WO1997048725A1 - Novel peptides for prevention and treatment of infection - Google Patents

Abstract

Peptides of 8 to 15 amino acids in length are described possessing neutrophil and/or monocyte/macrophage stimulatory activity. The peptides may be used in methods of treatment of various diseases and conditions in which enhancement of neutrophil and/or monocyte/macrophage function is desirable. The peptide is of the general formula: X1-X2-X3-X4-Ser-Thr-X5-Val-X6-Ile-Thr-X7-X8-X9-X10 in which, X1 is absent, Cys or R1; X2 is absent, Ala, Arg, Glu or Gly; X3 is absent, Ala, Arg, Asn, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Trp, η-Abu, βAla, Dbu, Sar, Suc or N-Me-Ala; X4 is absent, Ala, Arg, Asn, Glu, His, Leu, Lys, Met, Pro, Ser, Trp, βAla or Nip; X5 is Ala or His; X6 is Ala, Gly, Ile, Leu, Phe, Pro, Ser, Thr, Trp, Val, D-Ala, D-Ile, D-Pro, D-Ser, D-Thr, D-Val or βAla; X7 is His or Ala; X8 is absent, Ile, Ley, Thr or D-Ile; X9 is absent, Ile, D-Ile or Aib; and X10 is absent, Cys or R2.

Description

NOVEL PEPTIDES FOR PREVENTION AND TREATMENT OF INFECTION

Field of the Invention:

The present invention relates to peptides having neutrophil and/or monocyte/macrophage stimulating activity, and to the use of these peptides as therapeutic agents.

Background to the Invention:

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. antibodies, the complement system, cytokines) sources. These molecules bind to receptors on the phagocyte plasma membrane and thus trigger signal transduction elements that lead to functional cellular responses. Initial contact with certain mediators, e.g. tumour necrosis factor (TNF), results in priming of the neutrophil, (for example, through up regulation of receptors), so that subsequent challenge elicits an enhanced response. 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.

Various defects in neutrophil function have been described as acquired disorders in some patients with thermal injury, bacterial or viral infections (see below), rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, malnutrition, disseminated malignancy, bone marrow transplantation, and a number of other problems including age (i.e. the elderly and neonates) and alcoholism. In particular, depressed 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). In addition, depressed neutrophil function has been described in Acquired Immune Deficiency syndrome (Thorsen et al, 1984. AIDS. 3; 651-653; Ellis et al. 1988, J. Infect. Dis. 158; 1268-1276; Murphy et al, 1988. J. Infect. Dis. 158; 627-630), cancer patients, (who may also suffer neutropaenia as a result of radio and chemotherapy, consequently leaving them more susceptible to viral, bacterial and fungal infections: Bodey et al, 1966. Ann. Intern Med. 64; 328), and patients with acute leukemia undergoing bone marrow transplant (especially those with depleted T cell subsets, who are particularly at risk of fungal infection: Pirsch and Maki, 1986. Ann. Intern. Med. 104; 619-631). Also, it is known that improved phagocytic cell function may protect against infection in graft vs host disease and in patients treated with cyclosporin A (Kennedy et al 1994. J. Clin. Oncol. 12; 249-57). In patients with phagocyte dysfunction syndromes and conditions, early recognition and aggressive treatment of infections may be crucial. Unfortunately, microbial resistance to antibiotics is an increasing world-wide problem, thus new approaches to therapy are required. Immunotherapy has the advantage that the body's own defenses are upregulated, potentially leading to a reduction in antibiotic use. For example, interferon-γ, a macrophage stimulating agent, decreases the frequency of serious infections in patients with CGD. CONDITIONS AND DISEASES CAUSED BY BACTERIA:

Agents which upregulate the ability of neutrophils and/or monocytes/macrophages to treat bacterial infection would be desirable.

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). The greatest risk factor for tuberculosis is coincident infection with Human Immunodeficiency virus (HTV) which destroys CD4 positive T cells which act to control infection. 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. Frequently, breaks in the skin from burns, scratches, bites and surgical wounds allow bacteria to bypass the normal defense of intact skin. Many pathogenic bacteria are carried by normal individuals, e.g. up to 30% of healthy people carry S. pneumoniae and up to 40% of healthy people carry S. aureus. Pneumonia caused by these organisms may arise during viral respiratory infection. Similarly, in chronic obstructive pulmonary disease (COPD), such as chronic bronchitis, the initial insult, resulting in increased cough and expectoration, is probably viral. While no infective organism is found in all patients, Haemophilus influenzae and Streptococcus pneumoniae are the most commonly cultured organism. In addition, Mycoplasma is occasionally present. A growing body of evidence relates recurrent childhood respiratory infections to COPD in later life (Higgins 1984, Chest 85(Suppl.):35). Most community acquired pneumonia's are due to S. pneumoniae.

However. 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. TNFα 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-γ treatment of human monocytes inhibits its growth in vitro.

Patients with Cystic Fibrosis (CF) are particularly vulnerable to bacterial colonization of the lung despite apparently normal phagocytic cell function and cell-mediated immunity. Over 95% of the deaths of CF patients are caused by lung diseases. At birth, before infection, the lungs are histologically normal. Usually early in life, lung infection occurs and obstruction of the small airways by mucus, inflammatory infiltrates, and mucosal oedema begins. Early in the course, bacterial infection becomes established and can not be eradicated despite prolonged and intensive antibiotic therapy. S. aureus predominates early in the course but later Pseudomonas aeruginosa becomes predominate. H. influenzae and Pseudomonos cepacia are also common. Mycobacterial infection is rare. Specific immunotherapy as an adjunct to antibiotic therapy may be useful in reducing antibiotic use and hence prevent or delay the emergence of antibiotic resistant strains.

