[go: up one dir, main page]

WO1997033604A1 - Traitement de l'infection a vih par action sur le recepteur de la cyclophiline de cellules hotes - Google Patents

Traitement de l'infection a vih par action sur le recepteur de la cyclophiline de cellules hotes Download PDF

Info

Publication number
WO1997033604A1
WO1997033604A1 PCT/US1997/003819 US9703819W WO9733604A1 WO 1997033604 A1 WO1997033604 A1 WO 1997033604A1 US 9703819 W US9703819 W US 9703819W WO 9733604 A1 WO9733604 A1 WO 9733604A1
Authority
WO
WIPO (PCT)
Prior art keywords
cyclophilin
hiv
cyclosporin
infection
cells
Prior art date
Application number
PCT/US1997/003819
Other languages
English (en)
Inventor
Michael Bukrinsky
Barbara A. Sherry
Peter C. Ulrich
Anthony Cerami
Original Assignee
Picower Institute For Medical Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/615,933 external-priority patent/US5840305A/en
Application filed by Picower Institute For Medical Research filed Critical Picower Institute For Medical Research
Priority to AU25811/97A priority Critical patent/AU723905B2/en
Priority to JP09532773A priority patent/JP2000510097A/ja
Priority to EP97917517A priority patent/EP0944394A4/fr
Publication of WO1997033604A1 publication Critical patent/WO1997033604A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/52—Isomerases (5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • the present invention provides pharmaceutical compositions for the treatment of HIV-infection targeting cyclophilin A and its corresponding human cellular binding partner or receptor as a target for intervention.
  • the present invention provides anti-cyclophilin antibodies, derivatized cyclosporin A, and soluble forms of cyclophilin-binding partners and act presumably by interrupting the binding of cyclophilin A with its cellular binding partner(s) or receptor(s), as a treatment for HIV-infection.
  • the present invention further provides genetic constructs designed to interfere with production and release of cyclophilin and its cognate cellular binding partner.
  • the present invention further provides a screening assays for the identification of compounds which inhibit the interaction of cyclophilin and its cellular receptor.
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • CD4 cell surface protein acts as the cellular receptor for the HIV-1 virus (Dalgleish et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348).
  • Viral entry into cells is dependent upon g ⁇ l20 binding the cellular CD4 receptor molecules (McDougal et al., 1986, Science 231 :382-385; Maddon et al., 1986, Cell 47:333-348), explaining HIV's tropism for CD4 + cells, while gp41 anchors the envelope glycoprotein complex in the viral membrane.
  • HIV infection is pandemic and HIV-associated diseases represent a major world health problem. Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection. Here, the focus has thus far been on CD4, the cell surface receptor for HIV.
  • Recombinant soluble CD4 for example, has been shown to inhibit infection of CD4 + T cells by some HIV-1 strains (Smith et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar et al., 1990, Proc. Natl. Acad. Sci.
  • Cyclophilin A is a 19 KD protein, which is expressed in a wide variety of cells. Cyclophilin A binds the immunosuppressive agent, cyclosporin A, and possesses peptidyl- prolyl cis-trans isomerase (PPIase), and protein folding or "chaperone" activities. Cyclophilin A is a member of the "immunophilin family", i.e., a group of related cellular factors involved in regulating immunity. At least four types of mammalian cyclophilins have been cloned, cyclophilins A, B, C and hCyP3 (Friedman et al., 1993, Proc. Natl. Acad. Sci.
  • a glycoprotein with a N-terminal signal sequence has been identified which binds cyclophilin C, but not cyclophilin A or B (Friedman et al., supra). The functional significance of this interaction has yet to be elucidated.
  • a homologous protein was found in mouse macrophages, MAC-2 binding protein, and is postulated to be involved in the cascade of immunoregulatory events resulting from the absorption of the immunosuppressive drug cyclosporin A (Chicheportiche et al., 1994, Proc. Natl. Acad. Sci. USA, 269:5512-5577).
  • Cyclophilin is recognized to be one of the host cell receptors for cyclosporin A ⁇ a potent immunosuppressive drug which is widely used in prevention of graft rejection.
  • Cyclosporin A is a member of a family of hydrophobic cyclic undecapeptides that exhibits potent immunosuppressive, antiparasitic, fungicidal and chronic anti-inflammatory properties. Cyclosporin A is thought to exert its immunosuppressive effects by inhibiting the early stages in T cell activation. Cyclosporin A has been found to block RNA transcription of the T cell growth factor, interleukin 2 (IL-2) and to inhibit expression of the IL2 receptor by precursor cytolytic T lymphocytes (Palacios, 1982, J. Immunol.
  • IL-2 interleukin 2
  • Cyclophilin A binds to cyclosporin A with a dissociation constant in the range of 10 "8 mol L, a value consistent with levels needed to elicit immunosuppression (Handschumacher et al., 1984, Science 226:544). Cyclosporin A has been shown to interfere with Gag-cyclophilin interactions in vitro, block cyclophilin incorporation into virions, and inhibit the replication of HIV- 1 in cell culture (Franke et al., supra; Thali et al., supra; Billich et al., 1995, J. Virol. 69:2451-2461).
  • Cyclosporin A has not been shown to interact directly with the HIV-1 virus. It is hypothesized that cyclosporin A, and its analogs, interfere at two stages in the HIV life cycle by interacting with cyclophilin A — during establishment of infection where cyclosporin A treatment inhibits HIV infection prior to integration into the genome of the infected cell as measured by formation of circular HIV DNA and integration of proviral DNA into the host genome; and at a late stage of virus replication, when a reduction in shed virus particles but not viral antigen expression is observed (Billich et al., supra). Therefore, researchers have been focused on disrupting the interaction between the viral protein, Gag, and the cellular protein, cyclophilin A, in their attempts to develop an anti-HIV agent.
  • cyclosporin A is a potent immunosuppressor, therefore its use in HIV-infected patients, who will become immunocompromised, is contraindicated.
  • the noted HIV-inhibitory effects of cyclosporin A and its analogs required drug concentrations that are 10 to 100-fold higher than those necessary to effectuate immunosuppression with cyclosporin A.
  • the ability of cyclosporin to generally suppress the systemic immune system has been proposed as a treatment for HIV infection (see U.S. Patent No. 4,814,323). This method of treatment however has severe drawbacks, given the severely immunosuppressed state of HIV-infected patients.
  • the present invention provides a method for inhibiting HIV infection, comprising administering a cyclosporin derivative which is not internalized by cells, to a subject in an amount sufficient to inhibit HIV infection.
  • the cyclosporin derivative is PEG- cyclosporin.
  • the present invention further provides an immunotherapeutic method for inhibiting HIV infection, comprising administering to an individual a cyclophilin immunogen at a dose sufficient to induce an immune response to cyclophilin.
  • the cyclophilin immunogen is administered with an adjuvant, and is glycated.
  • the present invention further provides an immunotherapeutic method for inhibiting HIV infection, comprising administering to an individual an antibody that neutralizes cyclophilin, at a dose sufficient to inhibit HIV infection.
  • the antibody is a single- chain antibody, chimeric antibody, humanized antibody, or Fab fragment.
  • the present invention further provides a method for inhibiting HIV infection comprising administering to a subject exogenous cyclophilin in an amount sufficient to inhibit HIV infection.
  • the present invention further provides a pharmaceutical composition comprising a derivatized form of cyclosporin, wherein the cyclosporin is derivatized by reaction with polyethylene glycol to form a PEG-cyclosporin.
  • the present invention provides a drug screening method to identify compounds or compositions which inhibit the interaction of cyclophilin and its cognate cellular binding partner or receptor in vitro, which drug screen method comprises: (a) adding a test compound, cyclophilin and a cyclophilin binding partner to a reaction mixture; and (b) detecting any cyclophilin-cyclophilin binding partner complexes in the reaction mixture.
  • the invention is based, in part, on the applicants' discovery that an extracellularly supplied cyclophilin, or neutralizing antibody for cyclophilin A, as well as cyclosporin A derivatives modified to inhibit their internalization by host cells, each inhibit HIV replication.
  • This discovery is described in examples which demonstrate that exogenous cyclophilin A, an extracellularly supplied neutralizing antibody for cyclophilin A, and an extracellularly supplied cyclosporin A derivative modified so as to be excluded from cell entry, each inhibit HIV replication.
  • cyclophilin A a cellular protein having immunomodulatory activity, is believed to be incorporated into HIV virions by binding to HIV-Gag and is thought to be required for productive viral infection (e.g.
  • Gag-cyclophilin A complex contained in the core of the virion is surrounded by a lipid envelope bearing HIV-envelope glycoproteins.
  • the Gag-cyclophilin complex is generally understood not to be exposed to exogenous cyclophilin, to the extracellular neutralizing antibody, or to cyclosporin A derivatized to inhibit internalization by the cells.
  • antibodies that neutralize cyclophilin can be used to inhibit HIV infection.
