Filociclovir inhibits adenovirus

Abstract

The present invention relates to the development of therapeutic and prophylactic agents for the treatment and / or prevention of adenovirus infections in humans and other mammals. A method for treating or preventing adenovirus infections in mammals by administering an effective amount of filocyclovir (FCV) is disclosed.

Description

[Technical Field]

[0001] The present invention relates to therapeutic and prophylactic agents for treating adenovirus infections and diseases. In particular, the present invention relates to the use of filociclovir (FCV) in the treatment and / or prevention of adenovirus infections in mammals, and especially in the treatment and / or prevention of adenovirus infections in humans. [Background technology]

[0002] Adenovirus (AdV) is a ubiquitous DNA virus most commonly associated with childhood illnesses of the respiratory tract, gastrointestinal tract, and conjunctiva. Other rare signs of AdV infection include hepatitis and genitourinary and neurological symptoms (Non-Patent Literature 1). While most adenovirus infections are self-limiting in immunocompetent hosts, disseminated forms of the disease and subsequent high mortality rates can occur in immunocompromised patients, as well as in other vulnerable populations in closed or crowded environments, such as schoolchildren and military recruits (Non-Patent Literature 2). Infection in immunocompromised individuals is similar in scope but more severe, particularly in transplant recipients (Non-Patent Literature 3). HIV infection is also a risk factor for AdV infection, and serotypes have recently been isolated from this population (Non-Patent Literature 4). Adenovirus transmission can occur through aerosol droplet inhalation, fecal-oral transmission, and contaminated vectors (Non-Patent Literature 5). More than 50 serotypes of adenovirus have been identified and are classified into six distinct groups (A-F) based on their biochemical, immunological, and morphological criteria (Non-Patent Literature 6). Approximately one-third of the described serotypes are associated with human disease. Globally, serotypes 1-7, 21, and 41 are the most commonly associated with human disease. Different serotypes exhibit different histotropies that correlate with the clinical manifestations of infection (Non-Patent Literature 7). The clinical scope of disease varies by age, immune status, and population characteristics. Upper respiratory tract infections (URIs) are among the most common, but pneumonia and diarrhea have the highest mortality rates in infants. Epidemic keratoconjunctivitis and acute respiratory distress syndrome (ARDS) are major concerns in young military recruits. Adenoviruses account for at least 5-10% of respiratory tract infections in children and 1-7% of respiratory tract infections in adults (Non-Patent Literature 8). Pneumonia occurs in up to 20% of neonates and infants (Non-Patent Literature 9). Adenoviruses also account for more than 50% of pneumonia cases among unvaccinated military recruits, not only in the United States but globally (Non-Patent Literature 10). In immunocompromised individuals, disseminated and / or severe respiratory failure develops in 10–30% of cases, and the mortality rate for severe adenoviral pneumonia can exceed 50% (Non-Patent Literature 11).Very recently, an outbreak of AdV3 infection was reported at a nursing and rehabilitation center in New Jersey, affecting 54 people and resulting in 11 deaths. (See https: / / nj.gov / health / cd / topics / adenovirus.shtml).

[0003] Options for controlling adenovirus infection are very limited. Live oral vaccines reduce the risk of respiratory illness and are routinely used by the U.S. military, but are currently not available to civilians and only provide protection against types 4 and 7 (Non-Patent Literature 12). No antiviral drugs for treating adenovirus are approved. Previous reports have shown specific antiviral activity of several nucleoside analogs, including ribavirin, ganciclovir (GCV), cidofovir (CDV), and its prodrug brincidofovir (BCDV), in cell cultures and animal models (Non-Patent Literature 13). Ribavirin is teratogenic, causes hemolytic anemia, and is active only against group C adenovirus (Non-Patent Literature 14). Ribavirin treatment of adenovirus infection in immunocompromised hosts has yielded a variety of results that may correlate with its serotype specificity (Non-Patent Literature 15). The efficacy of GCV against AdV5 is very mild (IC50 = 66 ± 15.1 μM), and there are no controlled trials demonstrating the clinical efficacy of GCV use (Non-Patent Literature 16). Long-term use of CDV is associated with nephrotoxicity and CNS side effects (Non-Patent Literature 17). Brincidofovir (BCDV), a lipid-bound derivative of CDV, is orally bioavailable, is very potent in vitro, and is currently in clinical trials (Non-Patent Literature 18). However, these trials have shown only slight benefits for AdV disease or viremia (Non-Patent Literature 19). A recent randomized, placebo-controlled phase 2 trial showed that preemptive treatment of AdV infection with BCDV rapidly reduces the viral load (Non-Patent Literature 20). However, GI toxicity and acute kidney injury in solid organ transplants (SOTs), as well as graft-versus-host disease in hematopoietic stem cell transplants (HSCTs), have been reported (Non-Patent Literature 21). Two Phase 3 AdV trials (NCT01143181, NCT02087306) were completed several years ago, but the company has not sought approval. Development of BCDV for HCMV was discontinued due to an increased frequency of serious adverse events (primarily graft-versus-host disease) and a 24-week all-cause mortality rate observed during Phase 3 (Non-Patent Literature 22).Furthermore, two Phase 3 clinical trials of CDV in kidney transplant patients (NCT02439957, NCT02439970) recently concluded. These safety issues raise questions about whether the drug can be safely administered in any population. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Khanal et al., Biomedicines, 6(1)(2018) [Non-Patent Document 2] Hendrix et al., Emerg.Infect.Dis., 5(6):798~801 (1999); Munoz et al., Clin.Infect.Dis.27(5):1194~1200 (1998); Trei et al., Emerg.Infect.Dis., 16(5):769~775 (2010) [Non-Patent Document 3] Lion, T., Clin.Microbiol.Rev., 27(3):441~462(2014) [Non-Patent Document 4] De Jong et al., J. Clin. Microbiol., 37(12):3940~3945 (1999) [Non-Patent Document 5] Lynch, JP and Kajon, AE, Seminars in Respiratory and Critical Care Medicine, 37(4):586~602 (2016) [Non-Patent Document 6] Robinson et al., Scientific Reports, 3:1812 (2013) [Non-Patent Document 7] Lynch and Kajon, 2016 (previously cited) [Non-Patent Document 8] Lee et al., Journal of Medical Virology, 82(4):624–631 (2010) [Non-Patent Document 9] Zhang et al., Infectious Diseases, 48(1):40-47 (2016); Jain et al., The New England Journal of Medicine, 372(9):835-845 (2015) [Non-Patent Document 10] Lynch and Kajon, 2016 (previously cited) [Non-Patent Document 11] Low et al., Respiratory Medicine, 107(11):1810~1813(2013); Ison, MG, Clinical Infectious Diseases, 43(3):331~339(2006) [Non-Patent Document 12] Radin et al., Clinical Infectious Diseases, 59(7):962–968 (2014) [Non-Patent Document 13] Hartline et al., The Journal of Infectious Diseases, 191(3):396~399 (2005); Naesens et al., Antimicrobial Agents and Chemotherapy, 49(3):1010~1016 (2005); Ying et al., Antimicrobial Agents and Chemotherapy, 58(12):7171~7181 (2014); Toth et al., Antiviral Research, 153:1~9 (2018); Toth et al., Proc.Nat.Acad.Sci.USA, 105(20):7293~7297 (2008) [Non-Patent Document 14] Feld et al., Liver International, 37(1):5~18 (2017); Morfin et al., Antiviral Therapy, 10(2):225~229 (2005) [Non-Patent Document 15] Ljungman, P., Eur.J.Clin.Microbiol.Infect.Dis., 23(8):583~588(2004); Lankester et al., Clinical Infectious Diseases, 38(11):1521~1525(2004) [Non-Patent Document 16] Kinchington et al., The Journal of Antimicrobial Chemotherapy, 55(4):424–429 (2005) [Non-Patent Document 17] Bhadri et al., Transplant Infectious Disease, 11(4):373–379(2009); Vora et al., Journal of the Pediatric Infectious Diseases Society, 6(4):399–402(2017) [Non-Patent Document 18] Hartline et al., 2005, previously cited, Waye, MMY and Sing, CW, Pharmaceuticals, 3(10):3343~3354(2010) [Non-Patent Document 19] Lopez et al., Current opinion in organ transplantation, 23(4):395~399(2018) [Non-Patent Document 20] Grimley et al., Biology of Blood and Marrow Transplantation, 23(3):512–521 (2017) [Non-Patent Document 21] Detweiler et al., Journal of Pediatric Hematology / Oncology, 40(6):e364~e368(2018); Faure et al., Medicine, 95(44):e5226(2016) [Non-Patent Document 22] Marty et al., Biology of Blood and Marrow Transplantation, 25(20:369~381(2018)) [Overview of the project]

Problems to be Solved by the Invention

[0005] Currently, there are no drugs approved for the prevention or treatment of adenovirus infections, and the most advanced drug in development has safety issues that can hinder its progress. Therefore, there is an urgent and unmet medical need for a safe and effective anti-adenovirus drug for use in methods of treating and / or preventing adenovirus infections.

