WO2007103368A2 - Methods for regulating inflammatory mediators and peptides useful therein - Google Patents
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- WO2007103368A2 WO2007103368A2 PCT/US2007/005688 US2007005688W WO2007103368A2 WO 2007103368 A2 WO2007103368 A2 WO 2007103368A2 US 2007005688 W US2007005688 W US 2007005688W WO 2007103368 A2 WO2007103368 A2 WO 2007103368A2
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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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
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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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
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
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- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
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- A—HUMAN NECESSITIES
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- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
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- A61P19/00—Drugs for skeletal disorders
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- 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
- A61P25/04—Centrally acting analgesics, e.g. opioids
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- A—HUMAN NECESSITIES
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- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention relates to methods of modulating cellular secretory processes. More specifically the present invention relates to modulating the release of inflammatory mediators. The present invention also relates to the intracellular signaling mechanism regulating the secretion of inflammatory mediators from membrane-bound vesicles or granules in inflammatory cells.
- Hypersecretion of mucus contributes to the pathogenesis of a large number of airway inflammatory diseases in both human and non-human animals. Increased mucus secretion is seen in chronic disease states such as asthma, COPD and chronic bronchitis; in genetic diseases such as cystic fibrosis; in allergic conditions (atopy, allergic inflammation); in bronchiectasis; and in a number of acute, infectious respiratory illnesses such as pneumonia, rhinitis, influenza or the common cold.
- Airway mucus acts as a physical barrier against biologically active inhaled particles, and may help prevent bacterial colonization of the airways and inactivate cytotoxic products released from leukocytes. King et al, Respir. Physiol. 62:47-59 (1985); Vishwanath and Ramphal, Infect. Immun. 45:197 (1984); Cross et al, Lancet 1 :1328 (1984).
- mucus maintains the tear film, and is important for eye health and comfort.
- Mucus secretion in the gastrointestinal tract also has a cytoprotective function. The role of mucus as a chemical, biological and mechanical barrier means that abnormally low mucus secretion by mucous membranes is undesirable.
- Mucus are a family of glycoproteins secreted by the epithelial cells including those at the respiratory, gastrointestinal and female reproductive tracts.
- Mucins are responsible for the viscoelastic properties of mucus and at least eight mucin genes are known. Thornton, et al., J. Biol. Chem. 272, 9561-9566 (1997). Mucociliary impairment caused by mucin hypersecretion and/or mucus cell hyperplasia leads to airway mucus plugging that promotes chronic infection, airflow obstruction and sometimes death. Many airway diseases, such as chronic bronchitis, chronic obstructive pulmonary disease, bronchiectacis, asthma, cystic fibrosis and bacterial infections are characterized by mucin overproduction. E. Prescott, et al., Eur. Respir. J., 8:1333-1338 (1995); K. C.
- mucin granules are released via an exocytotic process in which the granules translocate to the cell periphery where the granule membranes fuse with the plasma membrane, allowing for luminal secretion of the contents.
- MARCKS a protein of approximately 82 kD
- MARCKS has three evolutionarily-conserved regions (Aderem et al., Nature 1988; 332:362-364; Thelen et al., Nature 1991 ; 351 :320-322; Hartwig et al., Nature 1992; 356:618-622; Seykora et al., J Biol Chem 1996; 271 :18797- 18802): an N-terminus, a phosphorylation site domain (PSD), and a multiple homology 2 (MH2) domain.
- PSD phosphorylation site domain
- MH2 multiple homology 2
- the N-terminus a 24 amino acid sequence with a myristic acid moiety attached to a terminal glycine residue is involved in binding of MARCKS to membranes (Seykora et al., J Biol Chem 1996; 271 :18797-18802) and possibly to calmodulin (Matsubara et al., J Biol Chem 2003; 278:48898-48902).
- This 24 amino acid sequence is known as the MANS peptide.
- the MANS peptide and active fragments thereof can compete with native MARCKS in cells for membrane binding.
- MARCKS protein in release of inflammatory mediators from the granules of infiltrating leukocytes is relevant to inflammation in diseases in all tissues and organs, including lung diseases characterized by airway inflammation, such as asthma, COPD and cystic fibrosis.
- inflammation and mucus secretion in the airways are two separate and independent processes (Li et al., J Biol Chem 2001 ; 276:40982-40990; Singer et al., Nat Med 2004; 10:193-196).
