EP0584244A1 - Peptide inhibitors of inflammation - Google Patents

Peptide inhibitors of inflammation

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Publication number
EP0584244A1
EP0584244A1 EP92912609A EP92912609A EP0584244A1 EP 0584244 A1 EP0584244 A1 EP 0584244A1 EP 92912609 A EP92912609 A EP 92912609A EP 92912609 A EP92912609 A EP 92912609A EP 0584244 A1 EP0584244 A1 EP 0584244A1
Authority
EP
European Patent Office
Prior art keywords
gln
ala
ile
asp
val
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.)
Withdrawn
Application number
EP92912609A
Other languages
German (de)
French (fr)
Inventor
George A. Heavner
Rodger P. Mcever
Jian-Guo Geng
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.)
University of Oklahoma
Janssen Biotech Inc
Original Assignee
Centocor Inc
University of Oklahoma
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Filing date
Publication date
Application filed by Centocor Inc, University of Oklahoma filed Critical Centocor Inc
Publication of EP0584244A1 publication Critical patent/EP0584244A1/en
Withdrawn legal-status Critical Current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
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C07K14/70564—Selectins, e.g. CD62
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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 adherence of platelets and leukocytes to vascular surfaces is a critical component of the inflammatory response, and is part of a complex series of reactions involving the simultaneous and
  • the complement proteins collectively play a leading role in the immune system, both in the
  • activated endothelial cells express tissue factor on the cell surface and decrease their surface expression of thrombomodulin, leading to a net
  • a single receptor can be involved in both inflammatory and coagulation
  • Leukocyte adherence to vascular endothelium is a key initial step in migration of leukocytes to tissues in response to microbial invasion.
  • a class of inducible leukocyte receptors the CD11-CD18 molecules, are thought to have some role in adherence to endothelium, mechanisms of equal or even greater importance for leukocyte adherence appear to be due to inducible changes in the endothelium itself.
  • Activated platelets have also been shown to interact with both neutrophils and monocytes in vitro.
  • the interaction of platelets with monocytes may be mediated in part by the binding of thrombospondin to platelets and monocytes, although other mechanisms have not been excluded.
  • the mechanisms for the binding of neutrophils to activated platelets are not well understood, except that it is known that divalent cations are required.
  • platelets In response to vascular injury, platelets are known to adhere to subendothelial surfaces, become activated, and support coagulation. Platelets and other cells may also play an important role in the recruitment of leukocytes into the wound in order to contain microbial invasion.
  • necrosis factor and interleukin-1 becomes adhesive after one to six hours.
  • the rapid endothelialdependent leukocyte adhesion has been associated with expression of the lipid mediator platelet activating factor (PAF) on the cell surface, and presumably, the appearance of other endothelial surface receptors.
  • PAF lipid mediator platelet activating factor
  • the slower cytokine-inducible endothelial adhesion for leukocytes is mediated, at least in part, by an
  • ELAM-1 endothelial cell receptor
  • a peripheral lymph node homing receptor also called “the murine Mel 14 antigen”, “Leu 8”, the “Leu 8 antigen” and “LAM-1” is another structure on neutrophils, monocytes, and lymphocytes that binds lymphocytes to high endothelial venules in peripheral lymph nodes.
  • GMP-140 granule membrane protein 140
  • PADGEM protein glycoprotein
  • GMP-140 (called PADGEM) has also been reported to mediate the interaction of activated platelets with neutrophils and monocytes by Larsen, et al., in Cell 59, 305-312 (October 1989) and Hamburger and McEver, Blood 75:550-554 (1990).
  • the cDNA-derived amino acid sequence reported by Johnston, et al., in Cell 56, 1033-1044 (March 24 1989), and in U.S. Serial No. 07/320,408 filed March 8, 1989, indicates that it contains a number of modular domains that are likely to fold independently. Beginning at the N-terminus, these include a "lectin” domain, an "EGF” domain, nine tandem consensus repeats similar to those in complement binding proteins, a transmembrane domain (except in a soluble form that appears to result from differential splicing), and a cytoplasmic tail.
  • GMP-140 membrane bound GMP-140 is presented within seconds on the cell surface.
  • glycoprotein could play an important role at sites of inflammation or vascular disruption.
  • GMP-140 is a receptor for neutrophils (Geng et al., Nature 343:757-760 (1990); Hamburger and McEver, Blood 75:550-554 (1990)), monocytes (Larsen, et al. Cell 59:305-312 (1989); Moore, et al., J. Cell Biol. 112:491-499 (1991)), and a subset of lymphocytes (Moore, et al. J. Cell Biol. 112:491-499 (1991) and Moore, et al., Blood (Suppl 1) 78:439a (1991)).
  • GMP-140 can serve as a receptor for leukocytes
  • thrombin agonists such as thrombin. This role in leukocyte recruitment may be important in hemostatic and
  • neutrophil adhesion to purified GMP-140 can therefore be used in diagnostic assays of patients and diseases characterized by altered binding by these molecules, in screening assays for compounds altering this binding, and in clinical applications to inhibit or modulate interactions of leukocytes with platelets or endothelial cells involving coagulation and/or inflammatory processes.
  • ELAM-1 the homing receptor, and GMP-140 have been termed "selectins", based on their related structure and function.
  • ELAM-1 is not present in unstimulated endothelium. However, when endothelium is exposed to cytokines such as tumor necrosis factor or interleukin-1, the gene for ELAM-1 is transcribed, producing RNA which in turn is translated into
  • ELAM-1 is expressed on the surface of endothelial cells one to four hours after exposure to cytokines, as reported by Bevilacqua et al., Proc.Natl.Acad.Sci.USA 84:9238-9242 (1987) (in contrast to GMP-140, which is stored in granules and presented on the cell surface within seconds after activation). ELAM-1 has been shown to mediate the adherence of neutrophils to cytokine-treated
  • ELAM-1 cDNA-derived primary structure indicates that it contains a "lectin" domain, an EGF domain, and six (instead of the nine in GMP-140) repeats similar to those of complement-regulatory proteins, a transmembrane domain, and a short cytoplasmic tail.
  • GMP-140 lectin-derived primary structure indicates that it contains a "lectin" domain, an EGF domain, and six (instead of the nine in GMP-140) repeats similar to those of complement-regulatory proteins, a transmembrane domain, and a short cytoplasmic tail.
  • Homing receptors are lymphocyte surface
  • the lymphocytes to migrate across the endothelium into the lymphatic tissues where they are exposed to processed antigens. The lymphocytes then re-enter the blood through the lymphatic system.
  • the homing receptor contains a lectin domain, an EGF domain, two
  • the homing receptor also shares extensive sequence homology with GMP-140, particularly in the lectin and EGF domains.
  • platelets in response to vascular injury, platelets are known to adhere to subendothelial surfaces, become activated, and support coagulation. Platelets and other cells may also play an important role in the recruitment of leukocytes into the wound in order to contain microbial invasion. Conversely, leukocytes may recruit platelets into tissues at sites of
  • coagulation and inflammatory pathways are regulated in a coordinate fashion in response to tissue damage.
  • activated endothelial cells express tissue factor on the cell surface and decrease their surface expression of thrombomodulin, leading to a net facilitation of coagulation reactions on the cell surface.
  • thrombomodulin a single receptor can be involved in both inflammatory and coagulation
  • Proteins are usually expensive to produce in quantities sufficient for administration to a patient. Moreover, there can be a reaction against the protein after it has been administered more than once to the patient. It is therefore desirable to develop peptides having the same, or better, activity as the protein, which are inexpensive to synthesize, reproducible and relatively innocuous.
  • peptides which can be prepared synthetically, having activity at least equal to, or greater than, the peptides derived from the protein itself.
  • inventions to provide peptides interacting with cells recognized by selectins, including GMP-140, ELAM-1, and lymphocyte homing receptor.
  • inventions to provide peptides for use in diagnostic assays relating to GMP-140, ELAM-1, and lymphocyte homing receptor.
  • Peptides derived from three regions of the lectin domain of GMP-140 and the related selectins, ELAM-1 and the lymphocyte homing receptor, have been found to inhibit neutrophil adhesion to GMP-140.
  • X in Formula (I) and P in Formula (II) are the N-terminus amino acids, and R 1 is a moiety attached to the function (NHR 1 ) ,
  • Y in Formula (I) and Z in Formula (II) are the C-terminus amino acids, and R is the moiety attached to the singly-bonded oxygen in the carboxy function (C(O)OR 2 ),
  • P is D- or L-tyrosine, D- or L-phenylalanine, D- or L-lysine, D- or L-glutamic acid, D- or L-arginine, D- or L-cysteine, D- or L- O-R 3 -tyrosine, D- or L-N ⁇ -R 3 -tyrosine, D- or L-4-amino phenylalanine, D- or L-R 4 -phenylalanine, D- or L-pyridylalanine, D- or L-naphthylalanine, or D- or L-tetrahydroisoquinoline carboxylic acid, where R 3 is lower alkyl or aryl and R 4 is halogen (fluorine, chlorine, bromine or iodine),
  • Q is D- or L-threonine, D- or L-lysine, D- or L-glutamic acid, D- or L-cysteine , or glycine,
  • S is D- or L-aspartic acid, D- or L-histidine, D- or L-glutamic acid, D- or L-asparagine, D or L-glutamine, D- or L-alanine, D- or L-phenylalanine, D- or L-lysine, or glycine,
  • T, U, V and W are independently D- or L-leucine, D- or L-isoleucine, D- or L-alanine, D- or L-valine, D- or L-alloisoleucine, glycine, D- or L-glutamic acid, D- or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either
  • Z is D- or L-glutamine, D- or L-glutamic acid and D- or L-asparagine,
  • R 1 is H (signifying a free N-terminal group), formyl, lower alkanoyl, aroyl or desamino (meaning the amino acid adjacent to the group R 1 , either X in formula I or P in formula 2 lacks the ⁇ -amino group of the amino acid, and is replaced with H),
  • R 2 is H (signifying in a free C-terminal
  • X and Y are linear chains of from one to ten amino acids.
  • Peptides of the Formula I and II have as their core region portions of the 23-30 amino acid sequence of GMP-140, with residue 1 defined as the N-terminus of the mature protein after the cleavage of the signal peptide. Examples demonstrate the inhibition of the binding of neutrophils to GMP-140 of peptides of Formula I or II in concentrations ranging from 5 to 1500 ⁇ M. It has been found that alterations within the core sequence, as well as N-terminal and C- terminal flanking regions, do not result in loss of biological activity. It has also been found that certain of these modifications can significantly increase the stability of peptides of Formula I or II against degradation by the enzymes found in human serum.
  • the peptides are useful as diagnostics and, in combination with a suitable pharmaceutical carrier, for clinical applications in the modulation or inhibition of coagulation processes or inflammatory processes.
  • Figure 1 shows the activity of several peptides of Formulas I and II in inhibiting the binding of neutrophils to GMP-140, % inhibition versus
  • Figure 2 shows the significant increase in stability against enzymes found in human serum that can be achieved by the modifications set down in Formula I and II, graphing percent of peptide remaining versus time (minutes) (dark triangle, Ac-YTDLVAIQ-NH 2 , O, YTDLVAIQ-NH 2 ).
  • X in Formula (I) and P in Formula (II) are the N-terminus amino acids, and R is a moiety attached to the amine function (NHR 1 ),
  • Y in Formula (I) and Z in Formula (II) are the C-terminus amino acids, and R 2 is the moiety attached to the singly-bonded oxygen in the carboxy function (C(O)OR 2 ),
  • P is D- or L-tyrosine, D- or L-phenylalanine, D- or L-lysine, D- or L-glutamic acid, D- or L-arginine,
  • D- or L-cysteine D- or L- O-R-tyrosine, D- or L-N ⁇ -R-tyrosine, D- or L-4-amino phenylalanine, D- or L-R -phenylalanine, D- or L-pyridylalanine, D- or L-naphthylalanine, or D- or L-tetrahydroisoquinoline carboxylic acid, where R 3 is lower alkyl or aryl and R 4 is halogen (fluorine, chlorine, bromine or iodine),
  • Q is D- or L-threonine, D- or L-lysine, D- or L-glutamic acid, D- or L-cysteine, or glycine,
  • S is D- or L-aspartic acid, D- or L-histidine, D- or L-glutamic acid, D- or L-asparagine, D or L-glutamine, D- or L-alanine, D- or L-phenylalanine, D- or L-lysine, or glycine,
  • T, U, V and W are independently D- or L-leucine, D- or L-isoleucine, D- or L-alanine, D- or L-valine, D- or L-alloisoleucine, glycine, D- or L-glutamic acid, D- or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either
  • Z is D- or L-glutamine, D- or L-glutamic acid and D- or L-asparagine,
  • R 1 is H (signifying a free N-terminal group), formyl, lower alkanoyl, aroyl or desamino (meaning the amino acid adjacent to the group R 1 , either X in formula I or P in formula 2 lacks the ⁇ -amino group of the amino acid, and is replaced with H),
  • R 2 is H (signifying in a free C-terminal
  • X and Y are linear chains of from one to ten amino acids.
  • Preferred peptides are those of Formula I wherein R 1 is H and R 2 is NR 3 R 4 , and Formula II wherein R 1 is H or acetyl and R 1 is NR 3 R 4 , wherein S is
  • aspartic acid glutamic acid or histidine.
  • peptides are Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH 2 ; Tyr-Thr-His-Leu-Val-Ala-Ile-Gln-NH 2 ; Acetyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH 2 ; Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH 2 ; Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH 2 ; Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH 2 ; Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH 2 ; Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-L
  • lower alkyl includes branched, straight-chain, and cyclic saturated
  • hydrocarbons having from one to six carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, and hexyl.
  • lower alkanoyl means RC(O), wherein R a lower alkyl group.
  • aroyl means where ArC(O), wherein Ar is an aryl group, an aromatic or
  • heteroaromatic structure having between one and three rings, which may or may not be ring fused structures, and are optimally substituted with halogens, carbons, or other heteroatoms such as nitrogen (N), sulfur (S), phosphorus (P), and boron (B).
  • the peptides of formula I can be used in the form of the free peptide or a pharmaceutically
  • Amine salts can be prepared by mixing the peptide with an acid according to known methods.
  • Suitable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, and sulfanilic acid.
  • Carboxylic acid groups in the peptide can be converted to a salt by mixing the peptide with a base according to known methods.
  • Suitable bases include inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases such as mono-, di-, and tri-alkyl and aryl amines (e.g., triethylamine, disopropylamine, methylamine, and dimethylamine and optionally substituted mono-, di, and tri-ethanolamines.
