WO2000056350A9 - Methods of use of beta 1-integrin inhibitors - Google Patents
Methods of use of beta 1-integrin inhibitorsInfo
- Publication number
- WO2000056350A9 WO2000056350A9 PCT/US2000/007680 US0007680W WO0056350A9 WO 2000056350 A9 WO2000056350 A9 WO 2000056350A9 US 0007680 W US0007680 W US 0007680W WO 0056350 A9 WO0056350 A9 WO 0056350A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- peptide
- integrin inhibitor
- aritgyiiy
- wqpprariy
- Prior art date
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- 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
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Definitions
- Integrins are a family of cell surface proteins that mediate the interactions of cells with their environment. They are heterodimeric proteins that include two membrane glycoproteins, a larger ⁇ subunit and a smaller ⁇ subunit. The ⁇ 2 subunits are largely involved in cell-cell interactions, while ⁇ j, subunits are associated with mediating cell adhesion to extracellular matrix constituents, including extracellular matrix (ECM) macromolecules reactive with ⁇ l subunit containing integnn molecules, such as fibronectin, lamiriin, tenascin, and Type IN collagen. See, for example, International Publication No. WO 99/37669.
- ECM extracellular matrix
- integrins regulate a variety of cellular interactions including cellular adhesion to extracellular matrices, cell-cell association, and cellular motility/migration wit n tissues/organs. They also are considered important mediators of a variety of pathological conditions, including acute inflammation, cancer, and osteoporosis, and serve to localize and/or promote development/differentiation of cells witJrin specific tissue and/or organ compartments, e.g., stem cell localization and differentiation.
- Inflammation Inflammation is required to orchestrate the recovery of tissue integrity, immune competence, and homeostasis following severe injury. Acute inflammation represents the first step of this biological process; however, if the inflammatory response is excessive in initial magnitude or duration, this otherwise beneficial process can contribute to the further deterioration rather than the restoration of tissue/organ homeostasis. Acute inflammation induced by CNS ischemic injury and thermal/cold injury represent two clinical conditions wherein this two-edged sword of i iflammation operates. In each of these situations, similar events occur subsequent to the reperfusion ofthe injured tissue(s). These events determine whether the injury extends outward from the initial focus ofthe injury to include additional tissue, or recover, thus containing the injury to the initial site and limiting any additional loss of functional tissue.
- leukocytes have been implicated in the pathogenesis and progression of micro vascular injury and extravascular tissue damage. To reach the site of injury, leukocytes must adhere to the vessel/capillary wall and then migrate to the injured tissue. Thus, leukocyte-mediated injury is dependent in part on polymo ⁇ honuclear neutrophil adherence to endothelial cell surface and leukocyte aggregation in extracellular tissue.
- Leukocyte adherence to endothelial cells and to extracellular matrix components is mediated by multiple adhesion receptor systems.
- leukocyte rolling into inflammatory sites is mediated by the selectin family of adhesion receptors.
- Additional cellular recognition receptors, the integrins then mediate leukocyte binding to the endothelium. Once leukocytes migrate across the endothelial cell membrane their adherence to the extracellular matrix is directed by many factors, including integrins.
- integrins represent one important set of mediators ofthe pathological events associated with acute inflammation.
- ⁇ l -integrins have recently been determined to play a critical role in the adhesion and migration of leukocytes (PMNs) into tissues at the site of injury, as the expression of ⁇ l -integrins is elevated 25-fold on extravasated PMNs over the level of ⁇ l -integrins expressed on circulating PMNs, Similar elevation of ⁇ l-integrin expression is observed in other biological and/or pathological conditions.
- PMNs leukocytes
- Cancer ⁇ l -integrins also play important roles in cancer cell differentiation, tumor metastasis, angiogenesis, and regulation of tumor cell apoptosis-modulators of cancerous tissue growth and spread.
- ⁇ 1 -integrins As increased expression of ⁇ 1 -integrins has been demonstrated to correlate with prognosis and metastatic potential (i.e., higher levels of ⁇ l -integrins correlate with higher metastatic potential), ⁇ l -integrins appear to coordinately regulate critical parameters within cancerous tissue.
- the levels of ⁇ l-integrin are often increased on cancerous cells/tissues, thus altering the interactions of cancer cells with their surroundings and inducing changes in the cancer cell's phenotype.
- Integrins mediate the interactions of osteoclasts with their environment and thus the actions of osteoclasts. Ofthe several integrins expressed on the surfaces of osteoclasts, ⁇ l -integrins are one of those expressed at the highest levels. Recent studies have determined ⁇ l-integrin activity to correlate with the adhesive and bone- resorptive capacity of osteoclasts.
- Bone marrow transplant (either allogeneic or autologous) represents an increasingly utilized approach to managing a variety of hematopoetic and cancerous disease states.
- Current methodologies require the direct harvesting of donor bone marrow, a painful and invasive medical procedure. This approach limits the availability of donor stem cell materials.
- agents that modulate integrin activity. Only two classes of agents, however, have been described that interact both selectively in the extracellular space with the ⁇ subunit of ⁇ l -integrins and possess the capacity to inhibit the activity of one or more integrin heterodimer complexes. These two classes include antibodies and the peptides disclosed in International Publication No. WO 99/37669, the latter of which are the only ones known to interact preferentially for activated ⁇ l -integrins. Inhibitory antibodies are known to possess the capability of binding and blocking the activity of integrin ⁇ l subunits; however, these tend to be large and not very accessible to pathological sites.
- agents that modulate (e.g., inhibit) integrin activity particularly those that have better access to pathological sites than antibodies.
- agents that can inhibit inflammatory leukocyte mediated destruction of tissues especially during ischemia reperfusion injury resulting from CNS ischemic injury (e.g., stroke), myocardial infarction, angioplasty, surgical incisions, injury-related trauma, and transplant reperfusion.