Means for Solving the Problems

[0006] The present invention relates to a method for treating or preventing adenovirus (AdV) infections through the administration of small molecule inhibitors. In particular, the present invention relates to a method for treating or preventing adenovirus infections by the administration of philocyclovir (FCV). The methods described herein are suitable for the treatment and / or prevention of adenovirus infections in mammals, particularly humans.

[0007] In particular, the method relates to treating or preventing infections caused by specific strains of adenovirus. Thus, in one embodiment, the present invention relates to a method for treating or preventing an infection in a mammal by adenovirus 5 (AdV5) by administration of a composition comprising philocyclovir or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the mammal is a human.

[0008] In another embodiment, the present invention relates to a method for treating or preventing an infection in a mammal by adenovirus 6 (AdV6) by administration of a composition comprising philocyclovir or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the mammal is a human.

[0009] In another embodiment, the present invention relates to a method for treating or preventing an infection in a mammal caused by adenovirus 8 (AdV8) by administration of a composition comprising filocyclovir or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the mammal is a human.

[0010] In another embodiment, the present invention relates to the use of filocyclovir in the manufacture of a pharmaceutical product for treating or preventing adenovirus infections in mammals. In a preferred embodiment, the present invention relates to the use of filocyclovir in the manufacture of a pharmaceutical product for treating or preventing infections caused by AdV5, AdV6 and / or AdV8.

[0011] Also disclosed are pharmaceutical compositions comprising a therapeutically effective amount of filocyclovir or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The pharmaceutical compositions are suitable for use in the disclosed manner for treating or preventing adenovirus infections in mammals, particularly humans. The pharmaceutical compositions may be formulated for both parenteral and / or non-parenteral administration to subjects or patients requiring it.

[0012] In another embodiment, filocyclovir may be administered to subjects requiring it, optionally in combination with one or more known antiviral agents. The additional one or more antiviral agents may be administered before, concurrently with, or after administration of filocyclovir.

[0013] In preferred embodiments, the filocyclovir compound / composition of the present invention provides an inhibitory concentration of 10 μM or less and a cytotoxicity of 100-150 μM or more against adenovirus infections (CC). 50 ) indicates.

[0014] (definition) The compositions or methods described herein as “comprising” (or “comprises”) one or more specified elements or steps are open-ended, meaning that while the specified elements or steps are essential, other elements or steps can be added within the scope of the composition or method. To avoid redundancy, any composition or method described herein as “comprising” one or more specified elements or steps also describes a more limited corresponding composition or method that “consistes essentially of” (or “consists essentially of” the same specified elements or steps, and the composition or method may include the specified essential elements and additional elements or steps that do not substantially affect the basic and novel features of the composition or method. Any composition or method described herein as “comprising” or “consisting essentially of” one or more specified elements or steps also describes a more limited closed-end composition or method that “consistes of” (or “consists of” the specified elements or steps), excluding any other elements or steps. In any composition or method disclosed herein, any known or disclosed equivalent of any specified essential element or step may be substituted for that element or step, respectively.

[0015] Phyllocyclovir (also known as cyclopropavir, 2-amino-9-{(Z)-[2,2-bis(hydroxymethyl)cyclopropyridene]methyl}-3,9-dihydro-6H-purine-6-one) and its salts are methylenecyclopropane nucleoside analogs represented by the following structure: [ka]

[0016] As used herein, the term “subject” may be human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cattle, cattle, guinea pig, or rodent. “Patient” or “subject in need” refers to a mammal suffering from a disease or disorder. The term “patient” includes human and veterinary subjects.

[0017] Terms such as "parenteral" or "parenterally" refer to routes or modes of administration of compounds or compositions to an individual other than along the gastrointestinal tract. Examples of parenteral administration routes include, but are not limited to, subcutaneous (sc), intravenous (iv), intramuscular (im), intra-arterial (ia), intraperitoneal (ip), transdermal (absorption through the skin or dermis), transnasal ("intranasal"; absorption through the nasal mucosa), or transpulmonary (e.g., inhalation for absorption through lung tissue), transvaginal, direct injection or infusion into body cavities or organs other than the gastrointestinal tract, and implantation into the body of any of the various devices (e.g., compositions, depots, or devices that enable the active or passive release of compounds or compositions into the body).

[0018] Terms such as “not parenteral,” “enteral,” “enterally,” “oral,” and “oral” refer to the administration of a compound or composition to an individual via a route or manner along the gastrointestinal tract. Examples of enteral administration routes include, but are not limited to, oral administration when swallowing solid (e.g., tablets) or liquid (e.g., syrup) dosage forms, sublingual administration (absorption via the mucous membrane covering the floor of the mouth, e.g., under the tongue), buccal administration (absorption via the mucous membrane covering the cheek), nasojejunal or gastrostomy tube administration (delivery to the stomach), intraduodenal administration, and rectal administration (e.g., suppositories for release of the drug composition into the lower intestinal tract and absorption by the lower intestinal tract). [Brief explanation of the drawing]