  • amino acid components of the peptides and certain materials used in their preparation are identified by abbreviations for convenience. These abbreviations are as follows:
  • the peptides can generally be prepared following known techniques, as described for example in the cited publications, the teachings of which are specifically incorporated herein. In a preferred method, the peptides are prepared following the solid- phase synthetic technique initially described by
  • N-terminal acetylation on the deprotected N ⁇ -amino group of peptides synthesized using either Boc or FMOC strategies is accomplished with 10% Ac 2 O and 5% DIEA in NMP, followed by washing of the peptide resin with NMP and/or CH 2 Cl 2 .
  • the peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art.
  • the peptide can be produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide in the presence of the required amino acids.
  • the peptide is then purified using chromatographic or electrophoretic techniques, or by means of a carrier protein which can be fused to, and subsequently cleaved from, the peptide by inserting into the expression vector in phase with the peptide encoding sequence a nucleic acid sequence encoding the carrier protein.
  • the fusion protein-peptide may be isolated using
  • the peptide can be cleaved using chemical methodology or enzymatically, as by, for example, hydrolases.
  • a peptide of Formula I or II or a base or acid addition salt thereof is combined as the active ingredient with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • This carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., sublingual, rectal, nasal, oral, or parenteral.
  • any of the usual pharmaceutical media may be employed, for example, water, oils, alcohols, flavoring agents, preservatives, and coloring agents, to make an oral liquid preparation (e.g., suspension, elixir, or solution) or with carriers such as
  • an oral solid preparation e.g., powder, capsule, or tablet.
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are employed. If desired, tablets may be sugar coated or enteric coated by standard techniques.
  • the carrier will usually be sterile water, although other ingredients to aid solubility or as preservatives may be included.
  • injectable suspensions may also be prepared, in which case appropriate liquid carriers and suspending agents can be employed.
  • the peptides can also be administered locally at a wound or inflammatory site by topical application of a solution or cream.
  • the peptide may be administered in liposomes or microspheres (or microparticles).
  • Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract
  • the peptide can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time, ranging from days to months. See, for example, U.S. Patent No. 4,906,474,
  • Peptides that are biologically active are those which inhibit binding of neutrophils, monocytes, subsets of lymphocytes or other cells to GMP-140, or which inhibit leukocyte adhesion to endothelium that is mediated by ELAM-1 and/or the homing receptor.
  • Peptides can be screened for their ability to inhibit adhesion to cells, for example, neutrophil adhesion to purified GMP-140 immobilized on plastic wells, using the assay described by Geng, et al.,
  • Human neutrophils are isolated from heparinized whole blood by density gradient centrifugation on Mono-Poly resolving media, Flow Laboratories.
  • Neutrophil suspensions are greater than 98% pure and greater than 95% viable by trypan blue exclusion.
  • neutrophils are suspended at a concentration of 2 ⁇ 10 6 cells/ml in Hanks' balanced salt solution containing 1.26 mM Ca 2+ and 0.81 mM Mg 2+ (HBSS, Gibco) with 5 mg/ml human serum albumin
  • GMP-140 is isolated from human platelet lysates by immunoaffinity chromatography on antibody S12-SepharoseTM and ion-exchange chromatography on a Mono-QTM column (FLPC, Pharmacia Fine Chemicals), as follows.
  • Outdated human platelet packs (100 units) obtained from a blood bank and stored at 4°C are pooled, adjusted to 5 mM EDTA at pH 7.5, centrifuged at 4,000 rpm for 30 min in 1 liter bottles, then washed three times with 1 liter of 0.1 M NaCl, 20 mM Tris pH 7.5 (TBS), 5 mM EDTA, 5 mM benzamidine.
  • the pellets are then resuspended in a minimum amount of wash buffer and made 1 mM in DIFP, then frozen in 50 ml screwtop tubes at -80°C.
  • the frozen platelets are thawed and resuspended in 50 ml TBS, 5 mM benzamidine, 5 mM EDTA pH 7.5, 100 M leupeptin.
  • the suspension is frozen and thawed two times in a dry ice-acetone bath using a 600 ml lyophilizing flask, then homogenized in a glass/teflon mortar and pestle and made 1 mM in DIFP.
  • the NaCl concentration is adjusted to 0.5 M with a stock solution of 4 M NaCl. After stirring the suspension at 4°C, it is
  • the soluble fraction (0.5 M NaCl wash) and the membrane extract (also adjusted to 0.5 M NaCl) are absorbed with separate pools of the monoclonal
  • Bound GMP-140 is eluted from the S12 Affigel with 100 ml of 80% ethylene glycol, 1 mM MES pH 6.0, 0.01% Lubrol PX. Peak fractions with absorbance at 280 nm are pooled. Eluates are dialyzed against TBS with 0.05% Lubrol, then applied to a Mono Q column (FPLC from Pharmacia). The concentrated protein is step eluted with 2 M NaCl, 20 mM Tris pH 7.5 (plus 0.05% Lubrol PX for the membrane fraction). Peak fractions are dialyzed into TBS pH 7.5 (plus 0.05% Lubrol PX for the membrane fraction).
  • GMP-140 is plated at 5 micrograms/ml and the control proteins: human serum albumin (Alb), platelet glycoprotein Hb/IIIa (IIb), von Willebrand factor (vWF), fibrinogen (FIB), thrombomodulin (TM), gelatin (GEL) or human serum (HS), are added at 50
  • myeloperoxidase activity Ley, et al., Blood 73, 1324- 1330 (1989).
  • the number of cells bound is derived from a standard curve of myeloperoxidase activity versus numbers of cells. Under all assay conditions, the cells release less than 5% of total
  • the subject peptides are generally active when administered parenterally in amounts above about 1 ⁇ g peptide/kg of body weight.
  • the peptides may be administered parenterally from about 0.01 to about 10 mg peptide/kg body weight.
  • the same range of dosage amounts may be used in treatment of the other diseases or conditions where inflammation is to be reduced. This dosage will be dependent, in part, on whether one or more peptides are administered.
  • a synergistic effect may be seen with combinations of peptides from different, or overlapping, regions of the lectin domain, or in combination with peptides derived from the EGF domain of GMP-140.
  • the peptides can be used to
  • An inflammatory response may cause damage to the host if unchecked, because leukocytes release many toxic molecules that can damage normal tissues. These molecules include proteolytic enzymes and free
  • leukocytes can cause tissue damage.
  • pathological situations in which leukocytes can cause tissue damage include injury from ischemia and reperfusion, bacterial sepsis and disseminated intravascular coagulation, adult respiratory distress syndrome, tumor metastasis, rheumatoid arthritis and atherosclerosis.
  • thrombolytic therapy with agents such as tissue plasminogen activator or streptokinase can relieve coronary artery obstruction in many patients with severe myocardial ischemia prior to irreversible myocardial cell death. However, many such patients still suffer myocardial neurosis despite restoration of blood flow. This "reperfusion injury” is known to be associated with adherence of leukocytes to vascular endothelium in the ischemic zone,
  • leukocytes in the pulmonary circulation This leads to extravasation of large amounts of plasma into the lungs and destruction of lung tissue, both mediated in large part by leukocyte products.
  • LAK cells lymphokine-activated lymphocytes
  • LAK cells adhere to endothelium could potentially release molecules that activate endothelium and then bind to endothelium by mechanisms similar to those operative in neutrophils.
  • Tumor cells from many malignancies can metastasize to distant sites through the vasculature.
  • Platelet-leukocyte interactions are believed to be important in atherosclerosis. Platelets might have a role in recruitment of monocytes into
  • Atherosclerotic plaques the accumulation of monocytes is known to be one of the earliest detectable events during atherogenesis. Rupture of a fully developed plaque may not only lead to platelet deposition and activation and the promotion of thrombus formation, but also the early recruitment of neutrophils to an area of ischemia. Another area of potential application is in the treatment of rheumatoid arthritis.
  • the criteria for the effective dosage to prevent extension of myocardial infarction would be determined by one skilled in the art by looking at marker enzymes of myocardial necrosis in the plasma, by monitoring the electrocardiogram, vital signs, and clinical response.
  • For treatment of acute respiratory distress syndrome one would examine improvements in arterial oxygen, resolution of pulmonary infiltrates, and clinical improvement as measured by lessened dyspnea and tachypnea.
  • the effective dosage For treatment of patients in shock (low blood pressure), the effective dosage would be based on the clinical response and specific measurements of function of vital organs such as the liver and kidney following restoration of blood pressure. Neurologic function would be monitored in patients with stroke. Specific tests are used to monitor the functioning of transplanted organs; for example, serum creatinine, urine flow, and serum electrolytes in patients
  • the peptides can also be used for the detection of human disorders in which the ligands for the selectins might be defective. Such disorders would most likely be seen in patients with increased
  • Detection systems include ELISA procedures, binding of radiolabeled antibody to immobilized activated cells, flow cytometry, or other methods known to those skilled in the arts.
  • Inhibition of binding in the presence and absence of the lectin domain peptides can be used to detect defects or alterations in selectin binding.
  • selectins such disorders would most likely be seen in patients with increased susceptibility to infections in which leukocytes would have defective binding to platelets and endothelium because of deficient
  • leukocyte ligands for GMP-140 The peptide is labeled radioactively, with a fluorescent tag, enzymatically, or with electron dense material such as gold for electron microscopy.
  • the cells to be examined usually leukocytes, are incubated with the labeled peptides and binding assessed by methods described above with antibodies to GMP-140, or by other methods known to those skilled in the art. If ligands for GMP-140 are also found in the plasma, they can also be measured with standard ELISA or radioimmunoassay procedures, using labeled GMP-140-derived peptide instead of antibody as the detecting reagent.
  • EXAMPLE 1 Preparation of Tyrosyl-threonyl-histidyl- leucyl-valyl-alanyl-isoleucyl-glutamine- amide.
  • the peptide was prepared on an ABI model 431A peptide synthesizer using Version 1.12 of the standard scale Boc software.
  • the amino acids used were Boc-(BrCBZ)Tyr, Boc-(Bzl)Thr, Boc-(Tos)His, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile and Boc-Gln.
  • 4-Methylbenzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 1.13 g.
  • the peptide was cleaved from the resin (1.03 g) using 11 mL of HF and 1.1 mL of anisole for 60 min at 0° C.
  • the crude peptide (55 mg) was purified on a Vydac C-18 column (10 ⁇ 2.2 ⁇ 25 cm), eluting with a gradient of 10 to 20% of acetonitrile in 0.1% aqueous TFA over 20 minutes at a flow rate of 8 mL per minute. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 3.9 mg of purified peptide. Amino acid analysis: Ala 0.98 (1.0), Glx 1.02 (1.0), His 1.06 (1.0), Ile 1.07 (1.0), Leu 1.07 (1.0), Thr 0.85 (1.0), Tyr 0.85 (1.0), Val 0.94 (1.0). FAB/MS: MH + 944 (calcd 944).
  • EXAMPLE 2 Preparation of Tyrosyl-threonyl-glutamyl- leucyl-valyl-alanyl-isoleucyl-glutamine- amide.
  • the peptide was prepared on a ABI model 431A peptides synthesizer using Version 1.12 of the
  • Boc-(BrCBZ)Tyr Boc-(Bzl)Thr, Boc-(Bzl)Glu, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ill, Boc-Gln.
  • 4-methylbenzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. Final weight of the resin was 1.20 g.
  • the peptide was cleaved from the resin (1.10 g) using 11 mL of HF and 1.1 mL of anisole for 60 minutes at 0° C. The hydrogen fluoride was removed using a stream of dry nitrogen, the residue triturated with ether and the ether removed by filtration.
  • EXAMPLE 3 Preparation of Acetyl-tyrosyl-threonyl- aspartyl-leucyl-valyl-alanyl-isoleucyl- glutamine amide.
  • the peptide was prepared on ABI model 431A peptide synthesizer using Version 1.12 of the standard scale Boc software modified for N-terminal acetylation according the instrument operations manual. 4-Methylbenzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. Final weight of the peptide resin was 1.23 g.
  • the peptide was cleaved from the resin (1.11 g) with 10 mL of HF and 1 mL of anisole for 60 minutes at 0° C. The HF was removed by a nitrogen stream. The resulting solid was triturated with ether, collected by filtration and washed with ether.
  • the peptide was extracted from the resin with 50% TFA and methylene chloride (5 ⁇ 20 mL). The resin was removed by filtration, the solvents removed under reduced pressure and the residue triturated with ether to give 0.50 g of crude peptide.
  • the crude peptide was purified by preparation HPLC using a Vydac C-18 column (10 ⁇ , 2.2 ⁇ 25 cm) eluting with a 20 to 30% gradient of acetonitrile and 0.1% aqueous TFA over 140 minutes at a flow rate of 3 mL per minute. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 60 mg of the purified peptide as a white solid.
  • Amino acid analysis Tyr 0.99 (1.0), Thr 0.91 (1.0), Asx 0.98 (1.0), Leu 1.03 (1.0), Val 1.05 (1.0), Ala 1.03 (1.0), Ile 1.00 (1.0), Glx 0.01 (1.0).
  • EXAMPLE 4 Preparation of Tyrosyl-threonyl-aspartyl- leucyl-valyl-alanyl-isoleucyl-glutamine amide.
  • the peptide was prepared by manual solid phase synthesis using Boc chemistry.
  • the amino acids used were Boc-(BrCBZ)Tyr, Boc-(Bzl)Thr, Boc-(Bzl)Asp, Boc- Leu, Boc-Val, Boc-Ile and Boc-Gln.
  • 4- Methylbenzhydrylamine resin (6.25 g, 5.0 mmol) was used in synthesis. 20 Mmol of each Boc-AA was
  • Coupling step (monitored by ninhydrin testing of a resin sample).
  • the final weight of the peptide-resin was 11.97 g.
  • the resin-peptide (11.8 g) was treated with 12 mL of anisole and 120 mL of HF for one hour at 0° to 4°C.
  • the HF was removed by nitrogen stream followed by aspiration.
  • the resultant solids were triturated with ether (1 ⁇ 100 mL then 1 ⁇ 80 mL), collected by
  • EXAMPLE 5 Preparation of Arginyl-tyrosyl-threonyl- aspartyl-leucyl-valyl-alanyl-isoleucyl- glutamine amide.
  • the peptide was prepared on a DuPont RAMPS system using the FMOC strategy.
  • the amino acids used for the synthesis were FMOC-(Mtr)Arg, FMOC-(t-Bu) Tyr, FMOC-(t-Bu) Thr, FMOC-(t-Bu)Asp, FMOC-Leu, FMOC-Val, FMOC-Ala, FMOC-Ile and FMOC-Gln.
  • DuPont rapid amide resin (0.1 mmol) was used in the synthesis.
  • the peptide was cleaved from the resin using a mixture of phenol (0.25 g), ethanedithiol (0.083 mL), thioanisole (0.66 mL), water (0.166 mL) and trifluoroacetic acid (3.33 mL) for 6 hours for 20° C.
  • the resin was removed by filtration and the peptide precipitated from the filtrate by the addition of ether.
