WO1997048725A1 - Nouveaux peptides pour la prevention et le traitement d'infections - Google Patents

Nouveaux peptides pour la prevention et le traitement d'infections Download PDF

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Publication number
WO1997048725A1
WO1997048725A1 PCT/AU1997/000395 AU9700395W WO9748725A1 WO 1997048725 A1 WO1997048725 A1 WO 1997048725A1 AU 9700395 W AU9700395 W AU 9700395W WO 9748725 A1 WO9748725 A1 WO 9748725A1
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thr
ile
ser
val
pro
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PCT/AU1997/000395
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English (en)
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Deborah Ann Rathjen
Joy Merilyn Sleigh
Philip On-Lok Mack
Fred Widmer
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Peptech Limited
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Priority claimed from AUPO0610A external-priority patent/AUPO061096A0/en
Priority claimed from AUPO2165A external-priority patent/AUPO216596A0/en
Priority claimed from AUPO3309A external-priority patent/AUPO330996A0/en
Application filed by Peptech Limited filed Critical Peptech Limited
Priority to AU30842/97A priority Critical patent/AU3084297A/en
Publication of WO1997048725A1 publication Critical patent/WO1997048725A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • 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

  • the present invention relates to peptides having neutrophil and/or monocyte/macrophage stimulating activity, and to the use of these peptides as therapeutic agents.
  • the phagocytic leukocytes include three types of granulocytes; neutrophils, eosinophils and basophils; and two types of mononuclear phagocytes; monocytes and macrophages.
  • Neutrophils predominate in the response to acute bacterial and fungal infections: monocytes and macrophages respond to chronic infections, especially those caused by intracellular pathogens; eosinophils are associated with allergic reactions and invasion by metazoan parasites and basophils are associated primarily with immunologic disorders.
  • the responses of phagocytic cells to invasion by microorganisms are mediated by humoral agents derived from both microbial (e.g. endotoxin, chemotactic factors) and host (e.g.
  • cytokines antibodies, the complement system, cytokines
  • TNF tumour necrosis factor
  • Phagocyte defects are either acquired or inherited disorders and are associated with either inadequate numbers of circulating cells (neutropenia, granulocytopenia) or impaired function (such as in chronic granulomatous disease (CGD), myeloperoxidase deficiency, adherence glycoprotein deficiency, Chediak-Higashi syndrome, specific granule deficiency and Job's syndrome). Secondary failure of phagocyte responses occurs in disorders of humoral mediator systems, such as agammaglobulinemia or complement deficiency syndromes. The major clinical manifestation of phagocyte disorders is recurrent infections, most often involving the skin, lymph nodes, and respiratory tract. Frequently encountered pathogens include S. aureus, Streptococci, gram negative bacilli, Candida, Aspergillus and Narcardia. Periodontal disease also has a high prevalence and poor wound healing is characteristic.
  • neutrophil function is known to occur in a number of viral, fungal, bacterial and parasitic infections (Abramson and Mills, 1988), Rev. Infect. Dis. 10; 326-341; Ferrante et al. 1989. Immunol. Letts. 22; 301-6).
  • Tuberculosis presents a major health problem throughout the world with a recent resurgence of tuberculosis in industrialised societies (Snide et al IN: Bloom B R, ed. Tuberculosis Washington; ASM Press, 1994: 3-12; Friedman et al 1996. Engl. J. Med. 334; 828-833; Frieden et al, 1993. N. Engl. J. Med. 328; 521-526).
  • HTV Human Immunodeficiency virus
  • Immunosuppression by either drug therapy or HTV co-infection also allows recrudescence of infection in a person who had previously controlled the infection.
  • Advanced HTV infection is also complicated by infection with environmental mycobacteria of the Mycobacterium avium complex (MAC). Infection with MAC is difficult to treat because of its resistance to multiple antibiotics. Agents which stimulate the host response to mycobacteria would be advantageous as they may shorten the period of chemotherapy necessary to eradicate the organism. They may also permit new approaches to the treatment of infection with drug resistant M. tuberculosis and M. avium. In most patients with recurrent infections however, no white blood cell defect, complement or immunoglobulin abnormalities can be identified.
  • Haemophilus influenzae once considered a relatively infrequent cause of adult pneumonia, is now estimated to cause 2% to 20% of acute community acquired pneumonia. Furthermore, up to 15% of cases of community acquired pneumonia in the United Kingdom is caused by Legionella pneumophila. Marginally compromised hosts such as nursing home patients, elderly alcoholic patients, and the chronically disabled at any age, represent an important subset of patients in whom the cause of community acquired bacterial pneumonia differs from that seen in younger, normal populations. S. aureus and gram negative bacilli including H. influenzae, Klebsiella pneumoniae and Enterobacter aerogenes are more common causes of pneumonia in this group.
  • Group B Streptococci is a common cause of severe neonatal infections, including pneumonia and meningitis.
  • Host defense, which is particularly lacking in neonates, against Group B Streptococci depends entirely on the phagocytic and oxygen-dependent killing of the organisms by neutrophils.
