WO2000033654A1 - Utilisation d'inhibiteurs de protease pour moduler les chemins cellulaires, immunite et therapies associees - Google Patents
Utilisation d'inhibiteurs de protease pour moduler les chemins cellulaires, immunite et therapies associees Download PDFInfo
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- WO2000033654A1 WO2000033654A1 PCT/US1999/028548 US9928548W WO0033654A1 WO 2000033654 A1 WO2000033654 A1 WO 2000033654A1 US 9928548 W US9928548 W US 9928548W WO 0033654 A1 WO0033654 A1 WO 0033654A1
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- the present invention hinges on the discovery that molecules that target proteases, especially viral or microbial proteases such as viral protease inhibitors, or proteasome inhibitors, may be used to modulate cellular pathways that regulate cell metabolism, cell activation, cell proliferation, cell differentiation, cell mutation, cell cycle and cell death. Because of the myriad of cellular pathways that may be modulated according to the invention using such inhibitors, the subject methods have numerous applications in the fields of human therapy and diagnosis, agriculture and veterinary medicine.
- the present invention relates to the use of HIN protease inhibitors or proteasome inhibitors to modulate cellular pathways.
- the invention relates to a method for enhancing the efficacy of vaccines and a method for modulating the immune system as well as modulating cellular immunity, cellular metabolism, cell proliferation, cell differentiation, cell maturation, cell cycle, and cell activation using HIN protease inhibitors, or inhibitors of proteasomes.
- HIV-1 Human Immunodeficiency Virus type I (HIV-1) (Emini et al., Journal of Virology, 64:3674-8, 1990; Emini et. al, Nature, 355:728-30, 1992; Conley et. al., Journal of Virology, 70:6751-8, 1996; Haigwood et. al., Immunology Letters, 51 : 107-14, 1996; Putkonen et. al., Nature, 352:436-8, 1991 ; Prince et. al., AIDS Research & Human Retroviruses, 7:971-3, 1991; Parren et.
- HIV-1 Human Immunodeficiency Virus type I
- HAART highly active anti-retroviral therapy
- a successful therapy or vaccine must be aimed at both blocking HIV replication, as well as eliminating the viral reservoir.
- One strategy is to significantly enhance HIV-specific immune responses to the "super natural" level such that HIV replication is blocked and latently infected cells are eliminated. This will, however, require enhancing the immune response beyond the level of that obtained by HIV infection itself.
- therapeutic vaccines have to overcome the limitations of immune responses to natural HIV infection.
- protease inhibitors can have a significant effect on opportunistic infections with HIV-1 infection and AIDS. While in some instances the use of protease inhibitors has a beneficial effect on the incidence and resolution of opportunistic infections, paradoxically the use of such inhibitors has in some instances had an adverse effect, i.e., resulted in worsening or increased incidence of such infections.
- Jacobsen et al Lancet 349: 1443-1445 (1997); Michelet et al, AIDS 12: 1815-1822 (12998); Rodriguez-Rosado et al, Antiviral Therapy 3:229-231 (1998); Narita et al, Am. J. Respor. Crit.
- CD4+ T-cells increase remarkably (Perrin et al, Science 280: 1871-1873 (1998)), whereas in some other patients, transient reactivation of other persistent viruses after treatment has been observed (Jacobsen et al, Lancet 349: 1443-1495 (1997); Carr et al, Lancet 349:993-996 (1997); and Rutschmann et al, J. Infect. Dis. 177:783-785 (1998)).
- CTLs cytotoxic T lymphocytes
- Ritonavir may modulate proteasome activity and major histocompatibility complex (MHC) class I-restricted presentation of several LMCV and MART-1 epitopes in vivo and in vitro and that these observations may help to explain some ofthe observations made in AIDS patients undergoing highly active anti-retroviral therapy.
- MHC major histocompatibility complex
- HIV-1 protease inhibitor or a proteasome inhibitor as a model to ascertain the effects of protease inhibitor, especially microbial or viral protease inhibitors such as cysteine or serine protease inhibitors on cellular pathways that regulate cell-metabolism, -activation, -proliferation, -differentiation, -maturation, -cycle and -death.
- protease inhibitor especially microbial or viral protease inhibitors such as cysteine or serine protease inhibitors on cellular pathways that regulate cell-metabolism, -activation, -proliferation, -differentiation, -maturation, -cycle and -death.
- an HIV-1 protease inhibitor or proteasome inhibitor as a model to evaluate the effects of protease inhibitors on systemic metabolic changes such as increases in blood sugar, lipids, and decreases in protein wasting. It is another object ofthe invention to provide novel treatments involving the administration of at least one protease inhibitor, e.g, an HIV protease, proteasome, serine protease or cysteine protease inhibitor, to a human or animal in need of such treatment, wherein such treatment is effected to modulate at least one cellular pathway that regulates cell-metabolism, -activation, -proliferation, -differentiation, -maturation, -cycle or -death.
- protease inhibitor e.g, an HIV protease, proteasome, serine protease or cysteine protease inhibitor
- at least one protease inhibitor e.g, an HIV protease, proteasome, serine protease, or cysteine protease inhibitor
- a protease e.g, a microbial or viral protease, such as HIV-1 protease inhibitor, proteasome inhibitor, serine protease inhibitor, or cysteine protease inhibitor.
- at least one protease inhibitor such as an HIV-1 protease or proteasome inhibitor
- a protease inhibitor such as an HIV- 1 protease or proteasome inhibitor.
- a protease inhibitor e.g, an HIV-1 protease, proteasome, cysteine protease, or serine protease inhibitor.
- a protease inhibitor e.g, an HIV-1 protease, proteasome, cysteine protease, or serine protease inhibitor.
- a protease inhibitor e.g, an HIV-1 protease, proteasome, cysteine protease, or serine protease inhibitor.
- a protease inhibitor e.g, an HIV-1 protease, proteasome inhibitor, cysteine protease, or serine protease inhibitor.
- a protease inhibitor e.g, an HIV-1 protease, proteasome inhibitor, cysteine protease, and serine protease inhibitor.
- a protease inhibitor e.g, an HIV-1 protease, proteasome, serine protease, or cysteine protease inhibitor.
- a protease inhibitor e.g, an HIV-1 protease, proteasome, cysteine protease, or o serine protease inhibitor.
- a protease inhibitor e.g, an HIV protease, proteasome, cysteine protease, or serine protease inhibitor.
- a protease inhibitor e.g, an HIV protease, proteasome, serine protease, or cysteine protease inhibitor.
- a protease inhibitor e.g, an HIV protease, proteasome, cysteine protease, or serine protease 5 inhibitor.
- a protease inhibitor e.g, an HIV protease, proteasome, cysteine protease, or serine protease inhibitor.
- a protease inhibitor e.g, an HIV protease, proteasome, serine protease, or cysteine protease inhibitor.
- protease inhibitor e.g, an HIV-1 protease inhibitor, proteasome inhibitor, serine protease inhibitor, or cysteine protease inhibitor.
- FIGURES Figure 1: Ritonavir related changes in susceptibility to cell death. Preactivated PBMC were exposed to IFN-alpha (300IU/ml), IFN-gamma (30ng/ml), and TNF (lOng/ml), or their combinations for 3 days in the presence or absence of Ritonavir. After three days, CD95 agonistic mAb CHI 1 (200ng/ml) was added for 16h. Cell viability as assessed by standardized WST-1 assays in quadruplicate cultures is depicted (Means ⁇ SD). Differences between Ritonavir treated and untreated groups are statistically significant (paired t-Test, p ⁇ 0.05). One representative experiment is illustrated out of four performed with similar results from different cell donors. Figure 2:
- HIV Protease Inhibitor related changes in susceptibility to cell death Preactivated PBMCs were exposed to IFN-alpha (50 ng/mL), IFN-gamma (50 ng/mL) and TNF-alpha (50 ng/mL) in combination in the presence or absence of HIV Protease Inhibitors at low, medium and high concentrations, before adding CD95 agonistic monoclonal antibody, CHI 1. Cell viability was assessed by standardized WST-1 assays.
- HIV Protease Inhibitor related changes in susceptibility to cell death Preactivated PBMCs were exposed to IFN-alpha (50 ng/mL), IFN-gamma (50 ng/mL) and TNF-alpha (50 ng/mL) in combination in the presence or absence of HIV Protease Inhibitors at low, medium and high concentrations, before adding CD95 agonistic monoclonal antibody, CHl l . Apoptosis Index was assessed by standardized TUNEL assays.
- HIV transgene expression as measured in RNA extracts from tail clippings, that were probed with full length HIV-1 probes (Northern blot), was decreased by Ritonavir treatment.
- Figure 9 Decrease in HIV transgene expression by Ritonavir. HIV transgene expression as measured in RNA extracts from tail clippings, that were probed with full length HIV-1 probes (Northern blot), was decreased by Ritonavir treatment.
- Viable CD3+ cells were gated and histogram overlays are shown for: A) Intracellular Caspl (ICE) staining; Ritonavir treated cells (lOnM, slim-lined curve) are compared to untreated controls (bold line). Controls for non-specific stain (dotted curve) were obtained by competition with specific peptides to anti-Caspl antibodies. B) Cell surface staining with Annexin V; intensity of staining in Ritonavir treated T-cells (slim line) is compared to that of untreated cells (bold). C) Cell membrane staining for CD95 (Fas)-ligand in CD3+ CD4+ cells; Ritonavir treated cells (line) are compared with untreated cells (bold).
- NFAT activation by HHV8 orf-74 is inhibited by Ritonavir.
- the nuclear factor of activation in T cells represents an important element in the regulation of cellular functions.
- Viral genes such as the human herpes virus 8 (HHV-8) derived open reading frame 74(orf-74) are known to activate NFAT in order to gain control over infected or bystander cells so as to promote viral replication and spread.
- Protein lysates of orf-74 co-transfected cells were prepared from cultures after 12 hours of treatments as indicated in the figure, and luciferase activity was measured by luminescence. This represents yet another example for the regulation of cellular pathways by protease inhibitors that were designed to target a viral protease.
- Figure 23 Inhibition of HIV-Tat induced NFkB activity by Ritonavir. Ritonavir inhibits
- NFkB activity induced by HIV-1 Tat protein The nuclear factor kappa B (NFkB) translocates from the cytoplasmic compartment of a cell to the nucleus, where it binds to promotor elements of cytokine- and other regulatory genes, thus initiating changes in cellular activation and function.
- the HIV-encoded Tat protein is known to induce the activation ofthe cellular NFkB-pathway.