- agents that can inhibit inflammatory leukocyte mediated destruction of tissues subsequent to injuries induced by exposure to heat, cold, light, electricity, chemicals, or other agents that cause tissue injury and destruction.
- cyto-reductive therapies cancer chemo- and radio- therapeutics and the like
- the present invention provides methods that involve the use of ⁇ l-integrin inhibitors.
- a method of inhibiting inflammatory leukocyte mediated destruction of tissue in a patient is provided.
- the method includes administering to the patient a composition comprising a ⁇ l-integrin inhibitor.
- a ⁇ l-integrin inhibitor is an agent that inhibits ⁇ l subunit containing integrin irrespective ofthe associated ⁇ subunit.
- the inflammatory leukocyte mediated destruction of tissue can occur as a result of CNS ischemic injury, myocardial infarction, angioplasty, surgical incisions, injury-related trauma, transplant reperfusion, or a combination thereof.
- the inflammatory leukocyte mediated destruction of tissue can occur as a result of exposure to heat, cold, light, electricity, chemicals, or a combination thereof.
- the injury that results from any of these conditions is refened to herein as a "burn-type" injury.
- a method of treating a stroke patient includes administering to the patient a composition that includes a ⁇ l-integrin inhibitor in an amount effective to reduce infarct size, reduce neurological deficit, or both. Preferably, such administration occurs within about 3 hours after the stroke occurs.
- a method of treating a patient having a burn-type injury includes administering a composition that includes a ⁇ l-integrin inhibitor in an amount effective and over a period of time effective to reduce leukocyte-mediated tissue destruction.
- the period of time is preferably at least 1 hour.
- the administration is topical.
- the composition is administered periodically over a predetermined period of time.
- a method of treating a burn patient that involves mamt ⁇ ining a composition that includes an effective amount of a ⁇ 1 -integrin inhibitor on a burn-type injury for a period of time effective to reduce leukocyte-mediated tissue destruction and achieve a desired degree of healing.
- the present invention also provides a method of treating a cancer patient.
- the method involves administering to the patient a composition that includes a ⁇ l- integrin inhibitor in an amount effective to inhibit one or more of angiogenesis, cancer cell metastasis, cancer cell motility, or cancer cell migration.
- the method involves a ⁇ ninistering a ⁇ l-integrin inhibitor in an amount effective to induce programmed cell death in cancerous tissue or restore normal cellular phenotype to cancerous tissue.
- a method of treating a patient for osteoporosis involves administering to the patient a composition that includes a ⁇ 1 -integrin inhibitor in an amount effective to inhibit osteoclast adhesion and bone reso ⁇ tion.
- a method of peripheralizing stem cells involves administering to a patient a composition comprising a ⁇ l -integrin inhibitor.
- a composition that includes a ⁇ 1 -integrin inhibitor and a pharmaceutically acceptable carrier.
- a means one or more, such that combinations of inhibitors can be used in the compositions and methods ofthe invention.
- the ⁇ l-integrin inhibitor is a peptide that has a C-terminal LipAr motif. More preferably, the ⁇ l-integrin inhibitor is a peptide comprising an amino acid sequence selected from the group consisting of WQPPRARIY (SEQ ID NO: 1
- PRQAWRPIY SEQ ID NO:
- RPAPQRWIY SEQ ID NO:
- ARITGYIIY SEQ ID NO:
- PRARIY SEQ ID NO:
- RARIY SEQ ID NO:
- Figure 1 is a bar chart ofthe Doppler blood flow in marginal zones of burn study. This shows blood flow in burn zones at baseline, 24, 48, and 72 hours.
- Figure 3 is graph of data showing rabbit weight changes for seven days.
- Figure 4 is graph of data showing daily temperature changes for seven days.
- Figure 5 is graph of data showing body temperatures for seven days.
- Figure 6 is graph of data showing daily white blood cell count for seven days.
- Figure 7 is graph of data showing daily hematocrit for seven days.
- Figure 8 is a photograph ofthe zones of stasis in a group of control animals.
- Figure 9 is a photograph ofthe zones of stasis in a group of animals treated for 24 hours with T ⁇ -9-Tyr.
- Figure 10 is a photograph ofthe zones of stasis in a group of animals treated for 48 hours with T ⁇ -9-Tyr.
- Figure 11 is table of data showing abscess formation at day 7 in burn study.
- Figure 12 is table of data showing abscess size at day 7 in burn study.
- Figure 13 is a bar chart of infarction size of MCAO (stroke) study.
- Figure 14 is a bar chart of neurological index results of MCAO (stroke) study.
- TNF tumor necrosis factor alpha
- Figure 17 is a graph ofthe time course of WQPPRARIY (SEQ ID NO: ) in human plasma at 37°C with varying initial concentrations.
- Figure 18 is a graph of average (SD) concentrations of WQPPRARIY (SEQ ID NO:
- the present invention provides methods that use agents capable of one or more ofthe following: inhibiting leukocyte mediated destruction of tissues, especially after injuries to tissue due to heat or cold (i.e., burns or frostbite), light, electricity, chemicals, or other agents that cause tissue injury and destruction; inhibiting leukocyte mediated destruction of tissues after myocardial infarction, C ⁇ S ischemic injury (e.g., stroke), angioplasty, surgical incisions, injury-mediated trauma, and transplant reperfusion; inhibiting angiogenesis; inhibiting cancer cell metastasis, motility, and/or migration within tissues; restoring and/or potentiating the induction of programmed cell death in cancerous tissues or restoring normal cellular phenotype to cancerous tissue; inhibiting osteoclast adhesion and bone reso ⁇ tion; and or inhibiting stem cell adhesion to bone marrow.