  • Legionella pneumophila is a facultative gram-negative bacterium that has been identified as the eitologic agent of Legionnaires disease. Legionella pneumonia is believed to follow the inhalation of contaminated water aerosols. The bacteria are ingested by the resident alveolar macrophages within which Legionellae multiply. The multiplying bacteria eventually disrupt the alveolar macrophages and the released bacteria are in turn phagocytosed by mononuclear phagocytes and neutrophils recruited from the circulation. As mentioned above, Legionella may cause up to 15% of community acquired pneumonia as well as 1-40% of nosocomial pneumonias.
  • TNF ⁇ has been shown to protect mice against lethal Legionella pneumophila infection via activation of neutrophil function (Blanchard et al., 1988 J Leukocyte Biol. 43:429) while interferon- ⁇ treatment of human monocytes inhibits its growth in vitro.
  • CF Cystic Fibrosis
  • Most chronic bacterial skin infections occur in lower extremities and result from secondary infection of a break in the skin associated with a traumatic injury or underlying disease
  • the most common underlying diseases are diabetes mellitus, arterial insufficiency, venous stasis, vasculitis and haematologic problems such as sickle cell anaemia, leukemia and dysproteinemias.
  • Most local skin infections are caused by S. aureus, S. epidermidis, and gram negative bacilli such as P. aeruginosa and Aeromas hydrophila.
  • Bacteremia occurs in both community and hospital acquired infections.
  • Endocarditis isolated infection of the brain and other parenchymal organs, skin pustules and ocular infections are less frequent.
  • the gastrointestinal tract is considered to be the portal of entry.
  • Penetration of the intestinal villi, extension to the villous stroma, and ingestion and survival in non-immune macrophages provide access to the blood stream.
  • listeriosis in murine models suggest that resistance occurs in two phases. Bacterial multiplication is limited by nonimmune macrophages and neutrophils in the first phase and by immune macrophages in the second.
  • the microbiocidal activity of macrophages is associated with increased MHC following contact with interferon- ⁇ .
  • TNF interleukin-1
  • IL-2 interleukin-2
  • Those at risk of Listeria infection include patients immunosuppressed by lymphoreticular malignancies, immunosuppressive drugs (e.g. cortic ⁇ steroids, cyclosporin A and estrogens) and pregnancy. Neonates and the elderly are also susceptible to Listeria infection.
  • Up-regulation of phagocytic function may be of assistance in the treatment of fungal and protozoal diseases and conditions.
  • the dimorphic fungus H. capsulatum is an intracellular parasite that infects the host by deposition of microconidia into the terminal bronchioles and alveoli of the lungs.
  • Inhaled microconidia convert into yeasts that are phagocytosed by alveolar macrophages within which they multiply.
  • Dividing yeasts destroy the alveolar macrophages and then are ingested by other resident alveolar macrophages or monocytes recruited to the loci of infection.
  • Organisms may multiply intracellularly for 2 to 3 weeks before the development of cell mediated immunity (specifically armed macrophages), kill the fungus with the production of intense inflammation at the sites of infection.
  • necrosis occurs, and after several years the lesions may calcify. Where the development of cell-mediated immunity is delayed or fails to occur, progressive disseminated histoplasmosis occurs generally in individuals infected with HTV, taking glucocorticoids or other immunosuppressants or ongoing therapy for hematologic malignancies. However, apparently normal adults can also experience dissemination.
  • Cryptococcosis is an aerosol-borne mycosis. Of significance is the lack of any known host defect or pre-disposing condition in about half of all patients with cryptococcosis.
  • Factors pre-disposing to cryptococcal infections include AIDS, lymphoma, corticosteroids, sarcoidosis, diabetes, cytotoxic drug therapy for cancer, and renal transplantation. Suppression of the neutrophil and macrophage activating cytokines interferon ⁇ and TNF ⁇ increases susceptibility in a murine model of Cryptococcus neoformans infection reducing the ability of mice to survive central nervous system infection. (Aguirre et al., 1995, Infection and Immunity, 63: 1725-1731). Further, it has been demonstrated that administration of recombinant human TNF ⁇ protects mice from lethal C. neoformans lung infection (Kawakami et al., 1996, Clin Exp Immunol 106: 468:474).
  • the causative agents of human norcardiosis are members of the Actinomycetaceae.
  • N. asteroides is the most common species and is responsible for over 90% of pulmonary disease, disseminated disease or brain abscess.
  • N. brasiliensis is responsible for 60% to 80% of primary skin infections.
  • the basic tissue inflammatory response involves the neutrophil.
  • Cell-mediated immunity and macrophage function are also considered to be important in the pathogenesis of the disease.
  • fungi e.g. Candida, Aspergillus, and Phycomycetes .
  • Superficial chronic fungal infection of the skin is a common problem but invasion from the skin rarely occurs.
  • Fungi that produce nodular ulcerative skin lesions include blastomycosis, coccidiodmycosis and cryptococcosis.
  • the dygenetic protozoan Trypanosoma cruzi is the eitologic disease of Chagas' Disease in man.