- mucus production and secretion can be provoked by a number of factors, including mediators released by inflammatory cells, there is no known direct link between excess mucus and inflammation.
- the invention relates to a new use for the 24 amino acid, myristoylated polypeptide, also known as the MANS peptide.
- the invention also relates to a new method for blocking any cellular secretory process, especially those that involve the release of inflammatory mediators from inflammatory cells, whose stimulatory pathways involve the protein kinase C (PKC) substrate MARCKS protein and release of contents from intracellular vesicles or granules.
- PKC protein kinase C
- the present invention is directed to a method of inhibiting the exocytotic release of at least one inflammatory mediator from at least one inflammatory cell comprising contacting the at least one inflammatory cell, which cell comprises at least one inflammatory mediator contained within a vesicle inside the cell, with at least one peptide selected from the group consisting of a MANS peptide and an active fragment thereof in an effective amount to reduce the release of the inflammatory mediator from the inflammatory cell as compared to the release of the inflammatory mediator from the same type of inflammatory cell that would occur in the absence of the at least one peptide.
- the present invention is further directed to a method of inhibiting the release of at least one inflammatory mediator from at least one inflammatory cell in a tissue or fluid of a subject comprising the administration to the subject's tissue and/or fluid, which comprises at least one inflammatory cell comprising at least one inflammatory mediator contained within a vesicle inside the cell, a therapeutically effective amount of a pharmaceutical composition comprising at least one peptide selected from the group consisting of a MANS peptide and an active fragment thereof in a therapeutically effective amount to reduce the release of the inflammatory mediator from at least one inflammatory cell as compared to release of the inflammatory mediator from at least one of the same type of inflammatory cell that would occur in the absence of the at least one peptide. More specifically, inhibiting the release of an inflammatory mediator comprises blocking or reducing the release of an inflammatory mediator from the inflammatory cell.
- the present invention includes a method of reducing inflammation in a subject comprising the administration of a therapeutically effective amount of a pharmaceutical composition comprising a MANS peptide [i.e., N-myristoyl- GAQFSKTAAKGEAAAERPGEAAV (SEQ ID NO: 20)] or an active fragment thereof.
- the active fragment is at least six amino acids in length.
- an "active fragment" of a MARCKS protein is one that affects (inhibits or enhances) the MARCKS protein-mediated release.
- An active fragment can be selected from the group consisting of N-myristoyl- GAQFSKTAAKGEAAAERPGEAA (SEQ ID NO: 3); N-myristoyl- GAQFSKTAAKGEAAAERPGEA (SEQ ID NO: 4); N-myristoyl-
- GAQFSKTAAKGEAAAERPG SEQ ID NO: 6
- N-myristoyl-GAQFSKTAAKGEAAAERP SEQ ID NO: 7
- N-myristoyl-GAQFSKTAAKGEAAAER SEQ ID NO: 8
- N-myristoyl- GAQFSKTAAKGEAAAE SEQ ID NO: 9
- N-myristoyl-GAQFSKTAAKGEAAA SEQ ID NO: 10
- N-myristoyl-GAQFSKTAAKGEAA SEQ ID NO: 11
- N-myristoyl- GAQFSKTAAKGEA SEQ ID NO: 12
- N-myristoyl-GAQFSKTAAKGE SEQ ID NO: 13
- N-myristoyl-GAQFSKTAAKGE SEQ ID NO: 13
- N-myristoyl-GAQFSKTAAKG SEQ ID NO: 14
- N-myristoyl-GAQFSKTAAK SEQ ID NO: 15
- hydrophobic N-terminal myristate moiety in these peptides can enhance their compatibility with and presumably their permeability to plasma membranes, and potentially enable the peptides to be taken up by cells.
- the hydrophobic insertion of myristate into a bilayer can provide a partition coefficient or apparent association constant with lipids of up to 10 4 M "1 or a unitary Gibbs free binding energy of about 8 kcal/mol (see, for example, Peitzsch, R.M., and McLaughlin, S. 1993, Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins. Biochemistry.
- the fragments can each exhibit partition coefficients and membrane affinities that are representative of their respective structure.
- the fragments can be prepared by methods of peptide synthesis known in the art, such as by solid phase peptide synthesis (see, for example, the methods described in Chan, Weng C.
