WO2010019698A2 - Inhibition of calicivirus (norovirus) - Google Patents

Abstract

The invention provides a method of treatment or prophylaxis of Calicivirus infection, comprising administering a pharmaceutical composition comprising a compound selected from the group of compounds consisting of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), honokiol, magnolol, ribavirin, z-guggulsterone, and a combination thereof and a pharmaceutically-acceptable carrier to a patient having a Calicivirus infection, or at risk for becoming infected with a Calicivirus.

Description

INHIBITION OF CALICIVIRUS (NOROVIRUS)

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 61/088,187, filed August 12, 2008, which is incorporated by reference.

STATEMENT REGARDING

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This invention was made with government support under COBRE grant 2 P20 RRO 16443 -07 awarded by the National Institutes of Health and under Animal Health Project No. KS481846 awarded by the United States Department of Agriculture. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Caliciviruses (Family Caliciviridae) are small, nonenveloped viruses of 27-35 nm in diameter (Green, K. Y. Chancock, R.M. & Kapikian, A.Z. (2001) in Fields Virology, eds. Knipe, D.M. & Howley, P.M. (Lippincott, Williams & Wilkins, Philidelphia), Vol. 1, pp. 841-874)). They possess a single-strand, plus-sense genomic RNA of 7-8 kb. Calicivirus replication proceeds through a minus strand RNA intermediate, which is used as the template for the synthesis of positive-sense full length genome and subgenomic RNA. This replication is catalyzed by the nonstructural proteins, including the viral RNA dependent RNA polymerase (RdRp), and occurs in cytoplasmic membrane-associated replicase complexes (Green, K. Y. Chancock, R.M. & Kapikian, A.Z. (2001) in Fields Virology, eds. Knipe, D.M. & Howley, P.M. (Lippincott, Williams & Wilkins, Philidelphia), Vol. 1, pp. 841-874)). [0004] Four genera have been established in the family: Norovirus, Sapovirus, Lagovirus, and Vesivirus. (Green et al, J Infect. Dis. 181, Suppl. 2, S322-330 (2000)). These genera have a similar genomic structure, but differ significantly in sequence. Overall amino acid identities between capsid proteins of viruses from different genera are below 30%. There are at least five genogroups (genogroup 1 to 5) in the Norovirus genus. Overall amino acid identities between capsid proteins from same genogroup or different genogroup viruses are about 70% or about 40-50%, repectively. Recent studies showed that noroviruses are responsible for greater than 90% of non-bacterial gastroenteritis outbreaks and are associated with an estimated 23 million cases of gastroenteritis in the U.S. each year (Mead et al., Emerg Infect Dis 5, 607-25 (1999)).

[0005] Norwalk virus (NV, a prototype norovirus) was originally identified in stools collected from an outbreak of gastroenteritis at a school in Norwalk, Ohio in 1968 (Kapikian et al., J Virol 10, 1075-81 (1972)). The viral genome is -7.7 kb in length and organized into three open reading frames (ORPs 1, 2 and 3) that encode a ~1800-amino acid polyprotein, the major capsid protein (VPl), and a minor capsid protein (VP2), respectively. (Xi et al., Science 250, 1580-3 (1990)). The polyprotein from ORFl is processed by a viral proteinase into final products including the viral RdRp. The structural proteins, VPl and VP2, are synthesized from a subgenomic RNA containing ORFs 2 and 3. Studies of NV replication and the development of antiviral drugs have been severely hampered by the absence of cell culture systems and animal models (Duizer et al., J Gen Virol 85, 79-87 (2004)). Studies of noroviruses associated with disease in humans have been hampered by the continued absence of a cell culture system (Duizer et al., J Gen Virol 85, 79-87 (2004)). Recent advances in norovirus research include the discovery of a murine norovirus that grows in a murine macrophage-like cell line (Karst et al., Science 299(5612): 1575-8 (2003)) and the demonstration that transfection of a full-length cDNA clone of the NV genome (under control of the T7 promoter) into modified vaccinia Ankara (MVA)-T7 infected cells allowed the expression of viral proteins and subsequent NV RNA replication (Asanaka et al., Proc Natl AcadSci USA. 102(29): 10327-32 (2005)). Chang et al. reported the generation of a stable RNA replicon system for NV that functions in both human Huh-7 cells and hamster BHK21 cells, and that circumvents the need for a helper virus (Chang et al., Virology 353, 463-473 (2006)). The replicon-bearing cells expressed NV proteins and RNA, and could be maintained after multiple passages in the presence of G418. Importantly, the replicon- bearing cells could be examined for the effects of potential viral inhibitors, and they provided evidence that NV replication is sensitive to the effects of exogenous interferon (IFN). Virus replicon systems (especially viruses which are fastidious in cells) have proved an important tool in the investigation of virus-host interactions and anti-viral drug developments (Blight, et al., Science 290, 1972-4 (2000); Foy et al., Science 300(5622): 1145-8 (2003); Gale and Foy, Nature 436(7053):939-45 (2005)), and the availability of a NV replicon provides a new system in which such interactions can be assessed for the human noroviruses. NV replicon- bearing cells were shown to be an excellent platform to screen antivirals (Chang et al., Virology 353, 463-473 (2006)). [0006] Although norovirus infection is generally considered self-limiting and a short- term illness, recent findings showed that the infection could last longer than several days or even several months especially in immunocompromised patients. (Nilsson, M. et al., J Virol 11, 13117-24 (2003)). The treatment options for norovirus infection are limited partly due to the absence of screening systems for antiviral drugs.