  • the parasite infects a variety of host cell types including macrophages. Intracellular replication as amastigotes is followed by the release of trypomastigotes that can eventually reach the blood stream before infecting other host cells. Control of parasite load and host survival depend on effective T-cell mediated immunity via the cell-dependent protective antibody responses, macrophage activation for intracellular killing of the protozoan, and class I-dependent effector mechanisms.
  • Cytokines synthesized in the course of the inflammatory and/or immune responses can regulate in vitro killing of the parasite by macrophages.
  • the addition of TNF ⁇ or interferon ⁇ to cultures of T. cruzi - infected macrophages results in more efficient killing of amastigotes by the phagocytes.
  • Treatment of animals in a murine model of T. cruzi infection with either anti-interferon ⁇ or anti-TNF ⁇ antibodies has been shown to increase levels of parasitemia (Abrahamson and Coffman, 1996, Exp Parasitol, 84: 231-244).
  • Pneumocystis carinii has been regarded as a protozoan parasite that is passed from human to human by the respiratory route, however, more recent evidence suggests that it is a fungus.
  • P. ca ⁇ nii is usually not pathogenic until it has the opportunity to multiply and invade in a person who has a helper T cell defect.
  • Agents which upregulate the ability of neutrophils and/or monocytes/macrophages to kill virus-infected cells may also be beneficial.
  • herpes simplex type I following resolution of acute infection the immune response declines during the period of latency allowing the virus to be re-activated (Cantin et al., 1995, J. Virology, 69: 4898- 4905).
  • TNF ⁇ -mediated activation of macrophages in controlling acute herpes simplex virus infection has been established (Heise et al., 1995, J. Virology, 69: 904-909).
  • Influenza A infects alveolar macrophages and infection leads to a comparatively slow development of a specific immune response, i.e. antibody production and generation of cytoxic T lymphocytes. Therefore upregulation of macrophage function is particularly beneficial in the early phase of infection.
  • Neutrophils are the blood cells from which Cytomegalovirus (CMV) can usually be recovered in culture during viremia, although monocytes are also infected.
  • CMV is a member of the herpesvirus family and infections with CMV are extremely common, occurring throughout life. Similar to other herpesviruses, after primary infection CMV remains in the host in a latent state.
  • nosocomial infections may be reduced by immuno therapy with peptides such as those described herein.
  • Hospital-acquired infections involve most commonly the urinary tract, biliary tract, pneumonia and wound infections.
  • Candida sp is emerging as a highly important nosocomial pathogen, the two most important pre-disposing factors are exposure to broad-spectrum antimicrobial agents and neutropenia. Neutropenia is not however a pre- requisite for disseminated candidiasis. About half of cases occur in patients with complicated post-operative courses unrelated to underlying neoplastic diseases.
  • P. aeruginosa accounts for approximately 10% of all hospital acquired infections. It is the second most common cause of nosocomial pneumonia, third leading cause of urinary tract infection and the fourth most common cause of surgical wound infections.
  • S. aureus Methicillin-resistant S. aureus is a common cause of nosocomial disease. S. aureus is the cause of hospital acquired pneumonia in approximately 10% of cases. Wound infection follows urinary tract infection as the second most frequent type of nosocomial infection. In general, surgical wound infections that develop within 24 to 48 hours are due to group A ⁇ haemolytic
  • Streptococci or Clostridium pe ⁇ ingens whereas S. aureus, S. epidermidis, and gram negative bacilli wound infections occur 4 to 7 days after surgery.
  • Pressure scores decubitus ulcers are frequently nosocomial and are usually infected with two or more bacterial species.
  • the present inventors have previously identified peptides derived from the primary amino acid sequence of human tumour necrosis factor (TNF) which stimulate neutrophil activity. These peptides which are described in Australian patent specification Nos. 74762/91 and 44664/93 (the disclosures of which are to be regarded as incorporated herein by reference), are of considerable clinical significance since treatment with such peptides is expected to enhance neutrophil and monocyte/macrophage activity, but would not be expected to cause the severe side effects associated with therapeutic use of the whole TNF molecule. The present inventors have now identified further peptides exhibiting one or more improved properties over the abovementioned peptides.
  • TNF tumour necrosis factor
  • the present inventors have identified a "core" sequence within the previously described TNF-derived peptide 419 (PSTHVLITHTI), which possesses neutrophil and monocyte/macrophage stimulatory activity.
  • the core sequence (STXVXITX) is predicted to exhibit approximately 40% of the activity of peptide 419. Further, variation of this sequence has led to the identification of classes of peptides which have neutrophil stimulatory activity (“class 1”), equal neutrophil and monocyte/macrophage stimulatory activity (“class 2”), or preferentially enhanced monocyte/macrophage stimulatory activity (“class 3").
  • Peptides in class 3 typically retain neutrophil stimulatory activity which may either be equal or greater than that displayed by peptides in class 1 or class 2 with exceptions as indicated below.
