WO2015081199A1 - Compositions et méthodes pour traiter une infection par le virus de l'herpès - Google Patents

Compositions et méthodes pour traiter une infection par le virus de l'herpès Download PDF

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Publication number
WO2015081199A1
WO2015081199A1 PCT/US2014/067624 US2014067624W WO2015081199A1 WO 2015081199 A1 WO2015081199 A1 WO 2015081199A1 US 2014067624 W US2014067624 W US 2014067624W WO 2015081199 A1 WO2015081199 A1 WO 2015081199A1
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nhc
group
chromen
alkyl
alkenyl
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PCT/US2014/067624
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English (en)
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Yan Yuan
Jun Xu
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The Trustees Of The University Of Pennsylvania
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones

Definitions

  • Kaposi's sarcoma-associated herpesvirus also known as human herpesvirus 8 (HHV-8)
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • KS Kaposi's sarcoma
  • KS Kaposi's sarcoma
  • N. Engl. J. Med. 332 1181-1185, Dupin N, et al. 1995. Lancet. 345:761-762, Schalling M, et. 1995. Nat. Med.
  • the contemporary treatment modalities for KS and other KSHV- associated malignancies include conventional cancer therapies, such as radiation and chemotherapy, or AIDS treatment such as HAART (Antman K, et al. 2000. N. Engl. J. Med. 342: 1027-1038). These cancer therapies in general present serious side effects and tumor response to them is only transient.
  • HPV vaccine in preventing cervical cancer has proven that antiviral orientated therapies have the potential to be used to treat human malignant diseases that are caused by viruses.
  • an antiviral targeting KSHV could prove successful at treating KSHV-associated tumor (Moore PS, et al. 2011. Blood. 117:6973-6974).
  • KSHV-associated disease currently there is no therapy for KSHV-associated disease on the basis of targeting of KSHV.
  • KSHV has two types of replication cycle, latent and lytic replication.
  • latent and lytic replication In KS lesions, most KSHV-transformed spindle-shaped cells are in latent replication phase, but a small percentage of infected cells undergo spontaneous lytic replication (Staskus KA, et al. 1997. J. Virol. 71 :715-719, Sun R, et al. 1999. J. Virol. 73:2232-2242, Zhong W, et al. 1996. Proc. Natl. Acad. Sci. U. S. A. 93:6641- 6646).
  • Increasing evidences suggest that the lytic replication in these cells is necessary for maintaining the stable infection and viral pathogenicity.
  • the majority of KSHV genes including those responsible for malignant cell growth and the alteration of
  • Viral DNA replication is considered an ideal target for antivirals.
  • the mechanisms that control KSHV lytic DNA replication have been reported (Lin CL, et al. 2003. J. Virol. 77:5578-5588, Wang Y, et al. 2004. J. Virol. 78:8615-8629, Wang Y, et al. 2006. J. Virol. 80: 12171-12186, Wang Y, et al. 2008. J. Virol. 82:2867-2882).
  • Topo II topoisomerases I and II
  • MSH2/6, RecQL MSH2/6, RecQL
  • PARP-1 poly[ADP-ribose] polymerase 1
  • Topo II inhibitors namely catalytic Topo II inhibitor, was found to have marked inhibition of KSHV replication with minimal cytotoxicity as indicated by their high selectivity indices (e.g. 31.6 for novobiocin) (Gonzalez-Molleda L, et al. 2012. Antimicrob. Agents Chemother. 56:893-902).
  • Topo II can serve as effective targets for antivirals and catalytic inhibitors of Topo II represent promising antiviral agents for the treatment of malignancies associated with KSHV infection, but clinically useful therapeutics have not been developed. There is thus a need in the art for identifying and generating therapeutics that can be used clinically to treat a KSHV infection. The present invention addresses this unmet need in the art.
  • the invention provides a method of treating a herpesvirus infection. In one embodiment, the invention provides a method of treating a gamma-herpesvirus infection. In one embodiment, the invention provides a method of treating a Kaposi's sarcoma- associated herpes virus (KSHV) infection. In one embodiment, the invention provides a method of treating an Epstein-Barr virus (EBV) infection.
  • KSHV Kaposi's sarcoma- associated herpes virus
  • EBV Epstein-Barr virus
  • the invention provides a method of treating a herpesvirus infection in an individual in need thereof, the method comprising the step of: administering to the individual an effective herpesvirus antiviral amount of a
  • composition comprising at least one compound selected from the group consisting of:
  • each occurrence of R 7 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C 3 -Ci 0 heterocycloalkyl, and C 3 -C 6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted; and X is -CH or O; and
  • each occurrence of R 6 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C 3 -C 10 heterocycloalkyl, and C 3 -C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted;
  • the compound is selected from the group consisting of:
  • the compound of formula (I) is at least one compound of formula (III):
  • each occurrence of R 7 and R 8 is independently selected from the group consisting of H, -Ci-C 6 alkyl, -Ci-C 6 alkenyl, aryl, heteroaryl, cycloalkyl,
  • each occurrence of R 9 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C 3 -C 10 heterocycloalkyl, and C 3 -C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted;
  • the compound is selected from the group consisting of (5)-2-(6-(2-methylbut-3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)propan- 2-yl acetate, a salt, solvate, or N-oxide thereof, and combinations thereof.
  • the method further comprises administering to the individual an effective amount of a therapeutic agent.
  • the therapeutic agent is selected from the group consisting of a protease inhibitor, a cytokine, and an immunomodulator.
  • the method further comprises administering to the individual an effective amount of a chemotherapeutic agent.
  • the chemotherapeutic agent is selected from the group consisting of a topoisomerase II inhibitor, an antibiotic, a vinca alkaloid, an
  • the invention also provides a method for inhibiting replication of a herpesvirus infection. In one embodiment, the invention provides a method for inhibiting replication of a gamma-herpesvirus. In one embodiment, the invention provides a method for inhibiting replication of a KSHV. In one embodiment, the invention provides a method for inhibiting replication of an EBV.
  • Figure 1 is a series of images depicting the results for the compound screening experiments.
  • Figure 1 A Thirty-three compounds derived from a virtual screening were assayed for their effects on KSHV lytic DNA replication. BCBL-1 cells were induced with TPA and treated with each of the compounds at the concentration of 20 ⁇ . Forty-eight hours post-induction, the intracellular KSHV genomic copy numbers were determined using quantitative real-time PCR. Seven compounds (marked with *) exhibited considerable inhibitory effects on viral lytic DNA replication.
  • Figure IB Effects of the compounds on KSHV virion production.
  • Figure 1C Effects of the compounds on host cell viability. Uninduced BCBL-1 cells were treated with each of the compounds at 20 ⁇ for 48 hours and cell viability was assessed by trypan blue staining as described in Materials and methods. Compounds are shown with ascending molecular weights.
  • Figure 2 is a series of images demonstrating the effect of (+)-Rutamarin on KSHV replication and its associated cytotoxicity.
  • Figure 2A Chemical structure of (+)-Rutamarin.
  • Figure 2B The effect of (+)-Rutamarin on KSHV lytic replication and its associated cytotoxicity in BCBL-1 cells that were treated with a wide range of concentration of (+)-Rutamarin 3 hour after the lytic replication was induced by TPA. Intracellular KSHV genomic DNA replication (blue), extracellular virion production, and cell viability were determined as described elsewhere herein. Values were compared to those from the control cells (non-drug treatment).
  • Figure 3 is a graph depicting the effects of (+)-Rutamarin and novobiocin on BCBL-1 cell proliferation.
  • BCBL-1 cells starting with 2> ⁇ 10 5 cells/ml
  • (+)-Rutamarin and novobiocin were exposed to (+)-Rutamarin and novobiocin, respectively, at the concentrations of their IC 50 and 5 XIC50.
  • Data were obtained from three independent determinations and presented as means with standard deviations.
  • Figure 4 is a graph depicting the effects of (+)-Rutamarin and novobiocin on BCBL-1 cell cycle progression.
  • BCBL-1 cells starting with 2x l0 5 cells/ml
  • (+)-Rutamarin and novobiocin at the concentrations of their IC 50 and 5xICso for 48 hours.
  • Cell cycle progressions were measured by propidium iodide (PI) staining followed by flow cytometric analysis. The data are presented as means obtained from three independent experiments.
  • Figure 5 is an image showing inhibition of KSHV orz-Zyt-dependent DNA replication with (+)-Rutamarin.
  • BCBL-1 cells were transfected with KSHV ori-Lyt- containing plasmid (pOri-A) and RTA expression vector (pCR3.1-ORF50). The transfected cells were treated with increasing concentrations of (+)-Rutamarin and incubated for 72 hours. Hirt DNAs were extracted and digested with Kpnl/Sacl or Kpnl/Sacl/Dpnl. Dpnl-resistant viral replicated DNA (Rep'd DNA) was detected by Southern blotting with 32 P-labeled pBluescript plasmid.
  • Figure 6 is a series of images demonstrating that identification of (+)-Rutamarin as a catalytic inhibitor of Topo Ila.
  • Figure 6A The Topo II-mediated kinetoplast DNA decatenation assay was performed to evaluate the effect of testing compounds for Topo Ila activity at the concentration of 100 ⁇ . The catenated (cat) and decatenated (decat) DNA positions in gels were indicated. The Topo Ila activities in the reactions in ( Figure 6A) were quantitated and inhibition rates of compounds were calculated and presented in ( Figure 6B). The inhibition of Topo Ila (Figure 6C) and Topo Ila (Figure 6E) activities by (+)-Rutamarin in a wide range of concentration was determined.
  • Figure 7 is a graph depicting the effect of (+)-Rutamarin on ATPase activity of Topo Ila.
  • the inhibitory effect of (+)-Rutamarin on Topo Ila ATPase activity was measured using the malachite green assay as described elsewhere herein.
  • the OD 6 2o represents the ATP hydrolysis level and reflects the inhibition of ATPase activity by (+)- Rutamarin.
  • Figure 8 is a series of images demonstrating the stability properties of simulation system and binding model of (+)- Rutamarin with Topo Ila.
  • Figure 8A RMSD plot for the backbone atoms (light blue) and (+)-Rutamarin (light green) during the MD simulations after the equilibration.