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. Community-acquired infections commonly associated with blood stream invasion include pneumonia, pyelonephritis, meningitis and those associated with perforating injuries of abdominal and thoracic viscera and obstructing gastrointestinal or genitourinary tract lesions. Patients with soft tissue infections with diabetes, malnourished persons, and those receiving corticosteroid therapy or other forms of immunosuppression are also at risk. One of the most serious complications of bacteremia, especially in association with gram negative infections is circulatory collapse (septic shock). Infections with Listeria monocytogenes cause a wide variety of distinct clinical syndromes. The most frequent are meningoencephalitis, primary bacteremia without meningitis and perinatal listeriosis. 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. Studies of 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-γ. TNF, interleukin-1 (IL-1) and interleukin-2 (IL-2) also promote murine resistance. Those at risk of Listeria infection include patients immunosuppressed by lymphoreticular malignancies, immunosuppressive drugs (e.g. corticσsteroids, cyclosporin A and estrogens) and pregnancy. Neonates and the elderly are also susceptible to Listeria infection.

FUNGAL AND PROTOZOAL DISEASES AND CONDITIONS:

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. Like tuberculosis, 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 γ and TNFα 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 TNFα 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. Amongst other organisms responsible for skin infections are other 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 TNFα or interferon γ 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 γ or anti-TNFα 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. caήnii is usually not pathogenic until it has the opportunity to multiply and invade in a person who has a helper T cell defect. VIRAL DISEASES AND CONDmONS:

Agents which upregulate the ability of neutrophils and/or monocytes/macrophages to kill virus-infected cells may also be beneficial. For example, in 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). Further the role of TNFα-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. Infection of the immunocompetent host does not elicit severe clinical symptoms, however infection of the immunocompromised host can cause severe and even fatal disease, mainly CMV pneumonia. Morbidity and mortality associated with primary CMV infection or with reactivation from latency is common in immunosuppressed transplant recipients and in patients with immunodeficiency caused by HTV infection. Chemotherapy with the guanosine analog 9-(l,3-dihydroxy-2-propoxymethyl) guanine (ganciclovir) can limit viral spread but is associated with serious side effects principally neutropenia. In absence of an improvement of the immunological status, maintenance therapy with ganciclovir does not eliminate the virus and even progression of infection is possible. Combined prophylactic therapy with interferon-γ and TNFα significantly reduces mortality in a murine model of CMV infection (Anderson et al., 1993. Antiviral Research 21:343). NOSOCOMIAL INFECΗON:

In addition, the incidence and/or severity of 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.

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 β haemolytic

Streptococci or Clostridium peφingens, 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. In particular, 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.

Disclosure of the Invention:

Thus, in a first aspect, the present invention provides a peptide with neutrophil and/or monocyte/macrophage stimulatory activity, wherein the peptide is of the general formula:-

X₁-X₂-X_rX₄-Ser-Thr-X₅-Val-X₆-Ile-Thr-X₇-X_β-X₉-X₁₀ in which,

X_: is absent, Cys or R_α, X₂ is absent, Ala, Arg, Glu or Gly,

X₃ is absent, Ala, Arg, Asn, Cys, Glu, Gly, His, He, Leu, Lys, Met, Pro, Ser, Trp, γ-Abu, βAla, Dbu, Sar, Sue or N-Me-Ala, X₄ is absent, Ala, Arg, Asn, Glu, His, Leu, Lys, Met, Pro, Ser, Trp, βAla or Nip, X₅ is Ala or His,

X₆ is Ala, Gly, He, Leu, Phe, Pro, Ser, Thr, Trp, Val, D-Ala, D-Ile D-Pro, D-Ser, D-Thr, D-Val or βAla, X₇ is His or Ala,

X_fl is absent, He, Leu, Thr or D-Ile, X_g is absent, He, D-Ile or Aib, and X₁₀ is absent, Cys or R₂, 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

R_Λ is glycosyl, nucleosyl or lipoyl and R_t is absent when the amino acid adjacent is an unsubstituted desamino-derivative; R₂ is

-NR12R13, wherein R12 and R13 are independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for R, or R₂ is N- glycosyl or N-lipoyl, or R₂ is -OR14, where R14 is H straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R_α or R_z is

-O-glycosyl, or -O-lipoyl or R₂ 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-

Thr-Ile. γAbu represents γ-aminobutyric acid, Dbu represents diaminobutyric acid, Aib represents amino isobutyric acid, Nip represents nipocotic acid, βAla represents β-alanine, Sar represents sarcosine (alternatively known as

N-methyl glycine), Sue represents succinic acid, N-Me-Ala represents 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.