[0007] Caliciviruses are plus strand RNA viruses in the family Caliciviridae that consists of four genera, Norovirus, Sapovirus, Lagovirus, and Vesivirus (Green et al., J Infect. Dis. 181, Suppl. 2, S322-330 (2000)). Caliciviruses are important pathogens in humans and animals with wide variety of pathogenicity that ranges gastroenteritis and system infections (Green et al., J Infect. Dis. 181, Suppl. 2, S322-330 (2000)). Viruses in genera Vesivirus and Lagovirus include animal viruses such as vesicular exanthema swine virus, feline calicivirus and rabbit hemorrhagic disease virus. Viruses in the genera Norovirus and Sapovirus cause gastroenteritis in humans and animals (Green et al., Human caliciviruses, p. 841-874. In D. M. Knipe and P. M. Howley (ed.), Fields Virology, vol. 1. Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0008] Recent studies estimate that human enteric caliciviruses are responsible for more than 90% of non-bacterial gastroenteritis outbreaks (Fankhauser et al., J Infect Dis. 178(6):1571-8 (1998)) and as many as 23 million cases of gastroenteritis in the U.S. each year (Mead, P.S. et al., Emerg Infect Dis 5, 607-25 (1999)). Norwalk virus (NV) is a prototype strain of the noro viruses, and was associated with an outbreak of gastroenteritis in Norwalk, Ohio in 1968 (Kapikian et al., J Virol 10, 1075-81 (1972)). [0009] Studies of the replication of human enteric caliciviruses have been severely hampered by the absence of a cell culture system (Duizer, et al., J Gen Virol 85, 79-87 (2004)). Among the noroviruses, only murine norovirus (strain MNV-I) (Karst et al., Science. 299(5612):1575-8 (2003)) has been successfully propagated in cell culture (Wobus et al., PLoS BiolA2:e432 (2004)). Murine noroviruses present widely in laboratory mouse colonies without apparent clinical symptoms (Hsu et al., Comp Med. 56(4):247-51 (2006); Wobus et al., PLoS Biol Λ2:e432 (2004)). Interestingly MNV-I has a tissue tropism of macrophage-like cells in vivo and in vitro, but it is not clear if human noroviruses target to such cells at present.

[0010] Recently the generation of Norwalk virus (NV) replicon-bearing cells in BHK21 and Huh-7 cells was reported. Interferon (IFN)-α effectively inhibited the replication of NV in the (Chang et al., Virology 353, 463-473 (2006)) was demonstrated. The replicon-bearing cells were generated by transfecting RNA transcripts derived from a plasmid containing the full length NV genome and neomycin resistant gene (neomycin phosphotransferase II, NPT II) in the place of VPl region (pNV-Neo) (Chang et al., Virology 353, 463-473 (2006)). The replicon bearing cells provide an excellent tool to study the replication of noroviruses and serve a platform to screen potential antiviral drugs.

[0011] U.S. Patent 6,923,992 describes the use of compounds of Bao-Ji-Wan, for anti- diarrhea and relieving gastrointestinal symptoms. However, this patent does not disclose the treatment of any such condition due to Calicivirus. Accordingly, there remains a need for methods for treating or for prophylaxis of Calicivirus infections.

BRIEF SUMMARY OF THE INVENTION

[0012] The invention provides a method of treatment or prophylaxis of Calicivirus infection, comprising administering a pharmaceutical composition comprising a compound selected from the group of compounds consisting of interferon-alpha (IFN-α), interferon- gamma (IFN-γ), honokiol, magnolol, ribavirin, z-guggulsterone, and a combination of any or all thereof (such as 2, 3, 4, 5, or all 6 of the compounds) and a pharmaceutically- acceptable carrierto a patient having a Calicivirus infection, or at risk for becoming infected with a Calicivirus. In another aspect, the invention provides the use of such compounds or such combinations thereof for preparing a medicament for treatment or prophylaxis of Calcivirus infection.

[0013] In another aspect, the invention provides a method of making an inhibitor of a Calicivirus, brining into contact a Calicivirus-cell based replicon system with a compound, identifying a compound that has an inhibitory effect on the Calicivirus replicon, and synthesizing the compound with the inhibitory effect on the Calicivirus replicon. [0014] The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) [0015] The accompanying drawings and figures, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description, serve to explain the principles of the invention. [0016] Fig. 1. The effect of IFN-α and IFN-γ on the NV replication in HG23 cells (A). The effect of IFN-α and IFN-γ on the expression of NV genome. One-day old semi-confluent NV replicon-bearing HG23 cells were incubated with various concentrations of IFN-α and IFN-γ for 72 h, and then total RNA was prepared for real time qRT-PCR for detecting NV genome. The reduction of NV genome by IFNs was calculated by the comparison to that with mock-treatment. (B). The effect of IFN-γ on the expression of NV ProPol or neomycin phosphotranferase in HG23 cells. One-day old semi-confluent HG23 cells were incubated with 0 (mock) or 100 units/ml of IFN-γ for 72 h. The cells were then fixed with 100% methanol, and stained with antibodies to NV ProPol (upper panel) or neomycin phosphotranferase (lower panel) and FITC-conjugated secondary antibody. The negative control includes parental Huh-7 cells with the same staining.

[0017] Fig. 2. The effect of ribavirin on the NV replication in HG23 cells (A). The effect of ribavirin on the expression of NV genome. One-day old semi-confluent HG23 cells were incubated with various concentrations ranging from 0 (mock) to 100 μM of ribavirin for 48, 72 and 96 h, and then total RNA was prepared for real time qRT-PCR for detecting NV genome. The reduction of NV genome by IFNs was calculated by the comparison to that with mock-treatment. (B). The effect of ribavirin on the expression of neomycin phosphotranferase in HG23 cells. One-day old semi-confluent HG23 cells were incubated with various concentrations ranging from 0 (mock) to 200 μM of ribavirin for 72 h. The cells were then fixed with 100% methanol, and stained with antibody to neomycin phosphotranferase and FITC-conjugated secondary antibody. The negative control includes parental Huh-7 cells with the same staining.

