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WO1995026744A1 - Inhibiteurs du facteur de necrose tumorale - Google Patents

Inhibiteurs du facteur de necrose tumorale Download PDF

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Publication number
WO1995026744A1
WO1995026744A1 PCT/US1995/004018 US9504018W WO9526744A1 WO 1995026744 A1 WO1995026744 A1 WO 1995026744A1 US 9504018 W US9504018 W US 9504018W WO 9526744 A1 WO9526744 A1 WO 9526744A1
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WO
WIPO (PCT)
Prior art keywords
seq
amide
carboxy terminal
peptide
terminal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1995/004018
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English (en)
Inventor
George A. Heavner
Marian Kruszynski
Miljenko Mervic
Robert Walter Weber
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Biotech Inc
Original Assignee
Centocor Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/221,580 external-priority patent/US5519000A/en
Priority claimed from US08/221,583 external-priority patent/US5486595A/en
Priority claimed from US08/221,581 external-priority patent/US5506340A/en
Application filed by Centocor Inc filed Critical Centocor Inc
Publication of WO1995026744A1 publication Critical patent/WO1995026744A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to compounds that inhibit tumor necrosis factor-alpha (TNF ⁇ ) activity by binding to TNF- ⁇ and thereby preventing the TNF ⁇ from binding to TNF receptors.
  • TNF ⁇ tumor necrosis factor-alpha
  • TNF ⁇ activity which is mediated through its binding to its receptor on cells is inhibited.
  • TNF ⁇ tumor necrosis factor-
  • cachectin a protein secreted primarily by monocytes and macrophages as a soluble homotrimer of 17 kD protein subunits in response to endotoxin or other stimuli
  • a membrane-bound 26 kD precursor form of TNF ⁇ has also been described (Kriegler, M. et al., Cell 1988, 53, 45-53) .
  • TNF ⁇ was originally discovered in the serum of animals injected sequentially with a bacterial vaccine (bacillus Calmette-Guerin, BCG) and endotoxin (Carswell, E.A. et al. , Proc . Natl . Acad. Sci . USA 1975, 72, 3666) .
  • TNF ⁇ plays an integral role in destroying tumors, mediating responses to tissue injury, and protecting hosts from infections by various microorganisms. However, its activity appears to be excessive in some disease states and inflammatory reactions such as rheumatoid arthritis, cachexia, and septic shock (Pujol-Borrell et al.
  • TNF ⁇ granulocyte/macrophage-colony stimulating factor
  • TNF ⁇ is noted for its pro-inflammatory actions which result in tissue injury, such as induction of procoagulant activity on vascular endothelial cells (Pober, J.S. et al . , J. Immunol . 1986, 336, 1680) , increased adherence of neutrophils and lymphocytes (Pober, J.S. et al. , J " . Immunol . 1987, 138, 3319) , and stimulation of the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al. , J. Exp . Med. 1987, 166, 1390) .
  • TNF ⁇ in the pathogenesis of many infections (Cerami, A. et al. , Immunol . Today 1988, 9, 28) , immune disorders, neoplastic pathology, e.g., in cachexia accompanying some malignancies (Oliff, A. et al . , Cell 1987, 50, 555) , and in autoimmune pathologies and graft-versus host pathology (Piguet, P.-F. et al. , J. Exp . Med . 1987, 166, 1280) .
  • the association of TNF ⁇ with cancer and infectious pathologies is often related to the host's catabolic state.
  • a major problem in cancer patients is weight loss, usually associated with anorexia.
  • Cachexia The extensive wasting which results is known as "cachexia” (Kern, K.A. al. , J. Parent . Enter. Nutr. 1988, 12, 286-298) .
  • Cachexia includes progressive weight loss, anorexia, and persistent erosion of body mass in response to a malignant growth.
  • the fundamental physiological derangement may be related to a decline in food intake relative to energy expenditure.
  • the cachectic state is thus associated with significant morbidity and is responsible for the majority of cancer mortality.
  • TNF ⁇ is an important mediator of the cachexia in cancer, infectious pathology, and in other catabolic states.
  • TNF ⁇ is thought to play a central role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock (Michie, H.R. et al . , Br. J. Surg . 1989, 76, 670-671; Debets, J.M.H. et al . , Second Vienna Shock Forum, 1989, p.463-466; Simpson, S.Q. et al . , Crit. Care Clin . 1989, 5, 27-47) , including fever, malaise, anorexia, and cachexia.
  • Endotoxin is a potent monocyte/macrophage activator which stimulates production and secretion of TNF ⁇ (Kornbluth, S.K.
  • TNF ⁇ could mimic many biological effects of endotoxin, it was concluded to be a central mediator responsible for the clinical manifestations of endotoxin-related illness. TNF ⁇ and other monocyte-derived cytokines mediate the metabolic and neurohormonal responses to endotoxin (Michie, H.R. et al. , N. Eng. J. Med. 1988, 318, 1481-1486) . Endotoxin administration to human volunteers produces acute illness with flu-like symptoms including fever, tachycardia, increased metabolic rate and stress hormone release (Revhaug, A. et al., Arch . Surg.
  • T ⁇ F ⁇ circulating tumors
  • Treatment of cancer patients with T ⁇ F ⁇ revealed that doses greater than 545 ⁇ g/m 2 /24 hours caused alterations similar to those induced by injection of endotoxin (4 ng/kg) into healthy humans (Michie, H.R. et al., Surgery 1988, 104, 280-286), supporting T ⁇ F ⁇ 's role as the principal host mediator of septic and endotoxemic responses .
  • T ⁇ F ⁇ chronic intravenous T ⁇ F ⁇ infusion into humans or rats was associated with anorexia, fluid retention, acute phase responses, and negative nitrogen balance (i.e., classic catabolic effects) , leading to the conclusion that T ⁇ F ⁇ may be responsible for many of the changes noted during critical illness (Michie, H.R. et al., Ann . Surg. 1989, 209, 19-24) .
  • the invention relates to peptides that comprise an amino acid sequence that consist of 4 to 25 amino acids and that inhibit tumor necrosis factor-alpha activity.
  • the peptides comprise at least a four amino acid residue fragment of : SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ
  • the peptides comprise at least a four amino acid residue fragment of :SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO: 65, SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ
  • the invention relates to peptides that have an amino acid sequence selected from the group consisting of: SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:3 , SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:
  • the invention relates to methods of inhibiting tumor necrosis factor activity.
  • the methods comprise contacting tumor necrosis factor alpha with a peptide that comprises an amino acid sequence which consists of 4 to 25 amino acids and that inhibits tumor necrosis factor-alpha activity.
  • the peptide comprises at least a four amino acid residue fragment Of: SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ
  • the peptide comprises at least a four amino acid residue fragment of :SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO.-46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ
  • the invention relates to methods of inhibiting tumor necrosis factor activity.
  • the methods comprise contacting tumor necrosis factor alpha with a peptide that has an amino acid sequence selected from the group consisting of: SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,
  • the invention relates to methods of treating animals suspected of suffering from a disease or disorder mediated by tumor necrosis factor-alpha activity.
  • the methods comprise the step of administering to the individual a therapeutically effective amount of a peptide that comprises an amino acid sequence that consists of 4 to 25 amino acids and that inhibits tumor necrosis factor-alpha activity.
  • the peptide comprises at least a four amino acid residue fragment of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 , SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO.-20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:
  • the peptide comprises at least a four amino acid residue fragment of :SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO-.52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71
  • the invention relates to methods of treating animals suspected of suffering from a disease or disorder mediated by tumor necrosis factor-alpha activity.
  • the methods comprise the step of administering to the individual a therapeutically effective amount of a peptide that has an amino acid sequence selected from the group consisting of: SEQ ID N0:1, SEQ ID N0:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27,
  • Figure 1 is a graph which shows data generated from experiments using peptides which are embodiments of the invention. The experiments were performed to evaluate dose dependent inhibition of TNF ⁇ by the peptides tested.
  • Figure 2 is a graph which shows data generated from experiments using peptides which are embodiments of the invention. The experiments were performed to evaluate dose dependent inhibition of TNF ⁇ by the peptides tested.