  • the solids were removed by filtration, extracted with 20% acetic acid and lyophilized to give 0.147 g of crude peptide.
  • the peptide was purified by preparative reverse phase (C-18) HPLC using an acetonitrile-water gradient in 0. 1% in TFA. Fractions were collected and those containing pure peptide were pooled and
  • the peptide was prepared using a DuPont RAMPS system and the FMOC strategy.
  • the amino acids used were FMOC-Asn, FMOC-(Mtr)Arg, FMOC-(t-Bu)Tyr, FMOC- (t-Bu)Thr, FMOC- (t-Bu) Asp, FMOC-Leu, FMOC-Val, FMOC- Ala, FMOC-Ile and FMOC-Gln.
  • DuPont rapid amide resin (0.2 mmol) was used in the synthesis.
  • the peptide was cleaved in the resin using a mixture of TFA (2.85 mL), thioanisole (0.135 mL) and ethanedithiol (0.015 mL) for 16 hours at ambient temperature.
  • the resin was removed by filtration and the peptide precipitated from the filtrate by the addition of ether.
  • the peptide was removed by filtration, extracted with 20% acetic acid and lyophilized to give 50 mg of crude peptide.
  • the crude peptide was purified by
  • EXAMPLE 7 Preparation of Cysteinyl-glutaminyl- asparaginyl-arginyl-tyrosyl-threonyl- aspartyl-leucyl-valyl-alanyl-isoleucyl- glutaminyl-asparaginyl-lysyl-asparaginyl- glutamine.
  • the peptide was prepared on an ABI model 430A peptides synthesizer using the standard scale Boc software.
  • the amino acids used were Boc-(4-Me-Bzl)Cys, Boc-Gln, Boc-Asn, Boc-(Tos)Arg, Boc- (BrCBZ)Tyr, Boc-(Bzl)Thr, Boc-(Bzl) Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile, Boc-(Cl-CBz)Lys.
  • Boc-(Bzl)Glu-Pam resin (0.5 mmol) was used in the synthesis.
  • the peptide was cleaved from the resin using 10 mL of HF, 1.0 mL of anisole, 1.0 mL of dimethyl sulfide and 0.2 mL p-thiocresol for 30 minutes at -10° C followed by 30 minutes at 0°C.
  • the hydrogen fluoride was removed under reduced pressure and the residue triturated with ether. Solids were removed by filtration and the peptide extracted from the resin using 20% acetic acid. Removal of the resin by filtration and
  • EXAMPLE 8 Preparation of Tyrosyl-threonyl-D-aspartyl- leucyl-valyl-alanyl-isoleucyl-glutamine amide.
  • the peptide was prepared on an ABI Model 431A peptide synthesizer using Version 1.12 of the standard Boc software.
  • the amino acids used were Boc- (BrCBZ)Tyr, Boc-(Bzl)Thr, Boc-D-(Bzl)Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile and Boc-Gln.
  • 4-Methylbenzhydrylamine resin (0.63 g, 0.5 mmol) was used in the synthesis.
  • the final weight of the peptide resin was 1.43 g.
  • the peptide was cleaved from the resin (1.43 g) using 15 mL of HF and 1.5 mL of anisole for 60 min at 0°C.
  • the hydrogen fluoride was removed under reduced pressure and the residue triturated with ether. Solids were removed by
  • the peptide was prepared on an ABI Model 431A peptide synthesizer using Version 1.12 of the standard Boc software.
  • the amino acids used were Boc-Phe, Boc- (Bzl)Thr, Boc-(Bzl)Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile and Boc-Gln.
  • Boc-Phe Boc- (Bzl)Thr
  • Boc-(Bzl)Asp Boc-Leu
  • Boc-Val Boc-Ala
  • Boc-Ile Boc-Gln.
  • the peptide was prepared on a ABI Model 431A peptide synthesizer using Version 1.12 of the standard scale Boc software.
  • the amino acids used were Boc-D- (BrCBZ)Tyr, Boc-(Bzl) Thr, Boc-(Bzl)Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile, Boc-Gln.
  • 4-Methylbenzhydrylamine resin (0.665 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 2.10 g.
  • the peptide was cleaved from the resin (2.10 g) using 20 mL of hydrogen fluoride and 2 mL of anisole for 60 minutes at 0°C. The hydrogen fluoride was evaporated using a stream of nitrogen and the resulting mixture triturated with ether. The solids were removed by filtration and extracted with a 50% solution of trifluoroacetic acid in methylene
  • EXAMPLE 11 Preparation of Tyrosyl-D-threonyl- aspartyl-leucyl-valyl-alanyl- isoleucyl-glutamine amide.
  • the peptide was prepared on an ABI Model 431A peptide synthesizer using Version 1.12 of the standard Boc software.
  • the amino acids used were: Boc-(BrCBZ)Tyr, Boc-D-(Bzl)Thr, Boc-(Bzl)Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile and Boc-Gln.
  • 4- Methylbenzhydrylamine resin (0.685 g, 0.5 mmol) was used in the synthesis.
  • the final weight of the peptide resin was 1.63 g.
  • the peptide was cleaved from the resin (1.63 g) using 20 mL of hydrogen fluoride and 2 mL of anisole for 60 min at 0°C.
  • the peptides tested were Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Acetyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-amide; Tyr-Thr-His-Leu-Val-Ala-Ile-Gln-amide; Tyr-Thr-Asp-Leu- Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-amide;
  • Figure 2 shows the significant increase in stability against enzymes found in human serum that can be achieved by the modifications set down in Formula I and II, graphing percent of peptide
  • the half-life in serum increased from 20 minutes for the unmodified peptide to four hours, 37 minutes for the modified peptide.
  • NAME Pabst, Patrea L.

Abstract

On a découvert que les peptides dérivés de trois régions du domaine lectine de GMP-140 et des sélectines associées, ELAM-1 et le récepteur accepteur de lymphocytes, inhibent l'adhésion des neutrophiles à GMP-140. On a synthétisé ces peptides, ainsi que des peptides supplémentaires, dont la région centrale est constituée de parties de la séquence d'aminoacides 23-30 de GMP-140, le résidu 1 étant défini en tant que terminaison N de la protéine adulte après le clivage du peptide de signalisation. Des exemples démontrent l'inhibition de la fixation de neutrophiles au GMP-140 de peptides, selon des concentrations situées dans une plage de 5 à 1500 muM. On a découvert que des modifications se produisant à l'intérieur de la séquence centrale, ainsi que dans des régions contiguës à la terminaison N et à la terminaison C, ne provoquent pas une perte d'activité biologique. On a découvert également que certaines de ces modifications peuvent considérablement améliorer la stabilité des peptides de formule (I) ou (II) contre la dégradation provoquée par des enzymes trouvés dans le sérum humain. Les peptides permettent un diagnostic efficace et, combinés à un excipient pharmaceutique approprié, servent à des applications cliniques dans la modulation ou l'inhibition de processus de coagulation ou de processus inflammatoires.It has been discovered that peptides derived from three regions of the lectin domain of GMP-140 and associated selectins, ELAM-1 and the lymphocyte acceptor receptor, inhibit adhesion of neutrophils to GMP-140. These peptides have been synthesized, as well as additional peptides, the central region of which consists of parts of the amino acid sequence 23-30 of GMP-140, residue 1 being defined as the N terminus of the adult protein after the cleavage of the signaling peptide. Examples demonstrate the inhibition of binding of neutrophils to GMP-140 peptides, at concentrations in the range of 5 to 1500 muM. It has been discovered that changes occurring within the central sequence, as well as in regions contiguous with the N-terminus and the C-terminus, do not cause loss of biological activity. It has also been discovered that some of these modifications can considerably improve the stability of the peptides of formula (I) or (II) against the degradation caused by enzymes found in human serum. The peptides allow efficient diagnosis and, combined with an appropriate pharmaceutical excipient, serve clinical applications in the modulation or inhibition of coagulation or inflammatory processes.

Description

PEPTIDE INHIBITORS OF INFLAMMATION Background of the Invention
This invention is generally in the field of methods for the treatment and prevention of
inflammatory responses using peptides derived from selectins including GMP-140, ELAM-1, and lymphocyte-homing receptor.
The adherence of platelets and leukocytes to vascular surfaces is a critical component of the inflammatory response, and is part of a complex series of reactions involving the simultaneous and
interrelated activation of the complement,
coagulation, and immune systems.
The complement proteins collectively play a leading role in the immune system, both in the
identification and in the removal of foreign
substances and immune complexes, as reviewed by
Muller-Eberhard, H.J., Ann. Rev. Biochem. 57:321-347 (1988). Central to the complement system are the C3 and C4 proteins, which when activated covalently attach to nearby targets, marking them for clearance. In order to help control this process, a remarkable family of soluble and membrane-bound regulatory proteins has evolved, each of which interacts with activated C3 and/or C4 derivatives. The coagulation and inflammatory pathways are regulated in a
coordinate fashion in response to tissue damage. For example, in addition to becoming adhesive for
leukocytes, activated endothelial cells express tissue factor on the cell surface and decrease their surface expression of thrombomodulin, leading to a net
facilitation of coagulation reactions on the cell surface. In some cases, a single receptor can be involved in both inflammatory and coagulation
processes.
Leukocyte adherence to vascular endothelium is a key initial step in migration of leukocytes to tissues in response to microbial invasion. Although a class of inducible leukocyte receptors, the CD11-CD18 molecules, are thought to have some role in adherence to endothelium, mechanisms of equal or even greater importance for leukocyte adherence appear to be due to inducible changes in the endothelium itself.
Activated platelets have also been shown to interact with both neutrophils and monocytes in vitro. The interaction of platelets with monocytes may be mediated in part by the binding of thrombospondin to platelets and monocytes, although other mechanisms have not been excluded. The mechanisms for the binding of neutrophils to activated platelets are not well understood, except that it is known that divalent cations are required. In response to vascular injury, platelets are known to adhere to subendothelial surfaces, become activated, and support coagulation. Platelets and other cells may also play an important role in the recruitment of leukocytes into the wound in order to contain microbial invasion.
Endothelium exposed to "rapid" activators such as thrombin and histamine becomes adhesive for
neutrophils within two to ten minutes, while
endothelium exposed to cytokines such as tumor
necrosis factor and interleukin-1 becomes adhesive after one to six hours. The rapid endothelialdependent leukocyte adhesion has been associated with expression of the lipid mediator platelet activating factor (PAF) on the cell surface, and presumably, the appearance of other endothelial surface receptors.
The slower cytokine-inducible endothelial adhesion for leukocytes is mediated, at least in part, by an
endothelial cell receptor, ELAM-1, that is synthesized by endothelial cells after exposure to cytokines and then transported to the cell surface, where it binds neutrophils. The isolation, characterization and cloning of ELAM-1 is reviewed by Bevilacqua, et al., in Science 243, 1160-1165 (1989). A peripheral lymph node homing receptor, also called "the murine Mel 14 antigen", "Leu 8", the "Leu 8 antigen" and "LAM-1", is another structure on neutrophils, monocytes, and lymphocytes that binds lymphocytes to high endothelial venules in peripheral lymph nodes. The
characterization and cloning of this protein is reviewed by Lasky, et al., Cell 56, 1045-1055 (1989) (mouse) and Tedder, et al., J. Exp. Med. 170, 123-133 (1989).
GMP-140 (granule membrane protein 140), also known as PADGEM, is a cysteine-rich and heavily glycosylated integral membrane glycoprotein with an apparent molecular weight of 140,000 as assessed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). GMP-140 was first
purified from human platelets by McEver and Martin, J. Biol. Chem. 259:9799-9804 (1984). The protein is present in alpha granules of resting platelets but is rapidly redistributed to the plasma membrane following platelet activation, as reported by Stenberg, et al., (1985). The presence of GMP-140 in endothelial cells and its biosynthesis by these cells was reported by McEver, et al., Blood 70(5) Suppl. 1:355a, Abstract No. 1274 (1987). In endothelial cells, GMP-140 is found in storage granules known as the Weibel-Palade bodies. (McEver, et al. J. Clin. Invest. 84:92-99 (1989) and Hattori, et al., J. Biol. Chem. 264:7768-7771 (1989)). GMP-140 (called PADGEM) has also been reported to mediate the interaction of activated platelets with neutrophils and monocytes by Larsen, et al., in Cell 59, 305-312 (October 1989) and Hamburger and McEver, Blood 75:550-554 (1990).
The cDNA-derived amino acid sequence, reported by Johnston, et al., in Cell 56, 1033-1044 (March 24 1989), and in U.S. Serial No. 07/320,408 filed March 8, 1989, indicates that it contains a number of modular domains that are likely to fold independently. Beginning at the N-terminus, these include a "lectin" domain, an "EGF" domain, nine tandem consensus repeats similar to those in complement binding proteins, a transmembrane domain (except in a soluble form that appears to result from differential splicing), and a cytoplasmic tail.
When platelets or endothelial cells are
activated by mediators such as thrombin, the membranes of the storage granules fuse with the plasma membrane, the soluble contents of the granules are released to the external environment, and membrane bound GMP-140 is presented within seconds on the cell surface. The rapid redistribution of GMP-140 to the surface of platelets and endothelial cells as a result of
activation suggested that this glycoprotein could play an important role at sites of inflammation or vascular disruption.
This important role has been confirmed by the observation that GMP-140 is a receptor for neutrophils (Geng et al., Nature 343:757-760 (1990); Hamburger and McEver, Blood 75:550-554 (1990)), monocytes (Larsen, et al. Cell 59:305-312 (1989); Moore, et al., J. Cell Biol. 112:491-499 (1991)), and a subset of lymphocytes (Moore, et al. J. Cell Biol. 112:491-499 (1991) and Moore, et al., Blood (Suppl 1) 78:439a (1991)). Thus, GMP-140 can serve as a receptor for leukocytes
following its rapid mobilization to the surfaces of platelets and endothelial cells stimulated with
agonists such as thrombin. This role in leukocyte recruitment may be important in hemostatic and
inflammatory processes in both physiologic and
pathologic states.
Peptides derived from GMP-140 are described in U.S. Serial No. 07/554,199 entitled "Functionally
Active Selectin-Derived Peptides" filed July 17, 1990 by Rodger P McEver that are useful in diagnostics and in modulating the hemostatic and inflammatory
responses in a patient wherein a therapeutically effective amount of a peptide capable of blocking leukocyte recognition of GMP-140 is administered to the patient. U.S. Serial No. 07/554,199 filed July 17, 1990 also discloses that peptide sequences within the lectin domain of GMP-140, having homology with the lectin domains of other proteins, especially ELAM-1 and the homing receptor, selectively inhibit
neutrophil adhesion to purified GMP-140, and can therefore be used in diagnostic assays of patients and diseases characterized by altered binding by these molecules, in screening assays for compounds altering this binding, and in clinical applications to inhibit or modulate interactions of leukocytes with platelets or endothelial cells involving coagulation and/or inflammatory processes.