- Luciferase-reporter gene constructs that are responsive to NFkB activation were used to measure the effect of cotransfected HIV-1 tat-gene products in Cos cells on NFkB under the influence of Pl-treatment. As depicted in the figure, luciferase activity in protein extracts from PI treated cells was inhibited in a dose-dependent fashion, indicating that ritonavir directly affects cellular pathways and functions.
- Figure 24 luciferase activity in protein extracts from PI treated cells was inhibited in a dose-dependent fashion,
- the control group of animals was injected with Phosphate Buffered Saline (PBS, placebo treatment). After 2 weeks of treatment, a significant inhibition of tumor growth was observed in animals treated with ritonavir. Representative examples of the treatment groups (6 animals in each group) are depicted in the fotographs.
- the control group of animals was injected with Phosphate Buffered Saline (PBS, placebo treatment). Tumor size was measured and volumes calculated as illustrated (means+SD are calculated from 5 animals per group)
- Figures 26a and 26b
- FIG 28 Inhibition of cell adhesion by Ritonavir This figure contains the results of an experiment that measured the inhibition of cellular adhesion as a functional readout of Ritonavir and MG132 (proteasome inhibitor) mediated inhibition of cellular activation (relevant for inflammation, lymphocyte infiltration and neo-vascularization).
- Indicator cells HL-60 and U937 were loaded with intracellular fluorescent dye (Molecular Probes, Inc., calcein AM) and after application of defined sheer forces, the amount of adhering cells was measured by fluorescence intensity per culture (WALLACE systems).
- Figure 29 Re.storation of hematopoietic function by Rritonavir.
- Hematopoietic functions are restored by ritonavir treatment in TG26 mice after 3 weeks of treatment as assessed in (hemato-poietic progenitor cell) colony formation assays (CFU-assay) using standard methylcellulose cultures (StemCell Technologies). HIV transgene expression as measured in RNA extracts from tail clippings, that were probed with full length HIV-1 probes (Northern blot) is decreased by ritonavir treatment.
- Molecules or compounds ofthe invention are molecules or compounds that target, bind to, or interact with a protease and thereby modulate at least one of its activities.
- a protease inhibitor preferably a microbial or viral protease inhibitor, and more preferably HIV-1 protease, proteasome, serine protease, or cysteine protease inhibitor.
- Examples thereof include, e.g, Ritonavir, Saquinavir, Nelfinavir and Indinavir, MG132, lactacystin, or cytochrome P450 inhibitor (terminal oxidase ofthe cellular microsomal mixed- function monoclonal- oxygenase system).
- HIV-1 Protease inhibitor - A class of molecules of the invention that inhibit function of HIV-1 function.
- HIV-1 Protease - A protease produced by HIV that functions to specifically cleave the HIV-1 Gag-Pol polypeptide into Gag and Pol proteins, which cleavage is essential for viral assembly and maturation of HIV-1.
- Proteasome - A multi-subunit complex that degrades specifically targeted proteins which is found in all eukaryotic cells and some bacteria. This protease complex is responsible for processing peptides for presentation on major histocompability complex 1 (MHC-1) molecules on the surface of cells.
- MHC-1 major histocompability complex 1
- Cysteine Protease - A protease that cleaves a peptide or protein at a site that comprises a cysteine residue.
- Host- A host includes humans, non-human primates, non-human mammals, and ungulates. Especially included are agricultural animals, and domestic animals, such as dogs and cats.
- the present invention is broadly directed toward the use of molecules that target proteases, especially microbial or viral proteases, or proteasome to modulate cellular pathways, e.g, pathways involved in cell metabolism, cell activation, cell proliferation, cell differentiation, cell maturation, cell cycle and cell death, or to induce systemic changes in metabolism such as changes in sugars, lipids, or protein metabolism.
- proteases especially microbial or viral proteases, or proteasome to modulate cellular pathways, e.g, pathways involved in cell metabolism, cell activation, cell proliferation, cell differentiation, cell maturation, cell cycle and cell death, or to induce systemic changes in metabolism such as changes in sugars, lipids, or protein metabolism.
- Such modulation of cellular pathways or systemic metabolism using a protease inhibitor will be utilized to effect human or animal therapy, e.g, in agricultural, veterinary, or therapeutic applications.
- the present invention is directed toward the use of microbial or viral protease inhibitors or HIV-1 protease, proteasome, serine protease, or cysteine protease inhibitors in particular to treat diseases and conditions including cancer, autoimmune disorders, keratinization disorders, infection (including non-HIV infection), transplantation, graft- vs-host or host-vs- graft disease, as an adjunct to radiation, immuno- or chemotherapy, acute ischemia, allergic disorders, inflammatory disorders such as arthritis, psoriasis, chronic inflammation, inflammatory bowel syndrome, conditions associated with aging, wasting syndrome, disorders associated with abnormal metabolism, such as those that affect sugar, lipid and protein metabolism.
- diseases and conditions including cancer, autoimmune disorders, keratinization disorders, infection (including non-HIV infection), transplantation, graft- vs-host or host-vs- graft disease, as an adjunct to radiation, immuno- or chemotherapy, acute ischemia, allergic disorders, inflammatory disorders such as arthritis, ps
- HIV protease inhibitors are in the context of HIV protease inhibitors or proteasome inhibitors
- other molecules ofthe invention can be used without altering the scope of the invention, i.e., inhibitors of other proteases, or proteasome inhibitors.
- serine protease inhibitors and cysteine protease inhibitors are intended to fall within the scope ofthe present invention. Therefore, in all instances HIV-1 protease inhibitor should be construed to be exemplary of protease inhibitors which are suitable for use in the claimed methods.
- the protease inhibitor will be a molecule that inhibits cellular proteasome or HIV-1 protease.
- HIV-1 proteases function to specifically cleave the HIV-1 Gag-Pol polypeptide into Gag and Pol proteins. This post-translational cleavage step is essential for viral assembly and maturation of the virion (Fields et. al. Fields Virology, 3 rd ed, Lippincott-Raven Publishers, Philadelphia, 1996).
- the crystal structure and binding site specificity of the HIV-1 protease have been defined, allowing for the design of protease inhibitors which interfere with substrate binding and protease function.
- these inhibitors In addition to inhibiting HIV proteases, it has also been shown that these inhibitors also inhibit the functions of cellular proteasome, a multi-subunit complex of proteases that degrades specifically targeted proteins.
- Proteasome which are found in all eukaryotic cells and in some bacteria such as archaebacteria, are responsible for producing peptides for presentation on major histocompatibility complex I (MHC-I) molecules on the surface of cells (Janeway et. al, Immunobiology: The immune system in health and disease, 2 nd ed. Garland Publishing, New York, 1996). Thus, inhibitors of proteasome prevent antigen presentation on MHC class I molecules resulting in a partial loss of immune function.
- MHC-I major histocompatibility complex I
- HIV protease inhibitors and proteasome inhibitors are well known in the art.
- examples of such inhibitors include, but are not limited to, the following compounds including derivatives or analogs thereof: HIV protease inhibitors- Saquinavir (Invirase and Fortovase), Ritonavir (Norvir), Indinavir (Crixivan) (Lacy et. al. Conn Med, 60(12): 723-7, 1996), Nelfinavir (Viracept) (Kaldor et. al, JMed Chem, 40(24):3979-85, 1997), Amprenavir (Agenerase) (No Authors Listed, Med. Lett. Drugs.
- Palinavir Lamarre et. al, Antimicrob Agents Chemother, 41(5):965-71, 1997), nonpeptidic HIV protease inhibitors (Vara Prasad et. al, Bioorg. Med. Chem. Lett., 9 (11): 1481-6, 1999), for example, Tipranavir (PNU- 140690) (Poppe et.
- HIV protease inhibitors small dipeptide-based HIV protease inhibitors, HIV protease inhibitors containing allophenylnorstatine (an unnatural amino acid), piperazine hydroxyethylamine based HIV protease inhibitors, hydroxyethylamine based HIV protease inhibitors, tetrahydropyrimidinone containing HIV protease inhibitors, cyclic HIV protease inhibitors such as cycloalkylpyranones and cycloalkyldihydropyrones (Romines et.
- HIV-1 protease inhibitors PR-I
- proteasome inhibitors are potent immune modulators.
- the ability of PR-I to modulate the immune system is independent of HIV-1 protease inhibition and is not related to HIV infection, since similar effects were observed in HIV free experimental systems.
- the inventors have found that exposure of cells (human and murine) to HIV-protease inhibitors, more specifically Ritonavir (Norvir), at concentrations that are achieved in humans under therapy for HIV disease effect changes in cell activation, proliferative capacity, and function. Further, these changes include but are not limited to an enhanced ability of cells to survive differently induced stress conditions (e.g, metabolic, apoptotic and the like).
- protease inhibitors may elicit effects on cellular pathways that affect immune and other cellular reactions.
- protease inhibitors such as HIV-1 protease or proteasome inhibitors affect cytokine expression, i.e., the specific cytokines which are expressed and the levels of expression thereof are cytokines well known to have profound effects on immunity, e.g, they are involved in inflammatory responses, and whether a host elicits a Thl (cellular) or Th2 (humoral ) immune response against a particular antigen. Based on such effects, protease inhibitor administration affords a means of modulating cytokine expression, and thereby the various cellular and immune effects that are affected thereby. For example, it may provide for the activation of different types of T-cells.
- protease inhibitors such as proteasome inhibitors may interact with chaperones such as Heat Shock Protein (HSP70, HSP72) resulting in the activation of expression thereof.
- HSP70, HSP72 Heat Shock Protein
- protease inhibitors modulate apoptosis and neovascularization.
- the present invention is directed toward the use of a protease inhibitor such as an HIV-1 protease or proteasome inhibitor to modulate cellular pathways, in particular those that regulate cell metabolism, cell activation, cell proliferation, cell differentiation, cell mutation, cell cycle and cell death. Accordingly, the invention will be used to treat or prevent any disease or condition wherein modulation of any of the foregoing cellular pathways provides an effective means of treatment or prophylaxis.
- a protease inhibitor such as an HIV-1 protease or proteasome inhibitor to modulate cellular pathways, in particular those that regulate cell metabolism, cell activation, cell proliferation, cell differentiation, cell mutation, cell cycle and cell death.
- the present invention is directed toward the use of protease inhibitors to treat disorders or conditions involving abnormal cell metabolism such as cancer or dermatological conditions.
- the present invention is directed toward the use of a protease inhibitor to treat disorders or conditions involving aberrant cell activation, proliferation or differentiation.