  • Figure 3 is a graph which shows data generated from experiments using peptides which are embodiments of the invention. The experiments were performed to evaluate dose dependent inhibition of TNF ⁇ by the peptides tested.
  • compounds which prevent TNF ⁇ from binding to p55 and p75 receptors.
  • the compounds of the invention inhibit the biological activity of TNF ⁇ .
  • the compounds of the invention prevent TNF ⁇ from producing the biological effect associated with the TNF ⁇ -TNF receptor binding.
  • the peptides of the present invention may be selected from the group consisting of: SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:
  • the peptides of the invention are peptides consisting of 4 to 25 amino acids. In some embodiments, the peptides consist of 20 amino acids or less. In some embodiments, the peptides consist of at least 8 amino acids. In some embodiments, the peptides consist of 8-20 amino acids. In some embodiments, the peptides consist of 10-15 amino acids.
  • peptides comprise of amino acid sequences selected from the group consisting of: fragments of SEQ ID NO:21 that have at least four amino acid residues, fragments of SEQ ID NO:22 that have at least four amino acid residues, fragments of SEQ ID NO:23 that have at least four amino acid residues, fragments of SEQ ID NO:42 that have at least four amino acid residues, fragments of SEQ ID NO:43 that have at least four amino acid residues, fragments of SEQ ID NO:44 that have at least four amino acid residues, fragments of SEQ ID NO:45 that have at least four amino acid residues, fragments of SEQ ID NO:46 that have at least four amino acid residues, fragments of SEQ ID NO:47 that have at least four amino acid residues, fragments of SEQ ID NO:48 that have at least four amino acid residues, and fragments of SEQ ID NO:49 that have at least four amino acid residues, fragments of SEQ ID NO:50 that have at least four amino acid residues, fragments of SEQ ID NO
  • fragments of SEQ ID NO:65 that have at least four amino acid residues
  • fragments of SEQ ID NO:66 that have at least four amino acid residues
  • fragments of SEQ ID NO:67 that have at least four amino acid residues
  • fragments of SEQ ID NO:68 that have at least four amino acid residues
  • fragments of SEQ ID NO:69 that have at least four amino acid residues
  • fragments of SEQ ID NO:70 that have at least four amino acid residues, fragments of SEQ ID - 11 -
  • the peptides may further comprise additional amino acid residues.
  • peptides consist of amino acid sequences selected from the group consisting of: fragments of SEQ ID NO:21 that have at least four amino acid residues, fragments of SEQ ID NO:22 that have at least four amino acid residues, fragments of SEQ ID NO:23 that have at least four amino acid residues, fragments of SEQ ID NO:42 that have at least four amino acid residues, fragments of SEQ ID NO:43 that have at least four amino acid residues, fragments of SEQ ID NO:44 that have at least four amino acid residues, fragments of SEQ ID NO:45 that have at least four amino acid residues, fragments of SEQ ID NO:46 that have at least four amino acid residues, fragments of SEQ ID NO:47 that have at least four amino acid residues, fragments of SEQ ID NO:48 that have at least four amino acid residues, and fragments of SEQ ID NO:49 that have at least four amino acid residues, fragments of SEQ ID NO:50 that have at least four amino acid residues, fragments of SEQ ID NO
  • peptides comprise amino acid sequences selected from the group consisting of: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO: 7, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:
  • peptides consist of amino acid sequences selected from the group consisting of: SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 , SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:32, SEQ ID NO:33,
  • the peptides are conformationally restricted such as those which are cyclicized, circularized or otherwise restricted by peptide and/or non-peptide bonds to limit conformational variation and/or to increase stability and/or half-life of the peptides.
  • peptides are provided as linear peptides.
  • peptides of the present invention comprise one or more D amino acids.
  • D amino acid peptides is meant to refer to peptides according to the present invention which comprise at least one and preferably a plurality of D amino acids.
  • D amino acid peptides consist of 4-25 amino acids.
  • D amino acid peptides retain the biological activity of the peptides of the invention that consist of L amino acids, i.e. D amino acid peptides inhibit TNF ⁇ .
  • the use of D amino acid peptides is desirable as they are less vulnerable to degradation and therefore have a longer half life.
  • derivatives refers to peptides of the invention which have the amino terminal and/or the carboxy terminal blocked, particularly those in which the amino group of the N terminal residue is acetylated and/or the carboxy group of the C terminal residue is amidated.
  • Peptides that comprise an amino acid sequence which consists of 4 to 25 amino acids and which comprise at least a four amino acid residue fragment of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO: 55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO: 60, SEQ ID NO:61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO
  • amino acid sequences selected from the group consisting of: SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO.-31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35
  • the peptides of the invention are selected from the group consisting of: SEQ ID NO:l in which the carboxy terminal arginine is arginine amide; SEQ ID NO:2 in which the carboxy terminal glutamic acid is glutamic acid amide; SEQ ID NO:3 in which the carboxy terminal serine is serine amide; SEQ ID NO:4 in which the carboxy terminal alanine is alanine amide; SEQ ID NO:5 in which the carboxy terminal asparagine is asparagine amide; SEQ ID NO:6 in which the carboxy terminal arginine is arginine amide; SEQ ID NO:7 in which the carboxy terminal arginine is arginine amide; SEQ ID NO: 8 in which the carboxy terminal valine is valine amide; SEQ ID NO: 9 in which the carboxy terminal tryptophan is tryptophan amide; SEQ ID NO:10 in which the carboxy terminal glutamine is glutamine amide; SEQ ID NO:
  • Contemplated equivalents include conservative analogs and mimetics.
  • Conservative analogs inhibit TNF ⁇ in the same manner as the peptides of the invention. By interacting with TNF ⁇ in such a way and thereby inhibiting TNF ⁇ activity, conservative analogs perform essentially the same function by essentially the same means to achieve essentially the same result as the peptides of the invention.
  • the present invention contemplates compounds which display substantially the same surface as the peptides of the invention.
  • the term “mimetics” is meant to refer to compounds that are not peptides but that comprise a similar surface as the peptides of the invention and can thus interact with the TNF receptor in a similar fashion as the peptides of the invention.
  • Mimetics inhibit TNF ⁇ by interacting with TNF ⁇ in the same manner as the peptides of the invention.
  • mimetics perform essentially the same function by essentially the same means to achieve essentially the same result as the peptides of the invention.
  • Peptides of the invention may be prepared using the solid-phase synthetic technique initially described by Merrifield, in J. Am . Chem. Soc , 15:2149-2154 (1963) .
  • Other peptide synthesis techniques may be found, for example, in M. Bodanszky et al . , (1976) Peptide Synthesis, John Wiley & Sons, 2d Ed.; Kent and Clark- Lewis in Synthetic Peptides in Biology and Medicine, p. 295- 358, eds. Alitalo, K. , et al . Science Publishers, (Amsterdam, 1985) ; as well as other reference works known to those skilled in the art.
  • these synthetic methods involve the sequential addition of one or more amino acid residues or suitable protected amino acid residues to a growing peptide chain.
  • amino acid residues or suitable protected amino acid residues Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group.
  • a different, selectively removable protecting group is utilized for amino acids containing a reactive side group, such as lysine.
  • Block synthesis techniques may also be applied to both the solid phase and solution methods of peptide synthesis. Rather than sequential addition of single amino acid residues, preformed blocks comprising two or more amino acid residues in sequence are used as either starting subunits or subsequently added units rather than single amino acid residues.
  • the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group.
  • the protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted with the residue already attached to the solid support.
  • the protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final peptide.
  • the peptide of the invention are preferably devoid of benzylated or methylbenzylated amino acids .
  • Such protecting group moieties may be used in the course of synthesis, but they are removed before the peptides are used. Additional reactions may be necessary, as described elsewhere, to form intramolecular linkages to restrain conformation.
