WO2011089166A1 - Semicarbazone proteasome inhibitors for treating hiv and hepatitis infection - Google Patents

Abstract

The invention relates to semicarbazone proteasome inhibitors, pharmaceutical compositions comprising the same, uses of said semicarbazone proteasome inhibitors and pharmaceutical compositions comprising the same for use in the treatment of heptatis virus infections, particularly in the treatment of HCV infections and/or HIV infections.

Description

VIROLOGIK GMBH Munich, 19 January 2011 Our ref.: V 8448 / DJB

ViroLogik GmbH

HenkestraBe 91, 91052 Erlangen, Deutschland

SEMICARBAZONE PROTEASOME INHIBITORS

FOR TREATING HIV AND HEPATITIS INFECTION

Technical Field of the Invention

The invention relates to semicarbazone proteasome inhibitors for treating retroviral infections, e.g. Human Immunodeficiency virus (HIV) infections and/or viral hepatitis infections, in particular for treating Hepatitis C Virus (HCV) infections. The present invention further concerns pharmaceutical compositions and kits of pharmaceutical compositions which may be used for treating HIV infections and or viral hepatitis infections, in particular for treating HCV infections as well as methods and uses of said proteasome inhibitors and pharmaceutical compositions comprising the same.

Background of the Invention HCV infections

Pegylated interferon (PEG-IFN) alpha-2a or alpha-2b in combination with nucleoside analogon ribavirin when being administered for 24 or 48 weeks currently is the standard therapy for patients suffering from chronic HCV infections. The aim of the standard therapy is to achieve elimination of the HC virus, meaning that no HCV- R A is detectable after treatment. The guanosine analogon ribavirin in combination with interferons has been authorized for therapy of chronic HCV infections since 1999. However, the mode of action of this medicament is only partially understood. A complete elimination of HCV after administration of ribavirin without IFN is not to be expected.

The standard therapy of IFN and ribavirin is frequently associated with side effects which can be partially attributed to the specific substance respectively. The most frequently observed side effects of an IFN therapy are flu- like symptoms such as fever, headache, muscle pain, joint pain as well as fatigue, loss of appetite and loss of weight. Moreover mood swings including depression have been described. A frequent side effect observed with ribavirin treatment is anemia, which necessitates continuous control of blood parameters during therapy.

In principle, patients undergoing treatment for viral hepatitis, and particularly for HCV infections, can be categorized as "responder", "non-responder" and "patients showing a relapse/therapy refractory patients". A response is typically understood to refer to a sustained decrease of the virus load below the detection limit for at least 6 months after standard therapy has ceased ("sustained virological response", SVR). A relapse typically refers to a complete virological response up until the 24th week of treatment at the latest. However, after the standard therapy has ceased, a renewed increase of virus load may be observed (therapy refractory). Non-responders are patients for which typically no decrease of virus load by a factor of at least 2 log steps is observed during 24 weeks or for which up to week 24, HCV-RNA are still detectable during standard therapy (therapy resistant). For example, more than 50% of patients infected with HCV of Genotype 1 do not react towards standard therapy ("non-responder") or suffer from a setback after therapy has ended ("relapser"). Treatment options, especially for non-responders and relapsed patients, are sparse (Kronenberger, B., Zeuzem, S., Annals of

Hepatology 2009; 8: 103). Evidently, a considerable medical need exists to develop new or improved treatment options for the treatment of hepatitis caused by viral infection, and specifically for HCV.

HIV infections

Furthermore, Human Immunodeficiency Virus type 1 (HIV-1) and type 2 (HIV-2) are the etiological agents that are responsible for the acquired immune deficiency syndrome (AIDS) and related disorders (Barre-Sinoussi, et al, Science, 220:868-871 (1983); Gallo, et al, Science, 224:500-503 (1984); Levy, et al, Science. 225:840-842 (1984); Popovic, et al, Science. 224:497-500 (1984); Sarngadharan, et al, Science. 224:506-508 (1984); Siegal, et al, Nl Engl. J. Med.. 305: 1439-1444 (1981); Clavel, F., AIDS, 1 : 135-140). HIV is transmitted through direct contact of a mucous membrane or the bloodstream with a bodily fluid containing HIV, such as blood, semen, vaginal fluid, preseminal fluid, and breast milk. This transmission can involve anal, vaginal or oral sex, blood transfusion, contaminated hypodermic needles, exchange between mother and baby during pregnancy, childbirth, breastfeeding or other exposure to one of the above bodily fluids.

AIDS occurs after an asymptomatic period following infection with HIV-1 or HIV-2. Main characteristics of the syndrome are the progressive degeneration of the immune system and the central nervous system. The infection with HIV itself, does not necessarily cause death. However, infected individuals will eventually suffer from severe immunosuppression, so that various other diseases , such as opportunistic infections, e.g viral infections (e.g. HCV, HSV, CMV, EBV, HHV6), bacterial and fungal infections, or malignancies, especially Kaposi's sarcoma, occur. HIV-infected individuals eventually succumb to these secondary conditions. In as far as a treatment is available, such secondary conditions may of course be treated.

However, treatment of secondary infections may adversely affect the already weakened immune system, and the combination of weak immune response with often a multitude of secondary adverse conditions can lead to fatal outcomes even for conditions otherwise easily cured in non-HIV-infected individuals. AIDS is now a pandemic. In 2007, it was estimated that 33.2 million people lived with the disease worldwide, and that AIDS killed an estimated 2.1 million people, including 330,000 children. Over three-quarters of these deaths occurred in sub- Saharan Africa. Although treatments for AIDS and HIV can slow the course of the disease, there is currently no known cure or vaccine leading to the elimination of the virus.

Antiretro viral treatment reduces both the mortality and the morbidity of HIV infection, but these drugs are expensive and routine access to antiretroviral medication is not available in all countries.

HIV virology

The family of retroviruses which also includes the human immune deficiency viruses (HIV) belongs to the large group of eukaryotic retrotransposable elements (for a review, see Doolittle, R F; et al. (1990) Curr. Top. Microbiol. Immunol. 157: 1-18.). Said elements are distinguished by the ability to transcribe RNA genomes into DNA intermediates by using the enzyme reverse transcriptase. Retroviruses are divided into five subfamilies: (i) spumaviruses; (ii) mammalian type C oncoviruses; (iii) BLV (bovine leukemia virus)/HTLV (human T-cell leukeumia virus) leukemia viruses; (iv) a heterogeneous group of RSV (Rous sarcoma virus), type A, B and D viruses; and (v) lentiviruses (for a review, see Doolittle et al, 1990). Lentiviruses replicate predominantly in lymphocytes and fully differentiated macrophages and usually cause long-lasting and/or incurable diseases. Retroviruses contain at least three characteristic genes: gag (groupspecific antigen), pol

(polymerase) and env (envelope proteins). Apart from structural and enzymatically active viral proteins, various retroviruses encode additional, usually small proteins with regulatory functions. The lentivirus subfamily includes, in addition to HIV, SIV (simian immunodeficiency virus), EIAV (equine infectious anemia virus), BIV (bovine immunodeficiency virus), FIV (feline immunodeficiency virus) and Visna virus. HIV in turn is divided into the two subtypes HIV-1 and HIV-2 (for a review, see Doolittle et al, 1990).

HIV Replication Cycle

The HIV replication cycle starts with the virus binding to various cell receptors among which the glycoprotein CD4 acts as the primary receptor and various cellspecific chemokine receptors act as co-receptors, after binding to CD4. After the virus has entered, the viral RNA genome is transcribed by means of reverse transcriptase (RT), RNase H and polymerase into double-stranded DNA which, in association with the preintegration complex, is then transported into the nucleus and incorporated as provirus genome into chromosomal DNA by means of viral integrase. After transcription and translation, Gag/Gag-Pol polyproteins and envelope proteins are transported to the cell membrane where virions are being assembled. After budding and detachment, virus particles mature due to proteolytic processing of said Gag/Gag-Pol polyproteins (for a review, see Swanstrom, R; Wills, J; In: Coffin, J., et al. (Eds.), Retroviruses, Cold Spring Harbor Press, Plainview, N.Y., 1997, pp. 263-334).

Assembly, Release and Maturation of HIV Particles

The main components of HIV structural proteins are translated in the form-of three polyproteins: Gag and Gag-Pol for the inner core proteins and viral enzymes and Env for proteins of the viral envelope proteins. In the case of HIV- 1, complete proteolytic processing of the Gag polyprotein Pr55 results in the formation of the matrix (MA), capsid (CA) and nucleocapsid (NC) and of the C-terminal p6Gag- protein. In general, HIV-1 virions are detached from the plasma membrane as mature

noninfectious virus particles, this process being referred to as virus budding.

Immediately after or else during budding, proteolytic processing of Gag and Gag-Pol polyproteins commences with the activation of PR. The proteolytic maturation of the virions is accompanied by morphological changes. A characteristic feature is the condensation of the inner core, resulting in the formation of a conical core cylinder typical for the mature virus (for a review, see Swanstrom and Wills, 1997).

Ubiquitin/Proteasome Pathway and Retrovirus Replication

Information on the importance of the UPS for particular sections of HIV replication is also known: on the one hand, the system is utilized for proteolysis of de novo synthesized virus receptor CD4. This pathway is mediated by the HIV-1 -specific protein Vpu which directs CD4 from the membrane of the endoplasmic reticulum (ER) to the site of proteosomal degradation in the cytoplasm (Schubert et al, 1998, J. Virol, 72:2280). Moreover, monoubiquitinated forms of Gag have been described for HIV-1 and Mo-MuLV Gag proteins (Ott et al, 1998, J. Virol, 72:2962). Although the catalytic activities of the 26S proteasome are completely different from the very specific aspartate- protease activity of the HIV-l/HIV-2 viral proteases, it was observed that a specific inhibitor of the HIV-1 protease, referred to as "ritonavir" (but none of the other previously known HIV protease inhibitors) can inhibit chymotrypsin activity of the 20S proteasome in vitro (Schmidtke et al., 1999, J. Biol. Chem., 274:35734) and proteasome-mediated MHC-I antigen expression in vivo (Andre et al, 1998, Proc. Natl. Acad. Sci. USA, 95: 1312; WO 00/33654).

Accordingly, there is a need for new anti-retroviral therapies in infected mammals, particularly in humans. New therapeutic approaches intend to prevent the spread of infectious viruses, the development of immunodeficiencies, such as AIDS, HIV- dementia, or other retrovirus-related diseases or conditions, etc.

The present invention also provides new therapies for both, retroviral infections, e.g. infection by HIV and hepatitis virus infections, particularly HCV infections.

Hepatitis C virus (HCV) Hepatitis C virus (HCV) is a small (55-65 nm in size), enveloped, positive-sense single- stranded RNA virus of the family Flaviviridae. Hepatitis C virus is the cause of hepatitis C in humans. The hepatitis C virus particle consists of a core of genetic material (RNA), surrounded by an icosahedral protective shell of protein, and further encased in a lipid (fatty) envelope of cellular origin. Two viral envelope

glycoproteins, El and E2, are embedded in the lipid envelope. Hepatitis C virus has a positive sense single-stranded RNA genome. The genome consists of a single open reading frame that is 9600 nucleotide bases long. This single open reading frame is translated to produce a single protein product, which is then further processed to produce smaller active proteins. At the 5' and 3' ends of the R A are the UTR, that are not translated into proteins but are important to translation and replication of the viral RNA. The 5' UTR has a ribosome binding site (IRES - Internal ribosome entry site) that starts the translation of a very long protein containing about 3,000 amino acids. This large pre-protein is later cut by cellular and viral proteases into the 10 smaller proteins that allow viral replication within the host cell, or assemble into the mature viral particles. Structural proteins made by the hepatitis C virus include Core El and E2; nonstructural proteins include p7, NS2, NS3, NS4, NS4A, NS4B, NS5A, and NS5B.

HCV replication

Replication of HCV involves several steps. The virus replicates mainly in the hepatocytes of the liver. The virus may also replicate in peripheral blood

mononuclear cells, potentially accounting for the high levels of immunological disorders found in chronically-infected HCV patients. HCV has a wide variety of genotypes and mutates rapidly due to a high error rate on the part of the virus' RNA- dependent RNA polymerase. The mutation rate produces so many variants of the virus it is considered a quasispecies rather than a conventional virus species. Entry into host cells occur through complex interactions between virions and cell-surface molecules, e.g. CD81, Claudin-1, Occludin-1, Scavenger Receptor Bl . Inside the hepatocyte, HCV takes over portions of the intracellular machinery to replicate. The HCV genome is translated to produce a single protein of around 3011 amino acids. The polyprotein is then proteolytically processed by viral and cellular proteases to produce three structural (virion-associated) and seven nonstructural (NS) proteins. Alternatively, a frameshift may occur in the Core region to produce an Alternate Reading Frame Protein (ARFP). HCV encodes two proteases, the NS2 cysteine autoprotease and the NS3-4A serine protease. The NS proteins then recruit the viral genome into an RNA replication complex, which is associated with rearranged cytoplasmic membranes. RNA replication takes places via the viral RNA-dependent RNA polymerase NS5B, which produces a negative-strand RNA intermediate. The negative strand RNA then serves as a template for the production of new positive- strand viral genomes. Nascent genomes can then be translated, further replicated, or packaged within new virus particles. New virus particles are thought to bud into the secretory pathway and are released at the cell surface.

Summary and detailed description of the Invention

It is an objective of the invention to provide pharmaceutical compositions, uses, kits, and methods of treatment which allow treatment of retroviral, particularly HIV infections and/or viral hepatitis infections, in particular of Hepatitis C Virus (HC V) infections.

These and other objectives as they will become apparent from the ensuing description are attained by the subject matter of the independent claims. The dependent claims refer to some of the preferred embodiments. In one embodiment the invention relates to a semicarbazone proteasome inhibitor for use in the treatment of hepatitis virus-infected individuals and/or retrovirally, e.g. HIV-infected individuals.

In one particular embodiment, the semicarbazone proteasome inhibitor is used for treatment of hepatitis virus infected individuals are infected with Hepatitis C Virus (HCV). In a specific embodiment of the invention, the semicarbazone proteasome inhibitor for use in the treatment of hepatitis virus-infected individuals and/or retrovirally, e.g. HIV-infected individuals is selected from [l-[l-{l-[(2,4-Dioxo-imidazolidin-l- ylimino)-methyl] -2-phenyl-ethylcarbamoyl} -2-( 1 H-indo 1-3 -yl)-ethylcarbamoyl] -2- (lH-indol)], pharmaceutically acceptable salts thereof, or structural and/or functional analogues thereof.

In specific embodiments, the invention pertains to the use of semicarbazone proteasome inhibitors such as [l-[l-{l-[(2,4-Dioxo-imidazolidin-l-ylimino)- methyl] -2-phenyl-ethylcarbamoyl} -2-( 1 H-indo 1-3 -yl)-ethylcarbamoyl] -2-( 1 H- indol)], pharmaceutically acceptable salts thereof, or structural and/or functional analogues thereof as the sole pharmaceutical agent in the therapy for hepatitis virus infections, such as infections by HCV, or in the treatment and/or prevention of HIV infections, within a given defined treatment period. Such a defined treatment period may last for about 1 to 48 weeks or longer, e.g. for about 1 week to 12 months, 1 week to 1 week to 9 months, 1 week to 8 months, 1 week to 7 months, 1 week to 6 months, 1 week to 5 months, or for about 2 to 4 weeks, 2 to 6 weeks, 1 to 2 months, 1 to 3 months, 1 to 4 months, etc.. The duration of the treatment period depends on the medical practitioner's decision.

In particular embodiments of the invention, the semicarbazone proteasome inhibitor in the treatment of HCV is intended for use in treating HCV-infected individuals selected from the following groups of patients: (i) patients not refractory to interferon and/or ribavirin;

(ii) patients treated with the semicarbazone proteasome inhibitor as

defined herein as the sole active agent; (iii) patients responding to interferon and/or ribavirin therapy; (iv) patients not being treated with interferon and/or ribavirin; (v) patients treated with interferon and/or ribavirin that are not interferon- and/or ribavirin-refractory;

(vi) patients as defined in any one of (i) to (v) having a liver cirrhosis;

(vii) patients previously not treated with further anti-HCV medicaments;

(viii) patients as defined in any of (i) to (vii) having increased transaminase concentrations;

(ix) patients as defined in any one of (i) to (vii) not having increased

transaminase concentration;

(x) patients as defined in any one of (i) to (ix) not having a chronic HCV- infection;

(xi) patients as defined in any one of (i) to (ix) having a chronic HCV- infection and/or a chronic HCV-hepatitis;

(xii) patients showing a sustained viro logical response (SVR) to interferon- and/or ribavirin-therapy; (xiii) patients showing a rapid virological response interferon- and/or ribavirin-therapy;

(xiv) patients as defined in any of (i) to (x) and (xii) to (xiii) having an acute asymptomatic or mildly symptomatic HCV infection;

(xv) patients as defined in any of (i) to (xiii) having a persistent HCV- infection;

(xvi) patients as defined in any of (i) to (xv) having a positive anti-HCV immunoassay;

(xvii) patients as defined in any of (i) to (xvi) positively tested for the presence of HCV-R A;

(xviii) patients as defined in any of (i) to (xvii) having leukopenia and/or thrombocytopenia;

(xix) patients as defined in any of (i) to (xviii) having an anemia, a

cardiovascular or a cerebrovascular disease.

In a specific embodiment the invention relates to a pharmaceutical composition comprising a semicarbazone proteasome inhibitor for use in the treatment of retrovirally, particularly HIV infected patients, and/or hepatitis virus infected individuals, e.g. HCV infected individuals, wherein said semicarbazone proteasome inhibitor is selected from [l-[l-{l-[(2,4-Dioxo-imidazolidin-l-ylimino)-methyl]-2- phenyl-ethylcarbamoyl}-2-(lH-indol-3-yl)-ethylcarbamoyl]-2-(lH-indol)], pharmaceutically acceptable salts or structural and/or functional analogues. Moreover, the present invention also relates to a kit comprising the semicarbazone proteasome inhibitor as defined herein or a pharmaceutical composition comprising the same for use in the treatment of retrovirally, e.g. HIV-infected and/or hepatitis- virus-infected individuals, particularly HCV-infected, individuals.

Yet other embodiments of the invention relate to the uses in methods of treating human or animal individuals infected with retrovirus, e.g. HIV and/or hepatitis viruses, e.g. HCV, with at least one semicarbazone proteasome inhibitor, and optionally concomitantly with or subsequently or previously to said treatment herewith, with a pharmaceutical composition comprising at least one

pharmaceutically active agent in use againt retroviral infection (e.g. HIV infection) or at least one pharmaceutically active agent in use against hepatitis virus infection (e.g. HCV infection).

The methods of treatment in accordance with the invention are practiced on human or animal individuals which are in need of such treatment. These may be individuals suffering from HIV infections [or infections with other retroviruses] and/or a hepatitis viral infections. Specifically, said hepatitis virus infection is a HCV infection.

In any of the above-mentioned embodiments or in the subsequently mentioned embodiments, the viral hepatitis infection is preferably a Hepatitis C Virus (HCV) infection. Alternatively, it may be a Hepatitis B Virus (HBV) infection.

Before the above embodiments are described in further detail, the following definitions are provided. Terms as set forth hereinafter are generally to be understood in their common sense unless indicated otherwise.

Where the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of is considered to be a preferred embodiment of the term "comprising of. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated. In the context of the present invention the terms "about" or "approximately" denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%. The term "viral hepatitis" refers to hepatitis induced by viral infections. Such viral infections may be infections with one or more viruses that may infect hepatic cells, such as, for example, without limitation, Hepatitis Virus A, B, C, D, E and G.

Alternatively, the hepatitis may be induced by an infection with a virus which may infect and potentially damage the liver as part of a more generalized infection, such as, for example, without limitation, certain Retro-, Herpes, Adeno-, Entero-,

Paramyxo-, Toga-, Flavi-, Bunya-, Arena-, Filo-, und Parvoviruses (H. Dancygier, Klinische Hepatologie, Springer- Verlag Berlin Heidelberg 2003, S. 489). More specifically, the hepatitis may be induced by infection with one or more viruses chosen from the group of: Herpes Simplex Virus, HIV, Cytomegalovirus, Epstein- Barr virus, yellow fever virus, mumps virus, rubella virus. The treatment of a viral hepatitis caused by HCV infection is preferred in the context of the present invention.

The term "pharmaceutically active agent in use against viral hepatitis infections" refers to a pharmaceutically active compound for which the art recognizes that it can be used for treatment of viral hepatitis infections, for example for the treatment of HCV infections. The term particularly relates to pharmaceutically active agents as they are authorized by regulatory agencies such as the FDA or the European

Medicines Agency (EMA) for treating viral hepatitis infections, in particular HCV infections. The term for the purposes of the present invention may not refer to proteasome inhibitors. Recognition in the art of a pharmaceutically active compound as useful for hepatitis treatment may have occurred at the time the instant invention was made, but nothing herein shall be construed as limiting the instant invention to such compounds. Pharmaceutically active compounds, which are characterized as useful for the therapy of viral hepatitis at a later date, are well within the scope of the instant invention, and are expressly included. The terms "retroviral" or "retrovirus" relate to enveloped viruses belonging to the family Retroviridae. This family comprises RNA viruses having a positive-mRNA- stranded RNA genome which is reverse transcribed and replicated in a host cell via the enzyme reverse transcriptase (RT) to produce DNA from its RNA genome. The DNA is incorporated as a so-called "provirus" into the host's genome by the viral integrase enzyme. The Retroviridae family comprises such important members as HIV, HTLV, Xenotropic murine leukemia virus-related virus (XMRV), FIV, SIV etc. In the context of the present application an important aspect relates to the treatment of human immunodeficiency virus, i.e. HIV, which encompasses at least two types of HIV, e.g. HIV-1 and HIV-2. While HIV-1 is the dominant type in the Western World, and certain examples provided herein use viruses of this type, the scope of the instant invention shall not be understood as limited to HIV-1, and preferably includes not just types of HI viruses presently known, but also further types which may arise or be characterized and/or typified in the future.

The expression "pharmaceutically active agent in use against retroviral infections", interchangeably used herein with the term "anti-retro viral compound", refers to a pharmaceutically active compound for which the art recognizes that it can be used for treatment of retroviral infections, preferably for the treatment of HIV infections. Preferably the term relates to pharmaceutically active agents as they are authorized by regulatory agencies such as the FDA or the EMA for treating retroviral infections, in particular HIV infections. The term for the purposes of the present invention may not refer to proteasome inhibitors. Recognition in the art of a pharmaceutically active compound as useful for the treatment of retrovirus infection may have occurred at the time the instant invention was made, but nothing herein shall be construed as limiting the instant invention to such compounds. Pharmaceutically active compounds, which are characterized as useful for the therapy of retroviral infections at a later date, are well within the scope of the instant invention, and are expressly included.

The term retroviral disease refers to diseases or conditions caused by an infection with a retrovirus. The term "HIV-related disease" refers to diseases and conditions including those which are commonly attributed to, or are causally related to, infection with a Human Immunodeficiency Virus, and specifically may include, without limitation, Acquired Immuno-Deficiency Syndrome (AIDS), and all diseases and conditions commonly referred to thereunder, and/or the condition of infection with a Human Immunodeficiency Virus, including in the absence of any overt symptoms of disease or discomfort directly or indirectly linked thereto, and furthermore including before the presence of the virus may be detected with certain diagnostics test as customary for this indication. However, in some embodiments, HIV-related disease will preferably not refer to diseases and conditions ultimately brought about by HIV infection through the associated Immunodficiency, but which, by their nature, cannot be alleviated or cured by removing or eradicating the virus alone, e.g. certain malignancies which often occur in HIV-posistive or AIDS patients, e.g. Karposi Sarcoma. The term "standard therapy for HIV infection" shall mean a therapy of an HIV infection following the recommendations summarized in "Guidelines for the Use of Antiretro viral Agents in HIV- 1 -Infected Adults and Adolescents" (Panel on

Antiretro viral Guidelines for Adults and Adolescents; The U.S. Department of Health and Human Services. Washington, DC, USA, January 10, 2011; pages 1- 166), or in a respective future update of this document as indicated by a relevant authority. A "dose recommended for treatment of an HIV-related disease" refers to the doses recommended in the latter document for the respective anti-retroviral compound under applicable circumstances. "Administering", or "administration of, a compound or agent, e.g. an anti-retroviral compound or a proteasome inhibitor, as used herein, shall be interpreted as equivalent to "administering a pharmaceutical composition comprising" or

"administration of a pharmaceutical composition comprising" the respective compound or agent, wherein the pharmaceutical composition will usually further comprise such additional components as described herein below and which are necessary to render the respective compound or agent bioavailable and .to minimize adverse effects of administration The term "proteasome inhibitor" refers to a compound which is capable of inhibiting, reversibly or irreversibly, the proteasome-mediated degradation of ubiquitin- modified peptides and proteins. Proteasome inhibitors encompass semicarbazone proteasome inhibitors that are used according to the invention as detailed below. In specific embodiments of the invention other non-semicarbazone proteasome inhibitors may be used in addition to the semicarbazone inhibitor.