  • FIG. 8B Distance distributions between the Asn95-, Alal67- and conserved water 2 and (+)-Rutamarin in water at 310 K. The cutoff value for the formation of a hydrogen bond is 3.5 A.
  • Figure 8C The time dependence of distance between carboxyl oxygen of Glu87, amide nitrogen of Asn91, the two conserved water molecules and Mg 2+ during the 10 ns MD simulations.
  • Figure 8D The detail binding model between (+)-Rutamarin and the residues in the ATPase domain of Topo ⁇ . Hydrogen bonds are shown in red dot line and magenta sphere represents Mg cation.
  • Figure 9 is an image demonstrating the dinding free energy decomposition results based on MM-GBSA method.
  • the key residues are labeled.
  • the unit of the each residue's contribution to total binding energy is kcal/mol.
  • Figure 10 is an image showing the cytotoxicity of (+)-Rutamarin to peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Figure 11 is an image showing the effect of (+)-Rutamarin on RTA expression.
  • BCBL-1 cells were induced with 20ng/ml TPA. After three hours, the cells were treated with different concentrations of (+) Rutamarin as indicated. Forty-eight hours post-induction, whole cell extracts were prepared and analyzed by Western blot with antibodies against RTA and ⁇ -actin.
  • Figure 12 is a schematic showing the chemical structure of novobiocin, merbarone and (+)-rutamarin.
  • Figure 13 comprising Figures 13A through 13E is a series of images showing the effects of Topo II inhibitors on EBV replication and their associated cytotoxicity in P3HR-1 cells.
  • EBV lytic replication in P3HR-1 was induced with TPA and sodium butyrate.
  • Topo II catalytic inhibitors, novobiocin ( Figure 13 A), merbarone ( Figure 13B) and (+)-rutamarin ( Figure 13C) were added to the cell culture 3 h after the induction.
  • the concentration ranges tested for different inhibitors are: 0.1-1000 ⁇ (novobiocin), 0.1 -760 ⁇ (merbarone), and 0.01-150 ⁇ ((+)-rutamarin).
  • Intracellular EBV DNA, extracellular virion DNA, and cell viability were determined for each concentration point. These values were compared to those from the control cells (non- drug treatment). Mean values of results from at least three independent experiments and standard deviations are presented on the Faxes of dose-response curves. Topo II inhibitor doses are indicated on the x axes as logarithmic scales. The effect of (+)- rutamarin on cell viability of P3HR-1 cells on 2 and 5 days exposure were shown in Figure 13D and Figure 13E.
  • Figure 14 is a series of images showing the effects of Topo II inhibitors on EBV replication and their associated cytotoxicity in Akata-Bxl cells.
  • EBV lytic replication in Akata-Bxl was induced by treatment of cells with anti-IgG.
  • Topo II catalytic inhibitors, novobiocin ( Figure 13 A), merbarone ( Figure 13B) and (+)-rutamarin ( Figure 13C) in wide ranges were added to the cell culture 3 h after the induction.
  • Intracellular EBV DNA, extracellular virion DNA, and cell viability were determined for each concentration point. These values were compared to those from the control cells (non-drug treatment).
  • Figure 15 is an image showing the effects of Topo II inhibitors on P3HR-1 cell proliferation.
  • P3HR-1 cells starting with 2 x 10 5 cells/ml
  • IC 50 and 5 x IC 50 concentrations
  • Figure 16 is a series of images showing inhibition of EBV ori-Lyt-associated DNA replication with Topo II inhibitors.
  • P3HR-1 cells were transfected with an ori-Lyt-containing plasmid (pEBV-oriL) and ZTA expression vector (McZ).
  • Transfected cells were cultured in the absence or presence of increasing concentrations of novobiocin ( Figure 16A), merbarone ( Figure 16B), and (+)- rutamarin ( Figure 16C). After 72 h of incubation, hirt DNAs were extracted from the cells and digested with EcoRI or EcoRI/Dpn I. Dpnl -resistant products of DNA replication (Rep'd DNA) were detected by Southern blotting with digoxigenin - labeled EcoR I - Hind III fragment from the pEBV-oriL plasmid.
  • the present invention relates to the unexpected discovery that a ligand based virtual screening approach identified a set of coumarin derivatives that exhibited inhibitory activity against Kaposi's sarcoma-associated herpesvirus (KSHV) DNA replication.
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • compounds of the invention exhibit topoisomerase II (Topo II) inhibitor activity.
  • the compounds of the invention belong to the category of catalytic Topo II inhibitors.
  • the compounds of the invention block KSHV lytic DNA replication.
  • the compounds of the invention can be used to treat viral (such as herpesvirus) infection, as well treating and KSHV infection or related conditions.
  • viral such as herpesvirus
  • the invention should not be liminted to treating herpesvirus infection. Rather, the invention includes treating all types of herpesvirus, for example, gamma-herpesvirus infections including but not limited to two human pathogens Epstein-Barr virus (EBV) and KSHV.
  • EBV Epstein-Barr virus
  • KSHV Pharmaceutically acceptable salts and
  • Herpesvirus is an animal DNA virus and Herpesviridae viruses are classified into three subfamilies, that is, the alphaherpesvirinae, betaherpesvirinae and gammaherpesvirinae.
  • Alphaherpesvirinae is classified further into Herpes simplex virus, Varicellovirus, Mardivirus, and Ilto virus and typical ones include HSV-1, HSV-2, varicella/zoster virus, porcine herpes virus 1 (pseudorabies virus), and bovine herpesvirus.
  • Viruses belonging to the betaherpesvirinae include human cytomegalovirus (HCMV), while as viruses belonging to the gammaherpesvirinae, EB virus, Kaposi's sarcoma-associated herpesvirus, and the like are known.
  • Epstein-Barr virus (EB virus) of Lymphocryptovirus genus is typical.
  • the present invention also provides a method for inhibiting gamma- herpesvirus replication in a subject.
  • the present invention also provides a method for inhibiting KSHV replication in a subject.
  • the method comprises administering to the subject a composition of the invention by any suitable route of administration.
  • the method of the present invention is useful for administrating the
  • compositions of the invention to a subject exposed to a gamma-herpesvirus, such as KSHV, and to a subject who is at risk of contact with a gamma-herpesvirus, such as KSHV.
  • a gamma-herpesvirus such as KSHV
  • the compounds of the invention act as a topoisomerase II catalytic inhibitor.
  • Another aspect of the invention pertains to a composition comprising a compound as described herein and a pharmaceutically acceptable carrier or diluent.
  • Another aspect of the present invention pertains to a compound as described herein for use in a method of treatment of the human or animal body by therapy.
  • Another aspect of the present invention pertains to use of a compound, as described herein, in the manufacture of a medicament for use in treatment.
  • the present invention pertains to a method of inhibiting (e.g., catalytically inhibiting) topoisomerase II in a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of a compound of the invention.
  • the present invention pertains to a method of treating a gamma-herpesvirus infection, such as KSHV infection or related conditions, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound of the invention, preferably in the form of a pharmaceutical composition.
  • a gamma-herpesvirus infection such as KSHV infection or related conditions
  • the treatment is treatment of a disease or condition that is ameliorated by the catalytic inhibition of topoisomerase II.
  • the treatment is treatment of a proliferative condition.
  • the treatment is treatment of cancer.
  • an element means one element or more than one element.
  • abnormal when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal” (expected) respective characteristic. Characteristics that are normal or expected for one cell or tissue type might be abnormal for a different cell or tissue type.
  • the term "container” includes any receptacle for holding the pharmaceutical composition.
  • the container is the packaging that contains the pharmaceutical composition.
  • the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition.
  • packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical
  • composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product.
  • the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating or preventing a disease in a subject.
  • a "disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a disorder in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • a disease or disorder is "alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
  • the terms "individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, primates, including simians and humans.
  • herpesvirus or “herpes virus” or “HV” refers to human herpesviruses and may be used, depending on the context, to refer to one, more, or all of the human herpesviruses, including Human Herpesvirus- 1 (HHV-1, Herpes Simplex Virus- 1, HSV1, HSV-1), HHV-2 (Herpes Simplex Virus-2, HSV2, HSV-2), HHV-3 (Varicella Zoster Virus, VZV), HHV-4 (Epstein-Barr Virus, EBV), HHV-5
  • HHV-1 Human Herpesvirus- 1
  • HHV-2 Herpes Simplex Virus-2, HSV2, HSV-2
  • HHV-3 Varicella Zoster Virus, VZV
  • HHV-4 Epstein-Barr Virus, EBV
  • HHV-5 Human Herpesvirus- 1
  • HHV-2 Herpes Simplex Virus-2, HSV2, HSV-2
  • HHV-3 V
  • a herpesvirus is HHV-1.
  • a herpesvirus is HHV-2.
  • a herpesvirus is HHV-3.
  • a herpesvirus is HHV-4.
  • a herpesvirus is HHV-5.
  • a herpesvirus is HHV-6.
  • a herpesvirus is HHV-7.
  • a herpesvirus is HHV-8.
  • each of the terms HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8 may refer to all strains of each respective HHV.
  • each of the terms HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8 may refer to a single strain of that HHV.
  • each of the terms HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV- 8 may include mutants of that particular HHV.
  • HV is HSV (HHV- 1 and/or HHV-2).
  • KS Kaposi sarcoma
  • Classic (or Mediterranean) KS KS
  • endemic KS KS
  • AIDS-related (epidemic) KS KS
  • iatrogenic (transplant-associated) KS immunosuppression-associated KS.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient, subject or individual is a human.
  • composition refers to a mixture of at least one compound of the invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • the pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
  • “Pharmaceutically acceptable” refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability.
  • “Pharmaceutically acceptable carrier” refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered.
  • the term "pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function.
  • a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function.
  • Such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient.
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; ellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic s
  • pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions.
  • the "pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention.
  • Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in
  • salt embraces addition salts of free acids or free bases that are compounds useful within the invention.
  • Suitable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, phosphoric acids, perchloric and tetrafluoroboronic acids.
  • organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, ⁇ -hydroxybutyric, sal
  • Suitable base addition salts of compounds useful within the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, lithium, calcium, magnesium, potassium, sodium and zinc salts.
  • Acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl- glucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding free base compound by reacting, for example, the appropriate acid or base with the corresponding free base.
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs or symptoms of pathology, for the purpose of diminishing or eliminating those signs or symptoms.
  • treating a disease or disorder means reducing the frequency or severity with which a sign or symptom of the disease or disorder is experienced by a patient.
  • the terms "effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • an "effective amount" of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.
  • the term “potency” refers to the dose needed to produce half the maximal response (ED 50 ).
  • the term “efficacy” refers to the maximal effect (E max ) achieved within an assay.
  • (+)-rutamarin refers to (5)-2-(6-(2-methylbut- 3-en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)propan-2-yl acetate.
  • alkyl by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e. Ci- 6 means one to six carbon atoms) and including straight, branched chain, or cyclic substituent groups.
  • Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl.
  • Most preferred is (Ci-C 6 )alkyl, particularly ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.
  • substituted alkyls include, but are not limited to, 2,2-difluoropropyl,
  • heteroalkyl by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized.
  • the heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -0-CH 2 -CH 2 -CH 3 , -CH 2 -CH 2 -CH 2 -OH, -CH 2 -CH 2 -NH-CH 3 ,
  • Up to two heteroatoms may be consecutive, such as, for example, -CH 2 -NH-OCH 3 , or -CH 2 -CH 2 -S-S-CH 3
  • alkoxy employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homo logs and isomers.
  • oxygen atom such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homo logs and isomers.
  • halo or halogen alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.
  • cycloalkyl refers to a mono cyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom.
  • the cycloalkyl group is saturated or partially unsaturated.
  • the cycloalkyl group is fused with an aromatic ring.
  • Cycloalkyl groups include groups having from 3 to 10 ring atoms.
  • Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties:
  • Monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Dicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene.
  • Polycyclic cycloalkyls include adamantine and norbornane.
  • cycloalkyl includes "unsaturated nonaromatic carbocyclyl” or “nonaromatic unsaturated carbocyclyl” groups, both of which refer to a nonaromatic carbocycle as defined herein, which contains at least one carbon carbon double bond or one carbon carbon triple bond.
  • heterocycloalkyl refers to a heteroalicyclic group containing one to four ring heteroatoms each selected from O, Sand N.
  • each heterocycloalkyl group has from 4 to 10 atoms in its ring system, with the proviso that the ring of said group does not contain two adjacent O or S atoms.
  • the heterocycloalkyl group is fused with an aromatic ring.
  • the heterocycloalky group is fused to a second heterocycle.
  • the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quatemized.
  • the heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure.
  • a heterocycle may be aromatic or non-aromatic in nature.
  • the heterocycle is a heteroaryl.
  • An example of a 3-membered heterocycloalkyl group includes, and is not limited to, aziridine.
  • 4-membered heterocycloalkyl groups include, and are not limited to, azetidine and a beta lactam.
  • 5-membered heterocycloalkyl groups include, and are not limited to, pyrrolidine, oxazolidine and thiazolidinedione.
  • 6-membered heterocycloalkyl groups include, and are not limited to, piperidine, morpholine and piperazine.
  • Other non-limiting examples of heterocycloalkyl groups are:
  • non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1 ,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,
  • aromatic refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e. having (4n + 2) delocalized ⁇ (pi) electrons, where n is an integer.
  • aryl employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene.
  • aryl groups include phenyl, anthracyl, and naphthyl. Preferred examples are phenyl and naphthyl, most preferred is phenyl.
  • aryl-(Ci-C3)alkyl means a functional group wherein a one- to three-carbon alkylene chain is attached to an aryl group, e.g.,
  • aryl-CH 2 CH 2 -phenyl Preferred is aryl-CH 2 - and aryl-CH(CH 3 )-.
  • substituted aryl-(Ci-C 3 )alkyl means an aryl-(Ci-C 3 )alkyl functional group in which the aryl group is substituted. Preferred is substituted aryl(CH 2 )-.
  • heteroaryl- (Ci-C 3 )alkyl means a functional group wherein a one to three carbon alkylene chain is attached to a heteroaryl group, e.g. , -CH 2 CH 2 -pyridyl. Preferred is heteroaryl-(CH 2 )-.
  • substituted heteroaryl-(Ci-C 3 )alkyl means a heteroaryl-(Ci-C 3 )alkyl functional group in which the heteroaryl group is substituted. Preferred is substituted heteroaryl- (CH 2 )-.
  • heteroaryl or “heteroaromatic” refers to a heterocycle having aromatic character.
  • a polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include the following
  • heteroaryl groups also include pyridyl, pyrazinyl, pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.
  • polycyclic heterocycles and heteroaryls examples include indolyl (particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2- and 5 -quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1 ,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-difiydrobenzofuryl, 1 ,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7-benzothien
  • 2-benzimidazolyl benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.
  • substituted means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group.
  • substituted further refers to any level of substitution, namely mono-, di-, tri-, terra-, or penta-substitution, where such substitution is permitted.
  • the substituents are
  • the substituents vary in number between one and four. In another embodiment, the substituents vary in number between one and three. In yet another embodiment, the substituents vary in number between one and two.
  • the term "optionally substituted” means that the referenced group may be substituted or unsubstituted. In one embodiment, the referenced group is optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In another embodiment, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.
  • the substituents are independently selected from the group consisting of oxo, halogen, -CN, -NH 2 , -OH, -NH(CH 3 ), -N(CH 3 ) 2 , alkyl (including straight chain, branched and/or unsaturated alkyl), substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted
  • the substituents are independently selected from the group consisting of Ci_ 6 alkyl, -OH, Ci_ 6 alkoxy, halo, amino, acetamido, oxo and nitro. In yet another embodiment, the substituents are independently selected from the group consisting of Ci_ 6 alkyl, Ci_ 6 alkoxy, halo, acetamido, and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic, with straight being preferred.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description
  • the invention provides compositions and methods for modulating and treatment of a gamma-herpesvirus infection, such KSHV infection, or KSHV-mediated effects on cellular proliferation and phenotype, comprising inhibiting KSHV lytic replication.
  • the compounds of the invention can be used to treat or prevent KSHV infection, Kaposi's sarcoma (KS) and related cancers and conditions.
  • KS Kaposi's sarcoma
  • the compounds of the invention inhibit both viral DNA synthesis and virion production with relatively low cytotoxicity.
  • the compounds of the invention inhibit the enzymatic activity of Topo ⁇ in a dose-dependent manner. In one embodiment, the compounds of the invention function as a Topo ⁇ catalytic inhibitor.
  • the compounds of the present invention may be synthesized using techniques well-known in the art of organic synthesis.
  • the starting materials and intermediates required for the synthesis may be obtained from commercial sources or synthesized according to methods known to those skilled in the art.
  • the compound of the invention is a compound of formula (I), or a salt, solvate, or N-oxide thereof:
  • each occurrence of R 7 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C3-C10 heterocycloalkyl, and C 3 -C 6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted; and
  • X is -CH or O.
  • the compound of the invention is a compound of formula (II), or a salt, solvate, or N-oxide thereof:
  • each occurrence of R 6 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C3-C10 heterocycloalkyl, and C3-C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted.
  • the compound of the invention is a compound of formula (III), or a salt, solvate, or N-oxide thereof:
  • each occurrence of R 7 and R 8 is independently selected from the group consisting of H, -Ci-C 6 alkyl, -Ci-C 6 alkenyl, aryl, heteroaryl, cycloalkyl,
  • each occurrence of R 9 is independently selected from the group consisting of H, Ci-C 6 alkyl, Ci-C 6 alkenyl, C 3 -C 10 heterocycloalkyl, and C 3 -C6 cycloalkyl, wherein the alkyl, alkenyl, heterocycloalkyl, or cycloalkyl group is optionally substituted.
  • the compound of the invention is selected from the group consisting of:
  • the compound of formula (III) is 2-(6-(2-methylbut-3- en-2-yl)-7-oxo-3,7-dihydro-2H-furo[3,2-g]chromen-2-yl)propan-2-yl acetate, a salt, solvate, or N-oxide thereof, and any combination thereof.
  • the compounds of the invention may possess one or more stereocenters, and each stereocenter may exist independently in either the R or S configuration.
  • compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In one embodiment, a mixture of one or more isomer is utilized as the therapeutic compound described herein.
  • compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non- limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
  • N-oxides if appropriate, crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound of the invention, as well as metabolites and active metabolites of these compounds having the same type of activity.
  • Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like.
  • the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol.
  • the compounds described herein exist in unsolvated form.
  • the compounds of the invention may exist as tautomers. All tautomers are included within the scope of the compounds presented herein.
  • prodrugs In one embodiment, compounds described herein are prepared as prodrugs.
  • a "prodrug” refers to an agent that is converted into the parent drug in vivo.
  • a prodrug upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.
  • sites on, for example, the aromatic ring portion of compounds of the invention are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In one embodiment, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.
  • Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes suitable for inclusion in the compounds described herein include and are not limited to 2 H, 3 H, U C, 13 C, 14 C, 36 C1, 18 F, 123 1, 125 1, 13 N, 15 N,
  • isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies.
  • substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements).
  • substitution with positron emitting isotopes such as 11 C, 18 F, 15 O and 13 N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
  • Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.
  • the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
  • Protecting groups are used to block some or all of the reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed.
  • each protective group is removable by a different means.
  • Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal.
  • protective groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and/or oxidative conditions.
  • reducing conditions such as, for example, hydrogenolysis
  • oxidative conditions such as, for example, hydrogenolysis
  • Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile.
  • Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of amines that are blocked with acid labile groups, such as t-butyl carbamate, or with carbamates that are both acid and base stable but hydrolytically removable.
  • base labile groups such as, but not limited to, methyl, ethyl, and acetyl
  • carboxylic acid and hydroxy reactive moieties are blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc.
