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WO2003050136A2 - Peptide t stimulant les reponses des lymphocytes t cytotoxiques (ctl) - Google Patents

Peptide t stimulant les reponses des lymphocytes t cytotoxiques (ctl) Download PDF

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Publication number
WO2003050136A2
WO2003050136A2 PCT/US2002/039109 US0239109W WO03050136A2 WO 2003050136 A2 WO2003050136 A2 WO 2003050136A2 US 0239109 W US0239109 W US 0239109W WO 03050136 A2 WO03050136 A2 WO 03050136A2
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Prior art keywords
thr
virus
group
cancer
terminal residue
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PCT/US2002/039109
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English (en)
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WO2003050136A3 (fr
Inventor
Francis W. Ruscetti
Michael R. Ruff
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THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES, NATIONAL INSTITUTES OF HEALTHTH
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Priority to AU2002357093A priority Critical patent/AU2002357093A1/en
Publication of WO2003050136A2 publication Critical patent/WO2003050136A2/fr
Publication of WO2003050136A3 publication Critical patent/WO2003050136A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Cytotoxic T lymphocytes play a role in containing HIV spread. These effector lymphocytes are not fully functional in suppressing virus production, perhaps due to lack of CD4 help or HIV suppression of virus-specific CD8 + cells.
  • the ability of CTLs to secrete gamma interferon (IFN- ⁇ ) is accepted to be an indicator of cytotoxic capability in both antiviral settings (Altfeld et al., J. Immunol. 2001, 167:2743, Goulder et. al., J. Virol. 2001 75:1339; Belz et al., J. Immunol. 2001, 166:4627) and anti tumor settings (Ochsenbein et al., Nature 2001 , 411 : 1058).
  • the percentage of IFN- ⁇ secretingCD8 + T cells from subjects increased after treatment with the HIV entry inhibitor Peptide T (DAPTA).
  • the invention provides methods for treating diseases that call for increased CTL response, increased IFN- ⁇ or increased IL-2, by administering a peptide as described. Further objects and advantages of the invention are described below.
  • Figure 1 shows that Peptide T increases in IFN- ⁇ Secreting CD8+ T- lymphocytes. This indicates a restoration of immune function.
  • PBMC's from HIV-1 infected patients under treatment with peptide T (6 mgs/day) were isolated and stained for intracellular IFN- ⁇ by activation with PHA overnight, then with phorbol ester and calcium ionophore in the presence of a protein transport inhibitor.
  • the cells were stained with fluorescence-labeled control antibodies (immunoglobulins IgGl and IgG2) or with fluorescence-labeled antibodies (anti-CD8 and mouse mAb antibody and anti- human IFN- ⁇ , all from the Pharmingen) for 30 min on ice.
  • Figure 2 shows increases in IFN- ⁇ Secreting CD8+ T- lymphocytes from the PBMC's of HIV-1 infected patients under treatment with peptide T. Samples were taken at 8, 12, and 24 weeks following the initiation of treatment.
  • the invention provides a method of increasing cytotoxic T lymphocyte (CTL) activity in a subject comprising administering to the subject a CTL activity-stimulating amount of a peptide selected from peptides of the formula:
  • R a (Ser-Thr-Thr-Thr-Asn-Tyr (SEQ ID NO: 1))-R b or
  • R a is selected from the group consisting of amino terminal residue Ala- and D- Ala;
  • R b is selected from the group consisting of carboxy terminal residue -Thr, -Thr amide and -Thr-Cys; R, is Thr-; R 2 is selected from the group consisting of Thr and Asp; R 3 is selected from the group consisting of Thr, Ser and Asn:
  • R 4 is Tyr
  • R 5 is selected from the group consisting of carboxy terminal residue -Thr, -Thr amides and -Thr-Cys.
  • the invention provides a method of increasing gamma interferon (IFN- ⁇ ) secretion in a subject comprising administering to the subject an IFN- ⁇ secretion- increasing amount of a peptide selected from peptides of the formula:
  • R a is selected from the group consisting of amino terminal residue Ala- and D- Ala;
  • R b is selected from the group consisting of carboxy terminal residue -Thr, -Thr amide and -Thr-Cys;
  • R is Thr-
  • R 2 is selected from the group consisting of Thr and Asp;
  • R 3 is selected from the group consisting of Thr, Ser and Asn: R 4 is Tyr; and
  • R 5 is selected from the group consisting of carboxy terminal residue -Thr, -Thr amides and -Thr-Cys.
