Compounds pharmaceutical compositions and methods for treatment of bacteremia and/or septicemia Number:7,435,716 from the United States Patent and Trademark Office (PTO) owispatent Novel conjugates of bacterial outer membrane binding peptides, preferably having bacterial sensitization activity, and immune cells chemotactic peptides, and pharmaceutical compositions containing same useful in the treatment of bacteremia and/or septicemia following infection by gram negative bacteria administered alone or in combination with conventional antibiotics.<H pharmaceutical, compositions, Compounds, pharm, 7435716.html, Patent, pto, 7435716

Title: Compounds pharmaceutical compositions and methods for treatment of bacteremia and/or septicemia

Abstract: Novel conjugates of bacterial outer membrane binding peptides, preferably having bacterial sensitization activity, and immune cells chemotactic peptides, and pharmaceutical compositions containing same useful in the treatment of bacteremia and/or septicemia following infection by gram negative bacteria administered alone or in combination with conventional antibiotics.

Patent Number: 7,435,716 Issued on 10/14/2008 to Ofek, et al.

Inventors: Ofek; Itzhak (Givataim, IL), Fridkin; Matityahu (Rehovot, IL), Tsubery; Haim (Ramat Gan, IL)
Assignee: Ramot At Tel Aviv University Ltd. (Tel-Aviv, IL)
Yeda Research and Development Co. Ltd. (Rechovot, IL)

Appl. No.: 10/451,795

Filed: January 16, 2002

PCT Filed: January 16, 2002

PCT No.: PCT/IL02/00038

371(c)(1),(2),(4) Date: July 15, 2003

PCT Pub. No.: WO02/055543

PCT Pub. Date: July 18, 2002

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
60261212	Jan., 2001

Current U.S. Class: 514/2 ; 514/11; 514/13; 514/14; 514/15; 530/300; 530/319; 530/326; 530/327; 530/345

Current International Class: A61K 38/02 (20060101); A61K 38/08 (20060101); A61K 38/10 (20060101); A61K 38/12 (20060101); C07K 2/00 (20060101); C07K 7/00 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


4427660	January 1984	Schiffman et al.
5484885	January 1996	Pereira et al.
5780429	July 1998	Wainwright
5925659	July 1999	Patchett et al.
6733997	May 2004	Ding et al.
6861053	March 2005	Lin et al.
2001/0026810	October 2001	McGhee et al.
2002/0049205	April 2002	Li et al.

Other References

Bycroft et al. Antibacterial and Immunostimulatory Properties of Chemotactic . . . Antimicrobial Agents And Chemotherapy. Sep. 1989, vol. 33, No. 9, pp. 1516-1521. cited by examiner .
Appelmelk et al. Analytical Biochemistry. Dec. 1992, vol. 207, No. 2, pp. 311-316. cited by examiner .
Amoscato et al. Peptides. May-Jun. 1984. vol. 5, No. 4, pp. 489-494. cited by examiner .
Tsubery et al. Biochemistry. Oct. 2000, vol. 39, No. 39, pp. 11837-11844. cited by examiner .
Tsubery et al. Journal Of Medicinal Chemistry. Aug. 2000, vol. 43, No. 16, pp. 3085-3092. cited by examiner .
Tsubery et al. Advances In Experimental Medicine And Biology. 2000, vol. 479, pp. 219-222. cited by examiner .
Ofek et al. Antimicrobial Agents And Chemotherapy. Feb. 1994, vol. 38, No. 2, pp. 374-377. cited by examiner .
Tsubery et al. Peptides. Oct. 2001, vol. 22, No. 10, pp. 1675-1681. cited by examiner .
Appelmelk et al. "Polymyxin B-Horseradish Peroxidase Conjugates as Tools in Endotoxin Research". Analytical Biochemistry, 207(2): 311-316, 1992. cited by other .
Amoscato et al. "Synthesis and Biological Activity of [L-3,4-Dehydroproline3]-Tuftsin." Peptides, 5(3): 489-494, 1984. cited by other .
Tsubery et al. "The Functional Association of Polymyxin B With Bacterial Lipopolysaccharide Is Stereospecific: Studies on Polymyxin B Nonapeptide", Biochemistry, 39(39): 11837-11844, 2000. cited by other .
Tsubery et al. "Structure-Function Studies of Polymyxin B Nonapeptide: Implications to Sensitization of Gram-Negative Bacteria", Journal of Medicinal Chemistry, 43(16): 3085-3092, 2000. cited by other .
Tsubery et al. "Structure Activity Relationship Study of Polymyxin B Nonapeptide", Advances in Experimental Medicine and Biology, 479: 219-222, 2000. cited by other .
Tsubery et al. "N-Terminal Modifications of Polymyxin B Nonapeptide and Their Effect on Antibacterial Activity", Peptides, 22(10): 1675-1681, 2001. cited by other .
Athamna et al. "Enzyme-Linked Immunosorbent Assay for Quantitation of Attachment and Ingestion Stages of Bacterial Phagocytosis", Journal of Clinical Microbiology, 26(1): 62-66, 1988. cited by other .
Athamna et al. "Lectinophagocytosis of Encapsulated Klebsiella Pneumoniae Mediated by Surface Lectins of Guinea Pig Alveolar Macrophages and Human Monocyte-Derived Macrophages", Infection and Immunity, 59(5): 1673-1682, 1991. cited by other .
Becker "Some Interrelations of Neutrophil Chemotaxis, Lysosomal Enzyme Secretion, and Phagocytosis as Revealed by Synthetic Peptides", American Journal of Pathology, 85: 385-394, 1976. cited by other .
Bryan "Mechanisms of Resistance to Antibacterial Agents", In: `Bacterial Resistance and Susceptibility to Chemotherapeutic Agents`, Cambridge University Press, Chap.3: 69-80, 1982. cited by other .
Chihara et al. "Chemical Synthesis, Isolation and Characterization of .alpha.-N-Fattyacyl Colistin Nonapeptide With Special Reference to the Correlation Between Antimicrobial Activity and Carbon Number of Fattyacyl Moiety", Agricultural Biology and Chemistry, 38(3): 521-529, 1974. cited by other .
Chihara et al. "Enzymatic Degradation of Colistin Isolation and Identification of .alpha.,.gamma.-Diaminobutyric Acid and Colistin Nonapeptide", Agricultural Biology and Chemistry, 37(11): 2455-2463, 1973. cited by other .
Chin et al. "Resistance to Antibiotics", Science, 264: 359-393, 1994. cited by other .
Danner et al. "Purification, Toxicity, and Antiendotoxin Activity of Polymyxin B Nonapeptide", Anitmicrobial Agents and Chemotherapy, 33(9): 1428-1434, 1989. cited by other .
Duwe et al. "In Vitro Cytotoxicity and Antibiotic Activity of Polymyxin B Nonapeptide", Antimicrobial Agents and Chemotherapy, 30(2): 340-341, 1986. cited by other .
Freer et al. "Further Studies on the Structural Requirements for Synthetic Peptide Chemoattractants", Biochemistry, 19: 2404-2410, 1980. cited by other .
Freer et al. "Formyl Peptide Chemoattractants: Model of the Receptor on Rabbit Neutrophils", Biochemistry, 21: 257-263, 1982. cited by other .
Kitamura-Matsunaga et al. "Enzymic Preparation of Colistin Fragments and Their Biological Activity", Peptide Chemistry, 22: 333-338, 1984. Abstract. cited by other .
Lam et al. "Membrane-Disorganizing Property of Polymyxin B Nonapeptide", Journal of Antimicrobial Chemotherapy, 18: 9-15, 1986. cited by other .
Metcalf et al. "Functions Related to Microbicidal Activity", Laboratory Manual of Neutrophil Function, Chap.5: 87-90, 1986. cited by other .
Morrison et al. "Binding of Polymyxin B to the Lipid A Portion of Bacterial Lipopolysaccharides", Immunochemistry, 13: 813-818, 1976. cited by other .
Niedel et al. "Formyl Peptide Chemotactic Receptors of Leukocytes and Macrophages", Current Topics in Cellular Regulation, 17: 137-169, 1980. cited by other .
Ofek et al. "Antibacterial Synergism of Polymyxin B Nonapeptide and Hydrophobic Antibiotics in Experimental Gram-Negative Infections in Mice", Antimicrobial Agents and Chemotherapy, 38(2): 374-377, 1994. cited by other .
Ofek et al. "Nonopsonic Phagocytosis of Microorganisms", Annual Reviews in Microbiology, 49: 239-276, 1995. cited by other .
Rustici et al. "Molecular Mapping and Detoxification of the Lipid A Binding Site by Synthetic Peptides", Science, 259: 361-365, 1993. cited by other .
Schiffmann et al. "N-Formylmethionyl Peptides as Chemoattractants for Leucocytes", Proc. Natl. Acad. Sci. USA, 72(3): 1059-1062, 1975. cited by other .
Sogaard "The Pharmacodynamics of Polymyxin Antibiotics With special Reference to Drug Restistance Liability", J. Vet. Pharmacol. Therapy, 5: 219-231, 1982. cited by other .
Vaara "Agents That Increse the Permeability of the Outer Membrane", Microbiological Reviews, 56(3): 395-411, 1992. cited by other .
Vaara et al. "Binding of Polymyxin B Nonapeptide to Gram-Negative Bacteria", Antimicrobial Agents and Chemotherapy, 27(4): 548-554, 1985. cited by other .
Vaara et al. "Polycations Sensitize Enteric Bacteria to Antibiotics", Antimicrobial Agents and Chemotherapy, 24(1): 107-113, 1983. cited by other .
Voitenko et al. "Relationship Between Structure and Histamine Releasing Action of Polymyxin B and Its Analogues", Agents and Actions, 30(1/2): 153-156, 1990. cited by other .
Young "Gram-Negative Sepsis", In: `Principles and Practice of Infectious Diseases`, Wiley & Sons, Section E, Chap.54: 452-475, 1985. cited by other .
Chihara et al. "Chemical Synthesis, Isolation and Characterization of ?-N-Fattyacyl Colistin Nonapeptide With Special Reference to the Correlation Between Antimicrobial Activity and Carbon Number of Fattyacyl Moiety", Agricultural Biology and Chemistry, 38(3): 521-529, 1974. cited by other .
Chihara et al. "Enzymatic Degradation of Colistin Isolation and Identification of ?,?-Diaminobutyric Acid and Colistin Nonapeptide", Agricultural Biology and Chemistry, 37(11): 2455-2463, 1973. cited by other .
EP/Summons to Attend Oral Proceedings of EP Application 02715695.6 dated Feb. 8, 2008. cited by other .
EP Brief Communication of Apr. 22, 2008 on Oral Proceedings of May 7, 2008. cited by other .
Obrist et al. "Conjugation Behaviour of Different Monoclonal Antibodies to F-Methionyl-Leucyl-Phenylalanine", International Journal of Immunopharmacology, 8(6): 629-632, 1986. cited by other .
Rose et al. "Targeting Lipopolysaccharides by the Nontoxic Polymyxin B Nonapeptide Sensitizes Resistant Escherichia coli to the Bactericidal Effect of Human Neutrophils", The Journal of Infectious Diseases, 182: 191-199, 2000. cited by other .
Tsubery et al. "Neopeptide Antibiotics That Function as Opsonins and Membrane-Permeabilizing Agents for Gram-Negative Bacteria", Antimicrobial Agents and Chemotherapy, 49(8): 3122-3128, 2005. cited by other.