- agents capable of one or more ofthe following: inhibiting leukocyte mediated destruction of tissues, especially after injuries to tissue due to heat or cold (i.e.
- the agents useful in the methods ofthe present invention are those that interact selectively with activated ⁇ l-integrin molecules (i.e., in the conformational state that permits/promotes interaction with ligand, which in this case are components ofthe extracellular matrix, such as fibronectin, laminin, collagen and the like, and/or cell surface molecules) relative to resting (i.e., unactivated) ⁇ l- integrin molecules.
- ligand which in this case are components ofthe extracellular matrix, such as fibronectin, laminin, collagen and the like, and/or cell surface molecules.
- ligand which in this case are components ofthe extracellular matrix, such as fibronectin, laminin, collagen and the like, and/or cell surface molecules
- ligand which in this case are components ofthe extracellular matrix, such as fibronectin, laminin, collagen and the like, and/or cell surface molecules
- They are preferably smaller than antibodies to ⁇ 1 -integrins.
- monomeric IgG molecules are approximately 150 kDa while the active agents used herein are no greater than about 15 kDa, preferably no greater than about 5 kDa, and more preferably no greater than about 1000 daltons (1 kDa).
- agents useful in the methods ofthe present invention do not induce an immuno-suppressed state.
- selectivity for activated ⁇ l- integrin molecules may allow for systemic administration and the use of higher doses.
- such agents are advantageous because they are believed to induce fewer side effects. Examples of such agents are described in International Publication No. WO 99/37669. These agents, as well as others (which may or may not be peptides) that can be developed based on structure-activity relationships defined by these agents, are useful in the methods ofthe present invention.
- the methods ofthe present invention include treatments for inflammation (e.g., such as that resulting from burns), cancer, and osteoporosis, as well as methods of harvesting hemotopoietic stem cells.
- Integrins represent an important set of mediators of acute inflammation, with ⁇ l -integrins playing an important role in the adhesion and migration of leukocytes into tissues at the site of injury. Moreover, the expression of ⁇ l -integrins is elevated 25 -fold on extravasated leukocytes over the level of ⁇ l -integrins expressed on circulating leukocytes. As such, inhibition of ⁇ l-integrin activity would likely provide an approach to mitigating the adverse effects ofthe acute inflammatory response, as it would block the migration and/or inflammatory responses elicited by leukocytes within tissues.
- agents that modulate ⁇ l-integrin activity are believed to provide superior therapeutic regulation of acute inflammation processes.
- the agents ofthe present invention are believed to provide superior access to sites of inflammation. They also provide the structural basis/rationale for the development of small molecules (peptides, peptiomrmetics, and/or small organic molecules) possessing enhanced anti- ⁇ l -integrin inhibitory activity.
- a wide variety of clinical situations that share an inflammation-mediated progression of injury size and scope include surgical incisions, injury-mediated trauma, myocardial infarction, angioplasty, CNS ischemic injury (e.g., stroke), transplant reperfusion, heat, cold, light, electricity, chemicals, etc., can also be positively affected by the agents ofthe present invention.
- Other clinical situations can include injuries initiated by heat or cold (i.e., burns or frostbite), light, electricity, chemicals, or other agents that cause tissue injury and destruction.
- the present invention provides a method of inhibiting inflammatory leukocyte mediated destruction of tissue in a patient by a ⁇ jninistering to the patient an effective amount of a ⁇ l-integrin inhibitor.
- the present invention provides a method that utilizes one or more agents capable of ameliorating leukocyte-mediated tissue destruction without suppressing leukocyte activity against foreign agents such as bacterial or viral agents.
- Such method results from the discovery that inhibition of WBC adherence with T ⁇ -9-Tyr reduces the microvascular damage and progression of tissue necrosis following thermal injury.
- the synthetic fibronectin peptide (T ⁇ - 9-Tyr) has the amino acid sequence WQPPRARIY (SEQ ID NO: ).
- the present invention provides a method of treating a patient having a burn-type injury by administering a ⁇ l-integrin inhibitor in an amount effective and over a period of time effective to reduce leukocyte-mediated tissue destruction.
- the order of preference for the period of time is at least 1 hour, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hour, at least 36 hours, and at least 48 hours.
- the mode of administration is topical and the administration is periodic.
- ⁇ 1 -integrin activity has been demonstrated to induce phenotypic restoration in cancer cells in vitro. Furthermore, inactivating antibodies against ⁇ l -integrins have been demonstrated to block tumor cell mediated angiogenesis, a process involved in tumor growth and expansion. Also, inactivating antibodies against ⁇ l -integrins block the interaction of ⁇ l -integrins with the extracellular matrix, which is rich in ⁇ 1 -integrin binding molecules, such as fibronectin, laminin, collagen, and tenascin that surround cancers, with ⁇ l -integrins, thereby restoring the induction of programmed cell death (apoptosis) in the cancerous cells and blocking cellular migration within tissues.
- ⁇ 1 -integrin binding molecules such as fibronectin, laminin, collagen, and tenascin that surround cancers
- agents that modulate ⁇ l-integrin activity can serve as powerful regulators of cancer cell biology.
- small molecule inhibitors (peptides, peptiomimetics, and/or small molecules) of ⁇ l -integrins are believed to provide superior access to tumor sites and permit the therapeutic regulation of cancer cell phenotype. They also provide the structural basis/rationale for the development of small molecules (peptides, peptiomimetics, and/or small organic molecules) possessing enhanced anti- ⁇ 1 -integrin inhibitory activity. Induction/restoration of a normal cellular phenotype in tumor cells would provide for the development of superior cancer therapeutic regimens.
- Cancers that can be treated using the methods ofthe present invention include, but are not limited to, breast cancer, melanoma, mesothelioma, and myeloma.
