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WO1989011860A1 - Combinaisons de proteines t4 solubles et d'agents anti-retroviraux, ainsi que procedes pour traiter ou prevenir l'infection par le sida, l'arc et le vih - Google Patents

Combinaisons de proteines t4 solubles et d'agents anti-retroviraux, ainsi que procedes pour traiter ou prevenir l'infection par le sida, l'arc et le vih Download PDF

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Publication number
WO1989011860A1
WO1989011860A1 PCT/US1989/002453 US8902453W WO8911860A1 WO 1989011860 A1 WO1989011860 A1 WO 1989011860A1 US 8902453 W US8902453 W US 8902453W WO 8911860 A1 WO8911860 A1 WO 8911860A1
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Prior art keywords
protein
soluble
hiv
day
combination
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PCT/US1989/002453
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English (en)
Inventor
Richard A. Fisher
Robert T. Schooley
Martin S. Hirsch
Victoria A. Johnson
Bruce D. Walker
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Biogen, Inc.
The General Hospital Corporation
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Publication of WO1989011860A1 publication Critical patent/WO1989011860A1/fr

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    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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

  • This invention relates to combinations and methods useful for the treatment and prevention of acquired immunodeficiency syndrome, AIDS related complex, and HIV infection. More particularly, this invention relates to pharmaceutically effective combinations of a soluble T4 protein and an anti-retroviral agent for treating or preventing AIDS, ARC and HIV infection.
  • a soluble T4 protein is used in a pharmaceutically effective combination with an anti-retroviral agent, such as AZT or a glucosidase inhibitor, the dosage of the soluble T4 protein and the dosage of the anti-retroviral agent being less than that required for a desired therapeutic or prophylactic effect when either compound is administered as a monotherapy.
  • T cell lymphocytes can be divided into two broad functional classes, the first class comprising T helper or inducer cells - - which mediate T cell proliferation, lymphokine release and helper cell interactions for Ig release; and the second class comprising T cytotoxic or suppressor cells - - which participate in T cell-mediated killing and immune response suppression.
  • these two classes of lymphocytes are distinguished by expression of one of two surface glycoproteins: T4 (m.w. 55,000-62,000 daltons) which is expressed on T helper or inducer cells, probably as a monomeric protein, or T8 (m.w. 32,000 daltons) which is expressed on T cytotoxic or suppressor cells as a dimeric protein.
  • T4 lymphocytes In immunocompetent individuals, T4 lymphocytes interact with other specialized cell types of the immune system to confer immunity to or defense against infection [E. L. Reinherz and S. F. Schlossman, "The Differentiation Function Of Human T-Cells", Cell, 19, pp. 821-27 (1980)]. More specifically, T4 lymphocytes stimulate production of growth factors which are critical to a functioning immune system. For example, they act to stimulate B cells, the descendants of hemopoietic stem cells, which promote the production of defensive antibodies. They also activate macrophages (“killer cells”) to attack infected or otherwise abnormal host cells and they induce monocytes (“scavenger cells”) to encompass and destroy invading microbes .
  • T4 lymphocytes stimulate production of growth factors which are critical to a functioning immune system. For example, they act to stimulate B cells, the descendants of hemopoietic stem cells, which promote the production of defensive antibodies. They also activate macrophages (“killer cells”) to attack infected or otherwise abnormal host cells and
  • T4 surface protein the primary target of certain infective agents.
  • these agents include, for example, viruses and retroviruses.
  • T4 lymphocytes When T4 lymphocytes are exposed to such agents, they are rendered nonfunctional. As a result, the host's complex immune defense system is destroyed and the host becomes susceptible to a wide range of opportunistic infections.
  • AIDS acquired immunodeficiency syndrome
  • AIDS is a disease characterized by severe or, typically, complete immunosuppression and attendant host susceptibility to a wide range of opportunistic infections and malignancies.
  • AIDS infection is accompanied by central nervous system disorders.
  • Complete clinical manifestation of AIDS is usually preceded by AIDS related complex ("ARC"), a syndrome accompanied by symptoms such as persistent generalized lymphadenopathy, fever and weight loss.
  • ARC AIDS related complex
  • HIV HIV
  • ARC Cytopathic Retroviruses
  • human immunodeficiency virus ( "HIV” ) , the generic term adopted by the human retrovirus subcommittee of the International Committee On Taxonomy Of Viruses to refer to independent isolates from AIDS patients, including human T cell lymphotropic virus type III ("HTLV-III”), lymphadenopathy-associated virus (“LAV”), human immunodeficiency virus type 1 (“HIV-1”) and AIDS-associated retrovirus (“ARV”) will be used. J. Med., 313, p. 1352 (1985)].