- Such conditions include, by way of example, autoimmune disorders, inflammatory conditions, cancers, keratinization disorders, dermatological disorders or autoimmune diseases.
- the present invention is directed toward treatment of diseases and conditions wherein modulation of cell cycle or cell death is desirable.
- diseases and conditions include, by way of example, cancer, infection, metastasis, aging or wasting syndrome.
- a method to modulate the immune system of a host comprising administering a therapeutically effective amount of a molecule or compound ofthe mvention such as an HIV protease inhibitor or proteasome inhibitor.
- a therapeutically effective amount of a molecule or compound ofthe mvention such as an HIV protease inhibitor or proteasome inhibitor.
- an HIV protease inhibitor can be used to modulate one aspect ofthe immune system, immune cell activation.
- an effective amount ofthe protease inhibitor is administered to a patient with a condition or disease of the immune system in order to modulate immune cell activation.
- the dose of protease inhibitor to be administered can be determined by methods well known in the art.
- protease inhibitors of the invention may be administered to a human or other animal in an amount sufficient to produce a therapeutic, prophylactic, cosmetic or dermatological effect.
- Suitable protease inhibitors ofthe invention e.g, HIV protease inhibitor, or proteasome inhibitor, can be administered to such human or other animal in a conventional dosage form prepared by combining an HIV protease inhibitor or proteasome inhibitor, or other protease inhibitor, with a conventional pharmaceutically, cosmetically or dermatologically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
- the route of administration of the protease inhibitor (e.g, HIV protease inhibitor or a fragment thereof) of the invention may be oral, parenteral, by inhalation or topical.
- parenteral as used herein includes intravenous, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Subcutaneous and intramuscular forms of parenteral administration are generally preferred. However, the preferred mode of administration will vary, e.g, dependent upon the particular condition treated.
- the daily parenteral and oral dosage regimens for administering protease inhibitor according to the invention will generally be at concentrations that are achieved in humans under therapy for HIV disease.
- the daily parenteral and oral dosage regimens for employing compounds of the invention will be in the range of about 0.05 to 100, but preferably about 0.5 to 20, milligrams per kilogram body weight per day.
- the protease inhibitors ofthe invention e.g, HIV-1 protease inhibitor or proteasome inhibitor, may also be administered by inhalation.
- inhalation is meant intranasal and oral inhalation administration.
- Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler may be prepared by conventional techniques.
- the preferred dosage amount of a compound ofthe invention to be employed is generally within the range of about 10 to 100 milligrams.
- protease inhibitors ofthe invention may also be administered topically.
- topical administration non-systemic administration and includes the application of an HIV protease inhibitor (or a fragment thereof) ofthe invention externally to the epidermis, to the buccal cavity, a instillation of such an HIV protease inhibitor into the ear, eye and nose, where it does not significantly enter the blood stream.
- This mode of administration is particularly desirable in the context of cosmetic or dermatological applications of the invention, e.g, in skin creams, ointments, or other topically administrable forms for the treatment of the skin.
- systemic administration is meant oral, intravenous, intraperitoneal and intramuscular administration.
- protease inhibitor e.g, HIV-1 protease inhibitor
- amount of a protease inhibitor required for therapeutic or prophylactic effect will, of course, vary with the particular protease inhibitor chosen, the nature and severity of the disease or condition of the immune system being treated and the human or animal undergoing treatment, and is ultimately at the discretion of the physician.
- a suitable topical dose of an HIV protease inhibitor or proteasome inhibitor will generally be within the range of about 1 to 100 milligrams per kilogram body weight daily.
- the protease inhibitor is a type one HIV protease inhibitor, such as Ritonavir (Norvir).
- protease inhibitors such as HIV-1 protease or proteasome inhibitors
- an HIV protease inhibitor can be administered to a patient to increase or decrease immune cell proliferation of particular types of immune cells, e.g, particular T-cell subsets.
- Yet another embodiment ofthe invention involves modulating non-immune cell proliferation. This can be effected in vivo or in vitro, e.g, an inhibitor can be used to treat bone marrow during purging procedures, e.g, effected during treatment of B-cell lymphoma.
- a protease inhibitors can be used to support cell generation poiesis, including blood cell growth and generation (pro- hematopoietic effect) after depletion or destruction of cells, as caused by, for example, toxic agents, radiation, immunotherapy, growth defects, malnutrition, immune dysregulation, anemia and the like.
- the protease inhibitors ofthe invention can provide a therapeutic control of tissue generation and degradation, and a therapeutic modification of cell and tissue maintenance and blood cell homeostasis.
- HIV protease inhibitors In HIV infected patients, the therapeutic application of HIV protease inhibitors commonly leads to a substantial increases in CD4+ T-cell counts but, interestingly, not always to an immediate decrease, or to only insignificant decreases, in HIV viremia (Levitz et al. New England J. Med., 338/15, 1074, 1998). Even after the initial decrease of plasma HIV load, a viral "break-through" is frequently observed while T-cell counts remain stable and increased (as compared to the time of therapy-initiation). Thus, HIV-PR-I and other protease inhibitors may have additional beneficial effects on T-cell homeostasis.
- HIV-1 and other protease inhibitors are for the treatment of disorders involving T-cells such as T-cell related cancers, T-cell related autoimmune disorders, inflammatory conditions, infections, aplastic anemia, multiple sclerosis, and DiGeorge syndrome.
- the overall finding ofthe invention hinges on the discovery that HIV protease inhibitors, although specifically designed to block HIV protease activity, have additional and direct effects on immune cell functions. These "side effects" may represent an important part of HIV PR-I's therapeutic effects.
- HIV protease and proteasome inhibitors Ritonavir for example, applied at therapeutic or lower concentrations, increased the colony forming capacity (CFC) of human bone marrow derived mononuclear cells (BM-MNC).
- CFC colony forming capacity
- peripheral blood derived T-cells responded with phenotypic changes in cell surface marker expression including CD25, CD28, and CD95(Fas)-ligand, as well as with changes in susceptibility to CD95 mediated apoptosis.
- HIV protease inhibitors have immune enhancing properties.
- the invention is directed toward the use of PR-I treatment for modulating and/or enhancing the immune system and, more specifically, the cellular arm ofthe immune system in a subject in need of such modulation and/or enhancement.
- Suitable PR-Is include HIV protease inhibitors, proteasome inhibitors, serine protease inhibitors and cysteine protease inhibitors.
- Boosting of the immune system may be beneficial in the treatment of other disorders wherein the host's immune system is compromised or overburdened, e.g, infection, shock, trauma, starvation and metabolic disorders such as malabsorption.
- PR-I administration can be used during cancer treatment and/or prophylaxis. It is anticipated that PR-I administration to cancer patients, or persons at increased risk of developing cancer, e.g, because of genetic reasons and/or age, will be administered at least one PR-I in order to boost and/or modulate the immune system, thereby resulting in effective treatment and/or prophylaxis of cancers. It is anticipated that the administration of one or more PR- Is will enhance cellular immune processes and immune surveillance, thereby potentially providing for the eradication and/or reduced number of malignant cells. Also, the inhibitor may elicit an anti-cancer effect by inducing apoptosis, inhibition of neovascularization and by increasing host immune responses against cancer cells. Further, as discussed previously, it is hypothesized that proteasome inhibitor may in conjunction with chaperones, e.g, heat shock proteins such as hsp70, triggers an increase in antigen processing and presentation by antigen presenting cells.
- chaperones e.g, heat shock proteins such
- Such protease inhibitor can be used by itself or in conjunction with other anti-cancer treatments or prophylaxis, e.g, chemotherapeutics, radiation, other immune modulators, cytokines, and immunotherapeutics.
- Cancers which should be treatable and/or preventable according to the invention include, by way of example, breast, prostate, liver, bladder, lung, esophageal, stomach, skin, pancreatic, brain, uterine, colon, brain, head and neck, and ovarian cancer.
- PR-I treatment will be effected in the early stages of cancer, i.e., at a point wherein enhanced immune surveillance can potentially provide a cure.
- Another significant aspect ofthe invention is in the area of vaccines, both prophylactic and therapeutic vaccines. It is anticipated that PR-I treatment before, proximate, or after the administration of a vaccine composition will potentiate or alter the immune response of a particular subject, i.e., the cellular arm, for example by enhancing or resulting in a CTL response, thereby enhancing the efficacy of the vaccine composition.
- Protease inhibitors may act to shift the immune response, e.g, from a Th 2 to a Th t response, thereby enhancing cellular immune functions. This is particularly desirable in the context of vaccines against infectious agents such as viruses, bacteria, parasites such as plasmodia, trypanosomes, and fungi, among others.
- PR-I treatment should shorten the necessary time for the host to amount an appropriate immunological response, i.e., "defense", against the particular agent to which the vaccine is specific.
- PR-I treatment should broaden the immune response of a vaccine. This should reduce the risk of chronic adverse responses such as inflammatory or infectious responses.
- PR-1 treatment will be beneficial in treating any condition wherein mounting or maintaining a Th j response is therapeutically desirable.
- Infectious conditions wherein PR-I treatment should be beneficial include, by way of example, Lyme disease, herpes, hepatitis, parasite infection such as plasmodia, trypanosomes, schistosomiasis, and other chronic infections such as those induced by other viruses, bacteria and fungi.
- PR-I administration may be combined with vaccine administration or may be effected separately. Another embodiment of the invention comprises the administration of a
- PR-I for the treatment and/or prophylaxis of inflammatory diseases and conditions, e.g, psoriasis, arthritis, inflammatory bowel syndrome, chronic inflammation and chronic diarrhea.
- Another embodiment of the invention comprises the administration of a PR-I to bolster the immune system of a patient prior to surgery. PR-I administration and the resulting immune/nonimmune cell proliferation would avoid the need for transfusions and limit post surgical risks of infection.
- Yet another embodiment ofthe invention involves the treatment of persons who are immuno-compromised, for example because of disease or trauma.
- PR-I treatment will be beneficial in that it should alleviate symptoms ofthe disease or trauma associated with an impaired immune response, and also avert complications which may arise as a result of immuno-compromised states, such as post-operative infection, post-trauma infection, cancer, or other conditions which may occur in persons that are immuno-compromised.
- immuno-compromised states such as post-operative infection, post-trauma infection, cancer, or other conditions which may occur in persons that are immuno-compromised.
- persons who are immuno-compromised wherein PR-I administration should be beneficial include transplant recipients, persons receiving immunosuppressants, persons immuno-compromised because of genetic reasons, persons who have experienced shock or trauma, burn victims, and persons undergoing radiation or chemotherapy, or any treatment that results in an immuno-compromised state.