  • the present invention provides a peptide with neutrophil and/or monocyte/macrophage stimulatory activity, wherein the peptide is of the general formula:-
  • X is absent, Cys or R ⁇ , X 2 is absent, Ala, Arg, Glu or Gly,
  • X 3 is absent, Ala, Arg, Asn, Cys, Glu, Gly, His, He, Leu, Lys, Met, Pro, Ser, Trp, ⁇ -Abu, ⁇ Ala, Dbu, Sar, Sue or N-Me-Ala
  • X 4 is absent, Ala, Arg, Asn, Glu, His, Leu, Lys, Met, Pro, Ser, Trp, ⁇ Ala or Nip
  • X 5 is Ala or His
  • X 6 is Ala, Gly, He, Leu, Phe, Pro, Ser, Thr, Trp, Val, D-Ala, D-Ile D-Pro, D-Ser, D-Thr, D-Val or ⁇ Ala, X 7 is His or Ala,
  • X fl is absent, He, Leu, Thr or D-Ile
  • X g is absent, He, D-Ile or Aib
  • X 10 is absent
  • Cys or R 2 , Ri is H or R-CO, where R is H, straight, branched or cyclic alkyl up to C20, optionally containing double bonds and/or substituted with halogen, nitro, amino, hydroxy, sulfo, phospho or carboxyl groups which may be substituted themselves or aralkyl or aryl optionally substituted as listed for the alkyl or
  • R12 and R13 are independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for R, or R 2 is N- glycosyl or N-lipoyl, or R 2 is -OR14, where R14 is H straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R ⁇ or R z is
  • -O-glycosyl, or -O-lipoyl or R 2 is absent when the adjacent amino acid is a dicarboxy derivative of cysteine or a homologue thereof or the peptide is in a N-C cyclic form; with the proviso that the peptide is not Pro-Ser-Thr-His-Val-Leu-Ile-Thr-His-
  • ⁇ Abu represents ⁇ -aminobutyric acid
  • Dbu represents diaminobutyric acid
  • Aib represents amino isobutyric acid
  • Nip represents nipocotic acid
  • ⁇ Ala represents ⁇ -alanine
  • Sar represents sarcosine (alternatively known as
  • N-methyl glycine Sue represents succinic acid
  • N-Me-Ala N- methyl alanine
  • the peptides according to the present invention may exhibit one or more improved properties over the peptides described in Australian patent specification Nos. 74762/91 and 44664/93.
  • the improved property or properties may be increased potency, extended in vivo half life or, particularly, specificity of action.
  • X t and X 10 are absent.
  • X 6 is preferably He.
  • Particularly preferred peptides include :-
  • Trp-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile (1065)Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1066) Asn-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1067)Glu-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1068) Arg-Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1069) Lys-Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1070)His-Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,
  • the present invention provides a peptide with neutrophil stimulatory activity (i.e. inactive or only negligibly active on monocytes/macrophages), referred to herein as a class 1 peptide.
  • a class 1 peptide wherein the peptide is of the general formula:-
  • X 3 is Ala, Lys or Ser
  • X 5 is Ala or His
  • Xs is He or Leu
  • X 7 is Ala or His
  • X 8 is He, Thr or D-Ile
  • X 9 is He or D-Ile
  • X 10 is absent, Cys or R 2 , wherein R T and R 2 are as defined above.
  • class 1 peptides are:- (1073) Lys-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,
  • the present invention provides a peptide with neutrophil and monocyte/macrophage stimulatory activity, referred to herein as a class 2 peptide, wherein the peptide is of the general formula:- X r X 2 -X 3 -X 4 -Ser-Thr-X 5 -Val-X 6 -Ile-Thr-X 7 -X 8 -X 9 -X 10 in which,
  • X is absent, Cys or R lt
  • X 3 is absent, Asn, Lys or ⁇ Ala X 4 is Arg, His, Lys, Pro, Trp, Ala or Nip,
  • X 5 is Ala or His
  • X 6 is He or Leu
  • X 7 is Ala or His
  • X B is He, Leu, Thr or D-Ile
  • X 9 is He or D-Ile
  • X 10 is absent, Cys or R 2 , wherein R, and R 2 are as defined above; with the proviso that when X 3 is Lys, then X 4 is not Pro.
  • class 2 peptides are:-
  • Trp-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile (1069) Lys-Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1080) Asn-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1175) Nip-Ser-Thr-Ala-Val-Ile-Ile-Thr-His-Thr-Ile, (1176) Nip-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile, (1177) Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Leu-Ile,
  • Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-Ile (1179) Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-D-He-Ile, and (1180) Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-D-He.
  • Class 1 and 2 peptides may exert their stimulatory activity by, for example, eliciting superoxide production by neutrophils or by enhancing respiratory burst following treatment with N-formyl-L-methionyl-L-leucyl-L- phenylalanine (fMLP).
