated with indicated concentrations of AT-527 for 48 h. The effects on viral RNA were quantified by qRT-PCR (n =4). (D) Immunofluorescence analysis of viral ORF2-encoded capsid protein after 10 pM AT-527 48h treatment in human liver organoids. Uninfected liver organoids incubated with the anti-HEV capsid protein antibody serves as mock. Untreated HEV infected liver organoids serve as the positive control. DAPI (blue) and Ep CAM (green) were applied to visualize nuclei and cytomembrane. (Scale bar, 100 pm. 40x oil immersion objective). (E) The viral replication-related luciferase activity of GT1 HEV replicon in liver organoids (Sar55 clone) was assessed after treatment with AT-527 for 24, 48, or 72 hours (n = 4). Data are presented as means ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 4. AT-527 inhibited HEV variant in cell models and liver organoids.

(A) The infectious Huh7-p6G1634R cell model was treated with indicated concentrations of AT-527(n = 4) or ribavirin (n=4) for 48 h. The effects on viral RNA were quantified by qRT-PCR. (B) Immunofluorescence analysis of HEV ORF2-encoded capsid protein in Huh7-p6G1634R cells treated with 10 pM AT-527 for 48 h. (C) Naive Huh7 cells were inoculated with HEV particles performed from HEV infected Huh7 cells 48 h posttreatment with 10 pM of AT-527(untreated set as control group). After the inoculation for 48 h, the Huh7 cells were used for immunofluorescence analysis of HEV ORF2 protein. Huh7 cells incubated with the anti-HEV capsid protein antibody serves as Mock. HEV infected Huh7 cells untreated and incubated with the anti-HEV capsid protein antibody serves as the positive control, the nuclei were seen using DAPI (blue). (Scale bar, 100 pm. 40x oil immersion objective). (D) The infectious p6 human liver organoids were treated with indicated concentrations of AT-527 for 48 h. The effects on viral RNA were quantified by qRT-PCR (n =4). (E) Immunofluorescence analysis of viral ORF2-encoded capsid protein after 10 pM AT-527 48h treatment in human liver organoids. Uninfected liver organoids incubated with the anti-HEV capsid protein antibody serves as mock. Untreated HEV infected liver organoids serve as the positive control. DAPI (blue) and Ep CAM (green) were employed to visualize nuclei and cytomembrane (Scale bar, 100 pm. 40x oil immersion objective). The results are shown as means ± SEM. (*P < 0.05; **P < 0.01; ***P < 0.001). Figure 5. Combination of AT-527 with other antivirals and guanosine supplementation.

(A) Schematic representation of drug combination treatment on huh 7 harboring p6 luciferase model. (B) The antiviral effects of AT-527 in combination with ribavirin for two days, synergy distribution of pairwise combination of AT-527 and ribavirin (n = 4). (C) The antiviral effects of AT- 527 in combination with IFN-a for two days, synergy distribution of pairwise combination of IFN-a and AT-527(n = 4). (D, E) HEV RNA in Huh7-p6 cells and organoids harboring p6 was quantified by qRT-PCR after 48 h of treatment with 10 pM AT-527 by adding 1 pg/ml exogenous guanosine (n = 4). Data were normalized to the untreated control (set as 1) and presented as means ± SEM. (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 6. AT-511 and AT-752 inhibited HEV replication in Huh7- based cell models.

(A) The infectious Huh7-p6 cell model was treated with indicated concentrations of AT-511 (n = 4) for 48 hours. The effects on HEV viral RNA were quantified by qRT-PCR. *P<0.05. (B) The infectious Huh7-p6 cell model was treated with indicated concentrations of AT-752 (n = 4) for 48 hour. The effects on HEV viral RNA were quantified by qRT-PCR (n = 4). *P<0.05. Untreated (CTR) group serves as control (set as relative RNA level 1).

Figure 7. Comparison of long-term effects of AT-527 and Ribavirin on GT3 Huh7-p6 model.

The effects of 21 days treatment with AT 527 and Ribavirin on GT3 Huh7-p6 model were measured by qRT-PCR of HEV viral RNA levels; the untreated (CTR) group serves as control (set as relative RNA level 1) (n = 2). DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term ‘AT-527’, as used herein, refers to the hemi-sulfate salt of the B-D-2'-deoxy-2'-a-fluoro-2'-B-C-substituted-2-modified-N⁶-mono-methyl purine nucleotide of formula 1:

AT-527

The term ‘AT-527’, as used herein, includes reference to the dissolved free base compound (AT-511) as well as other pharmaceutically acceptable salts or prodrugs of the free base (AT-511) that metabolize in vivo to the active triphosphate metabolite (AT-9010). The compounds and methods for their manufacture are disclosed in WO2016/144918, US9,828,410, WO2018/048937, and WO2018/144640. AT-527 is actively metabolized in vivo to AT-9010 exhibiting viral RNA polymerase inhibitor activity against enveloped positive single-stranded RNA viruses, such as human coronaviruses and human flaviviruses, as described in Good et al., 2020 (PLOS ONE 15(1): e0227104). AT-527 is commercialized under the name bemnifosbuvir by Atea Pharmaceuticals Inc., Boston, USA.

The term ‘AT-752’, as used herein, refers to the hemi-sulfate salt of B-D-2'-deoxy-2'-a-fluoro-2'-B-C-substituted-2-modified-N⁶-mono-methyl purine nucleotide of formula 2:

The term ‘AT-752’, as used herein, includes reference to the dissolved free base compound (AT-281) as well as other pharmaceutically acceptable salts or prodrugs of the free base (AT-281) that metabolize in vivo to the active triphosphate metabolite (AT-9010).

The term ‘pharmaceutically acceptable salt’ or ‘prodrug’ is used to describe any pharmaceutically acceptable form (such as an ester, phosphoramidate, thiophosphoramidate, phosphate ester, salt of an ester, or a related group) of AT-511 or AT-281 which, upon administration to a patient, provides the desired active compound AT-9010. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids, which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a-ketoglutarate, and a-glycerophosphate. Suitable inorganic salts may also be formed, including sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium, or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made. Preferably, the pharmaceutically acceptable salt is a hemisulfate.