- PR-I should potentiate or modulate the immune system and mechanisms associated therewith, thereby averting the risk of infection or other immunologically related diseases and, moreover, should also potentiate the body's natural repair processes. It is hypothesized, based on the results observed by the present inventors, that this enhances the immune response, e.g, by altering cytokine expression, which act to drive the immune response or by affecting the interaction of proteasome with chaperones, e.g, hsp70, the expression of which triggers an enhance in antigen presentation by APCs.
- PR-I treatment in the context of the invention can be broadly construed to be an immune "adjuvant" which acts to boost or enhance the immune system of subjects in need of such treatment.
- immune suppression is a problem which may be treated according to the invention include retroviral infections, such as HIV-1 and HIV-2, cancer, papilloma viridae, herpes viridae, HTLV-I and -II.
- PR-I treatment may be used to effect specific types of immune processes, e.g, T-help type I responses (Thl) that may result in a specific cytotoxic lymphocyte response. This is in contrast to Th-2 responses which result in enhancement of humoral immune responses (enhanced antibody response).
- Thl T-help type I responses
- Th-2 responses which result in enhancement of humoral immune responses
- PR-I administration may actually alter the type of immune response that is elicited against a particular antigen.
- PR administration may actually change the means by which a vaccine or antigen composition affects the immune system.
- administration of a protease inhibitor may result in the immune system eliciting a cellular (e.g, CTL) response against a pathogen rather than a humoral immune response. This may be particularly beneficial in the context of diseases wherein humoral immune responses are thought to be of little benefit or perhaps even detrimental to the host's defense against a particular disease or infectious agent, e.g, parasite infection or anti- tumor immunity.
- protease inhibitor treatment because of its effect on cell proliferation, especially cell generation and poiesis, can be used in the treatment of persons wherein increased cell proliferation and poiesis is beneficial.
- examples thereof include subjects undergoing radiation or chemotherapy, persons with growth defects, shock and trauma victims, persons with other injuries, malnutrition, persons who have suffered significant blood loss, persons who have received a tissue graft, anemia, persons suffering from immune dysregulation, and other conditions or persons wherein increased cell proliferation is therapeutically desirable.
- PR-I treatment should also be useful for maintaining tissue regeneration and promoting tissue degradation, blood cell hemeostasis, and cell and tissue maintenance. Another usage is during wartime, e.g, to persons exposed to bioharzardous agents, chemicals, mutagens, or radiation.
- PR-I treatment will be beneficial in protecting cells or tissues from different kinds of death signals (e.g, elicited by toxins, starvation, radiation, hyperactivation, anergy, and apoptosis), for example, the result of trauma, inflammation, infection or transplantation.
- death signals e.g, elicited by toxins, starvation, radiation, hyperactivation, anergy, and apoptosis
- PR-I treatment should be beneficial is in the geriatric population wherein the immune system, because of age, may be compromised relative to younger persons.
- PR-I administration may provide for increased immune and other cell proliferation, decreased cell degradation, blood and tissue maintenance, which would otherwise potentially be reduced relative to younger persons.
- PR-I treatment may be used prophylactically or therapeutically in aged persons in order to enhance or reduce their immune systems, thereby reducing the risk of some cancers, infection, or other conditions and disorders that increase in incidence as a result of age.
- PR-1 administration may inhibit telomerase activity, which has been reported to play a significant role in aging and cell death.
- protease inhibitors are utilized to treat diseases or conditions ofthe immune system.
- autoimmune diseases such as lupus, Scleroderma, Sjogren's Syndrome, ITP, diabetes, multiple sclerosis, Graves' disease, IBD, and rheumatoid arthritis
- the HIV protease inhibitors of the invention are also applicable in treating other diseases or conditions which occur or persist due to an impaired or compromised immune system.
- cancers persist in hosts due to the immune system no longer recognizing antigens presented on the surface of tumor cells. Treatment in accordance with the invention, will enhance anti-cancer immune responses as mediated by HIV protease inhibitor-activated immune cells resulting in the restoration of immune surveillance.
- another embodiment ofthe invention comprises the use of protease inhibitors to modulate different aspects of cellular metabolism.
- cells and tissues can be protected from induced death signals (e.g, by toxins, starvation, radiation, hyperactivation, anergy and apoptosis) produced in situations such as trauma, inflammation, infection and transplantation.
- HIV protease inhibitors can be used to alter cellular metabolism in cancer to make cancer cells less neoplastic and more accessible for "conventional" anti- cancer drugs and host cell cycle control. This can be effected in vitro or in vivo.
- bone marrow can be treated in vitro during purging of bone marrow, e.g, by contacting with a protease inhibitor, to render tumor cells more susceptible to apoptosis.
- PR-1 administration may be used to treat subjects with wasting syndrome by triggering a shift in cellular metabolism. This is useful in the context of disorders or treatments that may have an adverse consequence on the subject's weight and overall metabolism, such as viral or parasitic infection, cancer, chemotherapy, radiotherapy and gene therapy such as bacterial or viral.
- protease inhibitors such as HIV-1 protease or proteasome inhibitors
- HIV-1 protease or proteasome inhibitors are utilized to modulate or decrease anti -rejection responses in hosts receiving transplants and/or grafts.
- patients who have undergone organ transplantation procedures typically reject the new organ due to the immune system recognizing the organ as "foreign” and attempting to eliminate or destroy it.
- Administration of a molecule or compound ofthe invention may inhibit the immune system from recognizing the "foreign" organ, thereby reducing the chance of organ rejection.
- protease inhibitors are to modulate apoptosis, i.e., programmed cell death. This is useful in various therapeutic contexts, e.g, treatment of cancer, or infection, wherein eradication of specific types of cells is therapeutically beneficial.
- T-lymphocyte apoptosis can be induced by signals mediated by the tumor necrosis factor (TNF) and nerve growth factor (NGF) receptor family and their respective ligands (Ashkenazi et. al. Science, 281 : 1305, 1998).
- TNF tumor necrosis factor
- NGF nerve growth factor
- Others have shown that peripheral blood CD4+ and CD8+ T-cells derived from HIV-infected individuals express abnormally high levels of both death receptors and ligands.
- susceptibility of these cells to apoptosis as mediated through T-cell receptor (TCR), CD95, and TNF receptor 1 (TNFR1) signals is increased, and can be further enhanced by interferon (IFN)-alpha and IFN-gamma (Sloand et. al.
- IFN interferon
- protease inhibitors and HIV protease inhibitors or proteasome inhibitors specifically, to modulate apoptosis, e.g, for the treatment of infection, cancer, metabolic disorders, malnutrition and mal- absorption.
- HIV-1 protease or proteasome inhibitors are for the treatment of HIV-2 infection.
- the efficacy of such treatment was shown in monkeys and clearly is a function ofthe immune stimulating properties ofthe protease inhibitors as HIV-2 is not cleaved by HIV-1 protease.
- PR-I administration can be used as an adjunct or treatment of any condition wherein the immune system is desirably boosted, enhanced, or altered.
- PBMCs peripheral blood samples were obtained from healthy volunteers according to the protocols approved by the Institutional Review Board.
- PBMCs were isolated by automated Ficoll/Hypaque density-gradient centrifugation using a CS-3000 Plus Blood Cell Separator (Fenwal Division, Baxter Healthcare Corp, Deerfield, IL).
- Monocytes were isolated from PBMCs using counterflow-centrifugal elutriation utilizing a Beckman JE-5.0 rotor and a type A chamber (Beckman Instruments, Inc., Palo Alto, CA).
- Purity of the separated monocyte fraction was approximately 95% as determined by cytomorphology in Pappenheim stain and approximately 96% as determined by the expression of CD 14 antigen (LeuM3; Becton Dickinson, Mountain View, CA) and measured by flow cytometry.
- T-cells were cultured in complete medium (RPMI 1640, containing 10% heat inactivated FCS, low endotoxin grade; all GIBCO BRL, Gaithersburg, MD) at an initial concentration of 0.5x10 6 cells/ml PBMC culture media was supplemented with IL-2 (20 U/ml, Boehringer Mannheim, Indianapolis, MN), 0.1 mg/ml penicillin G and 0.25 mg/ml streptomycin (both Gibco-BRL).
- IL-2 (20 U/ml, Boehringer Mannheim, Indianapolis, MN
- 0.1 mg/ml penicillin G and 0.25 mg/ml streptomycin (both Gibco-BRL).
- Ritonavir Abbott Laboratories, North Chicago, IL
- Medium and drug were replaced daily by exchanging 75% ofthe culture supernatants, and every four days the cultures were split equally as necessary to prevent overgrowth.
- IFN-alpha 300 IU/ml, recombinant IFN-alpha-2b, Schering Corporation, Kenilworth, NJ
- IFN-gamma 30 ng/ml, Biosource, Camarillo, CA
- TNF 10 ng/ml, BioSource
- anti-CD95 mAb CHl l (Kamyia, San Francisco, CA)
- an antibody that mimics Fas-L by cross-linking and triggering the CD95 receptor or mAb ZB4, a CD95 blocking antibody (AMAC, Westbrook, ME)
- AMAC a CD95 blocking antibody
- Elutriated monocytes were cultured in serum free medium (Cellgrow 40-101-LV, Mediatech Inc., Herndon, VA) at a density of 3x10° cells/ml Ritonavir (Abbott Laboratories) was added at concentrations ranging from 0.1 nM to 100 nM, before stimulation with lipopolysaccharide (LPS, 10 ng/ml, Sigma Chemicals, St. Louis, MO) or IFN-gamma (50 ng/ml, BioSource).
- LPS lipopolysaccharide
- IFN-gamma 50 ng/ml, BioSource
- IL-2 natural lymphocyte derived interleukin-2
- anti-CD3 mAb OKT3 for AICD experiments
- phytohemagglutinin PHA, Boehringer
- Anti-Fas mAb CHl l Kamyia, San Francisco, CA
- an antibody which mimics the Fas-L by cross-linking the Fas receptor or ZB4 a blocking anti-Fas mAb was used at 0.25 ⁇ g/ml.
- PBMCs were pre-incubated with HIV PR-I, or for comparison, with ICE inhibitors for 16 hours at 37° C in a 5% C0 2 humid atmosphere before being transferred to antibody-coated plates.
- Different human cell lines were used to identify those that closely resemble the effects of HIV-PR-I on primary human cells. Withdrawal of IL-2 and essential nutrients as well as treatment with IFN-alpha, IFN-gamma and TNF-alpha were used in other assays to induce apoptosis.