  • fMLP N-formyl-L-methionyl-L-leucyl-L- phenylalanine
  • the present invention provides a peptide with preferential monocyte/macrophage stimulatory activity (i.e. with enhanced monocyte/macrophage stimulatory activity as compared to class 1 and 2 peptides), referred to herein as a class 3 peptide, wherein the peptide is of the general formula:-
  • X is absent, Cys or R ⁇ , X 2 is absent, Ala, Arg, Glu or Gly,
  • X 3 is absent, Ala, Arg, Glu, His, Leu, Met, Trp, ⁇ -Abu, Dbu, Sar or N-methyl Ala
  • X 4 is Arg, Asn, Glu, Leu, Met or Pro
  • X 5 is Ala or His
  • X 6 is He or Leu
  • X 7 is Ala or His
  • X 8 is He, Leu, Thr or D-Ile
  • X 9 is He or D-Ile
  • X 10 is absent, Cys or R 2 , wherein R ⁇ and R 2 are as defined above; with the proviso that when X 2 is absent and X 3 is Ala, then X 4 is not Pro.
  • Particularly preferred class 3 peptides are:-
  • Peptides in this class 3 generally display 70-130% activity of peptide 419 at lO ⁇ M in the neutrophil assay described below. Those that have asterisks exhibit only 30-40% activity of peptide 419 on neutrophils.
  • the present invention provides a method of treating a subject in order to improve neutrophil and/or monocyte/macrophage function such that infection may be either prevented or treated more effectively, the method comprising administering to the subject a therapeutic amount of the peptide of any one of the first to fourth aspects of the present invention.
  • the peptide may be administered with a pharmaceutically acceptable carrier or adjuvant.
  • the subject is suffering from one or more of the following: acquired immunity deficiency syndrome (AIDS); cancer; diabetes; nosocomial infection; tuberculosis; cystic fibrosis; community acquired pneumonia; meningitis; Chlamydia; Legionnaires Disease; Listeriosis; coccidian parasitical (e.g. Toxoplasma gondii) infection; an inherited primary neutropenic disorder (e.g. Kostermann's syndrome, mild chronic neutropaenia and cyclic neutropaenia); an inherited primary defect of phagocytic cell function (e.g.
  • chronic granulomatous disease myeloperoxidase deficiency, Chediak-Higashi syndrome, specific granule deficiency and Job's syndrome
  • an inherited secondary defect of phagocytic cell function e.g. agammaglobulinemia or complement deficiency syndromes
  • an acquired defect of phagocytic cell function e.g. diabetes mellitus, malnutrition, disseminated malignancy, age (elderly or neonates) and alcoholism
  • immunosuppression due to administration of immunosuppressive drugs and other bacterial, fungal (e.g. Candida), viral or protozoan (e.g. Pneumocystis carni ⁇ ) infection.
  • the subject may be suffering from bacterial, fungal, viral (e.g. cytomegalovirus (CMV) infection of neutrophils) or protozoan infection, or cancer and the peptide may be used with myelosuppressive agents to provide an early beneficial enhancement of neutrophil function.
  • CMV cytomegalovirus
  • Class 1 peptides may also be beneficial in the treatment of a subject suffering from AIDS since monocyte/macrophage activation is undesirable in order to avoid an increase in viral replication.
  • Other conditions that may be beneficially treated with a class 1 peptide include: cancer; inherited or acquired neutropenic disorders; infectious mononucleosis, paroxysomal nocturnal hemoglobinuria; conditions where bone marrow infiltration occurs such as in leukemia, lymphoma and myelofibrosis; and inherited primary or secondary defects of phagocytic cell function.
  • the subject may be suffering from a bacterial infection with, for example, any of Pseudomonas, E. coli, S. aureus and S. pneumonia, a fungal (e.g. C. albicans and T. glabrata) infection, viral (e.g. CMV) infection, norcardiosis (e.g. N. asteroides and N. brasiliensis), or protozoan infection.
  • class 2 peptides may be particularly useful in preventing or treating chronic lung infection by these organisms (such as H.
  • class 2 peptides may be useful include: cancer; cystic firbrosis; Legionnaires disease; tuberculosis; diabetes; meningitis and nosocomial infection.
  • the subject may be at risk of or suffering from a condition caused by intracellular pathogens including Mycobacteria (e.g. M. leprae, M. kansasii, M. melonae, M. tuberculosis, M. avium complex, M. marinum and M. ulcerans), Chlamydia (e.g. C. pneumoniae, C. pstittaci and C. trachomatis); Brucellae, Francisella (e.g. F. tularensis), Pasteurellosis (e.g. P. moltocida), Legionellosis (e.g. L.
  • Mycobacteria e.g. M. leprae, M. kansasii, M. melonae, M. tuberculosis, M. avium complex, M. marinum and M. ulcerans
  • Chlamydia e.g. C. pneumoniae, C. pstittaci and C.
  • Another condition that may be beneficially treated with class 3 peptides is cancer and, particularly, Chronic lymphocytic leukemia where T cell numbers are depressed by chemotherapy with fludarabine and 2-cyclodeoxyadenosine (Girmenia et al., 1994, Brit. J. Haematol. 87: 4078) and macrophage function as measured by TNF ⁇ production induced by bacterial products is defective (Dahlke et al., 1995, J. Haemotol. 49: 76-82; Fleiger et al., 1990, Int. J. Cancer 45: 280-286).
  • the peptides having been shown to prevent relapse of mycobacterial infection when CD4 cells are depleted.