[0018] Fig. 3. The combination effects of IFN-α and ribavirin on NV replication in HG23 cells. (A). The effect of ribavirin alone or co-treatment with IFN-α and ribavirin on the expression of NV genome. One-day old semi-confluent HG23 cells were incubated with various concentrations ranging from 0 (mock) to 100 μM of ribavirin with or without 2 units/ml of IFN-α. After 72 h of the incubation, total RNA was prepared for real time qRT- PCR for detecting NV genome. The reduction of NV genome by the treatments was calculated by the comparison to that with mock-treatment. (B). The effect of ribavirin alone or co-treatment with IFN-α and ribavirin on the expression of NPT II detected by Western blot analysis. One-day old semi-confluent HG23 cells were incubated with various concentrations ranging from 0 (mock) to 100 μM of ribavirin with or without 2 units/ml of IFN-α. After 72 h of the incubation, cell lysate was prepared for Western blot analysis with antibody to neomycin phosphotransferase.

[0019] Fig. 4. The effect of ribavirin on MNV-I in RAW267.2 cells. Confluent RAW267.2 cells in 6- well plates were treated with varying concentrations (0-100 μM) of ribavirin for 6 hr before MNV-I was added to the same medium at a MOI of 5. The virus infected cells were further incubated for 24 h, and the virus replication was measured by the TCID50 assay after the plates were freezing and thawing 3 times.

[0020] Fig. 5. The effect of supplement of guanosine in ribavirin treatment and of MPA on NV replication in HG23 cells. (A) The effect of ribavirin with or without of guanosine (100 μM) on NV genome in HG23 cells. One-day old semi-confluent HG23 cells were incubated with various concentrations ranging from 0 (mock) to 100 μM of ribavirin with or without 100 μM of guanosine. After 72 h of the incubation, total RNA was prepared for real time qRT-PCR for detecting NV genome. The reduction of NV genome by the treatments was calculated by the comparison to that with mock-treatment. (B). The effects of MPA on NV replication. One-day old semi-confluent HG23 cells were incubated with various concentrations ranging from 0 (mock) to 2 μM of MPA for 72 h. After the incubation, total RNA was prepared for real time qRT-PCR for detecting NV genome. The reduction of NV genome by the treatments was calculated by the comparison to that with mock-treatment. [0021] Fig. 6. The effect of magnolol on the NV replication in HG23 cells (A). The effect of magnolol on the expression of NV genome. One-day old semi-confluent HG23 cells were incubated with various concentrations ranging from 0 (mock) to 20 μM of magnolol for 48, 72 and 96 h, and then total RNA was prepared for real time qRT-PCR for detecting NV genome. The reduction of NV genome by magnolol was calculated by the comparison to that with mock-treatment.

[0022] Fig. 7. Chemical structure of z-gugglesterone . [0023] Fig. 8. Chemical structure of rivabirin. [0024] Fig. 9 Chemical structure of IFN-γ. [0025] Fig. 10. Chemical structure of magnolol. [0026] Fig. 11. Chemical structure of honokiol.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0028] IFN-γ and ribavirin effectively inhibited the replication of NV in the replicon- bearing cells. Ribavirn (l-β-D-ribofuranosyl-l,2,4-triazole-3-carboxamide) is a synthetic guanosine analogue and has shown antiviral actions against a range of DNA and RJSfA viruses including hepatitis C virus (HCV) and respiratory syncytial virus (De Clercq E, J. Clin Virol. 30(2): 115-33 (2004)). In the chronic HCV infection, combination therapy of IFN-α and ribavirin is the most effective treatment. Similar studies were done in the HCV replicon- bearing cells for the antiviral effects of IFNs, ribavirin and other specific viral inhibitors (Bartenschlager, et al., Antiviral Res. 60(2):91-102 (2003); Pietschmann et al., Curr Opin Drug Discov Devel. 4(5):657-64 (2001)). Interferon-γ is synthesized by various cells including NK cells during the acute inflammations and plays an essential role as a frontier fighter against the invading microbes before specific immune responses elicit (Samuel, Clin Microbiol Rev. 14(4):778-809 (2001)). However, many viruses develop mechanisms against the IFN system to avoid its antiviral effects, which includes blocking STAT pathways and inhibiting the synthesis of IFNs by viral proteins or genome (Samuel, Clin Microbiol Rev. 14(4):778-809 (2001)). Using the replicon-bearing cells, Chang et al demonstrated that NV replicon lacked such anti-IFN mechanism, which may be a reason for the high sensitivity to IFN-α treatment (Chang et al., Virology 353, 463-473 (2006)). This is the first report that IFNs and ribavirin could be excellent antiviral drugs against norovirus replication. Also this report confirms that NV replicon-bearing cells as a significant tool for studying basic research and drug discovery relevant to the control of norovirus gastroenteritis. [0029] The development of effective therapies for noroviral gastroenteritis has been hampered by the absence of a cell culture system. Recently we reported the generation of Norwalk virus (NV) replicon bearing cells in BHK21 and Huh-7 cells, and demonstrated that interferon (IFN)-α effectively inhibited the replication of NV in the cells. In continuing studies for screening potential anti-noroviral agents, we tested IFN-γ, ribavirin, Z- gugglesterone, magnolol for their effects on NV replication in the cells. Like IFN-α, IFN-γ inhibited the replication of NV in the replicon bearing cells showing the reduction of NV genome and proteins in a dose-dependent manner. The effective dose for reducing 50% (ED50) of NV genome and protein was calculated as 50 units/ml. When ribavirin was applied to the cells, it effectively reduced NV genome and protein with the ED50 calculated as approximately 40 μM. When combined together, IFN-α and ribavirin showed additive effects on the inhibition of NV replication.