‘Pharmaceutically acceptable prodrug’ refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host to form the compound AT-9010 from AT-511 or AT-281. Typical examples of prodrugs include compounds that have biologically labile protecting groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated, thiophoshoramidated, dethiophoshoramidated, phoshoramidated or dephosphoramidated to produce the active compound. The compounds useful in this invention possess antiviral activity against HEV, or are metabolized to a compound that exhibits such activity.

The terms ‘treatment’ and ‘treating’, as used herein, include reference to the application of a form of therapy to a subject, with the object of e.g. curing the patient from a disease, halting or slowing down the development of a disease, prolonging the life of a subject, or relieving pain in a subject suffering from a disease or injury. Prophylactic treatment, therapy with the aim of preventing induction or onset of a disease, is also to be understood to be part of the term ‘treatment’.

The terms ‘prevention’ and ‘preventing’, as used herein, includes reference to the application of a form of therapy to a subject, with the object to hinder the outbreak of a disease, infection etc. The terms refer to the application of a form of therapy to a subject to prevent and/or reduce the likelihood of an occurrence of a disease, for example a HEV infection. The terms include proactive and prophylactic treatment and/or application of an active component in order to hinder outbreak of a disease, such as an infection in a subject and to immunize the subject.

The term ‘active component’ and ‘active compound’, as used herein, refers to an ingredient, drug, and/or molecule, which is able to trigger and/or facilitate a therapeutic effect in a subject.

The term ‘subject’, as used herein, refers to a human or animal susceptible to infection with HEV. The term ‘subject’, as used herein, includes reference to a recipient of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof or a pharmaceutical composition comprising the guanosine nucleotide analog AT-527, AT-752 or a combination thereof as described herein, i.e. a subject that is suffering, or suspected of suffering, from HEV. For example, ‘subject’ includes subjects in which HEV infection is prevented. For example, the subject is a mammal, more preferably a human. The terms ‘patient’ and ‘subject’ can be used interchangeably herein. The subject is for example a human, i.e., a human having a HEV infection and/or at risk getting a HEV infection. The subject is for example older or younger than 50 years old. For example the subject is older than 50 years old. For example, the subject is human suffering from an acute HEV infection. For example, the subject is a human suffering from a chronic HEV infection. For example the subject is male or female. For example the subject is immunocompromised. For example, the subject is pregnant, is suffering from cancer, is undergoing cancer treatment, such as chemotherapy, immunotherapy and/or radiation therapy, has an organ transplant, is undergoing an organ transplantation, is suffering from an autoimmune disease, or has a combination of those impheations.

The term ‘combination’ or ‘combination therapy’, as used herein, includes reference to using a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof as disclosed herein and additional antivirals, for example IFN-a and/or ribavirin, as disclosed herein in the same medical treatment. The term ‘combination’ or ‘combination therapy’, as used herein, includes reference to a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof, and additional antivirals, for example IFN-a and/or ribavirin. A guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof and additional antivirals, for example IFN-a and/or ribavirin, as disclosed herein is for example administered together at the same time (such as in the form of a single pharmaceutical composition), separately of each other at the same time (for instance in the form of separate pharmaceutical composition) or separately of each other staggered in time. Simultaneous, separate or sequential administration of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof and additional antivirals, such as IFN-a and/or ribavirin, as disclosed herein in the same treatment schedule are expressly envisaged. As an example, the time between administration of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof, and additional antivirals, such as IFN-a and/or ribavirin, is at least one minute, at least fifteen minutes, at least sixty minutes, at least four hours, at least one day, at least one week or at least one month or at least one year, or anywhere in between such as between one minute and one year. For example, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof is administered prior to administration of additional antivirals, such as IFN-a and/or ribavirin. Alternatively, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof, is administered together with, or after, administration of additional antivirals, such as IFN-a and/or ribavirin.

The term ‘antiviral’ or ‘antiviral drug’, as used herein, refers to a class of active compounds used for preventing and/or treating viral infections, such as HEV infections. For example, the ‘antiviral’ is a broadspectrum antiviral. Broad-spectrum antiviral means that the antiviral is effective against a wide range of viruses. For example, the antiviral is virus specific. For example the antiviral is specific to HEV. For example, the antiviral is selected from the group consisting of protease inhibitors, interferon alpha (IFN-a), non-substrate based inhibitors, helicase inhibitors, direct-acting antivirals, antisense oligodeoxynucleotides (S-ODN), aptamers, nuclease resistant ribozymes, iRNA, inducing microRNA, siRNA, viral antigens, nucleoside analogues, antibodies and a combination thereof. For example, the antiviral is a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof, IFN-a, ribavirin, telaprevir, boceprevir, simeprevir, paritaprevir, glecaprevir, ritonavir, ACH- 2684, AZD-7295, BMS-791325, danoprevir, filibuvir, GS-9256, GS-9451, MK-5172, setrobuvir, sovaprevir, tegobuvir, VX-135, VX-222, ALS-220, ACH-2928, ACH-3102, IDX-719, daclatasvir, ledispasvir, velpatasvir, elbasvir (MK-8742), grazoprevir (MK-5172), ombitasvir (ABT-267), AZD- 7295, clemizole, dasabuvir, ITX-5061, PPI-461, PPI-688, sofosbuvir, MK- 3682, mericitabine, ABT-333, MBX-700, GS-6624, or a combination thereof.

The term ‘therapeutically effective amount’, as used herein, means that the amount of active compounds administered is of sufficient quantity to achieve the intended purpose, such as, in this case, for a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof, to prevent and/or treat a HEV infection. The amount of compound included within therapeutically active formulations according to the present invention is an effective amount for treating the HEV infection, reducing the likelihood of a HEV infection or the inhibition, reduction, and/or abolition of HEV or its secondary effects, including disease states, conditions, and/or complications which occur secondary to a HEV infection. In general, a therapeutically effective amount of the present compound in pharmaceutical dosage form usually ranges from about 0.001 mg/kg to about 100 mg/kg per day or more, more often, slightly less than about 0.1 mg/kg to more than about 25 mg/kg per day of the patient or considerably more, depending upon the compound used, the condition or infection treated and the route of administration. The active nucleoside compound according to the present invention is often administered in amounts ranging from about 0.1 mg/kg to about 15 mg/kg per day of the patient, depending upon the pharmacokinetics of the agent in the patient. This dosage range generally produces effective blood level concentrations of active compound which may range from about 0.001 to about 100, about 0.05 to about 100 micrograms/cc of blood in the patient.