  • Carboxylic acid reactive moieties are protected by conversion to simple ester compounds as exemplified herein, which include conversion to alkyl esters, or are blocked with oxidatively-removable protective groups such as
  • Allyl blocking groups are useful in the presence of acid- and base- protecting groups since the former are stable and are subsequently removed by metal or pi-acid catalysts.
  • an allyl-b locked carboxylic acid is deprotected with a palladium-catalyzed reaction in the presence of acid labile t-butyl carbamate or base- labile acetate amine protecting groups.
  • Yet another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, that functional group is blocked and does not react. Once released from the resin, the functional group is available to react.
  • blocking/protecting groups may be selected from:
  • Kaposi's sarcoma-associated herpesvirus (KSHV) infection is a prerequisite for the development of Kaposi's sarcoma (KS) and related diseases. Without wishing to be bound by any particular theory, it is believed that blocking lytic KSHV replication hinders KSHV-induced hyperproliferative disorders such as cancer.
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • the invention includes methods for treating KSHV infection and related conditions (e.g., KS).
  • the methods for treating KS provided herein inhibit, reduce, diminish, arrest, or stabilize a tumor associated with KS or a symptom thereof.
  • the methods for treating KS provided herein inhibit, reduce, diminish, arrest, or stabilize the blood flow, metabolism, peritumoral inflammation or peritumoral edema in a tumor associated with KS or a symptom thereof.
  • the methods for treating KS provided herein reduce, ameliorate, or alleviate the severity of KS and/or a symptom thereof.
  • the methods for treating KS provided herein cause the regression of a KS tumor, tumor blood flow, tumor metabolism, or peritumoral edema and/or a symptom associated with KS.
  • the methods for treating KS provided herein reduce hospitalization (e.g., the frequency or duration of hospitalization) of a subject diagnosed with KS.
  • the methods for treating KS provided herein reduce hospitalization length of a subject diagnosed with KS.
  • the methods provided herein increase the survival of a subject diagnosed with KS.
  • the methods for treating KS provided herein inhibit or reduce the progression of one or more tumors or a symptom associated therewith.
  • the methods for treating KS provided herein enhance or improve the therapeutic effect of another therapy (e.g., an anti-cancer agent, radiation, chemotherapy, or surgery).
  • the methods for treating KS involve the use of a compound as an adjuvant therapy.
  • the methods for treating KS provided herein improve the ease in removal of tumors (e.g., enhance respectability of the tumors) by reducing vascularization prior to surgery.
  • the methods for treating KS provided herein reduce vascularization after surgery, for example, reduce vascularization of the remaining tumor mass not removed by surgery.
  • the methods for treating KS provided herein prevent recurrence, e.g., recurrence of vascularization and/or tumor growth.
  • the methods for treating KS provided herein reduce or eliminate one, two, or more of the following: skin lesions, nausea, vomiting, abdominal pain, bleeding, difficulty breathing and lymphedema.
  • the methods for treating KS provided herein reduce the growth of a tumor associated with KS.
  • the methods for treating KS provided herein eradicate, remove, or control primary, regional and/or metastatic tumors associated with KS.
  • the methods for treating KS provided herein decrease the number or size of lesions associated with KS.
  • the methods for treating KS provided herein reduce the mortality of subjects diagnosed with KS.
  • the methods for treating KS provided herein increase the tumor-free survival rate of patients diagnosed with KS.
  • the methods for treating KS provided herein increase relapse-free survival. In certain embodiments, the methods for treating KS provided herein increase the number of patients in remission or decrease the hospitalization rate. In other embodiments, the methods for treating KS provided herein maintain the size of the tumor so that it does not increase, or so that it increases by less than the increase of a tumor after administration of a standard therapy as measured by methods available to one of skill in the art, such as measurement of a lesion, photography, X-ray, CT Scan, MRI, PET Scan, bronchoscopy, and endoscopy. In other embodiments, the methods for treating KS provided herein prevent the development or onset of KS, or a symptom associated therewith.
  • the methods for treating KS provided herein increase the length of remission in patients. In particular embodiments, the methods for treating KS provided herein increase symptom-free survival of KS patients. In some embodiments, the methods for treating KS provided herein do not cure KS in patients, but prevent the progression or worsening of the disease.
  • the methods for treating KS achieve one or more of the following: (i) inhibition or reduction in pathological production of VEGF; (ii) stabilization or reduction of peritumoral inflammation or edema in a subject; (iii) reduction of the concentration of VEGF or other angiogenic or inflammatory mediators (e.g., cytokines or interleukins) in biological specimens (e.g., plasma, serum, cerebral spinal fluid, urine, or any other bio fluids); (iv) reduction of the concentration of P IGF, VEGF-C, VEGF-D, VEGF-R, IL-6, IL-8 and/or IL-10 in biological specimens (e.g., plasma, serum, cerebral spinal fluid, urine, or any other biofluids); (v) inhibition or decrease in tumor metabolism or perfusion; (vi) inhibition or decrease in angiogenesis or vascularization; and/or (vii) improvement in quality of life as assessed by methods well known in the art, for e.g, a
  • the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject as determined by methods well known in the art, e.g., MRI. Three dimensional volumetric measurement performed by MRI has been shown to be sensitive and consistent in assessing tumor size (see, e.g., Harris et al, Neurosurgery, June 2008, 62(6): 1314-9), and thus may be employed to assess tumor shrinkage in the methods provided herein.
  • the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by at least about 20% as assessed by methods well known in the art, e.g., MRI.
  • the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or 100%, relative to the tumor size prior to administration of a compound, as assessed by methods well known in the art, e.g., MRI.
  • the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by at least an amount in a range of from about 10% to about 100%, relative to the tumor size prior to administration of a compound, as assessed by methods well known in the art.
  • the methods for treating KS provided herein reduce the tumor size (e.g., volume or diameter) in a subject by an amount in a range of from about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, 40% to 100%, or any range in between, relative to the tumor size prior to administration of a compound, as assessed by methods well known in the art, e.g., MRI.
  • the methods for treating KS provided herein inhibit or decrease tumor perfusion in a subject as assessed by methods well known in the art, e.g., dynamic contrast-enhanced MRI (DCE-MRI).
  • DCE-MRI dynamic contrast-enhanced MRI
  • Standard protocols for DCE-MRI have been described (see., e.g., Morgan et al, J. Clin. Oncol, Nov. 1, 2003, 21(21):3955- 64; Leach et al, Br. J. Cancer, May 9, 2005, 92(9): 1599-610; Liu et al, J. Clin. Oncol, August 2005, 23(24): 5464-73; and Thomas et al, J. Clin. Oncol, Jun. 20, 2005,
  • the methods for treating KS provided herein inhibit or decrease tumor perfusion in a subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or 100%, relative to tumor perfusion prior to administration of a compound, as assessed by methods well known in the art, e.g., DCE-MRI.
  • the methods for treating KS provided herein inhibit or decrease tumor perfusion in a subject in the range of from about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, 40% to 100%, or any range in between, relative to tumor perfusion prior to administration of a compound, as assessed by methods well known in the art.
  • the methods for treating KS provided herein reduce or inhibit KSHV gene transcription or reduce KSHV mRNA levels in tumor biopsies from a subject with KS by about 5%, 10%, 15%, 20%, 25%, 35%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or any range in between, relative to the respective KSHV gene transcription level or KSHV mRNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • the methods for treating KS provided herein reduce or inhibit KSHV gene transcription or reduce KSHV mRNA levels in tumor biopsies from a subject with KS by 5% to 10%>, 10%> to 20%>, 10%> to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 99%, 30% to 100%, or any range in between relative to the respective KSHV gene transcription level or KSHV mRNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • the methods for treating KS reduce or inhibit KSHV replication or reduce the number of copies of KSHV in circulating peripheral blood mononuclear cells (PBMC) in a subject with KS by about 5%, 10%>, 15%, 20%, 25%, 35%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or any range in between, relative to the respective KSHV replication level or number of KSHV copies in circulating PBMC observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • PBMC peripheral blood mononuclear cells
  • the methods for treating KS provided herein reduce or inhibit KSHV replication or reduce the number of copies of KSHV in circulating PBMC in a subject with KS by about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 99%, 30% to 100%, or any range in between, relative to the respective KSHV replication level or number of KSHV copies in circulating PBMC observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • the methods for treating KS provided herein inhibit or reduce HIV plasma RNA levels in a subject with KS by about 5%, 10%, 15%, 20%, 25%, 35%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% in a subject with KS relative to the respective HIV plasma RNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • the methods for treating KS provided herein inhibit or reduce HIV plasma RNA levels in a subject with KS by about 5% to 10%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 99%, 30% to 100%, or any range in between, relative to the respective HIV plasma RNA levels observed prior to administration of a compound, as determined using an assay described herein or others known to one of skill in the art.
  • compositions can contain additional compatible pharmaceutically active materials for combination therapy (such as supplementary antimicrobials, antipruritics, astringents, local anesthetics, anticancer, or anti-inflammatory agents), or can contain materials useful in physically formulating various dosage forms of the preferred embodiments, such as excipients, dyes, perfumes, thickening agents, stabilizers, skin penetration enhancers, preservatives or antioxidants.
  • additional compatible pharmaceutically active materials for combination therapy such as supplementary antimicrobials, antipruritics, astringents, local anesthetics, anticancer, or anti-inflammatory agents
  • materials useful in physically formulating various dosage forms of the preferred embodiments such as excipients, dyes, perfumes, thickening agents, stabilizers, skin penetration enhancers, preservatives or antioxidants.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the subject either prior to or after the onset of a gamma-herpesvirus infection, such as KSHV infection, and related conditions. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions of the present invention may be carried out using known procedures, at dosages and for periods of time effective to treat a gamma-herpesvirus infection, such as KSHV infection, and related conditions in the patient.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a gamma- herpesvirus infection, such as KSHV infection, and related conditions in the patient.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be
  • an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • the compound in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of KSHV infection and related conditions a patient.
  • compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • compositions of the invention comprise a
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In another embodiment, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks.