[0030] Z guggulsteron as antiviral drugs to treat the gastroenteritis in humans and animals by enteric caliciviruses including noroviruses. Z guggulsteron is available freely at local vitamin stores as a natural product extracted from Indian guggl tree (named as guggul herb, guggul lipid or guggul gum). In this invention, Z gugglesteron which is an antagonist of bile acids on the farnesoid X receptor (FXR) can block the replication of norovirus. Z- gugglesteron inhibited the replication of NV in the replicon bearing cells showing the reduction of NV genome and proteins in a dose-dependent manner. The effective dose for reducing 50% (ED50) of NV genome and protein was calculated as 40 μM. [0031] Magnolol is a bioactive compound found in the bark of the Houpu magnolia (Magnolia officinalis) and is available commercially (e.g., from Sigma-Aldrich). It is known to act on the GABAA receptors in rats, as well as having antifungal properties. Magnolol is a bioactive plant component isolated from the root and stem bark of Magnolia officinalis. Magnolol displays an array of activities including antifungal, 1 antibacterial, and antioxidant effects.2 Magnolol has also been shown act as a natural inhibitor to acyl-CoA: cholesterol acyltransferase (ACAT).3 Magnolol has been shown to demonstrate anti-inflammatory activity by interfering with NF-kB signaling. It also appears to have anti-inflammatory properties related to vascular disorders such as atherosclerosis due to its ability to inhibit IL- 6-induced STAT3 activation.4 Magnolol inhibited the replication of NV in the replicon bearing cells showing the reduction of NV genome and proteins in a dose-dependent manner. The effective dose for reducing 50% (ED50) of NV genome and protein was calculated as 20 μM.

[0032] Honokiol is a structural isomer of Magnolol, the purification and synthesis of has been reported (Amblard et al., J Med Chem. 2006 Jun 1;49(11):3426-7; Esumi et al, Bioorg Med Chem Lett. 2004 May 17;14(10):2621-5; Takeya et al., Chemical & Pharmaceutical Bulletin (1986), 34(5), 2066-70) and is available commercially (e.g., from Sigma-Aldrich). Honokiol has shown pro-apoptotic effects in melanoma, sarcoma, myeloma, leukemia, bladder, lung, prostate, and colon cancer cell lines (Shigemura et al., Cancer. 2007 Apr 1;109(7): 1279-89; Ishitsuka et al., Blood. 2005 Sep l;106(5):1794-800; Battle et al., Blood. 2005 JuI 15;106(2):690-7; Bai et al., J Biol Chem. 2003 Sep 12;278(37):35501-7). Honokiol inhibits phosphorylation of Akt, p44/42mitogen-activated protein kinase (MAPK), and src. Additionally, honokiol modulates the nuclear factor kappa beta (NfKB) activation pathway, an upstream effector of vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX- 2), and MCLl, all significant pro-angiogenic and survival factors. Honokiol induces caspase- dependent apoptosis in a TRAIL-mediated manner, and potentiates the pro-apoptotic effects of doxorubicin and other etoposides. So potent is honokiol's pro-apoptotic effects that it overcomes even notoriously drug resistant neoplasms such as multiple myeloma and chronic B-cell leukemia. Honokiol has been shown to promote neurite outgrowth and have neuroprotective effects in rat cortical neurons. Additionally, honokiol increases free cytoplasmic Ca²⁺ in rat cortical neurons (Fukuyama et al., Bioorg Med Chem Lett. 2002 Apr 22;12(8):1163-6). Honokiol inhibits platelet aggregation in rabbits in a dose-dependent manner, and protects cultured RAEC against oxidized low density liproptein injury. Honokiol significantly increases the prostacyclin metabolite 6-keto-PGFl alpha, potentially the key factor in honokiol's anti-thrombotic activity (Hu et al., Acta Pharmacol Sin. 2005 Sep;26(9): 1063-8.).

[0033] In accordance with the purpose of this invention, as embodied broadly described herein, this invention, in one aspect, relates to the development for potential anti-noroviral agents.

[0034] In conjunction with the inventive method, a pharmaceutical composition comprising a compound selected from the group of compounds consisting of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), honokiol, magnolol, ribavirin, z-guggulsterone, and a combination thereof is administered to a patient for Calicivirus prophylaxis or treatment of a Calicivirus infection. The composition can be formulated for administration to the patient by any desired route. Thus, for example, the composition can be formulated for administration topically (e.g., a cream or ointment), via cubcutaneous, intramuscular, or parenteral injection, inhalation, orally, or via any other traditional route of administration. Where the compositions are formulated for oral administration, such forms can be immediate-release, sustained-release, or extended release forms, such as capsules, caplets, tablets, lozenges, etc. The composition also can be formulated as a powder for oral administration. [0035] It will be observed that standard practice in pharmaceutical compounding can be employed to make a composition suitable for use in the inventive method. Thus, the composition can include suitable binders, diluents, glidants, budders, flavoring agents, preservatives, and other common excipients. (see Rowe et al., "Handbook of Pharmaceutical Excipients" (Pharmaceutical Press), 5^th Ed. 2006). Moreover, to the extent that formulations containing IFN-α, IFN-γ, honokiol, magnolol, ribavirin, or z-guggulsterone (or any combination thereof) are commercially available, such can be employed in the inventive method.

[0036] The dose of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), honokiol, magnolol, ribavirin, z-guggulsterone (or combination thereof) can vary depending on the size of the patient and dosage form, and the selection of an appropriate dose is within the skill of treating physician. However, typically, the dose will range from about 1 μg/kg to about 100 mg/kg.

[0037] The inventive method can be employed prophylactically or therapeutically against any Calicivirus, such as Norovirus (e.g., Norwalk virus and others), Sapovirus, Lagovirus,

Vesivirus. The composition is administered to the patent suitable for effecting prophylaxis or therapeutic treatment of such infections. For prophylactic use, it is not necessary for the inventive method to completely prevent infection, although this is a desired outcome. It is sufficient for prophylaxis to reduce the risk of infection or reduce the severity of any infection. Similarly, therapeutic use of the inventive method need not cure or eliminate the

Calicivirus infection, although this is a preferred result. It is sufficient for the inventive method to reduce the severity or duration of the infection or its symptoms.

[0038] It is, of course, possible for the inventive method to be performd in repetition, such as daily dosing for a period of time (e.g., a week or so), or dosing 2, 3, or 4 times daily as directed by a health care professional.