The term "semicarbazone proteasome inhibitor" as the term is used herein refers particularly to [ 1 -[ 1 - { 1 -[(2,4-Dioxo-imidazolidin- 1 -ylimino)-methyl]-2-phenyl- ethylcarbamoyl}-2-(lH-indol-3-yl)-ethylcarbamoyl]-2-(lH-indol)], also referred to as S-2209, pharmaceutically acceptable salts, as well as structural and/or functional analogs thereof.

The term "structural and/or functional analogs of [l-[l-{l-[(2,4-Dioxo-imidazolidin- 1 -ylimino)-methyl] -2-phenyl-ethylcarbamoyl} -2-( 1 H-indo 1-3 -yl)-ethylcarbamoyl] -2- (lH-indol)]", as used herein, refers to the group of compounds, but excluding [1-[1- { 1 -[(2,4-Dioxo-imidazolidin- 1 -ylimino)-methyl]-2-phenyl-ethylcarbamoyl} -2-(lH- indo 1-3 -yl)-ethylcarbamoyl]-2-(l H-indo 1)] and its pharmaceutically acceptable salts, of the general formula (I), pharmaceutically acceptable salts or a pharmaceutically acceptable salt or a stereoisomer thereof,

Formula (I)

wherein Y is a group of formula (III)

R^a, R^b, R^c, R^d independently represent H. -CN, -OH, alkoxy, -SH, alkyl, alkenyl- or alkynylthio, -C0₂R^4', -C(0)R^4', -S0₂NR^4', -S0₂-alkyl, -alkenyl or -alkynyl, -S0₂R^4', - S0₃R^4', -NO₂, -NR^4'R^5*, alkyl-, alkenyl- or alkynyl amino, -N=CR⁴R^5*, - NR^4'C(0)R⁴", -NR^4'-CO-haloaIkyI, -alkenyl or -alkynyl , -NR^4'-S0₂-haloalkyl, - alkenyl or -alkynyl, -NR⁴ -S0₂-alkyl, -alkenyl or -alkynyl, -NR⁴ -CO-alkyl, -alkenyl or -alkynyl, -NR^4'(CH₂)_nheterocycle, -C(NR^4")NR^4'benzimidazolyl, - C(NR^4")NR^4'benzothiazolyl, -C(NR^4")NR^4'benzoxazolyl, alkyl, alkenyl or alkynyl, cycloalkyl, -alkenyl or -alkynyl, -0(CH₂)_n[0(CH₂)_n]_rOCH₃, hydroxyaIkyl(alkenyl, alkynyl)amino, hydroxycycloalkyl, -alkenyl or -alkynyl, hydroxyalkyl, -alkenyl or - alkynyl amino, halogen, haloalkyl, -alkenyl or -alkynyl, haloalkyl, -alkenyl or - alkynyl oxy, aryl, arylalkyl, -alkenyl or -alkynyl or a heterocycle;

R^4', R^4", R⁵ independently are H, halogen, alkyl, alkenyl or alkynyl , -C(NR⁷)NR⁷R⁸, -(CH₂)„aryl, -(CH₂)„NR⁷R⁸, -C(0) NR⁷R⁸, -N=CR⁷R⁸, -NR⁷C(0)R⁸, cycloalkyl, - alkenyl or -alkynyl, heterocycloalkyl, -alkenyl or -alkynyl, haloalkyl, -alkenyl or- alkynyl, hydroxyalkyl, -alkenyl or -alkynyl, hydroxyalkyl, -alkenyl or -alkynyl, aminoalkyl. -alkenyl or -alkynyl, heteroaryl, alkyl-, alkenyl- or alkynylairl, or aryl;

R⁷, R⁷ , R⁸ independently are H, halogen, alkyl, -alkenyl or -alkynyl, cycloalkyl, - alkenyl or -alkynyl, heterocycloalkyl, -alkenyl or -alkynyl, haloalkyl, -alkenyl or - alkynyl, hydroxyalkyl, -alkenyl or -alkynyl, -alkenyl or -alkynyl amino, alkyl-, alkenyl- or alkynylamino, heteroaryl, alkylaryl, or aryl; n is 1; m is 1; r is 1 ;t is 1; X is O; Z is C=0;

Zl is (CH₂)_tR²; Z² is (CH₂)_t-R³; Z³ is (CH₂)_t-R⁴; Z⁴ is H, or methyl;

R², R³, R⁴ are independently from each other H, Phenyl, Benzyl, 3-Benzothienyl, 2- Thienyl, 2-Thiazolyl, 4-Pyridyl, 3-Pyridyl, 2-Pyridyl, 2-Quinolyl, 2-Indolyl, 3- Indolyl, Ethylbenzene, 2-Naphtyl, 1-Naphtyl, p-Aminobenzyl, p-Azidobenzyl, p- Bromobenzyl, p-Hydroxyphenyl, p-tButyl-benzyl, p-Carboxybenzyl, p-Chloro- benzyl, p-Cyanobenzyl, 3,4-Dichlorobenzyl, p-Fluorobenzyl, p-Iodobenzyl, p- Nitrobenzyl, Pentafluorobenzyl, p-Phenylbenzyl, m-Fluorobenzyl, p-Methyl-benzyl, Tryptoline-3-carboxylic acid, 5-Methyl-tryptophan, 4-Methyl-tryptophan, 3 -Methyl - lH-indolyl, 2-Methyl-lH-indolyl, 2-Amino-4-ethyl-phenol, 2,6-Dibromo-4-ethyl- phenol, 4-Ethyl-2,6-diiodo-phenol, l-Ethoxy-4-ethyl-benzene, l-Ethyl-4-methoxy- benzene, 4-Ethyl-2-iodo-phenol, (4-Ethyl-phenyl)-phenyl-methanone, 1-Thiophen- 2-yl-ethanol, l,2,3,4-Tetrahydro-isoquinoline-3-carboxylic acid, 7-Hydroxy-l,2,3,4- tetrahydro-isoquinoline-3-carboxylic acid, Sulfuric acid mono-(4-ethyl-phenyl) ester, Phosphoric acid mono-(4-ethyl-phenyl) ester, 4-Ethyl-2-nitro-phenol, 1-tert-Butoxy- 4-ethyl-benzene and 4-(4-Ethyl-phenoxy)-phenol; R¹ is phenyl, 4-pyridyl, 3-pyridyl, 2-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5- pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 2-pyrazinyl, 1 -pyrazolyl, 3 -pyrazolyl, 4- pyrazolyl, 2-indolyl, 3-indolyl, 1-imidazolyl, 2- imidazolyl, 4-tetrahydro-thieno[3,4- ]imidazol-2-one-yl, 4-phenoxy-benz-l-yl, which are optionally substituted by halogen, alkoxy, haloalkyl, or haloalkoxy; provided the analogous compound is capable of inhibiting the activity of the proteasome to an extent which corresponds to at least 25% (50%, 60%, 70%, 80%, 90%, or 95%) of the proteasome inhibitory activity of S-2209.

In a preferred embodiment, R², R³, R⁴ are independently of each other H, benzyl,or indolyl optionally substituted by halogen.

Specific examples for structural/functional analogs are Compounds 1 to 6 and Compound 8 disclosed in Leban J., et al, Bioorg. Med. Chem. 2008, 16:4579), and/or Compounds 1 to 10 in WO 2007/017284. S-2209 is a particularly preferred proteasome inhibitor for use according to the present invention.

"Inhibition of proteasome activity" as the term is used herein, shall mean the reduction of proteasome activity inside or outside a cell by at least 20%>, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% compared to the situation where no compound or a compound which is known to not affect proteasome activity is administered. The proteasome that is inhibited by a proteasome inhibitor referred to herein thus has a residual activity of not more than about 80%, more particularly not more than about 75%, yet more particularly not more than about 70%, yet more particularly not more than about 65%, yet more particularly not more than about 60%, yet more particularly not more than about 55%, yet more particularly not more than about 50%, yet more particularly not more than about 45%, yet more particularly not more than about 40%, yet more particularly not more than about 35%, yet more particularly not more than about 30%, yet more particularly not more than about 20%, yet more particularly not more than about 5 to 15%, and particularly not more than about 10% residual activity.

However, the inhibition of proteasome activity may be limited by associated toxicity. Under certain circumstances, a minimum residual proteasome activity may be required for a cell to survive. Hence, the inventive proteasome inhibitors may inhibit the activity of the proteasome in a relevant cell, for example a cell in vitro that is a model for the cell type desired to be impacted by the proteasome inhibitory activity in vivo, by only a limited amount, e.g. by not more than about 20%, not more than about 25%, not more than about 30%, not more than about 35%, not more than about 40%, not more than about 45%, not more than about 50%, not more than about 55%, not more than about 60%, not more than about 70%, or not more than about 75%.

In certain embodiments of the invention, the proteasome inhibitor inhibits one or more of the catalytic activities of the 26-S proteasome, and more preferably one or more of the postglutamyl-peptide-hydrolyzing (caspase-like, Bl-subunit), trypsin- like (B2 subunit), and/or chymotrypsin-like (B5 subunit) catalytic activities. Yet more specifically, the proteasome inhibitor inhibits all three, yet more specifically not more than two, and most particularly only one of these 26-S proteasome catalytic activities. In another specific embodiment, the proteasome inhibitor inhibits at least two of these 26-S proteasome catalytic activities. In yet another specific embodiment, the proteasome inhibitor may inhibit at least one activity of the proteasome, but may actually activate another of the 26-S proteasome catalytic activities. In yet another specific embodiment, the proteasome inhibitor does not activate any of the 26-S proteasome catalytic activities.

In certain embodiments of the invention, the inhibitory activity is observed at nanomolar concentrations in cell culture in vitro, e.g. at concentrations ranging between 1 nM and 1 μΜ, or 10 nM to 1 μΜ, or 100 nM to 1 μΜ, or 1 nM to 100 nM, or 1 nM to 10 nM, or 10 nM to 100 nM, or 1 nM to 10 μΜ, or 10 nM to 10 μΜ, or lOO nM to 10 μΜ, or 1 μΜ to 10 μΜ.

The inhibitory activity on the proteasome of a candidate proteasome inhibitor may be assessed by, for example, the assay described in Adams, J., et al, Cancer Research 1999, 59:2615, or the commercially available Proteasome Glo™ Assay (Promega Corp. Madison WI, USA). These assays may be used to determine an IC₅₀ for the candidate proteasome inhibitor respective the one or more proteasome catalytic activities; any other suitable assay may then be used to determine an IC₅₀ for the candidate proteasome inhibitor respective another relevant activity in question; and these IC₅₀ values may hence be compared to obtain a measure of the specificity of the inhibitory activity of the candidate proteasome inhibitor. Further methods of testing the inhibitory activity of S-2209 and the like are disclosed in Baumann et al., Brit. J. Haematology 144: 875-886, 2009.

Supplementary to the semicarbazone proteasome inhibitor used according to the present invention, one may use all known proteasome inhibitors. These include, without limitation: naturally-occurring proteasome inhibitors e.g. epoxomicine, eponemicin, aclacinomycin A (also known as aclarubicin), lactacystin and its modified variants, e.g. clasto-lactacystin β-lactone);

synthetically prepared proteasome inhibitors (e.g. modified peptide aldehydes, such as N-carbobenzoxy-L-leucinyl-L-leucinyl-l-leucinal, sometimes referred to as MG132 or zLLL, its boronic acid derivative MG232, N-carbobenzoxy-Leu-Leu-Nva-H, sometimes referred to as

MG115), N-acetyl-L-leuzinyl-L-leuzinyl-L-norleuzinal, sometimes referred to as LLnL), N-carbobenzoxy-Ile-Glu(Obut)-Ala-Leu-H, sometimes referred to as PSI); synthetic peptides that carry at their C-terminal end an α, β- epoxyketone group vinyl- sulphone group,e.g. carbobenzoxy-L-leucinyl-L- leucinyl-L- leucine- vinyl sulfone or 4-hydroxy-5-iodo-3-nitrophenylacetyl-L- leucinyl-L-leucinyl-L-leucin-vinyl-sulfone, also referred to as NLVS), a glyoxal or boronic acid-residue, or Benzoyl(Bz)-Phe-boroLeu, Ph-acetyl- Leu-Leu-boroLeu, Cbz-Phe-boroLeu, or a pinacol-ester group, e.g.

benzyloxycarbonyl(CbZ)-Leu-Leu-boroLeu-pinacol-ester; and chemically modified derivatives of naturally occurring proteasome inhibitors, such as the β-lacton derivative PS-519 (1R-[1S, 4R, 5S]]-l-(l-hydroxy-2-methylpropyl)- 4-propyl-6-oxa-2-azabicyclo[3.2.0]heptanes-3,7-dione, (molecular formula: C12H19NO4), a derivative of lactacystine;

dipeptidyl-boronic acid derivatives such as PS-341 (N-(2,3- pyrazine)carbonyl-L-Phenylalanine-L-leucine-boronic acid, molecular formula: C19H25BN4O4).

In the context of the present invention, suitable proteasome inhibitors that may be used in addition to the semicarbazone proteasome inhibitor thus include PS-341, also commonly known as Bortezomib, and the active ingredient in the pharmaceutical preparation sold under the trade name Velcade^®, in use for the treatment of multiple myeloma; "PS-273" (Morpholin-CONH-(CH-napthyl)-CONH-(CH-isobutyl)- B(OH)₂ and its enantiomer "PS-293", "PS-296" (8-quinolyl-sulfonyl-CONH-(CH- napthyl)-CONH(-CH-isobutyl)-B(OH)₂); "PS-303" (NH₂(CH-Napthyl)-CONH-(CH- isobutyl)-B(OH)₂);"PS-321 " (morpholino-CONH-(CH-napthyl)-CONH-(CH- phenylalanine)-B(OH)₂); "PS-334" (CH₃-NH-(CH-napthyl-CONH-(CH-isobutyl)- B(OH)₂); "PS-325" (2-quinole-CONH-(CH-homo-phenylalanin)-CONH-(CH- isobutyl)-B(OH)₂); "PS-352" (Phenyalanine-CH₂-CH₂-CONH-(CH-phenylalanine)- CONH-(CH-isobutyl)l-B(OH)₂); "PS-383" (pyridyl-CONH-(CH/?F-phenylalanin2)- CONH-(CH-isobutyl)-B(OH)₂.

Further suitable proteasome inhibitors include (Groll, M., et al, J. Pept. Sci. 2009, 15:58): the aldehydes calpain inhibitor I, Mal- -Ala-Val-Arg-al and fellutamide B (Hines J., et al, Chem. Biol.2008, 15:501), syringolin A (Groll M, et al, J. Am. Chem. Soc. 2000, 122: 1237) and glidobactin A, members of the new class of Syrbactins; the natural lactones Omuralide, Homobelactosin C and Salinosporamide A (NPI-0052; Salinosporamide A is undergoing clinical Phase lb trials in drug combination therapy for patients with non-small cell lung cancer, pancreatic cancer, or melanoma), and the vinyl sulfone peptide Ac-Pro-Arg-Leu-Asn-vs; the analogue of epoxomocin carfilzomib, also known as PR-171 (Demo, SD., Cancer Res. 2007, 67:6383); the TMC-95 family of cyclic tripeptides from Apiospora montagnei

(Koguchi, Y., J. Antibiot. 2000, 53: 105, Kohno, J., Org. Chem. 2000, 65:990), such as TMC-95 A and its endocyclic oxindole-phenyl clamp (BIA-la) and endocyclic biphenyl-ether clamp (BIA-2a) derivatives; the natural cyclic octapeptide argyrin A derived from the myxobacterium Archangium gephyra (Vigneron, N., Science 2004, 304:587), the acetylated tripeptide aldehydes Ac-Leu-Leu-Nle-H and Ac-Arg-Val- Arg-H, the corresponding pegylated tripeptide aldehydes (PEG)i9-₂5-Leu-Leu-Nle-H and (PEG) i9_₂5-Arg-Val-Arg-H, as well as their bifunctional equivalents H-Nle-Leu- Leu-(PEG)i9-₂₅-Leu-Leu-Nle-H and H-Arg-Val-Arg-(PEG) i₉-₂₅-Arg-Val-Arg-H. Yet further suitable proteasome inhibitors include (Huang, L., Chen, CH, Current Medicinal Chemistry, 2009, 16:931): CEP1612, a dipeptide aldehyde proteasome inhibitor that is highly selective for the chymotrypsin-like proteolytic activity of the proteasome; ZLVS (ZLLL-vs) and YLVS (YLLL-vs), further examples of vinyl sulfones (herein, -vs is used as shorthand for a vinyl sulfone group); MG-262, a boronate analog of MG132, which exhibits a 100-fold increase in anti-proteasome activity compared to its parent compound; Tyropeptin A, a tripeptide aldehyde natural product isolated from Kitasatospora sp. MK993-dF2, preferentially inhibiting the chymotrypsin-like proteasome activity by binding to the β5 subunit of the proteasome (Momose, I.; et al, J. Antibiotics 2001, 54:997; Momose, I., et al, Bioorg. Med. Chem. Lett. 2005, 15:1867); Peptide epoxyketones, isolated from various microbials, are small peptides with a ketone epoxide functional group; for example, epoxomycin was derived from Streptomyces hygroscopicus (Hanada, M., et al, J. Antibiot. (Tokyo) 1992, 45 : 1746), TMC-86 and TMC-89 were isolated from Streptomyces sp. [Koguchi, Y., et al, J. Antibiot. (Tokyo) 2000, 53:63; Koguchi, Y, et al, J. Antibiot. (Tokyo) 2000, 53:967); peptide epoxyketones inhibit the proteasome by covalently modifying the catalytic sites of the β subunits; Carfilzomib (PR- 171), an epoxyketone peptide structurally related to epoxomicin, is in Phase 2 clinical trials for patients with relapsed solid tumors including non- small cell lung cancer, small cell lung cancer, ovarian cancer, and renal cancer (Kuhn, DJ, et al, Blood 2007, 110:3281); it is also in a phase 2 single-agent trial for patients with multiple myeloma and in a phase 1 study for lymphoma patients; some peptide epoxyketone derivatives, such as dihydroeponemycin analogs, were shown to preferentially target the immunoproteasome (Ho, Y.K., et al, Chem. Biol. 2007,

14:419); PR39 is a naturally occurring antibacterial peptide containing 39 amino acid residues isolated from pig intestine, and was shown to inhibit the proteasome; unlike small tripeptide proteasome inhibitors that bind to the proteolytic active site located at β5 subunit, PR39 binds to the nonproteolytic β7 subunit of the 20S proteasome. PR11 (first 11 residues of PR39 sequence: RRRPRPPYLPR) and its analogs exhibit similar activity to that of PR39; Inhibition of the proteasome by PR11 and PR39 results in accumulation of ΙκΒ, a factor that regulates the NF-KB-dependent gene expression pathways; natural products derived from plant sources, such as celastrol, isolated from the traditional herbal medicine "Thunder-god vine", and withaferin A, isolated from Indian winter cherry, which were shown to inhibit the proteasome at low micro molar concentrations (Celastrol is a triterpene and withaferin A is structurally related to steroids); Gliotoxin, a fungal metabolite structurally related to the epipolythiodioxo- piperazines; green tea polyphenolic catechins such as (-)- epigallocatechin- 3-gallate {(-)-EGCG} and its analogs have been widely studied for their possible benefits in cancer prevention; EGCG was reported to potently inhibit the chymotrypsin- like activity of the proteasome in vitro and in cultured tumor cells; Disulfiram, a drug for the treatment of alcohol dependence, was shown to inhibit the proteasome; certain acridine derivatives, a class of anti-cancer agents primarily targeting DNA and topoisomerase II, also having proteasome inhibiting activity, e.g. tetra-acridine; certain derivatives of betulinic acid, e.g. 3',3'-dimethylsuccinyl betulinic acid; in contrast to BA a proteasome activator, many BA derivatives inhibit the proteasome; similarly, certain derivatives of glycyrrhetinic acid (GLA) may be potent inhibitors of the proteasome, and such inhibitors are envisaged by the present invention.

Yet further suitable proteasome inhibitors may include: NEOSH-101, also known as OSH-101, a tetrapeptide aldehyde in clinical trials for androgenetic alopecia; CEP- Structure of (1 ), PR-047

18770, a P2 threonine boronic acid derivative under development for, e.g., multiple myeloma; IPSIOOl, IPSI007, as well as MLN2238 and its prodrug MLN9708, under development for indications in oncology by Millennium Pharmaceuticals/Takeda; ONX 0912 (formerly PR-047 (1) by Proteolix, Inc.; Peese, K., Drug Discovery

Today 2009, 14: 905), a proteasome inhibitor based on the same novel chemistry as carfilzomib, and ONX 0914 (formerly PR-957; Muchamuel, T., et al, Nature Medicine 2009, 15:7) an inhibitor of the immunoproteasome, both being developed by Onyx Pharmaceuticals; AA-102, an anticancer agent being developed by

Bionovo, Inc., 26 S PI, a proteasome inhibitor being developed by Ergon

Pharmaceuticals for oncology and other indications; AVR-147, a development candidate by Advanced Viral Research, Corp., in oncology; BU-32 (pyrazy 1-2,5 -bis- CONH(CHPhe)CONH(CHisobutyl)-B(OH)₂, NSC D750499-S; Aygin, JK, et al-., Breast Cancer Res. 2009, 11 :R74), 4E12, a non-peptidyl small molecule proteasome inhibitor identified by Telik, Inc., and intended for development in oncology; and Compound 13 and Compound 20 (Purandare, AS, et al, Am. Assoc. Cancer Res. Annual Meeting 2007, 98^th: April 15, Abs. 717), two lactam boronic acid proteasome inhibitors having high activity (low nM IC₅₀ values) as well as high specificity (>100 fold selective against chymotrypsin, trypsin, elastase and Factors Xa, Xia and Vila). Yet further suitable proteasome inhibitors may include: ALLnL, ALLnM; LLnV; DFLB (dansyl-Phe-Leu-boronate); Ada-(Ahx)₃-(Leu) ₃-vs; YU101 (Ac-hFLFL-ex), MLN519. Proteasome inhibitors may thus include di-, tri,- tetra-, penta-, hexa-, hepta-, octa-, nona-peptide aldehydes or peptide aldehydes having ten or more amino acids such as 15, 20, 30, 40 or more amino acids.

Such di-, tri,- tetra-, penta-, hexa-, hepta-, octa-, nona-peptide aldehydes or peptide aldehydes having ten or more amino acids may carry at their C-terminus and a,B- epoxyketone functionality, a vinyl- sulphone functionality, a glyoxal functionality, a boronic acid functionality, pinacol ester functionality or other functionalities.

Such di-, tri,- tetra-, penta-, hexa-, hepta-, octa-, nona-peptide aldehydes or peptide aldehydes having ten or more amino acids may comprise natural or non-natural amino acids.

Such di-, tri,- tetra-, penta-, hexa-, hepta-, octa-, nona-peptide aldehydes or peptide aldehydes having ten or more amino acids may also be chemically modified by hydrogenation, dehydrogenation, hydroxylation, dehydroxylation, acylation, deacylation, alkylation, dealkylation, pegylation, hesylation, glycosylation and the like.

Such di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-peptide aldehydes or peptide aldehydes having ten or more amino acids and which optionally may carry α,β- epoxyketone functionality, a vinyl- sulphone functionality, a glyoxal functionality, a boronic acid functionality, pinacol ester functionality or other functionalities at their C-terminus may also may comprise natural or non-natural amino acids. Such di-, tri,- tetra-, penta-, hexa-, hepta-, octa-, nona-peptide aldehydes or peptide aldehydes having ten or more amino acids and which optionally may carry α,β- epoxyketone functionality, a vinyl- sulphone functionality, a glyoxal functionality, a boronic acid functionality, pinacol ester functionality or other functionalities at their C-terminus may also be further chemically modified by hydrogenation,

dehydrogenation, hydroxylation, dehydroxylation, acylation, deacylation, alkylation, dealkylation, pegylation, hesylation, glycosylation and the like. Such di-, tri,- tetra-, penta-, hexa-, hepta-, octa-, nona-peptide aldehydes or peptide aldehydes having ten or more amino acids and which optionally may carry α,β- epoxyketone functionality, a vinyl- sulphone functionality, a glyoxal functionality, a boronic acid functionality, pinacol ester functionality or other functionalities at their C-terminus may also be further chemically modified by hydrogenation,

dehydrogenation, hydroxylation, dehydroxylation, acylation, deacylation, halogenations, alkylation, dealkylation, pegylation, hesylation, glycosylation and the like may comprise natural or non-natural amino acids.