  • Compounds of the invention for administration may be in the range of from about 1 ⁇ g to about 10,000 mg, about 20 ⁇ g to about 9,500 mg, about 40 ⁇ g to about 9,000 mg, about 75 ⁇ g to about 8,500 mg, about 150 ⁇ g to about 7,500 mg, about 200 ⁇ g to about 7,000 mg, about 3050 ⁇ g to about 6,000 mg, about 500 ⁇ g to about 5,000 mg, about 750 ⁇ g to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1 ,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
  • the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • the present invention is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a gamma-herpesvirus infection, such as KSHV infection, and related conditions in a patient.
  • a gamma-herpesvirus infection such as KSHV infection
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • routes of administration of any of the compositions of the invention include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.
  • the compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral
  • formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets.
  • excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • the tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
  • the compounds of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone,
  • the tablets may be coated using suitable methods and coating materials such as OPADRYTM film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRYTM OY Type, OYC Type, Organic Enteric OY- P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRYTM White,
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions.
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxy benzoates or sorb
  • the powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation.”
  • a binder material For example, solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.
  • Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents.
  • the low melting solids when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium.
  • the liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together.
  • the resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form.
  • Melt granulation improves the dissolution rate and bioavailability of an active (i.e. drug) by forming a solid dispersion or solid solution.
  • U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties.
  • the granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture.
  • certain flow improving additives such as sodium bicarbonate
  • only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) melt.
  • the present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the invention, and a further layer providing for the immediate release of a medication for treatment of KSHV infection and related conditions.
  • a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the invention, and a further layer providing for the immediate release of a medication for treatment of KSHV infection and related conditions.
  • a wax/pH-sensitive polymer mix a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.
  • the compounds of the invention may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion.
  • Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used. Additional Administration Forms
  • Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO
  • the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds.
  • the compounds for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • the therapeutically effective amount or dose of a compound of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of KSHV infection and related conditions in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
  • a suitable dose of a compound of the present invention may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.
  • the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
  • the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the administration of the inhibitor of the invention is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday").
  • the length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday includes from 10%- 100%, including, by way of example only, 10%, 15%,20%,25%,30%, 35%,40%,45%,50%,55%,60%,65%,70%,75%,80%,85%,90%, 95%, or 100%.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the viral load, to a level at which the improved disease is retained.
  • patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
  • the compounds for use in the method of the invention may be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD 50 and ED 50 .
  • Active compounds exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human.
  • the dosage of such active compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity.
  • the dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
  • reaction conditions including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
  • Example 1 Antiviral Activity of (+)-Rutamarin against SHV by Inhibiting Catalytic Activity of Human Topoisomerase II
  • Kaposi's sarcoma-associated herpesvirus is an etiological agent of several AIDS-associated malignancies including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castkeman's disease (MCD). Its lytic replication cycle has been proven to be critical for the pathogenesis of KSHV-associated diseases. In KS lesion, lytic viral replication, production of virion particles and reinfection of endothelial cells are essential to sustain the population of infected cells that otherwise would be quickly lost as spindle cells divide. Thus, antivirals that block KSHV replication could be a strategy in treatment of KSHV-associated diseases. However there is no effective anti-KSHV drug currently available.
  • (+)-Rutamarin exhibits the highest efficiency in blocking KSHV lytic replication.
  • the results presented herein demonstrated that (+)-Rutamarin has human Topo II inhibition activity and belongs to the category of catalytic Topo II inhibitor.
  • (+)-Rutamarin a novel catalytic inhibitor of human Topo ⁇ , namely (+)-Rutamarin.
  • the binding mode of (+)-Rutamarin to the ATPase domain of human Topo ⁇ was established by docking and validated by MD simulations. More importantly, (+)- Rutamarin efficiently inhibits KSHV lytic DNA replication in BCBL-1 cells with an IC 50 of 1.12 ⁇ and blocks virion production with an EC 50 of 1.62 ⁇ . It possesses low cytotoxicity as indicated by the selectivity index (SI) of 84.14. This study demonstrated a great potential of (+)-Rutamarin to become an effective drug for treatment of human diseases associated with KSHV infection.
  • SI selectivity index
  • the BCBL-1 and JSC-1 are two primary effusion lymphoma cell lines that are latently infected with KSHV (Renne R, et al. 1996. Nat. Med. 2:342-346, Cannon JS, et al. 2000. J. Virol. 74: 10187-10193).
  • BJAB cell line is a KSHV-negative B cell line isolated from Burkitt's lymphoma.
  • PBMC Peripheral blood mononuclear cells
  • Plasmid pOri-A contains an EcoRI-PstI fragment (nucleotides 22409 to 26491) of KSHV DNA in pBluescript at the EcoRI/PstI site as previously described (Lin CL, et al. 2003. J. Virol. 77:5578-5588).
  • pCR3.1-ORF50 is an RTA expression vector described previously (Lin CL, et al. 2003. J. Virol. 77:5578-5588).
  • JSC-1 cells were induced by 3 mM sodium butyrate (Cannon JS, et al. 2000. J. Virol. 74: 10187- 10193). Three hours post-induction, compounds in dilutions were added to BCBL-1 or JSC-1 cells and subsequently antiviral effects and cytotoxicity were assayed at different time points.
  • KSHV genomic DNA content was quantified using the DNeasy kit according to the manufacturer's protocol (Qiagen).
  • KSHV genomic copy number was quantified by realtime PCR on a Roche LightCycler instrument using the LightCycler FastStart DNA Master plus SYBR green kit with the primers for the detection of L ANA (forward, 5 '- CGCGAATACCGCTATGTACTCA-3'; SEQ ID NO: 1; reverse, 5'- GGAACGCGCCTCATACGA-3'; SEQ ID NO: 2).
  • the intracellular viral genomic DNA in each sample was normalized to GAPDH using the primers directed to GAPDH (forward, 5'-ACATCATCCCTGCCTCTAC -3'; SEQ ID NO: 3; reverse, 5'- TCAAAGGTGGAGGAGTGG -3'; SEQ ID NO: 4).
  • IC 50 half maximal inhibitory concentration
  • BCBL-1 cells after treated or untreated with chemicals were assessed by counting Trypan blue-stained cells 2 or 5 days post-treatment using a light microscope. Cell viabilities were defined relative to control cells (non-drug treated). The half maximal cytotoxic concentration (CC 50 ) was calculated from dose-response curves with Graphpad Prism software.
  • Cell proliferation assay BCBL-1 and BJAB cells (both starting with 2 l0 5 cells/ml) were treated with (+)-Rutamarin for 5 days at two different concentrations: IC 50 and 5xICso. The cells were stained by Trypan blue and counted every day for 5 days. To maintain the exponential growth, fresh medium (supplemented with or without the drug) was added to these cultures every 2 days.
  • BCBL-1 and BJAB cells (both starting with 4 l0 5 cells/ml) were treated with (+)-Rutamarin and novobiocin at two different concentrations: IC 50 and 5 x IC 5 o.
  • IC 50 and 5 x IC 5 o Two days post-treatment, cells were collected and fixed with cold 70% ethanol for 15 min, permeabilized, and stained with PI solution (50 g/ml of propidium iodide, 0.1 mg/ml of RNase A, and 0.05%> of Triton X-100) for 2 hours. Cell cycle progression was measured using a FACStar PLUS cell sorter flow cytometer (Becton Dickinson). ori-Lyt-dependent DNA replication assay
  • BCBL-1 cells were cotransfected with plasmids pOri-A (2.5 ⁇ g) and pCR3.1- ORF50 (2.5 ⁇ g) by nucleoporation (Amaxa) and cultured with different concentrations of (+)-Rutamarin. 72 hours post-transfection, extrachromosomal DNA was prepared from cells using the Hirt DNA extraction method as previously described (Gonzalez -Molleda L, et al.. 2012. Antimicrob. Agents Chemother. 56:893-902). The extracted
  • extrachromosomal DNA was treated with RNase A at 25°C for 30 min, followed by proteinase K at 50°C for 30 min. Five ⁇ g DNA was digested with Kpnl/Sacl or
  • the decatenation assay is designed to measure type II topoisomerase activity and inhibition of the enzyme by testing compounds.
  • the Kinetoplast DNA (kDNA, 200 ng) was incubated in 20 ⁇ reaction containing 50 mM Tris-HCl (pH 7.5), 85 niM KC1, 10 mM MgCl 2 , 0.5 mM Na 2 EDTA, 0.5 mM dithiothreitol, 1 mM ATP, 30 ⁇ g/ml BSA, and 2 unit of Topo ⁇ or Topo ⁇ for 30 min at 37°C in the absence or the presence of (+)-Rutamarin or control compounds (novobiocin and etoposide). The reactions were analyzed by electrophoresis on agarose gels. The decatenated kDNAs were measured with Gel DoxTM XR+ imaging system (BIO-RAD).
  • Malachite green reagent was prepared by mixing malachite green
  • ATP hydrolysis reaction (20 ⁇ ) containing 50 mM Tris-HCl, pH 7.4, 85 mM KC1, 10 mM MgCl 2 , 0.5 mM DTT, 0.5 mM Na 2 EDTA, 30 ⁇ g/mL BSA, 200 ng pBR322 DNA, 2 units Topo Ila, 500 ⁇ ATP and varying concentrations of (+)-Rutamarin was initiated by adding ATP to the reaction and incubated at 37°C for 2 hours. The reaction was stopped by adding 80 ⁇ of the malachite green reagent to the reaction, followed by adding 10 ⁇ of 34% sodium citrate to develop blue-green color change. The absorbance at 620 nm was measured using a plate reader. The OD 62 o was used for representing the ATP hydrolysis level. Molecular docking study
  • the docking program FlexX encoded in SYBYL 7.3 (Tripos Inc.) was applied to identify the potential binding of (+)-Rutamarin to the Topo Ila ATPase domain.
  • the receptor for docking simulation consists of protein chain A, two conserved water and Mg 2+ .
  • ADPNP was redocked into the crystal structure of the ATPase domain of Topo ⁇ .
  • the docked ADPNP has similar binding pose with the cocrystallized ligand, with a root mean square deviation of about 1.6 A.
  • the active sites were defined as all residues within 6.5 A radius of the bound ADPNP.
  • Other FlexX parameters were set to default values. Thirty poses were retained during the docking process. After running FlexX, all the poses were visually inspected, and the most suitable docking pose was selected on the basis of score and interactions with key residues of the active site.