- Efficacy of the individual peptides and of combinations of individual peptides in the methods of this disclosure can be readily determined without undue experimentation using the procedures described in the examples disclosed herein.
- a preferred combination will comprise two of the peptides; a preferred molar ratio of the peptides can be from 50:50 to 99.99 to 0.01, which ratio can be readily determined using the procedures described in the examples disclosed herein.
- the MANS peptide or active fragment thereof is contained in a pharmaceutical composition which is useful to block inflammation.
- the present invention also includes methods for regulating a cellular secretory process in a subject comprising the administration of a therapeutically effective amount of a compound comprising a MANS peptide or an active fragment thereof, that regulates an inflammatory mediator in a subject.
- the administration is generally selected from the group consisting of topical administration, parenteral administration, rectal administration, pulmonary administration, inhalation and nasal or oral administration, wherein pulmonary administration generally includes either an aerosol, a dry powder inhaler, a metered dose inhaler, or a nebulizer.
- compositions comprising a degranulation-inhibiting amount of the MANS peptide or a degranulation-inhibiting amount of an active fragment thereof, such as a pharmaceutical composition of the MANS peptide or an active fragment thereof, for human or animal use provides the MANS peptide or active fragment thereof at least to the site in or on a tissue or to a fluid-containing or mucus-containing layer in contact with the surface of a tissue where an inflammatory granulocytic cell resides or into which an inflammatory granulocytic cell will invade, thus enabling the MANS peptide or an active fragment thereof to contact the inflammatory granulocytic cell.
- administration of such a composition can be made at the first onset or first detection of inflammation or first perception of inflammation by the human or animal or at the first perceptible change in the level of inflammation in a human or animal to reduce the amount of inflammation that would otherwise occur in the absence of the MANS peptide or active fragment thereof.
- administration can be made during an ongoing inflammation of a tissue in the human or animal to reduce the amount of additional inflammation that would otherwise occur in the absence of the MANS peptide or active fragment thereof.
- dosing of a pharmaceutical composition can be repeated after 3 to 8 hours, preferably after 6 to 8 hours after the first administration of the pharmaceutical composition.
- the present invention also includes methods of reducing inflammation in a subject comprising the administration of a therapeutically effective amount of a compound that inhibits the MARCKS-related release of inflammatory mediators, whereby the release of at least one inflammatory mediator in the subject is reduced compared to that which would occur in the absence of said treatment.
- reducing generally means a lessening of the effects of inflammation.
- inflammatory mediators are inhibited or blocked by the methods disclosed.
- Another embodiment of the present invention includes methods of reducing inflammation in a subject comprising administering a therapeutically effective amount of a compound that inhibits the MARCKS-related release of inflammatory mediators, whereby the inflammation in the subject is reduced compared to that which would occur in the absence of said treatment.
- the present invention also discloses methods of reducing or inhibiting inflammation in a subject comprising the administration of a therapeutically effective amount of a MANS peptide or an active fragment thereof effective to modulate an inflammatory mediator at the inflammation site.
- the term “inhibiting” means a reduction in the amount of inflammatory mediator secretion.
- completely inhibiting means a reduction to zero in the amount of inflammatory mediator secretion.
- the active fragment is at least six amino acids in length.
- exocytotic process means exocytosis, i.e., a process of cellular secretion or excretion in which substances contained in a vesicle, which vesicle resides inside a cell, are discharged from the cell by fusion of the vesicular membrane of the vesicle with the outer cell membrane.
- Degranulation means the release of cellular granule contents.
- degranulation-inhibiting means a reduction in the release of the inflammatory mediators contained within the granules of the inflammatory cell.
- a degranulation-inhibiting amount of the MANS peptide and/or an active fragment thereof is the amount of these peptides that is sufficient to reduce the release of the inflammatory mediators contained in the granules as compared to release in the absence of the same peptide.
- MANS peptide and active fragments thereof can be useful in the prevention or reduction in amount of inflammation in a tissue in an animal caused by inflammatory mediators.
- MANS peptide and active fragments thereof can be useful in the prevention or reduction in amount of tissue damage in an animal produced or caused by inflammatory mediators.
- FIGS. IA-I D are bar graphs illustrating mucin hypersecretion by NHBE cells is maximized by activation of both PKC and PKG.