ELAM-1, the homing receptor, and GMP-140 have been termed "selectins", based on their related structure and function. ELAM-1 is not present in unstimulated endothelium. However, when endothelium is exposed to cytokines such as tumor necrosis factor or interleukin-1, the gene for ELAM-1 is transcribed, producing RNA which in turn is translated into
protein. The result is that ELAM-1 is expressed on the surface of endothelial cells one to four hours after exposure to cytokines, as reported by Bevilacqua et al., Proc.Natl.Acad.Sci.USA 84:9238-9242 (1987) (in contrast to GMP-140, which is stored in granules and presented on the cell surface within seconds after activation). ELAM-1 has been shown to mediate the adherence of neutrophils to cytokine-treated
endothelium and thus appears to be important in allowing leukocytes to migrate across cytokine-stimulated endothelium into tissues. The cDNA-derived primary structure of ELAM-1 indicates that it contains a "lectin" domain, an EGF domain, and six (instead of the nine in GMP-140) repeats similar to those of complement-regulatory proteins, a transmembrane domain, and a short cytoplasmic tail. There is extensive sequence homology between GMP-140 and ELAM- 1 throughout both proteins, but the similarity is particularly striking in the lectin and EGF domains.
Homing receptors are lymphocyte surface
structures that allow lymphocytes to bind to
specialized endothelial cells in lymphatic tissues, termed high endothelial cells or high endothelial venules (reviewed by Yednock and Rose, Advances in Immunology, vol. 44, F.I. Dixon,ed., 313-378 (Academic Press, New York 1989). This binding allows
lymphocytes to migrate across the endothelium into the lymphatic tissues where they are exposed to processed antigens. The lymphocytes then re-enter the blood through the lymphatic system. The homing receptor contains a lectin domain, an EGF domain, two
complement-binding repeats, a transmembrane domain, and a short cytoplasmic tail. The homing receptor also shares extensive sequence homology with GMP-140, particularly in the lectin and EGF domains.
Based on a comparison of the lectin domains between GMP-140, ELAM-1, and the homing receptor (LEU- 8), it may be possible to select those peptides inhibiting binding of neutrophils to GMP-140 which will inhibit binding of ELAM-1, the homing receptor, and other homologous selectins, to components of the inflammatory process, or, conversely, which will inhibit only GMP-140 binding.
The in vivo significance of platelet-leukocyte interactions has not been studied carefully. However, in response to vascular injury, platelets are known to adhere to subendothelial surfaces, become activated, and support coagulation. Platelets and other cells may also play an important role in the recruitment of leukocytes into the wound in order to contain microbial invasion. Conversely, leukocytes may recruit platelets into tissues at sites of
inflammation, as reported by Issekutz, et al., Lab. Invest. 49:716 (1983).
The coagulation and inflammatory pathways are regulated in a coordinate fashion in response to tissue damage. For example, in addition to becoming adhesive for leukocytes, activated endothelial cells express tissue factor on the cell surface and decrease their surface expression of thrombomodulin, leading to a net facilitation of coagulation reactions on the cell surface. In some cases, a single receptor can be involved in both inflammatory and coagulation
processes.
Proteins involved in the hemostatic and
inflammatory pathways are of interest for diagnostic purposes and treatment of human disorders. However, there are many problems using proteins
therapeutically. Proteins are usually expensive to produce in quantities sufficient for administration to a patient. Moreover, there can be a reaction against the protein after it has been administered more than once to the patient. It is therefore desirable to develop peptides having the same, or better, activity as the protein, which are inexpensive to synthesize, reproducible and relatively innocuous.
It is preferable to develop peptides which can be prepared synthetically, having activity at least equal to, or greater than, the peptides derived from the protein itself.
It is therefore an object of the present
invention to provide peptides interacting with cells recognized by selectins, including GMP-140, ELAM-1, and lymphocyte homing receptor.
It is another object of the present invention to provide methods for using these peptides to inhibit leukocyte adhesion to endothelium or to platelets. It is a further object of the present invention to provide methods for using these peptides to modulate the immune response and the hemostatic pathway.
It is yet another object of the present
invention to provide peptides for use in diagnostic assays relating to GMP-140, ELAM-1, and lymphocyte homing receptor.
Summary of the Invention
Peptides derived from three regions of the lectin domain of GMP-140 and the related selectins, ELAM-1 and the lymphocyte homing receptor, have been found to inhibit neutrophil adhesion to GMP-140.
These and additional peptides have been synthesized having the following formulae:
R1-X-P-Q-S-T-U-V-W-Z-Y-R2 (I)
RX-P-Q-S-T-U-V-W-Z-R2 (II) or a pharmaceutically acceptable salt thereof, wherein:
X in Formula (I) and P in Formula (II) are the N-terminus amino acids, and R1 is a moiety attached to the function (NHR1) ,
Y in Formula (I) and Z in Formula (II) are the C-terminus amino acids, and R is the moiety attached to the singly-bonded oxygen in the carboxy function (C(O)OR2),
P is D- or L-tyrosine, D- or L-phenylalanine, D- or L-lysine, D- or L-glutamic acid, D- or L-arginine, D- or L-cysteine, D- or L- O-R3-tyrosine, D- or L-Nα-R3-tyrosine, D- or L-4-amino phenylalanine, D- or L-R4-phenylalanine, D- or L-pyridylalanine, D- or L-naphthylalanine, or D- or L-tetrahydroisoquinoline carboxylic acid, where R3 is lower alkyl or aryl and R4 is halogen (fluorine, chlorine, bromine or iodine),
Q is D- or L-threonine, D- or L-lysine, D- or L-glutamic acid, D- or L-cysteine , or glycine,
S is D- or L-aspartic acid, D- or L-histidine, D- or L-glutamic acid, D- or L-asparagine, D or L-glutamine, D- or L-alanine, D- or L-phenylalanine, D- or L-lysine, or glycine,
T, U, V and W are independently D- or L-leucine, D- or L-isoleucine, D- or L-alanine, D- or L-valine, D- or L-alloisoleucine, glycine, D- or L-glutamic acid, D- or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either
residues T, U, V, or W from the peptide formulas I or
II,
Z is D- or L-glutamine, D- or L-glutamic acid and D- or L-asparagine,
R1 is H (signifying a free N-terminal group), formyl, lower alkanoyl, aroyl or desamino (meaning the amino acid adjacent to the group R1, either X in formula I or P in formula 2 lacks the α-amino group of the amino acid, and is replaced with H),
R2 is H (signifying in a free C-terminal
carboxylic acid), O(lower alkyl), O(aryl), NR3R4 where R3 and R4 are independently H or lower alkyl, or descarboxy (meaning the α-carboxylic acid group of the amino acid to which R1 is adjacent in formula I or 2, Y or Z, respectively, is replaced with H) ,
X and Y are linear chains of from one to ten amino acids.
Peptides of the Formula I and II have as their core region portions of the 23-30 amino acid sequence of GMP-140, with residue 1 defined as the N-terminus of the mature protein after the cleavage of the signal peptide. Examples demonstrate the inhibition of the binding of neutrophils to GMP-140 of peptides of Formula I or II in concentrations ranging from 5 to 1500 μM. It has been found that alterations within the core sequence, as well as N-terminal and C- terminal flanking regions, do not result in loss of biological activity. It has also been found that certain of these modifications can significantly increase the stability of peptides of Formula I or II against degradation by the enzymes found in human serum.
The peptides are useful as diagnostics and, in combination with a suitable pharmaceutical carrier, for clinical applications in the modulation or inhibition of coagulation processes or inflammatory processes.
Brief Description of the Drawings
Figure 1 shows the activity of several peptides of Formulas I and II in inhibiting the binding of neutrophils to GMP-140, % inhibition versus
concentration of peptide (mM), (dark squares, Cys- Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; open square, Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; dark diamond, Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; open diamond, Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; dark triangle, Acetyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; open triangle, Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-amide; X, Tyr-Thr-His-Leu-Val-Ala-Ile-Gln-amide; *, Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-amide; ╌, Leu-Gln-Thr-Ala-Tyr-Asp-Val-Ile-amide (negative control)).
Figure 2 shows the significant increase in stability against enzymes found in human serum that can be achieved by the modifications set down in Formula I and II, graphing percent of peptide remaining versus time (minutes) (dark triangle, Ac-YTDLVAIQ-NH2, O, YTDLVAIQ-NH2).
Detailed Description of the Invention
Peptides having GMP-140-like activity,
therapeutic compositions containing these peptides, methods for the preparation of these peptides and methods for use thereof, are disclosed. These
peptides have either of the following formulas:
R1-X-P-Q-S-T-U-V-W-Z-Y-R2 (I)
R1-P-Q-S-T-U-V-W-Z-R2 (II) or a pharmaceutically acceptable salt thereof,
wherein:
X in Formula (I) and P in Formula (II) are the N-terminus amino acids, and R is a moiety attached to the amine function (NHR1),
Y in Formula (I) and Z in Formula (II) are the C-terminus amino acids, and R2 is the moiety attached to the singly-bonded oxygen in the carboxy function (C(O)OR2),
P is D- or L-tyrosine, D- or L-phenylalanine, D- or L-lysine, D- or L-glutamic acid, D- or L-arginine,
D- or L-cysteine, D- or L- O-R-tyrosine, D- or L-Nα-R-tyrosine, D- or L-4-amino phenylalanine, D- or L-R -phenylalanine, D- or L-pyridylalanine, D- or L-naphthylalanine, or D- or L-tetrahydroisoquinoline carboxylic acid, where R3 is lower alkyl or aryl and R4 is halogen (fluorine, chlorine, bromine or iodine),
Q is D- or L-threonine, D- or L-lysine, D- or L-glutamic acid, D- or L-cysteine, or glycine,
S is D- or L-aspartic acid, D- or L-histidine, D- or L-glutamic acid, D- or L-asparagine, D or L-glutamine, D- or L-alanine, D- or L-phenylalanine, D- or L-lysine, or glycine,
T, U, V and W are independently D- or L-leucine, D- or L-isoleucine, D- or L-alanine, D- or L-valine, D- or L-alloisoleucine, glycine, D- or L-glutamic acid, D- or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either
residues T, U, V, or W from the peptide formulas I or
II,
Z is D- or L-glutamine, D- or L-glutamic acid and D- or L-asparagine,
R1 is H (signifying a free N-terminal group), formyl, lower alkanoyl, aroyl or desamino (meaning the amino acid adjacent to the group R1, either X in formula I or P in formula 2 lacks the α-amino group of the amino acid, and is replaced with H),
R2 is H (signifying in a free C-terminal
carboxylic acid), O(lower alkyl), O(aryl), NR3R4 where R3 and R4 are independently H or lower alkyl, or descarboxy (meaning the o;-carboxylic acid group of the amino acid to which R1 is adjacent in formula I or 2, Y or Z, respectively, is replaced with H),
X and Y are linear chains of from one to ten amino acids.
Preferred peptides are those of Formula I wherein R1 is H and R2 is NR3R4, and Formula II wherein R1 is H or acetyl and R1 is NR3R4, wherein S is
aspartic acid, glutamic acid or histidine.
Most preferred peptides are Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; Tyr-Thr-His-Leu-Val-Ala-Ile-Gln-NH2; Acetyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-LysAsn-Glu-NH2; D-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; Tyr-D-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; Tyr-Thr-D-Asp-Leu-Val-Ala-Ile-Gln-NH2; Phe-Thr-Asp-Leu-Val-Ala-Ile- Gln-NH2; Tyr-Thr-D-Asp-Leu-Val-Ala-Ile-Gln-NH2; Tyr-Thr-Asp-Leu-Val-Ala-D-Ile-Gln-NH2; Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln-NH2; Tyr-Thr-Ala-Leu-Val-Ala-Ile-GlnNH2; Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; Arg-Gly-His-Leu-Val-Ala-Ile-Gln-NH2; and Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2.
As used herein, the term "lower alkyl" includes branched, straight-chain, and cyclic saturated
hydrocarbons having from one to six carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, and hexyl. The term "lower alkanoyl" means RC(O), wherein R a lower alkyl group. The term aroyl means where ArC(O), wherein Ar is an aryl group, an aromatic or
heteroaromatic structure having between one and three rings, which may or may not be ring fused structures, and are optimally substituted with halogens, carbons, or other heteroatoms such as nitrogen (N), sulfur (S), phosphorus (P), and boron (B).
The peptides of formula I can be used in the form of the free peptide or a pharmaceutically
acceptable salt. Amine salts can be prepared by mixing the peptide with an acid according to known methods. Suitable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, and sulfanilic acid. Carboxylic acid groups in the peptide can be converted to a salt by mixing the peptide with a base according to known methods. Suitable bases include inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases such as mono-, di-, and tri-alkyl and aryl amines (e.g., triethylamine, disopropylamine, methylamine, and dimethylamine and optionally substituted mono-, di, and tri-ethanolamines.
As referred to herein, the amino acid components of the peptides and certain materials used in their preparation are identified by abbreviations for convenience. These abbreviations are as follows:
Amino Acid Abbreviations
L-alanine Ala A
D-alanine D-Ala a
L-allosoleucine Alle
P-alloisoleucine D-AIle
L-arginine Arg R
D-arginine D-Arg r
D-asparagine D-Asn N
L-asparagine L-Asn n
L-aspartic acid Asp D
D-aspartic acid D-Asp d
L-cysteine Cys C
D-cysteine D-Cys c
L-glutamic acid Glu E
D-glutamic acid D-Glu e
L-glutamine Gln K
D-glutamine D-Gln k glycine Gly G
L-histidine His H
D-histidine D-His h
L-isolelucine Ile I
D-isoleucine D-Ile i
L-leucine Leu L
D-leucine D-Leu l
L-lysine Lys K
D-lysine D-Lys k
L-phenylalanine Phe F
D-phenylalanine D-Phe f
L-proline Pro P
D-proline D-Pro P
L-pyroglutamic acid pGlu
D-pyroglutamic acid D-pGlu
L-serine L-Ser s
D-serine D-Ser s
L-threonine L-Thr T
D-threonine D-Thr t
L-tyrosine L-Tyr Y
D-tyrosine D-Tyr y
L-tryptophan Trp w
D-tryptophan D-Trp w
L-valine Val V
D-valine D-Val V Reagents Abbreviations
Trifluoroacetic acid TFA
Methylene chloride CH2Cl,
N,N-Diisopropylethylamine DIEA
N-Methylpyrrolidone NMP
1-Hydroxybenzotriazole HOBT
Dimethylsulfoxide DMSO
Acetic anhydride Ac2O
Methods of Preparation of Peptides
The peptides can generally be prepared following known techniques, as described for example in the cited publications, the teachings of which are specifically incorporated herein. In a preferred method, the peptides are prepared following the solid- phase synthetic technique initially described by
Merrifield in J.Amer.Chem.Soc., 85, 2149-2154 (1963). Other techniques may be found, for example, in M.
