Neutrophil stimulating peptides

Details Neutrophil stimulating peptides Neutrophil stimulating peptides

2000-06-21

2002-04-23

Russel, Jeffrey E. (Department: 1653)

Drug, bio-affecting and body treating compositions

In vivo diagnosis or in vivo testing

C424S009341, C424S009400, C424S009600

Reexamination Certificate

active

06375928

ABSTRACT:

BACKGROUND OF THE INVENTION
Tumour necrosis factor (TNF) was first identified as a factor found in the serum of Bacillus Calmette-Guerin treated mice which caused haemorrhagic regression of certain transplanted tumours and had cytolytic activity on several transformed cell lines in vitro (Carswell et al, PNAS 72, 3666-3670; Helson et al, 1975, Nature 258, 731-732). TNF, a product of activated macrophages, has subsequently been shown to be a primary mediator in the pathology of endotoxic shock (Tracey et al 1986, Science 234, 470-474). In addition to its pathological effects TNF also has a central role in host defences against viral, bacterial and parasitic pathogens.
The cellular targets of TNF important in host defence include neutrophils, eosinophils, monocyte/macrophages and lymphocytes. Within this context TNF is a major mediator of neutrophil activation. TNF stimulates enhanced phagocytosis (Shalaby et al 1985, J. Immunol., 135, 2069-2073), enhanced production of superoxide anions (Teujiimoto et al, 1986, Biochem. Biophys. Res. Commun., 137, 1094-1100), release of lysozyme and hydrogen peroxide and causes neutrophil degranulation (Klebanoff et al, 1986, J. Immunol., 136, 4220-4225). Neutrophils also show enhanced microbiocidal and tumouricidal activity when stimulated by TNF (Shalaby et al, 1985, J. Immunol., 135, 2069-2073; Djeu et al, 1986, J. Immunol., 137, 2980-2984; Blanchard et al, 1989, J. Leuk. Biol., 45, 538-545). It has been hypothesized that the cytostatic effect of TNF is mediated by high local concentrations of hydrogen peroxide produced by neutrophils (Shau 1986, J. Immunol., 141, 234-240).
TNF pretreatment enhances the response of neutrophils to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (F-met-leu-phe) and phorbol myristate acetate through specific receptors (Ferrante et al 1988, Int. Arch. Allergy AppI. Immunol., 86, 82-91). Neutrophils accumulate at sites of inflammation, caused in part by the increased expression of complement receptors by TNF (Berger et al 1988, Blood 71, 151-158). Further TNF causes neutrophil emigration into skin (Cybulsky et al 1988, J. Immunol. 140, 3144-3149).
Neutrophil function is known to be depressed in a number of viral, bacterial and parasitic infections (Abramson and Mills, 1988, Rev. Infect. Dis., 10, 326-341; Ferrante et al, 1989, Immunol. Letts., 22, 301-6). Depressed neutrophil function has, for example, been described in Acquired Immune Deficiency Syndrome (Thorsen et al, 1989, AIDS, 3, 651-653; Ellis et al, 1988, J. Infect. Dis., 158, 1268-1276; Murphy et al, 1988, J. Infect. Dis., 158, 627-630). Clearly TNF, which appears to play an important role in neutrophil activation both in vitro and in vivo as described above, given exogenously has the potential to overcome these neutrophil defects. The administration of TNF or indeed overproduction of TNF is, however, associated with severe side effects and the manifestation of pathology such as thrombocytopaenia, lymphocytopaenia, hepatotoxicity, renal impairment and hypertension.
SUMMARY OF THE INVENTION
The present inventors have identified novel peptides derived from the primary amino acid sequence of human TNF which stimulate neutrophil activity. These peptides have indicated that the region of amino acids 54 to 94 of human TNF has previously undiscovered neutrophil stimulating activity. This observation has important clinical applications as treatment with such peptides would be expected to restore depressed or aberrant neutrophil activity, but would not be expected to cause the severe side effects associated with-the therapeutic use of the whole TNF molecule.