[0039] While the inventive method is contemplated for use with human patients, the patent also can be a non-human animal, such as a mouse, rat, dog, cat, pig, horse, goat, cow, monkey or ape.

EXAMPLE

A. Materials and Methods

[0040] Cells, viruses, and reagents. The Huh-7, HG23 (the NV replicon-bearing cells, cells) and a murine macrophage-like RAW267.2 cells were maintained in Dulbecco's minimal essential medium containing 10% fetal bovine serum and antibiotics [chlortetracycline (25 μg/ml), penicillin (250 U/ml), and streptomycin (250 μg/ml)] (DMEM- C). Murine norovirus- 1 (MNV-I) was kindly provided by Dr. Virgin (Washington University at St Louis, MO), and maintained in RAW267.2 cells. Recombinant IFN type 1 (human IFN- αA+IFN-αD fusion protein) and recombinant human IFN-γ were purchased from Serotec Inc. (Raleigh, NC). Polyclonal Antibody specific to NV ProPol was described in previous report {Chang, 2006 #152}. Antibodies specific for NPT II or β-actin were obtained from Santa Cruz biotech (Santa Cruz, CA) or Cell Signaling Tech (Danvers, MA), respectively. Ribavirin, Z-gugglesteron, magnolol and MPA were purchased from Sigma (St. Louis, MO), dissolved in distilled water or DMSO, respectively.

[0041] Detection of Norwalk virus RNA and proteins. Immunofluorescence assay (IFA). The ProPol serum was added to methanol-fixed monolayers of cells, and the binding of antibodies was detected with fluorescein isothiocyanate (FITC)-conjugated, affinity- purified goat antibodies to guinea pig immunoglobulin G (IgG) (ICN Biomedicals, Aurora, OH) as described previously (Chang, Virology 304, 302-10 (2002)). We also used rabbit antiserum specific for the NPT II and FITC-conjugated goat antibodies to rabbit IgG for detecting the presence of NV replicon in cells.

[0042] Western blot analysis. Protein samples of Huh-7 and HG23 cells were prepared in SDS-PAGE sample buffer containing 2% 2-mercaptoethanol, and sonicated for 20 s. The proteins were resolved in a 10% No vex Tris-Bis gel (Invitrogen) and transferred to a nitrocellulose membrane. The membranes were probed with guinea pig antibodies specific for the ProPol protein, and the binding of the antibodies was detected with peroxidase- conjugated, goat anti-guinea pig IgG (Sigma, St .Louis, MO). Or the membranes were probed with rabbit antiserum specific for NPT II and peroxidase-conjugated, goat anti-rabbit IgG. Following incubation with a chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate, Pierce biotechnology, Rockford, IL), the signals were detected with X-ray film. We also used rabbit antiserum specific for the NPT II and peroxidase- conjugated goat antibodies to rabbit IgG for detecting the presence of NV replicon in cells. [0043] Real-Time qRT-PCR. The quantity of NV genome in the replicon-bearing cells was measured by real-time qRT-PCR with the One-step Platinum qRT-PCR kit (Invitrogen), following an established protocol with GI-specific primers and FAM-labeled Gl probes (Kageyama et al., J Clin Microbiol. 41(4): 1548-57 (2003)). For quantity control of cellular RNA, qRT-PCR for the β-actin was performed as described previously (Spann et al., J Virol. 78(8):4363-9 (2004)). For qRT-PCR, the total RNA in cells (in 6-well plates) was extracted with the RNeasy kit (Qiagen, Valencia, CA). A standard concentration curve was generated with serial dilutions of RNA transcripts derived from pNV-Neo in each experiment to calculate the total number of genome copies present. The relative genome levels in cells with various treatments were calculated after the RNA levels were normalized with those of β- actin.

[0044] Treatment of NV-harboring cells with IFN-α or IFN-γ. The effects of IFN-α or IFN -γ on the replication of NV in the replicon-bearing cells were examined at concentrations ranging from 1 to 20 units/ml (for IFN-α) or 1 to 100 units/ml (for IFN-γ). Varying concentrations of IFN-α or IFN-γ were added to one-day old, 80-90% confluent HG23, and the cells were analyzed for viral protein and genome expression at 24, 48, 72 or 96 h after treatment. The NV protein or genome expression levels were examined by IFA and Western blot analysis, or qRT-PCR, respectively, as described above. Western blot analysis included the detection of β-actin using the specific antibody for the loading control. The inhibitory effect of IFN-α or IFN-γ on the NV replicon was calculated as the concentration of IFN-α or IFN-γ that resulted in 50% reduction of NV genome (ED50) as detected by qRT-PCR. [0045] The effects of ribavirin, gugglesteron, magnolol on the replication of NV in the replicon-bearing cells. One-day old, 80-90% confluent HG23 cells were treated with varying concentrations of ribavirin (0-100 μM) to examine its effects on the replication of NV. After the desired time points, the NV protein or genome were analyzed with IFA and Western blot analysis, or qRT-PCR, respectively. To examine the combinatory effects of ribavirin and IFN-α, one-day old HG23 cells were treated with 2 units/ml (ED50) of IFN-α and varying concentrations of ribavirin (0-100 μM), and the reduction of NV protein and genome were compared to that by the ribavirin treatment alone. The nonspecific cytotoxic effects in HG23 cells by ribavirin were monitored by measuring the rates of cell propagation in the presence of various concentrations of ribavirin after 24, 48 and 72 hrs of incubation. The cells were digested with trypsin and counted after staining with trypan blue for examining the cell toxicity by ribavirin. In addition, we used to cell cytotoxicity assay kit (Promega) for the non-specific cytotoxic effects by ribavirin.