  • the complex of Topo ⁇ and the most suitable docking conformer of (+)-Rutamarin was used as initial coordinates for the subsequent molecular dynamics.
  • the simulation consisted of energy minimization, heat phase, equilibration and production.
  • first phase of the minimization only hydrogen atoms were relaxed for 1000 steps, holding all other atoms restrained.
  • second minimization phase is that the protein backbone atoms were restrained and hydrogen atoms, water molecules and ions were relaxed with 1000 steps.
  • a harmonic restraint of 5kcal mol-1 A-2 was applied.
  • all atoms were freely minimized with 2000 steps.
  • a combined steepest descent and conjugate gradient minimization steps was equal.
  • binding free energy calculations was performed on 1000 snapshot structures extracted at 4ps intervals over the last 4 ns stable MD trajectory. For each snapshot structure, the binding free energy was calculated for both enzyme-inhibitor complexes through the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and generalized Born (MM-GBSA) methods Kollman et al, 2000 Accounts of Chemical Research, 33, 889-897; Swanson et al., 2004 Biophysical Journal, 86, 67-74). In the MM -PBS A and MM-GBSA approach an interaction free energy is defined as
  • Gcomplex, Gprotein, and Gligand are the free energies of the complex, protein and the ligand, respectively.
  • Each free energy term in eq 1 was computed as sum of the absolute free energy in the gas phase (Egas), the solvation free energy (Gsolvation), and the entropy term (TS), using eq 2:
  • Egas was expressed as the sum of changes in the van der Waals energy (Evdw), electrostatic energy (Eele), and the internal energies (Eint) in the gas phase (eq 3).
  • Eint is the energy associated with vibration of covalent bonds and bond angels, rotation of single bond torsional angels (eq 4)
  • the polar contribution (GPB/GB) to the solvation energy was calculated either using the PB and GB model implemented in AMBER 12.
  • the grid size used is 0.5 A.
  • the dielectric constant was set to 1 for interior solute and 80 for exterior water.
  • (+)-Rutamarin inhibits the lytic replication of SHV
  • C31 which is identified as the natural product (+)-Rutamarin (Fig. 2A), exhibited significant inhibitions of both viral DNA synthesis and virion production with relatively low cytotoxicity at the concentration of 20 ⁇ . Therefore it was chosen for further investigation.
  • the half maximal inhibitory concentration (IC 50 ) values of (+)-Rutamarin were determined from dose-response curve of KSHV DNA content in TPA-induced BCBL-1 cells and found to be 1.12 ⁇ (Fig 2B).
  • the effect of (+)-Rutamarin on progeny virion production was also determined by quantification of encapsidated viral DNA in BCBL-1 culture media.
  • the half maximal antiviral effective concentration (EC 50 ) calculated from dose-response curve of extracellular virion is 1.62 ⁇ (Fig 2B).
  • (+)-Rutamarin inhibits KSHV lytic replication regardless of host cells and means of lytic cycle induction
  • experiments were designed to test the effect of (+)-Rutamarin on KSHV replication in JSC-1 cells induced by sodium butyrate for lytic replication.
  • the IC50, EC50 and CC50 of (+)-Rutamarin on butyrate- induced JSC-1 cells were found to be 2.29 ⁇ , 1.40 ⁇ and 94.34 ⁇ , respectively, that are very similar to those obtained with BCBL-1.
  • the cytotoxicity of (+)- Rutamarin to primary lymphocytes was also assessed with peripheral blood mononuclear cells (PBMC).
  • the CC 50 to PBMC was calculated to be 60.91 ⁇ (Fig. 10).
  • (+)-Rutamarin To further investigate the potential of (+)-Rutamarin to become an effective and safe antiviral, the effect of the compound on cell proliferation was assessed. KSHV-carrying BCBL-1 and virus-free BJAB cells were cultured in the presence and the absence of (+)-Rutamarin and live cell numbers were counted over 5 days. (+)-Rutamarin did not exhibit cell growth inhibitory effect in both cell lines at the IC 50 concentration. At an excess concentration (5 x IC 50 ), (+)-Rutamarin showed an adverse effect on cell growth but inhibition extent was less than that of novobioicin (Fig. 3). The effect of (+)- Rutamarin on cell cycle progression was also examined.
  • (+)-Rutamarin inhibits SHV lytic replication by blocking viral ori-L vt-dependent DNA replication
  • (+)-Rutamarin is capable of inhibiting KSHV DNA synthesis and virion production.
  • an ori-Lyt DNA replication assay was conducted. BCBL-1 cells were cotransfected with an on-Zyt-containing plasmid (pOri-A) and an RTA expression vector. Lytic DNA replication was induced by RTA expression (Sun R, et al. 1998. Proc. Natl. Acad. Sci. U. S. A. 95:10866-10871).
  • the transfected cells were cultured in the presence of (+)-Rutamarin at varying concentrations and the effect of (+)-Rutamarin on orz-Zyt-dependent DNA replication was measured by a Dpn I assay (Lin CL, et al. 2003. J. Virol. 77:5578-5588, Wang Y, et al. 2004. J. Virol. 78:8615-8629).
  • DNA was isolated from the treated cells 72 h post-transfection and digested with Kpnl/Sacl and Kpnl/Sacl/Dpnl.
  • Replicated plasmid DNA was distinguished from input plasmid by Dpnl restriction digest, which cleaves input DNA that has been dam + methylated in
  • (+)-Rutamarin blocks KSHV DNA replication through inhibiting RTA expression
  • the effect of (+)-Rutamarin on RTA expression in TPA-induced BCBL-1 cells was examined by Western analysis. The result showed no adverse effect of this compound on RTA expression up to 25 ⁇ (22 times excess over IC 50 ) (Fig. 11).
  • the reduced level of RTA in the treatment with 50 and 100 ⁇ of (+)-Rutamarin may result from the cytotoxicity of the compound in the concentration near its IC 50 (94.24 ⁇ ).
  • (+)-Rutamarin is an inhibitor of human topoisomerase ⁇ .
  • Topo II inhibitor that blocks KSHV on-Zyt-dependent DNA replication through inhibiting catalytic activity of host cell Topo II.
  • Topo II isoforms there are two Topo II isoforms in mammals, Topo ⁇ and Topo ⁇ .
  • a DNA decatenation-based topoisomerase II assay was established.
  • kinetoplast DNA consisting of a large network of interlocked DNA minicircles can be decatenated into separate minicircles in the presence of ATP and Topo II of either isoform.
  • (+)-Rutamarin in a wide range of concentration was added in the reaction and the effect of the compound on Topo II activity was measured by determining decatenated kDNA using agarose gel electrophoresis. Results showed that (+)-Rutamarin is able to inhibit the enzymatic activity of Topo ⁇ in a dose-dependent manner (Fig. 6A and 6B).
  • the IC 50 of (+)-Rutamarin on Topo ⁇ inhibition was calculated as 28.22 ⁇ 6.2 ⁇ (Fig. 6C and 6D).
  • the inhibition rate of (+)- Rutamarin is significantly higher than novobiocin and close to Topo II poison etoposide (Fig. 6A).
  • (+)-Rutamarin did not exhibit any inhibitory effect on Topo ⁇ activity (Fig. 6E).
  • (+)-Rutamarin is a catalytic inhibitor of Topo ⁇ and acts in blocking ATPase activity of the enzyme as novobiocin does
  • the inhibition effect of Rutamarin on ATP hydrolysis of Topo ⁇ was studied using the malachite green assay.
  • the APTase activity of Topo ⁇ was inhibited by (+)-Rutamarin in a dose-dependent manner, suggesting that, like novobiocin, (+)-Rutamarin inhibits human Topo ⁇ by binding to the ATP pocket of the enzyme and blocking its ATP hydrolysis.
  • Fig. 8A illustrates the time dependence of the RMSD values for X-ray reference enzyme structure of backbone atoms (C, CD, N, and O) over the production phase of simulation.
  • the RMSD values of simulation converged after ⁇ 3ns, indicating that the system is stable and equilibrated.
  • the RMSD value of ligand compared docking pose is swinging within 1.8 A, which illustrates that the docking pose is reliable.
  • (+)-Rutamarin forms a hydrogen bond with the side chain of Asn95, Ala 167 and conserved water 2, respectively, and hydrophobically interacts with He 125, Ilel41, Phel42, Alal67, Thr215, Glyl66 and Lysl23 in the catalytic site of Topo ⁇ (Fig. 8D).
  • the distance distributions analysis also showed that the hydrogen bonds between conserved water 2- and Alal67-(+)-Rutamarin are comparatively stable, whereas the hydrogen bond of Asn95-(+)-Rutamarin is unstable (Fig.
  • (+)-Rutamarin as an effective antiviral agent that inhibits SHV ori-L vt-dependent DNA replication with low cytotoxicity
  • (+)-Rutamarin was identified as an effective antiviral agent that inhibits KSHV on-Zyt-dependent DNA replication with low cytotoxicity. It blocks KSHV DNA replication through inhibiting catalytic activity of human Topo ⁇ .
  • KSHV has been proven to be the etiological cause of KS and other KSHV-associated malignancies.
  • the current treatment modalities for KSHV- associated diseases include only traditional cancer therapies.
  • the chemotherapeutics that have been approved by the FDA include liposomal anthracycline products (liposomal doxorubicin or liposomal daunorubicin), paclitaxel and interferon- alpha (Potthoff A, et al. 2007. J. Dtsch. Dermatol. Ges. 5: 1091-1094).
  • liposomal anthracycline products liposomal doxorubicin or liposomal daunorubicin
  • paclitaxel paclitaxel
  • interferon- alpha Pieris factor-alpha
  • IRIS immune reconstitution inflammatory syndrome
  • IRIS-KS is the result of responses by a recovered immune system to KS-causing pathogen, i.e. KSHV
  • KSHV antiretroviral
  • HIV- positive patients with a combination of antiretroviral (HAART) and anti-KSHV therapeutics is expected to yield positive results.
  • antiretroviral (HAART) antiretroviral
  • anti-KSHV therapeutics there are no effective drugs targeting KSHV available.