Primary Examiner: Russel; Jeffrey E

Parent Case Text

RELATED PATENT APPLICATION

This application is a National Phase Application of PCT/IL02/00038 International Filing Date 16 Jan. 2002, which claims priority from U.S. Provisional Patent Application No. 60/261,212 filed 16 Jan. 2001.

Claims

What is claimed is:

1. A compound comprising a bacterial outer membrane lipopolysaccharide-binding peptide conjugated to an immune cell chemotactic peptide, wherein said immune cell chemotactic peptide is selected from the group consisting of formyl chemotactic peptide, desformyl chemotactic peptide, chemotactic peptide with a urea derivative, and tuftsin, said outer membrane lipopolysaccharide-binding peptide and said immune cell chemotactic peptide acting in synergy to provide a bacterial sensitizing activity, wherein said formyl chemotactic peptide comprises an N-terminal N-formyl methionyl residue and said desformyl chemotactic peptide comprises an N-terminal methionyl residue.

2. The compound of claim 1, wherein said bacterial outer membrane lipopolysaccharide-binding peptide is a polymyxin derivative or a polymyxin analog.

3. A compound having the formula: ##STR00013## wherein X1 is a Thr or Ser residue or a covalent bond; X2 is selected from the group consisting of Dab and Ser; X3 is selected from the group consisting of Lys, Orn, Dap, Glu, Asp, Dab and Cys residues; X5 is a hydrophobic amino acid residue; X6 is a hydrophobic amino acid residue; X9 is selected from the group consisting of Ser, Tyr, Thr, Dab, Lys, Orn, Dap, Glu, Asp, Cys and n-butyric acid residues; L is a peptide cyclization linker moiety; R is selected from the group consisting of a covalent bond, an amino acid residue, a stretch of amino acid residues, an amino fatty acid residue having 1-12 carbon atoms in its carbon backbone and a polyethylene glycol derivative; Xa is a hydrophobic linear amino acid residue; Xb is selected from the group consisting of linear and branched aliphatic amino acid residues; Xc is an aromatic amino acid residue; A is selected from the group consisting of formyl and Z-NHCONH-, where Z is n-butyl, phenyl, 4-chlorophenyl, 4-methoxyphenyl or p-tolyl derivative; X0 . . . 5 is a stretch of 0-5 amino acid residues; with the provisions that said A-Xa-Xb-Xc-X0 . . . 5 has an immune cell chemotactic activity and that said ##STR00014## has a bacterial outer membrane lipopolysaccharide-binding activity, said immune cell chemotactic activity and said bacterial outer membrane lipopolysaccharide-binding activity acting in synergy to provide a bacterial sensitizing activity.

4. The compound of claim 3, wherein X5 is selected from the group consisting of DPhe, DTrp, DLeu, DNle, DMet, DNva and DVal residues.

5. The compound of claim 3, wherein X5 is selected from the group consisting of Phe, Trp, Leu, Nle, Met, Nva and Val residues.

6. The compound of claim 3, wherein X6 is selected from the group consisting of Leu, Thr, Nva, Val, Met and Ile residues.

7. The compound of claim 3, wherein said stretch of amino acid residues is selected from the group consisting of an oligoAla stretch, an oligoAla stretch interrupted by at least one Glu and/or Asp residue, an oligoGly stretch, and an oligoGly stretch interrupted by at least one Glu and/or Asp residue.

8. The compound of claim 3, wherein said amino fatty acid residue is HN(CH2)xCOOH, where x is 1-12.

9. The compound of claim 3, wherein said amino fatty acid residue is selected from the group consisting of aminocaproic acid residue and aminobutyric acid residue.