[0019] [Figure 1] This is a dose-response curve showing the antiviral activity (closed symbol) and cytotoxicity (open symbol) of filocyclovir against adenovirus 5 (AdV5). [Figure 2]This is a dose-response curve showing the antiviral activity and cytotoxicity of filociclovir against adenovirus 6 (AdV6). [Figure 3] This is an immunofluorescence assay of A549 cells infected with adenovirus 5 and treated with filociclovir. [Figure 4] This is an immunofluorescence assay of A549 cells infected with adenovirus 6 and treated with filociclovir. [Figure 5] This shows the bioavailability of filocyclovir administered orally (PO) or intravenously (IV) to hamsters. [Figure 6A] This shows the survival rate (6A) of Syrian hamsters infected with AdV6 and treated with 10 mg / kg, 30 mg / kg, 60 mg / kg, or 100 mg / kg of filociclovir. 6A: Survival. AdV6 + vehicle vs. AdV6 + 10 mg / kg or 30 mg / kg of filociclovir (p=0.0124) (log-rank). [Figure 6B] The mean weight change (6B) of Syrian hamsters infected with AdV6 and treated with 10 mg / kg, 30 mg / kg, 60 mg / kg, or 100 mg / kg of filociclovir is shown. 6B: Mean weight change. Group mean and standard error of the mean are shown. After euthanized animals were sacrificed from the group, no mean values ​​were calculated for that group. AdV6 + vehicle vs. AdV6 + 10 mg / kg or 30 mg / kg of filociclovir (p<0.0001) ((two-way ANOVA); AdV6 + 10 mg / kg of filociclovir vs. vehicle + vehicle or AdV6 + 30 mg / kg of filociclovir (p=0.0286). [Figure 7] This chart shows transaminase (ALT) levels in Syrian hamsters 5 days after the challenge. Treatment with filociclovir reduces liver lesions. The symbols indicate values ​​from individual animals, and the horizontal bars represent the geometric mean. The white symbols in the AdV6+ vehicle group indicate samples taken from dying animals. [Figure 8]This chart shows the level of AdV load in the liver of Syrian hamsters 5 days after the challenge. Treatment with filociclovir inhibits AdV6 replication in the liver. The symbols indicate values ​​from individual animals, and the horizontal bars represent the geometric mean. NQ: Unquantifiable; ND: Undetectable. [Figure 9A] The survival rates (9A) of Syrian hamsters infected with AdV6 and treated with 1 mg / kg, 3 mg / kg, or 10 mg / kg of filociclovir are shown. Filociclovir completely suppressed mortality at the 3 and 10 mg / kg qdpo dose levels and had a moderate but significant effect (p=0.0449) at the 1 mg / kg poqd dose level (see Figure 9A). No toxic effects were observed in filociclovir-treated hamsters. [Figure 9B] The mean weight change (9B) of Syrian hamsters infected with AdV6 and treated with filociclovir at 1 mg / kg, 3 mg / kg, or 10 mg / kg is shown. Filociclovir completely suppressed weight loss at 10 mg / kg, was moderately effective at 3 mg / kg, and was ineffective at 1 mg / kg (see Figure 9B). Symbols represent the group mean, and error bars represent the standard deviation. Weight data for a given group is shown only up to the point where no unplanned deaths occurred. [Figure 10] Serum alanine aminotransferase (ALT) levels in Syrian hamsters 5 days after the challenge are shown. Filociclovir alleviated lesions caused by intravenous injection of AdV6 at 3 and 10 mg / kg qdpo dose levels, while it was ineffective at the 1 mg / kg poqd dose level. Symbols represent data collected from individual hamsters, and horizontal bars represent the group mean. For the AdV6+ vehicle group and the AdV6+ 1 mg / kg filociclovir group, open symbols indicate that samples were taken from hamsters that were sacrificed earlier than planned. (*: p=0.0415; ***: p=0.0005) (One-sided Mann-Whitney U test) [Figure 11]This chart shows the level of AdV load in the liver of Syrian hamsters 5 days after the challenge. Treatment with 1, 3, or 10 mg / kg of filocyclovir inhibits AdV6 replication in the liver. The symbols indicate values ​​from individual hamsters, and the horizontal bars represent the geometric mean. NQ: Unquantifiable; ND: Undetectable. [Figure 12A] This study demonstrates that delayed treatment with 10 mg / kg of filociclovir is effective against AdV6 infection in hamsters. 12A. Survival. AdV6+ vehicle vs. AdV6+ filociclovir, -1 day; AdV6+ filociclovir, +1 day; AdV6+ filociclovir, +2 day; or AdV6+ filociclovir, 3 day (p=0.0005); and AdV6+ vehicle vs. AdV6+ filociclovir, +4 day (p=0.0023) (log-rank test). [Figure 12B] This study demonstrates that delayed treatment with 10 mg / kg of filocyclovir is effective against AdV6 infection in hamsters. 12B. Body weight change. The symbols represent the group mean, and the error bars represent the standard error of the mean. [Figure 13] Transaminases (ALT) levels in Syrian hamsters treated with 10 mg / kg filociclovir on days 1, 2, 3, and 4 after challenge are shown. Treatment with 10 mg / kg filociclovir up to day 4 after challenge reduces liver lesions. AdV6+ vehicle vs. AdV6+ filociclovir, -1 day or AdV6+ filociclovir, +1 day (p=0.0018); AdV6+ vehicle vs. AdV6+ filociclovir, +2 day (p=0.0087); AdV6+ vehicle vs. AdV6+ filociclovir, +3 day (p=0.0032); and AdV6+ vehicle vs. AdV6+ filociclovir, +4 day (p=0.1452) (two-sided Mann-Whitney test). Symbols indicate values ​​from individual animals, and horizontal bars represent the geometric mean. In the AdV6+ vehicle group, the white symbols indicate samples taken from dying animals. [Figure 14]This chart shows the levels of AdV load in the livers of Syrian hamsters treated with 10 mg / kg filocyclovir on days 1, 2, 3, and 4 after the challenge. Treatment with 10 mg / kg filocyclovir on day 4 after the challenge inhibits AdV6 replication in the liver. The symbols indicate values ​​from individual animals, and the horizontal bars represent the geometric mean. NQ: Unquantifiable; ND: Undetectable. [Modes for carrying out the invention]

[0020] Detailed description of the invention Human adenoviruses belong to the Adenoviridae family, which is distinct from the Herpesviridae family. For example, adenoviruses are double-stranded DNA viruses, but unlike herpesviruses, they are not enveloped by an icosahedral nucleocapsid, and instead carry elongated fibrous proteins on their outer surface. Adenoviruses are classified into seven species called human adenoviruses A through G. These viruses primarily infect the upper and lower respiratory tracts, conjunctiva, or gastrointestinal tract, causing a range of clinical symptoms distinct from those caused by herpesviruses. Most infections occur in children and immunocompromised individuals. It is known that adenoviruses do not encode the kinases that viruses encode, which suggests a novel mechanism for filociclovir activation.

[0021] Other rare signs of AdV infection include hepatitis and genitourinary and neurological symptoms (Khanal et al., 2018, cited above). Options for controlling AdV infection are very limited. Live oral vaccines reduce the risk of respiratory illness and are routinely used by the U.S. military, but are not currently available to civilians and only provide protection against types 4 and 7 (Radin et al., 2014, cited above). No antiviral drugs have been approved to treat adenoviruses.

[0022] In this specification, the inventors describe the discovery of a novel method for treating and / or preventing adenovirus infections in mammals by administering the small molecule inhibitor filociclovir (FCV). The inventors' results demonstrate that filociclovir is unexpectedly effective in use in a method for treating or preventing adenovirus infections, and is more effective than previously known treatments in treating or preventing adenovirus infections.

[0023] This method of using filociclovir to treat or prevent adenovirus infections is not limited to, adenovirus 3 (AdV3), adenovirus 4 (AdV4), adenovirus 5 (AdV5), adenovirus 6 (AdV6), adenovirus 7 (AdV7), adenovirus 8 (AdV8), adenovirus 19 (AdV19), and adenovirus 3 7 It is particularly effective against several strains of adenovirus, including (AdV37).

[0024] This method of using filociclovir to treat adenovirus infections is particularly suitable for the treatment or prevention of adenovirus infections, including those of the upper respiratory tract and adenovirus infections of the eye, such as conjunctivitis, especially keratoconjunctivitis affecting the ocular surface.

[0025] Therefore, the present invention provides a novel method for targeting anti-adenovirus therapeutics for treating or preventing adenovirus infections in mammalian cells. In particular, the present invention relates to the use of filociclovir for treating or preventing adenovirus infections in mammalian subjects. In preferred embodiments, the mammal is human.

[0026] The data described herein demonstrate that filociclovir is an effective pan-adenovirus treatment, and therefore, therapeutic administration of filociclovir is effective in treating or preventing infections caused by multiple strains of adenovirus. As described below, several assays have demonstrated that filociclovir is a potent, safe, and effective inhibitor of several adenovirus strains. For example, as described in Example 1, the inventors demonstrated using a cytopathic effect (CPE) reduction assay that filociclovir is a potent adenovirus 5 inhibitor compared to several other known viral inhibitors, including cidofovir and ganciclovir (see Table 1 and Figure 1).

[0027] Next, the inventors used a neutral red survival assay to demonstrate that phyllocyclovir is effective in reducing the cytotoxic effects of adenovirus 6. The results are shown in Figure 2. Essentially, healthy cells can take up the neutral red dye via active transport to lysosomes, while virus-infected cells cannot. The results shown in Figure 2 are 10 -1 We have demonstrated that a low phyllocyclovir concentration of μM was effective in improving the viability of AdV6-infected cells, specifically by demonstrating the active transport of neutral red dye to lysosomes in AdV6-infected cells.

[0028] As described in Example 3, an immunofluorescence assay was used to demonstrate that filocyclovir is effective in inhibiting "late" AdV replication in infected human A549 cells. In this assay, cells were infected with either AdV5 or AdV6, or falsely infected. After 1 hour, filocyclovir was added to final concentrations of 0, 4, 10, or 40 μM. The results are shown in Figures 3 and 4. As seen in Figures 3 and 4, the results of filocyclovir treatment were the same for AdV5 and AdV6 infections. Filocyclovir was highly effective in preventing late infection, as demonstrated by the absence of hexon protein, a viral coat protein produced in the later stages of the adenovirus replication cycle. No hexon-positive cells were observed in the 40 μM filocyclovir-treated wells, and very few hexon-positive cells were observed in the 10 μM filocyclovir-treated wells. Some hexon-positive cells are observed in the 4 μM phyllocyclovir-treated wells, but the 4 μM wells indicate substantially no progression of infection (as indicated by DBP staining patterns and nuclear size and shape) compared to the control (untreated) wells.