- FIGS. 2A-2B demonstrate that the MARCKS protein is a key component of the mucin secretory pathway.
- FIGS. 3A-3C depicts a gel illustrating that an antisense oligonucleotide directed against MARCKS down-regulates MARCKS expression and attenuates mucin hypersecretion.
- FIGS. 4A-4B illustrate that PKC-dependent phosphorylation releases MARCKS from the plasma membrane to the cytoplasm.
- FIGS. 5A-5C show that PKG induces dephosphorylation of MARCKS by activating PP2A.
- FlG. 6 depicts bar graphs that demonstrate that PP2A is an essential component of the mucin secretory pathway.
- FIG. 7 is a gel that illustrates that MARCKS associates with actin and myosin in the cytoplasm.
- FIG. 8 depicts a signaling mechanism controlling mucin secretion by human airway epithelial cells.
- FIG. 9 is a bar graph depicting the ability of MANS peptide to block secretion of myloperoxidase from isolated canine neutrophils.
- FIG. 10 is a bar graph depicting the ability of MANS peptide to block secretion of myloperoxidase from isolated human neutrophils.
- FIG. 11 is a bar graph showing that PMA stimulates a small increase in MPO secretion from LPS-stimulated human neutrophils which is enhanced in a concentration- dependent manner by co-stimulation with 8-Br-cGMP.
- FIG. 12 is a bar graph showing that 8-Br-cGMP simulation has little effect on MPO secretion from LPS-stimulated human neutrophils until a co-stimulation with PMA occurs in a concentration-dependent manner.
- FIG. 13 is a bar graph showing that PMA stimulates a small increase in MPO secretion from LPS-stimulated canine neutrophils which is enhanced in a concentration- dependent manner by co-stimulation with 8-Br-cGMP.
- FIG. 14 is a bar graph showing that 8-Br-cGMP simulation has little effect on MPO secretion from LPS-stimulated canine neutrophils until a co-stimulation with PMA occurs in a concentration-dependent manner.
- FIG. 15 is a bar graph showing that co-stimulation with PMA+8-Br-cGMP is required for maximal MPO secretion from LPS-stimulated canine neutrophils.
- the present invention is directed to a method of inhibiting the exocytotic release of at least one inflammatory mediator from at least one inflammatory cell comprising contacting the at least one inflammatory cell, which cell comprises at least one inflammatory mediator contained within a vesicle inside the cell, with at least one peptide selected from the group consisting of a MANS peptide and an active fragment thereof in an effective amount to reduce the release of the inflammatory mediator from the inflammatory cell as compared to the release of the inflammatory mediator from the same type of inflammatory cell that would occur in the absence of the at least one peptide.
- the present invention is further directed to a method of inhibiting the release of at least one inflammatory mediator from at least one inflammatory cell in a tissue or fluid of a subject comprising the administration to the subject's tissue and/or fluid, which comprises at least one inflammatory cell comprising at least one inflammatory mediator contained within a vesicle inside the cell, a therapeutically effective amount of a pharmaceutical composition comprising at least one peptide selected from the group consisting of a MANS peptide and an active fragment thereof in a therapeutically effective amount to reduce the release of the inflammatory mediator from at least one inflammatory cell as compared to release of the inflammatory mediator from at least one of the same type of inflammatory cell that would occur in the absence of the at least one peptide. More specifically, reducing the release of an inflammatory mediator comprises blocking or inhibiting the mechanism that releases an inflammatory mediator from the inflammatory cell.
- the MANS peptide used in the present methods described above comprises SEQ ID NO:1.