  • the invention provides a method of increasing interleukin (IL-2) secretion in a subject comprising administering to the subject an IL-2 secretion-increasing amount of a peptide selected from peptides of the formula: R a -Ser-Thr-Thr-Thr-Asn-Tyr-R b or
  • R a is selected from the group consisting of amino terminal residue Ala- and D- Ala;
  • R b is selected from the group consisting of carboxy terminal residue -Thr, -Thr amide and -Thr-Cys; R, is Thr-;
  • R 2 is selected from the group consisting of Thr and Asp;
  • R 3 is selected from the group consisting of Thr, Ser and Asn:
  • R 4 is Tyr;
  • R 5 is selected from the group consisting of carboxy terminal residue -Thr, -Thr amides and -Thr-Cys.
  • a method of treating a subject diagnosed as having a disease associated with reduced CTL activity comprising administering to the subject a CTL activity- stimulating amount of a peptide selected from peptides of the formula:
  • R Thallium is selected from the group consisting of amino terminal residue Ala- and D- Ala;
  • R b is selected from the group consisting of carboxy terminal residue -Thr, -Thr amide and -Thr-Cys;
  • R is Thr-
  • R 2 is selected from the group consisting of Thr and Asp;
  • R 3 is selected from the group consisting of Thr, Ser and Asn:
  • R 4 is Tyr
  • R 5 is selected from the group consisting of carboxy terminal residue -Thr, -Thr amides and -Thr-Cys.
  • a method of treating a subject diagnosed as having a disease associated with reduced IFN- ⁇ activity comprising administering to the subject an IFN- ⁇ activity-stimulating amount of a peptide selected from peptides of the formula:
  • R a is selected from the group consisting of amino terminal residue Ala- and D- Ala;
  • R b is selected from the group consisting of carboxy terminal residue -Thr, -Thr amide and -Thr-Cys;
  • R is Thr-
  • R 2 is selected from the group consisting of Thr and Asp;
  • R 3 is selected from the group consisting of Thr, Ser and Asn:
  • R 4 is Tyr
  • R 5 is selected from the group consisting of carboxy terminal residue -Thr, -Thr amides and -Thr-Cys.
  • R a is selected from the group consisting of amino terminal residue Ala- and D- Ala;
  • R b is selected from the group consisting of carboxy terminal residue -Thr, -Thr amide and -Thr-Cys; R, is Thr-;
  • R 2 is selected from the group consisting of Thr and Asp;
  • R 3 is selected from the group consisting of Thr, Ser and Asn:
  • R 4 is Tyr
  • R 5 is selected from the group consisting of carboxy terminal residue -Thr, -Thr amides and -Thr-Cys.
  • U.S. Patent No. 5,276,016 is hereby incorporated by reference in its entirety for its teachings of Peptide T and Peptide T based pharmaceutical compositions.
  • U.S. Patent No. 5,529,983 is hereby incorporated by reference in its entirety for its teachings on Peptide T treatment of Psoriasis.
  • U.S. Patent No. 5,834,429 is hereby incorporated by reference in its entirety for its teachings on methods of inhibiting HIV binding to T4 and inhibiting HIV infection of cells.
  • U.S. Patent No. 5,863,718 is hereby incorporated by reference in its entirety for its teachings on kits designed to identify antibodies to antigens that bind the CD4 receptor.
  • R a represents an amino terminal residue Ala- or D-Ala and R b represents a carboxy terminal residue -Thr or -Thr amide or a derivative thereof with an additional Cys- residue at one or both of the amino and carboxy terminals, or a peptide of formula (II):
  • R is an amino terminal residue Thr-, Ser-, Asn-, Glu-, Arg-, He- or Leu-,
  • R 2 is Thr, Ser or Asp
  • R 3 is Thr, Ser, Asn, Arg, Gin, Lys or Tip
  • R 4 is Tyr and R 5 is preferably a carboxy terminal residue -Thr, -Arg or -Gly or a derivative thereof with a corresponding D- amino acid as the amino terminal residue, and/or a corresponding amide derivative at the carboxy terminal residue and/or additionally a Cys- residue at one or both of the amino and carboxy terminals. While the preferred amino acids at R 5 have been designated, it is known the amino acid at this position can vary widely. In fact, it is possible to terminate the peptide with R 4 (Tyrosine) as the carboxy terminal amino acid wherein R 5 is absent. Such peptides retain the binding properties of the group taught herein. Serine and threonine appear to be interchangeable for purposes of biological properties taught herein.