[0029] As shown in Examples 4-7, using Syrian hamster models infected with adenovirus, filociclovir exhibits favorable bioavailability (see Figure 5), reduces morbidity and mortality (see Figure 6), mitigates liver damage (see Figure 7), and inhibits AdV virus replication (see Figure 8) when administered orally and intravenously to immunocompromised hamsters infected with AdV5 or AdV6 at doses ranging from 10 mg / kg to 100 mg / kg.

[0030] In Example 6, using the same Syrian hamster model as described above, we also demonstrated that even low doses of filociclovir (3 mg / kg and 10 mg / kg) were effective in suppressing morbidity and mortality (Figure 9), mitigating liver damage (Figure 10), and inhibiting AdV virus replication (Figure 11) after AdV infection.

[0031] Example 7 demonstrates that late administration of filociclovir (10 mg / kg) in Syrian hamsters infected with AdV6, for example, up to day 4 after the challenge, is effective in reducing morbidity and mortality (see Figure 12), mitigating liver damage (see Figure 13), and inhibiting AdV virus replication (see Figure 14).

[0032] Therefore, in one embodiment, the present invention relates to the use of filociclovir in a method for treating or preventing mammalian infections caused by adenovirus 5 (AdV5).

[0033] In another embodiment, the present invention relates to the use of filociclovir in a method for treating or preventing mammalian infections caused by adenovirus 6 (AdV6).

[0034] In another embodiment, the present invention relates to the use of filociclovir in a method for treating or preventing mammalian infections caused by adenovirus 7 (AdV7).

[0035] In yet another embodiment, the present invention relates to the use of filociclovir in a method for treating or preventing mammalian infections caused by adenovirus 8 (AdV8).

[0036] The results also demonstrate that filociclovir is effective in treating or preventing mammalian infections caused by adenovirus 3 (AdV3), adenovirus 4 (AdV4), adenovirus 19 (AdV19), and adenovirus 37 (AdV37) (data not shown).

[0037] In a preferred embodiment, the mammal is a human.

[0038] The present invention further relates to the use of filocyclovir in a method for producing a pharmacopoeci for treating or preventing adenovirus infections in mammals (e.g., humans), comprising combining filocyclovir or a composition comprising filocyclovir with a pharmaceutically acceptable carrier or diluent. Accordingly, in one embodiment, the present invention relates to a method for producing a pharmacopoeci for combining at least one disclosed compound (e.g., filocyclovir) or at least one disclosed product according to the present invention with a pharmaceutically acceptable carrier or diluent.

[0039] Individual dose requirements for administration to the target population are determined by the healthcare provider, such as a physician, primary care physician, or nurse.

[0040] As described herein, phyllocyclovir has an IC50 of 5 μM or less. 50 The value and preferably the minimum mammalian cytotoxicity (CC) of 100-150 μM or higher. 50 It has been demonstrated to show dose-dependent inhibition of adenovirus infection.

[0041] Filocyclovir may be administered as pharmaceutically acceptable salts. Such pharmaceutically acceptable salts include gluconates, lactates, acetates, tartrates, citrates, phosphates, maleates, borates, nitrates, sulfates, and hydrochlorides. Salts of the compounds described herein can be prepared, for example, by reacting a base compound with a desired acid in solution. After the reaction is complete, the salt is crystallized from the solution by adding an appropriate amount of a solvent in which the salt is insoluble. In some embodiments, hydrochlorides are prepared by passing hydrogen chloride gas through an ethanol solution of a free base.

[0042] In another embodiment, the compound is formulated into a pharmaceutically acceptable carrier or excipient for administration to a target requiring it. In yet another embodiment, the compound is formulated into a pharmaceutical formulation which may further contain additional antiviral compounds. In yet another embodiment, the pharmaceutical formulation may be formulated for oral, parenteral, or topical administration.

[0043] It is preferable to develop orally active therapeutic agents because this is the simplest and fastest method for administering drugs to large exposed populations in the event of a pandemic. Currently, oral live adenovirus vaccines for protection against serotypes 4 and 7 are used by the military but are not available to the general public. However, in the case of a spontaneous outbreak, it is anticipated that infected patients may require intravenous (IV) administration, and therefore it is hoped that filociclovir as described herein is suitable for intravenous (IV) administration. Thus, the methods described herein will provide an effective, safe, and easy therapeutic option for any newly emerging pandemic strain(s) of adenovirus.

[0044] The methods of the subject matter of this disclosure are useful in treating these symptoms in that they inhibit the onset, progression, or spread of symptoms, induce regression of symptoms, cure symptoms, or improve the general health of subjects who are affected with or at risk of being affected with symptoms. Accordingly, in accordance with the subject matter of this disclosure, the terms “treat,” “treating,” and their grammatical variations, as well as the phrase “method of treating,” mean to encompass any desired therapeutic intervention, which includes, but is not limited to, methods for treating existing adenovirus infections in subjects, and methods for preventing (i.e., preventing) adenovirus infections in subjects who have been exposed to or are expected to be exposed to the viruses disclosed herein.

[0045] The pharmaceutical composition according to the present invention comprises an adenovirus inhibitor, filocyclovir, or a pharmaceutically acceptable salt thereof as described herein as an “active ingredient,” and a pharmaceutically acceptable carrier (i.e., “vehicle”), which may be a liquid, solid, amorphous, or semi-solid compound.

[0046] In some embodiments, the subject matter of the present disclosure relates to a method for treating or preventing an adenovirus infection in a subject requiring treatment for an adenovirus infection, the method comprising administering to the subject a composition comprising an effective amount of filocyclovir. Filocyclovir may be administered alone or, optionally, in combination with one or more additional antiviral agents.

[0047] In another embodiment, filocyclovir may be formulated on a pharmaceutically acceptable carrier and administered to a subject requiring it by injection, including but not limited to intradermal, transdermal, intramuscular, intraperitoneal, and intravenous injection. According to another embodiment of the present invention, administration is oral, and the compound may be provided, for example, in the form of tablets, or encapsulated in gelatin capsules or microcapsules, thereby facilitating oral administration. The production of these dosage forms is within the scope of the general knowledge of technical experts. Multiple routes of administration are envisioned in the methods described herein, and highly cost-effective production strategies can be easily achieved.

[0048] In another aspect, the present invention relates to a pharmaceutical composition comprising phyllocyclovir, or a pharmaceutically acceptable salt, solvate, hydrate or polymorph thereof, and a pharmaceutically acceptable carrier.

[0049] In a further embodiment, the pharmaceutical composition is a solid dosage form selected from capsules, tablets, pills, powders, granules, effervescent granules, gels, pastes, lozenges, and pastilles. In yet another embodiment, the pharmaceutical composition is a liquid dosage form selected from emulsions, liquids, suspensions, syrups, and elixirs.

[0050] As used herein, the term “pharmaceutically acceptable salt” refers to a salt prepared from a pharmaceutically acceptable non-toxic base or acid. If a compound of the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic and organic bases. Salts derived from such inorganic bases include salts of aluminum, ammonium, calcium, copper (cupric and cuprous), ferric, ferrous, lithium, magnesium, manganese (manganese di and manganese 1), potassium, sodium, and zinc. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic and substituted amines, such as naturally occurring and synthetic substituted amines. Other pharmaceutically acceptable organic non-toxic bases that can form salts include ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydravamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, and tromethamine.

[0051] As used herein, the term “pharmaceutically acceptable non-toxic acid” includes inorganic acids, organic acids, and salts prepared therefrom, such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucinic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and the like. Citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, and tartaric acid are preferred.

[0052] When preparing compositions for oral dosage forms, any convenient pharmaceutical medium can be used. For example, water, glycol, oil, alcohol, flavoring agents, preservatives, colorants, etc., can be used to form oral liquid formulations such as suspensions, elixirs, and solutions. On the other hand, carriers such as starch, sugar, and microcrystalline cellulose, diluents, granulators, lubricants, binders, disintegrants, etc., can be used to form oral solid preparations such as powders, capsules, and tablets. Tablets and capsules are preferred oral dosage units due to their ease of administration, and thus solid pharmaceutical carriers are used. If necessary, tablets can be coated by standard aqueous or non-aqueous techniques.