- the active fragment useful in the present invention comprises at least one myristoylated N-terminal fragment of SEQ ID NO:1 which comprises at least six amino acids, wherein the first amino acid of said fragment begins at the N-terminal glycine of SEQ ID NO: 1. More specifically, the active fragment can be selected from the group consisting of N-myristoyl-GAQFSKTAAKGEAAAERPGEAA (SEQ ID NO: 3); N-myristoyl- GAQFSKTAAKGEAAAERPGEA (SEQ ID NO: 4); N-myristoyl- GAQFSKTAAKGEAAAERPGE (SEQ ID NO: 5); N-myristoyl-
- GAQFSKTAAKGEAAAERPG SEQ ID NO: 6
- N-myristoyl-GAQFSKTAAKGEAAAERP SEQ ID NO: 7
- N-myristoyl-GAQFSKTAAKGEAAAER SEQ ID NO: 8
- N-myristoyl- GAQFSKTAAKGEAAAE SEQ ID NO: 9
- N-myristoyl-GAQFSKTAAKGEAAA SEQ ID NO: 10
- N-myristoyl-GAQFSKT AAKGEAA SEQ ID NO: 1 1
- N-myristoyl- GAQFSKTAAKGEA SEQ ID NO: 12
- N-myristoyl-GAQFSKTAAKGE SEQ ID NO: 13
- N-myristoyl-G AQFSKTAAKG SEQ ID NO: 14
- N-myristoyl-G AQFSKTAAK SEQ ID NO: 6
- N-myristoyl-GAQFSKTAAKGEAAAERP SEQ ID NO: 7
- N-myristoyl-G AQFSKTAA SEQ ID NO: 16
- N-myristoyl-GAQFSKTA SEQ ID NO: 17
- N-myristoyl-GAQFSKT SEQ ID NO: 18
- N-myristoyl-GAQFSK SEQ ID NO: 19
- the present invention is directed to the contact and/or administration of the peptide described above and throughout the specification with any known inflammatory cell that may be contained in the tissue or fluid of a subject which contains at least one inflammatory mediator contained within a vesicle inside the cell.
- the inflammatory cell is preferably a leukocyte, more preferably a granulocyte, which can be further classified as a neutrophil, a basophil, an eosinophil or a combination thereof.
- the inflammatory cells contacted in the present method may also be a monocyte/macrophage.
- the present invention is directed to reducing the release of inflammatory mediators contained within the vesicles of inflammatory cells and these inflammatory mediators are selected from the group consisting of myeloperoxidase (MPO), eosinophil peroxidase (EPO), major basic protein (MBP), lysozyme, granzyme, histamine, proteoglycan, protease, a chemotactic factor, cytokine, a metabolite of arachidonic acid, defensin, bactericidal permeability-increasing protein (BPI), elastase, cathepsin G, cathepsin B, cathepsin D, beta-D-glucuronidase, alpha-mannosidase, phospholipase A 2 , chondroitin-4- sulphate, proteinase 3, lactoferrin, collagenase, complement activator, complement receptor, N-formylme
- these inflammatory mediators are selected from the group consisting of myeloperoxidase (MPO), eosinophil peroxidase (EPO), major basic protein (MBP), lysozyme, granzyme and a combination thereof.
- MPO myeloperoxidase
- EPO eosinophil peroxidase
- MBP major basic protein
- lysozyme granzyme and a combination thereof.
- the present invention contacts an effective amount of the peptide with an inflammatory cell, wherein the effective amount is defined as a degranulation-inhibiting amount of MANS peptide or an active fragment thereof that reduces the amount of an inflammatory mediator released from at least one inflammatory cell from about 1% to about 99% as compared to the amount released from at least one inflammatory cell in the absence of MANS peptide or an active fragment thereof.
- the effective amount is defined as a degranulation-inhibiting amount of MANS peptide or an active fragment thereof that reduces the amount of an inflammatory mediator released from at least one inflammatory cell from about 1% to about 99% as compared to the amount released from at least one inflammatory cell in the absence of MANS peptide or an active fragment thereof.
- this effective amount of the contacted peptide comprises a degranulation-inhibiting amount of MANS peptide or an active fragment thereof that reduces the amount of an inflammatory mediator released from at least one inflammatory cell from between about 5-50% to about 99% as compared to the amount released from at least one inflammatory cell in the absence of MANS peptide or an active fragment thereof.
- the present invention in one embodiment is directed to the administration of at least one peptide comprising a MANS peptide and an active fragment thereof in a therapeutically effective amount into tissue or fluid of a subject where the subject is afflicted by a respiratory disease, which is preferably asthma, chronic bronchitis or COPD.
- a respiratory disease which is preferably asthma, chronic bronchitis or COPD.
- the subject may be afflicted by a bowel disease, a skin disease, an autoimmune disease, a pain syndrome, and combinations thereof.
- the bowel disease may be ulcerative colitis, Crohn's disease or irritable bowel syndrome.
- the subject may be afflicted with a skin disease, such as rosacea, eczema, psoriasis or severe acne.