- Such treatment methods can involve, for example, the inhibition of tumor cell metastasis, the inhibition of tumor cell motility, the inhibition of tumor cell migration, the enhancement of tumor cell apoptosis, the induction of targeted paralysis and reversion of cancerous cell phenotype, and/or the inhibition of tumor mediated angiogenesis.
- the present invention provides a method of treating a cancer patient by admmistering a ⁇ l-integrin inhibitor in an amount effective to inhibit one or more of angiogenesis, cancer cell metastasis, cancer cell motility, or cancer cell migration.
- the present invention provides a method of treating a patient for osteoporosis by administering a ⁇ l-integrin inhibitor in an amount effective to inhibit osteoclast adhesion and bone reso ⁇ tion.
- CD34 cells Stem Cell Localization Hematopoetic stem cells express high levels of ⁇ l -integrins.
- agents that modulate ⁇ l-integrin activity are believed to induce the peripheralization (movement into the circulation) of hematopoetic stem cells.
- This allows stem cells to be harvested using standard cellular phoresis methodologies, for example, thus providing a readily available supply of donor hematopoetic stem cells.
- agents are believed to permit the development of enhanced hematopoetic stem cell harvesting techniques. It is further believed that this would increase the availability of donor materials, thus increasing the number and variety of procedures for which bone marrow transplantation could be employed.
- methods ofthe present invention include the use of agents that enhance the peripheralization of stem cells through their interaction with activated forms of ⁇ l -integrins on stem cells. Such methods can be used in conjunction with known methods for harvesting cells from the peripheral circulation system.
- cytokines such as G-CSF, GM-CSF and or Fit 3 ligand, can be used that enhance stem cell peripheralization.
- Stem cells obtained according to the present invention can be used to treat a variety of conditions, including, but not limited to, cancer, AIDS, and genetic disorders through gene therapy.
- Suitable agents for use in the methods ofthe present invention include peptides with a C-terminal amino acid residue having a side chain that includes an aromatic group ("-Ar-”) and an amino acid residue with a lipophilic alkyl side chain group (“-Lip-”) as the penultimate C-terminal residue, as exemplified in International Publication WO 99/37669.
- This C-terminal dipeptide sequence is referred to herein as a "LipAr motif.”
- These peptides with a C-te ⁇ ninal LipAr motif are typically capable of inhibiting ⁇ l-integrin subunit dependent cell adhesion and, in particular, of inhibiting ⁇ 4 ⁇ l -integrin dependent cell adhesion, and typically ⁇ 2 ⁇ l, ⁇ 3 ⁇ l, and/or ⁇ 5 ⁇ l integrin dependent cell adhesion.
- suitable amino acid residues having an aromatic group include tyrosine (“Tyr”), phenylalanine (“Phe”), histidine (“His”), and tryptophan (“T ⁇ ”).
- the penultimate C-te ⁇ ninal "Lip” residue is an amino acid residue that includes a lipophilic alkyl side chain group.
- the ⁇ -carboxyl group ofthe C-terminal amino acid residue ofthe present peptides is typically in the form of a carboxylic acid (-CO 2 H).
- the "Lip" and “Ar” residues are L-amino acid residues.
- amino acid residues that have a lipophilic alkyl side chain group include leucine ("Leu”), isoleucine ("He”), and valine (“Val”).
- the lipophilic alkyl side chain group has a SCDC (cyclohexane-water side chain distribution coefficient calculated as -RT In K D and expressed in kcal/mol) of at least about 3.0 and, preferably, at least about 4.0.
- SCDC is defined according to Radzicka et al., Biochemistry. 27, 1664 (1988).
- the SCDC value may be determined by measurement ofthe distribution coefficient between wet cyclohexane and water or by a comparison of a compound containing the same alkyl side chain group with other similar compounds using a hydrophobicity scale derived from HPLC retention according to the method of Parker et al., Biochemistry. 25, 5425 (1986).
- lipophilic alkyl side chain groups such as leucine, isoleucine, and valine
- insertion of a methionine residue at the penultimate position i.e., an "M Y" C-terminal motif
- Suitable peptides with a C-terminal Lip-Ar motif for use in the methods of the present invention include and WQPPRARIY (SEQ ID NO: ).
- the alanine knockout analogs i.e., an analog of a peptide in which a single residue has been substituted by an alanine residue
- the alanine knockout analogs can also be used in the methods ofthe present invention.
- WQPPRARIY (SEQ ID NO: ) have an alanine residue substituted for one ofthe arginine residues in the "PRARI" motif (Pro-Arg-Ala-Arg-Ile (SEQ ID NO: )). These alanine knockout analogs have the amino acid sequences WQPPRAAIY (SEQ ID NO: ) and WQPPAARIY (SEQ ID NO: ). Two ofthe other alanine knockout analogs, AQPPRARTY (SEQ ID NO: ), WAPPRARIY (SEQ ID NO:
- WQPPDADIY SEQ ID NO:
- WQPPDADIY SEQ ID NO:
- other suitable peptides include those that contain no more than 10 amino acid residues and have a sequence that does not correspond substantially to the amino acid sequence of WQPPRARIY (SEQ ID NO: ).
- sequence of a particular peptide does not correspond substantially to a reference amino acid sequence, if the particular peptide sequence has less than about 80% identity and preferably less than about 50% homology with the reference sequence.
- % homology refers to the percentage of amino acid residues of a peptide which are either identical to that of an original peptide sequence or differ from the original peptide sequence solely as a result of a conservative amino acid substitution.
- the peptide PAIFDRSCGS (SEQ ID NO: ) has 40% identity and 80% homology with respect to the peptide sequence
- PKVMERTCDS (SEQ ID NO: ).