[0053] Tablets containing the composition of the present invention may be prepared by compression or molding, optionally together with one or more auxiliary components or adjuvants. Compressed tablets may be prepared by mixing the active ingredient in a free-flowing form, such as powder or granules, optionally with a binder, lubricant, inert diluent, surfactant, or dispersant, and compressing it in a suitable machine. Molded tablets may be produced by molding a mixture of powder compounds moistened with an inert liquid diluent in a suitable machine.

[0054] The pharmaceutical compositions of the present invention, suitable for parenteral administration, can be prepared, for example, from lyophilized samples of the active compound as a solution or suspension of the active compound in water. Suitable surfactants, such as hydroxypropyl cellulose, may be included. Dispersants can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof in oil. Furthermore, preservatives may be included to prevent harmful microbial growth.

[0055] The pharmaceutical compositions of the present invention suitable for injection include sterile aqueous solutions, emulsions, or dispersions. Furthermore, the compositions may be in the form of sterile powders for the immediate preparation of such sterile injection solutions or dispersions. In all cases, the final injectable form must be sterile and substantially fluid for easy injection. The pharmaceutical compositions must be stable under manufacturing and storage conditions and, therefore preferably, should be protected from microbial contamination such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0056] The pharmaceutical compositions of the present invention may be in forms suitable for topical use, such as aerosols, creams, ointments, lotions, powders for application, mouthwashes, and gargles. Furthermore, the compositions may be in forms suitable for use in transdermal devices. These formulations can be prepared by conventional processing methods using the compounds of the present invention or pharmaceutically acceptable salts thereof. As an example, a cream or ointment may be prepared by mixing a hydrophilic material and water with about 5% to about 10% by weight of the compound to create a cream or ointment of the desired consistency.

[0057] The pharmaceutical composition of the present invention may be in a form suitable for rectal administration, wherein the carrier is solid. The mixture preferably forms a unit-dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppository can be conveniently formed by first mixing the composition with a softened or melted carrier(s), followed by cooling and molding in a mold.

[0058] In addition to the carrier components described above, the pharmaceutical formulation may optionally contain one or more additional carrier components such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, and preservatives (including antioxidants). Furthermore, other adjuvants may be included to make the formulation isotonic with the blood of the intended recipient. Compositions containing the compounds of the present invention and / or pharmaceutically acceptable salts thereof may also be prepared in powder or liquid concentrate form.

[0059] In one embodiment, the present invention relates to at least filocyclovir according to the present invention, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, and a) at least one additional agent known to have antiviral activity, if applicable; b) Instructions for treating adenovirus-related illnesses; c) Instructions for administering compounds related to the treatment of adenovirus infections; and / or d) Instructions for administering the compound together with at least one known drug to treat adenovirus-related illnesses. Regarding kits that include one or more of the following.

[0060] The kit may also include compounds and / or products that are packaged together with other components, formulated together, and / or delivered together. For example, a drug manufacturer, drug reseller, physician, dispensing shop, or pharmacist could provide a kit comprising the disclosed compounds and / or products of the present invention and other components for delivery to a patient.

[0061] In a further embodiment, the kit further comprises a plurality of dosage forms, each of which comprises one or more doses, each of which comprises an amount of a compound and a drug known to have antiviral activity. In another embodiment, the kit further comprises a plurality of dosage forms, each of which comprises one or more doses, each of which comprises an effective amount of a compound and a drug known to have antiviral activity.

[0062] In a further embodiment, the effective dose is the therapeutically effective dose. In yet another embodiment, the effective dose is the prophylactically effective dose. [Examples]

[0063] The following embodiments are included to illustrate the form of the subject matter of this disclosure. In light of this disclosure and the general level of skill in the art, those skilled in the art will understand that the following embodiments are for illustrative purposes only and that numerous variations, modifications, and changes can be made without departing from the present invention.

[0064] Example 1. Cytopathic effect (CPE) reduction assay to measure the inhibition of adenovirus 5 (AdV5) by filocyclovir.

[0065] Preparation and culture of human foreskin fibroblast (HFF) cells Human foreskin tissue was obtained from the University of Alabama Birmingham Tissue Procurement Facility with the approval of the institutional review board. The tissue was stored at 4°C in a cell culture medium consisting of minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS; HyClone, Inc., Logan, UT) and standard concentrations of L-glutamine, amphotericin B (Fungizone), and vancomycin. The tissue was then placed in phosphate-buffered saline, finely chopped, and rinsed to remove red blood cells. The tissue fragments were then resuspended in trypsin-EDTA solution and incubated at 37°C to disperse the cells, which were then collected by centrifugation. The cell pellet was then resuspended in 4 ml of culture medium and placed in a 25 cm² tissue culture flask, where it was incubated at 37°C for 24 hours. The culture medium was then replaced with fresh medium, and the cell condition was monitored daily until a confluent cell monolayer was formed. Next, HFF cells were grown by serial passage in standard growth medium of MEM containing R-salt supplemented with 10% FBS, L-glutamine, penicillin, and gentamicin. Each lot of cells was confirmed to be free of mycoplasma infection, regularly passaged, or used for assays at or before passage 10.

[0066] Antiviral assay To obtain accurate estimates of antiviral activity and statistical data, antiviral and cytotoxicity data were obtained for each virus in a series of 3–5 individual experiments. Each assay included positive and negative control compounds, as well as infected and uninfected controls, to ensure experimental integrity. Simultaneous assessment of cytotoxicity was performed on each assay plate using the same number of cells and equivalent levels of compound exposure to obtain accurate selection index (SI) values. All liquid handling steps were performed using BioMek4000, significantly improving assay efficiency and reducing analyst hands-on time.

[0067] The CPE reduction assay was performed on a monolayer (5000 cells / well) of human foreskin fibroblasts (HFFs) in a 384-well plate using assay medium consisting of Earl's salt, 2% FBS, and MEM containing standard concentrations of L-glutamine, penicillin, and gentamicin. 5000 cells were seeded in a 384-well microtiter plate and incubated at 37°C for 24 hours in a humidified 5% CO2 incubator to allow for the formation of a confluent monolayer. Dilutions of antiviral test compounds, including phyllocyclovir, were prepared directly in the plate in a series of 5-fold dilutions in double wells, resulting in final concentrations ranging from 0.1 to 300 μM or 0.003 to 10 μM. This allows for a broad dynamic range of compound concentrations to facilitate the detection of the antiviral activity of unknown compounds with weak or potent antiviral activity.

[0068] Cell monolayers were infected with virus strains containing AdV5 at an infection multiplicity (MOI) of approximately 0.005 PFU / cell. Infected cells were incubated in virus control wells at 37°C until 100% CPE was observed. Cell pathology was assessed by adding CellTiter-Glo® reagents (Promega, Madison, WI). The concentration of the antiviral test compound sufficient to reduce CPE by 50% (EC2) was determined. 50The values ​​were interpolated from experimental data using standard methods in Microsoft Excel. Cytotoxicity was also determined using the CellTiter-Glo® bioluminescent cell viability assay (Promega, Madison, WI), and the concentration of the test compound that reduced cell viability by 50% (CC) was determined. 50 ) is calculated from the data, and the selection index (SI) value is used as a measure of antiviral activity. 50 / EC 50 The calculation was performed as follows. The results are shown in Table 1 and Figure 1 below.

[0069] [Table 1]

[0070] Abbreviation: CDX-Sidofovir; PFA-Foscarnet; GCV-Ganciclovir; ACV-Acyclovir; PCV-Penciclovir; FIAU-Fluoriodoarabinosyladenine; IDU-Idoxuridine; BDCRB-Bromodichlororibobenzimidazole; CMX001-Brincydofovir; AZT-Azidothymidine; FCV-Filocyclovir; 4-Thio-IDU-4-Thioidoxuridine; N-MCT-N-Methanocarbatimidine; L-BHDU-L-Bromovinyl-Hydroxymethyl-Dioxolanuracil; PMEA-Adefovir

[0071] The results shown in Table 1 demonstrate that filociclovir (FCV) showed significantly improved inhibitory activity against adenovirus 5 (SI=43) compared to all other test compounds, was at least twice as potent as cidofovir (CDV) (SI=24), the next most effective antiviral agent, and significantly better than ganciclovir (GCV) (SI≧1.5).