- the subject may also be afflicted with arthritis, such as rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus.
- Subjects afflicted by cystic fibrosis may also be treated by the present method and peptides.
- the present method is preferably useful for the treatment of subjects, such as mammals, and preferably humans, canines, equines and felines.
- the present method of treatment of subjects is by the administration of one or more peptides including the MANS peptide or an active fragment described herein to include topical administration, parenteral administration, rectal administration, pulmonary administration, nasal administration, or oral administration. More specifically, pulmonary administration is selected from the group of aerosol, dry powder inhaler, metered dose inhaler, and nebulizer. Additionally, the disclosed method may further comprise the administration to the subject of a second molecule selected from the group consisting of an antibiotic, an antiviral compound, an antiparasitic compound, an anti-inflammatory compound, and an immunosuppressant.
- the invention relates to a method of administering a pharmaceutical composition.
- the pharmaceutical composition comprises a therapeutically effective amount of a known compound and a pharmaceutically acceptable carrier.
- a "therapeutically effective" amount as used herein is an amount of a compound that is sufficient to ameliorate symptoms exhibited by a subject.
- the therapeutically effective amount will vary with the age and physical condition of the patient, the severity of the condition of the patient being treated, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used and like factors within the knowledge and expertise of those skilled in the art.
- Pharmaceutically acceptable carriers are preferably solid dosage forms such as tablets or capsules.
- Liquid preparations for oral administration also may be used and may be prepared in the form of syrups or suspensions, e.g., solutions containing an active ingredient, sugar, and a mixture of ethanol, water, glycerol, and propylene glycol. If desired, such liquid preparations may include one or more of following: coloring agents, flavoring agents, and saccharin. Additionally, thickening agents such as carboxymethylcellulose also may be used as well as other acceptable carriers, the selection of which are known in the art.
- the present invention relates to methods for regulating cellular secretory processes, especially those releasing inflammatory mediators from inflammatory cells.
- regulating means blocking, inhibiting, decreasing, reducing, increasing, enhancing or stimulating.
- a number of cellular secretory processes involve the release of contents from membrane-bound vesicles or granules within cells
- a membrane- bound vesicle or granule is defined as an intracellular particle, which is primarily vesicular (or a vesicle inside a cell) and which contains stored material that can be secreted.
- This invention provides a means of blocking secretion from any membrane- bound vesicle, including those found in inflammatory cells, by targeting a specific molecule important in the intracellular secretory pathway with a synthetic peptide. This approach may be of therapeutic importance for the treatment of a wide variety of hypersecretory and inflammatory conditions in humans and animals.
- the present invention targets inflammatory cells that contain the inflammatory mediators in one or more granules or vesicles within the cells' cytoplasm.
- the cells are contacted with one or more peptides that are selected from the MANS peptide or an active fragment thereof, all of which are described in detail herein.
- the contact of the peptide with the inflammatory cell is via administration to a subject afflicted by or suffering from a disease in which these inflammatory cells are present in specific tissue or fluid within the tissue.
- the peptide Upon administration or contact of the peptide with the cell, the peptide competitively competes for and competitively inhibits the binding of the native MARCKS protein to the membrane of the intracellular granules or vesicles which contain the inflammatory mediators.
- these vesicles in these cells do not move to the plasma membrane of the cells as they would normally do when stimulated to exocytotically release their contents of inflammatory mediators out of the cells.
- the method of the present invention inhibits the movement of the vesicles to the cells' plasma membrane, which in turn, reduces the release of the inflammatory mediators from the inflammatory cells.
- the amount of inflammatory mediators released from the cells over time is reduced because both the rate of release and the amount of release of the mediators from the inflammatory cells is dependent upon the concentration of the peptide administered and contacted with the inflammatory cells.
- One benefit of the present invention is that it may combine a therapy that includes the direct blocking of mucus secretion with a unique anti-inflammatory therapy.
- a benefit of the present invention over current anti-inflammation therapies that affect a general suppression of the immune system is that the peptide is thought to block secretion of only intracellular components secreted from inflammatory cells. Thus, many aspects of the immune system should still function even with the inhibition of the inflammatory mediators.