  • the active compounds of the invention can exist as physiologically acceptable salts of the peptides.
  • peptides as well as peptide T above, are the following octapeptides of formula (I):
  • the compounds of the invention can be beneficially modified by methods known to enhance passage of molecules across the blood-brain barrier. Acetylation has proven to be especially useful for enhancing binding activity of the peptide.
  • the terminal amino and carboxy sites are particularly preferred sites for modification.
  • the peptides of this can also be modified in a constraining conformation to provide improved stability and oral availability.
  • amino acids are, of course, in the natural form of L-stereoisomers.
  • Position 4 and 6 were first restricted (with one exception) to S, T and N, all of which are amino acids containing uncharged polar groups with closely similar steric properties.
  • An assessment of general sequence concordance among 5 various AIDS viral isolates reveals that the region around and including the peptide T sequence is a highly variable area. Such variability may indicate specialization through strong selective diversification of the function(s), which may be defined at this locus.
  • these peptide T analogs seem to exist in multiple forms, reminiscent of met and leu enkephalin.
  • These pentapeptide sequences represented in these various AIDS virus isolates are biologically active and capable of interacting as agonists of the CD4 receptor-previously known largely as a surface "marker" of T helper cells.
  • peptide T and the pentapeptide which is a portion thereof, could be isolated from the virus
  • the peptides prepared in accord with Merrifield are free of viral and cellular debris. Hence, untoward reactions to contaminants do not occur when the synthesized peptides are used.
  • the solid phase method of Merrifield is particularly convenient. In this process the peptide is synthesized in a stepwise manner while the carboxy end of the chain is covalently attached to the insoluble support. During the intermediate synthetic stages the peptide remains in the solid phase and therefore can be conveniently manipulated.
  • the solid support is a chloromethylated styrene- divinylbenzene copolymer.
  • N-protected form of the carboxy terminal amino acid e.g. a t- butoxycarbonyl protected (Boc-) amino acid
  • Boc- t- butoxycarbonyl protected amino acid
  • the amino acid will generally be used in activated form, e.g. by use of a carbodiimide or active ester.
  • DAPTA D- Ala, -peptide T-amide
  • the present invention can treat a broad array of diseases that share a role for CTL.
  • CTLs cause elimination of virally infected cells which express viral antigens
  • HTLV-1 disease causing retroviral infections
  • HIV infection viral infections in general, such as Herpesviridae
  • HTLV-1 has the peptide T epitope STNYT (SEQ ID NO: 14)
  • HIV-2 has ETWYS (SEQ ID NO: 15) and thus these peptides can treat other viral diseases by activating CTLs or blocking viral entry or blocking binding of viral envelope proteins, which can cause neuronal apoptosis, to receptors.
  • the viral infection can be selected from the list of viruses consisting of Herpes simplex virus type-1, Herpes simplex virus type-2, Cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus, Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adeno virus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus,
  • the disease is a bacterial infection.
  • the bacterial infection can be selected from the list of bacterium consisting of M. tuberculosis, M. bovis, M. bovis strain BCG, BCG substrains, M. avium, M. intracellulare, M. africanum, M.
  • the disease is a fungal infection.
  • the fungal infection can be selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneomocystis carnii, Penicillium marneffi, and Alternaria alternata.
  • CTL activity in the immune response to protozoan infection is well recognized in the art. See, for example, Maranon C, Thomas MC, Planelles L, Lopez MC, The immunization of A2/K(b) transgenic mice with the KMP 11 -HSP70 fusion protein induces CTL response against human cells expressing the T. cruzi KMP11 antigen: identification of A2 -restricted epitopes, Mol. Immunol.
  • the parasitic infection can be selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species., Schistosoma mansoni, other Schistosoma species., and Entamoeba histolytica.
  • Generalized deficiency in CTL responses which occur during aging, can also be remedied by administration of the peptides described herein.
  • CTLs are also recognized to be important in immune surveillance of neoplastic targets and enhanced CTL activity is associated with tumor regressions and increased survival.
  • the role of CTL activity in tumor immunology is well established in the literature. For example, see Jager D, Jager E, Knuth A. Immune responses to tumor antigens: implications for antigen specific immuno therapy of cancer, J. Clin. Pathol. 2001 Sep;54(9):669-74 ; Mukerjee, T. et al., MUCl-specific cytotoxic T lymphocytes eradicate tumors when adoptively transferred in vivo, Clin. Cancer Res. 2001 Mar. 7(3 Suppl):848s-855s; and Gendler and Mukherjee, Trends Mol.