10. The compound of claim 3, wherein said cyclization linker moiety is selected from the group consisting of --(CH2)x-NH--CO--, --(CH2)x-NH--CO--(CH2)y, CO--NH--(CH2)x, --CH.sub.2--S--CH.sub.2--CO-- and --S--S--, where x and y are each independently 1-12.

11. The compound of claim 3, wherein said hydrophobic linear amino acid residue is selected from the group consisting of Met and Nle.

12. The compound of claim 3, wherein said linear and branched aliphatic amino acid residue is selected from the group consisting of Leu, Ala, Abu, Nva, Val, Ile, Cys(Me), Met and Nle.

13. The compound of claim 3, wherein said stretch of 0-5 amino acid residues is selected from the group consisting of Phe, Ile, Nle-Tyr-Lys and DLeu-Phe-DLeu-Phe residues.

14. The compound of claim 3, wherein said Xc is selected from the group consisting of Phe residue, N-methyl-Phe residue, 2-oxy-3-phenylpropionic acid derivative and 2-aminoxy-3-phenylpropionic acid derivative.

15. The compound of claim 3, wherein said A-Xa-Xb-Xc-X0 . . . 5- is selected from the group consisting of: Formyl-Met-Leu-Phe- (SEQ ID NO: 1) Formyl-Met-Leu-Phe-Phe- (SEQ ID NO: 13) Formyl-Met-Leu-Phe-Ile- (SEQ ID NO: 14) Formyl-Nle-Leu-Phe-Nle-Tyr-Lys- (SEQ ID NO: 15) Z-NH-CO-NH-Phe-DLeu-Phe-DLeu-Phe- (SEQ ID NO: 16) Formyl-Met-Leu-Phe-Lys- (SEQ ID NO: 17) Formyl-Met-Leu-N-methyl-Phe- (SEQ ID NO: 18) Formyl-Met-Leu-2-oxy-3-phenylpropionic acid- (SEQ ID NO: 19) and Formyl-Met-Leu-2-aminoxy-3-phenylpropionic acid- (SEQ ID NO: 20).

16. A compound having the formula: ##STR00015## wherein X1 is a Thr or Ser residue or a covalent bond; X2 is selected from the group consisting of Dab and Ser; X3 is selected from the group consisting of Lys, Orn, Dap, Glu, Asp, Dab and Cys residues; X5 is a hydrophobic amino acid residue; X6 is a hydrophobic amino acid residue; X9 is selected from the group consisting of Ser, Tyr, Thr, Dab, Lys, Orn, Dap, Glu, Asp, Cys and n-butyric acid residues; L is a peptide cyclization linker moiety; R is selected from the group consisting of a covalent bond, an amino acid residue, a stretch of amino acid residues, an amino fatty acid residue having 1-12 carbon atoms in its carbon backbone and a polyethylene glycol derivative; Xf is selected from the group consisting of Thr, Leu, Gly and Val residues; Xg is selected from the group consisting of Lys and Arg residues; Xh is selected from the group consisting of Pro, Sar and N-methyl non-polar aliphatic amino acid residues; Xi is selected from the group consisting of Arg and Lys residues; X0 . . . 5 is 0-5 amino acid residues; m is an integer selected from the group consisting of 1-8, whereby if m is greater than 1 [Xf-Xg-Xh-Xi-X0 . . . 5]m is a branched structure; with the provisions that said [Xf-Xg-Xh-Xi-X0 . . . 5]m has an immune cell chemotactic activity and that said ##STR00016## has a bacterial outer membrane lipopolysaccharide-binding activity, said immune cell chemotactic activity and said bacterial outer membrane lipopolysaccharide-binding activity acting in synergy to provide a bacterial sensitizing activity.

17. The compound of claim 16, wherein X5 is selected from the group consisting of DPhe, DTrp, DLeu, DNle, DMet, DNva and DVal residues.

18. The compound of claim 16, wherein X5 is selected from the group consisting of Phe, Trp, Leu, Nle, Met, Nva and Val residues.

19. The compound of claim 16, wherein X6 is selected from the group consisting of Leu, Thr, Nva, Val, Met and Ile residues.

20. The compound of claim 16, wherein said stretch of amino acid residues is selected from the group consisting of an oligoAla stretch, an oligoAla stretch interrupted by at least one Glu and/or Asp residue, an oligoGly stretch, and an oligoGly stretch interrupted by at least one Glu and/or Asp residue.

21. The compound of claim 16, wherein said amino fatty acid residue is HN(CH2)xCOOH, where x is 1-12.

22. The compound of claim 16, wherein said amino fatty acid residue is selected from the group consisting of aminocaproic acid residue and aminobutyric acid residue.

23. The compound of claim 16, wherein said cyclization linker moiety is selected from the group consisting of --(CH2)x-NH--CO--, --(CH2)x-NH--CO--(CH2)y, CO--NH--(CH2)x, --CH2--S--CH2--CO--and --S--S--, where x and y are each independently 1-12.

24. The compound of claim 16, wherein [Xf-Xg-Xh-Xi-X0 . . . 5]m- is selected from the group consisting of: ##STR00017## where X is selected from the group consisting of Dap, Lys and Orn residues.

25. The compound of claim 1, selected from the group consisting of SEQ ID NOs: 3, 4, 6 and 8-11.

26. A pharmaceutical composition for treatment of bacteremia and/or septicemia following infection by gram negative bacteria comprising, as an active ingredient a compound which comprises a bacterial outer membrane lipopolysaccharide-binding peptide conjugated to an immune cell chemotactic peptide, wherein said immune cell chemotactic peptide is selected from the group consisting of formyl chemotactic peptide, desformyl chemotactic peptide, chemotactic peptide with a urea derivative, and tuftsin, said outer membrane lipopolysaccharide-binding peptide and said immune cell chemotactic peptide acting in synergy to provide a bacterial sensitizing activity, wherein said formyl chemotactic peptide comprises an N-terminal N-formyl methionyl residue and said desformyl chemotactic peptide comprises an N-terminal methionyl residue.

27. The pharmaceutical composition of claim 26, wherein said bacterial outer membrane lipopolysaccharide-binding peptide is a polymyxin derivative or a polymyxin analog.

28. A pharmaceutical composition for treatment of bacteremia and/or septicemia following infection by gram negative bacteria comprising, as an active ingredient, a compound having the formula: ##STR00018## wherein X1 is a Thr or Ser residue or a covalent bond; X2 is selected from the group consisting of Dab and Ser; X3 is selected from the group consisting of Lys, Orn, Dap, Glu, Asp, Dab and Cys residues; X5 is a hydrophobic amino acid residue; X6 is a hydrophobic amino acid residue; X9 is selected from the group consisting of Ser, Tyr, Thr, Dab, Lys, Orn, Dap, Glu, Asp, Cys and n-butyric acid residues; L is a peptide cyclization linker moiety; R is selected from the group consisting of a covalent bond, an amino acid residue, a stretch of amino acid residues, an amino fatty acid residue having 1-12 carbon atoms in its carbon backbone and a polyethylene glycol derivative; Xa is a hydrophobic linear amino acid residue; Xb is selected from the group consisting of linear and branched aliphatic amino acid residues; Xc is an aromatic amino acid residue; A is selected from the group consisting of formyl and Z-NHCONH-, where Z is n-butyl, phenyl, 4-chlorophenyl, 4-methoxyphenyl or p-tolyl derivative; X0 . . . 5 is a stretch of 0-5 amino acid residues; with the provisions that said A-Xa-Xb-Xc-X0 . . . 5 has an immune cell chemotactic activity and that said ##STR00019## has a bacterial outer membrane lipopolysaccharide-binding activity, said immune cell chemotactic activity and said bacterial outer membrane lipopolysaccharide-binding activity acting in synergy to provide a bacterial sensitizing activity.