The term ‘administration’ or ‘administering’, as used herein, includes reference to the application of a substance or composition to a subject. Main routes of administration are parenteral administration, enteral or gastrointestinal administration and topical administration. The term ‘parenteral’, as used herein, includes reference to any form of administration that is not via the application onto the skin or via the gastrointestinal tract. Non-limiting examples of parenteral administration include epidural, intracerebral, intracerebroventricular, epicutaneous, sublingual, extra- amniotic, nasal, intra-arterial, intra- articular, intracardiac, intracavernous, intradermal, intralesional, intramuscular, intraocular, intraosseous, intraperitoneal, intrathecal, intrauterine, intravaginal, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular, transmucosal, or rectal administration. The term ‘intravenous’, as used herein, includes reference to a parenteral route of administration wherein a substance or composition is injected into the vein of a subject, for example using a hollow needle. The substance or composition that is administered intravenously will directly reach the blood stream of the subject. For example, the guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof, or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof is administered orally.

Throughout this specification and the claims, unless the context requires otherwise, the word ’comprise’, and variations such as ’comprises’ and ’comprising’, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The terms ’a’ and ’an’ and ’the’ and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., ’such as’, ’for example’), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

All documents cited or referenced herein (’herein cited documents’), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

In the following, the features of the present invention will be described in more detail. It should be understood that embodiments may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed features. Furthermore, any permutations and combinations of all described features in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

The present invention solves the problem of providing a novel and improved therapeutic agent for preventing and/or treating HEV infections. The inventors of the present invention surprisingly identified a therapeutic effect of a guanosine nucleotide analog according to formula 3, preferably the guanosine nucleotide analog AT-527, AT-752 or a combination thereof for preventing and/or treating HEV infections.

formula 3.

AT-527 was originally designed to inhibit hepatitis C virus (HCV) replication by targeting the viral RNA polymerase. Monotherapy of AT-527 has been investigated in clinical trials for treating HCV infection showing excellent safety and reasonable efficacy profiles, but failed to proceed further in developing into an anti-HCV therapeutics (Berliba et al., 2019, Antimicrob Agents Chemother 63). Currently, AT-527 is undergoing clinical trials for treating COVID- 19 patients infected with SARS-CoV-2 (Han et al., 2021, Theranostics 11, 1207-1231; Shannon et al., 2022, Nat Commun 13, 621). Largely clinical trials confirmed its safety profile, but failed to meet the primary goal of reduction from baseline in the amount of SARS-CoV-2 virus in patients with mild or moderate COVID- 19 compared to placebo in the overall study population.

Hepatitis E virus (HEV) and Hepatitis C virus (HCV) are distinct RNA viruses that differ in classification, viral structure, transmission, clinical outcomes, and epidemiology. HEV, a nonenveloped virus (33 nm, 7.2 kb genome), belongs to the Hepeviridae family, while the enveloped HCV (60 nm, 9.5 kb genome) is classified under Flaviviridae. These viruses have dissimilar life cycles, reflected in differences in viremia duration. HCV exclusively infects human, whereas many HEV strains are zoonotic that animals such as swine serve as reservoirs. Differences in the severity of hepatitis and in the magnitude and duration of elevations in levels of alanine aminotransferase (ALT) between the two infections confirm that the two viruses differ in their abilities to stimulate the adaptive immune response and related mechanisms to clear the virus (Yu et al., 2010, J Virol. 84(21): 11264-11278).

Accordingly, it is the surprising achievement of the inventors of the present invention to have identified a therapeutic effect of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating HEV infections.

The present invention provides a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof, for use in a method of preventing and/or treating a HEV infection. The present invention also provides a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752 or a combination thereof, for use in a method of preventing and/or treating a HEV infection. The present invention also refers to a method of preventing and/or a treating HEV infection in a subject comprising the step of administering a therapeutically effective amount of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof, to said subject. Further, the present invention refers to the use of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof, in the manufacture of a medicament for preventing and/or treating a HEV infection.

Surprisingly a guanosine nucleotide analog according to formula 3, preferably AT-527, AT752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT752 or a combination thereof, facilitates the therapeutic effect to prevent and/or treat a HEV infection in a subject. The guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT752, or a combination thereof is pharmaceutically safe and has no adverse side effects. Thus, the present invention provides a new and improved active compound for preventing and/or treating a HEV infection. A guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752 or a combination thereof according to the present invention solves the problem of an urgent need for further and improved therapeutics against HEV infections.

The present invention relates to a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of treating a HEV infection in a subject. The novel guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752 or a combination thereof, has a significant antiviral effect against HEV. A guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof according to the present invention provides a potent inhibition of HEV.

The present invention further relates to a method of treating HEV infection in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof.

HEV infection is for example a liver infection caused by the Hepatitis E virus. HEV infection can lead to acute and chronic hepatitis as well as to extrahepatic manifestations such as neurological and renal disease. HEV infection can even lead to the death of the infected subject. HEV has at least four different natural genotypes (GT): Genotypes 1 - 4. The GT3 HEV harbors a G1634R mutation.

In embodiments of this invention, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof is for use in preventing and/or treating a HEV infection caused by GT1 HEV, GT2 HEV, GT3 HEV, GT4 HEV or a combination thereof. In preferred embodiments, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof is for use in preventing and/or treating HEV infections caused by GT1 HEV and/or GT3 HEV.