- EXAMPLE III Apoptosis and Viability Assays
- Apoptosis and viability assays for individual cell subsets have been described in detail (Sloand et. al. Blood, 89: 1357-1363, 1997; Sloand et. al, J. Clin. Invest, 101/1, 1-7, 1998).
- commercially available Apoptosis ELISAs can be used.
- Cell viability was measured using a standard Trypan blue (Life Technologies) exclusion test or an automated colorimetric assay utilizing tetrazolium bromide (MTT) reduction reaction in which in situ reduction of tetrazolium bromide to a blue formazan product by viable cells.
- MTT tetrazolium bromide
- the reduction reaction was assayed spectro-photometrically based on a standard curve and correlated to trypan blue exclusion results.
- the WST-1 reagent can be used for assessment of metabolic activity, according to the manufacturer's instruction (Boehringer Mannheim). Assessment of DNA fragmentation and Caspase activity Assessment of DNA fragmentation was performed using the Cell Death
- CMXRos incorporates into mitochondria driven by the delta Tm and reacts with thiol residues to form covalent aldehyde-fixable thiol ester bonds. After fixation (4% paraformaldehyde in PBS for 15 min at room temperature), cells were washed and stained for the detection of chromatinolysis using the TUNEL method.
- cells were stained with the potential-sensitive dye DiOC6(3) (15 min, 37° C, 40 nM) together with a biotin-Annexin V conjugate (50 x dilution; revealed by streptavidine-phycoerythrine at 5 ⁇ g/ml, following the manufacturer's protocol; Boehringer Mannheim GmbH, Mannheim, Germany), followed by sorting of DiOC6(3)low Annexin V+, DiOC6 (3) Annexin V and DiOC6(3)high Annexin V+ cells on an Elite cytofluorometer (Coulter Corp, Miami, FL).
- DiOC6(3) 50 x dilution; revealed by streptavidine-phycoerythrine at 5 ⁇ g/ml, following the manufacturer's protocol; Boehringer Mannheim GmbH, Mannheim, Germany
- EXAMPLE V Flow Cytometry For qualitative and quantitative determination of cell surface molecules, flow cytometry was carried out by incubating cells at 4° C for 30 minutes in PBS containing 1% BSA, 5 mM EDTA, and the indicated antibodies at concentrations recommended by the manufacturer. Fluorescein-isothiocyanate (FITC)-labeled or ECD-labeled mAb to CD3 and CD25, phycoerythrin (PE)-conjugated or PE-Cy5-conjugated mAbs to CD4 and CD 14, and appropriate isotype control antibodies were obtained from Becton Dickinson (Mountain View, CA).
- FITC Fluorescein-isothiocyanate
- PE phycoerythrin
- FITC-conjugated anti-CD95 mAb (UB2), and appropriate isotype control antibodies were obtained from PharMingen.
- Biotinylated anti-CD95-Ligand (Coulter/Immunotech) was developed with Avidin-PE (Becton Dickinson). Cell surface staining for AnnexinV was performed using the Apoptosis Detection Kit from R&D Systems (Minneapolis, MN).
- cytokines that effect T-cell recruitment, activation and polarization were determined in PBMC and monocyte culture supernatants at different time points post-treatments and stimulation, over a time period of five days, utilizing enzyme linked immunosorbent assay (ELISA) systems (R&D Systems Inc., Minneapolis, MN) following the manufacturers recommendations. Samples were stored at -80 °C until assayed. Measurement of TNF levels Levels of TNF were determined in PBMC and monocyte culture supernatants at different time points post-stimulation, utilizing an enzyme linked immunosorbent assay (ELISA) system (R&D Systems Inc., Minneapolis, MN) following the manufacturers recommendations. Samples were stored at -80°C until assayed. EXAMPLE VII
- RNA expression levels of IFN-alpha, IL-12, IL-6, and TGF-beta, of RANTES, MIP-1 alpha and MCP-1, and IL-4, IL-5, IL10 that are of special interest in understanding the immune augmenting effects of proteasome modulation
- cells were harvested at different time points and total RNA was isolated using methods well known in the art.
- the mRNA was quantified using RiboQant multi-probe RNase protection assay systems (PharMingen) according to the manufacturers' protocol.
- the ribonuclease protection assay is a highly specific and sensitive method for the detection and quantitation of mRNA species.
- PharMingen developed multi probe RPA systems that generate a series of templates, each of distinct length and representing a sequence in a distinct mRNA species.
- the templates are assembled into biologically relevant sets. We have used the following template sets: hCKl-5, hCR5-6, hAPO-lc, hAPO-2, and hAPO-3.
- cell cytosols 10 7 cells/100 ⁇ l in cell-free system [CFS] buffer [220 mM mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM PO 4 H 2 K, 0.5 mM EGTA, 2 mM MgCl 2 , 5 mM pyruvate, 0.1 mM PMSF, 1 mM dithiotreitol, 10 mM Hepes-NaOH]) and pH 7.4 buffer (supplemented with additional protease inhibitors: 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin A, 50 ⁇ g/ml anti -pain, 10 ⁇ g/ml chymopapain) were prepared by five freeze/thaw cycles in liquid nitrogen, followed by centrifugation (1.5 x 10 5 g, 4° C, 1 h) as described (Abbas et.
- CFS cell-free system
- Mitochondria were isolated from differently treated cells or cell lines that were prepared for the assays. Mitochondria were purified on a Percoll
- Example VIII for up to four hours. Mitochondria were then washed and resuspended in CFS supplemented with 2 mM ATP. Nuclei from HeLa cells were purified on a sucrose gradient and conserved in 50% glycerol (Sigma Chemical Co.) in HeLa nuclei buffer at 20° C for a maximum of 15 d. Mitochondrial apoptosis inducing factor (AIF) was purified from cells treated with atractyloside (5 mM; Atr; Sigma Chemical Co.) to induce permeability transition (PT) and liberation of apoptosis inducing factor (AIF).
- AIF Mitochondrial apoptosis inducing factor
- the active fraction (eluting at 1 10 mM NaCl) was dialyzed against protein-free CFS buffer (4° C, overnight, 5,000x excess of CFS buffer), concentrated on Centricon 10 membranes, adjusted to a concentration of 30 ⁇ g/ml, and aliquoted to be snap frozen in liquid nitrogen and stored at -80° C.
- EXAMPLE X Determination of Mitochondrial PT For the induction of permeability transition (PT), mitochondria from different cell lines were incubated with cytosolic extracts from Fas-treated and/or HIV PR-I treated cells (standard dose of 30 ⁇ g protein/ml), purified recombinant ICE (50 ⁇ g/ml), the pro-oxidant ter-butylhydroperoxide (t-BHP; 30 ⁇ M), atractyloside (5 mM; Sigma Chemical Co.), the protonophore mClCCP (100 ⁇ l; Sigma Chemical Co.), bongkrekic acid (50 ⁇ M), monochlorobiman (30 ⁇ M; Sigma Chemical Co.), and/or the calpain inhibitor N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-tyrosine diazomethylketone (100 ⁇ M; Molecular Probes Inc.).
- Recombinant ICE was produced following standard procedures and allowed to partially (5%) autoactivate by incubation at 20° C for 2 hours, followed by storage on ice for a maximum of four hours.
- Two different consequences of PT were assessed: a) mitochondrial large amplitude swelling, and b) collapse of the Delta Tm.
- mitochondria were washed and resuspended in CFS buffer supplemented with 2 mM ATP at a concentration of 100 ⁇ g mitochondrial protein/ 10 ⁇ l buffer, followed by addition of 90 ⁇ M CFS containing 2 mM ATP and recording of adsorption at 540 nm in a spectrophotometer (DU 7400; Beckman Instruments, Carlsbad, CA).
- Nuclei from cells were purified on a sucrose gradient, washed two times (1,000 g, 10 min, 4° C), and resuspended in CFS buffer. In standard conditions, nuclei (10 3 nuclei/ ⁇ l) were cultured in the presence of mitochondrial preparations for 90 min at 37° C. Nuclei were stained with propidium iodide (10 g/ml; Sigma Chemical Co.) and the lipophilic dye 5-methyl-bodipy-3-dodecanoic acid (100 nM; Molecular Probes Inc.), followed by cytofluorometric analysis in an analyzer (EPICS Profile II Analyzer; Coulter Corp.).
- Apoptosis inducing factor-mediated cleavage of nuclear substrates was determined by the comparative analysis of SDS-PAGE of HeLa nuclei (5 x 10 6 /lane) cultured in the presence or absence of supernatant from Atr-treated mitochondria (10 ⁇ g protein ml, 90 min, 37° C) in the presence or absence ofthe protease inhibitor Z-VAD.fmk. Western blots of these nuclei were tested for degradation of poly (ADP-ribose) polymerase (PARP) using a monoclonal antibody (C2-10, Guy Poirier, Montreal University, Canada).
- PARP poly (ADP-ribose) polymerase
- Cleavage of CPP32 in cells (8 x 10 5 cells/lane) or in vitro (10 ng recombinant CPP32+ 10 ⁇ g protein of mitochondrial supernatant in 50 ⁇ l CFS buffer ⁇ 100 ⁇ M Z-VAD.fmk, 15 min at 37° C) was determined by using a polyclonal rabbit antiserum recognizing both CPP32 and the pi 7 fragment of proteolytically activated CPP32 (Martin et. al. Cell, 82:349-352, 1995).
- CD95 (Fas) remained unchanged, whereas the number of HLA-DR expressing cells decreased from about 25%o in untreated cell cultures to less than 13%> in cell cultures treated with 20 nM PR-I. These dose-dependent effects were more pronounced in the CD4+T- cell subpopulation and differed slightly under various pre-activation conditions ⁇ Blood, 90/10, Suppl. 1, 2568, 1997). Further, it has been demonstrated that the expression of CD95 (Fas- receptor) increased on both CD4+ and CD8+ T-cells derived from HIV-1 infected individuals (Sloand et al.
- CD4+ cells express active forms of Interleukin-lbeta converting enzyme (ICE, also Caspase 1) (Sloand et al, J. Clin. Invest, 101/1, 1-7, 1998). Since these molecules are known to play an important role in mediating T-cell apoptosis (Alderson et al, J. Exp. Med., 181 :71-77, 1995; Brunner et al, Nature, 373: 441-444, 1995; Dhein et al. Nature, 373: 438-441, 1995; Ju et al. Nature, 373: 444-448, 1995; Alderson et al, J. Exp.