  • one or more of several assays may be performed. Included among these are assays which measure the ability a TNF ⁇ inhibitor candidate, i.e. a test compound, to inhibit TNF ⁇ from binding to a fusion protein that is composed of a TNF receptor or a TNF ⁇ -binding portion thereof, fused to an immunoglobulin molecule or a portion thereof. In other assays, the ability a test compound to inhibit TNF ⁇ from binding to an isolated TNF receptor is measured.
  • Other assays include those which the ability of a TNF ⁇ inhibitor candidate, i.e. a test compound, to inhibit TNF ⁇ activity when TNF ⁇ is contacted with cells that react to the presence of TNF ⁇ . For example, TNF ⁇ is cytotoxic to some cells, such as WEHI cells, and assays can be used to measure the ability a test compound, to inhibit TNF ⁇ cytotoxicity.
  • TNF ⁇ inhibitory activity There are numerous other assays which can be used to determine a test compound's ability to inhibit TNF ⁇ .
  • specific non-lethal effects of TNF ⁇ on some cells is used as an end point to evaluate the TNF ⁇ inhibitory activity of a test compound.
  • Known effects of TNF ⁇ on fibroblast cells include effects on mitogenesis, IL-6 secretion and HLA class II antigen induction. Comparisons can be made between TNF ⁇ 's effect on fibroblasts in the presence or absence of a test compound using these detectable phenotypic changes as endpoints.
  • known effects of TNF ⁇ on monocyte cells include effects on secretion of cytokines such as GMCSF, IL-6 and IL-8.
  • TNF ⁇ Comparisons can be made between TNF ⁇ 's effect on cytokine secretion by monocytes in the presence or absence of a test compound. Additionally, TNF ⁇ is known to have effects on secretion of cytokine by endothelial cells and similar assays may be designed and performed. Further, TNF ⁇ is also known to effect adhesion molecule induction, ICAM-1, E- selectin, VCAM and tissue factor production in endothelial cells. Comparisons can be made between TNF ⁇ 's effect on endothelial cells in the presence or absence of a test compound using these detectable phenotypic changes as endpoints as well . Likewise, TNF ⁇ is known to effect neutrophils in specific ways.
  • Comparisons can be made between TNF ⁇ 's effect on neutrophils in the presence or absence of a test compound using activation, priming, degranulation and superoxide production as detectable endpoints for evaluation of TNF ⁇ inhibitory activity.
  • the TNF ⁇ inhibitors according to the invention are useful for treating a vertebrate having a pathology or condition associated with levels of a substance reactive with a TNF receptor, in particular TNF ⁇ , in excess of the levels present in a normal healthy subject.
  • pathologies include, but are not limited to: sepsis syndrome, including cachexia; circulatory collapse and shock resulting from acute or chronic bacterial infection; acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections; acute and chronic immune and autoimmune pathologies, such as systemic lupus erythematosus and rheumatoid arthritis; alcohol- induced hepatitis; chronic inflammatory pathologies such as sarcoidosis and Crohn' s pathology; vascular inflammatory pathologies such as disseminated intravascular coagulation; graft-versus-host pathology; Rawasaki's pathology; and malignant pathologies involving TNF ⁇ -secreting tumors.
  • Such treatment comprises administering a single or multiple doses of the compounds of the invention.
  • Preferred for human pharmaceutical use are pharmaceutical compositions that comprise the compounds of the present invention in combination with a pharmaceutically acceptable carrier or diluent .
  • compositions of the present invention may be administered by any means that enables the active agent to reach the agent's site of action in the body of a mammal.
  • the primary focus is the ability to reach and bind with TNF ⁇ .
  • parenteral administration i.e., intravenous, subcutaneous, intramuscular, would ordinarily be used to optimize absorption.
  • pharmaceutical compositions which comprise the compounds of the present invention are administered intravenously or subcutaneously.
  • Pharmaceutical compositions of the present invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • the dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • a daily dosage of active ingredient can be about 0.001 to 1 grams per kilogram of body weight, in some embodiments about 0.1 to 100 milligrams per kilogram of body weight.
  • ordinarily dosages are in the range of 0.5 to 50 milligrams per kilogram of body weight, and preferably 1 to 10 milligrams per kilogram per day.
  • the pharmaceutical compositions are given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.
  • Dosage forms (composition) suitable for internal administration generally contain from about 1 milligram to about 500 milligrams of active ingredient per unit.
  • the active ingredient will ordinarily be present in an amount of about 0.5-95 by weight based on the total weight of the composition.
  • the TNF ⁇ inhibitor can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used.
  • the vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives) .
  • the formulation is sterilized by commonly used techniques. Suitable pharmaceutical carriers are described in the most recent edition of Remington ' s Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
  • a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.
  • the present invention also provides the peptide-based inhibitors of TNF ⁇ including fragments, derivatives, and mimetics thereof which are detectably labeled, as described below, for use in diagnostic methods for detecting TNF ⁇ in patients known to be or suspected of having a TNF ⁇ -mediated condition. Identification of peptides useful as diagnostic reagents may be performed routinely.
  • the detectably labelled molecules of the present invention are useful for immunoassays which detect or quantitate TNF ⁇ in a sample.
  • An immunoassay for TNF ⁇ typically comprises incubating a biological sample in the presence of a detectably labeled high affinity molecule of the present invention capable of selectively binding to TNF, and detecting the labeled molecules which is bound in a sample .
  • a detectably labeled high affinity molecule of the present invention capable of selectively binding to TNF
  • detecting the labeled molecules which is bound in a sample e.g., Various clinical immunoassay procedures are described in Immunoassays for the 80 ' s, A. Voller et al. , Eds., University Park, 1981.
  • the molecule or a biological sample may be added to nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled TNF ⁇ -specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on said solid support may then be detected by conventional means.
  • solid phase support or “carrier” is intended any support capable of binding TNF ⁇ proteins or molecules of the present invention.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to TNF ⁇ or an anti-TNF ⁇ antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads.
  • the binding activity of a given lot of anti-TNF ⁇ compound may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • One of the ways in which the TNF ⁇ -specific molecule can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) , or enzyme-linked immunosorbent assay (ELISA) .
  • EIA enzyme immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • Enzymes which can be used to detectably label the TNF ⁇ - specific molecules of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • radioactively labeling the TNF ⁇ - specific molecules By radioactively labeling the TNF ⁇ - specific molecules, it is possible to detect TNF ⁇ through the use of a radioimmunoassay (RIA) (see, for example, Work, T.S., et al. , Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, N.Y. , 1978.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are: 3 H, 125 I, 131 I, 35 S, 14 C, and, preferably, 125 I .
  • TNF ⁇ -specific molecules can also be labeled with a fluorescent compound.
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
  • the TNF ⁇ -specific molecules can also be detectably labeled using fluorescence-emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the TNF ⁇ -specific molecule using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediamine-tetraacetic acid (EDTA) .
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediamine-tetraacetic acid
  • the TNF ⁇ -specific molecules also can be detectably labeled by coupling to a chemiluminescent compound.
  • the presence of the chemiluminescently labeled compound is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the TNF ⁇ -specific molecule, fragment or derivative of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Detection of the TNF ⁇ -specific compound, fragment or derivative may be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorescent material.
  • the detection can be accomplished by colorometric methods which employ a substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • the TNF ⁇ which is detected by the above assays may be present in a biological sample.
  • a biological sample such as, for example, blood, serum, lymph, urine, inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissue extract or homogenate, and the like.
  • the invention is not limited to assays using only these samples, it being possible for one of ordinary skill in the art to determine suitable conditions which allow the use of other samples .
  • si tu detection may be accomplished by removing a histological specimen from a patient, and providing the combination of labeled antibodies of the present invention to such a specimen.
  • the peptide is preferably provided by applying or by overlaying the labeled molecule (or fragment) to a biological sample.
  • the peptide, fragment or derivative of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site” or “sandwich” assay.
  • an immunometric assay also known as a "two-site” or “sandwich” assay.
  • a quantity of unlabeled peptide (or fragment of the peptide) is bound to a solid support that is insoluble in the fluid being tested and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase peptide, TNF ⁇ , and labeled anti-TNF ⁇ antibody.