  • neutralizing antibodies which have been generated outside the patient to be treated for instance using cyclophilin as an antigen, can be administered in a passive immunotherapy approach.
  • cyclophilin is used in conjunction with an adjuvant and is covalently modified, as by glycation, to improve antigenicity and the utility of the antibodies.
  • cyclophilin can be formulated as an immunogen, for instance in conjunction with an adjuvant, for use as a "vaccine" to generate an active immune response in HIV-infected patients.
  • cyclophilin is modified by advanced glycation to improve immunogenicity or to improve the characteristics of the elicited antibodies for the intended purposes.
  • Cyclosporins which would ordinarily result in immunosuppression due to their intracellular interaction with cyclophilin A, can be derivatized to inhibit the entry of the drug into cells, for instance by the covalent attachment of bulky or charged substituents and thereby to render these derivatives non-immunosuppressive.
  • a cyclosporin is modified by attachment of polyethylene glycol substituents. Such cyclosporin derivatives can be used to inhibit HIV-infection without inducing immunosuppression in HIV-infected patients.
  • the present invention further relates to the use of exogenous cyclophilin to interfere with HIV- infection, and to the use of cyclophilin derivatives that are unable to form complexes with HIV-Gag, yet are still able to bind the cyclophilin host receptor. Therefore, these cyclophilin derivatives are unable to be incorporated into virions, yet compete with or block the binding of the HIV-virion-associated cyclophilin to host binding proteins.
  • Figure 1 shows inhibition of HIV- 1 infection in H9 cell cultures treated with exogenous cyclophilin at 40 ⁇ g/ml.
  • Reverse transcriptase (RT) activity was assayed in culture supernatants 5 days post-infection, according to standard methods.
  • Figure 2 shows inhibition of HIV- 1 infection in H9 cell cultures treated with exogenous cyclophilin (at 1 or 40 ⁇ g/ml) or with whole antiserum to glycated cyclophilin ( ⁇ -AGE-CyP) or to non-modified cyclophilin ( ⁇ -CyP).
  • Reverse transcriptase (RT) activity was assayed in culture supernatants 3 days post-infection, according to standard methods.
  • Figure 3 shows a time course of inhibition of HIV- 1 infection in human PBL cultures treated with exogenous cyclophilin at 40 ⁇ g/ml. p24 antigen levels in culture supernatants were assayed by a commercially available ELISA.
  • FIGURE 4 Inhibition of HIV- 1 infection in human monocyte/macrophage cultures by cyclophilin, anti-cyclophilin antibodies, and cyclosporin A derivatized to inhibit cell entry. Inhibition was assessed as the percent inhibition of HIV pol retrotranscription measured in quantitative PCR assays by reference to appropriate controls.
  • Cyclophilin refers to the protein cyclophilin of which four types are known
  • CyPA CyPB, CyPC and hCyP3, which is the receptor for cyclosporin A, and any derivative of cyclophilin thereof, or fragments or peptides having an amino acid sequence corresponding to cyclophilin.
  • Cyclosporin refers to the immunosuppressive drug, cyclosporin, and any derivative of cyclosporin thereof, or fragments or peptides having an amino acid sequence corresponding to cyclosporin.
  • Cyclophilin binding partner as used herein means any cellular binding protein, cell surface binding protein, extracellular receptor, or intracellular binding protein, which has cyclophilin specific binding activity.
  • the present invention provides and pharmaceutical compositions for the treatment of
  • cyclophilin can be administered to inhibit HIV infection in patients.
  • This cyclophilin may be a protein having a sequence substantially identical to human cyclophilin A, B or C, or to a cyclophilin of another species (i.e., mouse, yeast) or the protein may be a mutant of any of the above native sequences.
  • a cyclophilin of another species i.e., mouse, yeast
  • such treatment will have utility as an anti-HIV-infection treatment, by interfering with the interaction between virus-associated cyclophilin and host cell proteins.
  • neutralizing antibodies which have been generated against cyclophilin can be administered in a passive immunotherapy approach.
  • the present invention also relates to the use of cyclophilin formulated as an immunogen.
  • the use of cyclophilin as a "vaccine” will generate an active immune response useful in HIV-infected patients.
  • cyclophilin for use as an immunogen is first modified by non- enzymatic glycosylation to improve antigenicity or the desired properties of the antigen- specific immune response.
  • Small molecules are cyclosporins, which would ordinarily result in immunosuppression due to their interaction with intracellular cyclophilin A, but which are derivatized so as to inhibit their entry into cells, for instance by the covalent attachment of bulky or charged substituents, such as polyethylene glycol.
  • the extracellularly presented cyclosporin derivatives can be used to inhibit HIV-infection. This is thought to be attributable to blocking the interaction between cyclophilin A and its cellular binding proteins, which by their modification are without significant immunosuppressive activity.
  • the present invention additionally provides screening assays to identify compounds which inhibit HIV infection. Such compounds are selected for their activity in blocking the interaction between cyclophilin A and its cellular binding proteins. Such identified compounds would have utility in the treatment and prevention of HIV-infection.
  • the present invention is based, in part, on the applicants' surprising discovery that, when provided extracellularly, exogenous cyclophilin A, a neutralizing antibody for cyclophilin A, and cyclosporin A derivatives, which are modified to inhibit their entry into host cells, each inhibit HIV replication and infection in cell culture.
  • the Gag-cyclophilin complex should not be exposed to the extracellularly supplied cyclophilin A, neutralizing antibody or derivatized cyclosporin A. While not limited to any theory or explanation, the Applicants' hypothesis, that a host cell binding factor or receptor for cyclophilin exists, and is required in addition to the host CD4 receptor, for efficient infection of CD4+ host cells by HIV (e.g., attachment, uncoating and translocation to the nucleus), was developed.
  • compounds which may be used in accordance with the present invention encompass any compound which interferes with the interaction between cyclophilin and its cellular binding partner or receptor, including, but not limited to neutralizing antibodies against cyclophilin and the use of cyclophilin as an immunogen to generate an active immune response in HIV-infected patients.
  • compounds which may be used in accordance with the present invention include cyclophilin A and advantageously nonimmunosuppressive derivatives of cyclosporin which inhibit the interaction between virus-associated cyclophilin A and its cellular binding partner(s) or receptor(s) and correspondingly inhibit HIV infection.
  • peptides such as, for example, peptides representing soluble portions of cyclophilin binding partner or receptor
  • phosphopeptides small organic or inorganic molecules
  • antibodies including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab'), and FAb expression library fragments, and epitope-binding fragments thereof).
  • the present invention provides a use for neutralizing antibodies against cyclophilin, optionally raised against derivatized cyclophilin, in a passive immunotherapy approach and the use of cyclophilin, optionally a derivatized form of cyclophilin, as an immunogen to generate an active immune response in HIV-infected patients.
  • the present invention provides neutralizing antibodies which interact with cyclophilin, or with its cellular binding partner, and inhibit HIV infection. Such antibodies disrupt the interaction between cyclophilin and its host cellular binding partner or receptor thereby inhibiting HIV-infection.
  • These neutralizing antibodies of the invention can be administered in a passive immunotherapy approach.
  • the neutralizing antibodies are generated using glycated cyclophilin. Methods for glycating cyclophilin are described in WO 93/23081 , which is incorporated herein in its entirety by reference.
  • Antibodies that are specific for cyclophilin A or its host cellular binding partner or receptor may be generated to inhibit HIV-infection, and these antibodies are thought to work in this regard by interfering with the interaction between virally-associated cyclophilin and its cellular binding partner or receptor.
  • Such antibodies may be generated using standard techniques against the proteins themselves or against peptides corresponding to portions of the proteins.
  • the antibodies include but are not limited to polyclonal, monoclonal, Fab fragments, single chain antibodies, chimeric antibodies, human antibodies, non-human antibodies or humanized antibodies.
  • fragments of the antibody are used, the smallest inhibitory fragment which binds to the target protein's binding domain is preferred.
  • peptides having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the target protein may be used. Such peptides can be synthesized chemically or produced via recombinant DNA technology.
  • single chain neutralizing antibodies which bind to intracellular target epitopes may also be administered. Such single chain antibodies may be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. ( Proc. Natl. Acad. Sci. USA 90:7889-7893, 1993).