29. The pharmaceutical composition of claim 28, wherein X5 is selected from the group consisting of DPhe, DTrp, DLeu, DNle, DMet, DNva and DVal residues.

30. The pharmaceutical composition of claim 28, wherein X5 is selected from the group consisting of Phe, Trp, Leu, Nle, Met, Nva and Val residues.

31. The pharmaceutical composition of claim 28, wherein X6 is selected from the group consisting of Leu, Thr, Nva, Val, Met and Ile residues.

32. The pharmaceutical composition of claim 28, wherein said stretch of amino acid residues is selected from the group consisting of an oligoAla stretch, an oligoAla stretch interrupted by at least one Glu and/or Asp residue, an oligoGly stretch, and an oligoGly stretch interrupted by at least one Glu and/or Asp residue.

33. The pharmaceutical composition of claim 28, wherein said amino fatty acid residue is HN(CH2)xCOOH, where x is 1-12.

34. The pharmaceutical composition of claim 28, wherein said amino fatty acid residue is selected from the group consisting of aminocaproic acid residue and aminobutyric acid residue.

35. The pharmaceutical composition of claim 28, wherein said cyclization linker moiety is selected from the group consisting of --(CH2)x-N--CO--, --(CH2)x-NH--CO--(CH2)y, CO--NH--(CH2)x, --CH.sub.2--S--CH.sub.2--CO-- and --S--S--, where x and y are each independently 1-12.

36. The pharmaceutical composition of claim 28, wherein said hydrophobic linear amino acid residue is selected from the group consisting of Met and Nle.

37. The pharmaceutical composition of claim 28, wherein said linear and branched aliphatic amino acid residue is selected from the group consisting of Leu, Ala, Abu, Nva, Val, Ile, Cys(Me), Met and Nle.

38. The pharmaceutical composition of claim 28, wherein said stretch of 0-5 amino acid residues is selected from the group consisting of Phe, Ile, Nle-Tyr-Lys and DLeu-Phe-DLeu-Phe residues.

39. The pharmaceutical composition of claim 28, wherein said Xc is selected from the group consisting of Phe residue, N-methyl Phe derivative, 2-oxy-3-phenylpropionic acid derivative and 2-aminoxy-3-phenylpropionic acid derivative.

40. The pharmaceutical composition of claim 28, wherein said A-Xa-Xb-Xc-X0 . . . 5- is selected from the group consisting of: Formyl-Met-Leu-Phe- (SEQ ID NO: 1) Formyl-Met-Leu-Phe-Phe- (SEQ ID NO: 13) Formyl-Met-Leu-Phe-Ile- (SEQ ID NO: 14) Formyl-Nle-Leu-Phe-Nle-Tyr-Lys- (SEQ ID NO: 15) Z-NH-CO-NH-Phe-DLeu-Phe-DLeu-Phe- (SEQ ID NO: 16) Formyl-Met-Leu-Phe-Lys- (SEQ ID NO: 17) Formyl-Met-Leu-N-methyl-Phe- (SEQ ID NO: 18) Formyl-Met-Leu-2-oxy-3-phenylpropionic acid- (SEQ ID NO: 19) and Formyl-Met-Leu-2-aminoxy-3-phenylpropionic acid- (SEQ ID NO: 20).

41. A pharmaceutical composition for treatment of bacteremia and/or septicemia following infection by gram negative bacteria comprising, as an active ingredient, a compound having the formula: ##STR00020## wherein X1 is a Thr or Ser residue or a covalent bond; X2 is selected from the group consisting of Dab and Ser; X3 is selected from the group consisting of Lys, Orn, Dap, Glu, Asp, Dab and Cys residues; X5 is a hydrophobic amino acid residue; X6 is a hydrophobic amino acid residue; X9 is selected from the group consisting of Ser, Tyr, Thr, Dab, Lys, Orn, Dap, Glu, Asp, Cys and n-butyric acid residues; L is a peptide cyclization linker moiety; R is selected from the group consisting of a covalent bond, an amino acid residue, a stretch of amino acid residues, an amino fatty acid residue having 1-12 carbon atoms in its carbon backbone and a polyethylene glycol derivative; Xf is selected from the group consisting of Thr, Leu, Gly and Val residues; Xg is selected from the group consisting of Lys and Arg residues; Xh is selected from the group consisting of Pro, Sar and N-methyl non-polar aliphatic amino acid residues; Xi is selected from the group consisting of Arg and Lys residues; X0 . . . 5 is 0-5 amino acid residues; m is an integer selected from the group consisting of 1-8, whereby if m is greater than 1 [Xf-Xg-Xh-Xi-X0 . . . 5]m is a branched structure; with the provisions that said [Xf-Xg-Xh-Xi-X0 . . . 5]m has an immune cell chemotactic activity and that said ##STR00021## has a bacterial outer membrane lipopolysaccharide-binding activity, said immune cell chemotactic activity and said bacterial outer membrane lipopolysaccharide-binding activity acting in synergy to provide a bacterial sensitizing activity.

42. The pharmaceutical composition of claim 41, wherein X5 is selected from the group consisting of DPhe, DTrp, DLeu, DNle, DMet, DNva and DVal residues.

43. The pharmaceutical composition of claim 41, wherein X5 is selected from the group consisting of Phe, Trp, Leu, Nle, Met, Nva and Val residues.

44. The pharmaceutical composition of claim 41, wherein X6 is selected from the group consisting of Leu, Thr, Nva, Val, Met and Ile residues.

45. The pharmaceutical composition of claim 41, wherein said stretch of amino acid residues is selected from the group consisting of an oligoAla stretch, an oligoAla stretch interrupted by at least one Glu and/or Asp residue, an oligoGly stretch, and an oligoGly stretch interrupted by at least one Glu and/or Asp residue.

46. The pharmaceutical composition of claim 41, wherein said amino fatty acid residue is HN(CH2)xCOOH, where x is 1-12.

47. The pharmaceutical composition of claim 41, wherein said amino fatty acid residue is selected from the group consisting of aminocaproic acid residue and aminobutyric acid residue.

48. The pharmaceutical composition of claim 41, wherein said cyclization linker moiety is selected from the group consisting of --(CH2)x-NH--CO--, --(CH2)x-NH--CO--(CH2)y, CO--NH--(CH2)x, --CH.sub.2--S--CH.sub.2--CO-- and --S S--S--, where x and y are each independently 1-12.

49. The pharmaceutical composition of claim 41, wherein [Xf-Xg-Xh-Xi-X0 . . . 5]m- is selected from the group consisting of: ##STR00022## where X is selected from the group consisting of Dap, Lys and Orn residues.

50. The composition of claim 26, wherein said compound is selected from the group consisting of SEQ ID NOs: 3, 4, 6 and 8-11.

51. The pharmaceutical composition of claim 26, further comprising an antibiotic compound.

52. The pharmaceutical composition of claim 51, wherein said antibiotic compound is selected from the group consisting of novobiocin, erythromycin, lincomycin, nafcillin, naladixic acid, rifabutin, rifampin,fusidic acid and vancomycin.