[0072] Figure 1 shows the percentage reduction in AdV5 replication as a function of phyllocyclovir concentration in the range of 0.01 to 100 μM. The results in Figure 1 demonstrate that even a low concentration of phyllocyclovir as 0.2 μM is effective in reducing AdV replication.

[0073] Similar experiments were conducted with philocyclovir against adenovirus 6, adenovirus 7, and adenovirus 8. The results are shown in Table 2 below. Philocyclovir showed strong inhibition against adenovirus 8 (SI ≥ 61).

[0074]

Table 2

[0075] Example 2. Neutral Red Survival Assay for Measuring the Inhibition of Adenovirus 6 (AdV6) by Philocyclovir

[0076] The neutral red cytotoxicity assay was used to detect cell viability or drug cytotoxicity. The principle of this assay is based on the detection of live cells by the uptake of the dye neutral red. Neutral red is a eurhodin dye that stains lysosomes in live cells. Live cells can take up neutral red via active transport and incorporate the dye into lysosomes, while non-live cells cannot take up this chromophore. As a result, after washing, live cells can release the incorporated dye under acidic extraction conditions. The amount of released dye can be quantified and used to determine the total number of live cells or drug cytotoxicity. Thus, cytotoxicity is expressed as a concentration-dependent decrease in the uptake of neutral red after exposure to the compound under investigation.

[0077] The neutral red survival assay was performed in a 96-well format. Briefly, A549 human alveolar basal epithelial adenocarcinoma cells were plated at a concentration of 6×10 3 cells per well 1 day prior to infection. AdV6 infection was at 5×10 3The procedure was performed at PFU / well (estimated 0.5 PFU / cell at the time of infection). Filocyclovir was serially diluted 1:3 on individual 96-well plates with a drug-free final column as a control. AdV6 infection was performed as nine replication wells for each drug concentration, with three non-infected drug control wells for each drug concentration. Immediately before AdV6 infection, the filocyclovir dilution was added to the cell plates. On day 6 post-infection (when viral infection was present and the viral cytopathic effect reached 70–90% in the drug-free wells), neutral red was added for 1 hour, followed by washing the plates three times with PBS to remove unbound cells, and neutral red was extracted from the remaining cells using 50% ethanol / 1% glacial acetic acid. The filocyclovir plates were read, and the results were graphed using GraphPad Prism graphing and statistical software (GraphPad Software, San Diego, CA).

[0078] The results are shown in Figure 2. As can be seen in Figure 2, 10 -1 Low phyllocyclovir concentrations of μM were effective in improving the survival rate of AdV6-infected cells.

[0079] Example 3. Immunofluorescence assay of phyllocyclovir-treated human A549 cells infected with AdV5 or AdV6.

[0080] Human A549 cells on coverslips in 6-well plates were infected with either AdV5 or AdV6 at an MOI of 5 PFU / cell, or pseudoinfected. After 1 hour, phyllocyclovir was added to a final concentration of 0, 4, 10, or 40 μM. 27 hours post-infection, A549 cells were fixed in paraformaldehyde (3.7% in PBS) and permeabilized with methanol. Cells were stained for adenovirus DNA-binding protein and adenovirus hexon. DBP staining is antinuclear and homogeneous during early AdV infection (before AdV DNA replication). DBP associates with replication centers as infection progresses. Replication centers are initially small “dots” (each dot originating from one next-generation AdV genome). Replication centers expand and “multiply” as DNA replication occurs. Nuclei enlarge and deform as infection progresses. The AdV hexon, the most abundant component of the AdV viral capsid, is not expressed until DNA replication occurs and is therefore considered a "late" AdV protein. The results are shown in Figures 3 and 4.

[0081] As shown in Figures 3 and 4, the results of filocyclovir treatment were the same for AdV5 and AdV6 infections. Filocyclovir was highly effective in preventing late-stage infection (as indicated by hexon staining). No hexon-positive cells were observed in the 40 μM filocyclovir wells, and very few hexon-positive cells were observed in the 10 μM filocyclovir wells. Some hexon-positive cells were observed in the 4 μM filocyclovir wells, but these wells indicated substantially inactive infection compared to the control (untreated) wells (indicated by DBP staining patterns and nuclear size and shape).

[0082] The results demonstrate that filociclovir is a potent inhibitor of human adenovirus 5 (AdV5), AdV6, and AdV8. This level of activity is similar to that of cidofovir (CDV) and far superior to that of ganciclovir (GCV), which has an IC50 value of 66 μM against AdV5. (See Table 1.) The range of AdV serotypes tested to date suggests that FCV could potentially be developed as a pan-adenovirus inhibitor.

[0083] Example 4. Pharmacokinetics of filocyclovir

[0084] To study the pharmacokinetics of filocyclovir, the inventors used an adenovirus-infected Syrian male hamster model. The Syrian hamsters were administered either oral (PO 50 mg / kg) or intravenous (IV 10 mg / kg) doses of filocyclovir. The results are shown in Figure 5, demonstrating that filocyclovir remained detectable 6 hours after PO or IV administration.

[0085] Syrian Hamster AdV Model The Syrian male hamster model is particularly advantageous because it mimics the pathology seen in human patients and can be used to test the effectiveness of antiviral compounds (as outlined in Wold, WSM and Toth, K., Advances in Cancer Research, 115:69-92 (2012)). The Syrian hamster is one of two rodent species (the other being the cotton rat) that tolerate infection with AdV species C (types 1, 2, 5, and 6).

[0086] This model can be used to conduct controlled in vivo experiments to test the effectiveness of anti-adenovirus compounds (Ying et al., Antimicrobial Agents and Chemotherapy, 58(12):7171~7181 (2014); Toth et al., Proc. Natl. Acad. Sci. USA, 105(20):7293~7297 (2008); Tollefson et al., 2014; Toth et al., Viruses, 7(3):1409~1428 (2015)).

[0087] For these experiments, young hamsters were immunosuppressed with cyclophosphamide (CP), a drug often used as part of pretreatment regimens for human transplant recipients. After the desired degree of immunosuppression was achieved, the hamsters were intravenously (iv) infected with AdV5, resulting in AdV5 replication in most organs, most notably in the liver (Toth et al., 2008, cited above).

[0088] Drug preparations For post-administration, filocyclovir was suspended at 5 mg / ml in 0.4% carboxymethylcellulose (Sigma C5678) and sonicated until visually homogeneous. For intravenous administration, filocyclovir was dissolved in DMSO (Sigma D2650) and then diluted with water to a final concentration of 5 mg / ml filocyclovir and 75% DMSO. The administration solution was prepared one day before use and stored at 4°C.

[0089] Experimental Design Male Syrian hamsters weighing approximately 100g were purchased from Envigo (Hackensack, NJ). All hamsters were immunosuppressed using cyclophosphamide (CP), administered intraperitoneally at a dose of 140mg / kg, followed by 100mg / kg twice weekly. The animals received three injections of CP prior to filocyclovir administration. Two groups of animals (12 hamsters / group) were used for two different administration routes. In addition, three untreated animals (the same three hamsters for both routes) were used as simulated controls. Po and iv experiments were performed over two consecutive days. As the hamsters weighed approximately 100g (±10%), 1ml and 0.2ml were administered via the po and iv routes, respectively, to achieve the desired dose. For both routes, three hamsters were sacrificed at 0.5, 1, 3, and 6 hours post-administration.

[0090] Plasma and liver samples were collected from all animals and stored at -80°C until analyzed by LC-MS as follows: All samples and standards were processed on ice; • Standard curves are constructed for all samples spiked with the true compound (DMSO reconstituted from fresh powder spiked in "blank" hamster plasma) and 500 ng / mL of carbamezapine (internal standard); • Initially, dilute the sample in "blank" hamster plasma (attempting to obtain a concentration on the standard curve); • Sample extracted with twice the sample volume using 0.1% ammonium formate in methanol (i.e., 30 μL of sample + 60 μL of 0.1% ammonium formate in methanol); • Extracted sample centrifuged using the maximum setting of a microcentrifuge; The supernatant is analyzed using LC / MS / MS dynamic multiple reaction monitoring (DMRM) with a custom method.