  • various host animals may be immunized by injection with the protein, including but not limited to rabbits, mice, and rats.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to modification of the antigen of interest by the process of advanced glycation or by advanced glycation products, or by administration of the antigen with Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, key hole limpet hemocyanin. dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • passive vaccine formulations include but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal routes. It may be preferable to introduce the vaccine formulation via the natural route of infection of the pathogen for which the vaccine is designed, or into the tissue where the pathogen resides within the body.
  • the amount of immunogen to be used and the immunization schedule will be determined by a physician skilled in the art and will be administered by reference to the immune response and antibody titers of the subject.
  • the present invention further provides a use of cyclophilin, formulated as an immunogen, and used as a "vaccine" to generate an active immune response in HIV-infected patients that are not severely immunocompromised or in populations at risk for HIV exposure and infection.
  • active immunization comprises reacting the immunogen, cyclophilin, with glucose or another reducing sugar under conditions which lead to the formulation of irreversible covalent adducts, known as Advanced Glycation Endproducts (AGEs).
  • AGEs Advanced Glycation Endproducts
  • a vaccine comprising an immunogen consisting of cyclophilin or its cellular binding partner or receptor, or fragments or peptides having an amino acid sequence corresponding to the domains required for cyclophilin binding to its cellular binding partner or receptor, may be utilized to generate an immune response in the HIV patient or a person at risk for HIV exposure.
  • immunogens optionally may be modified by advanced glycation or advanced glycation products and may be formulated with an adjuvant to increase the immunological response, such as potentially useful human adjuvants, i.e., BCG (bacille Calmette-Guerin) and Corynebacterium parvum .
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum a potentially useful human adjuvants
  • vaccine formulations described above include but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal routes. It may be preferable to introduce the vaccine formulation via the natural route of infection of the pathogen for which the vaccine is designed.
  • the amount of immunogen to be used and the immunization schedule will be determined by a physician.
  • the present invention further provides cyclosporin derivatives that are modified so as to inhibit their entry into or internalization by host cells, such that the derivatives of cyclosporin are correspondingly not interactive with intracellular cyclophilin A.
  • cyclosporin derivatives of the present invention are believed to act to inhibit HIV viral entry when presented extracellularly.
  • Such cyclosporin derivatives inhibit infection by HIV, yet should not result in immunosuppression of the HIV-infected patient. Therefore, the invention encompasses derivatives of cyclosporin which prevent or minimize internalization of the molecule by the cell, yet do not prevent interaction with the cyclophilin, thereby inhibiting HIV-infection.
  • Cyclosporin may be derivatized by the addition of a bulky substituent to the protein.
  • substituents include but are not limited to, charged substituents (e.g. , spermine or spermidine), polynucleotides with and without modified backbones, carbohydrates (e.g., polyacrylic acid, polysodium acrylate, polycesium acrylate, polymethacrylic acid), amphiphilic block copolymers (e.g., polystyrene poly) (sodium acrylate), and amphiphilic homopolymers.
  • charged substituents e.g. , spermine or spermidine
  • polynucleotides with and without modified backbones e.g., polyacrylic acid, polysodium acrylate, polycesium acrylate, polymethacrylic acid
  • amphiphilic block copolymers e.g., polystyrene poly
  • sodium acrylate sodium acrylate
  • amphiphilic homopolymers e.g., polystyrene poly
  • cyclosporin is derivatized by reaction with polyethylene glycol, resulting in a "pegylated" cyclosporin.
  • Pegylated cyclosporin may be prepared by standard chemical methods. The preferred method of preparing the pegylated cyclosporin of the invention, involves reacting methoxypolyethylene glycol-succinimidyl succinate with 8-amino-cyclosporin A and 4-dimethylamino pyridine in methylene chloride with stirring for two days at room temperature in the dark. To block any unreacted sites, ethanolamine was then added and the mixture incubated at room temperature with stirring for another 24 hours. The derivatized cyclosporin A was purified from the reaction mixture by normal phase HPLC.
  • the present invention further provides the use of exogenous cyclophilin to inhibit the interaction between cyclophilin and its cellular binding partner or receptor.
  • exogenous forms of cyclophilin include but are not limited to human cyclophilin A, other human cyclophilins, the cyclophilins of other species (i.e. , mouse, yeast) derivatized cyclophilin, recombinant cyclophilin, cyclophilin fusion proteins or peptide fragments expressing the domain of cyclophilin which binds to its host cell receptor.
  • a further embodiment of the invention relates to the use of exogenous cyclophilin binding protein(s) to inhibit the interaction between cyclophilin and its cellular binding partner.
  • exogenous forms of cyclophilin binding partners or receptors include but are not limited to derivatized cyclophilin binding partners, recombinant cyclophilin binding partners, fusion proteins or peptide fragments expressing the domain of the cyclophilin binding partner or receptor which binds to cyclophilin.
  • a therapeutic modality for HIV- infection embodying the genetic engineering of cells to produce or release cyclophilin constitutively or in response to suitable inducers and the supply of said cells to an HIV patient by engraftment of exogenously transfected cells or treatment of the patient by gene therapy to transfect endogenous cell populations is envisioned.
  • This therapeutic modality is likewise suitable to implement HIV treatment by expression and release of cyclophilin mutants, molecular decoys or other mimics of cyclophilin that inhibit HIV infection, for instance by interfering with the interactions between virus associated cyclophilin and such cellular binding proteins, therefore, as are required for productive infection.
  • Therapeutic modalities capitalizing on the genetic engineering of cells to express and release soluble forms of cyclophilin-binding proteins or the cyclophilin-binding domains thereof will likewise find utility in the practice of the present invention and are contemplated hereunder.
  • Cyclophilin may be derivatized by a variety of methods.
  • cyclophilin is pegylated by reacting cyclophilin with methoxypolyethylene glycol-succinimidyl succinate and 4-dimethylamino pyridine in methylene chloride with stirring for two days at room temperature in the dark. To block any unreacted sites, ethanolamine is added and the mixture incubated at room temperature with stirring for another 24 hours. The pegylated cyclophilin is purified from the reaction mixture by normal phase HPLC.
  • cyclophilin can be produced by synthetic techniques or via recombinant DNA technology.
  • Recombinant DNA can be used to construct expression vectors containing differentially expressed cyclophilin protein coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination.
  • RNA capable of encoding differentially expressed cyclophilin protein sequences can be chemically synthesized using, for example, synthesizers.
  • host-expression vector systems can be utilized to express the cyclophilin coding sequences.
  • Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, produce and release cyclophilin protein in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing cyclophilin protein coding sequences; yeast (e.g.
  • yeast expression vectors containing cyclophilin protein coding sequences
  • insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing cyclophilin protein coding sequences
  • plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • recombinant plasmid expression vectors e.g., Ti plasmid
  • mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278, in which the cyclophilin protein coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; and pIN vectors.
  • pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene protein can be released from the GST moiety.
  • Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • Successful insertion of cyclophilin coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus.
  • These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed.
  • a number of expression systems can be utilized and either the protein expressed on the cells or the cells expressing the protein may be utilized.
  • the cyclophilin coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing cyclophilin protein in infected hosts.
  • the following assays are designed to identify compounds or compositions that bind to cyclophilin A, its cellular binding partner or receptor and interfere with the interaction between cyclophilin A and its cellular receptor. Those compounds identified as inhibitors of the interaction between cyclophilin A and its cellular binding partner or receptor would have utility as anti-HIV agents.
  • the principle of the assays to identify compounds which inhibit the interaction of cyclophilin as the target gene or protein of interest and its cellular binding partner or receptor involves preparing a reaction mixture of the test compound and cyclophilin and its host receptor or a cellular preparation comprising, in part, said cyclophilin-binding activity and for a time sufficient to allow the components to interact and bind, thus forming a complex which can be removed and/or detected.