In preferred embodiments of the invention, X_t and X₁₀ are absent. X₆ is preferably He. Particularly preferred peptides include :-

(923) Ser-Ser-Thr-His-Val-Leu-He-Thr-His-Thr-Ile,

(925) Pro-βAla-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile,

(926) βAla-Pro-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile, (927) Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(933) Suc-Pro-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile, (934) Nip-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile, (966) βAla-Pro-Ser-Thr-His-Val- Ile-Ile-Thr-His-Thr-Ile, (967) Pro-βAla-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (968) Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1059) Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1060) Lys-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1061)His-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1062) Ala-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1063)Leu-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile. (1064) 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,

(1071) Ala-Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1072) Arg-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1073) Lys-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1074) His-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1075) Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1076)Leu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1077)Trp-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1078) Met-Pro-Ser-Thr-His-Val-Ile-He-Thr-His-Thr-Ile, (1079) Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1080) Asn-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1081)Glu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1083)γAbu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1084)Dbu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1085) Sar-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1086)N-Me-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1087) Arg-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1088) Ala-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1089) Gly-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1090) Glu-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1097) Ala-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (llOO)Pro-Ser-Thr-Ala-Val-He-Ile-Thr-His-Thr-Ile, (1105) Pro-Ser-Thr-His-Val-Ile-Ile-Thr-Ala-Thr-Ile, (1108)Pro-Ser-Thr-His-Val-D-Ile-Ile-Thr-His-Thr-Ile, (1109) Pro-Ser-Thr-His-Val-Phe-Ile-Thr-His-Thr-Ile,

(1161)Ser-Pro-Ser-Thr-Ala-Val-Ile-Ile-Thr-His-Thr-Ile, (1162 Ser-Pro-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile, (1163 Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Leu-Ile, (1164 Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-Ile, (1165 Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-D-Ile-Ile, (1166 Ser-Pro-Ser-Thr-His-Val-Ile-He-Thr-His-Thr-D-Ile, (1168 Met-Ser-Thr-Ala-Val-Ile-Ile-Thr-His-Thr-Ile, (1169 Met-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile, (1170 Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Leu-Ile, (1171 Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-Ile, (1172 Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-D-Ile-He, (1173 Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-D-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, (1178 Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-Ile, (1179 Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-D-Ile-Ile , and (1180 Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-D-Ile.

In a second aspect, 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. wherein the peptide is of the general formula:-

X₁-X₂-X₃-X₄-Ser-Thr-X₅-Val-X_B-Ile-Thr-X₇-X₈-X₉-X₁₀ in which, X_Ύ is absent, Cys or R.,,

X₂ is absent,

X₃ is Ala, Lys or Ser,

X₄ is Pro,

X₅ is Ala or His, Xs is He or Leu,

X₇ is Ala or His,

X₈ is He, Thr or D-Ile,

X₉ is He or D-Ile,

X₁₀ is absent, Cys or R₂, wherein R_T and R₂ are as defined above.

Particularly preferred class 1 peptides are:- (1073) Lys-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1075) Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1079) Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1161) Ser-Pro-Ser-Thr-Ala-Val-Ile-Ile-Thr-His-Thr-Ile,

(1162) Ser-Pro-Ser-Thr-His-Val-Leu-He-Thr-His-Thr-Ile,

(1163) Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Leu-Ile, (1164) Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-He, (1165) Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-D-Ile-Ile, and (1166) Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-D-Ile.

In a third aspect, 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_rX₂-X₃-X₄-Ser-Thr-X₅-Val-X₆-Ile-Thr-X₇-X₈-X₉-X₁₀ in which,

X, is absent, Cys or R_lt

X₂ is absent,

X₃ is absent, Asn, Lys or βAla X₄ is Arg, His, Lys, Pro, Trp, Ala or Nip,

X₅ is Ala or His,

X₆ is He or Leu,

X₇ is Ala or His,

X_B is He, Leu, Thr or D-Ile, X₉ is He or D-Ile,

X₁₀ is absent, Cys or R₂, wherein R, and R₂ are as defined above; with the proviso that when X₃ is Lys, then X₄ is not Pro.

Particularly preferred class 2 peptides are:-

(966) βAla-Pro-Ser-Thr-Has-Val-Ile-Ile-Thr-His-Thr-Ile, (968) Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1060) Lys-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-He, (1061) His-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1062) Ala-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1064) 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,

(1178) 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).

In a fourth aspect, 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:-

X₁-X₂-X₃-X₄-Ser-Thr-X₅-Val-X₆-Ile-Thr-X₇-X₈-X₉-X₁₀ in which,

X, is absent, Cys or R_α, X₂ is absent, Ala, Arg, Glu or Gly,

X₃ is absent, Ala, Arg, Glu, His, Leu, Met, Trp, γ-Abu, Dbu, Sar or N-methyl Ala, X₄ is Arg, Asn, Glu, Leu, Met or Pro,

X₅ is Ala or His, X₆ is He or Leu, X₇ is Ala or His, X₈ is He, Leu, Thr or D-Ile, X₉ is He or D-Ile,

X₁₀ is absent, Cys or R₂, wherein R_λ and R₂ are as defined above; with the proviso that when X₂ is absent and X₃ is Ala, then X₄ is not Pro.

Particularly preferred class 3 peptides are:-

(1059) Aig-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1063) Leu-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-He, (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-He,

(1070) His-Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1072) Arg-Pro-Ser-Thr-His-Val-Ile-He-Thr-His-Thr-He, (1074) His-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1076) Leu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1077) Trp-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1078) Met-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1081) Glu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1083) γAbu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, ( 1084) Dbu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1085) Sar-Pro-Ser-Thr-His-Val-IIe-Ile-Thr-His-Thr-Ile,

(1086) N-methyl Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1087) Arg- Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1088) *Ala-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1089) Gly-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, (1090) *Glu-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

(1168) Met-Ser-Thr-Ala-Val-Ile-Ile-Thr-His-Thr-He, (1169) Met-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile, (1170) Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Leu-Ile, (1171) Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-Ile , (1172) Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-D-Ile-Ile, and

(1173) Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-D-Ile.