  • lymphomas Hodgkins and non-Hodgkins
  • B cell lymphoma B cell lymphoma
  • T cell lymphoma myeloid leukemia, leukemias, mycosis fungoides
  • carcinomas carcinomas of solid tissues
  • squamous cell carcinomas adenocarcinomas, sarcomas
  • gliomas blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumors, myelomas, AIDS-related lymphomas or sarcomas
  • metastatic cancers bladder cancer, brain cancer, nervous system cancer, squamous cell carcinoma of head and neck, neuroblastoma/glioblastoma, ovarian cancer, skin
  • Compounds disclosed herein can also be used for the treatment of precancer conditions such as cervical and anal dysplasias, other dysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, and neoplasias.
  • the invention further provides a method of treating a subject diagnosed as having a disease associated with reduced IFN- ⁇ activity, comprising administering to the subject an IFN- ⁇ activity stimulating amount of a peptide described herein.
  • IFN- ⁇ activity stimulating amount of a peptide described herein In addition to the role of IFN- ⁇ in CTLs, increasing IFN- ⁇ can have beneficial effects on the pathogenic events during microbial invasion and neoplastic development.
  • IFN- ⁇ is both an activator and mediator of the innate immune system (natural killer cells and macrophages). In several microbial infections, IFN- ⁇ is protective through stimulation of macrophages to produce nitric oxide synthase and other inflammatory substances which are toxic for infected cells (Hesse et al., J. Immunol.
  • IFN- ⁇ can have the same mechanism effects in preventing tumor metastasis and stimulating tumor regression (Nastala et al., J. Immunol, 1994, 153:1697; Wigginton et al., J. Clin. Invest. 2001, 108:51, which are hereby incorporated by reference in their entirety).
  • IFN- ⁇ also inhibits tumor neovascularization (which is necessary for the growth and survival of the tumor) (Coughlin et al; Immunity 1998, 9:25; Wigginton et al., Cancer Res. 1996, 56:1 131, which is hereby incorporated by reference in its entirety).
  • the invention further provides a method of treating a subject diagnosed as having a disease associated with reduced IL-2 activity, comprising administering to the subject an IL-2 activity stimulating amount of a peptide described herein.
  • IL-2 is also an activator and mediator of the innate immune system (natural killer cells and macrophages). It also has an important role in maintaining the viability of immune effectors. It has been shown to be important in maintaining the immune function both in AIDS and cancer (Smith, AIDS, 2001, 15 suppl 2:S28; Liu and Rosenberg, J. Immunol. 2001, 167:6356, which are hereby incorporated by reference in their entirety).
  • compositions according to the present invention comprise a peptide of the invention in association with a pharmaceutically acceptable carrier or excipient, adapted for use in human or veterinary medicine.
  • the peptides and peptide-containing compositions of the invention can be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject, along with the composition, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • compositions can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, subcutaneously, extracorporeally, topically (including ophthalmically, vaginally, rectally, intranasally) or the like, although topical intranasal administration or administration by inhalant is typically preferred.
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the composition.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, the particular composition used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • Parenteral administration of the peptides or peptide composition is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • the materials can be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These can be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275- 281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin- coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
  • compositions of the invention can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • the compositions can contain from 0.001-99% of the active material.
  • Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.
  • the compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • Pharmaceutical compositions can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • compositions can also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.
  • Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders can be desirable.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • the daily dosage as employed for treatment of an adult human of approximately 70 kg body weight ranges from about 0.2 mg to 10 mg or 0.5 to 5 mg, which can be administered in 1 to 4 doses, for example, depending on the route of administration and the condition of the patient.
  • DAPTA was administered intra-nasally by metered nasal spray at 2 mg three times a day.
  • the formulation was 5 mg/ml peptide T in 0.45% NaCl with a preservative such as benzyl alcohol.
  • Dose optimization can be routinely done. For example doses up to lOOmg or as low as 0.05mg could be more efficacious or be easier to administer, or require less frequent dosing.
  • Affinity constants are similar to those of morphine. On the basis of this affinity, dosage of 0.33-0.0003 mg/kg per day can be administered.
  • a blood concentration 10 6 to 10" molar blood concentration is suggested.
  • Peptide T was administered by nasal spray at three divided doses of 6 mg/day.