  • the apparent annual rate of diagnosis for those infected with HIV virus is between 1 and 2% - - a rate which may increase significantly in future years.
  • the host range of the HIV virus is associated with cells which bear the T4 surface glycoprotein.
  • Such cells include T4 lymphocytes and brain cells [P. J. Maddon et al., "The T4 Gene Encodes The AIDS Virus Receptor And Is Expressed In The Immune System And The Brain", Cell, 47, pp. 333-48 (1986)].
  • T4 lymphocytes Upon infection of a host by HIV virus, the T4 lymphocytes are rendered non-functional.
  • the progression of AIDS/ARC syndromes can be correlated with the depletion of T4 + lymphocytes, which display the T4 surface glycoprotein.
  • This T cell depletion, with ensuing immunological compromise, may be attributable to both recurrent cycles of infection and lytic growth and from cell-mediated spread of the virus.
  • clinical observations suggest that the HIV virus is directly responsible for the central nervous system disorders seen in many AIDS patients.
  • T4 The tropism of the HIV virus for T4 + cells is believed to be attributed to the role of the T4 cell surface glycoprotein as the membrane-anchored virus receptor. Because T4 behaves as the HIV virus receptor, its extracellular sequence probably plays a direct role in binding HIV. More specifically, it is believed that HIV envelope selectively binds to the T4 epitope(s), using this interaction to initiate entry into the host cell [A. G. Dalgelish et al., "The CD4 (T4) Antigen Is An Essential Component Of The Receptor For The AIDS Retrovirus", Nature, 312, pp. 763-67 (1984); D.
  • T4 Molecule Behaves As The Receptor For Human Retrovirus LAV Nature, 312, pp. 767-68 (1984)]. Accordingly, cellular expression of T4 is believed to be sufficient for HIV binding, with the T4 protein serving as a receptor for the HIV virus.
  • T4 protein Therapeutics based upon soluble T4 protein have been proposed for the prevention and treatment of the HIV-related infections AIDS and ARC.
  • T4 protein is divided into the following domains: Amino Acid
  • Soluble T4 proteins have been constructed by truncating the full length T4 protein at amino acid 375, to eliminate the transmembrane and cytoplasmic domains. Such proteins have been produced by recombinant techniques [R. A. Fisher et al., "HIV Infection Is Blocked In Vitro By Recombinant Soluble CD4", Nature, 331, pp. 76-78 (1988)]. Soluble T4 proteins advantageously interfere with the T4/HIV interaction by blocking or competitive binding mechanisms which inhibit HIV infection of cells expressing the T4 surface protein. And soluble T4 proteins inhibit interaction between T4 lymphocytes and antigen presenting cells and targets of T4 lymphocyte mediated killing. By acting as soluble virus receptors, soluble T4 proteins are useful as anti-viral therapeutics to inhibit HIV binding to T4 + cells and virally induced syncytium formation.
  • agents target the reverse transcriptase enzyme of HIV as a unique step in the life cycle of the virus.
  • HIV reverse transcriptase inhibition as the mechanism of action.
  • agents include, for example, suramin, azidothymidine (“AZT”) and dideoxycytidine [H. Mitsuya et al., "3'-Azido-3' -Deoxythymidine (BW A509U): An Antiviral Agent That Inhibits The Infectivity And Cytopathic Effect Of Human T-Lymphotropic Virus Type III/Lymphadenopathy-Associated Virus In Vitro", Proc. Natl. Acad. Sci. USA, 82, pp.
  • AZT administration in effective amounts has been accompanied by undesirable and debilitating side effects, such as granulocytopenia and anemia. Over the long term, therefore, hematologic toxicity appears to be a significant limiting factor in the use of AZT in the treatment of AIDS and ARC [D. D. Richman et al., "The Toxicity Of Azidothymidine (AZT) In The Treatment Of Patients With AIDS And AIDS-Related Complex: A Double-Blind, Placebo-Controlled Trial", N. Eng. J. Med., 317, pp. 192-97 (1987)].
  • Proposed methods for treating AIDS and ARC have also focused on the development of agents exhibiting anti-retroviral activity against steps in the viral replicative cycle other than reverse transcription [PCT patent application WO 87/03903].
  • Such methods include the administration of glucosidase inhibitors, such as the plant alkaloid castanospermine, which modify glycosylation of envelope glycoproteins of HIV infected cells by interfering with the normal processing of N-linked oligosaccharide chains on those glycoproteins, leading to reduced expression of a functional envelope protein at the cell surface and inhibition of production of infectious virus particles.
  • glucosidase inhibitors such as the plant alkaloid castanospermine
  • Such anti-retroviral agents may exert toxic effects on cellular metabolism at higher doses when given as monotherapy.