Peptides in this class 3 generally display 70-130% activity of peptide 419 at lOμM in the neutrophil assay described below. Those that have asterisks exhibit only 30-40% activity of peptide 419 on neutrophils.

In a fifth aspect, 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.

In a preferred embodiment of the fifth aspect of the present invention, 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 carniϊ) infection. More particularly, where the subject is administered a class 1 peptide, 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. 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.

Where the subject is administered a class 2 peptide, 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. When used in combination with other agents (e.g. antibiotics), class 2 peptides may be particularly useful in preventing or treating chronic lung infection by these organisms (such as H. influenzae in COPD and CF), acute community acquired pneumonia, treatment of skin and soft tissue infections, and treatment of inherited primary or acquired neutropenic disorders and other inherited or acquired defects of phagocytic cell function. Other particular conditions for which class 2 peptides may be useful include: cancer; cystic firbrosis; Legionnaires disease; tuberculosis; diabetes; meningitis and nosocomial infection.

Finally, where the subject is administered a class 3 peptide, 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. pneumophila), Histoplasmosis (e.g. H. capsulum) Listeriosis, Pneumocystis carnii and Trypanosoma cruzi. 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 TNFα 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. GM-CSF or levamisole); 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. mitoxantrone, plicamycin and procarbazine HCl, fludarabine, 2-cyclodeoxyadenosine; antibiotic agents such as amikcan, gentamycin, tobramycin, netilmicin, cephalosporins (such as cephalothin, cefaclor, cefamandole, cefixime, ceftriaxone), penicillins (such as penicillin G), ampicillin, nafcillin, oxacillin ticascillin, tetracycline, deoxycycline, imipenem, aztreonam, erythromycin, clindamycin, vancomycin, metriomidazole, pyrazinamide, ethambutal, rifabutin. azithromycin, chlaritiromycin, amikacin, cefoxitin, rifampin and isoniazid; anti-fungal agents such as amphotericin B, ketoconazole and flucytosine; and anti-viral agents such as gangciclovir and acyclovir.

With regard to such combination therapies reference herein to "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.

It will be appreciated by those skilled in the art that a number of modifications may be made to the peptides according to the present invention without deleteriously affecting the biological activity of the peptides. Such 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. By 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, Nα-alkylamino acids.

It may also be possible to add various groups to the peptides of the present invention to confer advantages such as increased potency or extended half-life in vivo, without substantially altering the overall biological activity of the peptide.

The term 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.

Apart from substitutions, three particular forms of peptide mimetic and/or analogue structures of particular relevance when designating bioactive peptides, which have to bind to a receptor while risking the degradation by proteinases and peptidases in the blood, tissues and elsewhere, may be mentioned specifically, illustrated by the following examples: Firstly, the inversion of backbone chiral centres leading to D-amino acid residue structures may, particularly at the N-terminus, lead to enhanced stability for proteolytical degradation while not impairing activity. An example is given in the paper "Tritriated D-ala^Peptide T Binding", Smith, C. S. et al, Drug Development Res. 15, pp. 371-379 (1988). Secondly, 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. Finally, the introduction of 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. An example of the latter type is given in the paper "Biologically active retroinverse analogues of thymopentin", Sisto A. et al in Rivier, J. E. and Marshall, G. R. (Eds) "Peptides, Chemistry, Structure and Biology", Escom, Leiden (1990), p. 722-773.

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. 37-46 (1979), and Kullman, W.: "Enzymatic Peptide Synthesis", CRC Press Inc., Boca Raton, Florida (1987), and Widmer, F., Johansen, J. T. In "Synthetic Peptides in Biology and Medicine:, eds., Alitalo, K., Partanen, P., Vatieri, A., pp. 79-86, Elsevier, Amsterdam (1985), or by a combination of chemical and enzymatic methods if this is advantageous for the process design and economy.

It will be seen that one of the alternatives embraced in the general formula set out above is for a 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.

The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated component or feature or group of components or features with or without the inclusion of a further component or feature or group of components or features.

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described by way of the following non-limiting examples and accompanying figures.

Brief description of the accompanying figures:

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 γ.

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

(disseminated mycobacterial) levels following CD4 cell depletion - effect of peptide treatment.

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.

Production of human TNF peptides tested for neutrophil stimulatory activity:

The following peptides were synthesised according to well known methods and are described using the I.U.P.A.C. one-letter code abbreviations for amino acid residues.

presents acetyl, and Acm represents acetamidomethyl. EXAMPLE 1

Effect of Peptides on neutrophil function:

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.

Duration of action of Peptides in vivo:

All 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 10⁶ 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. As can be seen from Table 1, N and C-terminal truncations of peptide 419 significantly reduced activity, with only the truncated peptides 771 (STHVLITHTI), 757 (PSTHVLITHT) and 758 (PSTHVLITΗ) 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. Within this core sequence, it is apparent that the position 1 (Ser), position 2 (Thr), position 4 (Val), position 6 (He), position 7 (Thr) and position 8 (His) are essential for retaining significant activity (see Table 2). TABLE l: ACTIVITY OF TRUNCATED FORMS OF PEPTIDE 419 IN NEUTROPHIL CHEMILUMINESCENCE ASSAY

TABLE 2: CORE SEQUENCES OF PEPTIDE 419

With knowledge of the above core residues and sequences, modifications of peptide 419 were designed and synthesised. These peptides show similar activities to peptide 419 in vitro (Tables 2 to 9), however some (e.g. peptide 966 and 968) show a duration of action and level of cellular activation which is significantly enhanced (Tables 10 and 11). This effect would be expected to lead to enhanced antimicrobial activity as indicated by increased resistance to infection and/or enhanced clearance of invading organisms. In addition, less frequent administration and/or more favourable delivery of the peptide, e.g. via oral absorption, would be expected as a consequence of the increased stability and longer duration of action of the novel peptides.