  • Patient blood cells PBMCs
  • PBMCs Patient blood cells
  • Cells were purified by centrifugation over phycoll-hypaque and prepared for analysis of intracellular gamma interferon (IFN- ⁇ ).
  • IFN- ⁇ intracellular gamma interferon
  • brefeldin A 10 ⁇ g/ml
  • 37 °C to inhibit protein transport and removed by vigorous pipetting
  • stained with labeled an anti-IFN- ⁇ , or isotype control mAbs for 30 minutes and analyzed by flow cytometry.
  • PBMC's were isolated from whole blood by LSM (Sigma);were seeded in 1 x 10 6 cells/ml; and cultured overnight in 5.0 ml complete RPMI 1640 (Gibco BRL) medium, supplemented with 10% FBS, 2mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml Streptomycin.
  • PHA 1 ng/ml and rH IL-2 stimulate cell proliferation at 25 U/ml. 24-hours after seeding, 5 ng/ml PMA (stock solution 10ng/( ⁇ l), and 500 ng/ml Ionomycin (Sigma, Cat. # I - 0634) (stock solution 1 mg/ml in EtOH) was added.
  • the cells were incubated 1 hour at 37 °C, 5% CO 2 .
  • Brefeldin A Sigma, Cat. # B 7651
  • 10 ⁇ g/ml(Stock solution 1.0 mg/ml) was added.
  • the cells were incubated 4 hours, at 37°C, 5% CO 2 .
  • the cells were pelletted by centrifugation 1000 rpm 5 min. at room temperature (RT).
  • the cells were fixed with 4% Paraformaldehyde + 3% BSA - 0.5 ml at 6 °C 15 min. At this step cells can be fixed overnight at 4°C. The cells were washed 2 times with 1.0 ml Surface stain buffer - 5 min. at 1 000 rpm. Permiabilization was performed with Saponin containing buffer: 0.5 mg saponin/ 200 ml PBS, 10% human AB serum, 0.1% Na Azide. Add: x 0.1-ml permiabilization buffer. After gentle mixing, the cells were incubated 15 min. at room temperature. Blocking was performed with mouse serum.
  • Second antibodies were added as follows: To the Control tubes: 1.5 ⁇ l isotype mouse IgG2a (PharMingen, Cat. # 33035X). To the Samples: mouse MAb ⁇ Hu IFN- ⁇ , R- Phycoerythrine conjugate.
  • cytokines II- 1, 6, 8, TNF- ⁇ were measured at greater than 100 pgms/ml at baseline in all five patients.
  • the cytokines IL-2, IL-4, IL-10, IL-12, and IL-13 were low to barely detectable prior to treatment (baseline).
  • baseline baseline
  • highly significant reductions in the levels of IL-1, IL-6, IL-8, and TNF- ⁇ were observed (P ⁇ .005) (Table 2).
  • cytokines IL-2, IL-4, IL-10, IL-12, and IL-13 increased significantly (P ⁇ .005) (Table 2). Where significant changes occurred all five members of the cohort responded in the same direction. Elevated TNF- ⁇ [Gendelman et al., 1998] and IL-6, [Navikas et al., 1995] and reduced IL-2, IL-4 [Graziosi et al., 1996] levels are reported in AIDS patient lymph nodes and peripheral mononuclear cells. Those in vitro observations made on isolated cell populations were qualitatively consistent with the baseline in vivo levels observed in the current patients.
  • Cytokine action in the nervous system has been invoked as an explanation for the mental deficits and neurological impairments of AIDS.
  • the inflammatory cytokines IL-1, 11-6, and TNF- ⁇ have been implicated in HIV brain pathologies [Merrill et al., 1992, Yeung et al., 1995] and levels of these inflammatory cytokines decreased in plasma following DAPTA treatment.
  • the pro-inflammatory cytokines TNF- ⁇ , IL-1, and IL-6 up-regulate virus expression in vitro (Folks,T; Merrill, J. Virol, 1989) and the reduction of these cytokines with DAPTA treatment would favor less viral replication.
  • Th2 cytokines 11-4, 10,and 13 increased by DAPTA (Table 2), induce a potent virostatic state in infected macrophages in vitro [Montaner and Gordon, 1995] as well as inhibit production of the pro-inflammatory cytokines II- 1 and TNF- ⁇ , which up-regulate virus expression [Merrill and Chen, 1991].