53. The pharmaceutical composition of claim 26, further comprising a pharmaceutically acceptable carrier.

54. A method of treating bacteremia and/or septicemia following infection by gram negative bacteria in a subject, the method comprising: administering to the subject a therapeutically effective amount of a compound which comprises a bacterial outer cell membrane lipopolysaccharide-binding peptide conjugated to an immune cell chemotactic peptide, wherein said immune cell chemotactic peptide is selected from the group consisting of formyl chemotactic peptide, desformyl chemotactic peptide, chemotactic peptide with a urea derivative, and tuftsin, said outer membrane lipopolysaccharide-binding peptide and said immune cell chemotactic peptide acting in synergy to provide a bacterial sensitizing activity.

55. The method of claim 54, wherein said bacterial outer membrane lipopolysaccharide-binding peptide is a polymyxin derivative or a polymyxin analog.

56. A method of treating bacteremia and/or septicemia following infection by gram negative bacteria in a subject, the method comprising: administering to the subject a therapeutically effective amount of a compound having the formula: ##STR00023## wherein X1 is a Thr or Ser residue or a covalent bond; X2 is selected from the group consisting of Dab and Ser; X3 is selected from the group consisting of Lys, Orn, Dap, Glu, Asp, Dab and Cys residues; X5 is a hydrophobic amino acid residue; X6 is a hydrophobic amino acid residue; X9 is selected from the group consisting of Ser, Tyr, Thr, Dab, Lys, Orn, Dap, Glu, Asp, Cys and n-butyric acid residues; L is a peptide cyclization linker moiety; R is selected from the group consisting of a covalent bond, an amino acid residue, a stretch of amino acid residues, an amino fatty acid residue having 1-12 carbon atoms in its carbon backbone and a polyethylene glycol derivative; Xa is a hydrophobic linear amino acid residue; Xb is selected from the group consisting of linear and branched aliphatic amino acid residues; Xc is an aromatic amino acid residue; A is selected from the group consisting of formyl and Z-NHCONH-, where Z is n-butyl, phenyl, 4-chlorophenyl, 4-methoxyphenyl or p-tolyl derivative; X0 . . . 5 is a stretch of 0-5 amino acid residues; with the provisions that said A-Xa-Xb-Xc-X0 . . . 5 has an immune cell chemotactic activity and that said ##STR00024## has a bacterial outer membrane lipopolysaccharide-binding activity, acid immune cell chemotactic activity and said bacterial outer membrane lipopolysaccharide-binding activity acting in synergy to provide a bacterial sensitizing activity.

57. The method of claim 56, wherein X5 is selected from the group consisting of DPhe, DTrp, DLeu, DNle, DMet, DNva and DVal residues.

58. The method of claim 56, wherein X5 is selected from the group consisting of Phe, Trp, Leu, Nle, Met, Nva and Val residues.

59. The method of claim 56, wherein X6 is selected from the group consisting of Leu, Thr, Nva, Val, Met and Ile residues.

60. The method of claim 56, wherein said stretch of amino acid residues is selected from the group consisting of an oligoAla stretch, an oligoAla stretch interrupted by at least one Glu and/or Asp residue, an oligoGly stretch, and an oligoGly stretch interrupted by at least one Glu and/or Asp residue.

61. The method of claim 56, wherein said amino fatty acid residue is HN(CH2)xCOOH, where x is 1-12.

62. The method of claim 56, wherein said amino fatty acid residue is selected from the group consisting of aminocaproic acid residue and aminobutyric acid residue.

63. The method of claim 56, wherein said cyclization linker moiety is selected from the group consisting of --(CH2)x-NH--CO--, --(CH2)x-NH--CO--(CH2)y, CO--NH--(CH2)x, --CH.sub.2--S--CH.sub.2--CO-- and --S--S--, where x and y are each independently 1-12.

64. The method of claim 56, wherein said hydrophobic linear amino acid residue is selected from the group consisting of Met and Nle.

65. The method of claim 56, wherein said linear and branched aliphatic amino acid residue is selected from the group consisting of Leu, Ala, Abu, Nva, Val, Ile, Cys(Me), Met and Nle.

66. The method of claim 56, wherein said stretch of 0-5 amino acid residues is selected from the group consisting of Phe, Ile, Nle-Tyr-Lys and DLeu-Phe-DLeu-Phe residues.

67. The method of claim 56, wherein said Xc is selected from the group consisting of Phe residue, N-methyl Phe derivative, 2-oxy-3-phenylpropionic acid derivative and 2-aminoxy-3-phenylpropionic acid derivative.

68. The method of claim 56, wherein said A-Xa-Xb-Xc-X0 . . . 5- is selected from the group consisting of: Formyl-Met-Leu-Phe- (SEQ ID NO: 1) Formyl-Met-Leu-Phe-Phe- (SEQ ID NO: 13) Formyl-Met-Leu-Phe-Ile- (SEQ ID NO: 14) Formyl-Nle-Leu-Phe-Nle-Tyr-Lys- (SEQ ID NO: 15) Z-NH-CO-NH-Phe-DLeu-Phe-DLeu-Phe- (SEQ ID NO: 16) Formyl-Met-Leu-Phe-Lys- (SEQ ID NO: 17) Formyl-Met-Leu-N-methyl-Phe- (SEQ ID NO: 18) Formyl-Met-Leu-2-oxy-3-phenylpropionic acid- (SEQ ID NO: 19) and Formyl-Met-Leu-2-aminoxy-3-phenylpropionic acid- (SEQ ID NO: 20).

69. A method of treating bacteremia and/or septicemia following infection by gram negative bacteria in a subject, the method comprising: administering to the subject a therapeutically effective amount of a compound having the formula: ##STR00025## wherein X1 is a Thr or Ser residue or a covalent bond; X2 is selected from the group consisting of Dab and Ser; X3 is selected from the group consisting of Lys, Orn, Dap, Glu, Asp, Dab and Cys residues; X5 is a hydrophobic amino acid residue; X6 is a hydrophobic amino acid residue; X9 is selected from the group consisting of Ser, Tyr, Thr, Dab, Lys, Orn, Dap, Glu, Asp, Cys and n-butyric acid residues; L is a peptide cyclization linker moiety; R is selected from the group consisting of a covalent bond, an amino acid residue, a stretch of amino acid residues, an amino fatty acid residue having 1-12 carbon atoms in its carbon backbone and a polyethylene glycol derivative; Xf is selected from the group consisting of Thr, Leu, Gly and Val residues; Xg is selected from the group consisting of Lys and Arg residues; Xh is selected from the group consisting of Pro, Sar and N-methyl non-polar aliphatic amino acid residues; Xi is selected from the group consisting of Arg and Lys residues; X0 . . . 5 is 0-5 amino acid residues; m is an integer selected from the group consisting of 1-8, whereby if m is greater than 1 [Xf-Xg-Xh-Xi-X0 . . . 5]m is a branched structure; with the provisions that said [Xf-Xg-Xh-Xi-X0 . . . 5]m has an immune cell chemotactic activity and that said ##STR00026## has a bacterial outer membrane lipopolysaccharide-binding activity, said immune cell chemotactic activity and said bacterial outer membrane lipopolysaccharide-binding activity acting in synergy to provide a bacterial sensitizing activity.

70. The method of claim 69, wherein X5 is selected from the group consisting of DPhe, DTrp, DLeu, DNle, DMet, DNva and DVal residues.

71. The method of claim 69, wherein X5 is selected from the group consisting of Phe, Trp, Leu, Nle, Met, Nva and Val residues.

72. The method of claim 69, wherein X6 is selected from the group consisting of Leu, Thr, Nva, Val, Met and Ile residues.