TABLE 3: STIMULATION OF HUMAN NEUTROPHILS BY PEPTIDES

Peak chemiluminescence i (mV)

TABLE 4: STIMULATION OF HUMAN NEUTROPHILS BY PEPTIDES

Λll peptides were at lOμM final concentration.

Values represent the mean±SD of triplicate determinants.

TABLE 5: STIMULATION OF HUMAN NEUTROPHILS BY PEPTIDES

Λll peptides were at 10μM final concentration.

Values represent the mean±SD of triplicate detormiriants.

TABLE 6: STIMULATION OF HUMAN NEUTROPHILS BY PEPTIDES

All peptides were at lOμM final concentration.

Values represent the mean±SD of triplicate determinants.

32

TABLE 7: STIMULATION OF HUMAN NEUTROPHILS BY PEPTIDES

419, 1037, 966, 927, 1100, 1105

Λll peptides were at lOμM final concentration.

Values represent the mean±SD of triplicate determinants.

TABLE 8: EFFECT OF PEPTIDES ON NEUTROPHIL ACTIVATION

All peptides were at lOμM final concentration.

Values represent the mean±SD of triplicate determinants. TABLE 9: STIMULATION OF HUMAN NEUTROPHILS BY PEPTIDES

Peak Chemiluminescence (mV)

Ex t No.

TABLE 10: EX VIVO CHEMILUMINESCENCE RESPONSE OF

MURINE PERITONEAL CELLS FOLLOWING INTRAPERITONEAL INOCULATION OF PEPTIDE

Λll 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

EXAMPLE 2

Peptides and evaluation for neutrophil and monocyte/macrophage stimulatory activity:

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.

TABLE 12: PEPTIDES EVALUATED FOR NEUTROPHIL AND MONOCYTE/MACROPHAGE STIMULATORY ACTIVITY

These peptides did not activate monocytes, i.e. appoared to be neutrophil specitic. TABLE 13: STIMULATION OF HUMAN MONOCYTES BY PEPTIDES

Λll peptides were at lOμM final concentration.

Values represent the mean±SD of triplicate determinants. TABLE 14: EFFECT OF PEPTIDES ON NEUTROPHIL AND

MONOCYTE ACTIVATION

All peptides were at lOμM final concentration.

Values represent the mean±SD of triplicate determinants.

EXAMPLE 3

Peptides tested for effect on nitrite release by M. bovis BCG-infected bone marrow macrophage (BMM):

Peptides 419, 926, 927, 966, 968 and 1065 were assessed for their effect on nitrite release by M. bovis BCG-infected bone marrow macrophage.

Measurement of macrophage nitric oxide production and inhibition of BCG growth by peptides:

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 (0.6-60μM final) were added to the macrophage cultures (10⁵ 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 ³H-Uracil (l.OμCi/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.

Results are provided in Figures 1 and 2. EXAMPLE 4

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 (300μg 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 lOOμg 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).

EXAMPLE 5

Effect of peptides 966 and 968 on Pseudomonas aeruBJnosa lung infection:

Pseudomonas aeruginosa (stain #16800) was grown overnight on blood agar plates from frozen stock. The organisms were encapsulated in agarose (Calbiochem 2% w/v in sterile phosphate buffered saline). Mice were infected intracheally with 30μl of agarose beads containing 6,000-12,000 CFU. Peptide treatments were given at t=-24 and t=-2 hours relative to infection and then daily during the course of the study. On day 5 post infection mice were treated two hours prior to sacrifice for enumeration of lung bacteria. The lungs were excised and homogenised. Serial dilutions of the lung homogenate were then plated onto blood agar and incubated overnight at 37°C. The number of colonies in each plate was then counted. Infection-related weight loss was monitored during the course of the infection by daily weighing of all animals (Figures 5 and 6, and Table 15). TABLE 15

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A peptide with neutrophil and/or monocyte/macrophage stimulatory activity, wherein the peptide is of the general formula:- X₁-X₂-X₃-X₄-Ser-Thr-X₅-Val-X₆-Ile-Thr-X₇-X₀-X₉-X₁₀ in which,

X_Λ is absent, Cys or R_α, X₂ is absent, Ala, Arg, Glu or Gly,

X₃ is absent, Ala, Arg, Asn, Cys, Glu, Gly, His, He, Leu, Lys, Met. Pro, Ser, Trp, γ-Abu, βAla, Dbu, Sar, Sue or N-Me-Ala,

X₄ is absent, Ala, Arg, Asn, Glu, His, Leu, Lys, Met, Pro, Ser, Trp, βAla or Nip, X₅ is Ala or His,

X„ is Ala, Gly, He, Leu, Phe, Pro, Ser, Thr, Trp, Val, D-Ala, D-Ile D-Pro, D-Ser, D-Thr, D-Val or βAla,

X₇ is His or Ala,

X₈ is absent, He, Leu, Thr or D-Ile, X₉ is absent, He, D-Ile or Aib, and X₁₀ is absent, Cys or R₂, R_T 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 R} is glycosyl, nucleosyl or lipoyl and R_x is absent when the amino acid adjacent is an unsubstituted desamino-derivative; R₂ is

-NR12R13, wherein R12 and R13 are independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for R_t or R₂ is N- glycosyl or N-lipoyl, or R₂ is -OR14, where R14 is H straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R_α or R₂ is -0-glycosyl, or -0-lipoyl or R₂ 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-Thr-Ile.