  • ganciclovir or foscarnet could reduce the incidence of KS in AIDS patients (Glesby MJ, et al. 1996. J Infect Dis. 173: 1477-1480, Mocroft A, et al. 1996. AIDS. 10: 1101-1105).
  • severe side effects renal impairment and bone marrow suppression
  • rapid development of drug resistance have limited the use of these drugs in the treatment of KS. Therefore, there is a need for effective drugs targeting KSHV.
  • Topo II inhibitors are divided into two categories: Topo II poisons, which target the
  • Topo II catalytic inhibitors which disrupt catalytic turnover of the enzyme.
  • Topo II poisions in general exhibit very strong cytotoxicities to host cells.
  • Topo II catalytic inhibitors show less cytotoxicity (such as novobiocin with a CC50 values of 871 ⁇ and merbarone of 212.9 ⁇ to BCBL-1 cells). Both novobiocin and merbarone are effective in halting KSHV DNA synthesis with IC 50 of 27.55 ⁇ and 19.54 ⁇ , respectively. The low
  • Topo II catalytic inhibitors cytotoxicities and high inhibition rates for viral replication of catalytic Topo II inhibitors suggest potentials of Topo II catalytic inhibitors in becoming effective anti-KSHV drugs for treatment of KS and other KSHV-associated diseases (Gonzalez-Molleda L, et al.. 2012. Antimicrob. Agents Chemother. 56:893-902).
  • viruses have tendencies to mutate their genome and therefore develop drug resistance
  • targeting host cellular proteins that viruses rely on for their replication offers the advantage of minimizing drug resistance, and hence constitutes a novel therapeutic strategy.
  • (+)-Rutamarin as a lead which efficiently inhibits KSHV lytic DNA replication in BCBL-1 cells with the IC 50 of 1.12 ⁇ and EC 50 of 1.62 ⁇ , 25- and 17- fold lower than those of novobiocin.
  • (+)-Rutamarin exhibits very low cytotoxicity and the selective index (SI) on KSHV lytic replication was calculated as 84.14, three times better that that of novobiocin.
  • SI selective index
  • (+)-Rutamarin is a natural product found in plants such as ruta graveolens L (common rue). It was reported that (+)-Rutamarin possesses anti-proliferation and anti- cancer activities against a variety of tumor cell lines (Yang QY, et al. 2007. J. Asian Nat. Prod. Res. 9:59-65). It is also found to induce the expression and translocation of glucose transporter 4 (GLUT4). As impaired translocation or decreased expression of GLUT4 in response to insulin is one of the major pathological features of type 2 diabetes, the function of (+)-Rutamarin in ameliorating glucose homeostatsis suggests a potential of this compound in anti-type 2 diabetes drug development (Zhang Y, et al. 2012. PLoS
  • (+)-Rutamarin did not show perceptible effect on cell proliferation and cell cycle progression in BCLB-1 and BJAB cells.
  • (+)-Rutamarin exhibited a degree of suppression on cell proliferation with a delay in the S phase.
  • (+)-Rutamarin has an extent of antiproliferative property and may provide an additional benefit if it becomes a drug to treat KSHV-associated malignancies, the moderate anti-proliferation property may not be potent enough to provide anti-tumor activities. It may also be true for all Topo II catalytic inhibitors.
  • (+)-Rutamarin is a novel human Topo Ila catalytic inhibitor
  • Topo II is an ATPase and uses the energy derived from ATP hydrolysis to resolve the winding problem of double-stranded DNA.
  • the detailed binding mode of (+)- Rutamarin and human Topo Ila was identified using molecular docking and MD simulation. The significance of these studies is two-fold. First, the study provided insight into the binding of (+)-Rutamarin to Topo Ila and results confirmed that this compound binds to the ATP pocket of Topo Ila. Second, the result of the study help in designing modification to the compound for more potent antiviral candidates. Energy
  • the major contributing residues can be divided into three clusters, which are consistent with the three binding pockets sites, i.e. (i) Walker A consensus motif (residues 161-167), (ii) Mg 2+ binding area (residues 87, 91 and 94), and (iii) hydrophobic chamber (key residues, He 125, Ilel41, Phel42, Lysl23 and Thr215). All of these residues except Glu87 positively contribute to the binding affinity for (+)-Rutamarin.
  • Topo II topoisomerase II
  • Topo II catalytic inhibitors were evaluated for their potentials in blocking EBV replication and becoming efficacious antiviral agents.
  • Topo II catalytic inhibitors in general exhibited marked inhibition of EBV lytic replication and minimal cytotoxicity.
  • (+)-rutamarin with the best selectivity index (SI > 63) among the inhibitors tested in this study, is effective in inhibiting EBV DNA replication and virion production but shows little adverse effect on cell proliferation, suggesting its potential to become an efficacious and safe drug for the treatment of human diseases associated with EBV infection.
  • the P3HR-1 is a EBV -positive Burkitt lymphoma cell line, a clonally derived subline of Jiyoye (Hinuma et al, 1967, J. Virol, 1 :1045-1051).
  • Akata-Bxl cell line carries a recombinant EBV where the thymidine kinase gene was replaced by a CMV immediate early promoter driven GFP (Guerreiro-Cacais et al., 2007, J. Virol., 81 : 1390- 1400).
  • Cells were cultured in RPMI 1640 medium (Gibico-BRL, Gaithersburg, MD), supplemented with 10% FBS (Gibico-BRL), penicillin (100 U/ml), streptomycin
  • the EBV ori-Lyt plasmid pEBV-oriL was constructed by cloning a 1434 bp fragment carrying EBV ori-Lyt (nucleotides 52385-53819 of EBV genome) into pUC18 vector.
  • the plasmid McZ is a ZTA expression vector in the backbone of pBXGl . Chemicals and cell treatment
  • Novobiocin was purchased from Sigma-Aldrich (St. Louis, MO) and merbarone was purchased from Merck Millipore. (+)-Rutamarin (Figure 12) is extracted from natural plant Ruta graveolens L, supplied by the Guangdong Small Molecule Tangible Library (GSMTL) (Gu et al, 2010, Molecules, 15:5031-5044). Novobiocin was prepared as aqueous stock, while the others were dissolved in dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • P3HR-1 cells (4 x 10 5 cells/ml of culture) were treated with 20 ng/ml 12-O-tetradecanoylphorbol 13 -acetate (TP A; Sigma- Aldrich) and 3 mM sodium butyrate (Sigma-Aldrich).
  • TP A 12-O-tetradecanoylphorbol 13 -acetate
  • Sigma-Aldrich 3 mM sodium butyrate
  • Novobiocin, merbarone, and (+)- rutamarin in different concentrations was added to P3HR-1 cells three hours after induction.
  • Akata-Bxl cells were resuspended at 2 x 10 6 /ml in fresh medium and treated with 0.8%(V/V) goat anti-human IgG (Jackson ImmunoResearch).
  • Akata-Bxl cells were adjusted to the density of 4 x 10 5 cells/ml and treated with various concentrations of novobiocin, merbarone, and (+)-rutamarin. The effects of these inhibitors on viral DNA content and virion production were assayed at different time points.
  • EBV genomic DNA was quantified by real-time PCR on a Roche Light Cycler II instrument using the Lightcycler 480 SYBR green I Master kit with primers directed to EBNA1 (forward, 5'- CATTGAGTCGTCTCCCCTTTGGAAT-3 ', SEQ ID NO:6; reverse, 5'- TC AT AAC AAGGTCCTT AATCGC ATC-3 ', SEQ ID NO:7).
  • the intracellular viral genomic DNA in each sample was normalized with the amount of GAPDH determined also by real-time DNA PCR by using primers directed to GAPDH (forward, 5'- AGCCACATCGCTCAGACAC-3', SEQ ID NO:8; reverse, 5'- GCCCAATACGACCAAATCC-3', SEQ ID NO:9).
  • EBV DNA content value from induced cells was subtracted by that from uninduced cells. These corrected values were divided by those from the control, non-drug treatment and then represented on the y axes of dose-response curves: y axis
  • l x phosphate -buffered saline PBS
  • PBS l x phosphate -buffered saline
  • the concentrated viruses were treated with DNase I (TaKaRa) at 37 °C for 1 h followed by proteinase K digestion.
  • the amounts of virion particles from the media were determined by quantifying encapsidated viral DNA by real-time PCR, and values were corrected as described above.
  • the 50% antiviral effective concentration (EC 50 ) for each compound was calculated from the dose- response curve with the aid of GraphPad Prism software.
  • P3HR-1 cells (starting with 2 x 10 5 cells/ml) were treated with topoisomerase inhibitors for 5 days at two different concentrations: IC 50 and an excess concentration (5 x IC 50 ). Cell samples were daily collected, stained with Trypan blue, and counted. To provide a constant cellular growth, fresh medium (supplemented with or without the drug) was added to these cultures every 2 days.
  • P3HR-1 cells (1 x 10 7 ) were transfected with plasmids pEBV-oriL (2.5 ⁇ g) and McZ (2.5 ⁇ g) by nucleoporation (Amaxa) and cultured in the media with each drug in a wide range of concentration. Seventy-two hours post-transfection, extrachromosomal DNAs were prepared from cells using the Hirt DNA extraction method (Hirt, 1967, J. Mol. Biol, 26:365-369). Cells were lysed in 700 ⁇ lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM EDTA, and 0.6% SDS).
  • Chromosomal DNA was precipitated at 4 °C overnight by adding 5 M NaCl to a final concentration of 0.85 M. Cell lysates were centrifuged at 4 °C at 14,000 rpm for 30 min. The supernatant containing
  • extrachromosomal DNA was subjected to phenol-chloroform extraction, followed by ethanol precipitation.
  • the DNA was treated with RNase A at 25 °C for 30 min and then with proteinase K at 50 °C for 30 min. Twelve micrograms of DNA was digested with EcoR I or EcoR I/Dpnl (TaKaRa). The DNAs were separated by electrophoresis on 0.9% agarose gels and transferred onto GeneScreen PLUS membranes (Perkin Elmer, Boston, MA).