Some proteasome inhibitors act on one or more of the postglutamyl-peptide- hydro lyzing (caspase-like, Bl -subunit), trypsin- like (B2 subunit), and/or

chymotrypsin-like (B5 subunit) activities found within the 26S proteasome (Groll, M., et al, J. Pept. Sci. 2009; 15:58; Lowe J, et al, Science 1995, 268:533; Groll M, et al, Nature 1997; 386:463). As such caspase-like, trypsin-like, and chymotrypsin-like proteolytic activities can also be found in other cellular proteases, proteasome inhibitors may not only inhibit the proteasome as described above, but also further cellular proteases. However, in specific embodiments of the invention, a subgroup of proteasome inhibitors, which can be particularly suitable for the pharmaceutical compositions, the kits, the uses and the methods as described herein, comprises so called specific proteasome inhibitors.

"Specific proteasome inhibitor", or "specific inhibition" of an activity of the proteasome by an inhibitor, herein shall mean that the inhibitor reduces the said activity by 50% at a concentration (termed IC50 or IC₅₀) that is lower by at least a factor of 1/2, 1/3, 1/5, 1/10, 1/20, 1/50, 10^"2, 5 x 10^"3, 2 x 10^"3, 10^"3, or less, than the IC₅₀ of that same inhibitor for the inhibition of another, or many other, or, specifically, any other relevant activity in question, e.g. a proteolytic activity not associated with the proteasome.

For example, a specific proteasome inhibitor may be at least twice as potent with respect to the inhibition of any one, or more, of the 26S proteasome catalytic activities than with respect to the inhibition of non-proteasomal cellular proteases or microorganism derived proteases, e.g., a lysosomal protease or an HIV protease, or at least three times as potent, or at least five times as potent, etc.. Such specific proteasome inhibitors include, without limitation, the proteasome inhibitors PS-519, PS-341 (Bortezomib) and PS-273. These proteasome inhibitors are potent, specific for the proteasome and substantially do not block other cellular proteases. The proteasome inhibitors PS-341 and PS-519 have moreover been tested pre-clinically in animal models and in humans for clinical studies (cancer patients). Pharmaceutically active agents in use against viral hepatitis infections may be broadly classified in two categories. Pharmaceutically active agents in use against viral hepatitis infections of the first category support or assist the body's natural response in dealing with viral infections. Pharmaceutically active agents of the second category will interfere with the function of a viral target, such as a virus- specific protease or polymerase.

In principle such pharmaceutically active agents of the first category which will be administered in addition to semicarbazone proteasome inhibitors and preferably in addition to S-2209 include, without intent to limitation, cytokines, such as interferons and their derivatives, and interleukins, preferably IL-1, IL-2, IL-6, IL-8, IL-12, IL- 15, IL-18, IL-21, and IL-2, inhibitors of viral enzymes, e.g. protease inhibitors e.g. telaprevir, boceprevir, ITNM-191, SCH 900518, TMC435, BI201335 and MK-7009, nucleoside analoga, nucleotide analoga and non-nucleoside analogous inhibitors of viral enzymes which may, for example, inhibit a viral polymerase and/or a viral protease, steroids, thymosin alpha 1, vaccines including vaccines allowing for passive and active vaccination, therapeutic and prophylactic vaccination,

glycyrrhizin, immunomodulators, e.g. Thymosin, ME3738, SCV-07, Alinia, Oglufavide, IPH-1101, CYT 107, or EGS-21, and/or immunosuppressants and/or inhibitors of assisted protein folding, e.g. ciclosporin and derivatives thereof, e.g. SCY-635, DEBIO-025, NIM811, Silibinin, Nitazoxanide, A-831, KPE02001003, TCM700C, PYN-17, BIT225, JTK-652, BMS-791325, amantadine or rimantadine and derivatives thereof, and azathoprine.

Pharmaceutically active agents which support or assist the body's natural response in dealing with viral infections will specifically, and in some embodiments exclusively, be used for viral hepatitis infections. Such agents may comprise interferons, interleukins, steroids, immunomodulators, immunosuppressants and inhibitors of assisted protein folding. The skilled person will be aware that some of such pharmaceutically active agents which support or assist the body's natural response in dealing with viral infections (e.g. immunosuppressants) are potentially unsuitable for the treatment of an HIV-related disease. The use of interferons is one option for pharmaceutically active agents which support or assist the body's natural response in dealing with viral infections. The term "interferon" refers to the various forms of interferons including their derivatives. Thus the term includes interferon alpha- 1, alpha-2, beta, gamma, delta, lambda and omega as well as the glycosylated, pegylated and hesylated forms thereof, and other forms wherein the interferon is fused or otherwise conjugated to another moiety conveying desirable properties to the overall molecule, e.g.

albinterferon, a fusion polypeptide of interferon with albumin, PEG-IFN alpha-2a, alpha-2b or lambda, Locteron, Omega IFN, Medusa IFN, DA-3021, EMZ702, Infradure, IL-29, Amarillo, Soluferon and Belerofon (see e.g. Thompson, A., et al, J. Hepatology , 50: 184 (2009)). For example, Albinterferon is a genetic fusion polypeptide of albumin and interferon alpha-2b with a longer half life than pegylated interferons. A phase 2 study comparing different doses of albinterferon alpha-2b and ribavirin with PEG- interferon alpha-2a and ribavirin indicated similar sustained viro logic response rates with a better tolerability of albinterferon alpha-2b based treatment. Based on the encouraging findings from the phase 2 study, the efficacy and safety of albinterferon alpha-2b administered every two weeks in combination with ribavirin for 48 weeks and 24 weeks in patients infected with HCV genotype 1 and 2/3, respectively, was investigated in two phase 3, randomized, active controlled, multi-center studies. Both studies ACHIEVE- 1 and ACHIEVE-2 were designed to demonstrate non- inferiority of the albinterferon alpha-2b regimes compared with PEG-interferon alpha-2a. Both studies achieved the primary objective. Locteron is a controlled-release interferon alpha-2b which is injected every 2 weeks. In a short term study controlled release interferon alpha-2b showed less flu-like symptoms than PEG-interferon alpha-2b injected every week indicating that the controlled-release formulation may have a better tolerability.

PEG-interferon lambda is a pegylated type III interferon that binds to a unique receptor with more limited distribution than the type I interferon receptor. In a phase lb study the mean decline of HCV-RNA in patients with relapsed HCV genotype 1 infection was 1.9-3.6 loglO IU/mL after 4 weeks of re-treatment withPEG-interferon lambda. PEG-interferon lambda is currently investigated in combination with ribavirin.

Further interferon varieties under development for hepatitis treatment include Omega IFN, Medusa IFN, DA-3021, EMZ702, Infradure, IL-29, Amarillo, and Belerofon. Also envisaged is Soluferon.

If in the context of the present invention an interferon is mentioned, the use of pegylated interferon alpha (PEG-interferon-alpha) is particularly preferred for all embodiments described hereinafter. This may be PEG-interferon-alpha 2a or 2b.

Pharmaceutically active agents of the second category, which interfere with the function of a viral target may be agents that inhibit viral enzymes such as, for example, a virus-specific protease or polymerase or virus envelope protein. In case of HCV infections this may be inhibitors of e.g. the HCV NS3/4A protease and/or the HCV NS5B polymerase.

Nucleoside analoga, nucleotide analoga and non-nucleoside analogous inhibitors of viral enzymes include lamivudine, cidovir, ribavarin, viramidine, didanosine, vidarabine, cytarabine, emtricitabine, zalcitabine, abacavir, stavudine, zidovudine, idoxuridine, trifhiridine, valopiticabine, R1626, R7128, IDX184, HCV-796, Filibuvir (PF 00868554), VCH-916, ANA598, BI 207127, VCH-222 PSI- 6130, ANA773, MK-3281, ABT-072, ABT-333, R1728, VCH-759, GS9190, BMS-650032, BE- 868554. The nucleoside analogon ribavirin is particularly preferred.

For example, the structure identification of the NS3/4A protease and the HCV NS5B polymerase and the development of a (sub) genomic replicon system have enabled the development and testing of HCV specific compounds. Further attractive targets within the HCV genome for antiviral therapy are the envelope proteins which are involved in HCV entry and NS5A which is involved in replication and in interferon alpha resistance. The clinical development of NS3/4A protease inhibitors is currently most advanced. HCV Protease inhibitors

The NS3/4A protease has key functions in the hepatitis C virus replication cycle. NS3/NS4A cleaves at four downstream sites in the polyprotein to generate the N- termini of the NS4A, NS4B, NS5A, and NS5B proteins. The NS3/4A serine protease has also been shown to cleave and inactivate the host proteins Trif and Cardif. Both proteins have important roles in the interferon response mediated by TLR3 and RIG-I, respectively.

Furthermore, it has been shown that NS3 is not only a protease but also an integral part of the viral RNA replication complex, functions as a RNA helicase and a nucleotide triphosphatase (NTPase). Due to the multiple functions, NS3 is an attractive target for anti-HCV therapy. Several protease inhibitors were investigated in clinical trials. Monotherapy with protease inhibitors ciluprevir, telaprevir and boceprevir was shown to be effective in lowering the viral load. Clinical evaluation of telaprevir and boceprevir is most advanced. Both protease inhibitors showed a rapid occurrence of drug resistant HCV strains within 2 weeks of therapy indicating that protease monotherapy is not sufficient for treatment of patients with chronic hepatitis C.

The peptidomimetic inhibitor of the NS3/4A serine protease telaprevir showed a 3 log 10 IU/mL decline of HCV RNA during the first 2 days of monotherapy in patients infected with HCV genotype 1 and previous non response to interferon based antiviral treatment. Combination therapy of telaprevir with PEG-interferon alpha-2a and ribavirin was effective in preventing the rapid occurrence of resistance. The combination therapy of PEG-interferon alpha-2a/ribavirin/telaprevir was investigated in the PROVE 1 and 2 studies. Both studies are completed and telaprevir is one of the first STAT-C compound for which sustained virologic response rates have been reported for the combination therapy with PEG-interferon alpha-2a and ribavirin. In both trials triple therapy was given for 12 weeks. The sustained virologic response rates in PROVE 1 and PROVE2 were 67% and 69% in patients treated with PEG- interferon alpha-2a/ribavirin/ telaprevir for 12 weeks followed by PEG- interferon/ribavirin for 36 or 12 weeks, respectively. The sustained virologic response rates in these telaprevir arms were significantly higher compared with the sustained virologic response rates in the standard of care control arms (41% and 46% in PROVE 1 and PROVE2 respectively). Overall, the PROVE-studies confirm that protease inhibitors are able to increase sustained virologic response rates in patients with HCV genotype 1 infection. Furthermore, the PROVE2 study indicates that by addition of telaprevir to standard therapy higher sustained virologic response rates can be achieved with shorter treatment duration. The results of the PROVE2- trial provide evidence that ribavirin has additive antiviral activity to telaprevir and PEG-interferon alpha-2a. Boceprevir, another NS3/4A serine protease inhibitor, binds reversibly to the NS3 protease active site and has potent activity in the Replicon system alone and in combination with interferon alpha-2b. Recently, the final results of the HCV

SPRINT- 1 study assessing safety and efficacy of boceprevir in combination with PEG-interferon alpha-2b (1.5 μg/kg/week) and ribavirin in treatment naive patients with chronic hepatitis C genotype 1 infection were presented. The triple combination arms with a total treatment duration of 48 weeks with or without a 4 weeks PEG- interferon-alpha2b/ribavirin lead-in were associated with significantly higher sustained viro logic response rates than the low dose ribavirin arm and the standard of care control arm (75% and 67% vs 36%> and 38%, respectively).

ITNM-191, SCH 900518, TMC435, BI201335 and MK-7009 are novel NS3/4A protease inhibitors currently in clinical trials. ITMN-191 is a potent HCV NS3/4A protease inhibitor that achieves high liver concentrations following oral

administration. ITNM-191 in combination with PEG-interferon alpha-2a/ribavirin showed a stronger decline of HCV RNA compared with PEG-interferon alpha- 2a/ribavirin standard of care after two weeks of treatment (4.7-5.7 loglO IU/mL vs 2.0 loglO IU/mL). After 2 weeks, 13-57% of patients in the triple therapy arm while no patient in the standard of care arm showed undetectable HCV RNA.

SCH-900518 with and without ritonavir boostering showed robust reductions in HCV RNA levels in both treatment-experienced and naive HCV genotype 1 -infected patients (4.01 loglO IU/mL and 4.5 loglO IU/mL vs 0.09-0.19 loglO IU/mL after 8 days in patients treated with SCH 900518 400 mg twice/day plus PEG-interferon alpha-2a/ribavirin plus ritonavir 100 mg/d and SCH 900518 800 mg thrice/day plus PEG-interferon alpha-2a/ribavirin, respectively, vs. patients receiving PEG- interferon alpha-2a/ribavirin alone). TMC435 administered for 4 weeks in combination with PEG-interferon-alpha2a/ribavirin was well tolerated and demonstrated potent antiviral activity in HCV genotype 1 infected treatment- experienced patients (4.3-5.3 loglO IU/mL in the TMC435 arms vs 1.5 loglO IU/mL in the control arms). BI 201335 was investigated as monotherapy for 14 days and in combination with PEG-interferon alpha-2a/ribavirin for 28 days in experienced patients and showed a median HCV R A decline of 3-4.2 loglO IU/mL in monotherapy and 4.8-5.3 loglO IU/ml in combination therapy. MK-7009 is a noncovalent competitive inhibitor of HCV NS3/4A protease. In treatment naive patients MK-7009 was administered for 28 days in combination with pegylated interferon-alpha/ribavirin. The rapid virologic rate was higher in patients treated with triple therapy than in patients treated with standard of care (68.8-82.4% vs

5.6%). All new compounds were relatively safe and well tolerated in monotherapy as well in combination with standard of care and will be further developed for HCV treatment (Kronenberger, B., Zeuzem, S., Annals of Hepatology 2009; 8: 103). Further protease inhibitors under development for hepatitis treatment include

BMS790052, VBY-376, and TMC-435350 (Thompson, A., et al, J. Hepatology, 50: 184(2009)).

HCV Polymerase inhibitors

Two classes of NS5B polymerase inhibitors, nucleoside and non-nucleoside polymerase inhibitors, have been developed. Nucleoside analogue polymerase inhibitors are converted into triphosphates by cellular kinases and incorporated into the elongating RNA strand as chain terminators. Generally, they show similar efficacy against all HCV genotypes. The mechanisms of action of non-nucleoside polymerase inhibitors are different from that of nucleoside polymerase inhibitors. Therefore, cross resistance between these two classes is unlikely to occur. Several structurally distinct non-nucleoside inhibitors of the HCV RNA-dependent RNA- polymerase NS5B have been reported to date, including benzimidazole,

benzothiadiazine, and disubstituted phenylalanine/ thiophene or dihydropyranone derivatives. They target different sites within the polymerase. Different resistance profiles due to distinct target sites can be expected for the class of non-nucleoside inhibitors. As with protease inhibitors a single mutation may already confer resistance to non-nucleoside polymerase inhibitors. In contrast to nucleoside polymerase inhibitors, a restricted spectrum of activity of non-nucleoside polymerase inhibitors against different HCV genotypes and subtypes has been described. Nucleoside analogues

Valopicitabine was the first nucleoside analogue polymerase inhibitor tested in patients with chronic hepatitis C. Valopicitabine showed antiviral activity in monotherapy (mean HCV-RNA decline 0.15-1.21 loglO IU/mL after 14 days in patients infected with HCV genotype 1 and prior non response to interferon based antiviral treatment) and in combination therapy with interferon alpha (mean HCV- RNA decline 3.75-4.41 loglO IU/mL after 36 weeks in treatment na^'ive patients infected with HCV genotype 1). The nucleoside analogue R1479 (4'-azidocytidine) is a potent inhibitor of NS5Bdependent RNA synthesis and hepatitis C virus replication in cell culture. Rl 626 is a prodrug of Rl 479. Rl 626 was investigated in treatment na^'ive patients with HCV genotype 1 infection in combination with PEG- interferon alpha-2a and ribavirin. After 48 weeks (4 weeks R1626 plus PEG- interferon alpha-2a with or without ribavirin followed by 44 weeks of PEG- interferon alpha-2a plus ribavirin) the viro logic response rates were 52-84% in the R1626 treatment arms and 65% in the control arm with PEG-interferon alpha-

2a/ribavirin. Remarkably, end of treatment response was higher in the ribavirin arm than in the non-ribavirin arm (84% vs 52-66%) indicating that ribavirin has additional effects on treatment antiviral activity to polymerase inhibitors. R1626, a prodrug of a cytidine analog, is a nucleoside inhibitor currently in phase 2 development. When used in combination with PEG-IFN and RBV for 4 weeks, the mean maximal viral load reduction from baseline was 5.2 loglO IU/mL. R7128 is another nucleoside analogue NS5B polymerase inhibitor. Non responders treated with R7128 1,500 mg twice daily showed a mean viral decline of 2.7 loglO IU/mL after 14 days of therapy. R7128 is currently evaluated in combination with PEG- interferon alpha-2a and ribavirin in treatment naive patients with chronic

HCVgenotype 1 infection. The week 4 rapid viro logic response rates in patients treated with PEG-interferon alpha- 2a, ribavirin plus R7128 500 mg or 1,500 mg twice daily were 30% and 85%, respectively, and 10%> in patients treated with PEG- interferon alpha-2a and ribavirin without R7128. R7128 also showed antiviral activity against HCV genotype 2/3 in vitro. Nucleotide analogues

IDX184 is a liver-targeted nucleotide prodrug designed to enhance formation of its active triphosphate in the liver while minimizing systemic exposure of the parent drug and its nucleoside metabolite. Oral administration of IDX184 to HCV-infected chimpanzees resulted in potent antiviral activity (mean HCV-RNA decline after 4 days 1.4 to 3.8 log 10 copies/mL). The antiviral activity was achieved with low systemic levels of the parent drug and its nucleoside metabolite.

Non-nucleoside-analogous inhibitors of viral enzymes

HCV-796 is a non-nucleoside polymerase inhibitor that has demonstrated potent antiviral activity in vitro and in patients with chronic hepatitis C. Monotherapy showed a maximum antiviral effect after 4 days of treatment with a mean HCV RNA reduction of 1.4 loglO IU/ mL. The combination of HCV-796 and PEG-interferon alpha-2b produced a mean viral reduction of 3.3-3.5 loglO IU/mL after 14 days of treatment compared to 1.6 loglO IU/mL with PEG-interferon alpha-2b alone. Filibuvir (PF 00868554) showed in monotherapy of patients with chronic HCV genotype 1 infection a dose-dependent inhibition of viral replication, with maximum reductions in HCV RNA ranging from 0.97 to 2.13 loglO IU/mL. In treatment naive patients with HCV genotype 1 infection triple therapy with PEG-interferon alpha-2a and ribavirin was associated with a rapid viro logic response rate of 60-75% while no patient in the placebo arm achieved a rapid viro logic response.

The non-nucleoside polymerase inhibitors VCH-916, ANA598, BI 207127 and VCH-222 were investigated only in monotherapy so far. VCH-916 showed a maximum HCV-RNA decline ranging between 0.2 and 2.5 loglO IU/mL within 14 days of treatment. ANA598 showed a decline of HCVRNA after 3 days of monotherapy ranging between 0.4 and 3.4 loglO IU/mL.43 ANA598 was combined in vitro with interferon alpha, the HCV NS3/4 protease inhibitor telaprevir, the NS5B nucleoside polymerase inhibitor PSI- 6130, and the TLR7 agonist ANA773, respectively. The in vitro combination studies demonstrated additive to synergistic antiviral effects of ANA598 in combination with other anti-HCV agents having distinct mechanisms of action and non-overlapping resistance profiles. The study indicates that combination therapy may produce a greater viral load reduction and potentially delay the emergence of drug resistance in vivo. BI 207127 monotherapy showed an HCV-RNA decline after 5 days ranging between 0.6 and 3.1 loglO IU/ mL in patients with chronic hepatitis C genotype 1 infection. Similar to ANA598, no increase in HCV RNA levels was observed during short term BI 207127

monotherapy. For VCH-222 only preliminary efficacy results on the first 4 treatment-na^'ive patients with chronic HCV genotype 1 infection treated for 3 days are available showing a decline of HCV-RNA ranging between 3.2 and 4.2 loglO IU/mL. MK-3281, ABT-072 and ABT-333 are additional nonnucleoside polymerase inhibitors in development. Further polymerase inhibitors under development for hepatitis treatment include

R1728, VCH-759, GS9190, BMS-650032, BE-868554, MK-3281 (Thompson, A., et al, J. Hepatology, 50: 184(2009)).

HCV-Entry inhibitors

Chronic hepatitis C is characterized by a high turnover of infected cells and continuous de novo infection of target cells. Due to the vital role of de novo infection in maintenance of HCV infection, blocking of de novo infection is a potential target for antiviral therapy. A target to block de novo infection is the HCV envelope protein E2. A fully humanized monoclonal antibody to a linear epitope of HCV E2 glycoprotein MBL-HCV1 that neutralizes pseudoviruses from multiple HCV genotypes was developed. The antibody was shown to completely neutralize infectious HCV particles in cell culture. Three chimpanzees received a single dose of the Anti-E2 antibody intravenously before challenge with HCV la strain H77. No HCV RNA was detected in the serum of 250 mg/kg dosed chimpanzees through week 20 while the 0 mg/kg and 50 mg/kg dosed chimpanzees both became infected by day 14. These findings indicate that a human monoclonal antibody directed to a conserved epitope of the HCV E2 glycoprotein has the potential to neutralize infectious, replication competent HCV and may prevent infection. The blocking of viral entry into a target cell by an agent may herein be referred to as "entry inhibition" or "envelope protein inhibition", regardless of the mechanism of the blocking action. Agents using other approaches of interfering with a viral target and combinations thereof

One may also consider using combinations of inhibitors of viral targets in the context of the present invention. Thus one may use e.g. an inhibitor of an HCV protease together with inhibitors of HCV polymerase and/or an HCV envelope protein. Of course these agents may be additionally combined with e.g. active agents which support or assist the body's natural response in dealing with viral infections such as interferons, interleukins, steroids, immunomodulators, immunosuppressants and inhibitors of assisted protein folding.

The nucleoside polymerase inhibitor R7128 and the protease inhibitor ITNM-191 showed substantial antiviral activity in patients with chronic hepatitis C. The INFORM- 1 trial is the first trial to investigate the combination of a nucleoside polymerase inhibitor and a protease inhibitor in patients with chronic hepatitis C. Both compounds have different resistance profiles and thus are good candidates for combination therapy. After 14 days of combination therapy (with yet lower doses for both compounds), a decline of HCV-RNA ranging between 2.9 and 5.0 to loglO IU/mL was observed. One patient had undetectable HCV-RNA. No viral rebound was observed.

Other compounds which may be used as pharmaceutically active agents aside from proteasome inhibitors may act both on viral targets and host cell factors. For example, cyclophilins are ubiquitous proteins in human cells that are involved in protein folding. Moreover, cyclophilins participate in HCV replication. It was shown that cyclophilin B binds to the HCV NS5B polymerase and stimulates its RNA-binding activity. Cyclophilin inhibitors show strong antiviral activity in vitro and in vivo. The cyclophilin inhibitor DEBIO-025 showed dual antiviral activity against HCV and HIV in a phase 1 trial with HCV/HIV co-infected patients.

DEBIO-025 was investigated in combination with PEG-interferon alpha-2a and ribavirin in HCV genotype 1 null responders to previous PEG-interferon/ribavirin combination therapy. Triple combination therapy showed a HCV RNA decline after 29 days of 0.88-2.38 loglO IU/mL in the different dosing arms while no antiviral activity was observed in patients receiving DEBIO-025 monotherapy.

NIM811 is another oral non- immunosuppressive cyclophilin inhibitor which has in vitro activity against HCV. In patients with HCV genotype 1 infection with previous relapse to PEG-interferon/ribavirin therapy, NIM811 in combination with PEG- interferon alpha-2a showed a mean HCV RNA decline of 2.78 loglO IU/mL after 14 days compared with a 0.58 loglO decline of HCVRNA in the PEG-interferon alpha- 2a monotherapy arm.

SCY-635 is also a non-immunosuppressive analog of cyclosporine A that exhibits potent suppression of HCV RNA replication in vitro. SCY 635 binds to human cyclophilin A at nanomolar concentrations. Different doses of SCY-635 were investigated in patients infected with HCV genotype 1 and viral load above 100,000 IU/mL. Consistent decreases in plasma HCV RNA were observed in the highest dose group (mean nadir values were 2.20 log 10 IU/mL).