  • a soluble T4 protein is used in a pharmaceutically effective combination with an anti-retroviral agent, such as azidothymidine (“AZT”) or a glucosidase inhibitor, for treating or preventing AIDS, ARC and HIV infection, the dosage of the soluble T4 protein and the dosage of the anti-retroviral agent each being less than that required for a desired therapeutic or prophylactic effect when either compound is administered as a monotherapy.
  • an anti-retroviral agent such as azidothymidine (“AZT”) or a glucosidase inhibitor
  • a soluble T4 protein in the combinations and methods of this invention increases the effectiveness of conventional anti-retroviral agents in the treatment of AIDS, ARC and HIV infection.
  • Combination therapies according to this invention exert a synergistic effect in inhibiting HIV replication, because each component agent of the combination acts at a different site of HIV virus replication. These combinations demonstrate greater anti-retroviral activity than the sum of their separate effects.
  • the use of the combinations and methods of this invention reduces the dosages which would be required by therapies based on the use of conventional anti-retroviral agents alone. Accordingly, the combinations and methods of this invention reduce or eliminate the side effects of conventional single anti-retroviral agent therapies, while not interfering with the anti-retroviral activity of those agents. And the combinations and methods of this invention reduce potential resistance to single agent therapies, while minimizing any associated toxicity.
  • Combinations of soluble T4 protein and anti-retroviral agents that attack the HIV-1 replicative cycle at multiple sites permit the interception of several events early in that cycle, prior to integration. Accordingly, the likelihood of subsequent productive and latent HIV-1 infections of T4 cells is diminished or avoided. Furthermore, in view of the conserved nature of T4 binding by diverse isolates of HIV-1 and HIV-2 and the activity of an anti-retroviral agent such as AZT, against both viruses [Q. J. Sattentau and R. A. Weiss, "The CD4 Antigen: Physiologic Ligand and HIV Receptor", Cell, 52, pp. 631-33 (1988); H. Mitsuya and S.
  • Figure 1 depicts the nucleotide sequence and the derived amino acid sequence of T4 cDNA of plasmid p170-2.
  • Figure 2 depicts the nucleotide sequence and the derived amino acid sequence of T4 cDNA of plasmid pBG381.
  • the amino acids are represented by single letter codes as follows:
  • the T4 protein translation start (AA -23 ) is located at the methionine at nucleotides 1199-1201 and the mature N-terminus is located at the asparagine (AA 3 ) at nucleotides 1274-1276.
  • the T4 protein translation start (AA -23 ) is located at the methionine at nucleoides 1207-1209 and the mature N-terminus is located at the asparagine (AA 3 ) at nucleotides 1282-1285.
  • Figure 3 depicts, in tabular form, the parameters of various assays demonstrating the effects of the combinations of this invention in inhibiting HIV replication in vitro.
  • Figure 4 depicts, in tabular form, the combination index values for various combinations of this invention.
  • Figure 5 depicts, in graphic form, the results of various assays demonstrating the effects of the combinations of this invention in inhibiting HIV replication in vitro.
  • Figure 6 depicts, in graphic form, the effects of the combinations of this invention in inhibiting HIV replication in vitro over a 14 day period.
  • This invention relates to therapeutic or prophylactic combinations and methods for treating AIDS, ARC and HIV infection. More particularly, this invention relates to pharmaceutically effective combinations comprising a soluble T4 protein and an anti-retroviral agent, wherein the dosage of each compound is less than that required for a desired therapeutic or prophylactic effect when either agent is administered as a monotherapy. Such combinations advantageously avoid the side effects of high level dosages of anti-retroviral agents.
  • the method of this invention comprises the step of treating a patient in a pharmaceutically acceptable manner with a dosage of a soluble T4 protein together with a dosage of an anti-retroviral agent, such as AZT or a glucosidase inhibitor, said dosages each being less than that required for a desired therapeutic or prophylactic effect when either compound is used alone, for a. period of time sufficient to lessen the immunocompromising effects of HIV infection or to prevent intracellular spread of HIV infection.
  • an anti-retroviral agent such as AZT or a glucosidase inhibitor
  • a soluble T4 protein in the compositions and methods of this invention advantageously enhances the antiviral activity of the anti-retroviral agent, AZT or the glucosidase inhibitor, in the treatment of AIDS, ARC and HIV infection. Additionally, the use of a soluble T4 protein in combination with such anti-retroviral agents may reduce the dosage of treatment which would be required by therapies based upon those anti-retroviral agents alone. Finally, combination therapies according to this invention advantageously permit administration of anti-retroviral agents in dosages formerly considered too low to result in anti-retroviral effects if given alone.