  • Southern blot was performed according to an optimized protocol using the DIG high prime DNA labeling and detection starter kit I (Roche). Probes were prepared by excising insert DNA from pEBV-oriL plasmid by restriction digestion and labeled with digoxigenin by random primed DNA synthesis with digoxigenin-dUTP. The membranes were prehybridization in 10 ml of the DIG Easy Hybridization solution, with 50 mg/ml denatured salmon sperm DNA for 4 h at 68 °C. Then, DIG labeled probe was added and hybridization was carried for overnight. The membranes were washed three times for 15 min at 65 °C with washing buffer (2* SSC, 0.1% SDS). Subsequently the membranes were blocked for 1 h and incubated in anti-DIG-AP solution for 1 h and then incubated in 10 ml color substrate solution in the dark.
  • DIG labeled probe was added and hybridization was carried for overnight. The membranes were washed three times for
  • Topo II catalytic inhibitors including novobiocin and merbarone, have potent antiviral activity halting KSHV DNA replication and virion production (Gonzalez-Molleda et al., 2012, Antimicrob. Agents
  • (+)-rutamarin revealed the highest potency in halting EBV DNA replication with a half-maximal inhibitory concentration (IC 50 ) of 3.78 ⁇ and in blocking virion production with a half-maximal antiviral effective concentration (EC50) of 5.40 ⁇ ( Figure 13C and Table 3 A).
  • Table 1 Antiviral activities of Topo II inhibitors and their associated cytotoxicities a in P3HR-1 cells induced with TPA/butyrate and Akata-Bxl cells induced with Anti-IgG.
  • aIC 5 o represents the half maximal inhibitory concentration for EBV DNA replication.
  • EC50 denotes the half maximal effective concentration for blocking EBV virion production.
  • CC50 refers to the concentration of the compound that causes 50% cell death after specific exposure time. They were determined by nonlinear regression analysis of dose response curves and are expressed as mean values of results from at least three independent experiments. All these parameters are presented in ⁇ units.
  • R 2 correlation coefficient.
  • c SI selectiveivity index
  • (+)-rutamarin again exhibited the highest antiviral potency in Akata-Bxl cells with the IC50 and EC50 of (+)-Rutamarin of 2.38 ⁇ and 2.94 ⁇ , respectively ( Figure 14 and Table 3B).
  • Cytotoxicities of these three inhibitors were examined in parallel with their inhibition of EBV DNA replication and virion production in P3HR-1 and Akata- Bxl cells. Cells treated with these inhibitors at different concentrations were subjected to the trypan blue exclusion method to assess the numbers of viable cells and nonviable cells in culture. The half-maximal cytotoxic concentrations (CC 50 ) were determined based on the results. Novobiocin and merbarone exhibited relatively low cytotoxicity to P3HR-1 cells with CC 50 values of 518.5 ⁇ and 137.6 ⁇ , respectively ( Figure 13A and B).
  • (+)-rutamarin the highest concentration used in this study (150 ⁇ ) showed little cytotoxicity in 2 and 5 days culture, suggesting that the CC 50 value of (+)-rutamarin to P3HR-1 cell is greater than 150 ⁇ ( Figure 13D and E). Similar results were obtained with Akata-Bxl cells ( Figure 14D and 14E).
  • topoisomerase II inhibitors tested in this study have been demonstrated to inhibit EBV DNA synthesis and virion production.
  • Experiments where designed to assess whether these inhibitors indeed block viral ori-Lyt-dependent DNA replication To address this question, P3HR-1 cells were cotransfected with an EBV ori- Lyt-containing plasmid (pEBV-oriL) and a ZTA expression vector (McZ). Expression of ZTA sufficiently drives latent EBV into lytic replication cycle (Countryman and Miller, 1985, Proc. Natl. Acad. Sci. U.S.A., 82:4085-4089 and Rooney et al, 1988, Proc. Natl. Acad. Sci. U.S.A., 85:9801-9805).
  • the transfected cells were cultured in the absence or the presence of each inhibitor at various concentrations, usually with two concentration lower than IC 50 , and two higher concentrations.
  • the ori-Lyt-dependent DNA replication and the drug effects were measured by a Dpn I assay (Gonzalez-Molleda et al., 2012, Antimicrob. Agents Chemother, 56:893-902).
  • DNA was isolated from the treated cells 72 h post-transfection and digested with EcoR I and EcoR I/Dpn I.
  • Replicated plasmid DNA can be distinguished from input plasmid by Dpnl restriction digest, which cleaves input DNA that has been dam + methylated in Escherichia coli but leaves intact the DNA that has been replicated at least one round in eukaryotic cells
  • Human topoisomerase II catalytic inhibitors are potent in inhibiting EBV replication
  • EBV is associated with a number of human diseases.
  • the lytic replication of EBV is directly linked to infectious mononucleosis, chronic active EBV infection (CAEBV), oral hairy leukoplakia and to an increased risk of EBV-associated
  • CAEBV nasopharyngeal carcinoma
  • Chronic active EBV infection is characterized by chronic or recurrent infectious mononucleosis-like symptoms that persist for a long time and by an unusual pattern of anti-EBV antibodies, with life-threatening complications, such as virus-associated hemophagocytic syndrome and lymphoma (Kimura et al., 2001, Blood, 98:280-286).
  • the patients usually have a markedly elevated EBV DNA level in the blood (10 3 - 10 7 copies/ml), indicating active lytic viral replication. So far no satisfactory therapy is available for CAEBV.
  • Antiviral or immunomodulatory agents such as acyclovir, ganciclovir, vidarabine, interferon-a, and interleukin-2
  • Acyclovir ACV
  • Acyclovir which inhibits herpesviral DNA polymerase, is commonly used for treatment of CAEBV (Gershburg and Pagano, 2005, J. Antimicrob. Chemother, 56:277-281) but found generally inefficient for this disease.
  • drug resistance frequently occurs as a major problem among immunosuppressed hosts, mainly those who have received prolonged ACV therapy (Andrei et al, 2012, J.
  • topoisomerases could be therapeutic targets for blocking replication of herpesviruses including EBV and treatment of the infection-associated human diseases. It has been reported previously that Topo I inhibitor camptothecin is able to inhibit replication of herpes simplex virus type 2 (Yamada et al, 1990, Arch. Virol., 110: 121-127).
  • Topo II catalytic inhibitor ICRF193 and Topo II poison teniposide were found to be able to block herpes simplex virus type 1 from replication (Ebert et al, 1990, J. Virol, 64:4059-4066 and Hammarsten et al, 1996, J. Virol, 70:4523-4529).
  • the results presented herein demonstrate that KSHV replication can be effectively blocked by Topo I inhibitor camptothecin, Topo II poisons etoposide and ellipticine as well as Topo II catalytic inhibitors novobiocin, merbarone and (+)- rutamarin (Gonzalez-Molleda et al, 2012, Antimicrob.
  • Topo I inhibitors and Topo II poisons in general possess considerable toxicities to host cells (Gonzalez-Molleda et al, 2012, Antimicrob. Agents Chemother. 56:893-902).
  • viruses have tendencies to mutate their genome and therefore develop drug resistance.
  • An antiviral that targets a cellular protein such as Topo II offers the advantage of minimizing drug resistance and hence constitutes an important, novel therapeutic strategy.
  • Novobiocin is an old antibiotic drug against staphylococcal infection (Kirby et al, 1956, AMA Arch. Intern. Med., 98: 1-7). Novobiocin has also been used in combination with chemotherapeutical agents for the treatment of several cancers (Eder et al, 1991, Cancer Res., 51 :510-513 and Kennedy et al, 1995, J. Clin. Oncol, 13: 1136- 1143). The results presented herein reveal its antiviral potency against EBV, suggesting a new usage for an old drug.
  • (+)-Rutamarin is a natural product and newly identified Topo II catalytic inhibitor (Xu et al., 2014, Antimicrob. Agents Chemother, 58:563-573). Among three inhibitors tested in this study, (+)-Rutamarin exhibited the highest potency in anti-EBV activity. (+)-Rutamarin effectively inhibits EBV replication with an IC 50 of 2.38 ⁇ and an EC 50 of 2.94 ⁇ (in Akata-Bxl cells). It possesses relative low cytotoxicity to P3HR- 1 cells with a CC50 greater than 150 ⁇ . Without wishing to be bound by any particular theory, experiments can be desgined to optimize the (+)-Rutamarin structure in order to further elevate its antiviral activity and improve its solubility, making it an anti-EBV drug lead.

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Abstract

L'invention concerne des compositions et des méthodes pour traiter une infection par le virus de l'herpès. L'invention concerne également des compositions et des méthodes pour traiter le sarcome de Kaposi ou une infection par le virus de l'herpès associé au sarcome de Kaposi par administration à un individu nécessitant un tel traitement d'une dose efficace d'un composé qui inhibe les topoisomérases IIα. Par exemple, le composé qui inhibe les topoisomérases IIα est le (+)-rutamarin, un analogue ou un dérivé de celui-ci.
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CN106543197A (zh) * 2016-11-09 2017-03-29 中国科学院新疆理化技术研究所 一种补骨脂素席夫碱类衍生物及用途
CN106565734A (zh) * 2016-11-09 2017-04-19 中国科学院新疆理化技术研究所 一种补骨脂素酯类衍生物及用途
EP3601294A4 (fr) * 2017-03-22 2020-09-30 Taipei Medical University Composés à induction atf3

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105541859A (zh) * 2016-02-23 2016-05-04 湖南大学 二氢呋喃并色满酮衍生物及其制备方法与医药用途
CN106543197A (zh) * 2016-11-09 2017-03-29 中国科学院新疆理化技术研究所 一种补骨脂素席夫碱类衍生物及用途
CN106565734A (zh) * 2016-11-09 2017-04-19 中国科学院新疆理化技术研究所 一种补骨脂素酯类衍生物及用途
CN106565734B (zh) * 2016-11-09 2018-07-03 中国科学院新疆理化技术研究所 一种补骨脂素酯类衍生物及用途
EP3601294A4 (fr) * 2017-03-22 2020-09-30 Taipei Medical University Composés à induction atf3
US11365200B2 (en) 2017-03-22 2022-06-21 Taipei Medical University ATF3 induction compounds

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