Oral silibinin is widely used for treatment of hepatitis C, but its efficacy is unclear. Intravenous silibinin was investigated in non-responders to prior interferon-based antiviral therapy and showed a significant decline in HCV RNA between 0.55 to 3.02 log 10 IU/mL after 7 days and a further decrease after additional 7 days in

combination with PEG-interferon alpha-2a and ribavirin in the range between 1.63 and 4.85 loglO IU/mL. Next, intravenous silibinin was investigated as rescue treatment for patients with chronic hepatitis C who were still HCV-RNA positive after 24 weeks of treatment with PEG-interferon alpha-2a/ribavirin. After 24 weeks of treatment with standard of care the patients received additionally 20 mg/kg/d silibinin intravenously for 15 days. Thereafter PEG-interferon/ribavirin was continued. After 15 days of intravenous silibinin therapy HCV-RNA decreased in all patients and 7 out of 9 patients achieved undetectable HCV RNA plasma levels. After the end of silibinin administration patients were followed for at least 12 weeks. In one patient HCV-RNA increased to 100 IU/mL, and a second course of intravenous silibinin for 15 days was given. HCV-RNA became negative again and remained negative so far.

Nitazoxanide is a thiazolide anti- infective with activity against a number of protozoa, bacteria, and viruses. It is FDA approved for treatment of Cryptosporidium and giardia. Nitazoxanide inhibits replication of hepatitis C virus, hepatitis B virus, and rotavirus in vitro. Based on its broad antiviral activity, the mechanism of action is likely through cellular processes rather than specific anti- viral targets. Rossignol et al. recently reported that the use of nitazoxanide in combination with PEG-interferon alpha-2a with or without ribavirin among treatment-naive hepatitis C patients infected with genotype 4 significantly improved viral response rates compared to the standard of care (PEG-interferon alpha-2a plus ribavirin). The sustained virologic response rates were 79% and 64% in patients treated with PEG-interferon alpha- 2a/ribavirin/nitazoxanide and PEG-interferon alpha-2a/nitazoxanide, respectively, versus 45% in patients treated with PEG-interferon alpha-2a/ribavirin. Other active ingredients under development for HCV therapy and not falling in any of the above categories are A-831, KPE02001003, TCM700C, PYN-17, BIT225, JTK-652, BMS-791325. A patient which does not respond to viral hepatitis treatment, and in particular to HCV treatment, may be designated as a ,,ηοη-responder" or "therapy resistant" patient. A response is understood to refer to a decrease of the virus load below the detection limit for at least 6 months after standard therapy has ceased ("sustained virological response", SVR). The therapy usually is a combination of pegylated interferon alpha and ribavirin. Non-responders are patients for which no decrease of virus load by a factor of least 2 log steps is observed during 24 weeks or for which up to week 24, HCV-RNA is still detectable during therapy. The terms "therapy" and "treatment" can be used interchangeably.

A relapse refers to a complete virological response up until the 24th week of treatment at the latest. However, after the standard therapy has ceased, a renewed increase of virus load is observed (therapy refractory). Such patients are designated as "relapser" or "therapy refractory" patients. The terms "therapy" and "treatment" can be used interchangeably.

A patient that is "resistant or refractory to therapy with at least one pharmaceutically active agent in use against viral hepatitis infection" is a patient that has undergone therapy with at least one pharmaceutically active agent in use against viral hepatitis infection, and preferably has undergone Standard of Care (SOC) therapy for his condition, but was either found resistant to such therapy, or who relapsed after such therapy. The at least one pharmaceutically active agent in use against viral hepatitis infection to be used in the inventive methods, kits etc. for the treatment of such a patient may be the same as the one or several pharmaceutically active agents in use against viral hepatitis infection which he was found resistant or refractory to, or it, or they, may be different. It should be noted here, that where it is referred to a "first different pharmaceutically active agent in use against viral hepatitis" or a "second different pharmaceutically active agent in use against viral hepatitis", these agents are meant to differ from each other and from the proteasome inhibitor, not from the agent that a patient has shown to be resistant or refractory to.

If patients are mentioned in the context of the present invention, this term preferably relates to patients suffering from a hepatitis viral infection which are selected according to inclusion and exclusion criteria in accordance with the guidelines of the International Conference of Harmonization (ICH) as they are practiced e.g. by the Food and Drug Administration (FDA) or the European Medicines Agency (EMEA) for clinical trials being concerned with hepatitis viral infections. Patients may thus be of Caucasian origin, of average weight, male or female and may be 20 to 60 years of age.

Common viral hepatitis infection treatment schedules include:

Hepatitis A: dietary and lifestyle adjustments (no alcohol, reduced lipid intake) until the infection is cleared; these adjustments are usually considered to have a significant negative impact on quality of life by affected patients;

• Acute Hepatitis B: symptomatic treatment (bed rest, reduction of intake of agents causing hepatic stress), until symptoms abate; in severe cases, recovery is assisted by lamivudine treatment (typically 2 mg/kg body weight, twice daily);

• Inactive chronic Hepatitis B: lifestyle adjustment, continuous monitoring;

Active chronic Hepatitis B: administration of interferon-cc (typically 5-6 Mio. Units 3 times per week), Peg-Interferon alpha-2a (typically 180 μg once per week) Peg-Interferon alpha-2b (typically 50-100 μg once per week), lamivudin (typically 100 mg daily), entecavir (typically 0,5 mg to 1 mg once daily), telbivudin (typically 600 mg once daily), or adefovir (typically 10 mg to 30 mg once daily), for at least several months and up to several years; combination therapies do apparently not improve primary outcome, but are employed if and when resistance emerges;

Hepatitis C: The standard of care (SOC) for patients with chronic hepatitis C is pegylated interferon alpha in combination with ribavirin (herein also referred to as "standard therapy" in the context of Hepatitis C treatment). Two pegylated interferons, alpha-2a (40 kD) and -2b (12 kD), are approved. The aim of antiviral therapy is the sustained elimination of the hepatitis C virus. The HCV genotype is the most important predictive factor for treatment response of patients with chronic hepatitis C and has become an important decision criterion for treatment duration and ribavirin dosage. The SOC treatment duration is 48 weeks and 24 weeks for patients infected with HCV genotype 1 and 2/3, respectively. The SOC ribavirin dosage is 1,000- 1,200 mg and 800 mg for patients infected with HCV genotype 1 and 2/3, respectively. The initial virologic response of patients with chronic hepatitis C shows large individual variation and can be classified into rapid virologic response (HCV undetectable after 4 weeks of therapy), complete early virologic response (HCV undetectable after 12 weeks of therapy), partial early virologic response (> 2 loglO decline of HCV RNA after 12 weeks of treatment but still HCV RNA positive), slow virologic response (> 2 log 10 decline of HCV RNA after 12 weeks of treatment but still HCV RNA positive followed by undetectable HCV RNA by week 24) and non-response (detectable HCV RNA 24 weeks after start of antiviral treatment). The faster a patient develops undetectable HCV RNA, the higher is his/her probability to achieve a sustained virologic response. Based on the rapid virologic response criterion, individualization of treatment duration is possible without reduction of the overall sustained viro logic response rate;

Patients infected with HCV genotype 1 who start with a low baseline viral load (< 600.000 IU/mL) and who achieve a rapid virologic response were shown to have favorable sustained virologic response rates after 24 weeks of antiviral treatment indicating that a shorter treatment duration can be considered in this group of patients. The possibility of shorter treatment duration was also investigated in patients with HCV genotype 2/3 infection. Smaller trials showed that a shorter treatment duration of 12-14 weeks is equally effective as the standard treatment duration in patients infected with HCV genotype 2/3 who achieve a rapid virologic response after 4 weeks of therapy. However, the large ACCELERATE trial comparing 16 versus 24 weeks of treatment in patients with HCV genotype 2/3 infection showed that a shorter treatment duration of 16 weeks results in lower sustained virologic response rates compared with the standard treatment duration. In the ACCELERATE trial, a shorter course of therapy over 16 weeks has been shown to be as effective as a 24 week course in those patients with genotype 2/3 infection who have a baseline viral load < 400.000 IU/mL and rapid virologic response. In patients with genotype (2 and) 3 infection without a rapid virologic response (< 50 IU/mL) at week 4, a longer than 24 weeks treatment duration may be necessary to optimize sustained virologic response rates.

Patients infected with HCV genotype 1 and slow virologic response have a high risk to relapse after 48 weeks of treatment with PEG-interferon and ribavirin. An approach to reduce the relapse rates is treatment extension to 72 weeks. In a German multicenter study, patients infected with HCV genotype 1 were randomized for treatment with PEG-interferon alpha-2a/ribavirin 800 mg for 48 weeks and 72 weeks, respectively. In this study, the overall sustained viro logic response rate was not superior in patients infected with genotype 1 treated for 72 weeks with PEG-interferon alpha-2a/ribavirin 800 mg compared with patients treated for 48 weeks. However, the subgroup analysis of patients infected with HCV genotype 1 and a slow viro logic response showed a significantly lower relapse rate in patients treated for 72 weeks compared with patients treated for 48 weeks indicating that a subgroup of patients may benefit from extended treatment duration. The SUCCESS study was the first prospective study to compare 48 weeks of treatment with 72 weeks of treatment in slow responders. In this trial slow responders were randomized at week 36 of treatment to receive PEG-interferon alpha-2b (1.5 μg/kg/week) plus weight-based dosed ribavirin (800-1400 mg/day) for a total of 48 or 72 weeks. Of the 1,427 patients included in the trial 157 (11%) were slow responders. In the intent to- treat analysis, sustained viro logic rates were not different between the two groups (43% and 48% in the 48 and the 72 week treatment arms, respectively). Patients, however, who showed 80/80/80 compliance had sustained viro logic response rates of 44% and 57% in the 48 weeks and 72 weeks treatment arms, respectively. Overall, these studies indicate that extended treatment duration can be considered in slow responders, however, adherence to treatment is crucially important.

The treatment options for patients with chronic hepatitis C and non-response to antiviral treatment are sparse. It was hypothesized that long term maintenance therapy with interferon may reduce progression to liver cirrhosis and its complications. In the HALT-C trial 1,050 patients with prior non response to PEG-interferon alpha/ribavirin and advanced fibrosis/cirrhosis were randomized for treatment with low dose PEG-interferon alpha-2a 90 μg/ week or no treatment for 3.5 years. The primary end point was progression of liver disease defined as liver related death, hepatocellular carcinoma, hepatic decompensation or increase of the ISHA fibrosis score of 2 or more points. The level of aminotransferases, HCV-R A and necroinflammatory scores decreased significantly, however, there was no difference between the groups in the rate of any primary outcome. Similar results were observed in the CoPilotlO and the EPIC311 trials investigating long term PEG-interferon alpha-2b 0.5 mg/kg vs colchicine for 4 years or PEG-interferon alpha-2b 0.5 mg/kg vs no treatment in patients for 5 years with prior non-response to PEG- interferon/ribavirin, respectively.

Hepatitis D: as HDV virions are incapable of replication absent

infection, therapy is as given above for HBV;

Hepatitis E: symptomatic therapy until symptoms abate;

Hepatitis G: GB virus C (GBV-C), formerly known as Hepatitis G virus (HGV), is a virus in the Flaviviridae family which has not yet been assigned to a genus. Hepatitis G virus and GB virus C (GBV-C) are RNA viruses that were independently identified in 1995, and were subsequently found to be two isolates of the same virus. Although GBV-C was initially thought to be associated with chronic hepatitis, extensive investigation failed to identify any association between this virus and any clinical illness. So far, no therapy has been approved;

Hepatitis as a complication of Epstein Barr Virus infection: Infectious mononucleosis, also known as EBV infectious mononucleosis, Pfeiffer's disease or Filatov's disease, and colloquially as kissing disease, mono (in North America) and glandular fever (in other English-speaking countries) is an infectious, very widespread viral disease caused by the Epstein-Barr virus (EBV); hepatitis occurs as a rare (<5%) complication of EBV infectious mononucleosis. Infectious mononucleosis is generally self-limiting and only symptomatic and/or supportive treatments are used

(acetaminophen/paracetamol or non-steroidal anti- inflammatory drugs may be used to reduce fever and pain; Prednisone is commonly used as an antiinflammatory to reduce symptoms of pharyngeal pain, odynophagia, or enlarged tonsils). There is little evidence to support the use of aciclovir, although it may reduce initial viral shedding. However, the antiviral drug valacyclovir has recently been shown to lower or eliminate the presence of the Epstein-Barr virus in patients afflicted with acute mononucleosis, leading to a significant decrease in the severity of symptoms;

Hepatitis as a complication of Cytomegalovirus infection: Cytomegalovirus is a herpes viral genus of the Herpesviruses group; in humans it is commonly known as HCMV or Human Herpesvirus 5 (HHV-5). Most healthy people who are infected by HCMV after birth have no symptoms. Some of them develop an infectious mononucleosis/glandular fever- like syndrome, with prolonged fever, and a mild hepatitis. A sore throat is common. After infection, the virus remains latent in the body for the rest of the person's life. Overt disease rarely occurs unless immunity is suppressed either by drugs, infection or old-age. Initial HCMV infection, which often is asymptomatic is followed by a prolonged, inapparent infection during which the virus resides in cells without causing detectable damage or clinical illness. However, in patients with a depressed immune system, CMV-related disease may be much more aggressive. CMV hepatitis may cause fulminant liver failure in such patients. Cytomegalovirus Immune Globulin Intravenous (Human) (CMV- IGIV), is an immunoglobulin G (IgG) containing a standardized amount of antibody to Cytomegalovirus (CMV). It may be used for the prophylaxis of cytomegalovirus disease associated with transplantation of kidney, lung, liver, pancreas, and heart. Ganciclovir treatment is used for patients with depressed immunity who have either sight-related or life-threatening illnesses. Valganciclovir may be applied effectively by orally administration, yet its therapeutic efficacy is frequently compromised by the emergence of drug-resistant virus isolates. Foscarnet or cidofovir are only given to patients with CMV resistant to ganciclovir, as, e.g., foscarnet often causes

nephrotoxicity;

Hepatitis as a complication of yellow fever virus infection: Yellow fever virus is a 40 to 50 nm enveloped R A virus with positive sense of the Flaviviridae family. The virus is transmitted by the bite of mosquitos. A safe and efficient vaccine exists, yet official estimations of the WHO still amount to 200,000 cases of disease and 30,000 deaths a year. The disease presents itself in most cases with fever, nausea and pain and it disappears after several days. In some patients, a toxic phase follows, in which liver damage with jaundice (giving the name of the disease) can occur and lead to death.

Currently, there is no causative cure for yellow fever. Hospitalization is advisable and intensive care may be necessary because of rapid deterioration in some cases. Different methods for acute treatment of the disease have been shown to of limited success; passive immunisation after emergence of symptoms is probably without effect. Ribavirin and other antiviral drugs as well as treatment with interferons do not have a positive effect in patients. A symptomatic treatment includes rehydration and pain relief with drugs like paracetamol; • Hepatitis as a complication of mumps virus and rubella virus infection: Rare incidences of acute or fulminant hepatitis have been reported in conjunction with mumps virus and rubella virus (Matsunaga, T., et al, Journal of the Japan Pediatric Society 2003, 107: 1645; Masao, A., et al, Journal of

Gastroenterology 1995, 30: 539); no treatment exists or is under

development.

HIV treatment The main focus of retroviral therapy currently relates to the treatment of HIV- infected individuals. Other currently known retrovirus infections in humans are rare, e.g. infections with HTLV-I causing blood cancer. The infection with HTLV-I is believed to occur from mother to child. HTLV-I infected individuals are frequented treated with anti-cancer drugs, but the treatment with the nucleoside analogue reverse transcriptase inhibitor azidothymide and cyclophosphamide, hydroxydaunorubicin (doxorubicin), Oncovin (vincristine), and prednisone/prednisolone has also been attempted (Taylor GP, Matsuoka M (September 2005), Oncogene 24 (39): 6047-57).

In HIV-infections, current treatment frequently consists of highly active

antiretro viral therapy, or HAART. This has been highly beneficial to many HIV- infected individuals since its introduction in 1996 when the protease inhibitor-based HAART initially became available (Palella FJ Jr, et al, (1998), N. Engl. J. Med 338 (13): 853-860). Current optimal HAART options consist of combinations consisting of at least three drugs (herein also referred to as anti-retroviral compounds) belonging to at least two types, or classes, of antiretroviral agents. Typical regimens consist of two nucleoside analogue reverse transcriptase inhibitors (NARTIs or NRTIs) plus either a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor ( NRTI). Standard goals of HAART include improvement in the patient's quality of life, reduction in complications, and reduction of HIV viremia below the limit of detection, but it does not cure the patient of HIV nor does it prevent the return, once treatment is stopped, of high blood levels of HIV, often HAART resistant.

For some patients, which can be more than fifty percent of patients, HAART achieves far less than optimal results, due to medication intolerance/side effects, prior ineffective antiretroviral therapy and infection with a drug-resistant strain of HIV. Non-adherence and non-persistence with therapy are the major reasons why some people do not benefit from HAART. The reasons for non-adherence and non- persistence are varied and include poor access to medical care, inadequate social supports, psychiatric disease and drug abuse. HAART regimens can also be complex and thus hard to follow, with large numbers of pills taken frequently. A huge number of anti-retrovirus compounds has been authorized in various countries or is clinically investigated. These compounds may be broadly classified by the phase of the retrovirus life-cycle that is inhibited:

Nucleoside and nucleotide reverse transcriptase inhibitors (NRTI) inhibit reverse transcription by being incorporated into the newly synthesized viral DNA and preventing its further elongation.

Non-nucleoside reverse transcriptase inhibitors (NNRTI) inhibit reverse transcriptase directly by binding to the enzyme and interfering with its function. Protease inhibitors target viral assembly by inhibiting the activity of protease, an enzyme used by HIV to cleave nascent proteins for final assembly of new virions. Integrase inhibitors inhibit the enzyme integrase, which is responsible for integration of viral DNA into the DNA of the infected cell. There are several integrase inhibitors currently under clinical trial, and raltegravir became the first to receive FDA approval in October 2007.

Entry inhibitors (or fusion inhibitors) interfere with binding, fusion and entry of HIV- 1 to the host cell by blocking one of several targets. Maraviroc and enfuvirtide are the two currently available agents in this class.

Maturation inhibitors inhibit the last step in gag processing in which the viral capsid polyprotein is cleaved, thereby blocking the conversion of the polyprotein into the mature capsid protein (p24). Because these viral particles have a defective core, the virions released consist mainly of non- infectious particles. There are no drugs in this class currently available, though two are under investigation, bevirimat and Vivecon.

AV-HALTs (Antiviral HyperActivation Limiting Therapeutics or

'virostatics') combine immunomodulating and antiviral properties to inhibit a specific antiviral target while also limiting the hyper-elevated state of immune system activation driving disease progression.

Broad spectrum inhibitors. Some natural antivirals, such as extracts from certain species of mushrooms like Shiitake and Oyster mushrooms, may contain multiple pharmacologically active compounds, which inhibit the virus at various different stages in its life cycle. Researchers have also isolated a protease inhibitor from the Shiitake mushroom.

Anti-retroviral compounds to be combined with (a) proteasome inhibitor(s) according to the invention include, but are not limited to, HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhbitors, inhibitors of HIV TAT, budding/maturation inhibitors, entry inhibitors, including fusion inhibitors, inhibitors of the CD4 receptor, inhibitors of the CCR5 co-receptor and inhibitors of the CXCR4 coreceptor.

Reverse transcriptase inhibitors include, but are not limited to BCH-189, AzdU, carbovir, ddA, d4C, d4T (stavudine), 3TC (lamivudine), DP-AZT, FLT

(fluorothymidine), BCH-189, 5-halo-3'-thia-dideoxycytidine, PMEA, bis- POMPMEA, Retrovir (AZT), zidovudine (AZT), MSA-300, trovirdine, R82193, L- 697,661, R82150, U-87201E, amdoxovir, alovudine, cidovir, ribavarin, viramidine, vidarabine, cytarabine, idoxuridine, trifluridine, valopiticabine, BIT225, R1728, BE- 868554, ITMN-5489, SPD754, capravirine, emivirine, calanolide A, GW5634, BMS- 56190 (DPC-083), DPC-961, MTV- 150, MTV- 160, MIV-210, MIV-310, MIV-410, alovudine (FLT), zalcitabine (ddC), didanosine (ddl), abacavir, ABC, emtricitabine (FTC), racivir (racemic FTC), adefovir (ADV), entacavir (BMS 30 200475), alovudine (FLT), tenofovir (TNF), amdoxavir (DAPD), D-d4FC (DPC-817), -dOTC (SPD754), elvucitabine (ACH-126443), BCH 10681, SPD-756, D-FDOC, GS7340, INK-20 (thioether phospholipid AZT), 2'3'-dideoxy-3'-fluoroguanosine (FLG) & its prodrugs such as MIV-210 and reverset (RVT, D-D4FC, DPC-817), delavirdine, efavirenz (DMP-266), (+)calanolide A and B, capravirine (AG1549f S-l 153), GW- 695634 (GW-8248), MV026048 (R-1495), NV-05 2 2, R-278474, RS-1588, UC-781, YM215389, RDEA900, CMX157, Festinavir, APD ([-]beta-d-2-Aminopurine Dioxolane), DXG (2'-Deoxyguanosine analog), Fosalvudine Tidoxil / HDP 99.0003, KP-1461, KP-1212, 3-fluoro-2,3-Dideoxyguanosine (FLG), Amdoxovir (DAPD), Apricitabine, Hydroxyurea, VS411, Stavudine, Aciclovir / Acyclovir, Lamivir, Lamivir-S, Truvada, Zidovudine Transdrug, Dexelvucitabine, RDEA640, IDX-989, RDEA806, IDX899, UK-453,061, Dapivirine (TMC-120), Etravirine (TMC125, R- 165335), Rilpivirine (TMC-278), Nevirapine (BI-RG-587), Cipla-Nevirapine, RDEA427, Non-Nucleoside Reverse Transcriptase Inhibitor NUMERATE, oxetanocin, oxetanocin-G, BV ara-U, penciclovir, L-697,639 and the like.

Retroviral protease inhibitors include, but not limited to, ritonavir, lopinavir, saquinavir, Saquinavir Transdrug, amprenavir (VX-478), fosamprenavir, nelfmavir (AG 1343), tipranavir, indinavir, atazanavir, TMC-126, Darunavir (TMC-114), mozenavir (DMP-450), JE-2147 (AG1776), L-756423, RO0334649, KNI-272, DPC- 681, DPC-684, GW640385X, SC-52151, BMS 186,318, SC-55389a, BILA 1096 BS, DMP-323, KNI-227, HEPT compounds, PA-457, KPC-2, AG-1859, GS224338, R- 944, VX-385, GW640385, P-1946, CTP-518, Doxovir-M, DG17, DG35, (Nar DG35), Amprenavir, DG43 (Nar DG43), PPL- 100. and the like.

HIV integrase inhibitors include, but are not limited to, S-1360, zintevir (AR-177), L-870812, L-870810, L-c-2507 and S(RSC)-1838 ), TG-10, INH001, S-247303, S- 265744, S-349572, Raltegravir, Elvitegravir, HIV Integrase Inhibitors by

AMBRILIA (e.g. AMBRILIA Compound 1, AMBRILIA Compound 2, AMBRILIA Compound 3), HIV Integrase Inhibitors by AVEXA, HIV Integrase Inhibitors by BIOALLIANCE (e.g. derivatives of styrylquino lines), HIV Integrase Inhibitors by CRITICAL OUTCOME, HIV Integrase Inhibitors by VIROCHEM, and the like

TAT inhibitors include, for example, RO-24-7429 and the like. HIV budding/maturation inhibitors include, but are not limited to, PA-457 and the like.

HIV entry/fusion inhibitors include, but are not limited to, AMD-070 (AMD 11070), BlockAide/CR, BMS 806 (BMS-378806), Enfurvirtide (T-20, R698, Fuzeon), KRH1636, ONO-4128 (GW-873140, AK-602, E-913), PRO-140 , PRO-542, Schering C (SCH-C), SCH-D (SCH-417690), T-1249 (R724), TAK-220, TNX-355 and UK 427,857, PRO 2000, AMD-3100, Peptide T, FP21399 and the like. CCR5 antagonists include, but are not limited to: TAK-779, Vicriviroc (SCH- 417690), Aplaviroc (GW871340), Maraviroc, INCB9471, PF-232798, and

Compounds 1 and 3-96 as disclosed in Yang, H., Expert opinion on therapeutic patents 2010, 20:325 Other anti-retroviral compounds to be administered in combination with at least one proteasome inhibitor according to the present invention may include AL-721, polymannoacetate, HPA-23, trisodium phosphono formate, foscarnet, eflonithine, Reticulose, UA001, cylobut-G, cyclobut-A, ara-M, BW882C87, BW256U87, L- 693,989, FIAC, HOE-602, ganciclor, rCD4/CD4-IgG, CD4-PE40, butyl-DNJ, oxamyristic acid, and dextran sulfate..