  • the combinations and methods of this invention may be used to treat humans having AIDS, ARC, HIV infection or antibodies to HIV. These cominations and methods may also be used for treating AIDS-like diseases caused by retroviruses, such as simian immunodeficiency viruses, in mammals including humans.
  • patients are treated by the pharmaceutically acceptable administration of a pharmaceutically effective combination of a soluble T4 protein and an anti-retroviral agent, such as AZT or castanospermine, the dosage of the soluble T4 protein and the dosage of the anti-retroviral agent each being less than that required for a desired therapeutic or prophylactic effect when either compound is administered as a monotherapy, for a period of time sufficient to reduce the effects of retroviral infection or to prevent intracellular spread of a retrovirus.
  • an anti-retroviral agent such as AZT or castanospermine
  • Glucosidase inhibitors useful in the combinations and methods of this invention include, but are not limited to, castanospermine and deoxynojirimycin and deoxynojirimycin derivatives.
  • soluble T4 protein includes all proteins, polypeptides and peptides which are natural or recombinant soluble T4 proteins, or soluble derivatives thereof, and which are characterized by the immunotherapeutic (anti-retroviral) activity of soluble T4 protein. They include soluble T4-like compounds from a variety of sources, such as soluble T4 protein derived from natural sources, recombinant soluble T4 protein and synthetic or semi-synthetic soluble T4 protein. Such soluble T4-like compounds advantageously interfere with the T4/HIV interaction by blocking or competitive binding mechanisms which inhibit HIV infection of cells expressing the T4 surface protein.
  • Soluble T4 proteins include polypeptides selected from the group consisting of a polypeptide of the formula AA -23 -AA 362 of Figure 1, a polypeptide of the formula AA 1 -AA 362 of Figure 1, a polypeptide of the formula Met-AA 1 -AA 362 of Figure 1, a polypeptide of the formula AA 1 -AA 374 of Figure 1, a polypeptide of the formula Met-AA 1-374 of Figure 1, a polypeptide of the formula AA 1 -AA 377 of Figure 1, a polypeptide of the formula Met-AA 1-377 of Figure 1, a polypeptide of the formula Met-AA 1-377 of Figure 1, a polypeptide of the formula AA -23 -AA 374 of Figure 1, a polypeptide of the formula AA -23 -AA 377 of Figure 1, or portions thereof.
  • soluble T4 proteins include polypeptides selected from the group consisting of a polypeptide of the formula AA -23 -AA 182 of Figure 1, a polypeptide of the formula Met-AA 1 -AA 182 of Figure 1, a polypeptide of the formula AA 1 -AA 182 of Figure 1, a polypeptide of the formula AA -23 -AA 182 of Figure 1, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA 1 -AA 182 of Figure 1, followed by the amino acids asparagine-leucine-glutamine-histidine- serine-leucine, a polypeptide of the formula Met-AA 1-182 of Figure 1, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA -23 -AA 113 of Figure 1, a polypeptide of the formula AA
  • AA -23 -AA 145 of Figure 1 a polypeptide of the formula AA 1 -AA 145 of Figure 1, a polypeptide of the formula Met-AA 1-145 of Figure 1, a polypeptide of the formula AA -23 -AA 166 of Figure 1, a polypeptide of the formula AA 1 -AA 166 of Figure 1, a polypeptide of the formula Met-AA 1- 1 66 of Figure 1 , or portions thereof.
  • soluble T4 proteins include polypeptides selected from the group consisting of a polypeptide of the formula AA -23 -AA 362 of mature T4 protein, a polypeptide of the formula AA 1-362 of mature T4 protein, a polypeptide of the formula Met-AA 1-362 of mature T4 protein, a polypeptide of the formula AA 1-374 of mature T4 protein, a polypeptide of the formula Met-AA 1-374 of mature T4 protein, a polypeptide of the formula Met-AA 1-374 of mature T4 protein, a polypeptide of the formula Met-AA 1-377 of mature T4 protein, a polypeptide of the formula Met-AA 1-377 of mature T4 protein, a polypeptide of the formula AA -23 -AA 374 of mature T4 protein, a polypeptide of the formula AA -23 -AA 377 of mature T4 protein, or portions thereof.
  • soluble T4 proteins include a polypeptide selected from the group consisting of a polypeptide of the formula AA -23 -AA 182 of mature T4 protein, a polypeptide of the formula AA 1 -AA 182 of mature T4 protein, a polypeptide of the formula Met-AA 1-182 of mature T4 protein, a polypeptide of the formula AA -23 -AA 182 of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA 1 -AA 182 of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula Met-AA 1-182 of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula Met-AA 1-182
  • soluble T4 proteins also include polypeptides of the formula AA 3 -AA 377 of Figure 1, or portions thereof.