  • the reaction mixture is prepared in the presence and absence of the test compound.
  • the test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of cyclophilin A and its cellular binding partner or receptor.
  • Control reaction mixtures are incubated without the test compound or with a control agent. The formation of any complexes between the target gene protein and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene protein and the interactive binding partner.
  • complex formation within reaction mixtures containing the test compound and normal target gene protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant target gene protein.
  • the assay for compounds that interfere with the interaction of the target gene products and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested.
  • test compounds that interfere with the interaction between the target gene products and the binding partners can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the target gene protein and interactive cellular or extracellular binding partner.
  • test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.
  • the various formats are described briefly below.
  • either the target gene protein or the interactive cellular or extracellular binding partner is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly.
  • the anchored species may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the target gene product or binding partner, and optionally drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance.
  • the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non- immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
  • the antibody in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody.
  • test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.
  • the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes.
  • test compounds which inhibit complex or which disrupt preformed complexes can be identified.
  • a homogeneous assay can be used.
  • a preformed complex of the target gene protein and the interactive cellular or extracellular binding partner is prepared in which either the target gene product or its binding partners is labeled, but the signal generated by the label is quenched due to complex formation (U.S. Patent 4,109,496).
  • the addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt target gene protein/cellular or extracellular binding partner interaction can be identified.
  • the target gene product can be prepared for immobilization using recombinant DNA techniques routinely used in the art.
  • the target gene coding region can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-l, in such a manner that its binding activity is maintained in the resulting fusion protein.
  • GST glutathione-S-transferase
  • the interactive cellular or extracellular binding partner can be purified and used to raise a monoclonal antibody. This antibody can be labeled with the radioactive isotope 125 I.
  • the GST-target gene fusion protein can be anchored to glutathione-agarose beads.
  • the interactive cellular or extracellular binding partner can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur.
  • unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components.
  • the interaction between the target gene protein and the interactive cellular or extracellular binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.
  • the GST-target gene fusion protein and the interactive cellular or extracellular binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads.
  • the test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the target gene product/binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.
  • Such cyclophilin fusion protein constructs are similarly useful to identify and isolate cellular cyclophilin binding partners and mutants thereof.
  • the compounds of the present invention including but not limited to, anti-cyclophilin antibodies, recombinant cyclophilin, derivatized cyclosporin and derivatized cyclophilin A, have utility as pharmacological compositions for the treatment and prevention of HIV - infection.
  • a compound of the invention can be administered to a human patient by itself or in pharmaceutical compositions where it is mixed with suitable carriers or excipients at doses to treat or ameliorate various conditions involving HIV-infection.
  • a therapeutically effective dose further refers to that amount of the compound sufficient to inhibit HIV infection.
  • Therapeutically effective doses may be administered alone or as adjunctive therapy in combination with other treatments for HIV infection or associated diseases.
  • Techniques for the formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, PA, latest addition.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, and optionally in a depot or sustained release formulation.
  • a targeted drug delivery system for example in a liposome coated with an anti-CD4 antibody.
  • the liposomes will be targeted to and taken up selectively by cells expressing CD4.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked poly vinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafiuoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafiuoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form in ampoules or in multi-dose containers, with an added preservative.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • Many of the compounds of the invention identified as inhibitors of the interaction between cyclophilin A and its host receptor may be provided as salts with pharmaceutically compatible counterions.
  • Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.; or bases. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • salts examples include, but are not limited to, sodium, potassium, lithium, calcium, magnesium, iron, zinc, hydrochloride, hydrobromide, hydroiodide, acetate, citrate, tartrate, malate sales, and the like.
  • RNA was reverse transcribed, and resulting cDNA amplified by PCR using a set of cyclophilin-specific primers (5'-CCA-TGG-TCA-ACC-CCA-CC-3' and 5'-ACG-CTC-TCC- TGA-GCT-ACA-GA-3') which span the cyclophilin coding region.
  • a single DNA amplification product of the expected size was obtained, cloned into the plasmid pT7Blue and transformed into Nova Blue competent E. coli using the pT7Blue T-Vector Kit (Novagen; Madison, WI).
  • the recombinant pT7Blue clone containing murine cyclophilin cDNA was digested with the restriction enzymes Ndel and BamHl, and the cyclophilin insert was isolated and ligated in the ⁇ f ⁇ el/Zf ⁇ wjHI-digested pET14b vector (Novagen) allowing for its expression as a histidine fusion protein.
  • the cyclophilin insert was placed behind the T7 promoter which was under the control of the lac repressor (allowing for induction by the addition of IPTG to the medium).
  • the vector also contained a gene for ampicillin resistance (allowing for selection in carbenicillin-containing medium).
  • the cyclophilin-containing vector was used to transform E. coli DH5 ⁇ cells.
  • a bacterial pellet (corresponding to 100 mis of culture) was thawed and resuspended in 4.0 ml Binding Buffer in preparation for subsequent purification using His-Bind Resin (Novagen).
  • the bacteria were lysed by adding an equal volume of washed glass beads (106 ⁇ m; Sigma) and vortexing the mixture vigorously for 10 minutes. Glass beads were removed by centrifugation at 1000 x g for 10 minutes, and the bacterial extract clarified by centrifugation at 38000 x g for 30 min.
  • the cleared bacterial lysate was sterile-filtered through a 0.22 micron syringe filter, and immediately loaded onto a column containing 2.5 ml His-Bind Resin (Novagen). The column was washed extensively according to the manufacturers directions.
  • thrombin 0.5 Units per mg recombinant protein
  • thrombin 0.5 Units per mg recombinant protein
  • the recombinantly expressed peptide were incubated in Thrombin Cleavage Buffer (20 mM Tris-HCl, pH 8.4, 150 mM NaCl and 2.5 mM CaCl 2 ) for 2 hours at 20 °C.
  • the mixture was then concentrated using a Centriprep-10 concentration device (Amicon Corp., Danvers, MA) at 4 °C.
  • a Centriprep-10 concentration device Analog Chemical System, Inc., Danvers, MA
  • the concentrated mixture was subjected to high performance gel filtration chromatography over a Superose 12 column (Pharmacia, Piscataway, NJ) equilibrated in PBS, pH 7.4. Cyclophilin eluted as a sharp peak with an apparent molecular mass of 19 kDa.
  • the H9 T cell line was cultured in RPMI medium supplemented with 10% fetal calf serum and 1% pen/strep. Cells were seeded at a density of 0.5xl0 6 cells/ml and, every 2nd day, one-half of the cell suspension was removed and replaced with fresh medium, to keep cell density below lxlO 6 cells/ml. Cells were infected with HIV-I RP strain at a multiplicity index of 10 ng p24 per lxlO 6 cells. After a 2 hr adsorption at 37 °C, 5% CO 2 , non-bound virus was washed out, and incubation was continued in fresh medium with or without rCyP at the indicated doses. Samples were removed for RT and p24 analysis (according to standard methods) every 2nd day, and half of the cell suspension substituted with fresh medium at that time.
  • Figure 1 illustrates that on day 5 after infection, as assayed by RT activity in the culture supernatants, experimental HIV infection was inhibited in H9 T cell cultures treated with recombinant murine cyclophilin at 40 ⁇ g/ml.
  • Figure 2 shows in part that the presence of cyclophilin at 40 ⁇ g/ml, but not at 1 ⁇ g/ml, inhibited HIV infection in H9 cell cultures, as measured by RT activity post-infection.