The present invention provides a method of detecting a site of inflammation in a subject which method comprises administering to the subject a detectably labeled peptide, the peptide comprising a sequence selected from the group consisting of LFKGQGCPSTHVLLTHTISRI (SEQ ID NO. 6), GLYLIYSQVLFKGQG (SEQ ID NO. 10), HVLLTHTISRIAVSYQTKVNLL (SEQ ID NO. 11), PSTHVLLTHTI (SEQ ID NO. 15), PSAHVLLTHTI (SEQ ID NO. 17), and PSTHVLITHTI (SEQ ID NO. 18); and detecting the labeled peptide.
The present invention also provides a method of diagnosing a disease state selected from the group consisting of a bacterial infection, a fungal infection, a parasitic infection, a viral infection, Group B streptococcal disease, osteomyelitis, pneumonia, bronchitis, chronic obstructive pulmonary disease, inflammatory bowel disease, atypical appendicitis, Acquired Immune Deficiency Syndrome, a granulomatous disease, respiratory distress syndrome, cystic fibrosis and rheumatoid arthritis, which method comprises administering to a subject a detectably labeled peptide, the peptide comprising a sequence selected from the group consisting of LFKGQGCPSTHVLLTHTISRI (SEQ ID NO. 6), GLYLIYSQVLFKGQG (SEQ ID NO. 10), HVLLTHTISRIAVSYQTKVNLL (SEQ ID NO. 11), PSTHVLLTHTI (SEQ ID NO. 15), PSAHVLLTHTI (SEQ ID NO. 17), and PSTHVLITHTI (SEQ ID NO. 18); and detecting the labeled peptide.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a peptide which primes neutrophils for superoxide production and an enhanced respiratory burst following treatment with N-formyl-L-methionyl-L-leucyl-L-phenylalanine, wherein the peptide is of the general formula:
X
1
-X
2
-X
3
-X
4
-X
5
-X
6
-X
7
in which
X
1
is null, Cys or R
1
X
2
is null, Cys, R1, or A1-A2-A3-A4-A5-A6-A7
 in which A1 is Leu, Ile, Val or Met
A2 is Phe, Tyr, Trp or His
A3 is Lys, Arg or His
A4 is Gly or Ala
A5 is Gln or Asn
A6 is Gly or Ala
A7 is Cys
X3 is Cys, R1 or A8-A9-A10
 in which A8 is Pro or N
&agr;
-alkylamino acid
A9 is Ser or Thr
A10 is Thr or Ser or Ala or Gly
X4 is A11-A12-A13-A14-A15-A16-A17-A18
 in which A11 is His, Lys or Arg
A12 is Val, Ile, Leu or Met
A13 is Leu, Ile Val or Met
A14 is Ile, Leu, Val or Met
A15 is Thr or Ser
A16 is His, Lys or Arg
A17 is Thr or Ser
A18 is 16, Leu, Val or Met
X5 is Cys, R2 or A19-A20-A21
 in which A19 is Ser or Thr
A20 is Arg, Lys or His
A21 is Ile, Leu, Val or Met
X6 is null, Cys, R2 or A22-A23-A24-A25-A26-A27-A28-A29-A30-A31-A32
 in which A22 is Ala or Gly
A23 is Val, Ile, Leu or Met
A24 is Ser or Thr
A25 is Tyr, Phe, Trp or His
A26 is Glu or Asp
A27 is Thr or Ser
A28 is Lys, Arg or His
A29 is Val, Ile, Leu or Met
A30 is Asn or Gln
A31 is Leu, Ile, Val or Met
A32 is Leu, Ile, Val or Met
X7 is null, Cys or R2
R1 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 R1 is glycosyl, nucleosyl or lipoyl and R1 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 R1 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 R1 or R2 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:
X1 is always and only null when X2 is R1, Cys or null
X2 is always and only null when X3 is R1 or Cys
X6 is always and only null when X5 is R2 or Cys
X7 is always and only null when X6 is R2 or Cys or null.
In a preferred embodiment of the present invention X1 is null, X2 is R1, X3 is A8-A9-A10, X5 is R2 and X6 and X7 are null. It is further preferred that A8 is Pro, A9 is Ser, A10 is Thr, A11 is His, A12 is Val, A13 is Leu, A14 is Leu, A15 is Thr, A16 is His, A17 is Thr and A18 is Ile, or A8 is Pro, A9 is Ser, A10 is Thr, A11 is His, A12 is Val, A13 is Leu, A14 is Ile, A15 is Thr, A16 is His, A17 is Thr and A18 is Ile, or A8 is Pro, A9 is Ser, A10 is Ala, A11 is His, A12 is Val, A13 is Leu, A14 is Leu, A15 is Thr, A16 is His, A17 is Thr and A18 is Ile.
In another preferred embodim

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