  • Typical and preferred immunometric assays include "forward" assays in which the peptide of the invention bound to the solid phase is first contacted with the sample being tested to extract the TNF ⁇ from the sample by formation of a binary solid phase peptide-TNF ⁇ complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted TNF ⁇ , if any, and then contacted with the solution containing a known quantity of labeled peptide (which functions as a "reporter molecule”) .
  • This type of forward sandwich assay may be a simple "yes/no" assay to determine whether TNF ⁇ is present or may be made quantitative by comparing the measure of labeled peptide with that obtained for a standard sample containing known quantities of TNF ⁇ .
  • Such "two-site” or “sandwich” assays are described by Wide, Radioimmune Assay Method, Kirkham, Ed., E. & S. Livingstone, Edinburgh, 1970, pp. 199-206) .
  • a simultaneous assay involves a single incubation step wherein the peptide bound to the solid support and labeled peptide are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled peptide. The presence of labeled peptide associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
  • stepwise addition first of a solution of labeled peptide to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period, is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled peptide.
  • the determination of labeled peptide associated with a solid support is then determined as in the "simultaneous" and "forward” assays.
  • a combination of peptide of the present invention specific for separate epitopes may be used to construct a sensitive three-site immunoradiometric assay.
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using version 1.12 of the standard Boc software. 4-Methyl benzhydrylamine resin (0.58 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 1.64 g.
  • the peptide was cleaved from the resin (1.6 g) using 16 mL of HF and 1.6 mL of anisole for 60 min at 0°C.
  • the resin was washed with ether and the peptide extracted with trifluoroacetic acid/methylene chloride (1:1, v/v) (3 x 15 mL) to give 720 mg of crude peptide.
  • the crude peptide (720 mg) was dissolved in 150 mL of 50% acetic acid and purified on a Vydac C-18 column (15 ⁇ , 10 x 30 cm) eluting with a 10-45% gradient of 80% ethanol in 0.1% trifluoroacetic acid over 120 min at a flow rate of 60 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 72 mg of white solid.
  • the peptide was cleaved from the resin (1.89 g) using 19 mL of HF and 1.9 mL of anisole for 60 min at 0°C.
  • the resin was washed with ether and the peptide extracted with trifluoroacetic acid/methylene chloride (1:1, v/v) (3 x 15 mL) to give 960 mg of crude peptide.
  • the crude peptide (960 mg) was dissolved in 80 mL at 30% acetic acid and purified on a Vydac C-18 column (15x, 10 x 30 cm) eluting with a 10-30% gradient of 80% ethanol in 0.1% trifluoroacetic acid over 120 min at a flow rate of 60 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 340 mg of white solid.
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using version 1.12 of the standard Boc software.
  • 4-methyl benzhydrylamine resin (0.58 g, 0.5 mmol) was used in the synthesis.
  • the final weight of the resin was 1.91 g.
  • the peptide was cleaved from the resin (1.91 g) using 19 mL of HF and 1.9 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with 50% trifluoroacetic acid in methylene chloride to give 660 mg of crude peptide.
  • the crude peptide (660 mg) was purified on a Vydac C-18 column (15 ⁇ , 5 x 25 cm) eluting with a 10-45% gradient of 80% acetonitrile in 0.1% trifluoroacetic acid over 120 min at a flow rate of 15 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 151 mg.
  • Example 4 Glutamyl-alanyl-glutamyl-seryl-glycyl-seryl- phenylalanyl-threonyl-alanyl-seryl-glutamyl- asparaginyl-histidyl-leucyl-arginine amide
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using version 1.12 of the standard Boc software. 4-Methyl benzhydrylamine resin (0.58 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 2.12 g.
  • the peptide was cleaved from the resin (2.1 g) using 21 mL of HF and 2.1 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with 50% trifluoroacetic acid in methylene chloride to give 783 mg of crude peptide.
  • the peptide was prepared on an ABI Model 431A Peptide
  • the peptide was cleaved from the resin (2.1 g) using 21 mL of HF and 2.1 mL anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with 50% trifluoroacetic acid/methylene chloride to give 1.19 g of crude peptide.
  • the crude peptide (1.1 g) was purified on a Vydac C-18 column (15 ⁇ , 10 x 30 cm) eluting with a 10-40% gradient of 80% ethanol in 0.1% trifluoroacetic acid over 120 min at a flow rate of 60 mL per min.
  • Semipure fractions were pooled and rechromatographed (same column) with a 15-40% gradient of 80% ethanol in 0.1% trifluoroacetic acid. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 172 mg.
  • the peptide was cleaved from the resin (1.5 g) using 15 mL of HF and 1.5 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with trifluoroacetic acid/methylene chloride (1:1, v/v) (3 x 15 mL) to give 780 mg of crude peptide.
  • the crude peptide (780 mg) was dissolved in 110 mL of 80% acetic acid and purified on a Vydac C-18 column (15 ⁇ , 10 x 30 cm) eluting with a 20-80% gradient of 80% ethanol in 0.1% trifluoroacetic acid over 120 min at a flow rate of 60 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 235.3 mg of white solid.
  • the peptide was cleaved from the resin (1.385 g) using
  • the peptide was prepared on an ABI Model 431A Peptide
  • the peptide was cleaved from the resin (1.333 g) using
  • the crude peptide (500 mg) was purified on a Vydac C-18 column (15 ⁇ , 10 x 30 cm) eluting with a 20-60% gradient of 80% acetonitrile in 0.1% trifluoroacetic acid over 120 min at a flow rate of 60 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 34 mg.
  • Amino acid analysis Ala 3.08 (3) , Asx 2.03 (2) , Gly 0.99 (1) , His 0.97 (1) , Leu 3.38 (3) , Phe 0.88 (1) , Ser 1.79 (2) , Thr 0.74 (1) , Val 0.67 (1) .
  • the crude peptide (610 mg) was purified on a Vydac C-18 column (15 ⁇ , 10 x 30 cm) eluting with a 25-70% gradient of 80% ethanol in 0.1% trifluoroacetic acid over 120 min at a flow rate of 60 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 127 mg of peptide that is a mixture of N-terminal glutamine and pyroglutamine.
  • the peptide was cleaved from the resin (1.67 g) using 17 mL of HF and 1.7 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with 30% acetic acid to give 743 mg of crude peptide.
  • the crude peptide (390 mg) was purified on a Vydac C-18 column (15 ⁇ , 5 x 25 cm) eluting with a 23-35% gradient of 80% acetonitrile in 0.1% trifluoroacetic acid over 120 min at a flow rate of 15 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 90 mg of pure peptide.
  • the peptide was cleaved from the resin (2.7 g) using 27 mL of HF and 2.7 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with 50%
  • the crude Acm-Cys protected peptide (652 mg) was purified on a Vydac C-18 column (15 ⁇ , 5 x 25 cm) eluting with a 10-50% gradient of 80% acetonitrile in 0.1% trifluoroacetic acid over
  • the crude linear peptide was purified on a Vydac C-18 (lO ⁇ , 2.5 x 25 cm) column using a 10-50% gradient of 80% acetonitrile in 0.1% trifluoroacetic acid over 60 min at 10 mL/min. Fractions were analyzed by HPLC and pure fractions were pooled to give 14 mg.
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using version 1.12 of the standard Boc software.
  • 4-methyl benzhydrylamine resin (588 mg, 0.5 mmol) was used in the synthesis. The final weight of the resin was 1.66 mg.
  • the peptide was cleaved from the resin (1.66 g) using 17 mL of HF and 1.7 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with 20% acetic acid to give 815 mg of crude peptide.
  • the crude peptide (305 mg) was purified on a Vydac C-18 column (15 ⁇ , 5 x 25 cm) eluting with a 37-60% gradient of 80% ethanol in 0.1% trifluoroacetic acid over 120 min at a flow rate of 15 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 133 mg of pure peptide.
  • Amino acid analysis Ala 2.12 (2) , Asx 1.01 (1) , Glx 2.94 (3) , He 0.96 (1) , Leu 2.93 (3) , Lys 1.01 (1) , Pro 1.00 (1), Ser 0.71 (1), Thr 0.86 (1), Val 1.02 (1) .