2. A peptide according to claim 1, wherein X_B is He. 3. A peptide according to claim 1 or 2, wherein X^ and X₁₀ are absent.

4. A peptide according to claim 1 selected from the group consisting of:-

Ser-Ser-Thr-His-Val-Leu-He-Thr-His-Thr-He,

Pro-βAla-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile, βAla-Pro-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-He,

Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Suc-Pro-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile, Nip-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile, βAla-Pro-Ser-Thr-His-Val- Ile-Ile-Thr-his-Thr-Ile,

Pro-βAla-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, Lys-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

His-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Ala-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Leu-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Trp-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Asn-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Glu-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Arg-Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Lys-Arg-Ser-Thr-His-Val-He-Ile-Thr-His-Thr-Ile, His-Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Ala-Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Arg-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Lys-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

His-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, Ala-Pro-Ser-Thr-His-Val-Ile-He-Thr-His-Thr-Ile,

Leu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Trp-Pro-Ser-Thr-His-Val-Ile-He-Thr-His-Thr-Ile,

Met-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Ser-Pro-Ser-Thr-His-Val-He-Ile-Thr-His-Thr-Ile, Asn-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Glu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, γAbu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Dbu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Sar-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

N-methyl Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, Arg-Ala-Pro-Ser-Thr-His-Val-He-Ile-Thr-His-Thr-Ile ,

Ala-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Gly-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Glu-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Ala-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, Pro-Ser-Thr-Ala-Val-Ile-Ile-Thr-His-Thr-Ile,

Pro-Ser-Thr-His-Val-Ile-He-Thr-Ala-Thr-Ile,

Pro-Ser-Thr-His-Val-D-Ile-Ile-Thr-His-Thr-Ile,

Pro-Ser-Thr-His-Val-Phe-Ile-Thr-His-Thr-Ile,

Ser-Pro-Ser-Thr-Ala-Val-Ile-Ile-Thr-His-Thr-He, Ser-Pro-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile,

Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Leu-Ile,

Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-Ile,

Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-D-He-He,

Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-D-Ile, Met-Ser-Thr-Ala-Val-He-Ile-Thr-His-Thr-Ile,

Met-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile,

Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Leu-Ile,

Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-Ile,

Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-D-Ile, Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-D-Ile,

Nip-Ser-Thr-Ala-Val-Ile-Ile-Thr-His-Thr-Ile,

Nip-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile,

Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Leu-Ile,

Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-Ile, Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-D-Ile-Ile, and

Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-D-Ile.

5. A peptide with neutrophil stimulatory activity, wherein the peptide is of the general formula:- X₁-X₂-X₃-X₄-Ser-Thr-X₅-Val-X₆-Ile-Thr-X₇-X₈-X₉-X₁₀ in which, X₁ is absent, Cys or R_t,

X₂ is absent,

X₃ is Ala, Lys or Ser,

X₄ is Pro, X₅ is Ala or His,

X₆ is He or Leu,

X₇ is Ala or His,

X₈ is He, Leu, Thr or D-Ile,

X_g is He or D-Ile, X₁₀ is absent, Cys or R₂, wherein R_t 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 R_x is glycosyl, nucleosyl or lipoyl and R_t is absent when the amino acid adjacent is an unsubstituted desamino-derivative; R₂ is -NR12R13, wherein R12 and R13 are independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for R, or R₂ is N- glycosyl or N-lipoyl, or R₂ is -OR14, where R14 is H straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R_x or R₂ is -0-glycosyl, or -0-lipoyl or R₂ 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.

6. A peptide according to claim 5, wherein X_α and X₁₀ are absent.

A peptide according to claim 5 selected from the group consisting of:-

Lys-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Ser-Pro-Ser-Thr-Ala-Val-Ile-Ile-Thr-His-Thr-Ile,

Ser-Pro-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile,

Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Leu-Ile.

Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-He,

Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-D-Ile-Ile, and Ser-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-D-Ile.

8. A peptide with neutrophil and monocyte/macrophage stimulatory activity wherein the peptide is of the general formula:- Xj-Xz-Xs-X^-Ser-Thr-X_ϋ-Val-Xβ'Ile-Thr-Xy-Xβ-Xβ-Xm in which,

X, is absent, Cys or R_v X_z is absent,

X₃ is absent, Asn, Lys or βAla, X₄ is Arg, His, Lys, Pro, Trp, Ala or Nip,

X₅ is Ala or His, X₆ is He or Leu, X₇ is Ala or His, X₈ is He, Leu, Thr or D-Ile, X₉ is He or D-Ile,

X₁₀ is absent, Cys or R₂, wherein Rj 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 R, is glycosyl, nucleosyl or lipoyl and R_α is absent when the amino acid adjacent is an unsubstituted desamino-derivative; R₂ is -NR12R13, wherein R12 and R13 are independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for R_t or R₂ is N- glycosyl or N-lipoyl, or R₂ is -OR14, where R14 is H straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R₅ or R₂ is -0-glycosyl, or -0-lipoyl or R₂ 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 when X₃ is Lys, then X₄ is not Pro.