  • polypeptides include polypeptides selected from the group consistng of a polypeptide of the formula AA 3 -AA 362 of Figure 1, a polypeptide of the formula AA 3 -AA 374 of Figure 1, a polypeptide of the formula AA 3 -AA 182 of Figure 1, a polypeptide of the formula AA 3 -AA 113 of Figure 1, a polypeptide of the formula AA 3 -AA- 131 of Figure 1, a polypeptide of the formula AA 3 -AA 145 of Figure 1, a polypeptide of the formula AA 3 -AA 166 of Figure 1.
  • Soluble T4 proteins also include the above-recited polypeptides preceded by an N-terminal methionine group.
  • Soluble T4 proteins useful in the combination and methods of this invention may be produced in a variety of ways.
  • the T4 cDNA of p170-2 is almost identical to the approximately 1,700 bp sequence reported by Maddon et al., supra.
  • the T4 cDNA of p170-2 contains three nucleotide substitutions that, in the translation product of this cDNA, produce a protein containing three amino acid substitutions compared to the sequence reported by Maddon et al. These differences are at amino acid position 3, where the asparagine of Maddon et al.
  • Soluble T4 protein constructs may be produced by truncating the full length T4 sequence at various positions to remove the coding regions for the transmembrane and intracytoplasmic domains, while retaining the extracellular region believed to be responsible for HIV binding. More particularly, soluble T4 proteins may be produced by conventional techniques of oligonucleotide directed mutagenesis, restriction digestion, followed by insertion of linkers, or chewing back full-length T4 protein with enzymes.
  • the cDNA coding sequence of a full length T4 clone may be modified in sequential steps of site-directed mutagenesis and restriction fragment substitution to modify the amino acids at positions 64 and 231.
  • site-directed mutagenesis For example, one may employ oligonucleotide-directed mutagenesis to modify amino acid 64.
  • restriction fragment substitution with a fragment including the serine 231 codon of a partial T4 cDNA isolated from a T4 positive lymphocyte cell line [O. Acuto et al., Cell, 34, pp. 717-26 (1983)] library in ⁇ gt 11 may be used to modify the amino acid at position 231 [Fisher et al., supra].
  • DNA sequences coding for soluble T4 proteins may be used to transform eukaryotic and prokaryotic host cells by conventional recombinant techniques to produce recombinant soluble T4 proteins in clinically and commercially useful amounts.
  • soluble T4 proteins include those produced according to the processes set forth in United States patent application 094,322, filed September 4, 1987, United States patent application 141,649, filed January 7, 1988, and PCT patent application WO 89/01940, filed September 1, 1988, the disclosures of which are hereby incorporated by reference.
  • Microorganisms and recombinant DNA molecules characterized by DNA sequences coding for soluble T4 proteins are exemplified by cultures deposited in the In Vitro International, Inc. cul ture collection in Linthicum, Maryland, and identified as:
  • soluble T4 proteins may be chemically synthesized by conventional peptide synthesis techniques, such as solid phase synthesis. [R. B. Merrifield, "Solid Phase Peptide Synthesis. I. The Synthesis Of A Tetrapeptide", J. Am. Chem. Soc, 83, pp. 2149-54 (1963)].
  • compositions used in therapies according to this invention may be in a variety of conventional depot forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, capsules, suppositories, injectable and infusable solutions. The preferred form depends upon the intended mode of administration and therapeutic application.
  • the compositions of this invention also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art.
  • carriers and adjuvants include, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosebased substances and polyethylene glycol.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride
  • Adjuvants for topical or gel base forms may be selected from the group consisting of sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropyleneblock polymers, polyethylene glycol and wood wax alcohols.
  • the compositions of the invention are in the form of a unit dose and will usually be administered to the patient one or more times a day.
  • the antiretroviral agent such as AZT or castanospermine
  • the antiretroviral agent may be administered to the patient in any pharmaceutically acceptable dosage form including those which may be administered to a patient intravenously as bolus or by continued infusion over a period of hours, days, weeks or months, intramuscularly - - including paravertebrally and periarticularly - - subcutaneously, intracutaneously, intra-articularly, intrasynovially, intrathecally, intralesionally, periostally or by oral or topical routes.
  • any pharmaceutically acceptable dosage form including those which may be administered to a patient intravenously as bolus or by continued infusion over a period of hours, days, weeks or months, intramuscularly - - including paravertebrally and periarticularly - - subcutaneously, intracutaneously, intra-articularly, intrasynovially, intrathecally, intralesionally, periostally or by oral or topical routes.
  • the soluble T4 protein may be formulated and administered to the patient using methods and compositions similar to those employed for other pharmaceutically important polypeptides (e.g., ⁇ -IFN).