  • the peptide was cleaved from the resin (1.87 g) using 19 mL of HF and 1.9 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with 30% acetic acid to give 943 mg of crude peptide.
  • the crude peptide (460 mg) was purified on a Vydac C-18 column (15 ⁇ , 5 x 25 cm) eluting with a 32-45% gradient of 80% ethanol in 0.1% trifluoroacetic acid over 120 min at a flow rate of 15 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 260 mg of pure peptide.
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using version 1.12 of the standard Boc software. 4-Methyl benzhydrylamine resin (588 mg, 0.5 mmol) was used in the synthesis. The final weight of the resin was 1.88 g.
  • the peptide was cleaved from the resin (1.88 g) using 19 mL of HF and 1.9 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with 30% acetic acid to give 790 mg of crude peptide.
  • the crude peptide (502 mg) was purified on a Vydac C-18 column (15 ⁇ , 5 x 25 cm) eluting with a 12-23% gradient of 80% ethanol in 0.1% trifluoroacetic acid over 120 min at a flow rate of 15 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 227 mg of pure peptide.
  • This peptide is a derivative of SEQ ID NO:63 having the same amino acid sequence as SEQ ID NO:63 except that in this derivative, the N terminal leucine residue is blocked, forming acetyl-leucine and the C terminal isoleucine is blocked, forming isoleucine amide.
  • the peptide was prepared on an ABI
  • the peptide was cleaved from the resin (1.5 g) using 15 mL of HF, 2-mercaptopyridine (1.0 g) and 1.5 mL of anisole for 60 min at 0°C.
  • the resin was washed with ether and the peptide extracted with trifluoroacetic acid/methylene chloride (1:1, v/v) (3 x 15 mL) to give 610 mg of crude peptide.
  • the crude peptide (600 mg) was purified on a Vydac C-18 column (15 ⁇ , 10 x 30 cm) eluting with a a) 0-25%; b) 25-65%% gradient of 80% acetonitrile in 0.1% trifluoroacetic acid over a) 15 min; b) 45 min at a flow rate of 120 mL per min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 195 mg of white solid.
  • the peptide was cleaved from the resin (1.5 g) using 15 mL of HF, dithiothreitol (1.0 g) and anisole (1.5 mL) for 60 min at 0°C.
  • the resin was washed with ether and the peptide extracted with trifluoroacetic acid/methylene chloride (1:1, v/v) (3 x 15 mL) to give 1.03 g of crude peptide.
  • the crude peptide was dissolved in dimethylformamide (100 mL) , diluted with water (20 mL) , stirred with 2 mL of piperidine over 60 min at room temperature then evaporated to dryness (1.7 g, yellow oil) .
  • Example 17 Acetyl-tyrosyl-alanyl-glutamyl-seryl-valyl-lysyl- glycyl-arginyl-phenylalanyl-threonyl-isoleucyl- seryl-arginyl-aspartyl-aspartyl-seryl-lysyl-seryl- alanyl-valyl-tyrosyl-leucine-amide
  • This peptide is a derivative of SEQ ID NO:65 having the same amino acid sequence as SEQ ID NO:65 except that in this derivative, the N terminal tyrosine residue is blocked, forming acetyl-tyrosine and the C terminal leucine is blocked, forming leucine amide.
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using version 1.12 of the standard Boc software. 4-Methyl benzhydrylamine resin (0.46 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 1.730 g.
  • the peptide was cleaved from the resin (1.730 g) using
  • the crude peptide (795 mg) was purified on a Vydac C-18 column (15 ⁇ , 5 x 25 cm) eluting with a 20-60% gradient of 80% ethanol in 0.1% trifluoroacetic acid over 120 min at a flow rate of 15 mL per min. Fractions were collected, analyzed by
  • Example 19 Arginyl-glutaminyl-seryl-prolyl-glutamyl-lysyl- glycyl-leucyl-glutamyl-tryptophyl-valyl-alanyl- glutamyl-isoleucyl-arginyl-seryl-lysyl-seryl- isoleucine-amide.
  • This peptide is a derivative of SEQ ID NO: 66 having the same amino acid sequence as SEQ ID NO:66 except that in this derivative, the C terminal isoleucine is blocked, forming isoleucine amide.
  • the peptide was prepared on an ABI Model 431A peptide synthesizer using version 1.12 of the standard Boc software. The synthesis was done on 4 methylbenzyhydrylamine resin (Bachem 0.637 g, 0.51 mmol) . The final weight of the dried resin was 2.217 g.
  • the peptide was cleaved from the resin (2.059 g) using anisole (2.0 mL) and HF (18 mL) at 0° C for 60 min. The peptide and resin were precipitated with ether and dried. The peptide was extracted with a mixture of trifluoroacetic acid and dichloromethane (1/1, 100 mL) . The solvents were removed in vacuo to give an oil, and the oil triturated with ether to give the crude peptide as a gummy off-white solid (0.56 g) . The solid was dissolved in 30 mL of 2,2,2-trifluoroethanol and treated with 75 mL of 2% piperidine in water for 1 hr at room temperature.
  • the peptide was purified on the Vydac C-18 column (15 ⁇ m,
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using version 1.12 of the standard Boc software. 4-Methyl benzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 2.473 g.
  • the peptide was cleaved from the resin (2.267 g) using
  • the crude peptide (1.07 g) was purified on a Vydac C-18 column (15 ⁇ , 2.2 x 25 cm) eluting with a 15-80% gradient of 80% ethanol in 0.1% trifluoroacetic acid over 415 min at a flow rate of 5 mL per min. Fractions were collected, analyzed by
  • Example 21 Acetyl-aspartyl-isoleucyl-leucyl-leucyl-threonyl- glutaminyl-seryl-prolyl-alanyl-isoleucyl-leucyl- seryl-valyl-seryl-prolyl-glycyl-glutamyl-arginyl- valyl-seryl-phenylalanyl-serine-amide
  • This peptide is a derivative of SEQ ID NO:68 having the same amino acid sequence as SEQ ID NO:68 except that in this derivative, the N terminal aspartic acid residue is blocked, forming acetyl-aspartic acid and the C terminal serine is blocked, forming serine amide.
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using version 1.12 of the standard Boc software. 4-Methyl benzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 2.0 g.
  • the peptide was cleaved from the resin (2 g) using 20 mL of HF and 2 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with trifluoroacetic acid/methylene chloride (1:1, v/v) (3 x 15 mL) to give 1.06 g of crude peptide.
  • the crude peptide (0.6 g) was purified on a Vydac C-18 column (15 ⁇ , 10 x 30 cm) eluting with a 0-30% over 10 min and
  • Example 22 Acetyl-tyrosyl-tyrosyl-seryl-glutaminyl-glutaminyl- seryl -histidyl -seryl -tryptophyl- prolyl - phenylalanyl-threonyl-phenylalanine-amide
  • This peptide is a derivative of SEQ ID NO:69 having the same amino acid sequence as SEQ ID NO:69 except that in this derivative, the N terminal tyrosine residue is blocked, forming acetyl-tyrosine and the C terminal phenylalanine is blocked, forming phenylalanine amide.
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using version 1.12 of the standard Boc software. 4-Methyl benzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 1.87 g.
  • the peptide was cleaved from the resin (1.8 g) using 18 mL of HF and 1.8 mL of anisole for 60 min at 0°C.
  • the resin was washed with ether and the peptide extracted with trifluoroacetic acid/methylene chloride (1:1, v/v) (3 x 15 mL) to give 592 mg of crude peptide.
  • the crude peptide (590 mg) was purified on a Vydac C-18 column (15 ⁇ , 10 x 30 cm) eluting with a a) 0-25%; b) 25-65% gradient of 80% acetonitrile in 0.1% trifluoroacetic acid over a) 15 min; b) 45 min at a flow rate of 120 mL per min.