  • these inhibitory effects seen with a cultured T cell line were confirmed and extended to normal human peripheral blood lymphocyte cultures, reinforcing the utility of this therapeutic modality against HIV infection in humans.
  • Figure 3 shows a time course of the inhibitory effect of exogenously supplied recombinant cyclophilin at a dose of 40 ⁇ g/ml in PBL cultures, as measured by the appearance of p24 antigen in culture supernatants as assayed by a p24 ELISA kit.
  • This example provides in vitro experiments and assays demonstrating the effectiveness of antibodies raised against cyclophilin (anti-CyP) or against an experimentally glycated preparation of CyP (anti-AGE-CyP).
  • anti-AGE-CyP antibodies recognize soluble CyP in its native configuration as indicated by immunoprecipitation studies; the anti-CyP antiserum did not.
  • reaction mixture was dialyzed to remove non-covalently attached low molecular weight reactants, aliquoted into eppendorf tubes (0.125 ⁇ g per tube), and stored frozen at -20 °C until used for immunization of rabbits.
  • Polyclonal rabbit anti-cyclophilin antisera were raised.
  • Female New Zealand White rabbits were immunized with cyclophilin which had been emulsified in 1.0 ml saline plus 1.0 ml complete Freund's adjuvant.
  • the immunogen (20 ⁇ g human cyclophilin) was administered by subcutaneous injection at four sites on the dorsum of the rabbit. Animals were administered booster injections monthly with the same antigen emulsified in 1.0 ml saline and 1.0 ml incomplete Freund's adjuvant. Blood was withdrawn 7 days and 14 days following each booster injection, and the serum fraction was recovered and stored frozen at -20 °C.
  • Pre- immune and immune sera were tested for reactivity by Western blotting and immunoprecipitation against both recombinant human cyclophilin and recombinant murine cyclophilin.
  • Pre-immune sera were negative in both assays.
  • On western blots the immune sera was positive against both antigens. Immunoprecipitation experiments were negative.
  • the recombinant murine cyclophilin used for immunization and boosting was produced in E. coli by the procedure described in example 1. At each time point, rabbits were immunized and boosted with 50 ⁇ g murine cyclophilin. Pre-immune and immune sera were tested for reactivity by Western blotting and immunoprecipitation against both recombinant human cyclophilin and recombinant murine cyclophilin. Pre-immune sera were negative in both assays. On western blots the immune sera was positive against both antigens. Immunoprecipitation experiments were negative.
  • rabbits were immunized and boosted with 125 ⁇ g AGE-modified murine cyclophilin.
  • Pre-immune and immune sera were tested for reactivity by Western blotting and immunoprecipitation against both recombinant human cyclophilin and recombinant murine cyclophilin.
  • Pre-immune sera were negative in both assays.
  • recombinant murine cyclophilin was coupled to Sepharose beads according to a protocol recommended by the manufacturer.
  • three grams of freeze-dried CNBr-activated Sepharose 4B (Pharmacia; Piscataway, NJ) was rehydrated in 1 mM HCl (yielding 10.5 ml swollen gel), and washed for 15 minutes with 1 mM HCl (200 ml/g freeze dried powder) on a sintered glass funnel.
  • the gel was then transferred to a sintered glass funnel and washed with three alternate cycles of 0.1 M sodium acetate (pH 4.0) containing 0.5 M NaCl and 0.1 M Tris-HCl (pH 8.0) containing 0.5 M NaCl (40 ml each wash).
  • the final volume of swollen gel was about 5.0 ml.
  • Murine cyclophilin affinity resin (5.0 ml packed volume) was transferred to a 10 ml glass walled chromatography column.
  • the column was washed with 10 bed-volumes 20 mM Tris (pH 7.5), followed by 10 bed-volumes 10 mM Tris/0.5 M NaCl (pH 7.5), and then cyclophilin specific antibodies were eluted by washing the column with 100 mM glycine-HCl (pH 2.5) and collecting 1.0 ml fractions.
  • H9 T cell line was cultured in RPMI medium supplemented with 10% fetal calf serum and 1% pen/strep. Cells were seeded at a density of 0.5x10 6 cells/ml and, every 2nd day, one- half of the cell suspension was removed and replaced with fresh medium, to keep cell density below lxlO 6 cells/ml. Cells were infected with HIV-1 RF strain at a multiplicity index of 10 ng p24 per lxl 0 6 cells. After a 2 hr adsorption at 37 °C, 5% CO 2 , non-bound virus was washed out, and incubation was continued in fresh medium. Samples were removed for RT and p24 analysis every 2nd day, and half of the cell suspension substituted with fresh medium at that time. Monocyte cultures were prepared from the whole blood of HIV -negative donors.
  • PBMCs Peripheral blood mononuclear cells
  • Recombinant mouse cyclophilin at various concentrations ranging from 1 to 200 ⁇ g/ml
  • anti-cyclophilin antibodies (1 :40 dilution) were added to cells together with the virus, and were likewise present throughout all subsequent incubations.
  • HIV-1 pol PCR primers sense, 5'-GAAGCTCTATTAGATACAGG-3'; anti-sense, 5'- TCCTGGCTTTAATTTTACTGG-3'; probe, 5 , -GGAATTGGAGGTTTATCAAAGT-3 , ; HIV-1 2-LTR circle DNA, or cellular ⁇ -tubulin gene as a control (ATI and AT2).
  • PCR reactions in 50 ⁇ l were prepared as follows: 25 ml cell lysate; 1 ⁇ l of 10 mM dNTP mixture; 1 ⁇ l of each primer; 12.5 ⁇ l of 2xPCR buffer without Proteinase K; 0.25 ⁇ l of Taq polymerase (5 U/ml or 1.25 U per reaction). 35 cycles of PCR were performed, each composed of 30 sec, 95 °C denaturation; 30 sec, 60 °C annealing; 45 sec, 72 °C extension. Cycles were preceded by a 6 min, 95 °C denaturation, and followed by a 7 min, 72 °C final extension. PCR products were visualized by hybridization to a 32 P-labeled probe after a Southern transfer, and results were quantitated on a direct imager system (Packard).
  • Packard direct imager system
  • This example provides in vitro experiments and assays demonstrate the effectiveness of pegylated cyclosporin as an inhibitor of HIV-infection.
  • Human monocyte/macrophage cell cultures are challenged with an inoculum of HIV in the presence and absence of a "pegylated" cyclosporin analogue.
  • This cyclosporin analogue has been modified by a reaction with polyethylene glycol so as to prevent cell entry.
  • This pegylated CsA is not immunosuppressive in a standard T cell proliferation assay. Monitoring the progression of the infection will identify the effective dose range of pegylated cyclosporin to interfere with HIV infection.
  • CsA cyclosporin A
  • the H9 T cell line was cultured in RPMI medium supplemented with 10% fetal calf serum and 1% pen/strep. Cells were seeded at a density of 0.5x10 6 cells/ml and, every 2nd day, one-half of the cell suspension was removed and replaced with fresh medium, to keep cell density below lxlO 6 cells/ml. Cells were infected with HIV-I RF strain at a multiplicity index of 10 ng p24 per lxlO 6 cells. After a 2 hr adsorption at 37 °C, 5% CO 2 , non-bound virus was washed out, and incubation was continued in fresh medium. Samples were removed for RT and p24 analysis every 2nd day, and half of the cell suspension substituted with fresh medium at that time.
  • PBMCs Peripheral blood mononuclear cells
  • DMEM normal human serum
  • NHS heat-inactivated normal human serum
  • adherent cells were washed 3 times with DMEM, and re-fed with DMEM+10%NHS+1 ng/ml M-CSF (Sigma).
  • Example 4 This example provides cross-linking studies to determine the identity of cyclophilin- binding partners on H9 cell membranes.
  • DSS Disuccinimidyl suberate
  • H9 cells are harvested, washed in ice-cold PBS, 2.5 x 10 7 cells transferred to a clean tube, and cells pelleted by centrifugation. This pellet was resuspended in 0.5 ml cross-linking buffer, and 125 I -cyclophilin added. After a 60 minute incubation on ice, cells were pelleted at 500 x g, and supernatant discarded.
  • the pellet was resuspended to 2.5 x 10 6 cells/ml in cross-linking buffer, and DSS added to a final concentration of 20 ug/ml.
  • This reaction mixture was rocked at 4 °C for 20 minutes, at which time 1 volume of TE buffer, pH 7.4 was added to stop the reaction.
  • Cells were washed 3 times in ice-cold TE buffer (pH 7.4), and membranes extracted by homogenization in an NP40 lysis buffer.
  • NP40 extracts were analyzed by SDS-PAGE electrophoresis and autoradiography to identify proteins crosslinked to iodinated cyclophilin. Additional analyses, in which cells were fractionated into membrane, cytosolic, and/or nuclear fractions further characterized the subcellular localization of cyclophilin-binding partners.
  • Bolton-Hunter reagent N-succinimidyl-3-(4- hydroxy-5-([ U5 I] iodophenyl-propionate) was be purchased from NEN.
  • This example provides an immunization protocol to generate monoclonal anti-AGE- modified cyclophilin begins with immunization of mice.
  • mice Four BALB/C mice were immunized by i.p. injection with 25 ⁇ g AGE-modified murine cyclophilin in 1 : 1 mixture of saline and adjuvant (Ribi). Twenty-one days post-immunization, mice were boosted with 25 ⁇ g AGE-modified murine cyclophilin in RIBI adjuvant, and mice were boosted with this amount of antigen in adjuvant every 21 days.
  • mice were tail-bled (approximately 50 ⁇ l) and the serum from each mouse analyzed for immunoreactivity against cyclophilin in a direct ELISA in which the plate was coated with recombinant murine cyclophilin.
  • a mouse screened positive for anti-cyclophilin IgG the mouse was given a final boost of antigen and then euthanized for spleen removal and hybridoma production. Resulting hybridomas were screened for production of cyclophilin-specific monoclonal antibodies.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • AIDS & HIV (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