Bodanszky, et al., Peptide Synthesis, second edition, (John Wiley & Sons, 1976), as well as in other
reference works known to those skilled in the art.
Appropriate protective groups usable in such syntheses and their abbreviations will be found in the above text, as well as in J.F.W. McOmie, Protective Groups in Organic Chemistry. (Plenum Press, New York, 1973). The common protective groups used herein are t-butyloxycarbonyl (Boc), fluorenylmethoxycarbonyl (FMOC), benzyl (Bzl), tosyl (Tos), o-bromo-phenylmethoxycarbonyl (BrCBZ) , phenylmethoxycarbonyl (CBZ), 2-chloro-phenylmethoxycarbonyl, (2-Cl-CBZ), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), trityl (Trt), formyl (CHO), and tertiary butyl (t-Bu).
General synthetic procedures for the synthesis of peptides of Formula I and II by solid phase
methodology are as follows: A. General Synthetic Procedures for Solid Phase Peptide Synthesis Using Nα-Boc Protection.
REPETITIONS TIME
1. 25% TFA in CH2Cl2 1 3 min
2. 50% TFA in CH2Cl2 1 16 min
3. CH2Cl2 5 3 min
4. 5% DIEA in NMP 2 4 min
5. NMP 6 5 min
6. Coupling step 1 57 min a . Preformed BOC-Amino Acid- 36 min
HOBT active ester in NMP
b. DMSO 16 min c. DIEA 5 min
7. 10% Ac2O, 5% DIEA in NMP 1 9 min
8 . CH2Cl2 5 3 min
B. General Synthetic Procedure For Solid
Phase Peptide synthesis Using N**- FMOC Protection
REPETITIONS TIME
1. 20% piperdine in NMP 1 3 min
2. 20% piperdine in NMP 1 15 min
3. NMP 6 9 min
4. Coupling 1 71 min
Preformed FMOC-Amino AcidHOBT active ester in NMP
5. NMP 6 7 min
N-terminal acetylation on the deprotected Nα-amino group of peptides synthesized using either Boc or FMOC strategies is accomplished with 10% Ac2O and 5% DIEA in NMP, followed by washing of the peptide resin with NMP and/or CH2Cl2.
The peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art. For example, the peptide can be produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide in the presence of the required amino acids. The peptide is then purified using chromatographic or electrophoretic techniques, or by means of a carrier protein which can be fused to, and subsequently cleaved from, the peptide by inserting into the expression vector in phase with the peptide encoding sequence a nucleic acid sequence encoding the carrier protein. The fusion protein-peptide may be isolated using
chromatographic, electrophoretic or immunological techniques (such as binding to a resin via an antibody to the carrier protein). The peptide can be cleaved using chemical methodology or enzymatically, as by, for example, hydrolases.
Methods of Preparation of Pharmaceutical Compositions
To prepare the pharmaceutical compositions containing these peptides, a peptide of Formula I or II or a base or acid addition salt thereof is combined as the active ingredient with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. This carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., sublingual, rectal, nasal, oral, or parenteral. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, for example, water, oils, alcohols, flavoring agents, preservatives, and coloring agents, to make an oral liquid preparation (e.g., suspension, elixir, or solution) or with carriers such as
starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents, to make an oral solid preparation (e.g., powder, capsule, or tablet).
Controlled release forms or enhancers to
increase bioavailability may also be used. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are employed. If desired, tablets may be sugar coated or enteric coated by standard techniques.
For parenteral products, the carrier will usually be sterile water, although other ingredients to aid solubility or as preservatives may be included. Injectable suspensions may also be prepared, in which case appropriate liquid carriers and suspending agents can be employed.
The peptides can also be administered locally at a wound or inflammatory site by topical application of a solution or cream.
Alternatively, the peptide may be administered in liposomes or microspheres (or microparticles).
Methods for preparing liposomes and microspheres for administration to a patient are known to those skilled in the art. U.S. Patent No. 4,789,734 describe methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary. A good review of known methods is by G. Gregoriadis, Chapter 14. "Liposomes", Drug Carriers in Biology and Medicine pp. 287-341 (Academic Press, 1979).
Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract
directly into the bloodstream. Alternatively, the peptide can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time, ranging from days to months. See, for example, U.S. Patent No. 4,906,474,
4,925,673, and 3,625,214.
Methods for Demonstrating Binding
Peptides that are biologically active are those which inhibit binding of neutrophils, monocytes, subsets of lymphocytes or other cells to GMP-140, or which inhibit leukocyte adhesion to endothelium that is mediated by ELAM-1 and/or the homing receptor.
Peptides can be screened for their ability to inhibit adhesion to cells, for example, neutrophil adhesion to purified GMP-140 immobilized on plastic wells, using the assay described by Geng, et al.,
Nature 343. 757-760 (1990).
Human neutrophils are isolated from heparinized whole blood by density gradient centrifugation on Mono-Poly resolving media, Flow Laboratories.
Neutrophil suspensions are greater than 98% pure and greater than 95% viable by trypan blue exclusion. For adhesion assays, neutrophils are suspended at a concentration of 2 × 106 cells/ml in Hanks' balanced salt solution containing 1.26 mM Ca2+ and 0.81 mM Mg2+ (HBSS, Gibco) with 5 mg/ml human serum albumin
(HBSS/HSA). Adhesion assays are conducted in
triplicate in 96-well microtiter plates, Corning, incubated at 4°C overnight with 50 microliters of various protein solutions.
GMP-140 is isolated from human platelet lysates by immunoaffinity chromatography on antibody S12-Sepharose™ and ion-exchange chromatography on a Mono-Q™ column (FLPC, Pharmacia Fine Chemicals), as follows.
Outdated human platelet packs (100 units) obtained from a blood bank and stored at 4°C are pooled, adjusted to 5 mM EDTA at pH 7.5, centrifuged at 4,000 rpm for 30 min in 1 liter bottles, then washed three times with 1 liter of 0.1 M NaCl, 20 mM Tris pH 7.5 (TBS), 5 mM EDTA, 5 mM benzamidine.
The pellets are then resuspended in a minimum amount of wash buffer and made 1 mM in DIFP, then frozen in 50 ml screwtop tubes at -80°C. The frozen platelets are thawed and resuspended in 50 ml TBS, 5 mM benzamidine, 5 mM EDTA pH 7.5, 100 M leupeptin. The suspension is frozen and thawed two times in a dry ice-acetone bath using a 600 ml lyophilizing flask, then homogenized in a glass/teflon mortar and pestle and made 1 mM in DIFP. The NaCl concentration is adjusted to 0.5 M with a stock solution of 4 M NaCl. After stirring the suspension at 4°C, it is
centrifuged in polycarbonate tubes at 33,000 rpm for 60 min at 4°C. The supernatant (0.5 M NaCl wash) is removed and saved; this supernatant contains the £ soluble form of GMP-140. Care is taken not to remove the top part of the pellet with the supernatant. The pellets are then homogenized in extraction buffer (TBS, 5 mM benzamidine, 5 mM EDTA, pH 7.5, 100 μM leupeptin, 2% Triton X-100). After centrifugation at 19,500 rpm for 25 min at 4°C, the supernatant is removed. The extraction procedure is repeated with the pellet and the supernatant is combined with the first supernatant. The combined extracts, which contain the membrane form of GMP-140, are adjusted to 0.5 M NaCl.
The soluble fraction (0.5 M NaCl wash) and the membrane extract (also adjusted to 0.5 M NaCl) are absorbed with separate pools of the monoclonal
antibody S12 (directed to human GMP-140) previously coupled to Affigel (Biorad) at 5 mg/ml for 2 h at 4°C. After letting the resins settle, the supernatants are removed. The S12 Affigel containing bound GMP-140 is then loaded into a column and washed overnight at 4°C with 400 ml of 0.5 M NaCl, 20 mM Tris pH 7.5, 0.01% Lubrol PX.
Bound GMP-140 is eluted from the S12 Affigel with 100 ml of 80% ethylene glycol, 1 mM MES pH 6.0, 0.01% Lubrol PX. Peak fractions with absorbance at 280 nm are pooled. Eluates are dialyzed against TBS with 0.05% Lubrol, then applied to a Mono Q column (FPLC from Pharmacia). The concentrated protein is step eluted with 2 M NaCl, 20 mM Tris pH 7.5 (plus 0.05% Lubrol PX for the membrane fraction). Peak fractions are dialyzed into TBS pH 7.5 (plus 0.05% Lubrol PX for the membrane fraction).
GMP-140 is plated at 5 micrograms/ml and the control proteins: human serum albumin (Alb), platelet glycoprotein Hb/IIIa (IIb), von Willebrand factor (vWF), fibrinogen (FIB), thrombomodulin (TM), gelatin (GEL) or human serum (HS), are added at 50
micrograms/ml. All wells are blocked for 2 h at 22°C with 300 microliters HBSS containing 10 mg/ml HSA, then washed three times with HBSS containing 0.1% Tween-20 and once with HBSS. Cells (2 × 105 per well are added to the wells and incubated at 22°C for 20 min. The wells are then filled with HBSS/HSA, sealed with acetate tape (Dynatech), and centrifuged inverted at 150 g for 5 min. After discarding nonadherent cells and supernates, the contents of each well are solubilized with 200 microliters 0.5%
hexadecyltrimethylammonium bromide, Sigma, in 50 mM potassium phosphate, pH 6.0, and assayed for
myeloperoxidase activity, Ley, et al., Blood 73, 1324- 1330 (1989). The number of cells bound is derived from a standard curve of myeloperoxidase activity versus numbers of cells. Under all assay conditions, the cells release less than 5% of total
myeloperoxidase and lactate dehydrogenase. Inhibition is read as a lower percent adhesion, so that a value of 5% means that 95% of the specific adhesion was inhibited.
Clinical Applications.
The subject peptides are generally active when administered parenterally in amounts above about 1 μg peptide/kg of body weight. For treatment to prevent organ injury in cases involving reperfusion, the peptides may be administered parenterally from about 0.01 to about 10 mg peptide/kg body weight. Generally, the same range of dosage amounts may be used in treatment of the other diseases or conditions where inflammation is to be reduced. This dosage will be dependent, in part, on whether one or more peptides are administered. A synergistic effect may be seen with combinations of peptides from different, or overlapping, regions of the lectin domain, or in combination with peptides derived from the EGF domain of GMP-140.
Since the selectins have several functions related to leukocyte adherence, inflammation, and coagulation, clinically, compounds which interfere with binding of GMP-140, ELAM-1 or LEU-8 can be used to modulate these responses.
For example, the peptides can be used to
competitively inhibit leukocyte adherence by
competitively binding to GMP-140 receptors on the surface of leukocytes. This kind of therapy would be particularly useful in acute situations where
effective, but transient, inhibition of leukocyte-mediated inflammation is desirable. Chronic therapy by infusion of the peptides may also be feasible in some circumstances.
An inflammatory response may cause damage to the host if unchecked, because leukocytes release many toxic molecules that can damage normal tissues. These molecules include proteolytic enzymes and free
radicals. Examples of pathological situations in which leukocytes can cause tissue damage include injury from ischemia and reperfusion, bacterial sepsis and disseminated intravascular coagulation, adult respiratory distress syndrome, tumor metastasis, rheumatoid arthritis and atherosclerosis.
Reperfusion injury is a major problem in
clinical cardiology. Therapeutic agents, that reduce leukocyte adherence in ischemic myocardium can
significantly enhance the therapeutic efficacy of thrombolytic agents. Thrombolytic therapy with agents such as tissue plasminogen activator or streptokinase can relieve coronary artery obstruction in many patients with severe myocardial ischemia prior to irreversible myocardial cell death. However, many such patients still suffer myocardial neurosis despite restoration of blood flow. This "reperfusion injury" is known to be associated with adherence of leukocytes to vascular endothelium in the ischemic zone,
presumably in part because of activation of platelets and endothelium by thrombin and cytokines that makes them adhesive for leukocytes (Romson et al.,
Circulation 67: 1016-1023, 1983). These adherent leukocytes can migrate through the endothelium and destroy ischemic myocardium just as it is being rescued by restoration of blood flow.
There are a number of other common clinical disorders in which ischemia and reperfusion results in organ injury mediated by adherence of leukocytes to vascular surfaces, including strokes; mesenteric and peripheral vascular disease; organ transplantation; and circulatory shock (in this case many organs might be damaged following restoration of blood flow).
Bacterial sepsis and disseminated intravascular coagulation often exist concurrently in critically ill patients. They are associated with generation of thrombin, cytokines, and other inflammatory mediators, activation of platelets and endothelium, and adherence of leukocytes and aggregation of platelets throughout the vascular system. Leukocyte-dependent organ damage is an important feature of these conditions.
Adult respiratory distress syndrome is a
devastating pulmonary disorder occurring in patients with sepsis or following trauma, which is associated with widespread adherence and aggregation of
leukocytes in the pulmonary circulation. This leads to extravasation of large amounts of plasma into the lungs and destruction of lung tissue, both mediated in large part by leukocyte products.
Two related pulmonary disorders that are often fatal are in immunosuppressed patients undergoing allogeneic bone marrow transplantation and in cancer patients suffering from complications that arise from generalized vascular leakage resulting from treatment with interleukin-2 treated LAK cells (lymphokine-activated lymphocytes). LAK cells are known to adhere to vascular walls and release products that are presumably toxic to endothelium. Although the
mechanism by which LAK cells adhere to endothelium is not known, such cells could potentially release molecules that activate endothelium and then bind to endothelium by mechanisms similar to those operative in neutrophils.
Tumor cells from many malignancies (including carcinomas, lymphomas, and sarcomas) can metastasize to distant sites through the vasculature. The
mechanisms for adhesion of tumor cells to endothelium and their subsequent migration are not well
understood, but may be similar to those of leukocytes in at least some cases. The association of platelets with metastasizing tumor cells has been well
described, suggesting a role for platelets in the spread of some cancers.
Platelet-leukocyte interactions are believed to be important in atherosclerosis. Platelets might have a role in recruitment of monocytes into
atherosclerotic plaques; the accumulation of monocytes is known to be one of the earliest detectable events during atherogenesis. Rupture of a fully developed plaque may not only lead to platelet deposition and activation and the promotion of thrombus formation, but also the early recruitment of neutrophils to an area of ischemia. Another area of potential application is in the treatment of rheumatoid arthritis.