- peptides with less than 50% homology with the corresponding C- terminal portion of WQPPRARIY exhibit the capability of inhibiting ⁇ l integrin subunit dependent adhesion, and thus are suitable for use in the methods ofthe present invention.
- Examples of such peptides include ARITGYIIY (SEQ ID NO: ), RARITGYIY (SEQ ID NO: ), PRQAWRPIY
- peptides of the invention are those which include a C-terminal "IIY" motif, i.e., the sequence ofthe three C-te ⁇ ninal most amino acid residues is Ile-Ile-Tyr.
- One such peptide contains 9 amino acid residues and has the sequence
- ARITGYIIY SEQ ID NO:
- Another group of particularly advantageous peptides ofthe invention include the C-terminal IY motif and contain no more than ten and, preferably, no more than six amino acid residues.
- suitable examples of this group include PRARIY (SEQ ID NO: ), RARIY (SEQ ID NO: ), ARTY (SEQ ID NO: ), and RIY.
- conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one ofthe following classes of residues: Class I: Ala, Gly, Ser, Thr, and Pro (representing small aliphatic side chains and hydroxyl group side chains); Class II: Cys, Ser, Thr and Tyr (representing side chains including an -OH or -SH group); Class III: Glu, Asp, Asn and Gin (carboxyl group containing side chains): Class IN: His, Arg and Lys (representing basic side chains); Class V: lie, Nal, Leu, Phe and Met (representing hydrophobic side chains); and Class Nl: Phe, T ⁇ , Tyr and His (representing aromatic side chains).
- the classes also include related amino acids such as 3Hyp and 4Hyp in Class I; homocysteine in Class II; 2-aminoadipic acid, 2- aminopimelic acid, ⁇ -carboxyglutamic acid, ⁇ -carboxyaspartic acid, and the corresponding amino acid amides in Class III; ornithine, homoarginine, ⁇ -methyl lysine, dimethyl lysine, trimethyl lysine, 2,3-diaminopropionic acid, 2,4- diaminobutyric acid, homoarginine, sarcosine and hydroxylysine in Class IN; substituted phenylalanines, norleucine, norvaline, 2-aminooctanoic acid, 2- aminoheptanoic acid, statine and ⁇ -valine in Class N; and naphthylalanines, substituted phenylalanines, tetrahydroisoquinoline-3 -carboxylic acid, and hal
- the peptides ofthe invention may be synthesized by the solid phase method using standard methods based on either t-butyloxycarbonyl (BOC) or 9- fluorenylrnethoxy-carbonyl (FMOC) protecting groups. This methodology is described by G.B. Fields et al. in Synthetic Peptides: A User's Guide. W.M. Freeman & Company, New York, NY, pp. 77-183 (1992).
- the present peptides may also be synthesized via recombinant techniques well known to those skilled in the art. For example, U.S. Patent No.
- 5,595,887 describes methods of forming a variety of relatively small peptides through expression of a recombinant gene construct coding for a fusion protein which includes a binding protein and one or more copies ofthe desired target peptide. After expression, the fusion protein is isolated and cleaved using chemical and/or enzymatic methods to produce the desired target peptide.
- the peptides used in the methods ofthe present invention may be employed in a monovalent state (i.e., free peptide or a single peptide fragment coupled to a carrier molecule).
- the peptides may also be employed as conjugates having more than one (same or different) peptide fragment bound to a single carrier molecule.
- the carrier may be a biological carrier molecule (e.g., a glycosaminoglycan, a proteoglycan, albumin or the like) or a synthetic polymer (e.g., a polyalkyleneglycol or a synthetic chromatography support).
- a synthetic polymer e.g., a polyalkyleneglycol or a synthetic chromatography support
- ovalburnin, human serum albumin, other proteins, polyethylene glycol, or the like are employed as the carrier.
- Such modifications may increase the apparent affinity and/or change the stability of a peptide.
- peptide/carrier molecule conjugates may be prepared by treating a mixture of peptides and carrier molecules with a coupling agent, such as a carbodiimide.
- the coupling agent may activate a carboxyl group on either the peptide or the carrier molecule so that the carboxyl group can react with a nucleophile (e.g., an amino or hydroxyl group) on the other member ofthe peptide/carrier molecule, resulting in the covalent linkage ofthe peptide and the carrier molecule.
- the conjugate includes at least one peptide fragment which is not linked to the carrier molecule through an amide bond with the ⁇ - carboxyl group ofthe C-terminal aromatic amino acid residue of the LipAr- terminated fragment.
- conjugates of a peptide coupled to ovalbumin may be prepared by dissolving equal amounts of lyophilized peptide and ovalbvmiin in a small volume of water.
- l-ethyl-3-(3-dimemylamino-propyl)- carboiimide hydrochloride (EDC; ten times the amount of peptide) is dissolved in a small amount of water.
- EDC l-ethyl-3-(3-dimemylamino-propyl)- carboiimide hydrochloride
- the EDC solution was added to the peptide/ovalbumin mixture and allowed to react for a number of hours.
- the mixture may then dialyzed (e.g., into phosphate buffered saline) to obtain a purified solution of peptide/ovalburnin conjugate.
- Peptide/carrier molecule conjugates prepared by this method typically contain about 4 to 5 peptide fragments per ovalbumin molecule.
- the peptides described herein are degraded enzymatically.
- a compound that inhibits the enzymatic degradation of the ⁇ l- integrin inhibitor can be coadministered with the ⁇ 1 -integrin inhibitor.
- the present invention also provides a composition that includes one or more active agents (i.e., compound such as a peptide) ofthe invention and one or more pharmaceutically acceptable carriers.
- the methods ofthe invention include administering to a patient, preferably a mammal, and more preferably a human, the composition ofthe invention in an amount effective to produce the desired effect.