On décrit des compositions pharmaceutiques pour traiter l'infection à VIH, ce traitement consistant à utiliser la cyclophiline A ainsi que le partenaire de liaison cellulaire, humain et correspondant de celle-ci, en tant que cible d'intervention. On décrit en outre un procédé d'utilisation de cyclophilines, d'anticorps dirigés contre les cyclophilines, et de la cyclosporine A polyéthylèneglycolée en tant que traitement de l'infection à VIH. La présente invention concerne encore des méthodes de criblage destinées à identifier des composés inhibant l'interaction entre la cyclophiline et le récepteur cellulaire de celle-ci.
PCT/US1997/003819 1996-03-12 1997-03-12 Traitement de l'infection a vih par action sur le recepteur de la cyclophiline de cellules hotes WO1997033604A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU25811/97A AU723905B2 (en) 1996-03-12 1997-03-12 Treatment of HIV-infection by interfering with host cell cyclophilin receptor activity
JP09532773A JP2000510097A (ja) 1996-03-12 1997-03-12 宿主細胞シクロフィリンレセプター活性を妨げることによるｈｉｖ感染の治療
EP97917517A EP0944394A4 (fr) 1996-03-12 1997-03-12 Traitement de l'infection a vih par action sur le recepteur de la cyclophiline de cellules hotes