The criteria for assessing response to
therapeutic modalities employing these peptides are dictated by the specific condition and will generally follow standard medical practices. For example, the criteria for the effective dosage to prevent extension of myocardial infarction would be determined by one skilled in the art by looking at marker enzymes of myocardial necrosis in the plasma, by monitoring the electrocardiogram, vital signs, and clinical response. For treatment of acute respiratory distress syndrome, one would examine improvements in arterial oxygen, resolution of pulmonary infiltrates, and clinical improvement as measured by lessened dyspnea and tachypnea. For treatment of patients in shock (low blood pressure), the effective dosage would be based on the clinical response and specific measurements of function of vital organs such as the liver and kidney following restoration of blood pressure. Neurologic function would be monitored in patients with stroke. Specific tests are used to monitor the functioning of transplanted organs; for example, serum creatinine, urine flow, and serum electrolytes in patients
undergoing kidney transplantation.
Diagnostic Reagents.
The peptides can also be used for the detection of human disorders in which the ligands for the selectins might be defective. Such disorders would most likely be seen in patients with increased
susceptibility to infections in which leukocytes might not be able to bind to activated platelets or
endothelium. Cells to be tested, usually leukocytes, are collected by standard medically approved
techniques and screened. Detection systems include ELISA procedures, binding of radiolabeled antibody to immobilized activated cells, flow cytometry, or other methods known to those skilled in the arts.
Inhibition of binding in the presence and absence of the lectin domain peptides can be used to detect defects or alterations in selectin binding. For selectins, such disorders would most likely be seen in patients with increased susceptibility to infections in which leukocytes would have defective binding to platelets and endothelium because of deficient
leukocyte ligands for GMP-140. The peptide is labeled radioactively, with a fluorescent tag, enzymatically, or with electron dense material such as gold for electron microscopy. The cells to be examined, usually leukocytes, are incubated with the labeled peptides and binding assessed by methods described above with antibodies to GMP-140, or by other methods known to those skilled in the art. If ligands for GMP-140 are also found in the plasma, they can also be measured with standard ELISA or radioimmunoassay procedures, using labeled GMP-140-derived peptide instead of antibody as the detecting reagent.
The following examples are presented to
illustrate the invention without intending to
specifically limit the invention thereto. In the examples and throughout the specifications, parts are by weight unless otherwise indicated.
EXAMPLE 1: Preparation of Tyrosyl-threonyl-histidyl- leucyl-valyl-alanyl-isoleucyl-glutamine- amide.
The peptide was prepared on an ABI model 431A peptide synthesizer using Version 1.12 of the standard scale Boc software. The amino acids used were Boc-(BrCBZ)Tyr, Boc-(Bzl)Thr, Boc-(Tos)His, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile and Boc-Gln. 4-Methylbenzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 1.13 g. The peptide was cleaved from the resin (1.03 g) using 11 mL of HF and 1.1 mL of anisole for 60 min at 0° C. The hydrogen fluoride was evaporated using a stream of nitrogen and the resulting mixture triturated with ether. The solids were removed by filtration and extracted with 25 mL of a 50% solution of TFA in methylene chloride. Removal of the resin by filtration, evaporation of the solvent and trituration of the residue with ether gave 0.52 g of crude
peptide.
The crude peptide (55 mg) was purified on a Vydac C-18 column (10 μ 2.2 × 25 cm), eluting with a gradient of 10 to 20% of acetonitrile in 0.1% aqueous TFA over 20 minutes at a flow rate of 8 mL per minute. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 3.9 mg of purified peptide. Amino acid analysis: Ala 0.98 (1.0), Glx 1.02 (1.0), His 1.06 (1.0), Ile 1.07 (1.0), Leu 1.07 (1.0), Thr 0.85 (1.0), Tyr 0.85 (1.0), Val 0.94 (1.0). FAB/MS: MH+ 944 (calcd 944).
EXAMPLE 2: Preparation of Tyrosyl-threonyl-glutamyl- leucyl-valyl-alanyl-isoleucyl-glutamine- amide.
The peptide was prepared on a ABI model 431A peptides synthesizer using Version 1.12 of the
standard scale Boc software. The amino acids used were Boc-(BrCBZ)Tyr, Boc-(Bzl)Thr, Boc-(Bzl)Glu, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ill, Boc-Gln. 4-methylbenzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. Final weight of the resin was 1.20 g. The peptide was cleaved from the resin (1.10 g) using 11 mL of HF and 1.1 mL of anisole for 60 minutes at 0° C. The hydrogen fluoride was removed using a stream of dry nitrogen, the residue triturated with ether and the ether removed by filtration. The remaining solids were triturated with 25 mL of a 50% solution of TFA in methylene chloride. The resin was removed by filtration, the solution evaporated under reduced pressure and the residue triturated with ether to give 0.40 g of the crude peptide, isolated by filtration. The crude peptide (0.31 g) was purified by HPLC (multiple injections) on a Vydac C-18 column (10 μ, 2.2 × 25 cm) eluting with 30% acetonitrile in 0.1% aqueous TFA over 90 minutes at a flow rate of 8 mL/min. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 47 mg of pure peptide. Amino acid analysis: Ala 1.01 (1.0), Glx 2.00 (2.0), Ile 1.02 (1.0), Leu 1.06 (1.0), Thr 0.78 (1.0), Tyr 0.98 (1.0), Val 0.93 (1.0).
FAB/MS: MH+ 936 (calcd 936).
EXAMPLE 3: Preparation of Acetyl-tyrosyl-threonyl- aspartyl-leucyl-valyl-alanyl-isoleucyl- glutamine amide.
The peptide was prepared on ABI model 431A peptide synthesizer using Version 1.12 of the standard scale Boc software modified for N-terminal acetylation according the instrument operations manual. 4-Methylbenzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. Final weight of the peptide resin was 1.23 g. The peptide was cleaved from the resin (1.11 g) with 10 mL of HF and 1 mL of anisole for 60 minutes at 0° C. The HF was removed by a nitrogen stream. The resulting solid was triturated with ether, collected by filtration and washed with ether. The peptide was extracted from the resin with 50% TFA and methylene chloride (5 × 20 mL). The resin was removed by filtration, the solvents removed under reduced pressure and the residue triturated with ether to give 0.50 g of crude peptide. The crude peptide was purified by preparation HPLC using a Vydac C-18 column (10 μ, 2.2 × 25 cm) eluting with a 20 to 30% gradient of acetonitrile and 0.1% aqueous TFA over 140 minutes at a flow rate of 3 mL per minute. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 60 mg of the purified peptide as a white solid. Amino acid analysis: Tyr 0.99 (1.0), Thr 0.91 (1.0), Asx 0.98 (1.0), Leu 1.03 (1.0), Val 1.05 (1.0), Ala 1.03 (1.0), Ile 1.00 (1.0), Glx 0.01 (1.0).
EXAMPLE 4: Preparation of Tyrosyl-threonyl-aspartyl- leucyl-valyl-alanyl-isoleucyl-glutamine amide.
The peptide was prepared by manual solid phase synthesis using Boc chemistry. The amino acids used were Boc-(BrCBZ)Tyr, Boc-(Bzl)Thr, Boc-(Bzl)Asp, Boc- Leu, Boc-Val, Boc-Ile and Boc-Gln. 4- Methylbenzhydrylamine resin (6.25 g, 5.0 mmol) was used in synthesis. 20 Mmol of each Boc-AA was
activated by dicyclohexylcarbodiimide and
hydroxybenzotriazole (20 mmol of each) and coupled to the resin. The sequence used was as follows:
WASH REPETITIONS TIME(mins)
25% TFA/CH2Cl2 1 3
50% TFA/CH2Cl2 1 16
CH2Cl2 1 13
5% N-methylmorpholine/CH2Cl2 1 4
CH2Cl2 1 3
Coupling step (monitored by ninhydrin testing of a resin sample).
The final weight of the peptide-resin was 11.97 g. The resin-peptide (11.8 g) was treated with 12 mL of anisole and 120 mL of HF for one hour at 0° to 4°C. The HF was removed by nitrogen stream followed by aspiration. The resultant solids were triturated with ether (1 × 100 mL then 1 × 80 mL), collected by
filtration and washed with ether (3 × 100 mL). The residue was then extracted with 50% trifluoroacetic acid/methylene chloride (4 × 50 mL), and the solvents removed by vacuum. The residue was triturated with 500 mL of diethyl ether. The solids were collected by filtration and air-dried overnight at ambient temperature followed by drying in vacuo at room temperature for 1 hour. The yield of crude peptide was 4.08 g. The crude peptide (106 mg) was purified in two 53 mg runs by reverse phase HPLC using Vydac 22 × 250 mm C-18, 10 μ 300 Angstrom pore packed column. Elution with a gradient of 25% to 40% B over 72 minutes at a flow rate of 6 mL/min was carried out (solvent A = 0.1% TFA; solvent B = 0.1% TFA in 50% acetonitrile/Water). Fractions were collected and the appropriate fractions pooled to give 56.8 mg of wtlvte solid. Amino Acid Analysis: Asx 1.02 (1.00), Thr 0.89 (1.00), Glx 0.99(1.00, Ala 1.02 (1.00), Val 0.96 (1.00), Ile 1.03 (1.00), Leu 1.06 (1.00), Tyr 0.91 (1.00). FAB/MS: MH+ = 922 (calcd 922).
EXAMPLE 5: Preparation of Arginyl-tyrosyl-threonyl- aspartyl-leucyl-valyl-alanyl-isoleucyl- glutamine amide.
The peptide was prepared on a DuPont RAMPS system using the FMOC strategy. The amino acids used for the synthesis were FMOC-(Mtr)Arg, FMOC-(t-Bu) Tyr, FMOC-(t-Bu) Thr, FMOC-(t-Bu)Asp, FMOC-Leu, FMOC-Val, FMOC-Ala, FMOC-Ile and FMOC-Gln. DuPont rapid amide resin (0.1 mmol) was used in the synthesis. The peptide was cleaved from the resin using a mixture of phenol (0.25 g), ethanedithiol (0.083 mL), thioanisole (0.66 mL), water (0.166 mL) and trifluoroacetic acid (3.33 mL) for 6 hours for 20° C. The resin was removed by filtration and the peptide precipitated from the filtrate by the addition of ether. The solids were removed by filtration, extracted with 20% acetic acid and lyophilized to give 0.147 g of crude peptide. The peptide was purified by preparative reverse phase (C-18) HPLC using an acetonitrile-water gradient in 0. 1% in TFA. Fractions were collected and those containing pure peptide were pooled and
lyophilized. EXAMPLE 6: Preparation of Asparaginyl-arginyl-tyrosyl- threonyl-aspartyl-leucyl-valyl-alanyl- isoleucyl-glutamine amide.
The peptide was prepared using a DuPont RAMPS system and the FMOC strategy. The amino acids used were FMOC-Asn, FMOC-(Mtr)Arg, FMOC-(t-Bu)Tyr, FMOC- (t-Bu)Thr, FMOC- (t-Bu) Asp, FMOC-Leu, FMOC-Val, FMOC- Ala, FMOC-Ile and FMOC-Gln. DuPont rapid amide resin (0.2 mmol) was used in the synthesis. The peptide was cleaved in the resin using a mixture of TFA (2.85 mL), thioanisole (0.135 mL) and ethanedithiol (0.015 mL) for 16 hours at ambient temperature. The resin was removed by filtration and the peptide precipitated from the filtrate by the addition of ether. The peptide was removed by filtration, extracted with 20% acetic acid and lyophilized to give 50 mg of crude peptide. The crude peptide was purified by
preparative reverse phase (C-18) HPLC using a gradient of acetonitrile water in 0.1% TFA. Fractions were collected and those containing the pure peptide were pooled and lyophilized.
EXAMPLE 7: Preparation of Cysteinyl-glutaminyl- asparaginyl-arginyl-tyrosyl-threonyl- aspartyl-leucyl-valyl-alanyl-isoleucyl- glutaminyl-asparaginyl-lysyl-asparaginyl- glutamine.
The peptide was prepared on an ABI model 430A peptides synthesizer using the standard scale Boc software. The amino acids used were Boc-(4-Me-Bzl)Cys, Boc-Gln, Boc-Asn, Boc-(Tos)Arg, Boc- (BrCBZ)Tyr, Boc-(Bzl)Thr, Boc-(Bzl) Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile, Boc-(Cl-CBz)Lys. Boc-(Bzl)Glu-Pam resin (0.5 mmol) was used in the synthesis. The peptide was cleaved from the resin using 10 mL of HF, 1.0 mL of anisole, 1.0 mL of dimethyl sulfide and 0.2 mL p-thiocresol for 30 minutes at -10° C followed by 30 minutes at 0°C. The hydrogen fluoride was removed under reduced pressure and the residue triturated with ether. Solids were removed by filtration and the peptide extracted from the resin using 20% acetic acid. Removal of the resin by filtration and
lyophilization of the filtrate gave 25 mg of crude peptide. The crude peptide was purified by the preparative HPLC using a 2.2 × 25 cm Synchrom C-18 column (6.5 μ), eluting with a gradient of 5 to 25% acetonitrile in 0.1% TFA over 20 minutes at a flow rate of 6 mL per minute. Fractions were collected and the fractions containing the pure peptide and pooled lyophilized.
EXAMPLE 8: Preparation of Tyrosyl-threonyl-D-aspartyl- leucyl-valyl-alanyl-isoleucyl-glutamine amide.
The peptide was prepared on an ABI Model 431A peptide synthesizer using Version 1.12 of the standard Boc software. The amino acids used were Boc- (BrCBZ)Tyr, Boc-(Bzl)Thr, Boc-D-(Bzl)Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile and Boc-Gln. 4-Methylbenzhydrylamine resin (0.63 g, 0.5 mmol) was used in the synthesis. The final weight of the peptide resin was 1.43 g. The peptide was cleaved from the resin (1.43 g) using 15 mL of HF and 1.5 mL of anisole for 60 min at 0°C. The hydrogen fluoride was removed under reduced pressure and the residue triturated with ether. Solids were removed by
filtration and the peptide extracted from the resin using a 50% solution of trifluoroacetic acid in methylene chloride. Removal of the resin by
filtration and precipitation with ether gave 1.09 g of crude peptide. The crude peptide (0.5 g) was purified by preparative HPLC using a Vydac C-18 column (15 μ, 5.0 × 25 cm) eluting with a 0-100% gradient of 50% acetonitrile in 0.1% TFA over 120 minutes at a flow rate of 15 mL per minute. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 200 mg of the desired product. Amino acid analysis: Ala 0.99 (1.00), Asx 1.01
(1.00), Glx 1.02 (1.00), Ile 0.97 (1.00), Leu 1.02
(1.00), Thr 0.91 (1.00), Tyr 0.96 (1.00), Val 1.04
(1.00). FAB/MS: MH+ = 921
EXAMPLE 9: Preparation of Phenylalanyl-threonyl- aspartyl-leucyl-valyl-alanyl-isoleucyl- glutamine amide.