  • Example 23 Ac e tyl - i s o l eucyl - a sparaginyl - threonyl - valyl - glutamyl - seryl -glutamyl - aspar tyl - isoleucyl - alanyl - aspar tyl - tyrosyl - tyrosyl - cys teinyl - glutaminyl - glutaminyl - seryl -his tidyl - seryl - tryptophan- amide
  • This peptide is a derivative of SEQ ID NO:70 having the same amino acid sequence as SEQ ID NO:70 except that in this derivative, the N terminal isoleucine residue is blocked, forming acetyl-isoleucine and the C terminal tryptophan is blocked, forming tryptophan amide.
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using version 1.12 of the standard Boc software. 4-Methyl benzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 2.2 g.
  • the peptide was cleaved from the resin (2.2 g) using 22 mL of HF and 2.2 mL of anisole for 60 min at 0°C. The resin was washed with ether and the peptide extracted with trifluoroacetic acid/methylene chloride (1:1, v/v) (3 x 15 mL) to give 1.1 g of crude peptide.
  • This peptide is a derivative of SEQ ID NO:71 having the same amino acid sequence as SEQ ID NO:71 except that in this derivative, the N terminal phenylalanine residue is blocked, forming acetyl-phenylalanine and the C terminal tryptophan is blocked, forming tryptophan amide.
  • the peptide was prepared on an ABI Model 431A Peptide Synthesizer using version 1.12 of the standard BOC software. 4-Methyl benzhydrylamine resin (0.58 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 1.3 g.
  • the peptide was cleaved from the resin (1.3 g) using 13 mL of HF, 1.3 g of 2-mercaptopyridine and 1.3 mL of anisole for 60 min at 0°C.
  • the resin was washed with ether and the peptide extracted with 50% trifluoroacetic acid in methylene chloride to give 1.3 g of crude peptide and salts..
  • the crude peptide (1.3 g) was first purified on a Vydac C-18 column (15 ⁇ , 5 x 25 cm) eluting with a 25-70% gradient of 80% ethanol in 0.1% trifluoroacetic acid over 120 min at a flow rate of 15 mL per min.
  • the semipure peptide (30 mg) was purified on a Vydac C-18 column (lO ⁇ , 2.2 x 25 cm) using a 20- 60% gradient of 80% acetonitrile in 0.1% trifluoroacetic acid over 60 min at 10 mL/min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 18 mg.
  • an assay In order to screen compounds for their ability to block TNF ⁇ binding to the TNF p55 receptor, an assay has been designed using TNF ⁇ and a p55/IgG fusion protein in place of monovalent, non-fusion p55 TNF receptor protein. This assay was designed to identify peptides which bind to human TNF ⁇ and thereby prevent the capture of the TNF ⁇ by a microtiter plate coated with p55-Ig fusion protein. A constant concentration of human TNF ⁇ is preincubated with the test peptide and then incubated on the p55-Ig coated microtiter wells. Bound TNF ⁇ is detected using a specific antisera and an alkaline phosphatase- conjugated probe. An active peptide will reduce the amount of human TNF ⁇ bound to the well relative to control wells in which TNF ⁇ but no peptide was added.
  • a 96-well, U-bottom polyvinylchloride microtiter plate was coated with 50 ⁇ l/well of p55-Ig fusion protein at 5 ⁇ g/ml in 0.01 M sodium phosphate, 0.15 M sodium chloride (PBS) by incubation overnight at 4°C or 2 hours at 37°C.
  • the fusion protein which consists of a p55 TNF receptor protein portion and an IgG portion, can be produced as disclosed in U.S. Application Serial Number 08/010,406 filed January 29, 1993 which is incorporated herein by reference.
  • the plate was washed three times with 0.05% Tween-20 in PBS, then blocked, by adding 150 ⁇ l/well of assay buffer (10 mM N-2- hydroxyethylpiperazine-N' -3-propanesulfonic acid (HEPES) pH 7.2, containing 0.1% porcine gelatin, 0.1% Tween-80, and 0.01% sodium azide) and continuing incubation for 1 hour at 37°C or at 4°C for 1-7 days.
  • assay buffer (10 mM N-2- hydroxyethylpiperazine-N' -3-propanesulfonic acid (HEPES) pH 7.2, containing 0.1% porcine gelatin, 0.1% Tween-80, and 0.01% sodium azide
  • Lyophilized peptides to be tested were weighed in tared 12 x 75 mm polystyrene tubes and reconstituted to a concentration of 1.2 mM with assay buffer. Each suspension was sonicated in a water bath for 1-5 minutes and vortexed 15-30 seconds to disperse large particles. Serial dilutions of each peptide suspension were prepared using assay buffer in a 96- well polystyrene microtiter plate. Additional wells received Fab fragment of the mouse/human monoclonal anti-human TNF ⁇ antibody cA2 (positive control) or assay buffer (negative control) .
  • TNF ⁇ Human recombinant TNF ⁇ (Biosource, Camarillo, CA) was added to all wells to give a final TNF ⁇ concentration of 25 ng/ml, final peptide concentrations of 1.0, 0.33 and 0.11 mM and a final cA2 Fab concentration of 500 ng/ml.
  • the polystyrene dilution plate was then sealed and incubated 1 hour at room temperature on an orbital mixer set at moderate speed.
  • TNF ⁇ (Genzyme, Boston, MA; 1:500 in assay buffer, 50 ⁇ l/well)
  • biotinylated goat anti-rabbit Ig H&L
  • streptavidin-alkaline phosphatase conjugate Piereptavidin-alkaline phosphatase conjugate
  • % inhibition 100 - ( (mean OD peptide/mean OD negative control) x 100) .
  • Example 26 The inhibition of binding of TNF ⁇ to p55TNFr-IgG chimeric construct was performed as described above using several embodiments of the invention. The peptides were tested at a concentration of ImM. The following data were generated.
  • ImM of SEQ ID NO:l in which the carboxy terminal arginine is arginine amide resulted in 46% inhibition.
  • ImM of SEQ ID NO:2 in which the carboxy terminal glutamic acid is glutamic acid amide resulted in 62% inhibition.
  • ImM of SEQ ID NO:3 in which the carboxy terminal serine is serine amide resulted in 53% inhibition.
  • ImM of SEQ ID NO:4 in which the carboxy terminal alanine is alanine amide resulted in 53% inhibition.
  • ImM of SEQ ID NO:5 in which the carboxy terminal asparagine is asparagine amide resulted in 58% inhibition.
  • ImM of SEQ ID NO:6 in which the carboxy terminal arginine is arginine amide resulted in 53% inhibition.
  • ImM of SEQ ID NO:7 in which the carboxy terminal arginine is arginine amide resulted in 42% inhibition.
  • ImM of SEQ ID NO: 8 in which the carboxy terminal valine is valine amide resulted in 56% inhibition.
  • ImM of SEQ ID NO:10 in which the carboxy terminal glutamine is glutamine amide resulted in 49% inhibition.
  • ImM of SEQ ID NO:11 in which the amino terminal glutamine is acetyl glutamine and the carboxy terminal asparagine is asparagine amide resulted in 27% inhibition.
  • ImM of SEQ ID NO:12 in which the amino terminal leucine is acetyl leucine and the carboxy terminal valine is valine amide resulted in 40% inhibition.
  • ImM of SEQ ID NO:13 in which the amino terminal alanine is acetyl alanine and the carboxy terminal serine is serine amide resulted in 34% inhibition.
  • ImM of SEQ ID NO:14 in which the carboxy terminal serine is serine amide resulted in 29% inhibition.
  • ImM of SEQ ID NO:15 in which the carboxy terminal glutamic acid is glutamic acid amide resulted in 48% inhibition.
  • ImM of SEQ ID NO:16 in which the carboxy terminal arginine is arginine amide resulted in 48% inhibition.
  • ImM of SEQ ID NO:17 in which the carboxy terminal glutamic acid is glutamic acid amide resulted in 59% inhibition.
  • ImM of SEQ ID NO:18 in which the carboxy terminal serine is serine amide resulted in 68% inhibition.
  • ImM of SEQ ID NO:19 in which the carboxy terminal asparagine is asparagine amide resulted in 53% inhibition.