73. The method of claim 69, wherein said stretch of amino acid residues is selected from the group consisting of an oligoAla stretch, an oligoAla stretch interrupted by at least one Glu and/or Asp residue, an oligoGly stretch, and an oligoGly stretch interrupted by at least one Glu and/or Asp residue.

74. The method of claim 69, wherein said amino fatty acid residue is HN(CH2)xCOOH, where x is 1-12.

75. The method of claim 69, wherein said amino fatty acid residue is selected from the group consisting of aminocaproic acid residue and aminobutyric acid residue.

76. The method of claim 69, wherein said cyclization linker moiety is selected from the group consisting of --(CH2)x-NH--CO--, --(CH2)x-NH--CO--(CH2)y, CO--NH--(CH2)x, --CH.sub.2--S--CH.sub.2--CO-- and --S--S--, where x and y are each independently 1-12.

77. The method of claim 69, wherein [Xf-Xg-Xh-Xi-X0 . . . 5]m- is selected from the group consisting of: ##STR00027## where X is selected from the group consisting of Dap, Lys and Orn residues.

78. The method of claim 54, wherein the compound is selected from the group consisting of SEQ ID NOs: 3, 4, 6 and 8-11.

79. The method of claim 54, further comprising administering an antibiotic compound.

80. The method of claim 79, wherein said antibiotic compound is selected from the group consisting of novobiocin, erythromycin, lincomycin, nafcillin, naladixic acid, rifabutin, rifampin, fusidic acid and vancomycin.

81. The compound of claim 4, wherein X5 is selected from the group consisting of DPhe and DLeu.

82. The compound of claim 81, wherein X6 is selected from the group consisting of Leu, Thr, Nva, Val, Met and Ile residues.

83. The compound of claim 82, wherein said cyclization linker moiety is selected from the group consisting of --(CH2)x-NH--CO--, --(CH2)x-NH--CO--(CH2)y, CO--NH--(CH2)x, --CH.sub.2--S--CH.sub.2--CO-- and --S--S--, where x and y are each independently 1-12.

84. The compound of claim 82, wherein said A-Xa-Xb-Xc-X0 . . . 5- is selected from the group consisting of: Formyl-Met-Leu-Phe- (SEQ ID NO: 1) Formyl-Met-Leu-Phe-Phe- (SEQ ID NO: 13) Formyl-Met-Leu-Phe-Ile- (SEQ ID NO: 14) Formyl-Nle-Leu-Phe-Nle-Tyr-Lys- (SEQ ID NO: 15) Z-NH-CO-NH-Phe-DLeu-Phe-DLeu-Phe- (SEQ ID NO: 16) Formyl-Met-Leu-Phe-Lys- (SEQ ID NO: 17) Formyl-Met-Leu-N-methyl-Phe- (SEQ ID NO: 18) Formyl-Met-Leu-2-oxy-3-phenylpropionic acid- (SEQ ID NO: 19) and Formyl-Met-Leu-2-aminoxy-3-phenylpropionic acid- (SEQ ID NO: 20).

85. The pharmaceutical composition of claim 28, wherein X5 is selected from the group consisting of DPhe and DLeu.

86. The pharmaceutical composition of claim 85, wherein X6 is selected from the group consisting of Leu, Thr, Nva, Val, Met and Ile residues.

87. The pharmaceutical composition of claim 86, wherein said cyclization linker moiety is selected from the group consisting of --(CH2)x-NH--CO--, --(CH2)x-NH--CO--(CH2)y, CO--NH--(CH2)x, --CH.sub.2--S--CH.sub.2--CO-- and --S--S--, where x and y are each independently 1-12.

88. The pharmaceutical composition of claim 86, wherein said A-Xa-Xb-Xc-X0 . . . 5- is selected from the group consisting of: Formyl-Met-Leu-Phe- (SEQ ID NO: 1) Formyl-Met-Leu-Phe-Phe- (SEQ ID NO: 13) Formyl-Met-Leu-Phe-Ile- (SEQ ID NO: 14) Formyl-Nle-Leu-Phe-Nle-Tyr-Lys- (SEQ ID NO: 15) Z-NH-CO-NH-Phe-DLeu-Phe-DLeu-Phe- (SEQ ID NO: 16) Formyl-Met-Leu-Phe-Lys- (SEQ ID NO: 17) Formyl-Met-Leu-N-methyl-Phe- (SEQ ID NO: 18) Formyl-Met-Leu-2-oxy-3-phenylpropionic acid- (SEQ ID NO: 19) and Formyl-Met-Leu-2-aminoxy-3-phenylpropionic acid- (SEQ ID NO: 20).

89. The method of claim 56, wherein X5 is selected from the group consisting of DPhe and DLeu.

90. The method of claim 89, wherein X6 is selected from the group consisting of Leu, Thr, Nva, Val, Met and Ile residues.

91. The method of claim 90, wherein said cyclization linker moiety is selected from the group consisting of --(CH2)x-NH--CO--, --(CH2)x-NH--CO--(CH2)y, CO--NH--(CH2)x, --CH.sub.2--S--CH.sub.2--CO-- and --S--S--, where x and y are each independently 1-12.

92. The method of claim 90, wherein said A-Xa-Xb-Xc-X0 . . . 5- is selected from the group consisting of: Formyl-Met-Leu-Phe- (SEQ ID NO: 1) Formyl-Met-Leu-Phe-Phe- (SEQ ID NO: 13) Formyl-Met-Leu-Phe-Ile- (SEQ ID NO: 14) Formyl-Nle-Leu-Phe-Nle-Tyr-Lys- (SEQ ID NO: 15) Z-NH-CO-NH-Phe-DLeu-Phe-DLeu-Phe- (SEQ ID NO: 16) Formyl-Met-Leu-Phe-Lys- (SEQ ID NO: 17) Formyl-Met-Leu-N-methyl-Phe- (SEQ ID NO: 18) Formyl-Met-Leu-2-oxy-3-phenylpropionic acid- (SEQ ID NO: 19) and Formyl-Met-Leu-2-aminoxy-3-phenylpropionic acid- (SEQ ID NO: 20).

93. The pharmaceutical composition of claim 28, further comprising an antibiotic compound.

94. The pharmaceutical composition of claim 93, wherein said antibiotic compound is selected from the group consisting of novobiocin, erythromycin, lincomycin, nafcillin, naladixic acid, rifabutin, rifampin, fusidic acid and vancomycin.

95. The pharmaceutical composition of claim 28, further comprising a pharmaceutically acceptable carrier.

96. The pharmaceutical composition of claim 41, further comprising an antibiotic compound.

97. The pharmaceutical composition of claim 96, wherein said antibiotic compound is selected from the group consisting of novobiocin, erythromycin, lincomycin, nafcillin, naladixic acid, rifabutin, rifampin, fusidic acid and vancomycin.

98. The pharmaceutical composition of claim 41, further comprising a pharmaceutically acceptable carrier.

99. The method of claim 56, further comprising administering an antibiotic compound.

100. The method of claim 99, wherein said antibiotic compound is selected from the group consisting of novobiocin, erythromycin, lincomycin, nafcillin, naladixic acid, rifabutin, rifampin, fusidic acid and vancomycin.

101. The method of claim 69, further comprising administering an antibiotic compound.

102. The method of claim 101, wherein said antibiotic compound is selected from the group consisting of novobiocin, erythromycin, lincomycin, nafcillin, naladixic acid, rifabutin, rifampin, fusidic acid and vancomycin.