A patient which does not respond to retroviral treatment, and in particular to HIV treatment, may be designated as a ,,ηοη-responder" or "therapy resistant" patient. Resistances may not only be determined in clinical trials but also in cell culture tests in vitro. For example, the antiviral activity of a drug in cell culture systems may be determined using the inhibition of replication as a read-out parameter. The concentration of a compound under investigation to inhibit virus replication by 50 percent (EC₅₀ for cell-based assays; IC₅₀ for biochemical or subcellular assays) should be determined. A large number of tools to determine this value are known to the person skilled in the art. A well-characterized wild-type HIV laboratory strain should serve as a reference standard. Other parameters that may be used to determine the efficiency of a compound on HIV replication as a measure for the development of resistances are for example cytotoxicity and therapeutic indices, protein binding assays to human serum proteins, genotypic and phenotypic assays using, for example, nucleic acid sequencing methodology to determine mutations that have evolved in viruses under investigation, and standard virus assays, such as p24, viral RNA, RT assay, MTT cytotoxic assay and reporter gene expression assays. See, for example, Walter, H.,

Anti-retro viral compounds that are currently used in the treatment of HIV are, e.g., the multi-class combination products comprising efavirenz, emtricitabine and tenovir disoproxil fumarate; Nucleoside Reverse Transcriptase Inhibitors (NRTIs) such as lamivudine, zidovudine, emtricitabine, lamivudine, abacavir, zalcitabine,

dideoxycytidine, azidothymidine, enteric coated didanosine, stavudine, abacavir sulfate; Nonnucleoside Reverse Transcriptase Inhibitors (NNRTIs) such as etravirine, delaviridine, efavirenz, nevirapine; Protease Inhibitors such as

amprenavir, tipranavir, indinavir, saquinavir, saquinavir mesylate, lopinavir, ritonavir, fosamprenavir calcium, darunavir, atazanavir sulfate, nelfmavir mesylate; Fusion Inhibitors such as enfuvirtide; Entry inhibitors/CCR5 co-receptor antagonists such as maraviroc; HIV integrase strand transfer inhibitors such as raltegravir. Other drugs that are currently tested include monoclonal antibodies and fragments thereof, etc. Any of these pharmaceutical agents may be used in combination with the semicarbazone proteasome inhibitors described above. A preferred combination comprises methods, kits, compositions, uses etc., as described above which involve S-2209 or pharmaceutically acceptable salts or analogues thereof. Additionally, PS- 341 or related molecules as proteasome inhibitor(s) and darunavir or related active agents as a further anti-retroviral compound may be used.

HIV resistances are mainly due to its high rate of replication (often of a magnitude of 10⁹ to 10¹⁰ virions per person per day) and error-prone polymerase. Therefore, HIV can easily develop mutations that alter susceptibility to anti-retroviral compounds. As a result, the emergence of resistance to one or more anti-retroviral compound is one reason for therapeutic failure in the treatment of HIV. In addition, the emergence of resistance to one anti-retroviral compound sometimes confers a reduction in or a loss of susceptibility to other or all anti-retroviral compounds of the same class (U.S. Dept. of Health and Human Services, FDA, CDER, 2007, in "Guidance for Industry - Role of HIV resistance testing in antiretro viral drug development). The present invention provides a new strategy to fight against retroviral, e.g. HIV-infections. In one embodiment the therapy presented herein is particularly well-suited to fight against the development of resistances, or may be used in patients that have already developed resistances to one or more of the above described anti-retroviral compound.

The treatments may be initiated in patients which received therapy for retroviral infections and in particular for HIV infections such as HAART and which have been classified as non responders or refractory, or in patients which have recently received the diagnosis of a retrovirus infection, i.e. before the treatment with any

conventionally anti-retroviral compounds. In the latter case the treatment may prevent or delay the onset of disease/symptoms associated with the retroviral infection, e.g. AIDS.

In certain embodiments, the patient has never received therapy or prophylaxis for a retrovirus infection, more particularly an HIV infection. In further embodiments, the patient has previously received therapy or prophylaxis for a retroviral infection, or more particularly an HIV infection. For instance, in certain embodiments, the patient has not responded to treatment for a retroviral disease, or more particularly an HIV infection. In certain embodiments, the patient can be a patient that received therapy but continued to suffer from viral infection or one or more symptoms thereof. In certain embodiments, the patient can be a patient that received therapy but failed to achieve a sustained viro logic response. In certain embodiments, the patient has received therapy for a virally induced disease, or more particularly a disease induced by a HIV infection, but has failed to show, for example, a 2 log 10 (2 orders of magnitude, 100-fold) decline in viral R A levels after 12 weeks of therapy.

In certain embodiments, the patient is a patient that discontinued therapy for a retroviral disease, or more particularly conditions induced by HIV infection, because of one or more adverse events associated with the therapy. In certain embodiments, the patient is a patient where current therapy is not indicated.

The kits, methods and compositions provided herein may reduce or eliminate the need for exposing patients to the agents of current therapy, either by reducing the dose needed or reducing the required time of exposure to these agents, or by facilitating the replacement of certain agents of current therapy.

Accordingly, provided are methods of treating or preventing a retroviral disease, or more particularly an HIV-related disease. In one embodiment, provided are methods of treating or preventing a retroviral disease, more particularly an HIV-related disease. Further provided are methods of treating a retroviral infection, or AIDS induced by an HIV infection, in patients where a neuropsychiatric event, such as depression, or risk of such indicates a different treatment of the current HIV therapy. In further embodiments, the patient has received treatment for a retroviral disease, or more particularly AIDS or related conditions induced by HIV infection, and discontinued that therapy prior to administration of a method provided herein. In further embodiments, the patient has received therapy and continues to receive that therapy along with administration of a method provided herein.

In one embodiment, the proteasome inhibitors described herein may be used in the treatment or prevention of HIV infections, particularly in patients that have developed resistances to conventional therapy such as HAART. In the treatment forms disclosed herein, the protease inhibitor may be used before or after or simultaneously with at least one, or alternatively before, following or simultaneously with more than one conventionally used compounds that are administered to patients infected with or suspected of being infected, with retroviruses, e.g. HIV. In another embodiment, the present invention thus relates to the use of at least one semicarbazone proteasome inhibitor alone or together with at least one different pharmaceutically active agent in use against retroviral infections in the manufacture of a medicament for treating patients which do not respond or are refractory to treatment with a pharmaceutically active agent in use against retroviral infections (e.g. HIV infections) alone.

Yet another embodiment of the present invention relates to the use of at least one semicarbazone proteasome inhibitor alone or together with at least one first and at least one second pharmaceutically active agent in use against retroviral infections in the manufacture of a medicament for treating patients which do not respond or are refractory to treatment with a pharmaceutically active agent in use against retroviral infections alone. In these embodiments it can be preferred to use specific semicarbazone proteasome inhibitors such as S-2209 or pharmaceutically acceptable salts or analogs together with structurally different proteasome inhibitors such as PS-519, PS-341

(Bortezomib) and PS-273.

If only one additional different pharmaceutical agent is present, this may be selected from the above mentioned anti-retroviral compounds used in the treatment of retroviral, e.g. HIV-infection.

With the above mentioned pharmaceutical compositions, kits, uses and methods, in particular where they relate to a combination of a proteasome inhibitor, and another anti-retroviral compound it is possible to reduce the virus load or to even completely remove the virus.

The present invention in one embodiment relates to a kit of pharmaceutical compositions comprising:

a) at least one first pharmaceutical composition comprising at least one semicarbazone proteasome inhibitor, and optionally

b) at least one further pharmaceutical composition comprising at least one pharmaceutically active agent in use against retroviral infection and/or viral hepatitis infections; and

c) optionally instructions for use and/or devices for administration of the pharmaceutical compositions, such as syringes, needles, etc.

In these embodiments it can be preferred to use specific semicarbazone proteasome inhibitors such as S-2209, and in addition other proteasome inhibitors such as PS- 519, PS-341 (Bortezomib) and PS-273, and/or nucleoside or nucleotide analoga as further pharmaceutically active agent. Therein, the pharmaceutically active agent in use against viral hepatitis infections may specifically be an interferon preparation chosen from the list of: albinterferon, PEG-IFN alpha-2a, alpha-2b or lambda, Locteron, Omega IFN, Medusa IFN, DA- 3021, EMZ702, Iniradure, IL-29, Amarillo, Soluferon and Belerofon. The interferon may also be replaced by, or assisted by, additional administration of, for example, an interleukin, preferably IL-1, IL-2, IL-6, IL-8, IL-12, IL-15, IL-18, IL-21, and IL-2, a steroid, or an immunomodulator, e.g. Thymosin, ME3738, SCV-07, Alinia,

Oglufavide, IPH-1101, CYT 107, or EGS-21, ciclosporin and derivatives thereof. The pharmaceutically active agent in use against viral hepatitis infections may be one that, for example, stimulates or assists the body's own functions, and specifically the body's natural defenses, more specifically against viruses.

In certain emboiments, where the pharmaceutically active agent in use against viral hepatitis infections is one that, stimulates or assists the body's own functions, and specifically the body's natural defenses, it ma ybe advantageous to provide an additional pharmaceutically active agent in use against viral hepatitis infections, and specifically one which interferes with the function of a viral target. Such additional pharmaceutically active agent may be one of, or any combination of the elements of the following list: telaprevir, boceprevir, ITNM-191, SCH 900518, TMC435, BI201335,MK-7009, lamivudine, cidovir, ribavarin, viramidine, didanosine, vidarabine, cytarabine, emtricitabine, zalcitabine, abacavir, stavudine, zidovudine, idoxuridine, trifhiridine, valopiticabine, R1626, R7128, IDX184, HCV-796, Filibuvir (PF 00868554), VCH-916, ANA598, BI 207127,VCH-222 PSI- 6130, ANA773, MK-3281, ABT-072, ABT-333, R1728, VCH-759, GS9190, BMS-650032, BE- 868554, thymosin alpha 1, a vaccine, glycyrrhizin, ciclosporin and derivatives thereof, e.g. SCY-635, DEBIO-025, NIM811, Silibinin, Nitazoxanide, A-831, KPE02001003, TCM700C, PYN-17, BIT225, JTK-652, BMS-791325, amantadine, rimantadine and derivatives thereof, and azathoprine.

The PROVE2 and PROVE3 studies referenced above aptly demonstrated that a combination of, for example, standard therapy including interferon + ribavirin with, for example, telaprevir provides an additional benefit over standard therapy or interferon + telaprevir only. The rapid development of resistances against single therapeutic compounds in hepatitis-causing viruses and/or or HIV necessitates a multi-pronged approach if elimination of the pathologic agent is to be achieved.

Therefore, the inventive pharmaceutical composition preferably comprises at least one semicarbazone proteasome inhibitor and optionally at least one further active agent against HIV and/or viral hepatitis infections, particularly at least two, three or even at least 4 active agents in use against HIV infections or viral hepatitis infections. The semicarbazone proteasome inhibitor, is preferably S-2209 or an analog thereof.

The pharmaceutical compositions and kits may be used for treating patients suffering from retroviral, particularly HIV infections and/or viral hepatitis, in particular due to an infection with HCV.

One advantage of the kits in accordance with the present invention is that they contain the pharmaceutical compositions in separate form, e.g. as different solutions, tablets etc. This may allow for a timely ordered, i.e. subsequent administration of the separate pharmaceutical compositions which can be important when treating patients suffering e.g from HCV infections and/or HIV infections. The kits in accordance with the present invention may comprise instructions in paper or electronic form advising the user to first administer the semicarbazone proteasome inhibitor or a pharmaceutical composition comprising the same and to administer the additional active agent or a pharmaceutical composition comprising the same concommitantly, subsequently or before.

The instructions may further instruct the user to wait a specified time period between administering one active agent or pharmaceutical composition and administering one or more additional active agent(s) or pharmaceutical composition(s).

The instructions may for example advise to wait for about 2 to 4 days, about 4 to 6 days, about 1 week, about 1 to 2 weeks, about 2 to 3 weeks, about 3 to 4 weeks, or more than 4 weeks after treatment with the semicarbazone proteasome inhibitor has ended and before commencing treatment with at least one different pharmaceutical composition. The instructions may additionally advise that treatment with the semicarbazone proteasome inhibitor may on a three to four daily basis such as e.g. on day 1, 4, 8 and 11, or on day 1, 4, 7, 10 or on day 1, 5, 9 and 13 and the like.

Instructions for treatment with one or more pharmaceutical composition(s) in addition to the semicarbarzone proteasome inhibitor may follow the dosage regimen developed for these compositions. In case of a treatment of HCV infection, an additional use of interferon such as PEG-interferon-alpha and/or a nucleoside analogon such as ribavirin may be undertaken on a one to two weekly basis for 32 to 60 weeks such as 48 weeks in case of infections with HCV genotype 1 and on a one to two weekly basis for 12 to 36 weeks such as 24 weeks in case of infections with HCV genotype 2/3. The pharmaceutical compositions may be formulated for oral, subcutaneous, transdermal, rectal, peritoneal or intravenous administration and may contain suitable pharmaceutically acceptable excipients.

In specific embodiments, the present invention relates to the use of at least one semicarbazone proteasome inhibitor and at least one pharmaceutically active agent in use against HIV infection and/or viral hepatitis infections, particularly HCV infections, in the manufacture of a medicament for treating human or animal individuals, wherein:

a) a medicament with said at least one semicarbazone proteasome inhibitor is first administered to an individual infected with HIV and/or a hepatitis virus such as HCV;

b) a medicament with said at least one pharmaceutically active agent in use against hepatitis virus infection or at least one pharmaceutically active agent in use against retroviral infection is administered subsequent to treatment with the medicament with said at least one semicarbazone proteasome inhibitor.

In another embodiment the present invention relates to the use of least one semicarbazone proteasome inhibitor and at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis infections, particularly HCV infections, in the manufacture of a medicament for treating human or animal individuals, wherein:

a) a medicament with said at least one semicarbazone proteasome inhibitor is administered to an individual infected with HIV and/or a hepatitis virus such as HCV;

b) a medicament with at least one pharmaceutically active agent in use

against hepatitis virus infection or at least one pharmaceutically active agent in use against retroviral infection is administered before or concomittantly with the treatment with the medicament comprising said at least one semicarbazone proteasome inhibitor. In certain embodiments, the patient can be any patient infected with, or at risk for infection with, a virus inducing hepatitis, specifically HCV, or the patient may be infected with, or at risk of an infection with a retrovirus, specifically with HIV. An infection can be determined according to any technique deemed suitable by the practitioner of skill in the art. The risk of an infection may be given, when sufficient contact with a source might have occurred or has occurred, e.g. with a body fluid suspected of containing the above infectious agent(s), or a source known to contain the above infectious agent(s).

In certain embodiments, the patient has never received therapy or prophylaxis for a retrovirus infection or a virally induced hepatitis, or more particularly an HIV infection and/or HCV infection. In further embodiments, the patient has previously received therapy or prophylaxis for an HIV infection and/or a virally induced hepatitis, or more particularly an HCV infection. For instance, in certain

embodiments, the patient has not responded to treatment for an HIV infection and/or a virally induced hepatitis, or more particularly an HCV infection. In certain embodiments, the patient can be a patient that received therapy but continued to suffer from viral infection or one or more symptoms thereof. In certain

embodiments, the patient can be a patient who received therapy but failed to achieve a sustained viro logic response. In certain embodiments, the patient has received therapy for an HIV infection and/or a virally induced hepatitis, or more particularly a hepatitis induced by HCV infection, but has failed to show, for example, a 2 log 10 (2 orders of magnitude, 100 fold) decline in viral RNA levels after 12 weeks of therapy. In certain embodiments, the patient is a patient who discontinued therapy for a virally induced hepatitis, or more particularly an HIV infection and/or a hepatitis induced by HCV infection, because of one or more adverse events associated with the therapy. In certain embodiments, the patient is a patient where current therapy is not indicated. For instance, certain therapies for HCV are associated with neuropsychiatric events. Interferon (IFN)-alpha plus ribavirin is associated with a high rate of depression. Depressive symptoms have been linked to a worse outcome in a number of medical disorders. Life-threatening or fatal neuropsychiatric events, including suicide, suicidal and homicidal ideation, depression, relapse of drug addiction/overdose, and aggressive behavior have occurred in patients with and without a previous psychiatric disorder during HCV therapy. Interferon-induced depression is a limitation for the treatment of chronic hepatitis C, especially for patients with psychiatric disorders. Psychiatric side effects are common with interferon therapy and responsible for about 10% to 20% of discontinuations of current therapy for HCV infection.

The use of semicarbazone proteasome inhibitors according to the present invention, or a pharmaceutical compositions or kits comprising the same, preferably of S-2209, for preventing side effects associated with conventional HCV treatments and/or HIV treatments is contemplated herein.

The kits, methods and compositions provided herein may reduce or eliminate the need for exposing patients to the agents of current therapy, either by reducing the dose needed or reducing the required time of exposure to these agents, or by facilitating the replacement of certain agents of current therapy.

Accordingly, provided are methods of treating or preventing a virally induced hepatitis, or more particularly a hepatitis induced by HCV infection and/or HIV infection, in patients where the risk of neuropsychiatric events, such as depression, contraindicates treatment with current therapies. In one embodiment, provided are methods of treating or preventing a virally induced hepatitis, or more particularly a hepatitis induced by HCV and/or HIV infection, in patients where a neuropsychiatric event, such as depression, or risk of such indicates discontinuation of treatment with current therapy. Further provided are methods of treating a virally induced hepatitis, or more particularly a hepatitis induced by HCV and/or HIV infection, in patients where a neuropsychiatric event, such as depression, or risk of such indicates dose reduction, or reduction of time of exposure to, current HCV or HIV therapy.

Current therapy is also contraindicated in patients that are hypersensitive to interferon or ribavirin, or both, or any other component of a pharmaceutical product for administration of interferon or ribavirin. Current therapy is not indicated in patients with hemoglobinopathies (e.g., thalassemia major, sickle-cell anemia) and other patients at risk from the hematologic side effects of current therapy. Common hematologic side effects include bone marrow suppression, neutropenia and thrombocytopenia. Furthermore, ribavirin is toxic to red blood cells and is associated with hemolysis. Accordingly, in one embodiment, provided are methods of treating or preventing an HIV infection and/or a virally induced hepatitis, or more particularly a hepatitis induced by HCV infection, in patients hypersensitive to interferon or ribavirin, or both, patients with a hemoglobinopathy, for instance thalassemia major patients and sickle-cell anemia patients, and other patients at risk from the hematologic side effects of current therapy. In certain embodiments, the patient has received treatment for HIV infection and/or a virally induced hepatitis, or more particularly a hepatitis induced by HCV infection, and discontinued that therapy prior to administration of a method provided herein. In further embodiments, the patient has received therapy and continues to receive that therapy along with administration of a method provided herein.

Treatment with at least one semicarbazone proteasome inhibitor may include concentrations of the semicarbazone proteasome inhibitors used within the range of about 1 nM to about 50 μΜ, preferably about 10 nM to about 10 μΜ in the pharmaceutical composition. The semicarbazone proteasome inhibitors may be used at doses of about 0.25 to about 5, of about 0.4 to about 2.5, or of about 0.7 to about 1.5 mg/m², or at doses of about 2.5 to about 50, of about 4 to about 25, or of about 7 to about 15 mg/m², or at doses of about 25 to about 500, of about 40 to about 250, or of about 70 to about 150 mg/m² body surface.

Treatment with semicarbazone proteasome inhibitors may be performed over several days and weeks up to months. Usually a semicarbazone proteasome inhibitor may be administered every day, or every second day, or every third day, or twice a week, or once a week, or once every two weeks, or once every month

Subsequent to said treatment with a semicarbazone proteasome inhibitor, administration of the at least one different and optionally further different pharmaceutically active agents is commenced.

The treatment with a different pharmaceutical agent may follow the treatment with the semicarbazone proteasome inhibitor either rather directly (e.g. the following day) or after a break (i.e. a therapy free period) of one to several days or weeks, such as 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, more than 6 weeks etc. Starting with the therapy after a break of about 1, 2, 3, 4, 5, or 6 weeks may be

recommendable in order to await whether the semicarbazone proteasome inhibitor alone improved the patient's condition. Further, the treatment with a different pharmaceutical agent may be re-commenced with the same frequency and dosage as it would have been performed without the patients receiving semicarbazone proteasome inhibitors.

If e.g. proteasome specific inhibitors such as S-2209 or analogs thereof are applied during treatment of a viral Hepatitis C infection and if this treatment is followed by standard therapy with pegylated interferons and ribavirine, treatment with pegylated interferons and ribavirine may be undertaken on a twice a week, once a week, or two weekly basis for 48 weeks in case of infections with HCV genotype 1 and on a twice a week, once a week, or two weekly basis for 24 weeks in case of infections with HCV genotype 2/3.

A combination of at least one semicarbazone proteasome inhibitor together with at least one other pharmaceutically active agent which is known to be effective in treating HIV and/or viral hepatitis may also be administered simultaneously.

In another embodiment the present invention thus relates to the use of at least one semicarbazone proteasome inhibitor together with at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis infections in the manufacture of a medicament for treating patients infected with retroviruses, e.g. HIV and/or hepatitis viruses, e.g. HCV.

In some embodiments of the invention it can be preferred to use specific proteasome inhibitors such as PS-519, PS-341 (Bortezomib) and PS-273 in addition to the semicarbazone proteasome inhibitor. If additionally a different pharmaceutical agent is present, for hepatitis virus infected patients, e.g. HCV-infected patients, this may be preferably selected from interferons including their derivatives such as pegylated interferon alpha as first different pharmaceutically active agent or from nucleoside analoga such as ribavirin as second different pharmaceutically active agent. If, however, at least two additional different pharmaceutical agents are present, these may be preferably selected from interferons including their derivatives such as pegylated interferon alpha as first different pharmaceutically active agent and from nucleoside analoga such as ribavirin as second different pharmaceutically active agent. If the patient is HIV infected, conventionally administered drugs used in the HAART may be used alongside with the semicarbazone proteasome inhibitor according to the invention, .

With the above mentioned pharmaceutical compositions, kits, uses and methods, in particular where they relate to a combination of a semicarbazone proteasome inhibitor, optionally together with an interferon and a nucleoside analog, it is possible to reduce the virus load in chronic HCV patients referred to in the claims (over several orders of magnitude) or to even completely remove the virus.

In any of the embodiments of the present invention (kits, uses, pharmaceutical compositions, methods of treatment), the semicarbazone proteasome inhibitor may be selected from the above described compounds, preferably from S-2209 or analogs thereof.

In some embodiments the present invention does not include semicarbazone proteasome inhibitors such as S-2209, pharmaceutically acceptable salts thereof or structural or functional analogs thereof, uses thereof, pharmaceutical compositions comprising these pharmaceutically active agents, uses of such pharmaceutical compositions, methods involving such pharmaceutically active agents, methods involving such pharmaceutical compositions, methods involving such uses, kits involving such pharmaceutically active agents, kits involving such pharmaceutical compositions, etc. as far as such pharmaceutically active agents, such pharmaceutical compositions, such uses, such methods, such kits etc. have been disclosed in

European patent application EP 10151135.0, US patent application US61/296,363 and international patent application PCT/EP/2010/060796. In some embodiments, the present invention thus does not consider the subject matter which is disclosed by European patent application EP 10151135.0, US patent application US61/296,363 or international patent application PCT/EP/2010/060796 to form part of the present invention. In some embodiments, the present invention is directed to the afore-mentioned semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, or structural or functional analogs thereof as mentioned herein, the uses thereof as mentioned herein, the pharmaceutical compositions as mentioned herein, the uses of such pharmaceutical compositions as mentioned herein, the methods as mentioned herein, the kits as mentioned herein, etc. but excluding the semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, or structural or functional analogs thereof, the uses thereof, the pharmaceutical compositions, the uses of such pharmaceutical compositions, the methods, the kits as they are described in European patent application EP

10151135.0, US patent application US61/296,363 or international patent application PCT/EP/2010/060796.