[0046] The effects of ribavirin on the replication of MNV-I in RAW267.2 cells. Because MNV-I can be cultured in the murine macrophage-like cell line RAW267.2 cells, we used MNV-I as a surrogate system to examine the effects of ribavirin on norovirus replication in cells. Confluent RAW267.2 cells in 6-well plates were treated with varying concentrations (0-100 μM) of ribavirin for 6 hr before MNV-I was added to the same medium at a MOI of 5. The virus infected cells were further incubated for 24 and 48 h. The replication of MNV-I in the presence of ribavirin was measured by the TCID50 assay after the plates were freezing and thawing 3 times. The nonspecific cytotoxic effects in RAW267.2 cells by ribavirin were monitored by the method described above. [0047] The potential mechanisms of ribavirin on the replication of NV. Several mechanisms of actions for the antiviral effects of ribavirin have been suggested and those include the depletion of intracellular guanosine triphosphosphate (GTP) through the inhibition of the cellular inosine monophosphate (IMP) dehydrogenase and triggering catastrophic mutations on virus genome by ribavirin. To examine the potential mechanisms, we performed two experiments: 1) supplement of guanosine in the medium with ribavirin treatment; 2) sequence analysis of NV genome in the presence of absence of ribavirin. First, HG23 cells were pre-incubated with 100 μM of guanosine for 6 hr before ribavirin was added to the medium at various final concentrations (0 [mock-medium] -100 μM). After 24 or 48 hr of the treatment, the NV protein or genome was analyzed with Western blot analysis or realtime qRT-PCR, respectively, as described above. We also tested mycophenolic acid (MPA) which is a potent inhibitor of the cellular IMP dehydrogenase for the expression of NV genome in the replicon-bearing cells. Various concentrations ranging 0 (mock-medium) to 2 μM of MPA were added in the medium of semi-confluent HG23 cells for 48 h, and total RNA was extracted for real-time qRT-PCR. The comparable expressions of NV genome by MPA were calculated by that of mock treatment. To examine if the treatment of ribavirin triggered catastrophic mutagenesis, HG23 cells were incubated with 0 (mock-medium) or 100 μM of ribavirin for 48 hr. After the incubation, total RNA was extracted and RT-PCR was performed to amplify the region encoding NV pol (nucleotide number 4298-5101) with primers of NV-4kF (GAGAATGCTAAACATATGAAACCC (SEQ ID NO: I)) and NV-5kR (GCGGGTCCAGAAGATTTGGCGTTC (SEQ ID NO:2)). Sequence analysis of the amplicon was done either directly or after the products were cloned into pCR2.1 vector (Invitrogen). For the sequencing of the gene in the recombinant vector, we selected 18 clones per each group, and analyzed mutations in the region. The sequence analysis was performed using the GenomeLab DTCS-Quick Start kit (Beckman-Coulter, Fullerton, CA). Sequences were resolved on an automated sequencer (Beckman Coulter).

[0048] Statistical analysis. The effects of IFNs, ribavirin, MPA, and combination of IFN-α and ribavirin on NV replication were analyzed by Student's t test. Results were considered statistically significant when the P value was < 0.05. B. Results [0049] Effect of IFN-α and IFN-γ on the NV replicon. Previously we reported that IFN-α effectively reduced the expression of NV proteins and genome: the effective dose for IFN-α for reducing NV protein (ProPol) and genome copies in HG23 cells to 50% of that observed in the non-treated (mock) control at 72 h was calculated to be approximately 2 units/ml. Similarly, we examined the effects of IFN-γ on the replication of NV in the cells. The HG23 cells were treated with increasing concentrations of human IFN-γ (up to 200 units/ml), and its effect on NV genome and protein expression was monitored by real time qRT-PCR, and IFA and Western blot analysis, respectively. Like IFN-α, the addition of IFN- γ to HG23 cells inhibited NV genome and protein expression in a dose-dependent manner (Fig.l), while the cells themselves showed no toxic effects of the treatment (data not shown). The presence of 100 units /ml of IFN-γ for 72 h resulted in nearly complete clearance of the replicon proteins and RNA (Fig. IA and B). The reduction rates of NV genome in the presence of IFN-γ was 81%, 73%, 43% and 8% at 2, 10, 50 and 100 units/ml, respectively for 72 h incubation (Fig IA). The ED50 of IFN-γ for reducing NV protein (ProPol) and genome copies in HG23 cells at 72 h was calculated to be approximately 40 units/ml (Fig. 1). However, the recombinant human IFN-γ did not show any effects on the expression of NV proteins and genome in the BHK21 -based NV replicon-bearing cells (G3 cells) (data not shown). The expression of NPT II was also examined using IFA and Western blot analysis with the antibody against the protein for the evaluation of NV replication. By IFA, the expression of NPT II was well correlated with that of NV ProPol (Fig. IB), indicated that the commercial antibody could be a good tool to study the expression of NV replication in NV replicon bearing cells.