  • ImM of SEQ ID NO:20 resulted in 26% inhibition.
  • ImM of SEQ ID NO:21 in which the carboxy terminal valine is valine amide resulted in 50% inhibition.
  • ImM of SEQ ID NO:22 in which the carboxy terminal serine is serine amide resulted in 34% inhibition.
  • ImM of SEQ ID NO:23 in which the carboxy terminal threonine is threonine amide resulted in 52% inhibition.
  • ImM of SEQ ID NO:24 in which the carboxy terminal asparagine is asparagine amide resulted in 21% inhibition.
  • ImM of SEQ ID NO:25 in which the carboxy terminal arginine is arginine amide resulted in 69% inhibition.
  • ImM of SEQ ID NO:26 in which the carboxy terminal tyrosine is tyrosine amide resulted in 71% inhibition.
  • ImM of SEQ ID NO:27 in which the carboxy terminal threonine is threonine amide resulted in 78% inhibition.
  • ImM of SEQ ID NO:28 in which the carboxy terminal methionine is methionine amide resulted in 72% inhibition.
  • ImM of SEQ ID NO:29 in which the carboxy terminal serine is serine amide resulted in 53% inhibition.
  • ImM of SEQ ID NO:30 in which the carboxy terminal serine is serine amide resulted in 59% inhibition.
  • ImM of SEQ ID NO:31 in which the carboxy terminal aspartic acid is aspartic acid amide resulted in 68% inhibition.
  • ImM of SEQ ID NO:32 in which the carboxy terminal glutamic acid is glutamic acid amide resulted in 64% inhibition.
  • ImM of SEQ ID NO:33 in which the carboxy terminal threonine is threonine amide resulted in 67% inhibition.
  • ImM of SEQ ID NO:34 in which the carboxy terminal glutamine is glutamine amide resulted in 65% inhibition.
  • ImM of SEQ ID NO:36 in which the carboxy terminal proline is proline amide resulted in 77% inhibition.
  • ImM of SEQ ID NO:37 in which the carboxy terminal leucine is leucine amide resulted in 77% inhibition.
  • ImM of SEQ ID NO:38 in which the carboxy terminal glycine is glycine amide resulted in 64% inhibition.
  • ImM of SEQ ID NO:39 in which the carboxy terminal alanine is alanine amide resulted in 42% inhibition.
  • ImM of SEQ ID NO:40 in which the carboxy terminal aspartic acid is aspartic acid amide resulted in 72% inhibition.
  • ImM of SEQ ID NO:41 in which the carboxy terminal glutamic acid is glutamic acid amide resulted in 68% inhibition.
  • ImM of SEQ ID NO:42 in which the carboxy terminal asparagine is asparagine amide resulted in 40% inhibition.
  • ImM of SEQ ID NO:43 in which the carboxy terminal leucine is leucine amide resulted in 37% inhibition.
  • ImM of SEQ ID NO:44 in which the carboxy terminal isoleucine is isoleucine amide resulted in 28% inhibition.
  • ImM of SEQ ID NO:45 in which the carboxy terminal proline is proline amide resulted in 73% inhibition.
  • ImM of SEQ ID NO:46 in which the carboxy terminal isoleucine is isoleucine amide resulted in 66% inhibition.
  • ImM of SEQ ID NO:47 in which the carboxy terminal valine is valine amide resulted in 29% inhibition.
  • ImM of SEQ ID NO:48 in which the carboxy terminal isoleucine is isoleucine amide resulted in 27% inhibition.
  • ImM of SEQ ID NO:49 in which the carboxy terminal asparagine is asparagine amide resulted in 26% inhibition.
  • ImM of SEQ ID NO:50 in which the carboxy terminal serine is serine amide resulted in 29% inhibition.
  • ImM of SEQ ID NO:51 in which the carboxy terminal proline is proline amide resulted in 68% inhibition.
  • ImM of SEQ ID NO:52 in which the carboxy terminal serine is serine amide resulted in 63% inhibition.
  • ImM of SEQ ID NO:53 in which the carboxy terminal proline is proline amide resulted in 63% inhibition.
  • ImM of SEQ ID NO:54 in which the carboxy terminal valine is valine amide resulted in 52% inhibition.
  • ImM of SEQ ID NO:55 in which the carboxy terminal histidine is histidine amide resulted in 60% inhibition.
  • ImM of SEQ ID NO:56 in which the carboxy terminal proline is proline amide resulted in 67% inhibition.
  • ImM of SEQ ID NO:57 in which the carboxy terminal threonine is threonine amide resulted in 72% inhibition.
  • ImM of SEQ ID NO:58 in which the carboxy terminal serine is serine amide resulted in 72% inhibition.
  • ImM of SEQ ID NO:59 in which the carboxy terminal phenylalanine is phenylalanine amide resulted in 73% inhibition.
  • ImM of SEQ ID NO:60 in which the carboxy terminal proline is proline amide resulted in 73% inhibition.
  • ImM of SEQ ID NO:61 in which the carboxy terminal proline is proline amide resulted in 72% inhibition.
  • the IC 50 of SEQ ID NO:63 in which the amino terminal leucine is acetyl leucine and the carboxy terminal isoleucine is isoleucine amide was 1.88mM.
  • the IC 50 of SEQ ID NO:64 in which the amino terminal phenylalanine is acetyl phenylalanine and the carboxy terminal arginine is arginine amide was 0.41mM.
  • the IC 50 of SEQ ID NO:65 in which the amino terminal tyrosine is acetyl tyrosine and the carboxy terminal arginine is arginine amide was 0.29mM.
  • the IC 50 of SEQ ID NO:66 in which the carboxy terminal isoleucine is isoleucine amide was 1.02mM.
  • the IC 50 of SEQ ID NO:67 in which the amino terminal threonine is acetyl threonine and the carboxy terminal tyrosine is tyrosine amide was 1.13mM.
  • the IC 50 of SEQ ID NO:68 in which the amino terminal aspartic acid is acetyl aspartic acid and the carboxy terminal serine is serine amide was 2.53mM.
  • the IC 50 of SEQ ID NO:69 in which the amino terminal tyrosine is acetyl tyrosine and the carboxy terminal phenylalanine is phenylalanine amide was 1.08mM.
  • the IC 50 of SEQ ID NO:70 in which the amino terminal isoleucine is acetyl isoleucine and the carboxy terminal tryptophan is tryptophan amide was l.llmM.
  • the IC 50 of SEQ ID NO:71 in which the amino terminal phenylalanine is acetyl phenylalanine and the carboxy terminal tryptophan is tryptophan amide was 0.06mM.
  • the IC 50 of SEQ ID NO:72 in which the carboxy terminal phenylalanine is phenylalanine amide was 0.37mM.
  • the IC S0 of SEQ ID NO:73 in which the carboxy terminal tyrosine is tyrosine amide was 0.85mM.
  • the IC 50 of SEQ ID NO:74 in which the carboxy terminal alanine is alanine amide was 0.60mM.
  • the IC 50 of SEQ ID NO:75 in which the carboxy terminal glutamine is glutamine amide was 0.3ImM.
  • the IC 50 of SEQ ID NO:76 in which the carboxy terminal glutamine is glutamine amide was 0.48mM.
  • C-terminal blocked SEQ ID NO:45 (SEQ ID NO:45 in which the carboxy terminal proline is proline amide) ;
  • C-terminal blocked SEQ ID NO:24 (SEQ ID NO:24 in which the carboxy terminal asparagine is asparagine amide) ;
  • N- and C-terminal blocked SEQ ID NO:11 (SEQ ID NO:11 in which the amino terminal glutamine is acetyl glutamine and the carboxy terminal asparagine is asparagine amide) .