In embodiments of this invention, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT752 or a combination thereof, or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof is for use in preventing and/or treating a HEV infection in a subject having an impaired immune response. For example, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof is for use in preventing and/or treating a HEV infection in an immunocompromised subject. For example, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752 or a combination thereof is for use in preventing and/or treating a HEV infection in a subject which is at a high risk of developing a chronic HEV infection. In other embodiments, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-572, or a combination thereof is for use in preventing and/or treating a HEV infection in a subject which is pregnant, suffering from an autoimmune disease, suffering from cancer, having an organ transplant, undergoing an organ transplantation, undergoing an immunotherapy, chemotherapy, radiotherapy, performance of a surgery, or a combination thereof. For example, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, or AT-752, or combination thereof is for use in preventing and/or treating a HEV infection in a subject which is pregnant. Preferably, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or combination is for use in preventing and/or treating a HEV infection, e.g., caused by GT1 and/or GT3 HEV, in a subject which is pregnant. In embodiments of this invention, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof is for use in preventing and/or treating a HEV infection in an immunocompromised subject. Preferably, a guanosine nucleotide analog according to formula 3, preferably AT-527, or AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof is for use in preventing and/or treating a HEV infection, e.g., caused by GT1 and/or GT3 HEV, in an immunocompromised subject.

In embodiments of this invention, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or combination thereof is the sole active ingredient of a pharmaceutical composition of the invention.

In embodiments of this invention, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is administered in combination with an additional antiviral. For example, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752, or a combination thereof or a pharmaceutical composition a guanosine nucleotide analog according to formula 3, preferably comprising AT-527, AT- 752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is administered in combination with an additional antiviral selected from the group consisting of protease inhibitors, interferon alpha (IFN-a), non-substrate based inhibitors, direct-acting antivirals, helicase inhibitors, antisense oligodeoxynucleotides (S-ODN), aptamers, nuclease resistant ribozymes, iRNA, inducing microRNA, siRNA, viral antigens, nucleoside analogues, antibodies and a combination thereof. The additional antiviral is for example a protease inhibitor selected from the group consisting of telaprevir, boceprevir, simeprevir, paritaprevir, glecaprevir, ritonavir, ACH-2684, AZD-7295, BMS-791325, danoprevir, filibuvir, GS-9256, GS-9451, MK-5172, setrobuvir, sovaprevir, tegobuvir, VX-135, VX-222, ALS-220 and a combination thereof. The additional antiviral is also for example a NS5A inhibitor selected from the group consisting of ACH-2928, ACH-3102, IDX-719, daclatasvir, ledispasvir, velpatasvir, elbasvir (MK-8742), grazoprevir (MK-5172), ombitasvir (ABT- 267) and a combination thereof. The additional antiviral is for example a NS5B inhibitor selected from the group consisting of AZD-7295, clemizole, dasabuvir, ITX-5061, PPI-461, PPI-688, sofosbuvir, MK-3682, mericitabine, ABT-333, MBX-700 and a combination thereof. The additional antiviral is for example an antibody such as GS-6624. The additional antiviral is for example an IFN-a or an albuferon, preferably a human IFN-a subtype, preferably selected from the group consisting of IFN-a 1 to IFN-a-21. The additional antiviral is for example a nucleoside analogue such as ribavirin. In embodiments of this invention, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is administered in combination with an additional antiviral selected from the group consisting of IFN-a, ribavirin, telaprevir, boceprevir, simeprevir, paritaprevir, glecaprevir, ritonavir, ACH-2684, AZD-7295, BMS-791325, danoprevir, filibuvir, GS-9256, GS-9451, MK-5172, setrobuvir, sovaprevir, tegobuvir, VX-135, VX-222, ALS-220, ACH-2928, ACH-3102, IDX-719, daclatasvir, ledispasvir, velpatasvir, elbasvir (MK-8742), grazoprevir (MK- 5172), ombitasvir (ABT-267), AZD-7295, clemizole, dasabuvir, ITX-5061, PPI-461, PPI-688, sofosbuvir, MK-3682, mericitabine, ABT-333, MBX-700, GS-6624 and a combination thereof. For example, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is administered in combination with IFN-a and/or ribavirin. For example, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is administered in combination with IFN-a. For example, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is administered in combination with ribavirin. For example, a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752, or a combination thereof for use in a method of preventing and/or treating a HEV infection further comprises an additional antiviral selected from the group consisting of IFN-a, ribavirin, telaprevir, boceprevir, simeprevir, paritaprevir, glecaprevir, ritonavir, ACH-2684, AZD-7295, BMS- 791325, danoprevir, filibuvir, GS-9256, GS-9451, MK-5172, setrobuvir, sovaprevir, tegobuvir, VX-135, VX-222, ALS-220, ACH-2928, ACH-3102, IDX-719, daclatasvir, ledispasvir, velpatasvir, elbasvir (MK-8742), grazoprevir (MK-5172), ombitasvir (ABT-267), AZD-7295, clemizole, dasabuvir, ITX-5061, PPI-461, PPI-688, sofosbuvir, MK-3682, mericitabine, ABT-333, MBX-700, GS-6624 and a combination thereof. For example, a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection further comprises IFN-a and/or ribavirin. For example, a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection further comprises IFN-a. For example, a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection further comprises ribavirin.

A guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is for example administered in a pharmaceutical dosage form which ranges from 0.001 mg/kg to 100 mg/kg per day, optionally from 0.01 mg/kg to 25 mg/kg per day, optionally from 0.1 mg/kg to 15 mg/kg per day. A guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is for example administered as a dosage in an amount ranging from 5 mg to 2000 mg, optionally from 20 mg to 1500 mg, optionally from 50 mg to 1000 mg, optionally from 100 mg to 800 mg. For example, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is administered as a dosage of 5, 10, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1100, 1130, 1500, 1750 or 2000 mg. For example, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is administered as a dosage of 100 to 800 mg at least once, at least twice, at least three times per day.

A guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is for example administered at least once per day for at least 1 days, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, or at least 12 weeks. A guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752, or a combination thereof, or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is for example administered orally, parenterally, topically, or in suppository form, as well as intranasally, as a nasal spray. Preferably, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof is administered orally, preferably in solid dosage form such as a pill or tablet. A guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is for example administered in a tablet, capsule, injection, intravenous formulation, suspension, liquid, emulsion, implant, particle, sphere, cream, ointment, suppository, inhalable form, transdermal form, buccal, sublingual, topical, gel, mucosal, and the like.