[0091] Example 5. Evaluation of the therapeutic efficacy of high-dose filocyclovir against human AdV6 administered intravenously in male immunosuppressed Syrian hamsters.

[0092] As described above, filociclovir has been shown to be an effective inhibitor of the replication of several adenovirus strains in vitro and exhibits good oral bioavailability in hamsters.

[0093] Next, the inventors evaluated whether filociclovir exhibits anti-adenovirus efficacy in immunosuppressed Syrian hamsters infected with AdV6 intravenously (iv). Four dose levels of 10, 30, 60, and 100 mg / kg poqd were tested. The maximum dose was determined based on rat toxicological data.

[0094] Experimental Design All hamsters were immunosuppressed with cyclophosphamide (CP). CP was administered intraperitoneally at a dose of 140 mg / kg, followed by 100 mg / kg twice weekly.

[0095] Filocyclovir in powder form was suspended in 0.4% carboxymethylcellulose (Sigma C5678) at 1, 3, 6, and 10 mg / ml doses, sonicated until visually homogeneous, and the aliquots were stored at 4°C. The aliquots were equilibrated to room temperature before administration. Hamsters weighing approximately 100g were administered appropriate suspensions in 1 ml volumes at dose levels of 10, 30, 60, and 100 mg / kg. The reference to "vehicle" refers to 0.4% carboxymethylcellulose.

[0096] The hamsters were divided into eight groups of 15 hamsters per group (excluding group 2, which had only 5 hamsters; see Table 3), immunosuppressed, and then subjected to vehicle (groups 1-2) or 2 × 10⁶ hamsters. 10AdV6 (groups 5-9) at PFU / kg was administered intravenously. Groups 1 and 3 received a drug vehicle (POQD), group 4 received 10 mg / kg of filocyclovir (POQD), group 5 received 30 mg / kg of filocyclovir (POQD), group 6 received 60 mg / kg of filocyclovir (POQD), groups 2 and 7 received 100 mg / kg of filocyclovir (POQD), and group 8 received cidofovir (20 mg / kg, three times weekly). For all groups, drug administration was started one day before the challenge and continued throughout the study period according to the above schedule.

[0097] The animals' weight and signs of illness were recorded daily. Five days after the challenge, five hamsters (as designated at the start of the experiment) were sacrificed from each group (except group 2) and subjected to macroscopic pathological observation. Serum and liver samples were collected, and the viral load in the liver was determined by the TCID50 assay, and the serum was analyzed for transaminase levels. The remaining 10 hamsters were sacrificed 14 days after the challenge. Hamsters that appeared visually mortal before day 14 were sacrificed as needed. In addition to hamsters observed to be mortal, all hamsters that had lost more than 20% of their original body weight were also sacrificed. Serum and liver tissue were collected from hamsters sacrificed at the end of the experiment and from hamsters sacrificed in a mortal state, and the serum transaminase levels and viral load in the liver were banked to determine as much as possible. Liver tissue was preserved in formalin for possible histopathological examination.

[0098] [Table 3]

[0099] result

[0100] Lifespan observation In the experiment, there were 5 treatment-related deaths, all in the AdV6+ vehicle group (Figure 6A). Filociclovir at 10 or 30 mg / kg reduced mortality (Figure 6A). Initially, all animals in the AdV6-infected group experienced weight loss, which was reversed by treatment with 10 mg / kg or 30 mg / kg of filociclovir (Figure 6B). Weight gain in animals with AdV6+ 10 mg / kg of filociclovir was slightly lower than in uninfected animals and in the AdV6+ 30 mg / kg filociclovir group. Higher doses of filociclovir, particularly 100 mg / kg, were toxic to AdV6-infected animals (not shown), and these groups were excluded from the study.

[0101] autopsy Extensive renal lesions were observed in sacrificial samples on day 5 (D5) in animals treated with AdV6+ 60 mg / kg and 100 mg / kg of filociclovir.

[0102] Serum transaminase levels Five days after the challenge, serum was collected from five animals in each group and analyzed for transaminase levels. Two hamsters in the AdV6+ vehicle group had high transaminase levels, but neither the filocyclovir-treated nor the CDV-treated animals showed elevated serum transaminase levels (Figure 7; showing alanine aminotransferase [ALT]).

[0103] Viral load in the liver Five days after the challenge, untreated AdV6-infected hamsters had a high viral load in the liver (Figure 8). Treatment with 30 mg / kg of filociclovir suppressed AdV6 replication to undetectable levels (Figure 8). In the AdV6 + 10 mg / kg filociclovir group, there was one animal with a very low hepatic viral load, but the inventors were unable to detect AdV6 in the livers of any of the other animals in this group (Figure 8). CDV treatment reduced viral replication from very low levels to unquantifiable levels (Figure 8).

[0104] conclusion Data for two low doses of filociclovir (10 mg / kg and 30 mg / kg) are preferred. Treatment with either of these doses inhibited viral replication (Figure 8), reduced liver damage (Figure 7), and lowered morbidity and mortality (Figure 6). The 10 mg / kg filociclovir dose was slightly less effective than the 30 mg / kg dose (see slightly lower weight gain in Figure 6 and slightly higher viral load in the liver in Figure 8). The 60 mg / kg and 100 mg / kg filociclovir doses were toxic to AdV6-infected hamsters, and these two groups, as well as the vehicle + 100 mg / kg group, were excluded from the study.

[0105] Example 6. Evaluation of the therapeutic efficacy of low-dose filocyclovir against human AdV6 administered intravenously in male immunosuppressed Syrian hamsters.

[0106] Next, the inventors evaluated whether low doses of filocyclovir exhibit anti-adenovirus efficacy in immunosuppressed Syrian hamsters infected intravenously (iv) with AdV6.

[0107] Three dose levels, 1, 3, and 10 mg / kg POQD, were tested.

[0108] All hamsters were immunosuppressed using cyclophosphamide (CP), administered intraperitoneally at a dose of 140 mg / kg, followed by 100 mg / kg twice weekly. Filocyclovir in powder form was suspended in 0.4% carboxymethylcellulose (Sigma C 5678) at 0.1, 0.3, and 1.0 mg / ml and sonicated until visually homogeneous. Filocyclovir was prepared weekly, aliquoted daily, and stored at 4°C. Aliquots were equilibrated to room temperature before administration.

[0109] The hamsters were divided into 7 groups of 15 hamsters / group (see Table 4), immunosuppressed as described above, and given vehicles (groups 1-2) or 4 x 10 10AdV6 (groups 3-7) at PFU / kg was administered intravenously. Groups 1 and 3 received a drug vehicle (POQD), groups 2 and 6 received 10 mg / kg of filocyclovir (POQD), group 4 received 3 mg / kg of filocyclovir (POQD), group 5 received 1 mg / kg of filocyclovir (POQD), and group 7 received cidofovir (a single dose of 37 mg / kg followed by 20 mg / kg three times weekly). For all groups, drug administration was started one day before the challenge and continued throughout the study period according to the schedule described above.

[0110] [Table 4]

[0111] The animals' weight and signs of illness were monitored and recorded daily. Five days after the challenge, five hamsters from each group (as specified at the start of the experiment) were euthanized and subjected to macroscopic pathological observation. Serum and liver were collected, and the viral load in the liver was determined by the TCID50 assay, and the serum was analyzed for transaminase levels. The remaining ten hamsters were euthanized 14 days after the challenge. Hamsters that were near death before day 14 were euthanized as needed. In addition to animals judged to be near death by observation, the inventors euthanized all hamsters that had lost more than 20% of their original body weight. Liver tissue from all animals was preserved in formalin for histopathological examination.

[0112] result

[0113] Lifespan observation In this experiment, there were 14 treatment-related deaths: 9 in the AdV6+ vehicle group and 5 in the AdV6+ 1 mg / kg filociclovir treatment group (Figure 9A). Initially, all AdV6-infected hamsters lost weight, which was completely reversed by treatment with 10 mg / kg filociclovir and partially reversed by treatment with 3 mg / kg filociclovir. 1 mg / kg filociclovir did not mitigate weight loss (Figure 9B). Cidofovir (CDV) treatment completely reversed weight loss (Figure 9B). Treatment with 10 mg / kg filociclovir in uninfected hamsters did not result in weight loss (Figure 9A).