  • N- and C-terminal blocked SEQ ID NO:71 (SEQ ID NO:71 in which the amino terminal phenylalanine is acetyl phenylalanine and the carboxy terminal tryptophan is tryptophan amide)
  • N- and C-terminal blocked SEQ ID NO:65 (SEQ ID NO:65 in which the amino terminal tyrosine is acetyl tyrosine and the carboxy terminal arginine is arginine amide) ;
  • N- and C-terminal blocked SEQ ID NO: 68 (SEQ ID NO:68 in which the amino terminal aspartic acid is acetyl aspartic acid and the carboxy terminal serine is serine amide) ;
  • N- and C-terminal blocked SEQ ID NO:63 SEQ ID NO:63 in which the amino terminal leucine is acetyl leucine and the carboxy terminal isoleucine is isoleucine amide.
  • the data shown in Figure 3 demonstrates the dose dependent inhibition of TNF ⁇ of some embodiments of the invention.
  • the peptides used were:
  • C-terminal blocked SEQ ID NO:73 (SEQ ID NO:73 in which the carboxy terminal tyrosine is tyrosine amide) ;
  • C-terminal blocked SEQ ID NO:76 (SEQ ID NO:76 in which the carboxy terminal glutamine is glutamine amide) ;
  • Example 29 TNF ⁇ cytotoxicity assays
  • the ability of the compounds of the invention to bind to the TNF receptor and inhibit activity by human TNF ⁇ is tested in a TNF ⁇ -mediated cell killing assay.
  • WEHI-164 murine fibrosarcoma cells (Espevik et al. , J " . Immunol . Methods 1986, 95, 99-105) , or another cell line sensitive to the cytotoxic effects of TNF, are used in the following cytotoxicity assay to identify peptides with TNF ⁇ antagonist activity.
  • the cells are grown in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal bovine serum, glutamine, nonessential amino acids and sodium pyruvate (DMEM/FBS) .
  • the WEHI cells are harvested using a cell scraper and suspend in DMEM/FBS at 1 X 10 6 cells/mL. The cells are then seeded at 50 ⁇ L (-5 X 10 4 cells) per well in a 96-well microtiter place. The plate is then incubated for 3-4 hr at 37°C in 5% C0 2 or until 50% confluent.
  • the peptides to be tested are solubilized at approximately 2.5 mM in 10 mM HEPES pH 7.5 by vortexing and brief sonication. Each peptide is then 0.2 ⁇ filtered and the peptide concentration estimated by measuring the absorbance at 214 nanometers.
  • Two serial threefold dilutions of each peptide are prepared in lOmM HEPES pH 7.5.
  • Four serial twofold dilutions of an anti-TNF ⁇ FAB known to inhibit TNF ⁇ activity such as for example cA2 Fab are also prepared in lOmM HEPES pH 7.5 to serve as a positive inhibition control.
  • One-fourth volume of DMEM/FBS containing 10 ⁇ g/mL actinomycin D and 500 pg/mL of recombinant human TNF ⁇ is then mixed with each dilution of peptide and cA2 Fab, as well as with 10 mM HEPES pH 7.5 (TNF control) , and preincubated for 30 minutes at room temperature.
  • a cell control is also prepared in which one- fourth volume of DMEM/FBS containing 10 ⁇ g/mL of actinomycin D (but no TNF) is added to lOmM HEPES pH 7.5.
  • the peptides and controls are transferred (50 ⁇ L/well) in triplicate to the microtiter wells seeded with WEHI cells and incubated overnight at 37°C in 5% C0 2 .
  • Viable cells are detected using a 5 mg/mL solution of 3 (4, 5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) in 0.01 M sodium phosphate, 0.15 M sodium chloride pH 7.2. After 0.2 ⁇ filtration, 25 ⁇ L of the MTT solution is added to each well and incubation continued for 2 hours at 37°C in 5% C0 2 .
  • MTT 2,5-diphenyl tetrazolium bromide
  • the cells and the blue formazan precipitate are solubilized by adding 100 ⁇ L/well of 20% (w/v) sodium dodecyl sulfate dissolved in 50% (v/v) dimethylformamide in water.
  • the absorbance at 570 nm is a direct measure of the number of cells that survived in each well. Replicates are averaged and the fraction of cells that survive is calculated based on the absorbance obtained in the cell control wells .
  • the percent inhibition of TNF ⁇ activity is then calculated as:
  • TNF ⁇ is of major importance in the pathogenesis of rheumatoid arthritis.
  • TNF ⁇ is present in rheumatoid arthritis joint tissues and synovial fluid at the protein and mRNA level (Buchan G, Barrett K, Turner M, Chantry D, Naini RN, and Feldmann N. , Interleukin-1 and tumor necrosis factor mRNA expression in rheumatoid arthritis: prolonged production of IL- l ⁇ , Clin . Exp . Immunol . 1988, 73, 449-455) , indicating local synthesis.
  • TNF ⁇ HIV-infected cell lines. See Poli et al . , Proc . Natl . Acad. Sci . USA 1990 81, 782-785 and Butera et al . , J. Immunology 1993 150, 625-634. Butera et al . demonstrated a reduction of induced TNF ⁇ production and HIV expression in an infected cell line after treatment with soluble TNF receptors.
  • the molecules of the present invention may be used to decrease the expression of TNF ⁇ and thereby lessen the induction of HIV expression.
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO:30:
  • Val Ala lie Pro Gly Asn Ala Ser Arg Asp Ala Val Ala Thr Ser 1 5 10 15

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Abstract

L'invention porte sur des peptides constitués de 4 à 25 acides aminés inhibant l'activité du facteur alpha de nécrose tumorale et sur des méthodes d'inhibition de cette activité consistant à mettre en contact le facteur alpha de nécrose tumorale avec un peptide comprenant une séquence d'acides aminés constituée de 4 à 25 acides aminés et inhibant l'activité du facteur alpha de nécrose tumorale. L'invention porte également sur des méthodes de traitement d'animaux dont on pense qu'ils souffrent de maladies ou d'affections dues audit facteur et consistant à leur administrer une dose thérapeutiquement efficace d'un peptide comprenant une séquence d'acides aminés constituée de 4 à 25 acides aminés inhibant l'activité dudit facteur.
PCT/US1995/004018 1994-04-01 1995-03-31 Inhibiteurs du facteur de necrose tumorale Ceased WO1995026744A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US08/221,583 1994-04-01
US08/221,580 US5519000A (en) 1994-04-01 1994-04-01 Tumor necrosis factor inhibitors
US08/221,583 US5486595A (en) 1994-04-01 1994-04-01 Tumor necrosis factor inhibitors
US08/221,581 US5506340A (en) 1994-04-01 1994-04-01 Tumor necrosis factor inhibitors
US08/221,581 1994-04-01
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7592304B2 (en) 1999-10-01 2009-09-22 Dmi Life Sciences, Inc. Metal-binding compounds and uses therefor
US7632803B2 (en) 1999-10-01 2009-12-15 Dmi Life Sciences, Inc. Metal-binding compounds and uses therefor
WO2010058419A1 (fr) * 2008-11-20 2010-05-27 Panacea Biotec Ltd Peptides inhibant le facteur de nécrose tumorale alpha et leurs utilisations
WO2018130170A1 (fr) * 2017-01-12 2018-07-19 朱乃硕 Peptide à haute affinité pour le facteur alpha de nécrose tumorale et son application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Volume 49, issued 01 April 1989, FOON K.A., "Biological Response Modifiers: the New Immunotherapy", pages 1621-1638. *
JOURNAL OF IMMUNOLOGY, Volume 141, Number 5, issued 01 September 1988, EILAT et al., "V Region Sequences of Anti-DNA and Anti-RNA Autoantibodies From NZB/NZW F1 Mice", pages 1745-1753. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7592304B2 (en) 1999-10-01 2009-09-22 Dmi Life Sciences, Inc. Metal-binding compounds and uses therefor
US7632803B2 (en) 1999-10-01 2009-12-15 Dmi Life Sciences, Inc. Metal-binding compounds and uses therefor
WO2010058419A1 (fr) * 2008-11-20 2010-05-27 Panacea Biotec Ltd Peptides inhibant le facteur de nécrose tumorale alpha et leurs utilisations
WO2018130170A1 (fr) * 2017-01-12 2018-07-19 朱乃硕 Peptide à haute affinité pour le facteur alpha de nécrose tumorale et son application

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