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US1318596P 1996-03-12 1996-03-12
US60/013,185 1996-03-12
US08/615,933 1996-03-14
US08/615,933 US5840305A (en) 1996-03-14 1996-03-14 Treatment of HIV-Infection by interfering with host cell cyclophilin receptor activity

Publications (1)

Publication Number Publication Date
WO1997033604A1 true WO1997033604A1 (fr) 1997-09-18

Family

ID=26684543

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/003819 WO1997033604A1 (fr) 1996-03-12 1997-03-12 Traitement de l'infection a vih par action sur le recepteur de la cyclophiline de cellules hotes

Country Status (5)

Country Link
EP (1) EP0944394A4 (fr)
JP (1) JP2000510097A (fr)
AU (1) AU723905B2 (fr)
CA (1) CA2248192A1 (fr)
WO (1) WO1997033604A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003038032A3 (fr) * 2001-10-25 2003-10-30 Hans Will Produit inhibiteur de virus lents
US7060672B2 (en) 2001-10-19 2006-06-13 Isotechnika, Inc. Cyclosporin analog formulations
US7108988B2 (en) 2001-11-02 2006-09-19 The J. David Gladstone Institutes Methods of identifying agents for inhibiting lentivirus replication
US7332472B2 (en) 2001-10-19 2008-02-19 Isotechnika Inc. Cyclosporine analogue mixtures and their use as immunomodulating agents
US7358229B2 (en) 1997-10-08 2008-04-15 Isotechnika Inc. Deuterated cyclosporin analogs and their use as immunomodulating agents
WO2016054354A1 (fr) * 2014-10-02 2016-04-07 Dana-Farber Cancer Institute, Inc. Compositions et méthodes pour le traitement de tumeurs malignes
US20210277064A1 (en) * 2018-08-22 2021-09-09 Bacainn Biotherapeutics, Ltd. Cyclosporine compositions and methods of use

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SK278808B6 (sk) * 1990-11-02 1998-03-04 Novartis Ag Cyklosporíny, ich použitie pri príprave liečiva na
WO1993023081A1 (fr) * 1992-05-15 1993-11-25 The Rockefeller University Procedes et agents pour moduler la reponse immunitaire, et leurs utilisations
GB9318612D0 (en) * 1993-09-08 1993-10-27 Sandoz Ltd An assay