- ICE Interleukin-lbeta converting enzyme
- Ritonavir had growth promoting effect on T-cells in short-term (five days) culture (in RPMI 1040, supplemented with 10% heat inactivated FCS, L-glutamine, IL-2) at concentrations of 1.0 nM to 10.0 uM.
- CFC Immune Precursor Cells Colony forming capacity
- BM bone marrow
- Figure 4 As a positive control, caspase- 1 (ICE) inhibitors (Patel et al, FASEB J, 10:587-597,1996), known to enhance CFC in culture, were included and compared to the effects of PR-I. While ICE inhibitors had effects at concentrations higher than 100 ⁇ M, a significant increase in CFU was obtained with PR-I at concentrations of 10 nM. This data supports the pro-cell-survival effects of HIV-PR-I and its potential use to enhance immune responses.
- ICE caspase- 1
- mice with PR-I were treated with PR-I for three weeks and assessed changes in peripheral white blood cell counts (WBC), as preliminary data had indicated that the effects of PR-I on immune cells were similar in rodents ( Figures 5, 7 and 9).
- WBC peripheral white blood cell counts
- Data depicted in the left panel of Figure 5 represent the WBCs of five mice from the treatment group receiving a dose that is equivalent to that used therapeutically in humans, the right graph shows, for comparison, placebo treated mice.
- HIV protease inhibitors are potent immune modulators, which can change the susceptibility of immune cells to apoptosis as part of their therapeutic benefit.
- the mode of action is independent of HIV infection, since similar effects were seen in HIV free experimental systems.
- mice BALB/c mice, 6-8 weeks old, were housed in sterilized microisolator cages and maintained on sterile food and water. Mice were grouped at five mice per treatment regimen. Peripheral blood cell counts, serum IgG and weights were determined and reco rded as base line.
- groups were treated in the initial experiments (based on our preliminary data) with A) 200mg/kg, B) 20mg/kg, C) 2mg/kg proteasome inhibitor MG123 (Calbiochem), and in a parallel experiment with Ritonavir (Abbott), and D) a placebo (PBS), daily i.p.
- mice were similarly immunized with an antigen preparation (using purified fully glycosylated HIV-1-bal gpl40, 10 ⁇ g gpl40/mouse) in incomplete Freunds Adjuvant by s.c. injections.
- an antigen preparation using purified fully glycosylated HIV-1-bal gpl40, 10 ⁇ g gpl40/mouse
- complete Freunds Adjuvant was immunized using complete Freunds Adjuvant to compare these controls to groups A-D.
- Proteasome inhibitor treatment was discontinued on day five after immunization (Based on our preliminary data, it appears to be most important to change the "set point" of the immune system at the time of immunization.).
- Subsequent series of experiments determined the optimal duration of treatments that result in strongest immune responses.
- Additional experiments determined the effect of proteasome modulation on boosting injections. In such experiments, mice were boosted with antigen in incomplete Freunds Adjuvant (IFA) on days 15 and 30 ( Figures
- EXAMPLE XVII Characterization of CD4+ T cell responses To measure T cell proliferation, each MNC preparation was stimulated with purified fully glycosylated HIV-l Bal gpl40 (Intracel Inc.) and proliferation was quantitated by 3 H-TdR incorporation. Each proliferative assay included a mitogen (Con A) control, an ovalbumin control, and a dose-response curve to Env (0.01-10 ⁇ g/ml).
- MNCs are cultured in CM only, or CM containing fully glycosylated HIV-l Bal gpl40 at 0.1-10 mg/ml.
- the culture plates were incubated for 24 hours at 37° C in 5% CO 2 .
- CD4+ and CD8+ T-cells were isolated by flow cytometry and no fewer than 5x10 5 ofthe purified cells were placed directly into Trizol R reagent and cDNA was synthesized; the resultant samples were then used in quantitative-competitive (QC) PCR reactions to evaluate the relative levels of chemokine and cytokine cDNA sequences.
- QC quantitative-competitive
- MlP-l ⁇ , MlP-l ⁇ , RANTES, TNF ⁇ , IL-2, IL-4, IL-5, IL-6, and IFN- ⁇ sequence specific primers are described.
- Each QC PCR reaction was conducted in parallel with the same reactions containing of control plasmid DNA at a range of defined concentrations, which encode truncated MlP-l ⁇ , MlP-l ⁇ and RANTES cDNA sequences and served as competitive sequences (R&D Systems and National Biosciences Inc).
- the PCR fragments were separated by agarose gel electrophoresis, strained with ethidium bromide and scanned using a BioRad UV densitometer. The results were expressed as arbitrary mRNA units.
- EXAMPLE XVIII Characterization of CD8+ T cells To characterize HIV-specific chemokine-secreting CD8+ T-cell responses, a further group of mice were immunized as above. On days 7, 14, 28, 42, 56, and 70 after immunization, groups of five mice were sacrificed and MNCs from the spleens were prepared. The MNCs were divided into total, CD4+ T cell-depleted and CD8+ T cell-depleted cells, and were cultured in (i) medium only, (ii) medium containing PHA, (iii) medium containing BC-lacZ fibroblasts expressing ⁇ galactosidase (Aggarwal et. al.
- CTL activity was measured using targets 51 Cr-labeled (i) BC-lacZ fibroblast or (ii) BC-env fibroblast (Chada et. al. Journal of Virology, 67:3409-17, 1993; Fujihashi et. al. Journal of Immunology, 145:2010, 1990).
- targets 51 Cr-labeled i) BC-lacZ fibroblast or (ii) BC-env fibroblast.
- the effector cells were phenotyped using standard methods applied by our group previously (Aggarwal et. al. Journal of Experimental Medicine; 172: 1083-90, 1990).
- CTL responses were expressed in lytic units/10 6 cells.
- HIV transgenic mouse model was used (Dickie et. al. Virology, 185: 109-19,1991). These animals show a number of phenotypic changes and develop functional defects that are similar to pathophysiological changes that are commonly observed in HIV disease. These changes include chronically and significantly elevated levels of proinflammatory cytokines, as well as increased levels of apoptosis in a variety of tissues. Under these conditions, modulation of immune responses differed from that of a normal organism. The treatments and immunizations were performed, and immune response assessment was conducted as described above, and results were evaluated under similar criteria as described for Balb/C mice.
- mice For immunizations, 6-8 weeks old female BALB/c mice were grouped at 5 mice per treatment regimen. Peripheral blood cell counts, serum IgG and weights were determined and recorded as "base line". To assess dose-dependent effects, groups were treated with A) 200 mg/kg, B) 20 mg/kg (human therapeutic equivalent), C) 2 mg/kg Ritonavir and D) a placebo (PBS), twice a day via oral application (gastro lavage). On day 3 under treatment, all mice were similarly immunized with an antigen preparation (5 ⁇ g ovalbumin/mouse) in incomplete Freunds Adjuvant by s.c. injections, and on days 13 and 23 mice were boosted with antigen in incomplete Freunds Adjuvant.
- an antigen preparation (5 ⁇ g ovalbumin/mouse) in incomplete Freunds Adjuvant by s.c. injections
- mice were immunized using complete Freunds Adjuvant to compare these controls to groups A-D.
- eye-bleed derived serum were obtained from all animals and assayed for specific antibodies to the antigen utilizing ELISA techniques. This approach allowed us to get measurable antibody levels in the control (placebo) group and possibly statistically significant and dose dependent increases in the treatment groups A-C, when compared to C. All samples from individual mice were determined in duplicate determinations, and statistical significance analysis for the differences between groups was applied. Further studies were conducted to determine optimal dosing and specific immunization conditions for different HIV derived antigens that under current condition are poorly antigenic. Subsequent experiments defined the levels of cellular immune response utilizing standard CTL assays.
- VSF viral suppressive factors
- mice were maintained in the IHV Animal Facility.
- the animals were housed, cared for, and used strictly in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23. 1985).
- This institution has an Animal Welfare Assurance on file with the NIH Office for Protection from Research Risks (OPRR), Assurance Number A3200-01.
- the Animal Facility provided accredited veterinary support and resources for the murine studies. This included expertise for the care and handling ofthe animals, standard operating procedures for the inoculation of animals and for the collection of specimens, including tissue samples.
- the IHV animal facility is equipped with surgical rooms, laboratory equipment and biological safety cabinets, thereby allowing both Biosafety Level 2 and 3 on-site manipulation of animals.
- the temperature in the animal rooms was maintained at 23-25 ° C and humidity is maintained at 50-20%.
- the temperature and humidity levels were monitored continuously via internal sensors tied to a central computer monitoring system.
- the system was also monitored on a 24-hour basis at a console in the office of the Facility Manager.
- Audible and Visual alarms alerted the Manager if the temperature or humidity drifts outside the desired parameters.
- negative air pressure was maintained in these rooms. Air pressure gradients were continuously maintained with airlocks and positive air pressure flow to the hallways.
- the air handling system has independent capability control units designed to achieve Biosafety Level 2 and 3 and to maintain the airflow throughout the animal facility. The rate of non-recirculated air exchange in each animal module was 15-20 times per hour. To mimic natural day and night cycles, lighting was controlled automatically to provide twelve hours each of darkness and light. The light cycle period was adjustable if needed.
- Procedures for minimizing stress and discomfort included anesthesia (metaphane) during immunizations and any other invasive procedure. No restraining devices were used throughout the proposed study.
- Carbon-dioxide inhalation was used as a means for euthanasia in accordance with institutional guidelines.
- mice that received proteasome inhibitor treatments during immunization with recombinant HIV gpl40 developed significantly higher titer of specific and neutralizing antibodies than the control groups as measured by ELISA, 30 days after a single injection of Ag in IFA.
- Flow cytometry in conjunction with surface marker staining showed the changes in phenotype and cell activation ofthe different cell subsets in response to HIV-PR-I treatment of primary human and rodent cell cultures.
- HIV-free primary cell culture systems were utilized to obtain information.
- PBMCs were exposed in culture to different HIV-PR-I so as to determine dose- dependent effects on cellular activation as measured by cell surface marker multicolor FACS analysis.
- HIV-PR-I may enhance the production of cytokines or regulate the cellular susceptibility for cytokine signals that augment immunizations, such as IL-2, IL-12, or GM-CSF. It is possible that immune suppressive cytokines (such as IFN-alpha, TNF-alpha and the like) or their effects (known to be involved in HIV pathogenesis) are blocked by PR-I. In that context, some of our in vitro data indicated pro-hematopoietic effects that could be due to regulation of cytokine production by PR-I ( Figures 4, 16a- 16b, 17, 19b and 29). Additionally, we assessed cellular markers including: a) CD3, CD4, and
- CD8 for identification of T-cell subsets
- CD 14 and CD86 for identification of monocytes
- CD16 and CD56 for identification of NK cells.
