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.

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• 1992
  • 1992-05-14 EP EP92912609A patent/EP0584244A1/en not_active Withdrawn
  • 1992-05-14 AU AU20293/92A patent/AU2029392A/en not_active Abandoned
  • 1992-05-14 WO PCT/US1992/004016 patent/WO1992020708A1/en not_active Application Discontinuation
  • 1992-05-14 JP JP5500170A patent/JPH06511477A/ja active Pending
  • 1992-05-14 CA CA002103139A patent/CA2103139A1/en not_active Abandoned
• 1996
  • 1996-07-30 AU AU60827/96A patent/AU6082796A/en not_active Abandoned

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