  • Any pharmaceutically acceptable dosage route including, parenteral, intravenous, intramuscular, intralesional or subcutaneous injection, may be used to administer the soluble T4 protein.
  • An effective dose may be in the range of from less than about 0.1 to 1.0 mg/kg body weight, it being recognized that lower and higher doses may also be useful.
  • the anti-retroviral agent such as AZT or a glucosidase inhibitor and the soluble T4 protein are administered sequentially or concurrently to the patient.
  • the most effective mode of administration and dosage regimen of anti-retroviral agent and soluble T4 protein will depend upon the severity and course of infection, previous therapy, the patient's health status and response to treatment and the judgment of the treating physician.
  • the anti-retroviral agent and the soluble T4 protein may be administered to the patient at one time or over a series of treatments.
  • the soluble T4 protein and the anti-retroviral agent may be administered sequentially to the patient, with the anti-retroviral agent being administered before, after, or both before and after treatment with the soluble T4 protein.
  • Concurrent administration involves treatment with soluble T4 protein at least on the same day (within 24 hours) of treatment with the anti-retroviral agent and may involve continued treatment with the anti-retroviral agent on days that the soluble T4 protein is not administered.
  • Other dosage regimens of AZT or glucosidase inhibitor and soluble T4 protein are also useful.
  • AZT may be administered orally at a dosage of less than about 200-250 mg, six times a day, i.e., less than about 1.2 g to 1.5 g per day.
  • castanospermine may be administered at a dosage of less than about 100 mg per day. Dosages of the particular anti-retroviral agent may be titrated to the individual patient.
  • AZT, castanospermine and soluble T4 protein may be administered in amounts less than the conventional dosage, for example, less than about 75% of the conventional dosage, when each is administered as a monotherapy.
  • a maintenance dose of the combination is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • soluble T4 protein used was recombinant soluble T4 protein ("rsT4") supplied by Biogen Research Corp. (Cambridge,
  • That recombinant soluble T4 protein was derived from a Chinese hamster ovary cell transfected with animal cell expression vector pBG381 [R. A. Fisher et al., "HIV Infection Is Blocked ln Vitro By Recombinant Soluble CD4" , Nature, 331, pp. 76-78 (1988)].
  • pBG381 is characterized by DNA coding for AA -23 to AA 377 of T4 protein, as depicted in Figure 2.
  • the rsT4 was stored at a concentration of 125 ⁇ g/ml in PBS in aliquots of 0.5 ml/vial at -20°C prior to use.
  • the AZT was obtained in powder form from
  • uninfected H9 cells (2 x 10 6 cells) were suspended in 5 ml of R-20 medium in T-25 flasks.
  • the multiplicities of infection varied, ranging from 500-3500 tissue culture infectious doses TCID 50 of cell-free HIV-1 (HTLV-IIIB) (obtained from Dr. Robert C. Gallo) per 1 x 10 6 cells.
  • the culture medium was changed on days 3, 5, 7 and 10, with 2 ml of cell suspension being resuspended in 5 ml of replacement medium containing the original concentrations of antiretroviral agent(s).
  • Various graded concentrations of recombinant soluble T4 protein or AZT were added to the cell cultures simultaneously with the HIV-1 isolate.
  • PBMCs peripheral blood mononuclear cells
  • PHA-P phytohemagglutinin
  • IL-2 Interleukin-2
  • PBMCs Five x 10 cells in 5 ml of R-20 medium with 10% IL-2 ) from a single HIV-1 seronegative donor were exposed to 3000 TCID 50 of HIV-1 per 10 6 cells.
  • the culture medium was changed on days 4, 7 and 10, and a 2 ml aliquot of cell suspension was resuspended in 5 ml of replacement medium containing the original concentrations of antiretroviral agent(s).
  • BT4 cells provided by
  • HIV-1 per 10 6 cells.
  • the culture medium was changed on days 3, 5, 7 and 10, with 2 ml of cell suspension being resuspended in 5 ml replacement medium containing the original concentrations of antiretroviral agent(s).
  • cell-free supernatants of the cultures were harvested on one or more of days 5, 7, 10, 12, and 14 for determination of HIV-1 p24 antigen production, viral reverse transcriptase activity (RT) or yield of infectious virus to evaluate the effects of the agents alone, or in combination, on HIV replication in vitro.
  • RT viral reverse transcriptase activity
  • cell pellets from H9 cultures were prepared for determination of HIV antigen expression by indirect immunofluorescence assay and by virus yield assay essentially according to the protocol set forth in K. L. Hartshorn, "Synergistic Inhibition Of Human Immunodeficiency Virus In Vitro By Azidothymidine And Recombinant Alpha Interferon", Antimicrob. Ag. Chemother., 31, pp. 168-72 (1987).