  • DAPTA DAPTA treatment
  • Panel A Cytokine decreases in 5 AIDS patients after 7-fcl weeks of intranasal Peptide T treatment. Cytokine changes are in patients receiving Peptide T. Cytokine measurements were made at baseline and at end of the study period from blinded plasma samples after capillary electrophoresis followed by ELISA. Patients were not taking anti-retroviral therapies, except for traditional Chinese herbal medicines. Patients were maintained on their treatments for 1 month prior to, and during, the period of added peptide T (3mgs/day). All members of the cohort (five of five patients) showed changes (decrease or increase, as indicated).
  • Panel B Cytokine increases in 5 AIDS patients after 7 ⁇ 1 weeks of intranasal Peptide T treatment. Enhanced Intracellular IFN- ⁇ Secretion by CD8 + T Cells in Ongoing San Francisco Trial
  • Cytotoxic T lymphocytes play a role in containing HIV spread. These effector lymphocytes are not fully functional in suppressing virus production, perhaps due to lack of CD4 help or HIV suppression of virus-specific CD8 + cells.
  • the ability of CTLs to secrete gamma interferon (IFN- ⁇ ) is accepted to be an indicator of cytotoxic capability.
  • IFN- ⁇ gamma interferon
  • the percentage of IFN- ⁇ secreting CD8 + T cells of the total CD8+ cell population from six persons after 24 weeks treatment with the HIV entry inhibitor Peptide T (DAPTA) was measured (Table 3 and Figure 1).
  • PBMCs were isolated and activated with PMA/ionomycin and IFN- ⁇ secreting/CD 8 + cells were enumerated by flow cytometric methods.

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Abstract

L'invention porte sur un procédé visant à accroître l'activité des lymphocytes T cytotoxiques (CTL) chez un sujet, ce procédé consistant à administrer au sujet une quantité d'un peptide stimulant l'activité des CTL, ce peptide étant sélectionné parmi les peptides décrits. L'invention porte également sur un procédé visant à accroître la sécrétion de l'interféron η (IFN-η) chez un sujet en administrant à ce dernier une quantité accrue de sécrétion d'IFN-η d'un peptide sélectionné parmi les peptides décrits. L'invention porte également sur un procédé visant à accroître la sécrétion de l'interleukine (IL-2) chez un sujet et consistant à administrer à ce dernier une quantité d'un peptide augmentant la sécrétion d'IL-2, ce peptide étant sélectionné parmi les peptides décrits. L'invention porte en outre sur un procédé de traitement d'un sujet chez lequel on a diagnostiqué une maladie associée à l'activité réduite des CTL, ce procédé consistant à administrer au sujet une quantité d'un peptide stimulant l'activité des CTL, ce peptide étant sélectionné parmi les peptides décrits. L'invention porte sur un procédé de traitement d'un sujet chez lequel on a diagnostiqué une maladie associée à l'activité réduite d'IFN-η, ce procédé consistant à administrer au sujet une quantité d'un peptide stimulant l'activité d'IFN-η, ce peptide étant sélectionné parmi les peptides décrits. L'invention porte enfin sur un procédé de traitement d'un sujet chez lequel on a diagnostiqué une maladie associée à l'activité réduite d'IL-2, ce procédé consistant à administrer au sujet une quantité d'un peptide stimulant l'activité d'IL-2, ce peptide étant sélectionné parmi les peptides décrits.
PCT/US2002/039109 2001-12-07 2002-12-06 Peptide t stimulant les reponses des lymphocytes t cytotoxiques (ctl) WO2003050136A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101125199B (zh) * 2006-08-15 2010-07-21 北京四环生物制药有限公司 白介素2作为制备治疗鼻炎药物的应用
CN104897891A (zh) * 2014-03-06 2015-09-09 中国农业大学 一种弓形虫检测试剂盒

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834429A (en) * 1986-06-03 1998-11-10 The United States Of America As Represented By The Department Of Health And Human Services Small peptides which inhibit binding to T-4 receptors and act as immunogens

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834429A (en) * 1986-06-03 1998-11-10 The United States Of America As Represented By The Department Of Health And Human Services Small peptides which inhibit binding to T-4 receptors and act as immunogens

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101125199B (zh) * 2006-08-15 2010-07-21 北京四环生物制药有限公司 白介素2作为制备治疗鼻炎药物的应用
CN104897891A (zh) * 2014-03-06 2015-09-09 中国农业大学 一种弓形虫检测试剂盒

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