  • the peptides according to the invention may be co-administered (i.e. used in combination therapy) with other therapeutic agents such as cyclosporin A and prednisolone (i.e. for T cell immunosuppressive therapies for, for example, patients with graft vs host disease) or other agents which stimulate increased white blood cell numbers (e.g. G-CSF.
  • other therapeutic agents such as cyclosporin A and prednisolone (i.e. for T cell immunosuppressive therapies for, for example, patients with graft vs host disease) or other agents which stimulate increased white blood cell numbers (e.g. G-CSF.
  • cancer chemotherapy agents such as aminoglutethimide, amsacrine, azacitidine, busulfan; carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, cytarabine HCl, dacarbazine, dactinomycin, deoxycoformycin, doxorubicin. etoposide, floxuride, fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, lomustine, mechlorethamine, melphalan, mercaptopurine, mitomycin.
  • cancer chemotherapy agents such as aminoglutethimide, amsacrine, azacitidine, busulfan; carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, cytarabine HCl, dacarbazine, dactinomycin, deoxycoformycin, doxorubic
  • antibiotic agents such as amikcan, gentamycin, tobramycin, netilmicin, cephalosporins (such as cephalothin, cefaclor, cefamandole, cefixime, cef
  • anti-fungal agents such as amphotericin B, ketoconazole and flucytosine
  • anti-viral agents such as gangciclovir and acyclovir.
  • co- administration of a peptide according to the invention with other therapeutic agents is to be understood as including single administration/dosage with a formulation comprising the peptide and therapeutic agent, as well as sequential separate administration/dosage of the peptide and the therapeutic agent. Sequential administration/dosage may be in either order with the administration/dosage separated by, for example, several minutes or up to 24 hours or more.
  • modifications may be made to the peptides according to the present invention without deleteriously affecting the biological activity of the peptides.
  • modifications include various changes such as sulfation, phosphorylation, nitration, halogenation, and insertions, deletions, and substitutions, either conservative or non-conservative (eg to amino acids, desamino acids) in the peptide sequence where such changes do not substantially alter the overall biological activity of the peptide.
  • conservative substitutions the intended combinations are - G, A; V, I, L, M; D, E: N, Q; S, T; K, R, H; F, Y, W, H; and P, N ⁇ -alkylamino acids.
  • peptide is to be understood to embrace peptide bond replacements (isosteres) and/or peptide mimetics, ie pseudopeptides, as recognised in the art (see for example: Proceedings of the 20th European Peptide Symposium, edt. G. Jung, E. Bayer, pp. 289-336, and references therein), as well as salts and pharmaceutical preparations and/or formulations which render the bioactive peptide(s) particularly suitable for oral, topical, nasal spray, ocular pulmonary, IV, subcutaneous, as the case may be, delivery.
  • Such salts, formulations, amino acid replacements and pseudopeptide structures may be necessary and desirable to enhance the stability, formulation, deliverability (eg, slow release, prodrugs), or to improve the economy of production, and they are acceptable, provided they do not negatively affect the required biological activity of the peptide.
  • cyclic structures for stability such as N to C interchain imides and lactames (Ede et al in Smith and Rivier (Eds) "Peptides: Chemistry and Biology", Escom, Leiden (1991), p268-270), and sometimes also receptor binding may be enhanced by forming cyclic analogues.
  • An example of this is given in "Confirmationally restricted thymopentin-like compounds", US Patent No. 4,457,489 (1985), Goldstein, G. et al.
  • ketomethylene, methylsulfide or retroinverse bonds to replace peptide bonds, ie the interchange of the CO and NH moieties may both greatly enhance stability and potency.
  • the peptides of the invention can be synthesised by various methods which are known in principle, namely by chemical coupling methods (cf. Wunsch, E.: “Methoden der organischen Chemie", volume 15, Band 1 + 2, Syntheses von Peptiden, Theime Verlag, Stuttgart (1974), and Barrany, G.; Merrifield, R. B: "The Peptides”, eds. E. Gross, J. Meienhofer., Volume 2, Chapter 1, pp. 1-284, Academic Press (1980), or by enzymatic coupling methods (cf. Widmer, F., Johansen, J. T., Carlsberg Res. Commun., volume 44, pp.
  • cysteine residue to be positioned at both the amino and carboxyl terminals of the peptide. This will enable the cyclisation of the peptide by the formation of a di-sulphide bond.
  • Figure 1 Provides graphical results of nitrite release by BCG-infected bone marrow-derived macrophages activated with peptides 966, 927, 926, 419 or 968 and interferon ⁇ .
  • Figure 2 Provides graphical results showing stimulation of nitrite release by BCG-infected bone marrow-derived macrophages by peptide 1065.
  • Figure 3 Provides graphical results of recrudescence of splenic BCG
  • Figure 4 Provides graphical results of the mean number of liver granulomas following CD4 depletion in BCG (disseminated mycobacterial) infection.
  • Figure 5 Provides graphical results showing the effect of peptide 966 on infection-related weight loss in chronic Pseudomonas aeruginosa lung infection.
  • Figure 6 Provides graphical results showing the effect of peptide 968 on infection-related weight loss in chronic Pseudomonas aeruginosa lung infection.