The one or more additional antiviral which is administered in combination with a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof is for example administered in an amount ranging from 5 mg to 2000 mg, optionally from 20 mg to 1500 mg, optionally from 50 mg to 1000 mg, optionally from 100 mg to 800 mg. For example, the one or more additional antiviral is administered in combination with a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof as a dosage of 5, 10, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1100, 1200, 1300, 1400, or 1500mg. For example, the one or more additional antiviral compound is administered in combination with a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for at least 1 days, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, or at least 12 weeks. For example, the a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof or a pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT- 752, or a combination thereof for use in a method of preventing and/or treating a HEV infection is administered in combination with IFN-a and/or ribavirin, wherein a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof as well as IFN-a and/or ribavirin are administered in an amount ranging from 5 mg to 2000 mg, optionally from 20 mg to 1500 mg, optionally from 50 mg to 1000 mg, optionally from 100 mg to 800 mg.

The pharmaceutical composition comprising a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof for use in a method of preventing and/or treating a HEV infection for example further comprises a pharmaceutical acceptable carrier. The pharmaceutical acceptable carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral. The pharmaceutical acceptable carrier comprised in the pharmaceutical composition according to the present invention is for example selected from the group consisting of water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, starches, sugar carriers, such as dextrose, manifold, lactose, diluents, granulating agents, lubricants, binders, disintegrating agents and combinations thereof.

The invention further relates to a method of treatment of patients suffering from HEV infection, comprising administering a therapeutically effective dose of a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof to a subject in need thereof.

In embodiments of this invention, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof is administered orally twice daily (preferably about every 12 hours) in a single dose of 500 mg or 1000 mg to a subject with acute infection of HEV for a total of 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days.

In embodiments of this invention, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof is administered orally once daily (preferably about every 24 hours) in a single dose of 500 mg to a subject with chronic infection of HEV for a total of 1 to 12 months, in particular for a total of 1 month, 3 months, 6 months, or 12 months. Alternatively, a guanosine nucleotide analog according to formula 3, preferably AT-527, AT-752, or a combination thereof is administered orally twice daily (preferably about every 12 hours) in a single dose of 500 mg to a subject with chronic infection of HEV for a total of 1 to 12 months, in particular for a total of 1 month, 3 months, 6 months, or 12 months. The invention will now be exemplified in the following examples which are included by way of illustration only.

EXAMPLES

Material & Methods

Reagents and antibodies

AT-527 and AT-752 were obtained from MedchemExpress, Monmouth Junction, NJ, USA (Cat. No.: HY-137958) and dissolved in dimethyl sulfoxide (DMSO). Ribavirin and Human IFN-a (Interferon a (IFN- a) 2B human, H6166-10UG, Sigma-Aldrich Chemie BV) were purchased from Sigma- Aldrich, Saint Louis, USA, and dissolved in DMSO and PBS respectively. The anti-HEV ORF2 protein antibody (clone 2E2, MAB- 8002, mouse mAB, EMD Millipore), B-actin antibody ((C4): sc-47778, mouse mAB Santa Cruz Biotechnology), anti-EpCAM antibody (Rabbit mAB,[EPR20532-225] (ab223582) Abeam), anti-mouse IRDye-conjugated secondary antibodies (Li-Cor Biosciences), anti-rabbit IgG (H+L, Alexa Fluor® 488), anti-mouse IgG (H+L, Alexa Fluor® 594) and DAPI (4, 6- diamidino-2 -phenylin dole; Invitrogen) were used for immunofluorescence staining.

Viruses and HEV models

Plasmid constructs containing the full-length HEV genome (Kernow- C1 p6 clone; GenBank Accession Number JQ679013), variant harboring an RNA-dependent RNA polymerase mutation G1634R (p6G1634R), subgenomic GT3 HEV replicon with a Gaussia luciferase reporter gene (p6Luc), subgenomic GT1 HEV replicon harboring Gaussia luciferase reporter gene (GTILuc), were transcribed into genomic RNA respectively by enzyme-digested and linearized plasmid DNA using mMessage mMachine T7 RNA kits (Invitrogen) (Cordoba et al., 2012, J Gen Virol 93, 2183-2194; Li et al., 2019, Hepatol Commun 3, 160-172; Li et al., 2016, J Hepatol 65, 1104-1111; Shukla et al., 2012, J Virol 86, 5697-5707). Next, genomic RNA was introduced into huh7 or human-derived liver organoids by electroporation as previous protocol (Li et al., 2022a). Luciferase activity was detected by BioLux Gaussia Luciferase Flex Assay Kit (New England Biolabs, Ipswich, MA, USA) through measuring the secreted luciferase units of supernatants.

Cell culture

Huh 7 cell line was cultured in DMEM supplemented with 10% (v/v) fetal calf serum (FCS) (Hyclone, Logan, UT, USA), 100 lU/ml penicillin and 100 lU/ml streptomycin. For human liver-derived organoids, tissue samples used for organoids isolation and culture were collected during liver transplantation at the Erasmus Medical Center Rotterdam. The use of liver tissues for research purposes was approved by the Medical Ethical Council of the Erasmus MC and informed consent was given (MEC-2014-060). Liver- derived organoids were isolated and cultured as previously described (Huch et al., 2015, Cell 160, 299-312). Briefly, organoids were cultured in organoid expansion medium (EM), based on Advanced DMEM/F12 (Invitrogen), complemented with 1% penicillin/streptomycin (Life Technologies), 1 M HEPES (Life Technologies), 200 mM Ultraglutamine (Life Technologies), 1% (vol/vol) of N2 (Gibco), 2% (vol/vol) of B27 (Gibco), 1 mM N- acetylcysteine (Sigma-Aldrich), 10 mM Nicotinamide (Sigma-Aldrich), 5 jiM A83.01 (Tocris), 10 jiM Forskolin (Tocris), 10 nM Gastrin (Sigma-Aldrich), 50 ng/ml EGF (P eprotech), 10% (vol/vol) of R-spondin-1 (conditioned medium), 100 ng/ml FGF10 (Peprotech), 25 ng/ml HGF (Peprotech) and 10 jiM Y27632 (Si gm a - Al drich) .