In some embodiments, the present invention is directed to the afore-mentioned semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof as mentioned herein, the uses thereof as mentioned herein, the pharmaceutical compositions as mentioned herein, the uses of such pharmaceutical compositions as mentioned herein, the methods as mentioned herein, the kits as mentioned herein, etc. but excluding the specific embodiments and in particular the first to twenty ninth embodiments and the preferred embodiments thereof as they are described in European patent application EP 10151135.0, US patent application US61/296,363 or international patent application PCT/EP/2010/060796. In particular, such semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof, uses thereof, pharmaceutical compositions, uses of such pharmaceutical compositions, methods, kits etc. as mentioned herein are excluded where the emicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof, uses thereof, pharmaceutical compositions, uses of such pharmaceutical compositions, methods, kits etc. as disclosed in European patent application EP 10151135.0, US patent application US61/296,363 or international patent application PCT/EP/2010/060796 involve a combination of semicarbazone proteasome inhibitors such as S-2209, the

pharmaceutically acceptable salts, structural or functional analogs thereof with at least one first pharmaceutically active agent in use against viral hepatitis infections and optionally with at least one second pharmaceutically active agent in use against viral hepatitis infections when treating a hepatitis viral infection in a human or animal individual who does not respond or is refractory to treatment. A

pharmaceutically active agent in use against viral hepatitis infections may include pharmaceutically active agents which support or assist the body's natural response in dealing with viral infections and/or pharmaceutically active agents which interfere with a viral target. Examples of pharmaceutically active agents which support or assist the body's natural response in of dealing with viral infections include the interferons, pegylated versions thereof etc. Examples of pharmaceutically active agents which interfere with a viral target HCV protease inhibitors as mentioned herein, HCV polymerase inhibitors as mentioned herein, nucleoside analogs as mentioned herein, nucleotide analogs as mentioned herein, non-nucleoside-analogous inhibitors of viral enzymes as mentioned herein, HCV entry inhibitors as mentioned herein as well agents using other approaches of interfering with a viral target and combinations thereof as mentioned herein.

In some embodiments, the present invention is directed to the afore-mentioned semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof as mentioned herein, the uses thereof as mentioned herein, the pharmaceutical compositions as mentioned herein, the uses of such pharmaceutical compositions as mentioned herein, the methods as mentioned herein, the kits as mentioned herein, etc. but excluding the semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analgos thereof as mentioned herein, the uses thereof as mentioned herein, the pharmaceutical compositions as mentioned herein, the uses of such pharmaceutical compositions as mentioned herein, the methods as mentioned herein, the kits as mentioned herein, as far as these

semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof, the uses thereof, the pharmaceutical compositions, the uses of such pharmaceutical compositions, the methods, the kits etc. involve a combination of semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof and pharmaceutically active agents in use against viral hepatitis infections as mentioned herein. More preferably the semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof as mentioned herein, the uses thereof as mentioned herein, the pharmaceutical compositions as mentioned herein, the uses of such

pharmaceutical compositions as mentioned herein, the methods as mentioned herein, the kits as mentioned herein, are excluded as far as these semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof, the uses thereof, the pharmaceutical compositions, the uses of such pharmaceutical compositions, the methods, the kits etc. involve a combination of semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof and pharmaceutically active agents in use against viral hepatitis infections for treating a human or animal individual or patient who does not respond or is refractory to treatment with at least one pharmaceutically acceptable agent in use against viral hepatitis infection. Even more preferably such a human or animal individual or patient is affected by a HCV infection and does not respond or is refractory to treatment with at least one pharmaceutically acceptable agent in use against viral hepatitis infection.

In some embodiments, the present invention thus is directed to the afore-mentioned semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof as mentioned herein, the uses thereof as mentioned herein, the pharmaceutical compositions as mentioned herein, the uses of such pharmaceutical compositions as mentioned herein, the methods as mentioned herein, the kits as mentioned herein, etc. but excluding the semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof as mentioned herein, the uses thereof as mentioned herein, the pharmaceutical compositions as mentioned herein, the uses of such pharmaceutical compositions as mentioned herein, the methods as mentioned herein, the kits as mentioned herein, as far as these semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof, the uses thereof, the pharmaceutical compositions, the uses of such pharmaceutical compositions, the methods, the kits etc. involve a combination of semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof and pharmaceutically active agents in use against viral hepatitis infections as mentioned herein such as pharmaceutically active agents which support or assist the body's natural response in dealing with viral infections and/or pharmaceutically active agents which interfere with a viral target. Examples of pharmaceutically active agents which support or assist the body's natural response in of dealing with viral infections include the interferons, pegylated versions thereof etc. Examples of pharmaceutically active agents which interfere with a viral target HCV protease inhibitors as mentioned herein, HCV polymerase inhibitors as mentioned herein, nucleoside analogs as mentioned herein, nucleotide analogs as mentioned herein, non-nucleoside-analogous inhibitors of viral enzymes as mentioned herein, HCV entry inhibitors as mentioned herein as well agents using other approaches of interfering with a viral target and combinations thereof as mentioned herein. More preferably the semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof as mentioned herein, the uses thereof as mentioned herein, the

pharmaceutical compositions as mentioned herein, the uses of such pharmaceutical compositions as mentioned herein, the methods as mentioned herein, the kits as mentioned herein, are excluded as far as these semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analogs thereof, the uses thereof, the pharmaceutical compositions, the uses of such pharmaceutical compositions, the methods, the kits etc. involve a combination of semicarbazone proteasome inhibitors such as S-2209, the pharmaceutically acceptable salts, structural or functional analgos thereof with such pharmaceutically active agents in use against viral hepatitis infections for treating a human or animal individual or patient who does not respond or is refractory to treatment with at least one pharmaceutically acceptable agent in use against viral hepatitis infection. Even more preferably such a human or animal individual or patient is affected by a HCV infection and does not respond or is refractory to treatment with at least one pharmaceutically acceptable agent in use against viral hepatitis infection. Pharmaceutical Compositions and Methods of Administration

The semicarbazone proteasome inhibitors can be formulated into pharmaceutical compositions using methods available in the art and those disclosed herein. Such compounds can be used in some embodiments to enhance delivery of the drug to the liver.

The methods provided herein encompass administering pharmaceutical compositions containing at least one semicarbazone proteasome inhibitor as described herein, with one or more compatible and pharmaceutically acceptable carriers, such as diluents or adjuvants, and/or with at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis infection, e.g. HCV infections. In certain embodiments, the at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis infections can be formulated or packaged with the semicarbazone proteasome inhibitor. Of course, the at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis infections, such as HCV infections, will only be formulated with the semicarbazone proteasome inhibitor when, according to the judgment of those of skill in the art, such co- formulation should not interfere with the activity of either agent or the method of administration. In certain embodiments, the semicarbazone proteasome inhibitor and the at least one pharmaceutically active agent are formulated separately. They can be packaged together, or packaged separately, for the convenience of the practitioner of skill in the art.

In clinical practice the active agents provided herein may be administered by any conventional route, in particular orally, parenterally, rectally or by inhalation (e.g. in the form of aerosols). In certain embodiments, the semicarbazone proteasome inhibitor is administered orally.

Use may be made, as solid compositions for oral administration, of tablets, pills, hard gelatin capsules, powders or granules. In these compositions, the active product is mixed with one or more inert diluents or adjuvants, such as sucrose, lactose or starch.

These compositions can comprise substances other than diluents, for example a lubricant, such as magnesium stearate, or a coating intended for controlled release.

Use may be made, as liquid compositions for oral administration, of solutions which are pharmaceutically acceptable, suspensions, emulsions, syrups and elixirs containing inert diluents, such as water or liquid paraffin. These compositions can also comprise substances other than diluents, for example wetting, sweetening or flavoring products.

The compositions for parenteral administration can be emulsions or sterile solutions. Use may be made, as solvent or vehicle, of propylene glycol, a polyethylene glycol, vegetable oils, in particular olive oil, or injectable organic esters, for example ethyl oleate. These compositions can also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterilization can be carried out in several ways, for example using a bacteriological filter, by radiation or by heating. They can also be prepared in the form of sterile solid compositions which can be dissolved at the time of use in sterile water or any other injectable sterile medium. The compositions for rectal administration are suppositories or rectal capsules which contain, in addition to the active principle, excipients such as cocoa butter, semisynthetic glycerides or polyethylene glycols. The compositions can also be aerosols. For use in the form of liquid aerosols, the compositions can be stable sterile solutions or solid compositions dissolved at the time of use in apyrogenic sterile water, in saline or any other pharmaceutically acceptable vehicle. For use in the form of dry aerosols intended to be directly inhaled, the active principle is finely divided and combined with a water-soluble solid diluent or vehicle, for example dextran, mannitol or lactose.

In one embodiment, a composition provided herein is a pharmaceutical composition or a single unit dosage form. Pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., a proteasome inhibitor, or other prophylactic or therapeutic agent), and a typically one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment and in this context, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" includes a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water can be used as a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

Typical pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well-known to those skilled in the art of pharmacy, and non limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Whether a particular excipient is suitable for

incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient and the specific active ingredients in the dosage form. The composition or single unit dosage form, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

Lactose free compositions provided herein can comprise excipients that are well known in the art and are listed, for example, in the U.S. Pharmocopia (USP) SP(XXI)/NF (XVI). In general, lactose free compositions comprise an active ingredient, a binder/filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. Exemplary lactose free dosage forms comprise an active ingredient, microcrystalline cellulose, pre gelatinized starch, and magnesium stearate.

Further encompassed herein are anhydrous pharmaceutical compositions and dosage forms comprising active ingredients, since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long term storage in order to determine characteristics such as shelf life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379 80. In effect, water and heat accelerate the decomposition of some compounds. Thus, the effect of water on a formulation can be of great significance since moisture and/or humidity are commonly encountered during manufacture, handling, packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine can be anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions can be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

Further provided are pharmaceutical compositions and dosage forms that comprise one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, which are referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

The pharmaceutical compositions and single unit dosage forms can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions and dosage forms will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent, in certain embodiments, in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In a certain embodiment, the pharmaceutical compositions or single unit dosage forms are sterile and in suitable form for administration to a patient, for example, an animal patient, such as a mammalian patient, for example, a human patient.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, buccal, sublingual, inhalation, intranasal, transdermal, topical, transmucosal, intra-tumoral, intra-synovial and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to human beings. In an embodiment, a pharmaceutical composition is formulated in accordance with routine procedures for subcutaneous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection. Examples of dosage forms include, but are not limited to: tablets; cap lets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions;

suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non aqueous liquid suspensions, oil in water emulsions, or a water in oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

The composition, shape, and type of dosage forms provided herein will typically vary depending on their use. For example, a dosage form used in the initial treatment of viral infection may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the maintenance treatment of the same infection. Similarly, a parenteral dosage form may contain smaller amounts of one or more of the active ingredients it comprises than an oral dosage form used to treat the same disease or disorder. These and other ways in which specific dosage forms encompassed herein will vary from one another will be readily apparent to those skilled in the art. See, e.g. Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

It will be understood by the skilled person that, wherever there is mention herein of a pharmaceutical composition which may be provided according to the invention, such pharmaceutical composition may be provided in a container. Where there is mention of more than one pharmaceutical composition, these compositions may be provided in one container, or they may be provided in separate containers, e.g. one container for each pharmaceutical composition provided. Embodiments wherein different pharmaceutical compositions are provided in separate containers constitute preferred embodiments of the instant invention. However, nothing herein shall be interpreted as indicating that separate containers may not be packaged together to make up but a single product, including for manufacturing and/or sales purposes. Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile

pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

Typical dosage forms comprise a proteasome inhibitor, or a pharmaceutically acceptable salt, solvate or hydrate thereof lie within the range of from about 0.1 mg to about 1000 mg per day, given as a single once-a-day dose in the morning or as divided doses throughout the day taken with food. Particular dosage forms can have about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100, 200, 250, 400, 500, 600, 750, 800 1000, 1250 or 1500 mg of the active compound.

Oral Dosage Forms Pharmaceutical compositions that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa.

(2000). In certain embodiments, the oral dosage forms are solid and prepared under anhydrous conditions with anhydrous ingredients, as described in detail in the sections above. However, the scope of the compositions provided herein extends beyond anhydrous, solid oral dosage forms. As such, further forms are described herein.

Typical oral dosage forms are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional

pharmaceutical compounding techniques, xcipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.

For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Examples of excipients that can be used in oral dosage forms include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.

Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL PH 101, AVICEL PH 103 AVICEL RC 581, AVICEL PH 105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, PA), and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC 581. Suitable anhydrous or low moisture excipients or additives include AVICEL PH 103.TM. and Starch 1500 LM.

Disintegrants are used in the compositions to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of

disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form solid oral dosage forms. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, specifically from about 1 to about 5 weight percent of disintegrant. Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar agar, alginic acid, calcium carbonate,

microcrystalline cellulose, croscarmnellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pre gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Piano, Tex.), CAB O SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated.

Delayed Release Dosage Forms

Active ingredients such as the compounds provided herein can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719;

5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556;

5,639,480; 5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945;

5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363; 6,264,970;

6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; 6,699,500 each of which is incorporated herein by reference. Such dosage forms can be used to provide slow or controlled release of one or more active ingredients using, for example,

hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients provided herein. Thus encompassed herein are single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled release.

All controlled release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non controlled counterparts. Ideally, the use of an optimally designed controlled release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

In certain embodiments, the drug may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see, Sefton, CRC Crit. Ref. Biomed. Eng. 1987, 14:201; Buchwald et al, Surgery 1980, 88:507; Saudek et al, N. Engl. J. Med. 1989, 321 :574). In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in a patient at an appropriate site determined by a practitioner of skill, i.e., thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical

Applications of Controlled Release, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 1990, 249: 1527). The active ingredient can be dispersed in a solid inner matrix, e.g.,

polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene- vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydro lyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene,

polyvinylchloride, vinylchloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble in body fluids. The active ingredient then diffuses through the outer polymeric membrane in a release rate controlling step. The percentage of active ingredient in such parenteral compositions is highly dependent on the specific nature thereof, as well as the needs of the patient.

Parenteral Dosage Forms

In one embodiment, provided are parenteral dosage forms. Parenteral dosage forms can be administered to patients by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are typically, sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into the parenteral dosage forms.

Transdermal, Topical & Mucosal Dosage Forms

Also provided are transdermal, topical, and mucosal dosage forms. Transdermal, topical, and mucosal dosage forms include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 16.sup.th, 18th and 20.sup.th eds., Mack Publishing, Easton Pa. (1980, 1990 & 2000); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treating mucosal tissues within the oral cavity can be formulated as mouthwashes or as oral gels. Further, transdermal dosage forms include "reservoir type" or "matrix type" patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal, topical, and mucosal dosage forms encompassed herein are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels or ointments, which are non toxic and pharmaceutically acceptable. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 16.sup.th, 18th and 20.sup.th eds., Mack Publishing, Easton Pa. (1980, 1990 & 2000).

Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with active ingredients provided. For example, penetration enhancers can be used to assist in delivering the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve delivery of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients so as to improve delivery. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery enhancing or penetration enhancing agent. Different salts, hydrates or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.

Dosage and Unit Dosage Forms

In human therapeutics, the physician will determine the posology which he considers most appropriate according to a preventive or curative treatment and according to the age, weight, stage of the infection and other factors specific to the patient to be treated. In certain embodiments, doses are from about 1 to about 5000 mg per day for an adult, or from about 1 to about 2500 mg per day, or from about 1 to about 1000 mg per day, or from about 500 to about 5000 mg per day, or from 250 to about 2500 mg per day, or from about 5 to about 250 mg per day or from about 10 to 50 mg per day for an adult. In certain embodiments, doses are from about 5 to about 400 mg per day or 25 to 200 mg per day per adult. In certain embodiments, dose rates of from about 50 to about 500 mg per day are also contemplated.

In further aspects, provided are methods of treating or preventing HIV associated clinical symptoms and/or a viral hepatitis, e.g. hepatitis caused by an HCV infection, in a patient by administering, to a patient in need thereof, inter alia an effective amount of a semicarbazone proteasome inhibitor, or a pharmaceutically acceptable salt thereof. The amount of the compound or composition which will be effective in the prevention or treatment of a disorder or one or more symptoms thereof will vary with the nature and severity of the disease or condition, and the route by which the active ingredient is administered. The frequency and dosage will also vary according to factors specific for each patient depending on the specific therapy (e.g., therapeutic or prophylactic agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, and the past medical history of the patient. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. In certain embodiments, exemplary doses of a composition include milligram or microgram amounts of the active compound per kilogram of patient or sample weight (e.g., about 10 micrograms per kilogram to about 50 milligrams per kilogram, about 100 micrograms per kilogram to about 25 milligrams per kilogram, or about 100 microgram per kilogram to about 10 milligrams per kilogram). For

compositions provided herein, in certain embodiments, the dosage administered to a patient is 0.140 mg/kg to 35 mg/kg of the patient's body weight, based on weight of the active compound. In certain embodiments, the dosage administered to a patient is between 0.20 mg/kg and 20.0 mg/kg, or between 0.30 mg/kg and 15.0 mg/kg of the patient's body weight. Alternatively, an estimate of the surface area of the patient's body may be used to scale the dose, as the surface area is sometimes a more accurate predictor of certain properties related to drug distribution and clearance (see, for example, Pinkel, D., Cancer Res. 1958, 18:853). The proteasome inhibitors may be used at doses of about 0.25 to about 5, of about 0.4 to about 2.5, or of about 0.7 to about 1.5 mg/m², or at doses of about 2.5 to about 50, of about 4 to about 25, or of about 7 to about 15 mg/m²0, or at doses of about 25 to about 500, of about 40 to about 250, or of about 70 to about 150 mg/m² body surface. In certain embodiments, the recommended daily dose range of a composition provided herein for the conditions described herein lie within the range of from about 0.1 mg to about 5000 mg per day, given as a single once-a-day dose or as divided doses throughout a day. In one embodiment, the daily dose is administered twice daily in equally divided doses. In certain embodiments, a daily dose range should be from about 1 to about 2500 mg per day, or from about 1 to about 1000 mg per day, or from about 500 to about 5000 mg per day, or from 250 to about 2500 mg per day, or from about 5 to about 250 mg per day or from about 10 to 50 mg per day. It may be necessary to use dosages of the active ingredient outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with patient response.

Different therapeutically effective amounts may be applicable for different diseases and conditions, as will be readily known by those of ordinary skill in the art.

Similarly, amounts sufficient to prevent, manage, treat or ameliorate such disorders, but insufficient to cause, or sufficient to reduce, adverse effects associated with the composition provided herein are also encompassed by the above described dosage amounts and dose frequency schedules. Further, when a patient is administered multiple dosages of a composition provided herein, not all of the dosages need be the same. For example, the dosage administered to the patient may be increased to improve the prophylactic or therapeutic effect of the composition or it may be decreased to reduce one or more side effects that a particular patient is experiencing. In certain embodiment, the dosage of the composition provided herein, based on weight of the active compound, administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a patient is 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 35 mg/kg or 50 mg/kg or more of a patient's body weight. In another embodiment, the dosage of the composition or a composition provided herein administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a patient is a unit dose of 0.1 mg to 1500 mg, 0.1 mg to 1000 mg, 0.1 mg to 500 mg, 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg..

In certain embodiments, treatment or prevention can be initiated with one or more loading doses of a compound or composition provided herein followed by one or more maintenance doses. In such embodiments, the loading dose can be, for instance, about 40 to 4000 mg per day, 60 to about 2000 mg per day, or about 100 to about 1000 mg per day for one day to five weeks. The loading dose can be followed by one or more maintenance doses. In certain embodiments, each maintenance does is, independently, about from about 10 mg to about 2000 mg per day, between about 25 mg and about 1500 mg per day, or between about 25 and about 800 mg per day. Maintenance doses can be administered daily and can be administered as single doses, or as divided doses.

In certain embodiments, a dose of a compound or composition provided herein can be administered to achieve a steady-state concentration of the active ingredient in blood or serum of the patient. The steady-state concentration can be determined by measurement according to techniques available to those of skill or can be based on the physical characteristics of the patient such as height, weight and age. In certain embodiments, a sufficient amount of a compound or composition provided herein is administered to achieve a steady-state concentration in blood or serum of the patient of from about 300 to about 4000 ng/mL or higher, from about 400 to about 1600 ng/mL, or from about 600 to about 1200 ng/mL. In some embodiments, loading doses can be administered to achieve steady-state blood or serum concentrations of about 1200 to about 8000 ng/mL or higher, or about 2000 to about 4000 ng/mL for one to five days. In certain embodiments, maintenance doses can be administered to achieve a steady-state concentration in blood or serum of the patient of from about 300 to about 4000 ng/mL, from about 400 to about 1600 ng/mL, or from about 600 to about 1200 ng/mL.

In certain embodiments, administration of the same composition may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In other embodiments, administration of the same prophylactic or therapeutic agent may be repeated and the administration may be separated by at least at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.

In certain aspects, provided herein are unit dosages comprising a compound, or a pharmaceutically acceptable salt thereof, in a form suitable for administration. Such forms are described in detail above. In certain embodiments, the unit dosage comprises 1 to 5000 mg, 5 to 2500 mg or 10 to 1000 mg active ingredient. In particular embodiments, the unit dosages comprise about 1, 5, 10, 25, 50, 100, 125, 250, 400, 500, 600, 800, 1000, 1500, 2000, 2500, 3000, 4000, or 5000 mg active ingredient. Such unit dosages can be prepared according to techniques familiar to those of skill in the art. The dosages of the at least one different pharmaceutically active agent in use against HIV infections and/or viral hepatitis (e.g. HCV) infections are to be used in the combination therapies provided herein. In certain embodiments, dosages lower than those which have been or are currently being used to prevent or treat HIV infection and/or a viral hepatitis (e.g. by HCV) are used in the combination therapies provided herein. The recommended dosages of at least one pharmaceutically active agent in use against viral hepatitis (e.g. HCV) infections can be obtained from the knowledge of those of skill. For those at least one pharmaceutically active agent in use against viral hepatitis (e.g. HCV) infections that are approved for clinical use, recommended dosages are described in, for example, Hardman et al, eds., 1996, Goodman &

Gilman's The Pharmacological Basis Of Basis Of Therapeutics gth Ed, Mc-Graw- Hill, New York; Physician's Desk Reference (PDR) 57.sup.th Ed., 2003, Medical Economics Co., Inc., Montvale, N.J., which are incorporated herein by reference in its entirety.

In various embodiments, the therapies (e.g., a semicarbazone proteasome inhibitor and the at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis (e.g. HCV) infections) are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours part. In various embodiments, the therapies are administered no more than 24 hours apart or no more than 48 hours apart. In certain embodiments, two or more therapies are administered within the same patient visit. In other embodiments, the semicarbazone proteasome inhibitor and the at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis (e.g. HCV) infections are administered concurrently.

In other embodiments, the semicarbazone proteasome inhibitor and the at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis (e.g. HCV) infections are administered at about 12 h to 24 h apart, 24 h to 48 h apart, 2 to 4 days apart, at about 4 to 6 days apart, at about 1 week part, at about 1 to 2 weeks apart, at about 2 to 3 weeks apart, at about 3 to 4 weeks apart, or more than 4 weeks apart.

In certain embodiments, administration of the same agent may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.

In certain embodiments, a semicarbazone proteasome inhibitor and at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis infections are administered to a patient, for example, a mammal, such as a human, in a sequence and within a time interval such that the semicarbazone proteasome inhibitor can act together with the other agent(s) to provide an increased benefit than if they were administered otherwise. For example, the at least one pharmaceutically active agent can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. In one embodiment, the proteasome inhibitor and the at least one first and/or second different pharmaceutically active agent exert their effect at times which overlap. Each different pharmaceutically active agent can be administered separately, in any appropriate form and by any suitable route. In other embodiments, the semicarbazone proteasome inhibitor is administered before administration of the different pharmaceutically active agent(s).

In certain embodiments, the semicarbazone proteasome inhibitor and the at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis (e.g. HCV) infections are cyclically administered to a patient. Cycling therapy involves the administration of a first agent (e.g. a first prophylactic or therapeutic agents) for a period of time, followed by the administration of at least one second agent for a period of time and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improve the efficacy of the treatment.

In certain embodiments, the semicarbazone proteasome inhibitor and the at least one first and/or second different pharmaceutically active agent are administered in a cycle of less than about 6 weeks, about once every four weeks, about once every three weeks, about once every two weeks, about once every 10 days or about once every week. One cycle can comprise the administration of a semicarbazone proteasome inhibitor and the at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis (e.g. HCV) infections (e.g. by infusion) over about 480 minutes every cycle, about 360 minutes every cycle, about 240 minutes every cycle, about 180 minutes every cycle, about 120 minutes every cycle, about 90 minutes every cycle, about 1 hour every cycle, about 45 minutes every cycle. Each cycle can comprise at least 1 week of rest, at least 2 weeks of rest, at least 3 weeks of rest, or at least 4 weeks of rest. The number of cycles administered is from about 1 to about 12 cycles, more typically from about 2 to about 10 cycles, and more typically from about 2 to about 8 cycles.