9. A peptide according to claim 8, wherein X_Λ and X₁₀ are absent.

10. A peptide according to claim 8 selected from the group consisting of:-

βAla-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-He, Lys-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-He, His-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, Ala-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, Trp-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, Lys-Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Asn-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, Nip-Ser-Thr-Ala-Val-Ile-Ile-Thr-His-Thr-Ile, Nip-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile, Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Leu-Ile, Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-Ile,

Nip-Ser-Thr-His-Val-Ile-Ile-Thr-His-D-Ile-Ile, and Nip-Ser-Thr-His-Val-Ile-He-Thr-His-Thr-D-He.

11. A peptide with monocyte/macrophage stimulatory activity, wherein the peptide is of the general formula:-

X₁-X₂-X₃-X₄-Ser-Thr-X₅-Val-X₆-Ile-Thr-X₇-X₈-X₉-X₁₀ in which,

X-i is absent, Cys or R_v X₂ is absent, Ala, Arg, Glu or Gly, X₃ is absent, Ala, Arg, Glu, His, Leu, Met, Trp, γ-Abu, Dbu. Sar or

N-methyl Ala, X₄ is Arg, Asn, Glu, Leu, Met or Pro, X₅ is Ala or His, X_B is He or Leu, X₇ is Ala or His,

X₈ is He, Leu, Thr or D-Ile, X₉ is He or D-Ile, X₁₀ is absent, Cys or R₂, wherein R_Λ 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 R_x is glycosyl, nucleosyl or lipoyl and R_α is absent when the amino acid adjacent is an unsubstituted desamino-derivative; R₂ is -NR12R13, wherein R12 and R13 are independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for R_t or R₂ is N- glycosyl or N-lipoyl, or R₂ is -OR14, where R14 is H straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R_t or R₂ is -O-glycosyl, or -0-lipoyl or R₂ 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 when X₂ is absent and X₃ is Ala. then X₄ is not Pro.

12. A peptide according to claim 11, wherein X_x and X₁₀ are absent.

13. A peptide according to claim 11 selected from the group consisting of:-

Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Leu-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Asn-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Glu-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-He,

Arg-Arg-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

His-Arg-Ser-Thr-His-Val-He-Ile-Thr-His-Thr-Ile, Arg-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

His-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Leu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Trp-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Met-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-He, Glu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile, γAbu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Dbu-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Sar-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

N-methyl Ala-Pro-Ser-Thr-His-Val-Ile-He-Thr-His-Thr-Ile, Arg-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Ala-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Gly-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Glu-Ala-Pro-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-Ile,

Met-Ser-Thr-Ala-Val-Ile-Ile-Thr-His-Thr-Ile, Met-Ser-Thr-His-Val-Leu-Ile-Thr-His-Thr-Ile,

Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Leu-Ile, Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Ile-Ile, Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-He-D-Ile , and Met-Ser-Thr-His-Val-Ile-Ile-Thr-His-Thr-D-Ile.

14. 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 a peptide according to any one of the preceding claims.

15. A method according to claim 14, wherein the subject is suffering from bacterial, fungal, viral or protozoan infection.

16. A method according to claim 14 or 15, wherein the peptide is co- administered with an antimicrobial or antiviral agent.

17. A method according to claim 16, wherein the antimicrobial or antiviral agent is selected from the group consisting of: amikcan, gentamycin, tobramycin, netilmicin, cephalosporins, penicillins, ampicillin, nafcillin, oxacillin, ticascillin, tetracycline, deoxycycline, imipenem, aztreonam, erythromycin, clindamycin, vancomycin, metriomidazole, rifampin and isonazid, amphotericin B, ketoconazole, flucytosine, gangcilovir and acyclovir.

18. A method according to claim 14, wherein the subject is suffering from graft vs host disease or chronic lymphocytic leukemia.

19. A method according to claim 18, wherein the peptide is co- administered in combination with cyclosporin A, prednisolone, fludarabine, 2-cyclodeoxyadenosine or other immune suppressant.

20. A method according to claim 14, wherein the subject is suffering from cancer.

21. A method according to claim 20, wherein the peptide is co- administered with a cancer chemotherapy agent. 22. A method according to claim 21, wherein the cancer chemotherapy agent is selected from the group consisting of: 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. mitoxantrone, plicamycin and procarbazine HCl, fludarabine and 2-cyclodeoxyadenosine.

23. A method according to claim 14, wherein the peptide is co- administered with G-CSF, GM-CSF, levamisole or other agent which stimulates increased white blood cell numbers.

24. A method according to any one of claims 14 to 23, wherein the subject is suffering from an inherited primary neutropenic disorder.

25. A method according to any one of claims 14 to 23, wherein the subject is suffering from an inherited primary or secondary defect of phagocytic cell function.

26. A method according to any one of claims 14 to 23. wherein the subject is suffering from an acquired defect of phagocytic cell function.

27. A method according to claim 26, wherein the subject is elderly, neonate, alcoholic or malnourished.

28. A method according to claim 14, wherein the subject is suffering from one or more of the following: acquired immunity deficiency syndrome (ADDS); cancer; diabetes; nosocomial infection; tuberculosis; mycobacterium avium complex, cystic fibrosis; community acquired pneumonia; meningitis; Chlamydia; Legionnaires Disease; Listeriosis; and coccidian parasitical infection.