[0050] The effects of ribavirin on NV replication. Next, we examined a known antiviral agent, ribavirin on NV replicon in HG23 cells. The HG23 cells were treated with increasing concentrations of ribavirin (up to 400 μM) for up to 96 h, and its effect on NV protein expression was monitored by IFA, and Western blot analysis using antibody to NV ProPol and the neomycin phosphotransferase. Also qRT-PCR assay was used for detecting NV genome in mock-treated or ribavirin-treated NV replicon-bearing cells. Minimal cell toxicity was observed in HG23 cells treated with ribavirin up to 200 μM for up to 96 h. The treatment of ribavirin at the concentrations above 10 μM in HG23 cells reduced the expression of NV genome in a dose-dependent manner (Fig.2A). The reduction of NV genome was observed at 24 h of the treatment and increased to 72h. The ED50 was calculated to be approximately 40 μM at 72 h of the ribvirin treatment (Fig. 2A). Like NV genome, the expression of NV ProPol and the NPT II was reduced in the presence of ribavirin in a dose-dependent manner (Fig.2B and 3B) without nonspecific toxic effects up to 200 μM. The HG23 cells were incubated with ribavirin and IFN-α (2 unit/ml) to examining any enhanced effects by the co-treatment. The treatment of IFN-α alone at the concentration of 2 units/ml reduced the expression of NPT II and NV genome to about 50% (Fig. 3 A and B). While ribavirin treatment showed the reduction of NV geneome to 43%, 35% and 18% of the mock-treated cells at 20 μM, 50 μM and 100 μM, repectively, co-treatment with IFN-α (2 units/ml) resulted in the reduction to 38%, 28% and 10% at the same concentrations of ribavirin (Fig. 3A). In addition, the similar reduction rates of NTP II in Western blot analysis were observed by the co-treatment (Fig. 3B). These results indicated that there was additive effect by the co-treatment rather than synergistic effects in HG23 cells. [0051] The effects of Z-gugglesteron on NV replication. The HG23 cells were treated with increasing concentrations of Z-gugglesterone (up to 100 μM) for up to 96 h, and its effect on NV protein expression was monitored by IFA, and Western blot analysis using antibody to NV ProPol and the neomycin phosphotransferase. Also qRT-PCR assay was used for detecting NV genome in mock-treated or Z-gugglesterone -treated NV replicon-bearing cells. Minimal cell toxicity was observed in HG23 cells treated with Z-gugglesterone up to 100 μM for up to 96 h. The treatment of Z-gugglesterone at the concentrations above 10 μM in HG23 cells reduced the expression of NV genome in a dose-dependent manner. The reduction of NV genome was observed at 24 h of the treatment and increased to 72h. The ED50 was calculated to be approximately 40 μM at 72 h of the Z-gugglesterone treatment. Like NV genome, the expression of NV ProPol and the NPT II was reduced in the presence of Z-gugglesterone in a dose-dependent manner.

[0052] The effects of magnolol on NV replication. The HG23 cells were treated with increasing concentrations of magnolol (up to 200 μM) for up to 96 h, and its effect on NV protein expression was monitored by IFA, and Western blot analysis using antibody to NV ProPol and the neomycin phosphotransferase. Also qRT-PCR assay was used for detecting NV genome in NV replicon-bearing cells. Minimal cell toxicity was observed in HG23 cells treated with magnolol up to 100 μM for up to 96 h. The treatment of magnolol at the concentrations above 5 μM in HG23 cells reduced the expression of NV genome in a dose- dependent manner. The reduction of NV genome was observed at 24 h of the treatment and increased to 72h. The ED50 was calculated to be approximately 20 μM at 72 h of the magnolol treatment. Like NV genome, the expression of NV ProPol and the NPT II was reduced in the presence of magnolol in a dose-dependent manner.

[0053] The effects of ribavirin on the growth of MNV-I. Murine norovirus-1 can be cultured in RAW267.2 cells and we examined if ribavirin could reduce the replication of MNV-I. First, nonspecific cytotoxicity of ribavirin in RAW267.2 cells was measured, and there was minimal toxicity at the concentrations below 200 μM determined by the methods described in the Method section. The treatment of ribavirin above 100 μM, reduced the titer of MNV-I at least one log at 24 hr after the virus inoculation (Fig. 4). At 48 h of the treatment, however the virus titers of ribavirin and mock-treated groups were similar each other (data not shown).

[0054] Potential mechanisms of antinoroviral effects by ribavirin. When we examined if the replenishment of guanosine affected the antiviral effects of ribavirin, we found that the addition of 100 μM of guanosin in the ribavirin treated cells, moderately reversed the antiviral effects by ribavirin (Fig. 5A). While the incubation of ribavirin alone for 48 h resulted in the reduction of NV genome level to 90%, 42%, 37% and 8% at 10 μM, 20 μM, 50 μM and 100 μM, respectively, the addition of guanosine to ribavirin reduced the levels to 100%, 62%, 51% and 34% at the same concentrations of ribavirin (Fig. 5A). The potent IMP dehydrogenase inhibitor, MPA also effectively reduced the NV genome levels at the concentrations above 0.2 μM (Fig. 5B). Sequence analysis of NV genome (the Pol region) in HG23 cells treated and mock-medium or 100 μM ribavirin for 48 h showed that there is no mutation in the RT-PCR products detected by the direct sequence of the amplicon. The sequence analysis of the NV Pol region after the amplicon was cloned into pCR2.1 indicated there were no different mutation rates between ribavirin treated and non-treated groups. Among 18 recombinant plasmids each group, point mutations occurred 12 or 13 plasmids in the ribavirin treated or non-treated groups, respectively Also total numbers of mutations were 22 or 23 in the ribavirin treated or non-treated groups, respectively C. Discussion