The peptide was prepared on an ABI Model 431A peptide synthesizer using Version 1.12 of the standard Boc software. The amino acids used were Boc-Phe, Boc- (Bzl)Thr, Boc-(Bzl)Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile and Boc-Gln. 4-Methylbenzhydrylamine resin
(0.625 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 1.18 g. The peptide was cleaved from the resin (1.10 g) using 11 mL of HF and 1.1 mL of anisole for 60 min at 0°C. The hydrogen fluoride was evaporated using a stream of nitrogen and the resulting mixture triturated with ether. Solids were removed by filtration and extracted with 25 mL of a 50% solution of trifluoroacetic acid in methylene chloride. Removal of the resin by filtration,
evaporation of the solvent and trituration of the residue with ether gave 0.52 g of crude peptide. The crude peptide (82 mg) was purified on a Vydac C-18 column (10 μ, 2.2 × 25 cm) eluting with a 0-60% gradient of acetonitrile and 1% TFA over 60 minutes at a flow rate of 8 mL per minute. Fractions were collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 27 mg. Amino acid analysis: Ala 1.01, (1.0), Asx 1.02 (1.0), Glx 1.00 (1.0), Ile 0.96 (1.0), Leu 1.01 (1.0), Phe 0.94 (1.0), Thr 0.85 (1.0), Val 1.02 (1.0). FAB/MS: MH+ 906. EXAMPLE 10: Preparation of D-Tyrosyl-threonyl- aspartyl-leucyl-valyl-alanyl- isoleucyl-glutamine amide.
The peptide was prepared on a ABI Model 431A peptide synthesizer using Version 1.12 of the standard scale Boc software. The amino acids used were Boc-D- (BrCBZ)Tyr, Boc-(Bzl) Thr, Boc-(Bzl)Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile, Boc-Gln. 4-Methylbenzhydrylamine resin (0.665 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 2.10 g. The peptide was cleaved from the resin (2.10 g) using 20 mL of hydrogen fluoride and 2 mL of anisole for 60 minutes at 0°C. The hydrogen fluoride was evaporated using a stream of nitrogen and the resulting mixture triturated with ether. The solids were removed by filtration and extracted with a 50% solution of trifluoroacetic acid in methylene
chloride. Removal of the resin by filtration,
evaporation of the solvent and trituration of the residue with ether gave 1.96 g of crude peptide containing residual solvents. The crude peptide (200 mL) was purified on a Vydac C-18 column (10 μ, 2.2 × 25 cm) eluting with a 0-100% gradient of 50%
acetonitrile and 0.1% TFA over 120 minutes at a flow rate of 15 mL per minute. Fractions were collected, analyzed by HPLC and pure fractures pooled and
lyophilized to give 52 mg of pure product. Amino acid analysis: Ala 0.99 (1.0), Asx 1.04 (1.0), Glx 1.00 (1.0), Ile 0.96 (1.0), Leu 0.99 (1.0), Thr 0.92 (1.0), Tyr 0.99 (1.0), Val 1.04 (1.0). FAB/MS: MH+ 921.
EXAMPLE 11: Preparation of Tyrosyl-D-threonyl- aspartyl-leucyl-valyl-alanyl- isoleucyl-glutamine amide.
The peptide was prepared on an ABI Model 431A peptide synthesizer using Version 1.12 of the standard Boc software. The amino acids used were: Boc-(BrCBZ)Tyr, Boc-D-(Bzl)Thr, Boc-(Bzl)Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile and Boc-Gln. 4- Methylbenzhydrylamine resin (0.685 g, 0.5 mmol) was used in the synthesis. The final weight of the peptide resin was 1.63 g. The peptide was cleaved from the resin (1.63 g) using 20 mL of hydrogen fluoride and 2 mL of anisole for 60 min at 0°C. The hydrogen fluoride was removed under reduced pressure and the residue triturated with ether. The solids were removed by filtration and the peptide extracted from the resin with a 50% solution of trifluoroacetic acid in methylene chloride. Removal of the resin by filtration and precipitation with ether gave 0.52 g of crude peptide. The crude peptide (0.50 g) was
purified on a Vydac C-18 column (15 μ, 5.0 × 25 cm) in four injections, eluting with a 0-100% gradient of 50% acetonitrile in 0.1% TFA over 120 minutes at a flow rate of 15 mL per minute. Fractions were
collected, analyzed by HPLC and pure fractions pooled and lyophilized to give 254 mg of pure peptide. Amino acid analysis: Ala 1.01 (1.0), Asx 1.08 (1.0), Glx 0.98 (1.0), Ile 0.98 (1.0), Leu 0.97 (1.0), Thr 0.95 (1.0), Tyr 0.98 (1.0), Val 1.01 (1.0). FAB/MS: MH+ 922.
EXAMPLE 12: Inhibition of Neutrophil Binding to
GMP140 Coated Wells.
Binding of various peptides to GMP-140 coated wells, as described above, were compared. The results are shown in Figure 1.
Binding of the peptides at various
concentrations, ranging from 0 to 1.5 mM, were
compared. The peptides tested were Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Acetyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-amide; Tyr-Thr-His-Leu-Val-Ala-Ile-Gln-amide; Tyr-Thr-Asp-Leu- Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-amide; Leu-Gln-Thr-Ala-Tyr-Asp-Val-Ile-amide (negative control).
In subsequent studies, additional peptides were tested for activity in inhibiting neutrophil addition to immobilized P-selectin. The IC50 data is presented in mM concentration. The results are shown below in Table 1.
TABLE l: INHIBITION OF BINDING OF NEUTROPHILS BY
PEPTIDES
STRUCTURE IC50 (mM)
CRGDLVAIQ-NH2 .132
QNRYTDLVAIQ-NH2 .034
NRYTDLVAIQ-NH2 .039
NRYTDLVAIQNKNE-NH2 .615
CYTDLVAIQ-NH2 .364
YTDLVAIQ-NH, .262
Formyl-YTDLVAIQ-NH2 .931
4-Br-Phe-TDLVAIQ-NH2 .090
4-NH2-Phe-TDLVAIQ-NH2 .383
Ac-RGDLVAIQ-NH2 .179
YTALVAIQ-NH2 .184
YTDAVAIQ-NH2 .347
RGHLVAIQ-NH2 .033
RGDLVAIQ-NH2 .041
RTDLVAIQ-NH2 .622
YTNLVAIQ-NH2 .271
YTDLVAIN-NH2 .187
CGDLVAIQ-NH2 .304
YTDLVaIQ-NH2 .420
YTdLVAIQ-NH2 .385
YTDLVAIq-NH2 .146
YTDLVAiQ-NH2 .162
YTDlVAIQ-NH2 .869
YtDLVAIQ-NH2 .271 yTdLVAIQ-NH2 .216 yTDLVAIQ-NH2 .316
YTDLVIQ-NH2 .463
YTDVAIQ-NH2 .296 desamino-Arg-TDLVAIQ-NH2 .707 desamino-Tyr-TDVAIQ-NH2 .416
YTDLVAI-descarboxy-Gln-NH2 .391
YTQLVAIQ-NH2 .097 TABLE 1 CONTINUED
STRUCTURE IC50(mM)
YTDLVAIQ-NH-n-Bu .151
ETDLVAIQ-NH2 .704
YEDLVAIQ-NH2 .267
YTELVAIQ-NH2 .389
YTDEVAIQ-NH2 .668
YGDLVAIQ-NH2 .977
YTHLVAIQ-NH2 .081
KTDLVAIQ-NH2 .226
YKDLVAIQ-NH2 .210
YTKLVAIQ-NH2 .060
N-Me-Tyr-TDLVAIQ-NH2 .336
Nal-TDLVAIQ-NH2 .192
O-Me-Tyr-TDLVAIQ-NH2 .103
FTDLVAIQ-NH2 .384
YTFLVAIQ-NH2 .234
Pya-TDLVAIQ-NH2 .346
YSDLVAIQ-NH2 .202
YTDLTAIQ-NH2 .359
Tic-TDLVAIQ-NH2 .062
YTDVAAIQ-NH2 .893
YTGLVAIQ-NH2 .134
Ac-YtHLVAIq-NH-n-Bu .366
Ac-YtDLVAIQ-NH-n-Bu .676
YTDLVAIQN-NH2 .153
YTDLVAIQNK-NH2 .199
Abbreviations: Nal - Naphthylalanine
O-Me-Tyr - O-methyltyrosine
N-Me-Tyr - Nα-methyltyrosine
Pya - Pyridylalanine
n-Bu - n-butyl
Tic - tetrahydroisoquinoline
carboxylic acid
The results demonstrate that, with the exception of the negative control, the peptides all inhibit neutrophil binding to immobilized GMP-140. EXAMPLE 13: Modification of peptide stability in serum.
Figure 2 shows the significant increase in stability against enzymes found in human serum that can be achieved by the modifications set down in Formula I and II, graphing percent of peptide
remaining versus time, for two peptides:
Ac-Y-T-D-L-V-A-I-Q-NH2 and
Y-T-D-L-V-A-I-Q-NH2.
The half-life in serum increased from 20 minutes for the unmodified peptide to four hours, 37 minutes for the modified peptide.
Modifications and variations of the present invention, synthetic peptides and methods for
modulating binding reactions involving selectins, will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the appended claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Heavner, George A.
McEver, Rodger P.
Geng, Jian-Guo
(ii) TITLE OF INVENTION: Peptide Inhibitors of Inflammation
(iii) NUMBER OF SEQUENCES: 45
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Kilpatrick & Cody
(B) STREET: 100 Peachtree Street
(C) CITY: Atlanta
(D) STATE: Georgia
(E) COUNTRY: US
(F) ZIP: 30303
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/699693
(B) FILING DATE: 14-MAY-1991
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Pabst, Patrea L.
(B) REGISTRATION NUMBER: 31,284
(C) REFERENCE/DOCKET NUMBER: OMRF-CTC100
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 404-572-6508
(B) TELEFAX: 404-658-6555
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Tyr Thr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Tyr Thr His Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Tyr Thr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln 1 5 10
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln
1 5 10 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Arg Tyr Thr Asp Leu Val Ala Ile Gln 1 5
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Tyr Thr Glu Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys Asn Glu 1 5 10
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Tyr Thr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Phe Thr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Tyr Thr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Tyr Thr Asp Leu Val Ala Ile Gln
1 5 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Tyr Thr Phe Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Tyr Thr Lys Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Lys Thr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Tyr Thr Ala Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Phe Thr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Tyr Thr Asp Ala Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Tyr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: C-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Cys Arg Gly Asp Leu Val Ala Ile Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln 1 5 10
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE : N-terminal
(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 22 :
Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn LysAsn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Cys Tyr Thr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Arg Gly His Leu Val Ala Ile Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Arg Gly Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Arg Thr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Tyr Thr Asn Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Tyr Thr Asp Leu Val Ala Ile Asn
1 5
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Cys Gly Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Tyr Thr Asp Leu Val Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Tyr Thr Asp Val Ala Ile Gln
1 5 (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Tyr Thr Gln Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Glu Thr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Tyr Glu Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Tyr Thr Asp Glu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Tyr Gly Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Lys Thr Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Tyr Lys Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
Tyr Thr Lys Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Tyr Ser Asp Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Tyr Thr Asp Leu Thr Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Tyr Thr Asp Val Ala Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Tyr Thr Gly Leu Val Ala Ile Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Tyr Thr Asp Leu Val Ala Ile Gln Asn 1 5
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys 1 5 10

Claims

We claim:
1. A peptide derived from selectins selected from the group consisting of the structures:
R1-X-P-Q-S-T-U-V-W-Z-Y-R2 (I)
R1-P-Q-S-T-U-V-W-Z-R2 (II)
or a pharmaceutically acceptable salt thereof,
wherein:
X in Formula (I) and P in Formula (II) are the N-terminus amino acids, and R1 is a moiety attached to the amine function (NHR1),
Y in Formula (I) and Z in Formula (II) are the C-terminus amino acids, and R2 is the moiety attached to the singly-bonded oxygen in the carboxy function (C(O)OR2),
P is D- or L-tyrosine, D- or L-phenylalanine, D- or L-lysine, D- or L-glutamic acid, D- or L-arginine, D- or L-cysteine, D- or L- O-R3-tyrosine, D- or L-Nα-R-tyrosine, D- or L-4-amino phenylalanine, D- or L-R-phenylalanine, D- or L-pyridylalanine, D- or L-naphthylalanine, or D- or L-tetrahydroisoguinoline carboxylic acid, where R3 is lower alkyl or aryl and R4 is halogen (fluorine, chlorine, bromine or iodine),
Q is D- or L-threonine, D- or L-lysine, D- or L-glutamic acid, D- or L-cysteine, or glycine,
S is D- or L-aspartic acid, D- or L-histidine, D- or L-glutamic acid, D- or L-asparagine, D or L-glutamine, D- or L-alanine, D- or L-phenylalanine, D-or L-lysine, or glycine,
T, U, V and W are independently D- or L-leucine, D- or L-isoleucine, D- or L-alanine, D- or L-valine, D- or L-alloisoleucine, glycine, D- or L-glutamic acid, D- or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either
residues T, U, V, or W from the peptide formulas I or
II, Z is D- or L-glutamine, D- or L-glutamic acid and D- or L-asparagine,
R1 is H (signifying a free N-terminal group), formyl, lower alkanoyl, aroyl or desamino (meaning the amino acid adjacent to the group R1, either X in formula I or P in formula 2 lacks the α-amino group of the amino acid, and is replaced with H),
R2 is H (signifying in a free C-terminal carboxylic acid), 0(lower alkyl), O(aryl), NR3R4 where R3 and R4 are independently H or lower alkyl, or descarboxy (meaning the α-carboxylic acid group of the amino acid to which R1 is adjacent in formula I or 2, Y or Z, respectively, is replaced with H), and
X and Y are linear chains of from one to ten amino acids.
2. The peptide of claim 1 having the structure
R1-X-P-Q-S-T-U-V-W-Z-Y-R2,
wherein R1 is H, X is Cys-Xxx-Xxx-Xxx, P is Tyr, T is Leu, U is Val, V is Ala, W is Ile, and Y is Asn- Lys-Xxx-Glu, where Xxx is any amino acid, and R2 is not OH.
3. The peptide of claim 1 of the structure
R1-X-P-Q-S-T-U-V-W-Z-Y-R2; wherein the peptide is selected from the group consisting of a peptide wherein X is Cys-Gln-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Gln-Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is pGlu-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X is Cys-Gln-Asp-Arg, S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X is Glu-Asn-Arg, P is Arg or Tyr, S is Asp, Lys, His, Asn, Gln, or Ala; and a peptide wherein X is Asn-Arg, P is Tyr or Arg, Q is Gly, and S is Asp, Lys, His, Asn, Gln, or Ala.
4. The peptide of claim 1 having the structure R1-P-Q-S-T-U-V-W-Z-R2 wherein S is Asp or
His.