  • the tRNA used was type X-S, obtained from Sigma Chemical, St. Louis, Missouri.
  • the 3H-TTP was obtained from DuPont, New England Nuclear Research Products, Boston, Massachusetts.
  • Oligo dT template primers (# 27-7878 and # 27-7868, Pharmacia/P-L Biochemicals, Piscataway, New Jersey) were dissolved in 2.5 ml universal buffer to give a 10 unit/ml solution. The solution was stored in 0.5 ml aliquots at -20°C and thawed before use.
  • the assay samples may be prepared by first disrupting the virus particles by adding 0.9% Triton-X-199 in 1.5 M KCl (50-100 ⁇ l) and clarifying by low-speed centrifugation. Subsequently, the samples may be pelleted by ultracentrifugation at 100,000 Xg for 2 hours and the virus pellets may then be resuspended for PEG precipitation.
  • RT cocktail by multiplying the volume of reagents by the number of samples plus 2 for controls (the dA cocktail was an internal negative control) plus 2 for pipetting loss. Subsequently, we added 90 ⁇ l of rA or dA cocktail to eppendorf tubes kept in an ice-water bath (1 rA and 1 dA tube for each sample). We then added 10 ⁇ l of each sample to an rA and a dA tube, vortexed and then incubated in a 37°C water bath for 1 hour.
  • Enzyme Regul., 22, pp. 27-55 (1984)] was used to calculate combined agent effects. This method involves the plotting of dose-effect curves for each agent and for multiply-diluted fixed-ratio combinations of the agents using the median effect equation. Based on this method, we determined the combination index (CI) for various combinations according to this invention. CI values were determined from the median-effect plot parameters m( slope) and D (ED 50 ) of each agent and their combination based on the isobologram equation. A combination index value of less than 1 indicates synergy, while a value equal to 1 indicates additive effects and a value greater than 1 indicates antagonism. We also analyzed the data by the isobologram technique, which evaluates drug interactions by a dose-oriented geometric method.
  • Infection kinetics were dependent on the multiplicity of infection and the cell type utilized.
  • combination indices are presented for assays which met the following criteria; (1) an infected/uninfected cell number ratio ⁇ 30%, (2) an infected control RT activity value ⁇ 5 x 10 4 counts/mm/ml and (3) ongoing viral replication, such that p24 antigen and reverse transcription activity values on day x were ⁇ x-n, where day x-n was the previous harvest day.
  • the CI values indicated for the PBMC assays represent calculations based on data corrected for viable cell numbers. In the BT4 assays, only days 10 and 12 in culture were analyzed, due to slower replication kinetics in that cell type.
  • HIV-1 p24 Level (ng of protein/ml) At The Following Concentration Of rsT4 ( ⁇ g/ml)
  • HIV-1 p24 Level (ng of protein/ml) At The Following Concentration Of rsT4 ( ⁇ g/ml)
  • Figure 3 we have summarized the parameters of additional assays demonstrating the effects of combinations of soluble T4 protein and AZT according to this invention in inhibiting HIV replication in vitro.
  • Figure 4 depicts, in tabular form, the combination index values for rsT4 and AZT combinations in assays 1-8.
  • concentrations of soluble T4 protein and AZT required to fully inhibit HIV-1 replication, as single agents and in combination varied depending on the input virus inoculum, the cell type tested, and the sensitivity of the HIV-1 replicative assay utilized, as expected in biologic assays.
  • These assays clearly demonstrate the synergistic effects of soluble T4 protein and AZT in combinations according to this invention.
  • Assay 3 was carried out using a virus inoculum of 1500 TCID 50 of HIV-1 per 1 x 10 6 cells and concentrations of 0.002 - 0.125 ⁇ g/ml soluble T4 protein and 0.01 - 0.64 ⁇ M AZT.
  • concentrations of soluble T4 protein from 0.031 - 0.125 ⁇ g/ml in combination with 0.16 - 0.64 ⁇ M AZT inhibited HIV-1 synergistically, as measured by p24 antigen production and RT activity, with one exception, in which a Cl value of slightly greater than 1 was seen for p24 antigen production on day 10 (Figure 4).
  • HIV-1 antigen expression by immunofluorescence As shown in Figure 4, the Cl values for each of the p24 antigen production, reverse transcriptase, yield of infectious virus and HIV-1 antigen expression by immunofluorescence assays were less than 1, indicating that these agents act synergistically to inhibit virus replication.