Quantification of gene expression

Total RNA was isolated by using Macherey-Nagel NucleoSpinSRNA II kit (Bioke, Leiden, Netherlands) and quantified by Nanodrop ND- 1000 (Wilmington, DE, USA). cDNA was produced by cDNA Synthesis Kit (Takara Bio Inc, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as housekeeping gene to normalize relative gene expression by using the formula 2'^AACT(AACT = ACTsample - ACTcontrol). The HEV primer sequences were 5'- GGTGGTTTCTGGGGTGAC-3’ (sense) and 5'-AGGGGTTGGTTGGATGAA-3’ (antisense), and the primers of GAPDH were 5'-GTCTCCTCTGACTTCAACAGCG-3’ (sense) and 5'- ACCACCCTGTTGCTGTAGCCAA-3’ (antisense).

MTT assay

Cells were plated in 96-well plates and treated with different concentration of AT-57 for 72 hours. Then 10 pL 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolipM bromide (MTT) (Sigma) was added to each well for 3 hours. Subsequently, supernatants were replaced by 100 pL DMSO. The absorbance of each well was read on the microplate absorbance reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 490 nm.

Western blot

Samples were lysed on ice for 10 minutes, followed by heating at 95 °C for 5 min. Loading samples onto 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). First 20 minutes at 120V, afterwards 100V for running. And blotting samples onto a poly vinylidene difluoride (PVDF) membrane (pore size: 0.45 mm; Thermo Fisher Scientific Life Sciences) for 120 min with an electric current of 250 mA. Next, the membrane was blocked with blocking buffer (Li-Cor Biosciences) for 1 hour then incubated with primary antibody overnight. Afterwards, the membrane was washed 3 times and subsequently added secondary antibody at room temperature for 1 hour. After washing 3 times, protein bands were detected with Odyssey 3.0 Infrared Imaging System. Immunofluorescence assay

Cells and organoids were spun down onto slides and fixed in 4% paraformaldehyde solution at 4 °C for 10 min, followed by permeabilizing with PBS containing 0.2% (vol/vol) tritonXIOO for 5 min. The slides were rinsed three times in PBS for five minutes each time, then permeabilized in PBS containing 0.2% (vol/vol) tritonXIOO for 5 minutes. Then slides were then rinsed twice with PBS for 5 minutes before being incubated for 1 hour at room temperature with blocking solution (5 % donkey serum, 1 % bovine serum albumin, 0.2 % tritonXIOO in PBS). The slides were then incubated in a humidity chamber overnight at 4 °C with primary antibody diluted in blocking solution. Slides were washed 3 times for 5 min each in PBS before 1 h incubation with 1:1000 dilutions of the anti-mouse IgG and the antirabbit IgG secondary antibodies. Nuclei were stained with DAPI.

Re-infection assay

Infectious HEV particles were harvested from Huh 7 cells harboring p6 full-length HEV RNA by repeated freezing and thawing. Harvested viruses were added to inoculate Naive Huh 7 cells 24h for re-infection, followed by 3 times 1 x PBS washing. Then cells were cultured for 48 hours incubated with DMEM supplemented with 2% FCS.

Statistics

All graphs were shown as Mean ± SEM. The statistical significance of differences between means was assessed with the Mann-Whitney test (GraphPad Prism; GraphPad Software Inc., La Jolla, CA). The threshold for statistical significance was defined as P < 0.05. Synergistic scores of drug combinations were analyzed by SynergyFinder 2.0(Ianevski et al., 2020).

Results

AT-527, AT-752 and AT-511 exert potent anti-HEV activity in cell models To examine the antiviral activity of AT-527 against HEV replication, we first employed a GT3 HEV subgenomic replicon model (p6Luc) in huh 7 cells. We found AT-527 dose-dependently inhibited virus replication, by detecting HEV replicating-related luciferase activity at 24, 48 and 72 hours after treatment (Fig 1 A). The inhibition efficacy was slightly enhanced over time, with half maximal inhibitory concentration (IC50) of 6.978 pM after treating for 72 hours (Fig 1 B). Importantly, the wide window between IC50 and half maximal cytotoxic concentration (CC50; noticeable cytotoxicity was absent with the tested concentrations) highlights the favorable safety of AT- 527 (Fig 1 B). Consistently, AT-527, AT-752 and AT-511 significantly inhibited HEV replication at viral RNA level in huh7 cells harboring GT3 infectious full-length genome RNA (AT-527:Fig 1 C, AT-511: Fig 6 A; AT- 752: Fig 6B). At same concentrations, ribavirin exerted much less inhibition against HEV compared to AT-527. These results were further validated by western blotting and immunofluorescence (Fig 1 D and E). Similarly, the potent inhibition on GT1 HEV was demonstrated in huh7 cells harboring GT1 subgenomic replicon (GTILuc), with 10 pM inhibited over 50% replication at 72 hours post-treatment (Fig IF). Next, we performed a reinfection assay, by inoculating huh7 cells with HEV particles from a huh7- p6 model after AT-527 treatment for 48 hours. We observed HEV replication was largely blocked by AT-527, demonstrated by qPCR and immunostaining (Fig 1 G and H). Collectively, AT-527 is a potent inhibitor against GT1 and GT 3 HEV infection.

AT-527 completely inhibits HEV during a long-term treatment

The long-term efficiency of antiviral treatment is particularly important for treating chronic HEV infection in immunocompromised populations. In this aspect, we investigated the long-term anti-HEV performance of AT-527 in huh7 cells. By administrating 1 pM and 10 pM of AT-527 for 7 days in huh7 based HEV replication model, we found HEV replication was constantly inhibited by both concentrations (Figure 2 A). Surprisingly, this long-term inhibitory activity was more robust in huh 7 cells harboring full-length GT3 genome, with over 99% viral RNA replication being blocked from 6 days to 21 days post-treatment (Fig 2B). At protein level, we observed potent inhibition against HEV ORF2 expression at 10 days and 21 days post-treatment determined by western blotting (Fig. 2C). This long-term inhibitory effect was also observed in huh 7 based GT1 HEV replication model (Fig 2D). Comparison of AT-527 to ribavirin on GT3 Huh7-p6 model showed that AT-527 had superior long-term inhibitory activity compared to ribavirin (Fig 7).