- the agents ofthe present invention are formulated in pharmaceutical compositions and then, in accordance with the methods ofthe invention, aclministered to a mammal, such as a human patient, in a variety of forms adapted to the chosen route of administration.
- the formulations include those suitable for oral, rectal, vaginal, topical, nasal, ophmalmic, or parental (including subcutaneous, intramuscular, intraperitoneal, intratumoral, and intravenous) a ⁇ xninistration. They may be used as a perfusate for organ transplantation. Preferably, the route of a ⁇ ninistration is topical or intravenous, or as a perfusate for the preparation of organs for transplantation.
- the formulations may be conveniently presented in unit dosage form and may be prepared by any ofthe methods well known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.
- Formulations suitable for parenteral conveniently include a sterile aqueous preparation ofthe active agent, or dispersions of sterile powders of the active agent, which are preferably isotonic with the blood ofthe recipient.
- Isotonic agents that can be included in the liquid preparation include sugars, buffers, and sodium chloride.
- Solutions ofthe active agent can be prepared in water, optionally mixed with a nontoxic surfactant.
- Dispersions ofthe active agent can be prepared in water, ethanol, a polyol (such as glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, glycerol esters, and mixtures thereof.
- the ultimate dosage form is sterile, fluid, and stable under the conditions of manufacture and storage.
- the necessary fluidity can be achieved, for example, by using liposomes, by employing the appropriate particle size in the case of dispersions, or by using surfactants.
- Sterilization of a liquid preparation can be achieved by any convenient method that preserves the bioactivity ofthe active agent, preferably by filter sterilization. Prefened methods for preparing powders include vacuum drying and freeze drying ofthe sterile injectible solutions. Subsequent microbial contamination can be prevented using various antimicrobial agents, for example, antibacterial, antiviral and antifungal agents including parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Abso ⁇ tion ofthe active agents over a prolonged period can be achieved by including agents for delaying, for example, aluminum monostearate and gelatin.
- Formulations ofthe present invention suitable for oral administration may be presented as discrete units such as tablets, troches, capsules, lozenges, wafers, or cachets, each containing a predetermined amount ofthe active agent as a powder or granules, as liposomes containing the chemopreventive agent, or as a solution or suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, or a draught.
- Such compositions and preparations typically contain at least about 0J wt-% ofthe active agent.
- the amount of active agent is such that the dosage level will be effective to produce the desired result (e.g., suppress the development of cancer or tissue destruction by leukocyte mediated inflammation) in the subject.
- Nasal spray formulations include purified aqueous solutions ofthe active agent with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes. Formulations for rectal or vaginal aclministration may be presented as a suppository with a suitable carrier such as cocoa butter, or hydrogenated fats or hydrogenated fatty carboxylic acids. Ophthalmic formulations are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that ofthe eye. Topical formulations include the active agent dissolved or suspended in one or more media such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations.
- media such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations.
- Useful dosages ofthe active agents can be determined by comparing their in vitro activity and the in vivo activity in animals models. Methods for extrapolation of effective dosages in mice, and other animals, to humans are known in the art; for example, see U.S. Patent No. 4,938,949. Generally the concentration ofthe active agent in a liquid composition will be at least about 0.1 wt-% (wt-%, weight percent, means grams of compound per 100 mL liquid). For adult humans, single dosages for intravenous or topical administration will generally be about 0.005 mg to about 50 mg, and may be adrninistered, for example, about 3 to about 5 times per day, to yield levels of about 0.02 mg to about 200 mg per kg of body weight per day.
- Suitable doses to be administered are those that are sufficient to produce the desired result. This will typically not exceed 100 micromoles per kg of body weight per day, and may be much lower.
- the tablets, troches, pills, capsules, and the like may also contain one or more ofthe following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, fructose, lactose or aspartame; and a natural or artificial flavoring agent.
- a binder such as gum tragacanth, acacia, corn starch or gelatin
- an excipient such as dicalcium phosphate
- a disintegrating agent such as corn starch, potato starch, alginic acid and the like
- a lubricant such as magnesium stearate
- a sweetening agent such as sucrose, fructose, lactose or aspartame
- a natural or artificial flavoring agent such
- Various other materials may be present as coatings or to otherwise modify the physical form ofthe solid unit dosage form.
- tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar and the like.
- a syrup or elixir may contain one or more of a sweetening agent, a preservative such as methyl- or propylparaben, an agent to retard crystallization ofthe sugar, an agent to increase the solubility of any other ingredient, such as a polyhydric alcohol, for example glycerol or sorbitol, a dye, and flavoring agent.
- the material used in preparing any unit dosage form is substantially nontoxic in the amounts employed.
- the active agent may be inco ⁇ orated into sustained-release preparations and devices.
- New Zealand White rabbits (3-5 kg) (Myrtle's Rabbitry, Thompson Station, IN) were shaved and baseline cutaneous blood flow was measured using a laser
- T ⁇ -9-Tyr is a nine amino acid synthetic peptide ofthe fibronectin molecule (Peninsula Laboratories, San Carlos, CA) having the sequence WQPPRARIY (SEQ ID NO: ). It is stored in powder form at 4°C. Immediately prior to use the powder is dissolved in normal saline to a 10 mg/ml solution for intravenous injection.
- Tissue Necrosis Control animals had 16 of 40 (40%) of zones of stasis progressing to necrosis at 72 hours. Animals receiving T ⁇ -9-Tyr for 24 hours had 7 of 40 (17%) of zones progressing to necrosis, significantly less tissue destruction than controls (p ⁇ 0.05 chi-square). Ariimals treated for 48 hours had 4 of 40 (10%) of zones progressing to necrosis, which was significantly less tissue loss than controls (p ⁇ 0.05 chi-square, Mann- Whitney) (Table 1 and Figures 8-10).