29. A method according to any one of claims 14 to 28, wherein the peptide is co-administered with interferon gamma. 30. A method of treating a subject in order to improve neutrophil function, the method comprising administering to the subject a therapeutic amount of a peptide according to any one of claims 5 to 7.

31. A method according to claim 30, wherein the subject is suffering from bacterial, fungal, viral or protozoan infection.

32. A method according to claim 30, wherein the subject is suffering from cancer.

33. A method according to claim 32, wherein the peptide is administered with a cancer chemotherapy agent.

34. A method according to claim 33, wherein the cancer chemotherapy agent is selected from the group consisting of: 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, mitoxantrone, plicamycin procarbazine HCl, fludarabine and 2-cytodeoxyadenosine.

35. A method according to claim 30, wherein the peptide is co- administered with G-CSF, GM-CSF, levamisole or other agents which stimulate increased white blood cell numbers.

36. A method according to claim 30 or 35, wherein the subject is suffering from: acquired immunity deficiency syndrome (AIDS); or inherited or acquired neutropenic disorder.

37. A method according to claim 30, wherein the subject is suffering from one or more of the following: infectious mononucleosis, paraoxysomal nocturnal, hemoglobinuria, leukemia, lymphoma, myelofibrosis, inherited primary or secondary defect of phagocytic cell function, and CMV. 38. A method according to claim 30 or 31, where the peptide co- administered with an antimicrobial or antiviral agent.

39. A method according to claim 38, wherein the antimicrobial or antiviral agent is selected from the group consisting of: amikcan, gentamycin, tobramycin, netilmicin, cephalosporins, penicillins, ampicillin, nafcillin, oxacillin, ticascillin, tetracycline, deoxycycline, imipenem. aztreonam, erythromycin, clindamycin, vancomycin, metriomidazole, rifampin and isonazid, amphotericin B, ketoconazole, flucytosine, gangcilovir and acyclovir.

40. A method of treating a subject in whom enhanced neutrophil and/or monocyte/macrophage function is desirable, the method comprising administering to the subject a therapeutic amount of a peptide according to any one of claims 8 to 10.

41. A method according to claim 40 wherein the subject is suffering from bacterial, fungal, viral or protozoan infection.

42. A method according to claim 40 or 41, wherein the peptide is co- administered with an antimicrobial or antiviral agent.

43. A method according to claim 42, wherein the antimicrobial or antiviral agent is selected from the group consisting of: amikcan, gentamycin, tobramycin, netilmicin, cephalosporins, penicillins, ampicillin, nafcillin, oxacillin, ticascillin, tetracycline, deoxycycline, imipenem, aztreonam, erythromycin, clindamycin, vancomycin, metriomidazole, rifampin, isonazid, amphotericin B, ketoconazole, flucytosine, gangciclovir and acyclovir.

44. A method according to claim 40, wherein the subject is suffering from cancer.

45. A method according to claim 44, wherein the peptide is co- administered with a cancer chemotherapy agent. 46. A method according to claim 45, wherein the cancer chemotherapy agent is selected from the group consisting of: 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, mitoxantrone, plicamycin and procarbazine HCl, fludarabine and 2-cytodeoxyadenosine.

47. A method according to claim 40, wherein the peptide is co- administered with G-CSF, GM-CSF, levamisole or other agents which stimulate increased white blood cell numbers.

48. A method according to claim 40 or 47, wherein the subject is suffering from one or more of the following: cancer; cystic fibrosis; community-acquired pneumonia; Legionnaires disease; tuberculosis; diabetes and meningitis.

49. A method according to any one of claims 40 to 48, wherein the subject is suffering from an inherited primary neutropenic disorder.

50. A method according to any one of claims 40 to 48, wherein the subject is suffering from an inherited primary or secondary defect of phagocytic cell function.

51. A method according to any one of claims 40 to 48, wherein the subject is suffering from an acquired defect of phagocytic cell function.

52. A method according to any one of claims 40 to 48, wherein the subject is at risk of or is suffering from nosocomial infection.

53. A method according to any one of claims 40 to 52, wherein the peptide is co-administered with interferon gamma.

54. A method of treating a subject in whom enhanced neutrophil and/or monocyte/macrophage function is desirable, the method comprising administering to the subject a therapeutic amount of a peptide according to any one of claims 11 to 13.

55. A method according to claim 54, wherein the subject is suffering from a condition caused by an intracellular pathogen selected from the group consisting of: Mycobacteria, Chlamydia, Brucellae, Francisella, Pasteurellosis, Legionellosis, Histoplasmosis, Listeriosis, Pneumocystis camii and Trjφanosoma cruzi.

56. A method according to claim 55, wherein the subject is suffering from a condition caused by Mycobacteria.

57. A method according to claim 55 or 56, wherein the peptide is co- administered with an antibiotic agent.

58. A method according to claim 57, wherein the antibiotic agent is selected from the group consisting of: isoniazid, rifampin, pyrazinamide, ethambutol, rifabutin, azithromycin, chlarithromycin, amikacin, cefoxitin and erythromycin.

59. A method according to claim 54, wherein the subject is suffering from cancer.

60. A method according to claim 59, wherein the peptide is co- administered with a cancer chemotherapy agent.

61. A method according to claim 60, wherein the cancer chemotherapy agent is selected from 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, mitoxantrone, plicamycin procarbazine HCl, fludarabine and 2- cyclodeoxyadenosine.