- Markers of activation that were assessed in cell subsets include: CDI la, CD25, CD26, CD28, CD38, CD45RO, CD45RA, CD62L, CD69, CD95, Fas- ligand, CDwl 19, IFN-receptors, TNF-alpha receptors, and HLA-DR.
- cytokine profiles were screened for cytokine profiles, first for known immune augmenting cytokines such as IL-2, IL-12, GM-CSF, and for known immune suppressing cytokines such as IFN-alpha, TNF-alpha, Fas-ligand (Katsikis et al. J. Exp. Med., 186/8, 1365-1372, 1997), using ELISA technique. Since some chemokines were identified as viral suppressive factors in HIV infection and may therefore play a role in AIDS pathogenesis, commercially available chemokine assays were included in the evaluation of factors that are affected by PR-I treatment. To confirm or reevaluate these data, differential mRNA analysis on cells derived from differently treated cultures were performed.
- HIV-PR-I inhibited apoptosis.
- the regulation of immune cell apoptosis as a concept for a therapy that augments vaccination appeared reasonable since HIV infected cells escape apoptosis mediated immune surveillance; significantly increased levels of apoptosis in lymphoid organs and T-cells correlate with HIV disease progression; and highly-HIV-antigen-specific T-cells are most susceptible to apoptosis and get depleted prematurely.
- the different pathways that could be involved in immune augmentation by PR-I were studied in detail.
- Our first focus was the assessment of changes in apoptosis pathways. It is currently assumed that the apoptotic process can be divided into at least three functionally distinct phases.
- the heterogeneous initiation phase cells receive the death-inducing stimulus via certain receptors such as the TNF receptor or Fas/APO-l/CD95, shortage of obligatory growth factors, oxygen or metabolic supply, or subnecrotic physical and chemical damage.
- the biochemical events participating in the initiation phase constitute "private" pathways in the sense that they depend on the lethal stimulus. It is only during the subsequent phases that these initiating events are translated into a regular common pattern of metabolic reactions.
- the common pathway can be subdivided into an initial effector phase, during which the "central executioner of apoptosis" is still subject to regulatory mechanisms, and a later degradation phase, beyond the "point of no return", during which catabolic enzymes become activated in an irreversible fashion.
- the morphology and characteristic biochemistry of apoptosis e.g, step-wise DNA fragmentation, and specific proteolysis of cytoplasmic and nuclear substrates
- apoptosis is associated with the critical activation of a family of specific proteases that include interleukin-1 converting enzyme (ICEl/caspase 1), CPP32 (Yama Apopain/caspase 3), and other proteases homologous to the Caenorhabditis elegans protein Ced-3.
- ICEl/caspase 1 interleukin-1 converting enzyme
- CPP32 Yama Apopain/caspase 3
- other proteases homologous to the Caenorhabditis elegans protein Ced-3.
- delta Tm the disruption of the mitochondrial inner transmembrane potential
- PT permeability transition
- Caspase-3 One of the main effector of caspases in several apoptosis pathways is Caspase-3. Since CD95 triggering leads to an increase in Caspase-3 activity, we investigated the effect of Ritonavir treatment on this enzyme's activity in PBMC cultures, utilizing a spectro-fluorometric assay. Significantly less Caspase-3 activity was detected in cytoplasmic extracts of activated PBMC cultures that were treated with Ritonavir and exposed to anti-CD95 (CHI 1) mAb (under the standard conditions described above) when compared to untreated control cells (Figure 11a). Higher Caspase-3 activity in cultures that were not treated with Ritonavir peaked one hour after CD95 triggering and correlated to lower cell viability detectable after 16 hours.
- CHI 1 anti-CD95
- Ritonavir Decreases CD95 and CD95 Ligand Expression on T-cells Since both CD95-dependent and independent induction of T-cell apoptosis in AIDS were reported (Ashkenazi et. al. Science, 281 : 1305, 1998), we assessed the effect of Ritonavir on CD95 and CD95-ligand levels in vitro, utilizing flow cytometry. After 3 days of treatment of PBMC under the conditions described, a slight decrease in CD95 expression was associated with the application of Ritonavir (data not shown). CD95-ligand expression on T-cells was markedly decreased in cultures treated with 10 nM Ritonavir. These effects were detectable in both the CD4+ and CD8+ T-cell subset, but more pronounced in the CD4+ cell population (Figure 10 panel C).
- Cytosolic antigen processing and subsequent presentation by MHC-I are functions that classically have been linked to the proteasome pathway (Harding et. al, J Immunol, 155, 1767, 1995). Other proposed functions include cell cycle regulation (Di Cunto et. al. Science, 280, 1069, 1998) and regulation of inflammation (Conner et. al, J. Pharmacol. Exp. Ther., 282, 1615, 1997).
- EXAMPLE XXIX Proteasome Modulation Improves the Effectiveness of Vaccinations
- Differential regulation of cytokines and chemokines that affect immune cell trafficking and immune responses provided one possible explanation for immune adjuvant effects of systemic Pi-treatment in vivo.
- mice were pretreated for five days with Ritonavir systemically by i.p. injections of 300 ⁇ g/mouse/day. Pi-treatment in this group was continued for additional five days post immunization ( Figures 6a-6d). Serum for measurements of Ig levels specific for gpl40 was collected three weeks after immunization and antibody titers were determined by ELISA. Systemic application of PI during the time of immunization and initiation of immune response resulted in a marked increase of the effectiveness of vaccination.
- MNC Mononuclear cells
- mice Groups of ten transplanted mice were allocated to receive treatment for 1 week with either saline (placebo), or Ritonavir (30 mg/kg), or recombinant human interferon gamma (5 ug/kg) daily by intra peritoneal injections.
- placebo saline
- Ritonavir 30 mg/kg
- recombinant human interferon gamma 5 ug/kg
- mice were killed with an overdose of pentobarbital (intraperitoneally) and different cellular compartments were analyzed.
- a quantitative and qualitative assessment of human cell contents in the recipient mouse bone marrows was performed with Flow cytometry.
- Phenotyping of engrafted human cells was performed by staining cells collected from the femoral bone ma ⁇ ow of transplanted mice with an anti-CD45 MoAb and sorted with flow cytometry. These human-specific CD45+ cells were then stained with MoAbs (Becton Dickinson) raised against the following antigens and analyzed with flow cytometry: CD34 (hematopoietic progenitors, stem cells) CD38 (activation marker, absence indicates more primitive stem cells), CD33 and CD14 (myeloid markers), CD3 and CD 19 (lymphoid markers). Appropriate isotype-matched IgG were used as a negative controls. Analyses were performed on an EPICS-Profile II Flow Cytometer (Coulter Electronics, Hialeah, FL).
- hematopoietic progenitor assays were performed. Femoral bone marrow cells (10 5 cells per ml culture meduim) were grown for 14 days in methylcellulose under conditions that selectively promote colony formation by human, but not by mouse, progenitor cells (Sirard et al. Blood, 87:1539, 1996).
- Kaposi's Sarcoma is a highly angiogenic tumor of endothelial cell origin, characterized by spindle shaped cells and infiltrates of immune cells.
- Inflammatory cytokines as well as viral gene products such as HIV Tat and
- HHV8 orf74 contribute to the development of KS lesions associated with HIV-1 infection.
- HIV protease inhibitors such as Ritonavir
- cellular proteases directly affect cellular proteases, immune cell activation and susceptibility to apoptosis, independent from HIV protease inhibition.
- NFkB-dependent adhesion molecules VCAM-1, ICAM-1, and E-Selectin in HUVEC and KS cells that correlated to a functional decrease of monocyte and T-cell adhesion to endothelial cells treated with Ritonavir.
- NFkB dependent inflammatory cytokine production of IL-6, IL-8, and TNF was also suppressed ( Figures 15, 23 and 28). Since pharmacological interference with apoptosis may favor tumor cell growth and could result in the exacerbation of AIDS related malignancies, we tested the effects of Ritonavir on tumor formation in vivo.
- Application of Ritonavir at doses equivalent to human treatments significantly decreased the formation of subcutaneously implanted tumors in mice ( Figures 25-27).
- HIV protease inhibitors commonly leads to substantial increases in CD4+ cell counts but, interestingly, not always to an immediate decrease, or to only insignificant decreases, in HIV viremia (Levitz et al. New England J. Med. , 338/15, 1074, 1998).
- PR-I HIV protease inhibitors
- This phenomenon is in discordance to effects usually seen with other groups of anti-HIV drugs, where development of drug resistance and increase in HIV plasma levels (i.e. increase in viral "load", viral rebound) parallels with significant decreases of T-cell counts (Lederman, S, personal communication 01-1998).
- HIV-PR-I may have additional beneficial effects on T-cell homeostasis.
- HIV protease inhibitors although specifically designed to block HIV protease activity, have additional and direct effects on immune cell functions. These "side effects" likely represent an unappreciated and important part of HIV PR-I's therapeutic effects.
- the HIV-PR-I Ritonavir for example, applied at therapeutic or lower concentrations, increased the colony forming capacity (CFC) of human bone manow derived mononuclear cells (BM-MNC) in standard methylcellulose cultures up to 160% of that in untreated controls.
- CFC colony forming capacity
- Peripheral blood derived T-cells responded with phenotypic changes in cell surface marker expression including CD25, CD28, and CD95(Fas)-ligand, as well as with changes in susceptibility to CD95 mediated apoptosis.
- HIV-PR-I have, independent from direct HIV blocking, immune enhancing properties. It is commonly believed that the degree of reduction of apoptosis observed during highly active anti-retroviral therapy (HAART) correlates with decreased levels of HIV replication and that molecular mechanisms controlling susceptibility of T-cells to apoptosis are dependent on the level of HIV replication (or HIV gene product generation).
- HAART highly active anti-retroviral therapy
- apoptosis in HIV disease eliminates parts of the potential HIV target cell pool, i.e., highly specific, activated effector cells that are most sensitive to apoptotic stimuli. This is supported by in vitro experiments, where induction of CD95 mediated apoptosis limited HIV production in cell cultures by eliminating the infection-susceptible pool of activated cells (Sloand et. al, Blood, 89:1357, 1997; Kobayashi et. al, PNAS, 87:9620, 1990).