[0114] autopsy On day 5, four hamsters in the AdV6+ vehicle group and three hamsters in the AdV6+ 1 mg / kg filociclovir group exhibited mottled livers and enlarged gallbladders (signs of adenovirus lesions). Similar findings were observed in animals slaughtered as mortal. No other significant findings were observed in the 14-day slaughter.

[0115] serum chemistry A filocyclovir qd dose of 10 mg / kg suppressed AdV6-induced liver injury as determined by serum transaminase levels, but a filocyclovir qd level of 1 mg / kg had little effect (Figure 10, ALT shown). Treatment with 3 mg / kg of filocyclovir had a moderate but significant effect (p=0.0419) (Figure 10). CDV completely suppressed AdV6-induced liver lesions (Figure 10).

[0116] Viral load in the liver A 10 mg / kg filocyclovir qd dose inhibited AdV6 replication in the liver, but a 1 mg / kg qd dose was ineffective (Figure 11). A 3 mg / kg filocyclovir qdpo dose suppressed viral replication to an intermediate level. CDV completely suppressed AdV6 replication in the liver (Figure 11).

[0117] conclusion The data suggest that filociclovir at a dose of 10 mg / kg poqd was effective in suppressing lesions caused by intravenous infection in immunosuppressed hamsters induced by AdV6, while filociclovir at 3 mg / kg had a moderate effect, and filociclovir at 1 mg / kg was ineffective.

[0118] Example 7. Evaluation of therapeutic efficacy of delayed treatment with 10 mg / kg of filocyclovir after challenge with AdV in male immunosuppressed Syrian hamsters.

[0119] Experimental Design Immunosuppression was induced in male Syrian hamsters (Envigo, Hackensack, NJ) weighing 60g-80g by intraperitoneal administration of cyclophosphamide (CP) at a dose of 140mg / kg, followed by administration of 100mg / kg twice weekly.

[0120] Filocyclovir powder was suspended at 1 mg / ml in 0.4% carboxymethylcellulose (Sigma C5678) and sonicated until visually homogeneous. Filocyclovir was prepared weekly, aliquoted daily, and stored at 4°C. Aliquots were allowed to equilibrate to room temperature before administration.

[0121] The hamsters were divided into eight groups of 15 hamsters each (Table 5), immunosuppressed as described above, and then given 4 × 10 per kg of vehicle or food. 10 AdV6 plaque-forming units (PFUs) were administered intravenously. Filociclovir was administered po-10 mg / kg qd one day before initiation, or one, two, three, or four days after AdV6 injection. As controls, an AdV6-infected group that did not receive filociclovir and a group that received either a viral vehicle and / or drug vehicle alone or a viral vehicle plus drug (initiated one day before the challenge) were used.

[0122] [Table 5]

[0123] The hamsters' weight and signs of illness were recorded daily. Seven days after the challenge, five hamsters from each group (as specified at the start of the experiment) were euthanized and subjected to macroscopic pathological observation. Serum and liver samples were collected, and the viral load in the liver was determined by the TCID50 assay, and the serum was analyzed for transaminase levels. The remaining ten hamsters were euthanized 14 days after the challenge. Hamsters that were near death before day 14 were euthanized as needed. In addition to hamsters determined to be near death by observation, the inventors also euthanized all hamsters that had lost more than 20% of their original weight.

[0124] result

[0125] Lifespan observation In this experiment, there were 14 treatment-related deaths: 9 in the AdV6+ vehicle group and 5 in the AdV6+ 1 mg / kg filociclovir group (Figure 12A). Initially, all AdV6-infected hamsters lost weight, which was completely reversed by treatment with 10 mg / kg filociclovir and partially reversed by treatment with 3 mg / kg filociclovir. However, 1 mg / kg filociclovir was unable to mitigate weight loss (Figure 12B). CDV treatment completely reversed weight loss (Figure 12B). Treatment of uninfected animals with 10 mg / kg filociclovir did not result in weight loss (Figure 12B).

[0126] autopsy On day 5, four hamsters in the AdV6+ vehicle group and three hamsters in the AdV6+ 1 mg / kg filociclovir group exhibited mottled livers and enlarged gallbladders (signs of adenovirus lesions). Similar findings were observed in hamsters that were euthanized. No other significant findings were observed. No significant findings were observed in hamsters on day 14.

[0127] serum chemistry A filocyclovir dose of 10 mg / kg qd suppressed AdV6-induced liver injury as determined by serum transaminase levels, but filocyclovir had little effect at a dose level of 1 mg / kg qd (Figure 13, ALT shown). Treatment with 3 mg / kg filocyclovir had a moderate but significant effect (p=0.0419) (Figure 13). CDV completely suppressed AdV6-induced liver lesions (Figure 13).

[0128] Viral load in the liver A dose of 10 mg / kg qd filociclovir inhibited AdV6 replication in the liver, but 1 mg / kg qd had no effect (Figure 14). A dose of 3 mg / kg qdpo filociclovir suppressed viral replication to an intermediate level. CDV completely suppressed AdV6 replication in the liver (Figure 14).

[0129] conclusion The data demonstrate that a dose of 10 mg / kg poqd filocyclovir is safe and effective in suppressing lesions caused by intravenous infection in immunosuppressed hamsters with AdV6 up to 4 days after infection. A dose of 1 mg / kg filocyclovir was not effective, but a dose of 3 mg / kg filocyclovir had a moderate effect.

[0130] Considering the aforementioned data, filociclovir has been shown to be an effective antiviral agent for inhibiting a range of adenovirus types, leading to the conclusion that filociclovir is useful as a pan-adenovirus treatment.

[0131] All publications, patent applications, patents, and other documents cited herein are incorporated by reference in their entirety. The examples above are illustrative and not intended to limit the scope. Obvious variations of the disclosed methods and alternative embodiments of the present invention will be apparent to those skilled in the art in light of the foregoing disclosure. All such obvious variations and alternatives are considered to fall within the scope of the present invention as described herein.

Claims

1. A pharmaceutical composition for preventing or treating adenovirus infections in mammals (excluding adenovirus infections in skin tissue), comprising an effective amount of filocyclovir or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, wherein the adenovirus is adenovirus 3 (AdV3).

3. The pharmaceutical composition according to claim 1, wherein the adenovirus is adenovirus 4 (AdV4).

4. The pharmaceutical composition according to claim 1, wherein the adenovirus is adenovirus 5 (AdV5).

5. The pharmaceutical composition according to claim 1, wherein the adenovirus is adenovirus 6 (AdV6).

6. The pharmaceutical composition according to claim 1, wherein the adenovirus is adenovirus 7 (AdV7).

7. The pharmaceutical composition according to claim 1, wherein the adenovirus is adenovirus 8 (AdV8).

8. The pharmaceutical composition according to claim 1, wherein the adenovirus is adenovirus 19 (AdV19).

9. The pharmaceutical composition according to claim 1, wherein the adenovirus is adenovirus 37 (AdV37).

10. The pharmaceutical composition according to claim 1, wherein the mammal is a human.

11. Use of filocyclovir in the manufacture of pharmaceuticals for the treatment of adenovirus infections in mammals (excluding adenovirus infections in skin tissue).

12. The use according to claim 11, wherein the adenovirus is adenovirus 3 (AdV3).

13. The use according to claim 11, wherein the adenovirus is adenovirus 4 (AdV4).

14. The use according to claim 11, wherein the adenovirus is adenovirus 5 (AdV5).

15. The use according to claim 11, wherein the adenovirus is adenovirus 6 (AdV6).

16. The use according to claim 11, wherein the adenovirus is adenovirus 7 (AdV7).

17. The use according to claim 11, wherein the adenovirus is adenovirus 8 (AdV8).

18. The use according to claim 11, wherein the adenovirus is adenovirus 19 (AdV19).

19. The use according to claim 11, wherein the adenovirus is adenovirus 37 (AdV37).

20. The use according to claim 11, wherein the mammal is a human.

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