  • Chemiluminescence assay Human neutrophils were treated with peptides for 15 minutes according to the protocol described by Ferrante et al. 1988 ⁇ Int. Arch. Allergy Appl. Immunol.. 86; 82-91) and lucigenin-dependent chemiluminescence measured.
  • peptides were administered ip at 4mg/kg. At various times post inoculation peritoneal cells were harvested and chemiluminescence in response to the bacterial peptide fMLP measured in duplicate to quadruplicate determinations. The data represent the mean ⁇ SD of 3-15 animals.
  • the peritoneal cells were harvested by the following method: Sterile, pyrogen-free Hanks Balanced Salt Solution (5ml) was injected intraperitoneally following exposure of the peritoneal cavity. The contents were massaged for 30 seconds and the peritoneal fluid carefully withdrawn. Cells contained in the peritoneal fluid were washed, counted by trypan blue exclusion (>99% viability) and resuspended to 10 6 macrophages/ml.
  • Peptides 757-761, 771-778 and 1097-1107 were synthesised and tested for neutrophil stimulation in order to determine the "core" residues and sequence of peptide 419 required for neutrophil stimulatory activity.
  • N and C-terminal truncations of peptide 419 significantly reduced activity, with only the truncated peptides 771 (STHVLITHTI), 757 (PSTHVLITHT) and 758 (PSTHVLIT ⁇ ) retaining approximately 40% or more of the activity of peptide 419. Consequently, it is predicted that the core sequence STHVLITH would exhibit approximately 40% of the activity of peptide 419.
  • ⁇ ll peptides were at lO ⁇ M final concentration.
  • Values represent the mean ⁇ SD of triplicate determinants.
  • ⁇ ll peptides were at 10 ⁇ M final concentration.
  • Values represent the mean ⁇ SD of triplicate detormiriants.
  • Values represent the mean ⁇ SD of triplicate determinants.
  • ⁇ ll peptides were at lO ⁇ M final concentration.
  • Values represent the mean ⁇ SD of triplicate determinants.
  • ⁇ ll peptide treated animals received 4mg/kg peptide.
  • Control animals were injected with sterile, pyrogen free phosphate-buffered saline. Results are expressed as the mean ⁇ standard deviation of determinations from 3-15 animals. The number of animals used is indicated in brackets. TABLE 11
  • Peptides 419, 966, 968 and 1059-1081, 1083-1090 were assessed for neutrophil and monocyte/macrophage stimulatory activity. Results are presented in Tables 12 to 14. Neutrophil stimulatory activity was determined by the chemiluminescence assay described in Example 1. Monocyte/macrophage stimulatory activity was determined according to the method described below. Monocyte chemiluminescence assay:
  • Monocytes were purified from the blood of normal volunteers by centrifugation in Ficoll-Hypaque medium followed by overnight adherence to cytodex microcarriers (Kumaratilake and Ferrante, 1988, /. Immunol Methods 112: 183-190). Monocyte stimulation by peptides was measured by lucigenin-dependent chemiluminescence as described for neutrophils.
  • ⁇ ll peptides were at lO ⁇ M final concentration.
  • Values represent the mean ⁇ SD of triplicate determinants.
  • Peptides 419, 926, 927, 966, 968 and 1065 were assessed for their effect on nitrite release by M. bovis BCG-infected bone marrow macrophage.
  • Mouse macrophages were derived from the bone marrow following isolation of progenitor cells and subsequent growth for 6 days in RMI-1640 containing 5% horse serum, 10% foetal calf serum, 20% L929 cell supernatant, 2mM L-glutamine, 2-mercaptoethanol, streptomycin and penicillin. Peptides (0.6-60 ⁇ M final) were added to the macrophage cultures (10 5 macrophages/well) 24 hours prior to infection with 10° Bacillus Cametre - Guerin (BCG) organisms. Three days after infection the degree of growth inhibition was determined by a 24 hour pulse of 3 H-Uracil (l.O ⁇ Ci/well). Nitric oxide content of the cell supernatant was also measured three days after infection by reaction with the Greiss reagent and optical density of the reaction at 540nm determined.
  • Peptides tested for effect on recrudescence of infections shown by splenic BCG levels and granulomatous pathology following CD4 cell depletion Peptides 419, 966 and 968 were tested for their effect on necrudescence of BCG infection in vivo following CD4 depletion.
  • mice were infected with 1x10 s BCG iv. Thirteen weeks after infection when the infection had become chronic, mice were treated (300 ⁇ g Mab daily for three days and then at weekly intervals for 4 weeks) with monoclonal antibody GKI-54 which binds to CD4 + lymphocytes. All mice received ip injection of saline or lOO ⁇ g of peptide every second day for 4 weeks. Mice were then sacrificed and the number of splenic BCG organisms in each was determined by growth on 7HH agar plates ( Figure 3). The number of granulomas in liver, as a measure of immunopathology, was determined by staining of frozen sections with the P7/9 Mab which recognises murine MHC class 11. Quantification of staining was carried out using Image Analysis (Chromatic colour image analysis software package version 2.2 from Leading Edge) ( Figure 4).