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Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1112741A1 (fr) * | 1997-12-11 | 2001-07-04 | Biovail Technologies Ltd | Utilisaton d'inhibiteurs de protéases pour le traitement des infections nécrotisantes |
WO2002030455A2 (fr) * | 2000-10-12 | 2002-04-18 | Viromics Gmbh | Moyens pour le traitement d'infections a des virus |
DE10051716A1 (de) * | 2000-10-12 | 2002-04-25 | Ulrich Schubert | Mittel zur Hemmung der Freisetzung, Reifung und Replikation von Retroviren |
WO2002087583A2 (fr) * | 2001-04-18 | 2002-11-07 | Istituto Superiore Di Sanita' | Utilisation d'inhibiteurs de la protease du virus de l'immunodeficience humaine (vih) pour bloquer une migration et/ou une invasion cellulaire, une infiltration tissulaire et un oedeme permettant de traiter des maladies associees a cette protease |
WO2003051361A1 (fr) * | 2001-12-14 | 2003-06-26 | Cedars-Sinai Medical Center | Utilisation d'inhibiteurs de la protease du vih-1 et de leurs derives dans le traitement d'inflammations |
WO2004004749A1 (fr) * | 2002-07-03 | 2004-01-15 | Charite - Universitätsmedizin Berlin | Inhibiteurs du proteaseome pour le traitement de sujets infectes par un herpes-virus |
WO2006017346A1 (fr) * | 2004-07-13 | 2006-02-16 | GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH A ND HUMAN SERVICES OFFICE OF TECHNOLOGY TRANSFER | Traitement d'infections virales au moyen d'inhibiteurs de proteasome |
DE102006008321A1 (de) * | 2006-02-17 | 2007-08-30 | Virologik Gmbh | Mittel zur Behandlung von Infektionen mit Influenzaviren |
AU2003228793B2 (en) * | 2002-04-30 | 2008-01-03 | Trustees Of Tufts College | Smart Pro-Drugs of Serine Protease Inhibitors |
EP2189159A1 (fr) * | 2003-02-21 | 2010-05-26 | Jarrow Formulas, Inc. | Procédés pour le traitement du VIH ou du paludisme utilisant des combinaisons de chloroquine et d'inhibiteurs de la protéase |
EP2343293A1 (fr) | 2004-09-20 | 2011-07-13 | 4Sc Ag | Nouveaux analogues de piperidin-4-yl-thiazole-carboxamide en tant qu'inhibiteurs de proliferation de lymphocytes t et leurs utilisations |
WO2011089166A1 (fr) | 2010-01-19 | 2011-07-28 | Virologik Gmbh | Inhibiteurs du protéasome à base de semicarbazone pour traiter une infection par le vih et une infection par une hépatite |
US8168803B2 (en) | 2003-06-20 | 2012-05-01 | Nereus Pharmaceuticals, Inc. | Methods of using [3.2.0] heterocyclic compounds and analogs thereof |
FR2970179A1 (fr) * | 2011-01-11 | 2012-07-13 | Centre Nat Rech Scient | Moyens pour reduire ou eradiquer le pouvoir tumoral de cellules souches cancereuses et/ou metastasiques |
WO2016004917A1 (fr) | 2014-07-10 | 2016-01-14 | Immunologik Gmbh | Produit destiné au traitement des maladies infectieuses dues à des rétrovirus |
DE102014010218A1 (de) | 2014-07-10 | 2016-01-14 | Immunologik Gmbh | Mittel zur Behandlung retroviraler Infektionen |
WO2016116281A1 (fr) | 2015-01-22 | 2016-07-28 | Immunologik Gmbh | Agents antiviraux |
WO2016137844A1 (fr) * | 2015-02-23 | 2016-09-01 | Mayo Foundation For Medical Education And Research | Méthodes et matières pour traiter des infections au virus de l'immunodéficience humaine |
US11938098B2 (en) | 2015-04-20 | 2024-03-26 | Mayo Foundation For Medical Education And Research | Method for killing HIV-infected cells using Bcl-2 inhibitors |
Citations (1)
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US5624934A (en) * | 1994-02-02 | 1997-04-29 | Eli Lilly And Company | Protease inhibitors |
-
1999
- 1999-12-03 WO PCT/US1999/028548 patent/WO2000033654A1/fr active Application Filing
- 1999-12-03 AU AU19309/00A patent/AU1930900A/en not_active Abandoned
Patent Citations (1)
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US5624934A (en) * | 1994-02-02 | 1997-04-29 | Eli Lilly And Company | Protease inhibitors |
Cited By (31)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1112741A1 (fr) * | 1997-12-11 | 2001-07-04 | Biovail Technologies Ltd | Utilisaton d'inhibiteurs de protéases pour le traitement des infections nécrotisantes |
WO2002030455A2 (fr) * | 2000-10-12 | 2002-04-18 | Viromics Gmbh | Moyens pour le traitement d'infections a des virus |
DE10051716A1 (de) * | 2000-10-12 | 2002-04-25 | Ulrich Schubert | Mittel zur Hemmung der Freisetzung, Reifung und Replikation von Retroviren |
WO2002030455A3 (fr) * | 2000-10-12 | 2002-08-08 | Ulrich Schubert | Moyens pour le traitement d'infections a des virus |
EP1430903A1 (fr) * | 2000-10-12 | 2004-06-23 | Viromics Gmbh | Inhibiteurs du proteasome pour le traitement des infections virales |
EP2305291A1 (fr) | 2000-10-12 | 2011-04-06 | ViroLogik GmbH | Inhibiteurs du proteasome pour le traitement des infections HIV |
EP2301565A1 (fr) | 2000-10-12 | 2011-03-30 | ViroLogik GmbH | Inhibiteurs du proteasome pour le traitement des infections virales |
WO2002087583A2 (fr) * | 2001-04-18 | 2002-11-07 | Istituto Superiore Di Sanita' | Utilisation d'inhibiteurs de la protease du virus de l'immunodeficience humaine (vih) pour bloquer une migration et/ou une invasion cellulaire, une infiltration tissulaire et un oedeme permettant de traiter des maladies associees a cette protease |
WO2002087583A3 (fr) * | 2001-04-18 | 2002-12-19 | Ist Superiore Sanita | Utilisation d'inhibiteurs de la protease du virus de l'immunodeficience humaine (vih) pour bloquer une migration et/ou une invasion cellulaire, une infiltration tissulaire et un oedeme permettant de traiter des maladies associees a cette protease |
WO2003051361A1 (fr) * | 2001-12-14 | 2003-06-26 | Cedars-Sinai Medical Center | Utilisation d'inhibiteurs de la protease du vih-1 et de leurs derives dans le traitement d'inflammations |
AU2003228793B2 (en) * | 2002-04-30 | 2008-01-03 | Trustees Of Tufts College | Smart Pro-Drugs of Serine Protease Inhibitors |
WO2004004749A1 (fr) * | 2002-07-03 | 2004-01-15 | Charite - Universitätsmedizin Berlin | Inhibiteurs du proteaseome pour le traitement de sujets infectes par un herpes-virus |
EP2189159A1 (fr) * | 2003-02-21 | 2010-05-26 | Jarrow Formulas, Inc. | Procédés pour le traitement du VIH ou du paludisme utilisant des combinaisons de chloroquine et d'inhibiteurs de la protéase |
US8168803B2 (en) | 2003-06-20 | 2012-05-01 | Nereus Pharmaceuticals, Inc. | Methods of using [3.2.0] heterocyclic compounds and analogs thereof |
WO2006017346A1 (fr) * | 2004-07-13 | 2006-02-16 | GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH A ND HUMAN SERVICES OFFICE OF TECHNOLOGY TRANSFER | Traitement d'infections virales au moyen d'inhibiteurs de proteasome |
EP2343293A1 (fr) | 2004-09-20 | 2011-07-13 | 4Sc Ag | Nouveaux analogues de piperidin-4-yl-thiazole-carboxamide en tant qu'inhibiteurs de proliferation de lymphocytes t et leurs utilisations |
DE102006008321A1 (de) * | 2006-02-17 | 2007-08-30 | Virologik Gmbh | Mittel zur Behandlung von Infektionen mit Influenzaviren |
WO2011089166A1 (fr) | 2010-01-19 | 2011-07-28 | Virologik Gmbh | Inhibiteurs du protéasome à base de semicarbazone pour traiter une infection par le vih et une infection par une hépatite |
WO2011089167A1 (fr) | 2010-01-19 | 2011-07-28 | Virologik Gmbh | Inhibiteur du protéasome destiné à traiter le vih |
WO2012095804A1 (fr) * | 2011-01-11 | 2012-07-19 | Centre National De La Recherche Scientifique (Cnrs) | Moyens pour reduire ou eradiquer le pouvoir tumoral de cellules souches cancereuses et/ou metastasiques. |
FR2970179A1 (fr) * | 2011-01-11 | 2012-07-13 | Centre Nat Rech Scient | Moyens pour reduire ou eradiquer le pouvoir tumoral de cellules souches cancereuses et/ou metastasiques |
WO2016004917A1 (fr) | 2014-07-10 | 2016-01-14 | Immunologik Gmbh | Produit destiné au traitement des maladies infectieuses dues à des rétrovirus |
DE102014010218A1 (de) | 2014-07-10 | 2016-01-14 | Immunologik Gmbh | Mittel zur Behandlung retroviraler Infektionen |
DE102014010220A1 (de) | 2014-07-10 | 2016-01-14 | Immunologik Gmbh | Mittel zur Behandlung retroviraler Infektionen |
WO2016116281A1 (fr) | 2015-01-22 | 2016-07-28 | Immunologik Gmbh | Agents antiviraux |
DE102015201100A1 (de) | 2015-01-22 | 2016-07-28 | Friedrich-Alexander-Universität Erlangen-Nürnberg | Antivirale Mittel |
DE102015201100B4 (de) | 2015-01-22 | 2018-05-17 | Friedrich-Alexander-Universität Erlangen-Nürnberg | Antivirale Mittel |
WO2016137844A1 (fr) * | 2015-02-23 | 2016-09-01 | Mayo Foundation For Medical Education And Research | Méthodes et matières pour traiter des infections au virus de l'immunodéficience humaine |
US10786519B2 (en) * | 2015-02-23 | 2020-09-29 | Mayo Foundation For Medical Education And Research | Methods and materials for treating human immunodeficiency virus infections |
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US11938098B2 (en) | 2015-04-20 | 2024-03-26 | Mayo Foundation For Medical Education And Research | Method for killing HIV-infected cells using Bcl-2 inhibitors |
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