  • HIV-1 p24 Level (ng of protein/10 6 cells)
  • Figure 6 depicts the results of assay 5 over a 14 day period. Even though soluble T4 protein (0.02 - 0.32 ⁇ g/ml) and AZT (0.003 - 0.040 ⁇ M) as single agents were less effective against HIV-1 replication by day 14 of the assay, combinations of ⁇ 0.16 ⁇ g/ml soluble T4 protein and ⁇ 0.02 ⁇ M AZT demonstrated synergistic interactions which increased over time and was evident on day 14 ( Figures 4 and 6). In assay 6, which was carried out in duplicate, similar synergism was observed throughout the 10 day assay period ( Figure 4).
  • HIV- 1 p24 ASSAY OF HIV-1 REPLICATION INHIBITION Day 12
  • HIV- 1 p24 Level ng of protein/ 10 cells

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Abstract

La présente invention se rapporte à des combinaisons et à des procédés utiles pour le traitement ou la prévention de l'infection par le SIDA, le complexe apparenté au SIDA (ARC) et le VIH. Plus particulièrement, la présente invention concerne des combinaisons pharmaceutiquement efficaces d'une protéine T4 soluble et d'un agent anti-rétroviral pour traiter ou prévenir l'infection par le SIDA, l'ARC et le VIH. Selon la présente invention, une protéine T4 soluble est utilisée dans une combinaison pharmaceutiquement efficace avec un agent anti-retroviral, tel que l'AZT ou un inhibiteur de la glucosidase, le dosage de la protéine T4 soluble et le dosage de l'agent anti-rétroviral étant respectivement inférieurs à celui exigé pour obtenir l'effet thérapeutique ou prophilactique souhaité lorsque l'un ou l'autre des composés est administré sous forme de monothérapie.
PCT/US1989/002453 1988-06-10 1989-06-08 Combinaisons de proteines t4 solubles et d'agents anti-retroviraux, ainsi que procedes pour traiter ou prevenir l'infection par le sida, l'arc et le vih WO1989011860A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004909A1 (fr) * 1990-09-25 1992-04-02 M.L. Laboratories Plc Composition pharmaceutique contenant la glycoproteine cd4 et un agent polyanionique anti-vih et son utilisation
US7585508B1 (en) 1992-03-11 2009-09-08 Prendergast Kenneth F Fusion proteins comprising CD4 and the malaria parasite merozoite glycophorin binding protein 130 (GBP-130)

Citations (2)

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Publication number Priority date Publication date Assignee Title
US4724232A (en) * 1985-03-16 1988-02-09 Burroughs Wellcome Co. Treatment of human viral infections
WO1988001304A1 (fr) * 1986-08-21 1988-02-25 The Trustees Of Columbia University In The City Of Adn de codage de la proteine t4 de la surface des cellules t et utilisation de fragments de t4 pour le traitement du sida

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US4724232A (en) * 1985-03-16 1988-02-09 Burroughs Wellcome Co. Treatment of human viral infections
WO1988001304A1 (fr) * 1986-08-21 1988-02-25 The Trustees Of Columbia University In The City Of Adn de codage de la proteine t4 de la surface des cellules t et utilisation de fragments de t4 pour le traitement du sida

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Title
Biochemical and Biophysical Research Communications, Volume 148, published 14 October 1987, SUNKARA, pp. 206-210. *
Drugs, Volume 34, No. 3, published September 1987, SANDSTROM, pp. 289-410. *
Journal of the American Medical Association, Volume 259, published January 1988, MERZ, p. 463. *
Nature, Volume 330, published November 1987, GRUTERS, pp. 74-77. *
Nature, Volume 331, published January 1988, DEEN, pp. 82-86. *
Nature, Volume 331, published January 1988, FISHER, pp. 76-78. *
Nature, Volume 331, published January 1988, HUSSEY, pp. 78-81. *
Nature, Volume 331, published January 1988, TRAUNECKER, pp. 84-86. *
New England Journal of Medicine, Volume 316, No. 9, published February 1987, YARCHOAN, pp. 557-564. *
Proceedings of the National Academy of Science, Volume 84, published November 1987, WALKER, pp. 8120-8124. *
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Science, Volume 238, published 18 December 1987, SMITH, pp. 1704-1707. *
See also references of EP0378643A4 *
The Lancet, published 31 October 1987, TYMS, pp. 1025-1026. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004909A1 (fr) * 1990-09-25 1992-04-02 M.L. Laboratories Plc Composition pharmaceutique contenant la glycoproteine cd4 et un agent polyanionique anti-vih et son utilisation
US7585508B1 (en) 1992-03-11 2009-09-08 Prendergast Kenneth F Fusion proteins comprising CD4 and the malaria parasite merozoite glycophorin binding protein 130 (GBP-130)

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