Description

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to compounds, pharmaceutical compositions and methods for the treatment of bacteremia and septicemia and, more particularly, to novel conjugates of bacterial outer membrane binding peptides, preferably having bacterial sensitization activity, and immune cells chemotactic peptides, and pharmaceutical compositions containing same useful in the treatment of bacteremia and/or septicemia following infection by gram negative bacteria, administered alone or in combination with conventional antibiotics.

Blood infection caused by gram-negative bacteria is one of the major challenges facing modern medicine, despite treatment availability with conventional antibiotics (Young, 1985). Mortality rates in the range of 25-40% for gram-negative septicemia have been reported in some medical centers. Antibiotic treatment is often administered at late stages of the disease, usually when symptoms appear. The time required by the antibiotic to kill the pathogen is often too long, thus failing to prevent irreversible tissue damage. Moreover, in many cases the antibiotic is given before performing any sensitivity tests. The emergence of strains resistant to the conventional antibiotics and lack of rapid diagnosis and antibiotic sensitivity pattern of the infecting bacteria are probably among the major causes of the inadequate conventional therapy and high mortality (Cassel, 1995; Chin & Marks, 1994).

Polymyxins:

Polymyxins are basic cyclic peptides having a lipid moiety and antibiotic activity, naturally produced by various strains of Bacillus polymyxa. Of the many isolated and characterized polymyxins, only polymyxin B and E (the latter is also known as colistin) were in actual therapeutic use (Lambert and O'grade 1992). Polymyxin B is a polycationic amphipathic decapeptide, a potent bactericidal agent against most gram-negative bacteria. Emergence of resistant strains to polymyxin B has been reported to be rare (Soogard, 1982). Polymyxin B binds to the outer membrane of the bacteria (Morrison and Jacobs, 1976), and inserts its lipid moiety into the membrane to completely disorganize it (Bryan, 1982). The clinical use of polymyxins has been restricted, however, because polymyxins are highly toxic to animal cells.

Polymyxin Derived Peptides:

The removal, by deacylation, of the fatty-acid side chain from polymyxins, e.g., polymyxin B and E, significantly reduces the toxicity of the parent molecules (Vaara 1992). The deacylated product of polymyxin B, which is known as polymyxin B-derived peptide (PMBP) and alternatively as polymyxin B nonapeptide (PMBN), lacks bactericidal activity but retains its ability to bind specifically to the lipopolysaccharide (LPS) of the outer leaflet of the bacterial membrane and renders the gram-negative bacteria susceptible to several antibiotics by permeabilizing their outer membrane. The latter antimicrobial activity of PMBP is referred to in the art as sensitizing activity.

Because the parent polymyxin B molecule and its derivative PMBP bind to the same receptor on the bacterial surface, probably via the cyclic heptapeptide portion, the emergence of strains resistant to the sensitizing activity of PMBP is rare, as is the case for the bactericidal activity of the parent polymyxin molecule (Chihara et al., 1973; Vaara and Vaara, 1983; Duwe et al., 1986, Ofek et al., 1994).

Formyl Chemotactic Peptides (fCP):

Schifmann et al., (1975) have shown that chemotaxis (migration of cells toward a gradient of a chemoattractant molecule) of phagocytic cells is induced by short-chain N-formyl-methionyl peptides active at extremely low concentrations (e.g., 10.sup.-9-10.sup.-10 M) and referred to as formyl chemotactic peptides (fCP). Phagocytic cells contain specific receptors that bind the formyl Met-Leu-Phe (SEQ ID NO:1) peptide (fCP) with relatively high affinity, whereas the affinity of the desformyl derivative (dfCP) to the receptor is two orders of magnitude smaller (Freer et al., 1980, 1982). The fCP also induce degranulation of polymorphonuclear leukocytes (PMN) and cause the release of antimicrobial agents in the surrounding milieu (Becker, 1976; Niedel and Cuatrecasas, 1980). Most importantly, it has been shown that the formyl chemotactic peptide, when immobilized on particles, greatly enhances their phagocytosis by PMN (Becker, 1976).

SUMMARY OF THE INVENTION

While reducing the present invention to practice, it was found that a conjugate of a bacterial outer membrane binding peptide, such as a polymyxin derivative or a polymyxin analog, e.g., a polymyxin B-derived peptide, and a chemotactic peptide, such as formyl chemotactic peptide, has an improved anti-bacterial activity when acting alone and/or when administered together with a conventional antibiotic, because of the combination of bacterial binding and sensitization activities of the bacterial outer membrane binding peptide and the chemotactic activity of the chemotactic peptide, which act in synergy, via independent mechanisms, at killing and/or eradicating bacteria.

Hence, according to one aspect of the present invention there is provided a compound comprising a bacterial outer membrane binding peptide conjugated to an immune cells chemotactic peptide.

According to another aspect of the present invention there is provided a pharmaceutical composition for treatment of bacteremia and/or septicemia following infection by gram negative bacteria comprising, as an active ingredient a compound which comprises a bacterial outer membrane binding peptide conjugated to an immune cells chemotactic peptide.

According to still further features in the described preferred embodiments the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

According to still further features in the described preferred embodiments, the pharmaceutical composition further comprising a conventional antibiotic compound.

According to yet another aspect of the present invention there is provided a method of treating bacteremia and/or septicemia following infection by gram negative bacteria, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound which comprises a bacterial outer membrane binding peptide conjugated to an immune cells chemotactic peptide.

According to still further features in the described preferred embodiments the method further comprising administering a conventional antibiotic compound.

According to still further features in the described preferred embodiments the conventional antibiotic compound is selected from the group consisting of novobiocin, erythromycin, lincomycin, nafcillin, naladixic acid, rifabutin, rifampin, fusidic acid and vancomycin.

According to further features in preferred embodiments of the invention described below, the compound has a bacterial sensitization activity.

According to still further features in the described preferred embodiments the bacterial outer membrane binding peptide is a polymyxin derivative or a polymyxin analog.

According to still further features in the described preferred embodiments the immune cells chemotactic peptide is a selected from the group consisting of formyl chemotactic peptide, desformyl chemotactic peptide, chemotactic peptide with a urea derivative and tuftsin.

According to still further features in the described preferred embodiments the compound has the formula:

##STR00001## wherein X1 is a Thr or Ser residue or a covalent bond; X2 is selected from the group consisting of Dab and Ser X3 is selected from the group consisting of Lys, Orn, Dap, Glu, Asp, Dab and Cys residues; X5 is a hydrophobic amino acid residue; X6 is a hydrophobic amino acid residue; X9 is selected from the group consisting of Ser, Tyr, Thr, Dab, Lys, Orn, Dap, Glu, Asp, Cys and n-butiric acid residues; L is a peptide cyclization linker moiety;

R is selected from the group consisting of a covalent bond, an amino acid residue, a stretch of amino acid residues, an amino fatty acid residue having 1-12 carbon atoms in its carbon backbone and polyethylene glycol derivative;

Xa is a hydrophobic linear amino acid residue;

Xb is selected from the group consisting of linear and branched aliphatic amino acid residues;

Xc is an aromatic amino acid residue;

A is selected from the group consisting of formyl and Z-NHCONH-, where Z is n-butyl, phenyl, 4-chlorophenyl, 4-methoxyphenyl or p-tolyl derivative;

X0 . . . 5 is a stretch of 0-5 amino acid residues;

with the provisions that the A-Xa-Xb-Xc-X0 . . . 5 has an immune cells chemotactic activity and that the

##STR00002## has a bacterial outer membrane binding activity.

According to still further features in the described preferred embodiments X5 is selected from the group consisting of DPhe, DTrp, DLeu, DNle, DMet, DNva and DVal residues.