- Table 1 Tabulation of zones of stasis progressing to necrosis comparing the 24- and 48 hour-treatment groups to controls.
- Synthetic analogues ofthe RGD domain of fibronectin have been used to prevent acute and chronic experimental liver injury in mice. Animals receiving intravenous RGD mimetics had lower serum levels of liver enzymes and less liver damage by histology than untreated mice following induction of hepatitis from intravenous concanavalin A injection (Bruck et al., Yale J. Biol. Med.. 70. 391-402 (1997)).
- Synthetic fibronectin peptides derived from the 33-kD carboxyl-terminal heparin-binding domain of fibronectin have demonstrated efficacy in blocking neutrophil accumulation in models of acute inflammation.
- Two of the more potent synthetic peptides identified thus far are WQPPRARIY (SEQ ID NO: ) and CS-
- WQPPRARIY As a cationic hydrophilic peptide, WQPPRARIY (SEQ ID NO: ) is thought to adhere to cell surface proteoglycans (Woods et al., Mol. Biol. Cell.. 4, 605-13 (1993); and Wahl et al., J. Clin. Invest.. 94, 655-62 (1994)).
- Cell surface proteoglycans which mediate a spectrum of cell-binding activities may alter proteoglycan interaction with selectins and/or integrin-dependent leukocyte homing (Woods et al., Mol. Biol. Cell.. 4, 605-13 (1993); Hines et al., Proc. Natl. Acad. Sci.. USA.
- ⁇ 4 ⁇ integrin also serves as a receptor for vascular cell adhesion molecule- 1 (VCAM-1), which is expressed on endothelial cells (Wahl et al., J. Clin. Invest.. 94, 655-62 (1994)).
- VCAM-1 vascular cell adhesion molecule- 1
- T ⁇ -9-Tyr improved blood flow in the marginal zones of stasis sunoimding burn.
- the use ofthe synthetic fibronectin peptide was associated with less tissue destruction following thermal injury.
- leukocytes In addition to mediating responses to injury-mediated inflammation, leukocytes also play an import role in responses to pathogenic infections within tissues. As such, inhibitors of leukocyte adhesion can and have been demonstrated previously to inhibit cellular responses to tissue infection — in particular soft tissue infections elicited in association with, but not limited to, thermal/cold injury, surgical incisions, injury-mediated trauma, and transplant reperfusion.
- LAD leukocyte adherence deficiency
- New Zealand white rabbits (1.8-2.5 kg each) were the experimental subjects. Animals were prepared by insertion of a 24-gauge catheter in a marginal ear vein, and clipping of the hair on their dorsa.
- Baseline weights, temperatures, hematocrits and WBC counts were obtained. Then the animals were given two sets of paired subcutaneous injections with S. aureus ATCC 25923. Following these injections animals were given T ⁇ -9-Tyr as described below. In addition animals were given the antibiotic cefazolin (20 mg/kg intravenously every 8 hours for three doses). Weights, temperature, WBC counts and hematocrits were measured daily for 7 days. Bupreno ⁇ hine (0.05 mg/kg) was aclministered for analgesia as determined from a quantitative assessment of pain score.
- the animals were sacrificed with a lethal intravenous injection of pentobarbital (150 mg/kg) on day 7, and the dorsal skin was dissected from the muscular fascia to allow determination of the incidence of subcutaneous abscess formation and measurement of abscess size by planimetry.
- Standard clinical laboratory S. aureus ATCC 25923 was grown on tryptic soy agar blood plates at 37°C, harvested and resuspended in sterile saline at concentrations of 10 8 and 10 9 CFU/ml as dete ⁇ nined by A540 measurements.
- the animals were given two subcutaneous injections (1.0 ml each) ofthe 10 9 CFU suspension and two subcutaneous injections (1.0 ml each) ofthe 10 8 CFU suspension via a 25-gauge needle and 3.0 ml syringe.
- Quantitative correlations of bacterial inocula were performed on each day of bacterial preparation.
- Mean CFU of S. aureus were 1.02+/- 0.21 times the expected concentrations determined by optical density.
- Statistical analysis ofthe data was performed by analysis of variance for repeated measures, t test, chi-square test and Kaplan-Meier survival analysis. Significance was assigned to P ⁇ 0.05.
- T ⁇ -9-Tyr synthetic peptide (stored in powder form at 4°C) was dissolved in normal saline to a 10 mg/ml solution for intravenous injection immediately before use. There were two treatment groups, control and peptide treatment. Controls were given saline (1.0 ml/kg). Treatment group was given the peptide, T ⁇ -9-Tyr (5 mg/kg) immediately after burn, then 3, 6, 12, 24 and 48 hours post-burn.
- coronal cryostat sections 20 ⁇ m thickness, 900 ⁇ m interval were used for infarction size measurements (data shown in Figure 12); and 10 ⁇ m thick sections through infarction area were taken for immunohistochemical staining to detect leukocyte infiltration (by MPO antibody). Neurological analysis is presented in Figure 13.
- This experiment was conducted to determine the anti-adhesion effect ofthe peptide PRARIY (SEQ ID NO: ) administered via local infusion on brain ischemia/reperfusion injury.
- the results show that the peptide PRARIY (SEQ ID NO: ) protects neurons against brain ischemia/reperfusion injury as evidenced by the smaller infarct size and the rapid reversal of transient neurological deficit.
- With local infusion ofthe peptide near the site of injury at least 80% reduction in infarct size and at least 80% reduction in neurological deficit was observed.