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AMERICAN JOURNAL OF HOSPITAL PHARMACY, 15 January 1994, Vol. 51, No. 2, BURNHAM N.L., "Polymers for Delivering Peptides and Proteins", pages 210-218. *
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, August 1994, Vol. 38, No. 8, ROSENWIRTH et al., "Inhibition of Human Immunodeficiency Virus Type 1 Replication by SDZ NIM 811, A Nonimmunosuppressive Cyclosporine Analog", pages 1763-1772. *
CLIN. IMMUNOL. AND IMMUNOPATHOL., 1988, Vol. 46, ANDRIEU et al., "Effects of Cyclosporin on T-Cell Subsets in Human Immunodeficiency Virus Disease", pages 181-198. *
PROC. NATL. ACAD. SCI. U.S.A., June 1995, Vol. 92, BARTZ et al., "Inhibition of Human Immunodeficiency Virus Replication by Nonimmunosuppressive Analogs of Cyclosporin A", pages 5381-5385. *
See also references of EP0944394A4 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7358229B2 (en) 1997-10-08 2008-04-15 Isotechnika Inc. Deuterated cyclosporin analogs and their use as immunomodulating agents
US7538189B2 (en) 1997-10-08 2009-05-26 Isotechnika Inc. Methods of making deuterated cyclosporin analogs
US7521421B2 (en) 1997-10-08 2009-04-21 Isotechnika Inc. Deuterated cyclosporine analogs and methods of making the same
USRE48226E1 (en) 2001-10-19 2020-09-29 Aurinia Pharmaceuticals Inc. Cyclosporine analogue mixtures and their use as immunomodulating agents
US10472394B2 (en) 2001-10-19 2019-11-12 Aurinia Pharmaceuticals Inc. Cyclosporine analogue mixtures and their use as immunomodulating agents
US7429562B2 (en) 2001-10-19 2008-09-30 Isotechnika Inc. Cyclosporin analog formulations
US7060672B2 (en) 2001-10-19 2006-06-13 Isotechnika, Inc. Cyclosporin analog formulations
US9765119B2 (en) 2001-10-19 2017-09-19 Aurinia Pharmaceuticals Inc. Cyclosporine analogue mixtures and their use as immunomodulating agents
US7332472B2 (en) 2001-10-19 2008-02-19 Isotechnika Inc. Cyclosporine analogue mixtures and their use as immunomodulating agents
WO2003038032A3 (fr) * 2001-10-25 2003-10-30 Hans Will Produit inhibiteur de virus lents
US7108988B2 (en) 2001-11-02 2006-09-19 The J. David Gladstone Institutes Methods of identifying agents for inhibiting lentivirus replication
US10365280B2 (en) 2014-10-02 2019-07-30 Dana-Farber Cancer Institute, Inc. Compositions and methods for treating malignancies
WO2016054354A1 (fr) * 2014-10-02 2016-04-07 Dana-Farber Cancer Institute, Inc. Compositions et méthodes pour le traitement de tumeurs malignes
US11391739B2 (en) 2014-10-02 2022-07-19 Dana-Farber Cancer Institute, Inc. Compositions and methods for treating multiple myeloma
US20210277064A1 (en) * 2018-08-22 2021-09-09 Bacainn Biotherapeutics, Ltd. Cyclosporine compositions and methods of use
EP3840769A4 (fr) * 2018-08-22 2022-06-22 Bacainn Biotherapeutics, Ltd. Compositions de cyclosporine et méthodes d'utilisation
EP4438618A3 (fr) * 2018-08-22 2025-01-01 Bacainn Biotherapeutics, Ltd. Compositions de cyclosporine et méthodes d'utilisation
US12252556B2 (en) 2018-08-22 2025-03-18 Bacainn Biotherapeutics, Ltd. Cyclosporine compositions and methods of use

Also Published As

Publication number Publication date
EP0944394A4 (fr) 2003-05-14
AU723905B2 (en) 2000-09-07
JP2000510097A (ja) 2000-08-08
EP0944394A1 (fr) 1999-09-29
CA2248192A1 (fr) 1997-09-18
AU2581197A (en) 1997-10-01

Similar Documents

Publication Publication Date Title
US5840305A (en) Treatment of HIV-Infection by interfering with host cell cyclophilin receptor activity
Nemerow et al. Identification of an epitope in the major envelope protein of Epstein-Barr virus that mediates viral binding to the B lymphocyte EBV receptor (CR2)
US5480799A (en) Sperm antigen corresponding to a sperm zona binding protein autoantigenic epitope
CA1330038C (fr) Methode et compositions pour l'utilisation des polypeptides env de vih et anticorps diriges contre ces polypeptides
MXPA02010374A (es) Composiciones y metodos para tratar infecciones virales.
JP2001517069A (ja) 結核の免疫治療および診断のための化合物および方法
US5185441A (en) Dna sequences, recombinant dna molecules and processes for producing pi-linked lymphocyte function associated antigen-3
RU2337922C9 (ru) ИЗОЛИРОВАННЫЕ ПОЛИПЕПТИДЫ НА ОСНОВЕ НЕЙТРАЛИЗУЮЩЕГО ЭПИТОПА БЕЛКА p17 ВИРУСА ВИЧ, ИСПОЛЬЗУЕМЫЕ В КАЧЕСТВЕ ВАКЦИН, А ТАКЖЕ НЕЙТРАЛИЗУЮЩИЕ АНТИ-p17-АНТИТЕЛА, СПЕЦИФИЧЕСКИ РАСПОЗНАЮЩИЕ УКАЗАННЫЙ НЕЙТРАЛИЗУЮЩИЙ ЭПИТОП
EP0474764A1 (fr) Procede permettant de traiter le rhumatisme articulaire
AU723905B2 (en) Treatment of HIV-infection by interfering with host cell cyclophilin receptor activity
EP0719281B1 (fr) Constructions de peptides a branches multiples destines a etre utilises contre le vih
WO2000071147A1 (fr) Nouvelle utilisation therapeuthique de la proteine modulatrice de l'inflammation virale pour bloquer le rejet d'une xenogreffe
AU763900B2 (en) Treatment of HIV-infection by interfering with host cell cyclophilin receptor activity
JP2003507011A (ja) Ｉｌ−１６拮抗剤
CA2317107C (fr) Constructions lipidiques a plusieurs ramifications contenant des liposomes et destinees a etre utilisees contre le virus de l'immunodeficience humaine
JP4149713B2 (ja) 感染症治療剤
US6290963B1 (en) Anti-HIV compositions containing native and recombinant peptides
JP3364181B2 (ja) ヒト免疫不全ウイルスのエンベロープポリペプチドおよびその抗体
WO1997039020A2 (fr) Sequences antigeniques d'une proteine seminale et methodes immunocontraceptives
Syennerholm et al. Vahlne et al.
WO2000078790A2 (fr) Procedes pour inhiber la replication du vih
MXPA99003380A (en) Compositions and methods for treating viral infections

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 2248192

Country of ref document: CA

Ref country code: CA

Ref document number: 2248192

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1997917517

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1997917517

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1997917517

Country of ref document: EP