AT-527 effectively inhibits HEV in human liver organoids

We have previously established HEV infection in human liver- derived organoids, which authentically supports antiviral drug assessment (Li et al., 2022a). Human liver organoids harboring subgenomic GT3 HEV replicon or full-length genome were first developed as schematic illustration (Fig 3A). By treating 1 pM and 10 pM of AT-527 in both models, we found that AT-527 dose-dependently inhibited GT3 HEV replication by detecting HEV replication-related luciferase signal and quantifying viral RNA, with 1 pM AT-527 treatment already inhibited over 60% HEV replication (Fig 3B and C). This inhibition activity was further demonstrated by immunostaining HEV ORF2 protein, with absent ORF2 signal in organoids treated with 10 pM AT-527 for 48 h (Fig 3D). Similarly, AT-527 potently inhibited GT1 HEV replication in Ever organoids based GTILuc model, with over 70% inhibition efficacy observed at 72 hours post-treatment with 10 pM (Fig 3F).

AT-527 inhibits HEV variant in cell models and liver organoids

The emerging GT3 HEV variant that harboring a G1634R mutation in RNA-dependent RNA polymerase, has been indicated to deliver resistance to ribavirin therapy in patients with chronic HEV infection (Debing et al., 2014, Gastroenterology 147, 1008-1011. el007). We thus introduced this variant into both huh7 cells and liver organoids, and evaluated the antiviral activity of AT-527 against this variant. In huh7 cells based p6G1634R variant model, AT-527 at 1 pM concentration has already exerted a significant inhibitory effect, and 10 pM concentration inhibited over 80% replication (Fig 4A). This was further validated by immunostaining viral ORF2 protein (Fig 4B). Notably, ribavirin treatment at these low concentrations failed to exert anti-HEV activity (Fig 4A). In a re-infection assay, 10 pM AT-527 largely abrogated the infectivity of p6 1634R variant, with few viral ORF2 protein signals observed by immunofluorescence staining (Fig 4C). Consistently, this potent inhibition was further observed in liver organoids based p6G1634R model, demonstrated by qRT-PCR and immunofluorescence staining (Fig 4 D and E).

Drug combination and guanosine supplementation

Since combination therapy is a common strategy to enhance antiviral efficacy and circumvent drug resistance, we evaluated combinations of AT-527 with IFN-a and ribavirin respectively in huh7 based GT3Luc model (Fig 5A). We found that combination of 5 pM AT-527 with 50 pM ribavirin had a clear synergistic effect (Fig 5B). Comparatively, combining 1 to 10 pM of AT-527 with 0 to 100 IU of IFN-a exerts more robust synergistic effects, with double synergy score than combining with ribavirin (Fig 5C).

Nucleoside analogs have been shown to exert anti-HEV activity by disordering nucleotide pool (Wang et al., 2016). AT-527 as a guanosine nucleotide analog, is thus postulated to employ a similar mechanism. To validate this hypothesis, huh 7 cells and liver organoids based GT3 HEV infectious models were supplemented with guanosine. Intriguingly, treating with 1 pg/ml guanosine largely reversed the anti-HEV activity of AT-527 in both types of HEV models (Fig 5D and E). Collectively, AT-527 inhibits HEV replication through counteracting with nucleotide biosynthesis pathway.

Discussion

Ribavirin and IFN-a have been used as off-label therapy for treating some chronic HEV patients, but the contraindication in pregnant women, increasing treatment failures by ribavirin therapy and severe side effects of IFN-a treatment in specific populations collectively urge the discovery for new HEV therapeutics. Drug repurposing is a prominent strategy for identifying new use of FDA- approved or safe-in-human drug candidates that are outside the original treating scope. In this regard, many antivirals that originally aimed for treating cancers or other types of virus infections, have been widely tested for treating HEV infection. AT-527, an orally administrated antiviral drug candidate, originally developed for treating hepatitis C was recently repurposed for treating COVID-19 patients. AT-527 was found to be safe and well-tolerated in broad range of populations evidenced by multiple clinical trials. These encouraged us to test AT-527 on HEV infection, because a high safety profile is critical treating the vulnerable populations such as pregnant woman and immunocompromised patients with severe HEV infection.

Consistently, in this study, we found the absence of cytotoxicity of AT-527 with tested concentrations up to 100 uM in our cell culture models. We demonstrated the potent antiviral efficacy against GT1 and GT3 HEV replication in both 2D liver cells and 3D organoids based models. These results indicated its potential prospect for treating patients with acute HEV infection. More importantly, by treating huh7 cells based HEV infection model with AT-527 for 21 days, we observed a long-term antiviral activity with complete inhibition of HEV replication. These results support the application of AT-527 for preventing and/or treating chronic HEV infection. In the clinic, there is an increasing concern of antiviral drug resistance particularly in immunocompromised populations, where constant viral rephcation and prolonged drug exposure result in the emerging of resistant variants. In the case of hepatitis E, resistance to ribavirin therapy has been frequently observed in immunocompromised organ transplant patients. The G1634R mutation in RNA-dependent RNA polymerase of GT3 HEV genome has been shown to confer ribavirin resistance possibly through enhancing viral replications. We demonstrated that G1634R mutant is highly sensitive to AT-527 in both huh 7 cells and liver-derived organoids.

Antiviral drugs with different but complementary mechanisms of action are often combined to enhance antiviral efficacy, but also minimize side effects and prevent viral evolution. In this study, we found synergistic antiviral activity of combining AT-527 with ribavirin and IFN-a, respectively. We expect that the combination of AT-527 with ribavirin and IFN-a may enhance antiviral potency, limit toxicity, and avoid drug resistance for treating chronic hepatitis E patients. We have previously demonstrated that guanosine analogs such as ribavirin and MPA, can inhibit virus replication by impairing nucleotide pool. This antiviral effect can be reversed by exogenous addition of guanosine. Here we observed that the antiviral effect of the guanosine analog AT-527 was blocked after guanosine supplement, suggesting that the antiviral mechanism of AT-527 involves nucleotide depletion.

In conclusion, it is demonstrated that AT-527 potently inhibits HEV infection in multiple in vitro models. It is much more potent than ribavirin and IFN-a in the similar experimental setting. The anti-HEV activity functions through impairing nucleotide biosynthesis pathway, but the precise mechanism of action remains to be further investigated. Considering the high safety profile in clinical trials and the potent anti-HEV activity in our short-term and long-term experiments, the great potential of exploring AT-527 for treating acute and chronic HEV infections in various patient populations is emphasized.