- Soluble peptides or specific anti- ⁇ l-integrin antibody was mixed with lymphocytes prior to the start ofthe adhesion assay. Following a brief (15 minute) preincubation, the cells were added to culture wells containing confluent endothelial that had been activated with cytokines. Following a 30 minute incubation, the lymphocyte/endothelial cultures were washed to remove weakly and nonadherent cells. The data represent the percentage of input cells remaining after this washing step. See Figures 14 and 15. The results indicate that either T ⁇ -9-Tyr or a specific inhibitory anti-integrin antibody can achieve the same level of inhibition of adhesion in this assay.
- Blood samples were collected from femoral artery at 6, 9, 12 and 15 min after dose initiation in both phases. Additionally, from 3 to 6 samples were drawn in the 5 minutes following the termination ofthe high infusion rate. Blood samples were immediately treated with methanol to inactivate enzymes, and processed using the same procedure described for the plasma study performed in vitro.
- the blood samples from the pharmacokinetic study were analyzed by a validated high-performance liquid chromatographic assay with an UN detector at wavelength of 220 nm. Mobile phase was as described above. Within-run and between-run variability of the assay was characterized by CNs of less than 10%. The average analytical recovery was 70 ⁇ 4.2%. Similar results were found in rat plasma and rat blood. The inclusion of quality control samples demonstrated accuracy, precision, and reproducibility ofthe method.
- the peptide WQPPRARIY (SEQ ID NO: ) was stable in PBS (OA M, pH 7.4) and in water over 4 hours of incubation at 37°C but markedly unstable in rat plasma and human plasma. At initial concentrations of 10 to 40 mcg/ml inhuman plasma the peptide exhibits an effective half-life of less than 3 minutes; at 100 and 400 mcg/ml it exhibited half-lives of 5 minutes and 15 minutes, respectively (Figure 17).
- WQPPRARIY (SEQ ID NO: ) was performed in vivo using high dose-short infusion periods at two rates. Michaelis-Menten parameters, suggesting saturation of blood-borne peptidases were estimated. The results are consistent with the degradation ofthe peptide incubated in rat and human plasma, and suggest efficient processing by peptidases. Although not identified, intermediates that resulted from the degradation of WQPPRARIY (SEQ ID NO: ) were observed in the studies in vitro. However, their role in inhibiting the cell adhesion ascribed to the parent peptide is unknown.
- Transient cerebral ischemia and associated brain injury may be mediated by several factors, including inflammatory processes (Hallenbeck et al., Stroke- 17. 246-253 (1986)).
- Leukocyte infiltration into ischemic tissue is a pathophysiological response, which often further aggravates ischemic injury by attenuating microvascular blood flow, and releasing chemical mediators such as free oxygen radicals (Kochanek et al., Stroke. 23, 1367-1379 (1992); and Matsuo et al., J. Cereb. Blood Flow Met.. 15, 941-947 (1995)).
- Cell adhesion molecules play important roles in leukocyte-endothelial interactions: the selectins (Lasky, Science, 258, 964-969 (1992)), the integrins, and the immunoglobulin superfamilies (Springer, Nature. 346, 425-434 (1990)). Integrins which contain ⁇ ] subunits usually are associated with mediating adhesion to extracellular matrix constituents (Springer, Nature. 346, 425-434 (1990)) whereas ⁇ integrins are largely involved in cell-cell interactions.
- One of these extracellular matrix macromolecules is fibronectin, which is found in plasma, cell matrix, and on the cell surface. These molecules can support leukocyte adhesion to endothelial cells (Akiyama et al., Adv. Enzvmol.. 57, 1-57 (1987)).
- Fibronectin possesses multiple domains recognized by integrins, including arginyl-glycyl-aspartic acid (RGD). The latter interacts selectively with ⁇ 5 ⁇ l integrin, and the alternately spliced connecting segment domain (CS-1) which is recognized selectively by ⁇ 4 ⁇ l integrin (Akiyama et al., Adv. EnzymoL. 57, 1-57 (1987); and Guan et al., Cell. 60, 53-61 (1990)).
- RGD arginyl-glycyl-aspartic acid
- Peptide WQPPRARIY (SEQ ID NO: ) has been characterized over the last several years with regard to structure/activity relationships and its molecular target of this peptide. The studies have shown that this peptide inhibits adhesion by a unique molecular mechanism. The rninimum structure ofthe peptide
- PRQAWRPIY SEQ ID NO:
- RPAPQRWIY SEQ ID NO:
Abstract
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AU40217/00A AU4021700A (en) | 1999-03-22 | 2000-03-22 | Methods of use of beta1-integrin inhibitors |
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US12563499P | 1999-03-22 | 1999-03-22 | |
US60/125,634 | 1999-03-22 | ||
US16753899P | 1999-11-24 | 1999-11-24 | |
US60/167,538 | 1999-11-24 |
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US6849712B1 (en) | 1998-01-22 | 2005-02-01 | Regents Of The University Of Minnesota | Peptides with β1 integrin subunit dependent cell adhesion modulating activity |
US7910711B2 (en) | 2002-12-24 | 2011-03-22 | Peking University | Human cancer-relating genes, the products encoded thereby and applications thereof |
JP4570402B2 (en) * | 2004-06-25 | 2010-10-27 | 日本サプリメント株式会社 | Central function improver |
CN102223915A (en) * | 2008-11-20 | 2011-10-19 | 默克专利有限公司 | New therapy and medicament using integrin ligands for treating cancer |
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US5019646A (en) * | 1987-08-25 | 1991-05-28 | Regents Of The University Of Minnesota | Polypeptides with fibronectin activity |
EP0673384A4 (en) * | 1992-12-10 | 1996-10-09 | Univ Minnesota | Polypeptides useful for treating inflammatory disorders. |
JP2002501082A (en) * | 1998-01-22 | 2002-01-15 | リージェンツ・オブ・ザ・ユニバーシティ・オブ・ミネソタ | Peptide having β1 integrin subunit-dependent cell adhesion regulating activity |
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2000
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