In other embodiments, courses of treatment are administered concurrently to a patient, i.e., individual doses of the at least one pharmaceutically active agent in use against HIV infections and/or viral hepatitis (e.g. HCV) infections are administered separately yet within a time interval such that the semicarbazone proteasome inhibitor can have an additive and/or synergistic effect with the at least one first and/or second different pharmaceutically active agent. For example, one component can be administered once per week in combination with the other components that can be administered once every two weeks or once every three weeks. In other words, the dosing regimens are carried out concurrently even if the therapeutics are not administered simultaneously or during the same day. The at least one pharmaceutically active agent in use against viral hepatitis infections can act additively or synergistically with the semicarbazone proteasome inhibitor. In one embodiment, the semicarbazone proteasome inhibitor is administered concurrently with one or more at least one pharmaceutically active agent in use against retroviral, e.g. HIV infections and/or viral hepatitis (e.g. HCV) infections in the same pharmaceutical composition. In another embodiment, a semicarbazone proteasome inhibitor is administered concurrently with one or more pharmaceutically active agents in use against retroviral, e.g. HIV infections and/or viral hepatitis (e.g. HCV) infections in separate pharmaceutical compositions. In still another embodiment, a semicarbazone proteasome inhibitor is administered prior to administration of one or more pharmaceutically active agents in use against retroviral, e.g. HIV infections and/or viral hepatitis infections. Also contemplated are administration of a semicarbazone proteasome inhibitor and at least one pharmaceutically active agent in use against retroviral, e.g. HIV infections and/or viral hepatitis (e.g. HCV) infections by the same or different routes of administration, e.g., oral and parenteral. In certain embodiments, when the semicarbazone proteasome inhibitor is administered concurrently with a at least one

pharmaceutically active agent in use against retroviral, e.g. HIV infections and/or viral hepatitis (e.g. HCV) infections that potentially produces adverse side effects including, but not limited to, toxicity, the one or more different pharmaceutically active agents in use against retroviral, e.g. HIV infections and/or viral hepatitis (e.g. HCV) infections can advantageously be administered at a dose that falls below the threshold that the adverse side effect is elicited.

Kits

Also provided are kits for use in methods of treatment of an HIV infection and/or liver disorder, such as virally induced hepatitis, and more specifically HCV infections. The kits can include at least one semicarbazone proteasome inhibitor, optionally at least one pharmaceutically active agent in use against retroviral, e.g. HIV infections and/or viral hepatitis (e.g. HCV) infections, and instructions providing information to a health care provider regarding usage for treating the disorder. Instructions may be provided in printed form or in the form of an electronic medium such as a floppy disc, CD, or DVD, or in the form of a website address where such instructions may be obtained. A unit dose of the at least one semicarbazone proteasome inhibitor, and optionally of the at least one

pharmaceutically active agent in use against retroviral, e.g. HIV infections and/or viral hepatitis (e.g. HCV) infections, can include a dosage such that when

administered to a patient, a therapeutically or prophylactically effective plasma level of the active ingredients(s) can be maintained in the patient for at least 1 day. In some embodiments, a composition can be included as a sterile aqueous

pharmaceutical composition or dry powder (e.g., lyophilized) composition. In a further embodiment, the kit may comprise instructions to administer at least one proteasome inhibitor and at least one anti-retro viral compound or the pharmaceutical compositions comprising the same, to a human or animal in need of treatment for an HIV-related disease, and/or a viral hepatitis infection, wherein the dose of at least one of the compounds administered is lower than otherwise recommended doses of that compound for such human or animal when said compound is used either in a monotherapy or in standard therapy for HIV infections, or in standard of care therapy for viral hepatitis infections, for anti-retroviral compounds, whichever is the lower, or when it is used without anti-retroviral compound(s) for proteasome inhibitors.

Optionally, and for the case of proteasome inhibitors, said dose is lower by at least a factor of 1/4, by at least a factor of 1/3, or by at least a factor 1/2 than the otherwise recommended doses suggested in the treatment of proliferative diseases as defined in WO 2007/017284 (page 30, lines 1 to 24). Furthermore, pertaining to kits useful in the treatment of an HIV-related disease, for those anti-retroviral compounds having been authorized for marketing in the US, the said dose is optionally lower by at least a factor of 1/4, by at least a factor of 1/3, or by at least a factor 1/2 than the otherwise recommended doses suggested in, for example "Guidelines for the Use of

Antiretro viral Agents in HIV- 1 -Infected Adults and Adolescents" (Panel on

Antiretro viral Guidelines for Adults and Adolescents; The U.S. Department of Health and Human Services. Washington, DC, USA, January 10, 2011; pages 1- 166).

In some embodiments, suitable packaging is provided. As used herein, "packaging" includes a solid matrix or material customarily used in a system and capable of holding within fixed limits the at least one semicarbazone proteasome inhibitor and the optional at least one pharmaceutically active agent in use against retroviral, e.g. HIV infections and/or viral hepatitis (e.g. HCV) infections suitable for administration to a patient. Such materials include glass and plastic (e.g., polyethylene,

polypropylene, and polycarbonate) bottles, vials, paper, plastic, and plastic-foil laminated envelopes and the like. If e-beam sterilization techniques are employed, the packaging should have sufficiently low density to permit sterilization of the contents.

The invention is illustrated in the following with respect to an example which however is not be construed as being limiting. It will be clear that the scope of claimed subject matter may be practiced otherwise than as particularly described herein. Numerous modifications and variations of the subject matter are possible in view of the teachings herein and, therefore, are within the scope the claimed subject matter.

Examples Example 1: Semicarbazone proteasome inhibitors for suppressing replication of Hepatitis C Virus

The Huh-7 Luc-ubi-neo/ET replicon system: an in vitro model of HCV replication.

The subgenomic HCV replicon system has widely been used for testing of anti-HCV compounds. The Luc-ubi-neo/ET replicon cells represent a stable selected Huh-7 cell line harbouring a subgenomic HCV Conl isolate (EMBL data accession number AJ238799), supporting the autonomous HCV RNA replication in cell culture (Lohmann et al. Science 285: 110-113 (1999)). It contains the HCV 5' nontranslated region directing translation of a fusion protein that is composed of the firefly luciferase (luc), ubiquitin (ubi), and the selectable marker neomycin

phosphotransferase (neo). An EMCV IRES, upstream the nonstructural genes, mediates translation of NS3 to NS5B. The cell clone ET used in these studies consists of a replicon variant with cell culture adaptive mutations, permitting a high R A replication in cell culture (Lohmann et al.,. J.Virol. 77: 3007-3019 (2003)). As described recently the amount of luciferase activity correlates with HCV RNA replication and is therefore a useful in vitro tool for measurement anti- HCV activity of antivirals (Vrolijk et al.,. J.Virol. Methods 110:201-209 (2003)).

Example 1.1: Antiviral effect vs. toxicity for the proteasome inhibitor S-2209

Inhibition of HCV replication

Replicon cells were grown in Dulbecco's modified minimal essential medium

(DMEM; Invitrogen) supplemented with 2 mM L-glutamine, nonessential amino acids, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 10% fetal calf serum. Huh-7 Luc-ubi-neo/ET cells were seeded in 24-well plates at a density of 4 x 10⁴ cells per well and after overnight incubation at 37°C, cells were treated with escalating S-2209 concentrations. For what is referred to herein as 12h pulse treatment, cell culture medium was replaced by 1ml of fresh medium 12 hours post treatment. After incubation for an additional 60h, antiviral and cytotoxic effects were determined. For what is referred to herein as structured treatment, 24 hours after the initial treatment a repeated treatment for additional 48 hours was performed.

Quantification of luciferase reporter activity was used to determine the antiviral effects in Huh-7 Luc-ubi-neo/ET replicon cells. Cells, treated in duplicate for each individual drug- combination, were harvested at the given time. Cells were washed once with PBS, 250 μΐ of lysis buffer (0.1 % Triton X-100, 10% Glycerol, 25 mM glycylglycine, 15 mM MgS0₄, 4 mM EGTA and 1 mM DTT, pH 7.8) was added and freeze-thaw lysates were prepared. For each well, two times 100 μΐ lysate was mixed with 100 μΐ assay buffer (10% Glycerol, 25 mM glycylglycine, 15 mM MgS0₄, 4 mM EGTA, 1 mM DTT, 2 mM ATP and 15 mM K₂P0₄, pH 7.8) and, after addition of 40 μΐ of a luciferin solution (1 mM luciferin, 25 mM glycylglycine, pH 7.8), measured for 2 s in a lumino meter (Synergy 2 HT, Biotek). The antiviral effect was determined by normalizing the relative light units (RLU) of the different applications to the corresponding values obtained with untreated cells. Means and standard deviations of a representative experiment are shown in Fig. 1. Under the conditions of the experiment described above, the S-2209 concentration necessary to inhibit the HCV-replicon-associated luciferase activity by 50% (termed IC₅₀ or, alternatively, EC₅₀, when referring to cell-based assays) was about 1.3 μΜ for the structured treatment, and about 7.4 μΜ for the 12h pulse treatment. (See Fig. 1)

Cell Viability (WST-1 Assay) The colorimetric WST-1 assay (Roche) is based on the enzymatic cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases present in viable cells, and is therefore used as a read-out system for cell viability and/or toxicity. Cell culture medium of cells, treated in duplicate for each individual drug- combination, was replaced by 0.33 ml cell culture medium supplemented with 10%) WST-1 reagent, incubated for 1 to 3 hours at 37°C and optical density at 450 nm and 650 nm, last-mentioned for the determination of background, was measured in a microplate reader (Synergy 2, BioTek). Means and standard deviations of a representative experiment are shown in Fig. 2.

Under the conditions of the experiment described above, the S-2209 concentration necessary to suppress formazan formation by 50%> (termed CC₅₀) was about 3.1 μΜ for the structured treatment, but was found to lie above and beyond the

concentrations employed in the experiment (maximum S-2209 concentration 50 μΜ) for the 12h pulse treatment. (See Fig. 2) It was thus established that, for both the 12h pulse treatment and the structured treatment, there exists an antiviral activity of the compound that is independent of any cytotoxic effects.

Example 1.2: Cooperative antiviral effect of S-2209 in combination with IFN-a, IFN-J, and IL-6, respectively.

Huh-7 Luc-ubi-neo/ET cells were seeded in 24-well plates at a density of 4 x 10⁴ cells per well and, after overnight incubation at 37°C, cells were treated with a nontoxic concentration of S-2209 (1.85 μΜ) or fresh medium. Twenty four hours later cell culture medium was removed and cells were cultured for additional 24h with 1ml fresh medium or medium either supplemented with 0.75 U/ml IFN-a (Roferon A, Roche), 0.25 U/ml IFN-γ (Roche) or 100 U/ml IL-6 (Roche). Finally, HCV replicon activity was determined by luciferase activity measurement. Means and standard deviations of a representative experiment are shown in Fig. 3.

Under the conditions of the experiment described above, S-2209, IFN-γ and IL-6 both weakly inhibited replicon activity, IFN-a inhibited Replicon more strongly than either of IFN-γ or IL-6, S-2209 showed yet stronger replicon inhibition, and substances acted at least additively in combination, see Fig. 3. However, IFN-a and IFN-γ were used in the above experiment at comparatively low concentrations. A direct comparison of the magnitude of the effects of these three cytokines, hence, cannot be realistically drawn from the data presented here. However, the additional effect of combining these cytokines with S-2209 is evident.

It was thus established that S-2209 acts at least cooperatively, if not synergistically, with IFN-a, IFN-γ, and IL-6, to inhibit HCV replicon activity . - I l l -

Co-operative activation ofSTAT3, but not STAT 1, by S-2209 in combination with IL- 6 and/or IFN-a

Huh-7 Luc-ubi-neo/ET replicon cells were seeded onto glass cover slips in 24-well plates at a density of 6xl0⁴ cells per well. To test the effect of S-2209 alone or in combination with IL-6, cells were treated 24 h after seeding with a non-toxic concentration of S-2209 (1.85 μΜ) or fresh medium. After 24 hours cell culture medium was replaced by fresh medium or medium supplemented with 100 U/ml IL- 6 (Roche) and incubated for additional 3 hours at 37°C. To test the effect of S-2209 alone or in combination with IFN-a, twenty four hours after cell seeding cell culture medium was replaced either by fresh medium or medium supplemented with 50 μΜ S-2209, 100 U/ml IFN-a (Roferon A, Roche) or the combination of S-2209 & IFN- a, and cells were incubated under such conditions for additional 3 hours at 37°C.

For evaluation by immunofluorescence, cells were fixed by treatment with 3% PFA in PBS for 10 min., washed 3 times with PBS, incubated for 30 min in 5% bovine serum albumin (diluted in PBS) and incubated for 1 h at room temperature (RT) with one of the following primary antibodies: anti P-STATl (Y701) mouse antiserum (BD Biosciences) diluted 1 :400, or anti P-STAT3 (Y705) mouse antiserum (Cell

Signaling) diluted 1 : 100. After an extensive wash with PBS, cells were treated with Alexa Fluor 488 or 546 conjugated antibodies, targeting mouse IgG domains (dilution 1 : 1,000 in bovine serum albumin) for 1 h at RT in complete darkness.

Unbound secondary antibodies were removed by washing three times with PBS and once with water. DNA was stained with 4', 6'-diamidino-2-phenylindole (DAPI; Molecular Probes) for 1 min at RT. Finally samples were mounted on slides with FluormountG (Southern Biotechnology Associates) and analyzed by using confocal fluorescence microscopy. As evident from Figs. 4-6, S-2209 in combination with IL-6 (Fig. 4) and IFN-a (Fig. 5), respectively, activate at least cooperatively the STAT3 protein via

phosphorylation of Tyrosine 705. By contrast, S-2209 in combination with IFN-a, a known activator of STAT1, shows a slight or no additional STAT1 phosphorylation at Tyrosine 701 (Fig. 6).

Evidence for mediation of antiviral activity by STAT3

Huh-7 Luc-ubi-neo/ET cells were seeded in 24-well plates at a density of 4 x 10⁴ cells per well and after overnight incubation at 37°C cells were treated with a nontoxic concentration of Stattic (5 μΜ) (Merck) or fresh medium. One hour later cell culture medium was removed and cells were cultured for additional 24h with escalating concentrations of S-2209 or IFN-a (Roferon A, Roche). Subsequently the antiviral effect was determined by measurement of the luciferase activity.

Under the experimental conditions, an EC50 for the inhibition of replicon activity by S-2209 of 0.77 μΜ without addition of 5 μΜ Stattic was established, while an EC50 of 1.2 μΜ resulted from the addition of 5 μΜ Stattic (See Fig. 7). For IFN-a, an EC50 for the inhibition of replicon activity of 0.14 μΜ resulted without addition of 5 μΜ Stattic, while an EC50 of 0.37 μΜ was found after addition of 5 μΜ Stattic (See Fig. 8).

To determine the co-operative antiviral effect of S-2209 and IFN-a in Stattic treated vs. untreated cells, Huh-7 Luc-ubi-neo/ET cells were seeded in 24-well plates at a density of 4 x 10⁴ cells per well and after overnight incubation at 37°C cells were treated with a non-toxic concentration of Stattic (5 μΜ) (Merck) or fresh medium. Two hours later cell culture medium was removed and cells were cultured for additional 24 hours with fresh medium or medium supplemented with 1.85 μΜ S- 2209. Twenty four hours later cell culture medium was removed and cells were cultured for additional 24h with 1ml fresh medium or medium supplemented with 0.75 U/ml IFN-a. Subsequently the antiviral effect was determined by measurement of the luciferase activity. Evidently, the selective STAT3 Inhibitor, Stattic, at the non- toxic concentration of 5 μΜ partially rescues the antiviral effects of IFN-a and, to a lesser extent, S-2209 (See Fig. 9).

It was thus established, that the antiviral activity of IFN-a and, to a lesser extent, S- 2209 in the current experimental setting is at least partially mediated through activation of STAT3.

STAT proteins (signal transducers and activators of transcription) are latent cytoplasmic transcription factors that are phosphorylated by Janus kinases in response to cytokines after binding to their corresponding cell surface receptors. Phosphorylated STAT proteins translocate to the nucleus, where they transiently turn on specific sets of cytokine-inducible genes. IFN-α/β and IFN-y activate STAT1 proteins after receptor-binding and induce an antiviral (including anti-HCV) status in target cells. In contrast to interferons, IL-6 activates predominantly STAT3.

It has been previously described, that pre-treatment of melanoma cells with

Bortezomib leads to prolonged STAT1 phosphorylation and increased transcription of interferon-stimulated genes (Lesinski et al., Cancer Immunol Immunother 2009, 58:2031). S-2209 slightly induces STAT3 phosphorylation in Huh7 replicon cells, whereas STAT1 was unaffected in cells treated with S-2209 or with the combination of S- 2209 and IFN-a. However, more important is the observation, that S-2209 in combination with IL-6 or IFN-a cooperatively induce STAT3- phosphorylation. Stattic, a selective inhibitor of STAT3 activation (Schust et al, Chem. & Biol. 2006, 13: 1235-1242), can indeed partially rescue the antiviral activity of S-2209 and IFN-a, or IFN-α in S-2209 pre-treated cells. It is, therefore, reasonable to conclude, that the antiviral effect of S-2209/ IFN-CC in Huh7/ Luc-ubi-neo/ET cells is at least partially STAT3 mediated. Example 2: Semicarbazone proteasome inhibitors for suppressing replication of Human Immunodeficiency Virus

Example 2.1 General methods Preparation of peripheral blood mononuclear cell (PBMC) cultures

Pathogen-free human buffy coat preparations were obtained from a blood bank (lymphocyte concentrate from 500 ml whole blood, Institut fur Trans fusionsmedizin Suhl gemeinnutzige GmbH, Suhl, Germany), were transferred into sterile 50 ml tubes and diluted 1 : 1 with PBS. The diluted preparation was overlaid in another sterile 50 ml tube on 15 ml Ficoll-Histopaque (Sigma- Aldrich, Cat. H8889) and centrifuged at 2.200 rpm (Heraeus Multifuge 3SR) for 20 min without brakes. The white interphase layer formed by peripheral blood mononuclear cells (PBMC) was transferred in a fresh tube and washed twice with ice cold PBS. Subsequently, the cell count was determined by Trypan Blue staining and the cells were adjusted to 5 x 10⁶ per ml and cultured in supplemented RPMI 1640.

For viral infection, cells were pre-stimulated with 10 μg/ml PHA-P (Sigma- Aldrich,Cat. No. L1668-5mg) and 100 U/ml IL-2 (Proleukin, Roche) and the culture was allowed to grow for at least 72 h.

Preparation of human lymphoid aggregate cultures from fresh tonsils Tonsil tissue was removed during routine tonsillectomy from HIV, HBV, HCV- negative patients. Tonsils were washed carefully with PBS and capsula, brown necrotic and bloody regions were removed. To prepare a Human Lymphoid Aggregate Culture (HLAC), tonsil tissue was mechanically dispersed by cutting tissue in 2- to 3-mm blocks and passing them through 70-μιη cell strainers with the plunger of a 2-ml syringe. The cell strainer was washed with PBS and cells were spun down for 5 min at 500 rcf at room temperature. PBS was removed and cells were suspended in HLAC medium (RPMI 1640 containing 15% fetal bovine serum, 2 mM L-glutamine, 100 U/ml PenStrep, 2.5 μg/ml fungizone, 1 mM sodium pyruvate, 1% non-essential amino acids, 50 μg/ml gentamycin). Cells were counted and diluted with fresh HLAC medium to a final concentration of 1 x 10⁷ cells/ml. Cells were seeded in 96-well plates (200 μΐ/well; 2 x 10⁶ cells/well) and incubated overnight at 37°C. Cultures were used within 24 h of preparation.

HIV virus stock preparation by transfection of 293T cells

Virus stocks were generated by transient transfection of 293T cells using

Lipofectamine 2000™ according to manufacturer's protocols. One day before transfection, 0.5 x 10⁵ 293T cells were seeded in 15 ml DMEM in T75 flasks. At a confluence of 50-75% the cells were used for transfection. 48 h post transfection, supernatants were collected and centrifuged at 1200 rpm for 5 min (Heraeus Multifuge 3SR). After centrifugation, the supernatant was passed through a 45 μιη sterile filter. 1 ml supernatant was overlaid on 200 μΐ 2% sucrose in a eppendorf tube and centrifuged at 14000 rpm (Eppendorf Centrifuge, model 5417R) for 90 min at 4°C. Each virus pellet was re-suspended in 50 μΐ RPMI 1640 and pellets were collected to a final volume of 750 μΐ virus stock. The HIV-1 p24 antigen concentration of virus stocks was determined by p24 antigen enzyme-linked immunosorbent assay

(ELISA).

Staining of apoptotic/necrotic cells Apoptosis is a tightly regulated process, characterized by DNA fragmentation, shrinkage of cytoplasm, and reassembly of membranes. In viable cells, phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane. In apoptotic cells, PS is translocated from the inner to the outer leaflet of the membrane and is therefore exposed to the extracellular environment. Annexin V, a human anticoagulant conjugated to a fluorophore, binds to PS and can therefore identify apoptotic cells. Propidium iodide (PI) is impermeant to live and apoptotic cells, but stains dead cells with red fluorescence by binding tightly to nucleic acids made accessible to membrane impermeant agents only by cell death. Staining a population of cells with Annexin V and PI leads to different fluorescent cell populations, of which apoptotic cells show a green fluorescence, dead cells show a green and red fluorescence, whereas live cells appear unstained. Cells were stained following the protocols described in Immunol Cell Biol 1998, 76:1.

P24 ELISA:

The p24 antigen ELISA is a standard method for detection of released HIV viral particles in the supernatant of tissue cell culture or blood samples of patients. The assay was performed using a commercially available assay kit following

manufacturer's instructions (Aalto Bio Reagents, Dublin, Ireland; see also: Moore et al, Science 1990, 250: 1139-1142; Moore et al, J.Virol. 1991, 65:852-860).

Briefly, 15 μΐ of HIV-1 containing supernatants are inactivated with 135 μΐ 2% Empigen. After one hour, ΙΟΟμΙ are transferred onto a 96-well plate coated with a polyclonal sheep-anti-HIV-l-p24 gag antibody. After washing, the samples are incubated with horseradish peroxidase (HRP-) conjugated p24 antibody, a HRP substrate is added after repeat washing, and luminescence is quantitated in a standard ELISA reader. RT-Assay

The RT-Assay is a standard method for detecting the amount of mature retroviruses present in cell culture supematants (Willey RL, et al, J Virol 1988, 62: 139-147.). It is used to assess potential anti- viral effects of test substances. Briefly, supematants from cultures of cells infected with the HI virus are used for assessing HIV replication in such cells, by measuring the incorporation of ³²P-dTT into DNA generated by viral reverse transcriptase liberated by the lysis of infected cells. ³²P-incorporation is measured by the scinitillation counting of ³²P-radioactivity in supernatant samples of defined volume. An average reading obtained from uninfected cells is subtracted as background. Counts per minute (cpm) in these supernatant samples quantitatively reflects the amount of active viral reverse transcriptase as an indicator of mature viruses present in the supernatant.

To gauge an inhibitory effect of inhibitor candidates on HIV-1 replication, the area under the curve is calculated for a plot of ³²P-radioactivity in supernatant samples vs. time as an indicator for total viral replication. The average area under the curve obtained from untreated infected samples is defined as 100%, and the results for samples having been treated with test substances are expressed relative to this value.

Example 2.2: Inhibition of HIV replication in human lymphoid aggregate cultures

Within 24 h of preparation, HLAC were infected with HIV-1NL4/3 (1 ng p24 per well).

Example 2.2.1: Inhibitory potency and cytotoxicity of a proteasome inhibitor (S- 2209)

To first compare and contrast the potency of S-2209 in inhibiting HIV replication with its toxicity to HLAC, IC₅₀ (concentration yielding 50% inhibition, also referred to as EC₅₀ in cell-based assays) and CC₅₀ (concentration yielding 50% cytotoxicity) for the treatment of HLAC with S-2209 under the experimental conditions to be used further on were first determined. To this effect, HLAC medium (RPMI 1640 containing 15% fetal bovine serum, 2 mM L-glutamine, 100 U/ml PenStrep, 2,5 μg/ml fungizone, 1 mM sodium pyruvate, 1% non-essential amino acids, 50 μg/ml gentamycin) containing S-2209 at concentrations ranging from 0 nM (mock treated) to 100 μΜ was added to HLAC infected with HIV-1NL4/3 immediately post-infection (for S-2209 IC₅₀) or uninfected HLAC (for cytotoxicity assessment), cultures were incubated for 24 h, the medium exchanged for fresh medium again containing S- 2209 at the same concentration as before, and the cultures incubated for another 48 h. Subsequently, the medium containing S-2209 was exchanged for fresh HLAC medium, and the cultures incubated for another 12 days (up to day 15 post infection) under HLAC medium, exchanging the HLAC medium for fresh HLAC medium every 72 h. Culture supernatants were removed on days 1 , 3, 6, 9, 12 and 15, and HIV-1 replication in infected cultures assessed by RT assay. The amounts of radioactivity measured in these samples were summed up to yield a number roughly corresponding to an aggregate viral production. The co-treated uninfected cultures were subjected to Annexin/PI staining for assessment of cytotoxicity on the day of peak HIV-1 replication in the infected culture treated with the same inhibitor concentration. The average value obtained for mock treated cells was set to 100% replication and 100% viable cells, respectively. All other results were expressed relative to these values. Polynomial regression (4-parameter, GraphPad Prism, GraphPad Software, La Jolla, CA, USA) on the resulting data points yielded an IC₅₀ (EC₅₀) for inhibiting HIV-1 replication by S-2209 of approximately 375 nM (see Fig. 10), and a CC₅₀ of approximately 5500 nM (see Fig. 1 l)under the conditions of the experiment as described. It was therefore established, that the inhibition of HIV- 1 replication was not due to S- 2209 rendering the cells non-viable.