[0055] Without a cell culture system it is extremely difficult (if not impossible) to study the development of effective therapies for noroviral gastroenteritis. Recent development of NV-replicon bearing cells enabled us to pursue potential anti-noroviral agents. In previous works, we demonstrated that IFN-α was effective to inhibit NV replication, and in this invention, we discovered that IFN-γ also reduced the expression of NV genome and proteins in a dose-dependent manner. [0056] Type I (IFN-α) and II (IFN-γ) play a central role in innate immunity against virus infections before adapted immunity arises. The functions of IFNs (type I and II) mediated through the interaction with IFN receptors which are expressed in all nucleated cells. The binding of IFNs to their cognitive receptors triggers activation of signal transducers and activator of transcriptions (STATs) and cascade events, which results in the inductions of various antiviral proteins such as PKR and RNase L. However, many viruses armed with anti-IFN mechanism, such as those counteracting the STATs and inhibiting the IFN synthesis. Previously we demonstrated that NV did not have strong anti-IFN mechanism in the replicon-bearing cells and this may be a reason for its high sensitivity to the treatment of IFN-α or IFN-γ in this study. Recently, Marcello et al. reported that both IFN-α and IFN-γ effectively reduced the replication of HCV in the replicon-harboring cells, but with different pathways. They demonstrated that the anti-HCV effect of IFN-γ was independent of type 1 and II IFN receptors and the kinetics of IFN-γ-mediated STAT activation and induction of potential effector genes were distinct from those of IFN-α (Marcello et al., Interferons alpha and lambda inhibit hepatitis C virus replication with distinct signal transduction and gene regulation kinetics. Gastroenterology 131 : 1887-98 (2006)). We plan to conduct similar studies to elucidate the mechanism of anti-NV effects of IFN-α and IFN-γ in HG23 cells. [0057] Ribavirn is a synthetic guanosine analogue and has shown antiviral actions against a range of DNA and RNA viruses including hepatitis C virus and respiratory syncytial virus. In this investigation, we found that ribavirin was also very effective to reduce NV replication in the replicon-bearing cells, as ED50 was calculated as approximately 40 μM. Ribavirin also showed the inhibitory effects on the murine norovirsus-1 (MNV-I) in RAW267.2 cells. In the presence of 100 μM of ribvirin, the titer of MNV-I dropped approximately 10 fold at 24 h after virus infection. However, at 48 h of virus infection, the titers of MNV-I in the presence of absence ribavirin were similar. It was reported that ribavirin was not effective on the replication of feline calicivirus. We are not sure the reason of the discrepancy between MNV- 1 and FCV. It may be related to different viruses or cell types. Ribavirin requires to be metabolized in cells to elicit antiviral activity, and it is possible that there are different metabolite rates of ribavirin among different cells. It has been shown that the effects of combination treatment of IFN and ribavirin were synergistic in various viruses including HCV in the replicon-bearing cells (Kanda et al., J Viral Hepat 11:479-87 (2004)); Tanabe et al., J Infect Dis 189:1129-39 (2004)). However, in this study, we found that when combined together, IFN-α and ribavirin showed only additive effects on the inhibition of NV replication in the replicon-bearing cells. See Chang, K.O. and George D.W., J. Virol. 81, 12111-12118 (2007).

[0058] Several mechanisms of action for the antiviral activity of ribavirin have been suggested and these include: 1) depletion of intracellular GTP pool by inhibition of the cellular IMP dehydrogenase; 2) induction of catastrophe mutation on viral genome; 3) inhibition of viral polymerase activity, 4) inhibition of viral capping by inhibition of viral or cellular guanylyltransferase activity. The mechanisms of actions may be different in different viruses and it can also be combinational effects with one or two mechanisms predominant. To elucidate potential mechanism of antiviral activity of ribavirin in NV, we tested the first two possibilities in this study. First, we replenish GTP in cells by adding guanosin in the medium in the ribavirin treated cells. In certain viruses (yellow fever virus, human parainfluenza virus 3), addition of guanosin in the medium efficiently reversed the antiviral effects of ribavirin (Leyssen et al., J. Virol. 79:1943-7 (2005)).

[0059] In this investigation, the addition of guanosine in the ribavirin treatment moderately reversed the antiviral effects in the cells (Fig. 5A). We also examined MPA which is a potent uncompetitive inhibitor of IMPDH, and found that MPA effectively inhibit NV replication at the concentrations above 0.2 μM. While the active form of ribavirin is 5'- monophosphate to elicit antiviral activities, the action of MPA is not required to be metabolically activated in the cells. The strong inhibition of NV replication by MPA suggested that the antiviral effects by ribavirin are associated with the inhibition of IMPDH. It has been suggested that ribavirin could trigger catastrophic mutations (including fetal mutations) for the mechanism of the antiviral effect (Crotty et al., Nat Med 6:1375-9 (2000)). Sequencing analysis of the conserved polymerase region of NV in the ribavirin-treated (100 μM) or non-treated groups showed that both groups produced similar rates of mutation. Interestingly, although we did not find any mutations in RT-PCR products, we found high rates of mutations in the clones even without ribavirin. The NV polymerase without proofreading functions may be responsible for the high mutations. This data suggested that the antiviral effects of ribavirin on NV may not be associated with catastrophic mutations in the replicon-bearing cells.

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[0061] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate 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") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0062] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. Use of a compound selected from the group of compounds consisting of interferon- alpha (IFN-α), interferon-gamma (IFN-γ), honokiol, magnolol, ribavirin, z-guggulsterone, and a combination of any or all thereof for preparing a medicament for treatment or prophylaxis of Calcivirus infection.

2. The use of claim 1, wherein the medicament is formulated for oral administration.

3. The use of claim 2, wherein the medicament is an immediate-release or a sustained release formulation.

4. The use of claim 2 or 3, wherein the medicament is a tablet, capsule, caplet, or lozenge.

5. The use of any of claims 1-4, wherein the Calicivirus infection comprises Noro virus, Sapovirus, Lagovirus, Vesivirus or any combination thereof.

6. The use of claim 5, wherein the virus is Norwalk Virus.

7. A method for treatment of Calicivirus infection, comprising administering to a patient infected with a Calicivirus, a pharmaceutical composition comprising a compound selected from the group of compounds consisting of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), honokiol, magnolol, ribavirin, z-guggulsterone, and a combination of any or all thereof and a pharmaceutically-acceptable carrier.

8. A method of prophylaxis of Calicivirus infection, comprising administering to a patient at risk for becoming infected with a Calicivirus, a pharmaceutical composition comprising a compound selected from the group of compounds consisting of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), honokiol, magnolol, ribavirin, z-guggulsterone, and a combination of any or all thereof and a pharmaceutically-acceptable carrier.

9. The method of claim 7 or 8, wherein the composition is formulated for oral administration.

10. The method of claim 9, wherein the composition is an immediate-release or a sustained release formulation.

11. The method of claim 9 or 10, wherein the composition is a tablet, capsule, caplet, or lozenge.

12. The method of any of claims 7-11, wherein the Calicivirus infection comprises Norovirus, Sapovirus, Lagovirus, Vesivirus or any combination thereof.

13. The method of claim 12, wherein the virus is Norwalk Virus.

14. The method of any of claims 7-13, wherein the patient is human.