According to still further features in the described preferred embodiments X5 is selected from the group consisting of Phe, Trp, Leu, Nle, Met, Nva and Val residues.

According to still further features in the described preferred embodiments X6 is selected from the group consisting of Leu, Thr, Nva, Val, Met and Ile residues.

According to still further features in the described preferred embodiments the stretch of amino acid residues is selected from the group consisting of an oligoAla stretch, an oligoAla strech interupted by at least one Glu and/or Asp residues an oligoGly strech and an oligoGly strech interupted by at least one Glu and/or Asp residues.

According to still further features in the described preferred embodiments the amino fatty acid residue is HN(CH2)xCOOH, where x is 1-12.

According to still further features in the described preferred embodiments the amino fatty acid residue is selected from the group consisting of aminocaproic acid residue and aminobutyric acid residue.

According to still further features in the described preferred embodiments the cyclization linker moiety is selected from the group --(CH2)x--NH--CO--, --(CH2)x--NH--CO--(CH2)y, CO--NH--(CH2)x, --CH.sub.2--S--CH.sub.2--CO-- and --S--S--, where x and y are each independently 1-12.

According to still further features in the described preferred embodiments the hydrophobic linear amino acid residue is selected from the group consisting of Met and Nle.

According to still further features in the described preferred embodiments the linear and branched aliphatic amino acid residue is selected from the group consisting of Leu, Ala, Abu, Nva, Val, Ile, Cys(Me), Met and Nle.

According to still further features in the described preferred embodiments the stretch of 0-5 amino acid residues is selected from the group consisting of Phe, Ile, Nle-Tyr-Lys and DLeu-Phe-DLeu-Phe residues.

According to still further features in the described preferred embodiments Xc is selected from the group consisting of Phe residue, N-methyl derivative, 2-oxy-3-phenylpropionic acid derivative and 2-aminoxy-3-phenylpropionic acid derivative.

According to still further features in the described preferred embodiments the A-Xa-Xb-Xc-X0 . . . 5- is selected from the group consisting of: Formyl-Met-Leu-Phe-Phe-(SEQ ID NO: 13) Formyl-Met-Leu-Phe-Ile-(SEQ ID NO: 14) Formyl-Nle-Leu-Phe-Nle-Tyr-Lys-(SEQ ID NO: 15) Z-NH-CO-NH-Phe-DLeu-Phe-DLeu-Phe-(SEQ ID NO: 16) Formyl-Met-Leu-Phe-Lys-(SEQ ID NO: 17) Formyl-Met-Leu-N-methyl-Phe-(SEQ ID NO: 18) Formyl-Met-Leu-2-oxy-3-phenylpropionic acid-(SEQ ID NO: 19) and Formyl-Met-Leu-2-aminoxy-3-phenylpropionic acid-(SEQ ID NO: 20).

According to still further features in the described preferred embodiments the compound has the formula:

##STR00003## wherein X1 is a Thr or Ser residue or a covalent bond; X2 is selected from the group consisting of Dab and Ser X3 is selected from the group consisting of Lys, Orn, Dap, Glu, Asp, Dab and Cys residues; X5 is a hydrophobic amino acid residue; X6 is a hydrophobic amino acid residue; X9 is selected from the group consisting of Ser, Tyr, Thr, Dab, Lys, Orn, Dap, Glu, Asp, Cys and n-butiric acid residues; L is a peptide cyclization linker moiety;

R is selected from the group consisting of a covalent bond, an amino acid residue, a stretch of amino acid residues, an amino fatty acid residue having 1-12 carbon atoms in its carbon backbone and polyethylene glycol derivative;

Xf is selected from the group consisting of Thr, Leu, Gly and Val residues;

Xg is selected from the group consisting of Lys and Arg residues;

Xh is selected from the group consisting of Pro, Sar and N-methyl non-polar aliphatic amino acid residues;

Xi is selected from the group consisting of Arg and Lys residues;

X0 . . . 5 is 0-5 amino acid residues;

m is an integer selected from the group consisting of 1-8, whereby if m is greater than 1 [Xf-Xg-Xh-Xi-X0 . . . 5]m is a branched structure;

with the provisions that the [Xf-Xg-Xh-Xi-X0 . . . 5]m has an immune cells chemotactic activity and that the

##STR00004## has a bacterial outer membrane binding activity.

According to still further features in the described preferred embodiments [Xf-Xg-Xh-Xi-X0 . . . 5]m- is selected from the group consisting of:

##STR00005## where X is selected from the group consisting of Dap, Lys and Orn residues.

According to still further features in the described preferred embodiments the compound is selected from the group consisting of SEQ ID NOs: 2, 4 and 6-11.

The present invention successfully addresses the shortcomings of the presently known configurations by providing new therapeutics highly effective in the treatment of bacteremia and septicemia.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a graph showing displacement curves of Dansyl-PMBN with tuftsin-PMBN and fMLF-PMBN. Peptides were added to E. coli LPS solution (3 .mu.g/mL) bound to dansyl-PMBN (0.55 .mu.M). The fluorescence inhibition was measured 5 minutes after each addition at excitation and emission wavelengths of 340 and 485 nm, respectively.

FIG. 2 is a bar graph demonstrating the enhancement of phagocytosis with conjugates peptide. Open boxes--10 .mu.M, closed boxes--1 .mu.M, of the respective peptides.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of compounds, pharmaceutical compositions and methods which can be used in the treatment of bacteremia and septicemia. Specifically, the present invention is of novel conjugates of bacterial outer membrane binding peptides, preferably having bacterial sensitization activity, and immune cells chemotactic peptides, and pharmaceutical compositions containing same, useful in the treatment of bacteremia and/or septicemia following infection by gram negative bacteria administered alone or in combination with conventional antibiotics.

The principles and operation of the compounds, compositions and therapeutic methods according to the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Bacteremia infections caused by various bacteria species and by gram negative bacilli in particular constitute one of the major, if not the principal, problem related to infection diseases encountered in modern medical centers. Antibiotic treatment is often administrated at late stages of the disease, usually when symptoms appear. The extended time required by the antibiotic to kill the pathogen is often not sufficient to prevent tissue damage. Moreover, in many cases the antibiotic is given before performing adequate sensitivity tests to determine the type of antibiotic toward which the pathogen is sensitive. In the case of gram negative infections, there is an increasing probability that the invading strain of bacteria is resistant to such "blind" antibiotic treatment. These are the main reasons for the high mortality rate due to bacterial-septicemia, in spite of antibiotic treatment.

While reducing the present invention to practice, it was found that a conjugate of a bacterial outer membrane binding peptide, such as a polymyxin derivative or a polymyxin analog, e.g., a polymyxin B-derived peptide, and a chemotactic peptide, such as formyl chemotactic peptide, has an improved anti-bacterial activity when acting alone and/or when administered together with a conventional antibiotic, because of the combination of bacterial binding and sensitization activities of the bacterial outer membrane binding peptide and the chemotactic activity of the chemotactic peptide, which act in synergy, via independent mechanisms, at killing and/or eradicating bacteria.

Hence, according to one aspect of the present invention there is provided a compound comprising a bacterial outer membrane binding peptide, which preferably has also a bacterial sensitization activity, conjugated to an immune cells chemotactic peptide.

According to another aspect of the present invention there is provided a pharmaceutical composition for treatment of bacteremia and/or septicemia following infection by gram negative bacteria comprising, as an active ingredient a compound which comprises a bacterial outer membrane binding peptide conjugated to an immune cells chemotactic peptide. The pharmaceutical composition preferably further comprising a pharmaceutically