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Abstract

L'invention concerne des peptides ayant une longueur comprise entre 8 et 15 acides aminés. Lesdits peptides ont une activité stimulatoire neutrophile et/ou monocyte/macrophage et peuvent être utilisés dans des méthodes de traitement de diverses maladies ou affections dans lesquelles une amélioration de la fonction neutrophile et/ou monocyte/macrophage est souhaitable. Le peptide est de formule générale X1-X2-X3-X4-Ser-Thr-X5-Val-X6-Ile-Thr-X7-X8-X9-X10 dans laquelle X1 est absent ou est Cys ou R1; X2 est absent ou est Ala, Arg, Glu ou Gly; X3 est absent ou est Ala, Arg, Asn, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Trp, η-Abu, βAla, Dbu, Sar, Suc ou N-Me-Ala; X4 est absent ou est Ala, Arg, Asn, Glu, His, Leu, Lys, Met, Pro, Ser, Trp, βAla ou Nip; X5 est Ala ou His; X6 est Ala, Gly, Ile, Leu, Phe, Pro, Ser, Thr, Trp, Val, D-Ala, D-Ile, D-Pro, D-Ser, D-Thr, D-Val ou βAla; X7 est His ou Ala; X8 est absent ou est Ile, Ley, Thr ou D-Ile; X9 est absent ou est Ile, D-Ile ou Aib; et X10 est absent ou est Cys ou R2.
PCT/AU1997/000395 1996-06-21 1997-06-20 Nouveaux peptides pour la prevention et le traitement d'infections WO1997048725A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001034149A1 (fr) * 1999-11-12 2001-05-17 Merck & Co., Inc. Derives de diaryle piperidyle pyrrole en tant qu'agents antiprotozoaires
FR2829767A1 (fr) * 2001-09-14 2003-03-21 Gerard Papierok Composes peptidiques destines a la prevention et au traitement d'affections chez les mammiferes
FR2844511A1 (fr) * 2001-09-14 2004-03-19 Oridan Inc Composes peptidiques destines a la prevention et au traitement d'affections chez les mammiferes
US6844318B2 (en) 2000-03-15 2005-01-18 Bristol Myers Squibb Pharma Company Peptidase-cleavable, targeted antineoplastic drugs and their therapeutic use
US20140364378A1 (en) * 2013-06-05 2014-12-11 Industrial Technology Research Institute Method and pharmaceutical composition for hair growth

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7476291A (en) * 1990-03-12 1991-10-10 Cephalon Australia Pty Ltd Neutrophil stimulating peptides
AU4466493A (en) * 1993-08-13 1995-03-02 Cephalon Australia Pty Ltd Neutrophil stimulating peptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7476291A (en) * 1990-03-12 1991-10-10 Cephalon Australia Pty Ltd Neutrophil stimulating peptides
AU4466493A (en) * 1993-08-13 1995-03-02 Cephalon Australia Pty Ltd Neutrophil stimulating peptides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF CLINICAL INVESTIGATION, Vol. 95, No. 5, issued 1995, KUMARATILAKE et al., "A Synthetic Tumor Necrosis Factor-alpha. Agonist Peptide Enhances Human Polymorphonuclear Leukocyte Mediated Killing of Plasmodium Falciparum in Vitro and Suppresses Plasmodium Chabaudi Infection in Mice", p. 2315-2323. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001034149A1 (fr) * 1999-11-12 2001-05-17 Merck & Co., Inc. Derives de diaryle piperidyle pyrrole en tant qu'agents antiprotozoaires
US6291480B1 (en) 1999-11-12 2001-09-18 Merck & Co., Inc. Diaryl piperidyl pyrrole derivatives as antiprotozoal agents
US6384052B1 (en) 1999-11-12 2002-05-07 Merck & Co., Inc. Anticoccidial compounds
US6844318B2 (en) 2000-03-15 2005-01-18 Bristol Myers Squibb Pharma Company Peptidase-cleavable, targeted antineoplastic drugs and their therapeutic use
FR2829767A1 (fr) * 2001-09-14 2003-03-21 Gerard Papierok Composes peptidiques destines a la prevention et au traitement d'affections chez les mammiferes
WO2003025012A2 (fr) * 2001-09-14 2003-03-27 Oridan, Inc. Complexe peptidique vaccinal therapeutique destine a la prevention et au traitement d'affections chez les mammiferes
WO2003025012A3 (fr) * 2001-09-14 2003-12-11 Oridan Inc Complexe peptidique vaccinal therapeutique destine a la prevention et au traitement d'affections chez les mammiferes
FR2844511A1 (fr) * 2001-09-14 2004-03-19 Oridan Inc Composes peptidiques destines a la prevention et au traitement d'affections chez les mammiferes
US20140364378A1 (en) * 2013-06-05 2014-12-11 Industrial Technology Research Institute Method and pharmaceutical composition for hair growth
US9827181B2 (en) * 2013-06-05 2017-11-28 Industrial Technology Research Institute Method and pharmaceutical composition for hair growth

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