5. The peptide of claim 1 selected from the group consisting of peptides having the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-His-Leu-Val-Ala-Ile-Gln-NH2; the formula Acetyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2;
the formula Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile- Gln-Asn-Lys-Asn-Glu-NH2; the formula D-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-D-Thr-Asp- Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-D-Asp- Leu-Val-Ala-Ile-Gln-NH2; the formula Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu--Val-Ala-D-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Ala-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Cys-Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-NH2; the formula Cys-Tyr- Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Formyl- Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4- Br-Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4- NH2-Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Ac-Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2;the formula Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln-NH2; the formula Arg- Gly-His-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Gly- Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Thr- Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asn- Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu- Val-Ala-Ile-Asn-NH2; the formula Cys-Gly-Asp-Leu-Val- Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-D- Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala- Ile-D-Gln-NH2; the formula Tyr-Thr-Asp-D-Leu-Val-Ala- Ile-Gln-NH2; the formula D-Tyr-Thr-D-Asp-Leu-Val-Ala- Ile-Gln-NH2; the formula D-Tyr-Thr-Asp-Leu-Val-Ala- Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Val-Ala-Ile-Gln-NH2; the formula desamino-Arg-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula desamino-Tyr-Thr-Asp-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-descarboxy-Gln-NH2; the formula Tyr-Thr-Gln-Leu-Val-Ala-Ile-GlnNH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH-n-Bu; the formula Glu-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Glu-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Glu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Lys-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula N-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Nal-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula O-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; the formula Pya-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Ser-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Thr-Ala-Ile-Gln-NH2; the formula Tic-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Val-Ala-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Gly-Leu-Val-Ala-Ile-Gln-NH2; the formula Ac-Tyr-D-Thr-His-Leu-Val-Ala-Ile-D-Gln-NH-n- Bu; the formula Ac-Tyr-D-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH-n-Bu; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-NH2; and pharmaceutically acceptable acid- or base-addition salts thereof.
6. The peptide of claim 1 in combination with a pharmaceutical carrier selected from the group
consisting of carriers suitable for parenteral
administration, oral administration, topical
administration, and controlled release formulations.
7. A method for the preparation of peptides of the structure
R1-X-P-Q-S-T-U-V-W-Z-Y-R2 (I)
R1-P-Q-S-T-U-V-W-Z-R2 (II) or a pharmaceutically acceptable salt thereof,
wherein:
X in Formula (I) and P in Formula (II) are the N-terminus amino acids, and R1 is a moiety attached to the amine function (NHR1),
Y in Formula (I) and Z in Formula (II) are the C-terminus amino acids, and R2 is the moiety attached to the singly-bonded oxygen in the carboxy function (C(O)OR2),
P is D- or L-tyrosine, D- or L-phenylalanine, D- or L-lysine, D- or L-glutamic acid, D- or L-arginine, D- or L-cysteine, D- or L- O-R3-tyrosine, D- or L-Nα-R3-tyrosine, D- or L-4-amino phenylalanine, D- or L- R4-phenylalanine, D- or L-pyridylalanine, D- or L- naphthylalanine, or D- or L-tetrahydroisoquinoline carboxylic acid, where R3 is lower alkyl or aryl and R4 is halogen (fluorine, chlorine, bromine or iodine),
Q is D- or L-threonine, D- or L-lysine, D- or L- glutamic acid, D- or L-cysteine, or glycine,
S is D- or L-aspartic acid, D- or L-histidine, D- or L-glutamic acid, D- or L-asparagine, D or L- glutamine, D- or L-alanine, D- or L-phenylalanine, D- or L-lysine, or glycine,
T, U, V and W are independently D- or L-leucine, D- or L-isoleucine, D- or L-alanine, D- or L-valine, D- or L-alloisoleucine, glycine, D- or L-glutamic acid, D- or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either
residues T, U, V, or W from the peptide formulas I or
II,
Z is D- or L-glutamine, D- or L-glutamic acid and D- or L-asparagine,
R1 is H (signifying a free N-terminal group), formyl, lower alkanoyl, aroyl or desamino (meaning the amino acid adjacent to the group R1, either X in formula I or P in formula 2 lacks the α-amino group of the amino acid, and is replaced with H),
R2 is H (signifying in a free C-terminal
carboxylic acid), O(lower alkyl), O(aryl), NR3R4 where R3 and R4 are independently H or lower alkyl, or descarboxy (meaning the α-carboxylic acid group of the amino acid to which R1 is adjacent in formula I or 2, Y or Z, respectively, is replaced with H), and
X and Y are linear chains of from one to ten amino acids,
whereby the amino acids are added either singly or in preformed blocks of amino acids to an appropriately functionalized solid support.
8. The method of claim 7, wherein the peptide has the structure R1-X-P-Q-S-T-U-V-W-Z-Y-R2,
wherein R1 is H, X is Cys-Xxx-Xxx-Xxx, P is Tyr, T is Leu, U is Val, V is Ala, W is Ile, and Y is Asn-Lys-Xxx-Glu, where Xxx is any amino acid, and R2 is not OH.
9. The peptide of claim 7 of the structure R1-X-P-Q-S-T-U-V-W-Z-Y-R2; wherein the peptide is selected from the group consisting of a peptide wherein X is Cys-Gln-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Gln-Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is pGlu-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X is Cys-Gln-Asp-Arg, S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X is Glu-Asn-Arg, P is Arg or Tyr, S is Asp, Lys, His, Asn, Gln, or Ala; and a peptide wherein X is Asn-Arg, P is Tyr or Arg, Q is Gly, and S is Asp, Lys, His, Asn, Gln, or Ala.
10. The method of claim 7 wherein the peptide has the structure R1-P-Q-S-T-U-V-W-Z-R2 wherein S is Asp or His.
11. The method of claim 7 wherein the peptide is selected from the group consisting of peptides having the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln- NH2; the formula Tyr-Thr-His-Leu-Val-Ala-Ile-Gln-NH2; the formula Acetyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln- NH2;
the formula Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala- Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val- Ala-Ile-Gln-NH2; the formula Arg-Tyr-Thr-Asp-Leu-Val- Ala-Ile-Gln-NH2; the formula Tyr-Thr-Glu-Leu-Val-Ala- Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile- Gln-Asn-Lys-Asn-Glu-NH2; the formula D-Tyr-Thr-Asp- Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-D-Thr-Asp- Leu-Val-Ala-Ile-Gln-NH2 ; the formula Tyr-Thr-D-Asp- Leu-Val-Ala-Ile-Gln-NH2; the formula Phe-Thr-Asp-Leu- Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val- Ala-D-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Ala-Val- Ala-Ile-Gln-NH2; the formula Tyr-Thr-Ala-Leu-Val-Ala- Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile- Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Cys-Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-NH2; the formula Cys-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Formyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4-Br-Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4-NH2-Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Ac-Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2;the formula Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln-NH2; the formula Arg-Gly-His-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asn-Leu- Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Asn-NH2; the formula Cys-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-D-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-D-Gln-NH2; the formula Tyr-Thr-Asp-D-Leu-Val-Ala-Ile-Gln-NH2; the formula D-Tyr-Thr-D-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula D-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Val-Ala-Ile-Gln-NH2; the formula desamino-Arg-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula desamino-Tyr-Thr-Asp-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-descarboxy-Gln-NH2; the formula Tyr-Thr-Gln-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH-n-Bu; the formula Glu-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Glu-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Glu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Lys-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula N-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Nal-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula O-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; the formula Pya-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Ser-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Thr-Ala-Ile-Gln-NH2; the formula Tic-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Val-Ala-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Gly-Leu-Val-Ala-Ile-Gln-NH2; the formula Ac-Tyr-D-Thr-His-Leu-Val-Ala-Ile-D-Gln-NH-n-Bu; the formula Ac-Tyr-D-Thr-Asp-Leu-Val-Ala-Ile-Gln- NH-n-Bu; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln- Asn-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln- Asn-Lys-NH2; and pharmaceutically acceptable acid- or base-addition salts thereof.
12. The method for preparation of the peptide of claim 7 whereby the amino acids are assembled either singly or in preformed blocks in solution or suspension by chemical ligation techniques.
13. The method for preparation of a peptide of claim 7 whereby the amino acids are assembled either singly or in preformed blocks in solution or
suspension by enzymatic ligation techniques.
14. The method for preparation of a peptide of claim 13 whereby the peptide is produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide.
15. A method for modifying binding of a selectin comprising providing a peptide selected from the group consisting of the structures
R1-X-P-Q-S-T-U-V-W-Z-Y-R2 (I)
R1-P-Q-S-T-U-V-W-Z-R2 (II) or a pharmaceutically acceptable salt thereof,
wherein:
X in Formula (I) and P in Formula (II) are the N-terminus amino acids, and R is a moiety attached to the amine function (NHR1),
Y in Formula (I) and Z in Formula (II) are the C-terminus amino acids, and R2 is the moiety attached to the singly-bonded oxygen in the carboxy function (C(O)OR2),
P is D- or L-tyrosine, D- or L-phenylalanine, D- or L-lysine, D- or L-glutamic acid, D- or L-arginine, D- or L-cysteine, D- or L- O-R3-tyrosine, D- or L-Nα-R3-tyrosine, D- or L-4-amino phenylalanine, D- or L-R4-phenylalanine, D- or L-pyridylalanine, D- or L- naphthylalanine, or D- or L-tetrahydroisoquinoline carboxylic acid, where R 3 is lower alkyl or aryl and R4 is halogen (fluorine, chlorine, bromine or iodine),
Q is D- or L-threonine, D- or L-lysine, D- or L-glutamic acid, D- or L-cysteine, or glycine,
S is D- or L-aspartic acid, D- or L-histidine, D- or L-glutamic acid, D- or L-asparagine, D or L-glutamine, D- or L-alanine, D- or L-phenylalanine, D- or L-lysine, or glycine,
T, U, V and W are independently D- or L-leucine, D- or L-isoleucine, D- or L-alanine, D- or L-valine, D- or L-alloisoleucine, glycine, D- or L-glutamic acid, D- or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either
residues T, U, V, or W from the peptide formulas I or
II,
Z is D- or L-glutamine, D- or L-glutamic acid and D- or L-asparagine,
R1 is H (signifying a free N-terminal group), formyl, lower alkanoyl, aroyl or desamino (meaning the amino acid adjacent to the group R1, either X in formula I or P in formula 2 lacks the α-amino group of the amino acid, and is replaced with H),
R2 is H (signifying in a free C-terminal
carboxylic acid), O(lower alkyl), O(aryl), NR3R4 where R3 and R4 are independently H or lower alkyl, or descarboxy (meaning the α-carboxylic acid group of the amino acid to which R1 is adjacent in formula I or 2, Y or Z, respectively, is replaced with H), and
X and Y are linear chains of from one to ten amino acids,
in combination with a pharmaceutically
acceptable carrier.
16. The method of claim 15 wherein the peptide is of the structure R1-X-P-Q-S-T-U-V-W-Z-Y-R2,
wherein R1 is H, X is Cys-Xxx-Xxx-Xxx, P is Tyr, T is Leu, U is Val, V is Ala, W is Ile, and Y is Asn- Lys-Xxx-Glu, where Xxx is any amino acid, and R2 is not OH.
17. The method of claim 15 wherein the peptide is of the structure R1-X-P-Q-S-T-U-V-W-Z-Y-R2 and is selected from the group consisting of a peptide wherein X is Cys-Gln-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Gln-Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is pGlu-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X is Cys-Gln-Asp-Arg, S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X is Glu-Asn-Arg, P is Arg or Tyr, S is Asp, Lys, His, Asn, Gln, or Ala; and a peptide wherein X is Asn-Arg, P is Tyr or Arg, Q is Gly, and S is Asp, Lys, His, Asn, Gln, or Ala.
18. The method of claim 15 wherein the peptide is of the structure R1-P-Q-S-T-U-V-W-Z-R2 wherein S is Asp or His.
19. The method of claim 15 wherein the peptide is selected from the group consisting of peptides having the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-His-Leu-Val-Ala-Ile-Gln-NH2; the formula Acetyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln- NH2; the formula Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-NH2; the formula D-Tyr-Thr- Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-D-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-D-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-D-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Ala-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Cys-Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-NH2; the formula Cys-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Formyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4-Br-Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4-NH2-Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Ac-Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2;the formula Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln-NH2; the formula Arg-Gly-His-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asn-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Asn-NH2; the formula Cys-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-D-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-D-Gln-NH2; the formula Tyr-Thr-Asp-D-Leu-Val-Ala-Ile-Gln-NH2; the formula D-Tyr-Thr-D-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula D-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Val-Ala-Ile-Gln-NH2; the formula desamino- Arg-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula desamino-Tyr-Thr-Asp-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-descarboxy-Gln-NH2; the formula Tyr-Thr-Gln-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH-n-Bu; the formula Glu-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Glu-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Glu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Lys-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula N-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Nal-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula O-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; the formula Pya-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Ser-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Thr-Ala-Ile-Gln-NH2; the formula Tic-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Val-Ala-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Gly-Leu-Val-Ala-Ile-Gln-NH2; the formula Ac-Tyr-D-Thr-His-Leu-Val-Ala-Ile-D-Gln-NH-n-Bu; the formula Ac-Tyr-D-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH-n-Bu; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-NH2; and pharmaceutically acceptable acid- or base-addition salts thereof.
20. The method of claim 15 wherein the pharmaceutical carrier is selected from the group consisting of carriers suitable for parenteral administration, oral administration, topical
administration, and controlled release formulations,
EP92912609A 1991-05-14 1992-05-14 Peptide inhibitors of inflammation Withdrawn EP0584244A1 (en)

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US5643873A (en) * 1992-05-06 1997-07-01 Affymax Technologies N.V. Peptides and compounds that bind selectins including endothelial leukocyte adhesion molecule 1
US5728802A (en) * 1992-05-06 1998-03-17 Affymax Technologies N.V. Peptides and compounds that bind selectins including endothelium leukocyte adhesion molecule 1 (ELAM-1)
US5648458A (en) * 1992-05-06 1997-07-15 Affymax Technologies N.V. Peptides and compounds that bind to ELAM-1
EP0656904A1 (en) * 1992-05-28 1995-06-14 Centocor Inc. Peptide inhibitors of selectin binding
US5440015A (en) * 1992-07-21 1995-08-08 Glycomed Incorporated Selectin peptide medicaments for treating disease
US5750508A (en) * 1993-06-16 1998-05-12 Glycomed Incorporated Sialic acid/fucose based medicaments
DE19929410A1 (en) * 1999-06-26 2000-12-28 Merck Patent Gmbh New octapepide compounds as alpha v beta 6 integrin inhibitors useful for treating and diagnosing heart disease, tumors, osteoporosis, fibrosis, inflammation, infection and psoriasis

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