Treatment of patients suffering from HEV infection

In the vast majority of people, HEV infection results in a selflimited, acute illness. However, acute infection can become chronic in rare cases, primarily in people who have received solid-organ transplants and receive immunosuppressive treatment. Chronically infected subjects shed virus as long as they remain infected.

Definitive diagnosis of hepatitis E infection is usually based on the detection of specific anti-HEV immunoglobuline (IgM, IgA or IgG) antibodies to the virus in a subject's blood or serum, or the detection of HEV antigen or HEV RNA in the blood, stool, and other body fluids.

If a subject is diagnosed with an acute infection of hepatitis E virus, treatment may comprise orally administering to the subject a 500 mg tablet of the guanosine nucleotide analog having a structure according to formula (3) every ~12 hours (twice a day) for a total of 5 days, up to 14 days.

The dosage may also be increased. For instance, if a subject is diagnosed with an acute infection of hepatitis E virus, treatment may comprise orally administering to the subject two 500 mg tablets of the guanosine nucleotide analog having a structure according to formula (3) every ~12 hours (twice a day) for a total of 5 days, up to 14 days.

If a subject is diagnosed with a chronic/persistent infection of hepatitis E virus, treatment may comprise orally administering to the subject a 500 mg tablet of the guanosine nucleotide analog having a structure according to formula (3) every ~24 hours (once a day) for a total of 1 month, 3 months, 6 months or 12 months.

The frequency of administration may also be increased. For instance, if a subject is diagnosed with a chronic/persistent infection of hepatitis E virus, treatment may comprise orally administering to the subject a 500 mg tablet of the guanosine nucleotide analog having a structure according to formula (3) every ~12 hours (twice a day) for a total of 1 month, 3 months, 6 months or 12 months.

The subject is considered cured from the infectious disease when HEV is no longer detected in the subject’s body, or samples obtained therefrom.

Claims

1. A guanosine nucleotide analog having a structure according to formula (3) or a pharmaceutically acceptable salt or a prodrug thereof

formula (3) for use in a method of preventing and/or treating a Hepatitis E virus (HEV) infection.

2. The guanosine nucleotide analog according to claim 1, having a structure according to formula 1, a free base, a pharmaceutically acceptable salt, or a prodrug thereof

formula (1) or having a structure according to formula 2, a free base, a pharmaceutically acceptable salt, or a prodrug thereof

formula (2).

3. The guanosine nucleotide analog for use according to any one of the preceding claims, wherein the HEV infection is a GT1, GT2, GT3 and/or GT4 HEV infection, preferably a GT1 and/or GT3 infection, and/or wherein the HEV infection is an acute or chronic HEV infection.

4. The guanosine nucleotide analog for use according to any one of the preceding claims, wherein the guanosine nucleotide analog is administered in combination with at least one additional antiviral, preferably wherein the additional antiviral is selected from the group consisting of protease inhibitors, interferon alpha (IFN-a), non-substrate based inhibitors, helicase inhibitors, direct-acting antivirals, antisense oligodeoxynucleotides (S-ODN), aptamers, nuclease resistant ribozymes, iRNA, inducing microRNA, siRNA, viral antigens, nucleoside analogues, antibodies and a combination thereof.

5. The guanosine nucleotide analog for use according to claim 4, wherein the additional antiviral is IFN-a and/or ribavirin.

6. The guanosine nucleotide analog for use according to any one of the preceding claims, wherein the guanosine nucleotide analog is administered for at least 3 days.

7. The guanosine nucleotide analog for use according to any one of the preceding claims, wherein the guanosine nucleotide analog is administered daily, twice a day, three times a day, every two days or once a week.

8. The guanosine nucleotide analog for use according to any one of the preceding claims, wherein the guanosine nucleotide analog is administered in an amount ranging from 5 mg to 2000 mg, preferably from 50 mg to 1000 mg, more preferably from 100 mg to 800 mg.

9. A pharmaceutical composition comprising a guanosine nucleotide analog having a structure according to formula (3), a pharmaceutically acceptable salt, or a prodrug thereof for use in a method of preventing and/or treating a Hepatitis E virus (HEV) infection.

10. The pharmaceutical composition according to claim 9, wherein the guanosine nucleotide analog has a structure according to formula 1, a free base, a pharmaceutically acceptable salt, or a prodrug thereof or has a structure according to formula 2, a free base, a pharmaceutically acceptable salt or a prodrug thereof.

11. The pharmaceutical composition for use according to claim 9 or 10, wherein the HEV infection is a GT1, GT2, GT3 and/or GT4 HEV infection, preferably a GT1 and/or GT3 infection, and/or wherein the HEV infection is an acute or chronic HEV infection.

12. The pharmaceutical composition for use according to any one of claims 9 to 11, further comprising at least one additional antiviral, preferably wherein the additional antiviral is selected from the group consisting of protease inhibitors, NS5A inhibitors, NS5B inhibitors, interferon alpha (IFN-a), non-substrate based inhibitors, helicase inhibitors, antisense oligodeoxynucleotides (S-ODN), direct-acting antivirals, aptamers, nuclease resistant ribozymes, iRNA, inducing microRNA, siRNA, viral antigens, nucleoside analogues, antibodies and a combination thereof, more preferably wherein the additional antiviral compound is IFN-a and/or ribavirin.

13. The pharmaceutical composition for use according to any one of claims 9 - 12, further comprising a pharmaceutical acceptable carrier.

14. The pharmaceutical composition for use according to claim 13, wherein the pharmaceutical acceptable carrier is selected from the group consisting of water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, starches, sugar carriers, such as dextrose, manifold, lactose, diluents, granulating agents, lubricants, binders, disintegrating agents and a combination thereof.

15. The pharmaceutical composition for use according to any one of claims 9 - 14, wherein the pharmaceutical composition is administered for at least 3 days.

16. The pharmaceutical composition for use according to any one of claims 9 - 15, wherein the pharmaceutical composition is administered daily, twice a day, three times a day, every two days or once a week.

17. The pharmaceutical composition for use according to any one of claims 9 - 16, wherein the pharmaceutical composition is administered in an amount ranging from 5 mg to 2000 mg, preferably from 50 mg to 1000 mg, more preferably from 100 mg to 800 mg.