Example 2.2.2: Comparison of the time course of inhibition of a proteasome inhibitor (S-2209), an agent inhibiting a viral target (protease inhibitor darunavir) In a further set of experiments, the time course of the inhibitory efficacy of a concentration of S-2209 giving near 100% inhibition of peak replication in Example 2.1 (750 nM S-2209) was compared to the comparable activity of an agent inhibiting a viral target, which is used routinely in the therapy of HIV infection (darunavir, DRV). To this effect, HLAC infected with HIV-l_NL4/3 were treated with HLAC medium containing 750 nM S-2209 or 15 nM DRV (EC/IC₅₀ for DRV: 8 nM under the conditions of this experiment, data not shown) for 24 h, the HLAC medium containing the respective agent exchanged for fresh HLAC medium containing the respective agent, and the incubation continued for another 48 h. Subsequently, the medium containing S-2209 or DRV was exchanged for fresh HLAC medium, and the cultures incubated for another 18 days (up to day 21 post infection) under HLAC medium, exchanging the HLAC medium for fresh HLAC medium every 72 h.

Culture supernatants were removed on days 1, 3, 6, 9, 12, 15, 18, and 21, and HIV-1 replication in the culture assessed by RT assay. Mock infected cells served as negative controls, cells treated with HLAC medium not containing any agent as positive controls. In positive controls, HIV-1 replication sharply increased on day 12, and peak replication was observed on day 15 post infection, gradually decreasing thereafter, most likely due to the depletion of uninfected cells available for further infection. In cells treated with DRV for the first 72h post infection, replication was observed slightly shifted, with peak replication observed on day 18, and gradually decreasing thereafter. In cultures treated with S-2209, virtually no increase of radioactivity readings over those obtained for mock infected cells were observed. It was thus established, that S-2209 is capable of suppressing HIV-1 replication over the full time course of the experiment, while treatment with the protease inhibitor DRV merely delays HIV-1 replication until removal of the protease inhibitor.

Example 2.2.3: Synergistic action of treatment with a combination of proteasome inhibitor (S-2209) with an agent inhibiting a viral target (protease inhibitor darunavir)

In further sets of experiments, HLAC were treated with the following regimens (see also Fig. 12):

(i) Addition of HLAC medium containing DRV at 15 nM or 7.5 nM

concentrations, as indicated below, directly following infection, exchanging the HLAC medium for fresh HLAC medium containing DRV at the same concentration 24 h post infection, and again replacing the HLAC medium with plain HLAC medium (i.e. not containing DRV), 72 h post infection; the plain HLAC medium was subsequently exchanged for fresh plain HLAC medium every 72 h until day 21 post infection

(ii) Addition of HLAC medium containing DRV at 15 nM or 7.5 nM, as

indicated below, directly following infection, exchanging the HLAC medium for fresh HLAC medium containing DRV at the same concentration 24 h post infection, and again replacing the HLAC medium with HLAC medium comprising S-2209 at 500 nM or 100 nM concentrations, as indicated below, but not DRV, 72 h post infection; the HLAC medium comprising S-2209 was subsequently replaced with fresh HLAC medium comprising S-2209 every 72 h until day 12 post infection; it was then replaced by plain HLAC medium (i.e. not containing S-2209 or DRV), and the plain HLAC medium refreshed every 72 h until day 21 post infection (iii) Addition of plain HLAC medium directly following infection, exchanging the HLAC medium for fresh plain HLAC medium 24 h post infection, and again replacing the HLAC medium with HLAC medium comprising S-2209 at 500 nM or 100 nM concentrations, as indicated below, but not DRV, 72 h post infection; the HLAC medium comprising S-2209 was subsequently replaced with fresh HLAC medium comprising S-2209 every 72 h until day 12 post infection; it was then replaced by plain HLAC medium (i.e. not containing S- 2209 or DRV), and the plain HLAC medium refreshed every 72 h until day 21 post infection (iv) Addition of plain HLAC medium directly following infection, exchanging the HLAC medium for fresh plain HLAC medium 24 h post infection, and again replacing the HLAC medium with fresh plain HLAC medium every 72 h until day 21 post infection (i.e. no treatment with either DRV or S-2209).

Mock infected cells served as negative (background) controls. Two sets of experiments were performed. In the first, concentrations of 15 nM DRV and 500 nM S-2209 were used for the cells treated with the either DRV or S-2209, and 15 nM DRV and 100 nM S-2209 were used on cells sequentially treated with both substances; supernatant samples were harvested and stored at -80°C for subsequent analysis by RT -Assay or p24-ELISA with every medium exchange. The results of this set of experiments are shown in Fig. 13. In the supematants from cells infected with HIV-1_NL4/3 and not treated with either substance, viral loads increased rapidly from day 6 to day 12, returning to almost baseline by day 21 due to the lytic destruction of essentially all cells in the culture (Fig. 13, filled circles). In cells treated with DRV but not S-2209, supernatant viral loads showed a similar profile of increase followed by a decrease, but shifted to later time points by approximately 6 days (Fig. 13„ filled squares). Likely, viral replication was suppressed in the presence of 15 nM DRV, but could resume almost unhampered once DRV was removed. In cells treated with 500 nM S-2209 only under the above conditions (NB: onset of S-2209 treatment 72 h post infection, hence the EC₅₀/IC₅₀ determined above is not applicable here), the viral loads increased between day 6 and day 12, as in untreated cells, but did not reach the levels seen in untreated controls (Fig. 13„ unfilled triangles). When using S-2209 at the lower concentration of 100 nM under these conditions, no effect on viral loads was observed (data not shown). However, when cells were treated sequentially with both 15 nM DRV and 100 nM S-2209, viral loads in supernatants stayed essentially at baseline over the whole course of the experiment (Fig. 13„ filled triangles).

In the second set of experiments, a concentration of DRV was used that had previously been found to inhibit viral replication under the conditions of this experiment by about 50% (EC₅₀/IC₅₀) compared to untreated controls, up to about the point in time when viral loads start to decrease in untreated cells due to a lack of uninfected cells available for new productive infection, namely 7.5 nM DRV. Post infection, cells were hence treated with 7.5 nM DRV on days 1 to 3, 100 nM S-2209 on days 4 to 12, or a combination of both treatments, and supernatant samples were collected for ³²P analysis on days 1, 3, 6, 9, 12 and 15. The amounts of radioactivity measured in these samples were summed up to yield a number roughly

corresponding to an aggregate viral production. Here, neither the treatment with 7.5 nM DRV nor with 100 nM S-2209 had any effect on the sums over supernatant viral loads, but the combination treatment with 7.5 nM DRV followed subsequently by 100 nM S-2209 reduced supernatant viral loads to baseline (see Fig. 14).

Example 2.3: Eradication of HIV from human macrophages

Cells of macrophage lineage represent a key target of human immunodeficiency virus (HIV) in addition to CD4-lymphocytes. The absolute number of infected macrophages in the body is relatively low compared to CD4-lymphocytes.

Nevertheless, the peculiar dynamics of HIV replication in macrophages, their long- term survival after HIV infection, and their ability to spread virus particles to bystander CD4-lymphocytes, make evident their substantial contribution to the pathogenesis of HIV infection. In addition, infected macrophages are able to recruit and activate CD4-lymphocytes through the production of both chemokines and virus proteins (such as nef). In addition, the activation of the oxidative pathway in HIV- infected macrophages may lead to apoptotic death of not-infected bystander cells. Finally, macrophages are the most important target of HIV in the central nervous system. The alteration of neuronal metabolism induced by infected macrophages plays a crucial role in the pathogenesis of HIV-related encephalopathy. Taken together, these results strongly support the clinical relevance of therapeutic strategies able to interfere with HIV replication in macrophages. In vitro data show the potent efficacy of all nucleoside analogues inhibitors of HIV-reverse transcriptase in macrophages. Nevertheless, the limited penetration of some of these compounds in sequestered districts, coupled with the scarce phosphorylation ability of

macrophages, suggests that nucleoside analogues carrying preformed phosphate groups may have a potential role against HIV replication in macrophages. This hypothesis is supported by the great anti-HIV activity of tenofovir and other acyclic nucleoside phosphonates in macrophages that may provide a rationale for the remarkable efficacy of tenofovir in HIV-infected patients. Non-nucleoside reverse transcriptase inhibitors (NNRTI) do not affect HIV-DNA chain termination, and for this reason their antiviral activity in macrophages is similar to that found in CD4- lymphocytes. Interestingly, protease inhibitors, acting at post-integrational stages of virus replication, are the only drugs able to interfere with virus production and release from macrophages with established and persistent HIV infection (chronically- infected cells). Since this effect is achieved at concentrations and doses higher than those effective in de-novo infected CD4-lymphocytes, it is possible that lack of adherence to therapy, and/or suboptimal dosage leading to insufficient concentrations of protease inhibitors may cause a resumption of virus replication from chronically- infected macrophages, ultimately resulting in therapeutic failure. For all these reasons, therapeutic strategies aimed to achieve the greatest and longest control of HIV replication should inhibit HIV not only in CD4-lymphocytes, but also in macrophages. (Aquaro, S., et al, Antiviral Research 2002, 55:209-225).

It is therefore another objective of the invention to provide compounds capable of interfering with HIV replication in infected macrophages, and preferably of eradicating macrophages with the ability to infect other cells in an organism, potentially after a re-activation of dormant cells.

Macrophage differentiation

For differentiation to monocyte derived macrophages (MDM), monocytes were seeded in 24-well Nunc UpCell™ Surface plates (Nunc GmbH & Co KG,

Langenselbold, Germany) at a density of 10⁶ cells per well. Macrophage medium (DMEM containing 10% human serum, 5 mM glutamine, 5 mM PenStrep, 1 mM sodium pyruvate) was replaced 24h post seeding and every 3-4 days thereafter. After 10-14 days, completion of differentiation was determined visually by assessment of macrophage morphology.

For isolation, cells were detached by reducing the temperature below the threshold temperature of 32°C for the Nunc UpCell™ Surface. These plates are coated with a polymer surface having either hydrophobic (cell adhesion) or hydrophilic (cell detachment) character depending on the temperature, with a threshold temperature of 32°C. Cells were pelleted by centrifugation, 5 x 10⁴ cells seeded into 24 well plates and allowed to adhere for at least 48h. Determination of HIV replication in monocyte derived macrophages

Before infection with 1 ng macrophage-tropic HIV-1_NL4/3, the medium was replaced to get a final volume after infection of 250 μΐ. On day one post infection, 250 μΐ macrophage medium was added, 200 μΐ supernatant samples were transferred to 96 well plates, and the samples stored at -80°C until further analysis. In one set of experiments, the remaining supernatant was replaced by 500 μΐ fresh macrophage medium, optionally containing an inhibitor of HIV replication; on day 4, a 200 μΐ supernatant sample was taken and stored in a 96 well plates at -80°C until analysis, the remaining supernatant discarded and replaced by 500 μΐ fresh macrophage medium, again optionally containing an inhibitor of HIV replication. On day 8 post infection, another 200 μΐ supernatant sample was taken, the reminder of the supernatant discarded, and replaced by 500 μΐ of plain macrophage medium. Further supernatant samples were harvested every 3-4 days, terminating after 29 days (see Fig. 15), by transferring 200 μΐ supernatant to 96 well plates for storage at -80°C until analysis. Each time, the remaining supernatant was discarded, and 500 μΐ fresh macrophage medium added to each well. The results of this experiment are graphically represented in Fig. 16.

In a second set of experiments, the cells were incubated with plain medium between days 1 and 3, and 1 μΜ S-2209 was added to the medium for some cells only with medium changes on day 4 and on day 7, or only on day 7, the medium replaced by fresh plain macrophage medium on day 11 for all cells, and the experiment terminated on day 18. HIV replication was quantitated by performing RT-assays on the stored supernatant samples. Expression of green fluorescent protein tagged HIV-1-NL4/3-IRES-GFP in monocyte derived macrophages

HIV-1-NL4/3-IRES-GFP is a modified pBR-322 vector (Promega GmbH,

Mannheim, Germany) expressing the HIV-1 NL4-3 provirus with nef and eGFP from a bicistronic mR A with the help of an IRES element (Schindler, M., et al., J. Virol. 2003, 77: 10548-10556; Schindler, M., J. Virol. 2005, 79:5489-5498).

Macrophages were re-seeded in 24-well plates at a density of 5 x 10⁴ cells per well. Cells were infected with 20 ng HIV-1-NL4/3-IRES-GFP (M-tropic) in a final volume of 250 μΐ per well. On day one post infection, cells were washed three times, and 500 μΐ of fresh macrophage medium was added. S-2209 treatment was performed during infection and on day one post infection. On Days three and 10 post infection cells were analyzed by fluorescence microscopy.

A green fluorescence was easily detectable in untreated cells on day 3 and up to day 10 post infection, but no green fluorescence was observed in MDM infected with HIV- 1 -NL4/3-IRES-GFP and treated with 1 μΜ solutions of S-2209 up to 10 days post infection, at which point the experiment was terminated.

HIV infection may be eradicated from human macrophages by treatment with proteasome inhibitors

The luminescence activities found in supernatant samples of MDM cultures treated with 500 nM, 750 nM and 1 μΜ solutions of S-2209 between days 1 and 8 post infection are depicted in Fig. 16. Evidently, S-2209 severely represses HIV replication even at the lowest concentration employed, and fully disallows replication of HIV at the highest concentration of 1 μΜ under experimental conditions.

Furthermore, as is evident from Fig. 17, viral replication can be severely reduced, if not abolished, in macrophages by the addition of S-2209 to their culture medium even after the infection has established itself firmly, e.g. 4 and/or 7 days post in vitro infection. Corroborating evidence is found in the fact that when monocyte derived macrophage cultures were infected with the green fluorescent protein expressing HIV-1-NL4/3-IRES-GFP, the green fluorescence was easily detectable in untreated cells on day 3 and up to day 10 post infection, but no green fluorescence was observed in MDM infected with HIV- 1 -NL4/3-IRES-GFP and treated with 1 μΜ solutions of S-2209 up to 10 days post infection, at which point the experiment was terminated.

It was thus established, that HIV infection can effectively be suppressed in monocyte derived macrophages by treatment with a proteasome inhibitor, and preferably by treatment with S-2209, thereby reducing or eliminating the potential of infected macrophages to act as a viral reservoir.

Example 2.4: Inhibition of replication of an omni-resistant HIV isolate.

Finding the right combination of antiviral therapies for each patient is often the key to combating HIV, wherein an assessment of potential viral resistance to standard therapeutic regimens needs to be taken into account. However, HI viruses are emerging at an increasing rate that are resistant to multiple, and sometimes all available, drug therapies. This makes the disease more and more difficult to treat, and underscores the need for further treatment options.

Inhibition of replication of omni-resistant strain IVJ_LRI A recombinant viral vector, designated as 7LR1, was created using samples from patients showing multiple resistances towards HAART standard therapies (see: Walter, H., et. al, J Clin Virol, 1999, 13:71-80). In appropriate assays taken from the literature, the relative sensitivity of this viral isolate towards the agents of standard HIV therapy was assessed. The following relative sensitivities were established (numbers represent fold-resistance; e.g. where the number given is 25, the IC₅₀ for the relevant virus and for the relevant agent is 25 times higher than the IC₅₀ for a reference wild type virus (here: HIV-1_NL-₄/3) for the same agent)

Protease inhibitors:

IDV - Indinavir: >190,

SQV - Saquinavir: >90,

RTV - Ritonavir: >140,

NFV - Nelfmavir: >70,

APV - Amprenavir: >120,

LPV - Lopinavir: >105,

ATV - Atazanavir: >150,

TPV - Tipranavir: 35,

DRV - Darunavir: 13;

Nucleoside analog reverse transcriptase inhibitors (NRTIs):

TDF - Tenofovir: 5,

ABC - Abacavir: 4,

FTC - Emtricitabine: 13,

d4T - Stavudine: 2,

ddl - Didanosine: 2,

AZT - Zidovudine: >230 Non-nucleoside reverse transcriptase inhibitors (NNRTIs):

DLV - Delavirdine: >19,

EFV - Efavirenz: >38,

NVP - Nevirapine: >72. Inhibition of replication of omni-resistant strain HIVJ_LRI

In this experiment the inhibitory action of S-2209 against omni-resistant HIV_7LRI isolate was compared to standard antiviral agents (darunavir, DRV; saquinavir, SQV; nevirapin, NVP; lamivudine, 3TC). Tonsillary tissue was prepared and HLAC (Human Lymphocyte Aggregate Cultures cells) were adjusted to a final cell concentration of lxlO⁷ cell/ml. The cells were seeded in 96-well plates (200 μΐ/well; 2xl0⁶ cells/well) and infected on the following day with HIV-1_7LRI (2 ng p24 per 2xl0⁶ HLAC cells, 5% of total volume). Infection was allowed to progress overnight, cells were washed with three changes of HLAC medium on day 1 post infection and new HLAC medium provided, and the HLAC medium was again exchanged for fresh HLAC medium on day 3 post infection. From day 3 until termination of the experiment on day 24 post infection, the HLAC medium was exchanged for fresh medium every three days. With every medium change, supernatant samples (100 μΐ) were harvested and stored at -80°C for subsequent analysis by RT-assay. All experiments were done as triplicates.

To compare the efficacy of S-2209 in inhibiting HIV_7LRI replication with the respective efficacy of other anti- viral compounds, HLAC medium was supplemented with S-2209 to attain concentrations of 250 nM, 500 nM, 750 nM or 1 μΜ, or with darunavir (DRV) to a concentration of 15 nM, saquinavir (SQV) to a concentration of 20 nM, nevirapine (NVP) to a concentration of 200 nM, or lamivudine (3TC) to a concentration of 300 nM.

HLAC were treated with medium comprising S-2209 on the day of infection and again 24 h later, finally exchanging the medium comprising S-2209 for plain medium on day 3 (i.e. treatment with S-2209 lasted from the day of infection until the medium change on day 3), while DRV, SQV, NVP, 3TC treatment was performed over the whole course of the experiment until termination.

Values obtained for viral replication from RT assays performed on supematants were summed over the whole course of the experiment, and normalized to the result obtained from mock-treated controls (i.e. set as 100% replication). None of DRV, SQV, NVP, or 3TC was able to reduce HI V_7LRI -replication, while treatment with S- 2209 at a concentration of 250 nM between day 0 (day of infection) and day 3 postinfection reduced replication by 25%, and essentially allowed no HI V_7LRI -replication at higher concentrations.

This result establishes the potential usefulness of the semicarbazone proteasome inhibitor S-2209 in the treatment of resistant, multi-resistant, or even omni-resistant Human Immunodeficiency Viruses.

Claims

A semicarbazone proteasome inhibitor for use in the treatment of hepatitis virus-infected individuals and/or HIV-infected individuals.

The semicarbazone proteasome inhibitor for use in the treatment according to claim 1 , wherein the hepatitis virus infected individuals are infected with Hepatitis C Virus (HCV).

The semicarbazone proteasome inhibitor for use in the treatment according to any one of claims 1 and 2, wherein said semicarbazone is selected from [1-[1- { 1 -[(2,4-Dioxo-imidazolidin- 1 -ylimino)-methyl]-2-phenyl-ethylcarbamoyl} - 2-( 1 H-indo 1-3 -yl)-ethylcarbamoy 1] -2-( 1 H-indo 1)] , a pharmaceutically acceptable salt thereof or structural and/or functional analogues thereof.

The semicarbazone proteasome inhibitor for use according to any of claims 1 to 3, wherein the HCV-infected individuals are selected from the following groups of patients:

(i) patients not refractory to interferon and/or ribavirin;

(ii) patients treated with the semicarbazone proteasome inhibitor as

defined herein as the sole active agent;

(iii) patients responding to interferon and/or ribavirin therapy;

(iv) patients not being treated with interferon and/or ribavirin; (v) patients treated with interferon and/or ribavirin that are not interferon- and/or ribavirin-refractory; (vi) patients as defined in any one of (i) to (v) having a liver cirrhosis;

(vii) patients previously not treated with further anti-HCV medicaments; (viii) patients as defined in any of (i) to (vii) having increased transaminase concentrations;

(ix) patients as defined in any one of (i) to (vi) not having increased

transaminase concentration; (x) patients as defined in any one of (i) to (ix) not having a chronic HCV- infection;

(xi) patients as defined in any one of (i) to (ix) having a chronic HCV- infection; (xii) patients showing a sustained viro logical response (SVR) to interferon- and/or ribavirin-therapy; (xiii) patients showing a rapid viro logical response interferon- and/or

ribavirin-therapy;

(xiv) patients as defined in any of (i) to (x) and (xii) to (xiii) having an acute asymptomatic or mildly symptomatic HCV infection; (xv) patients as defined in any of (i) to (xiii) having a persistent HCV- infection;

(xvi) patients as defined in any of (i) to (xv) having a positive anti-HCV immunoassay;

(xvii) patients as defined in any of (i) to (xvi) positively tested for the

presence of HCV-R A;

(xviii) patients as defined in any of (i) to (xvii) having leukopenia and/or thrombocytopenia;

(xix) patients as defined in any of (i) to (xviii) having an anemia, a

cardiovascular or a cerebrovascular disease.

A pharmaceutical composition comprising semicarbazone proteasome inhibitor for use in the treatment according to any one of claims 1 to 4, wherein said semicarbazone is selected [l-[l-{l-[(2,4-Dioxo-imidazolidin-l- ylimino)-methyl] -2-phenyl-ethylcarbamoyl} -2-( 1 H-indo 1-3-yl)- ethylcarbamoyl]-2-(lH-indol)], a pharmaceutically acceptable salt thereof or structural and/or functional analogues thereof for use in the treatment of hepatitis virus infected and/or HIV-infected individuals.

The pharmaceutical composition according to claim 5 for use in the treatment of hepatitis virus infected individuals, wherein said hepatitis virus is HCV selected from the following groups of patients: ) patients not refractory to interferon and/or ribavirin; patients responding to interferon and/or ribavirin therapy; patients not being treated with interferon and/or ribavirin; patients treated with interferon and/or ribavirin that are not interferon- and/or ribavirin-refractory; patients as defined in any one of (i) to (iv) having a liver cirrhosis; patients previously not been treated with further anti-HCV

medicaments; patients as defined in any of (i) to (vi) having increased transaminase concentrations; patients as defined in any one of (i) to (vi) not having increased transaminase concentration; patients as defined in any one of (i) to (viii) not having a chronic HCV-infection; patients as defined in any one of (i) to (viii) having a chronic HCV- infection; patients showing a sustained viro logical response (SVR) to interferon- and/or ribavirin-therapy; (xii) patients showing a rapid viro logical response interferon- and/or ribavirin-therapy;

(xiii) patients as defined in any of (i) to (viii) and (xi) to (xii) having an acute asymptomatic or mildly symptomatic HCV infection;

(xiv) patients as defined in any of (i) to (viii) and (xi) to (xii) having a persistent HCV-infection;

(xv) patients as defined in any of (i) to (xiv) positive in anti-HCV

immunoassay;

(xvi) patients as defined in any of (i) to (xv) positively tested for the

presence of HCV-R A;

(xvii) patients as defined in any of (i) to (xvi) having leukopenia and/or thrombocytopenia;

(xviii) patients as defined in any of (i) to (xvii) who have an anemia,

cardiovascular or cerebrovascular disease.

A semicarbazone proteasome inhibitor for use in the treatment as defined in claims 1 to 4 or a pharmaceutical composition as defined in claim 5 or 6 for use in the treatment of an HIV infection and/or hepatitis virus infections, particularly an infection with HCV, wherein said semicarbazone and/or pharmaceutical composition is used in a combined treatment with at least one further medicament for treating HIV-infected and/or hepatitis-virus-infected, particularly HCV-infected, individuals. A kit comprising the semicarbazone proteasome inhibitor as defined in 3 or a pharmaceutical composition as defined in claim 5 for use in the treatment of HIV-infected and/or hepatitis- virus-infected individuals, particularly HCV-infected, individuals.