- the compounds of the invention may regulate, i.e. block, inflammatory mediator release from cells. This inhibition of release of inflammatory mediators is an attractive means for preventing and treating a variety of disorders, e.g., diseases and pathological conditions involving inflammation. Thus, the compounds of the invention may be useful for the treatment of such conditions. These encompass airway diseases and chronic inflammatory diseases including, but not limited to, osteoarthritis, multiple sclerosis, Guillain-Barre syndrome, Crohn's disease, ulcerative colitis, psoriasis, graft versus host disease, systemic lupus erythematosus and insulin-dependent diabetes mellitus.
- the compounds of the invention can also be used to treat other disorders associated with the activity of elevated levels of proinflammatory mediators and enzymes such as responses to various infectious agents and a number of diseases of autoimmunity such as rheumatoid arthritis, toxic shock syndrome, diabetes and inflammatory bowel diseases.
- Uses of the peptide and methods of the invention include therapies to combat inflammation along with therapies that will combine the anti-inflammatory activity of the peptide with its ability to block mucus secretion.
- Diseases that may be treated by the peptide's ability to block both inflammation and mucus secretion include but are not limited to inflammatory bowel diseases, digestive disorders (i.e., inflamed gall bladder, Menetier's disease) and inflammatory airway diseases.
- the peptide may also be used to block release of excess insulin from pancreatic islet cells.
- proinflammatory mediators have been correlated with a variety of disease states that correlate with influx of neutrophils into sites of inflammation or injury. Blocking antibodies have been demonstrated as useful therapies against the neutrophil-associated tissue injury in acute inflammation (Harada et al., 1996, Molecular Medicine Today 2, 482).
- Cells other than neutrophils that may release inflammatory mediators include other leukocytes, such as basophils, eosinophils, monocytes and lymphocytes, and therapies may be directed against secretion from these cells.
- Neutrophils, eosinophils, and basophils are each a type of granulocyte, i.e., a leukocyte that has granules in its cytoplasm.
- Leukocytes synthesize a number of inflammatory mediators that are packaged and stored in cytoplasmic granules.
- mediators for example, myeloperoxidase [MPO] in neutrophils (Borregaard N, Cowland JB. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997; 89:3503-3521), eosinophil peroxidase [EPO] and major basic protein [MBP] in eosinophils (Gleich G J. Mechanisms of eosinophil-associated inflammation.
- MPO myeloperoxidase
- EPO eosinophil peroxidase
- MBP major basic protein
- Mast cells which usually do not circulate in the blood stream, and basophils contain secretory cytoplasmic granules which store and can release, upon cell activation, preformed inflammatory (anaphylactic) mediators, such as histamine; proteoglycans, such as heparin and chondroitin sulphate; proteases such as tyrptase, chymase, carboxypeptidase, and cathepsin G-like protease; chemotactic factors, cytokines and metabolites of arachidonic acid that act on the vasculature, smooth muscle, connective tissue, mucous glands and inflammatory cells.
- inflammatory mediators such as histamine
- proteoglycans such as heparin and chondroitin sulphate
- proteases such as tyrptase, chymase, carboxypeptidase, and cathepsin G-like protease
- Neutrophils also known as polymorphonuclear leukocytes (PMN) comprise 50 to 60% of the total circulating leukocytes. Neutrophils act against infectious agents, such as bacteria, fungi, protozoa, viruses, virally infected cells, as well as tumor cells, that penetrate the body's physical barriers at sites of infection or injury. Neutrophils mature through six morphological stages: myeloblast, promyeloblast, myelocyte, metamyelocyte, non-segmented (band) neutrophil, and segmented (functionally active) neutrophil.
- infectious agents such as bacteria, fungi, protozoa, viruses, virally infected cells, as well as tumor cells, that penetrate the body's physical barriers at sites of infection or injury.
- Neutrophils mature through six morphological stages: myeloblast, promyeloblast, myelocyte, metamyelocyte, non-segmented (band) neutrophil, and segmented (functionally active) neutrophil.
- inflammatory mediators are stored in primary (azurophil), secondary (specific), and tertiary (gelatinase) granules, as well as in secretory vesicles.
- primary (azurophil) granules contain myeloperoxidase (MPO), lysozyme, defensins, bactericidal permeability-increasing protein (BPI), elastase, cathepsin G, cathepsin B, cathepsin D, beta-D-glucuronidase, alpha- mannosidase, phospholipase A 2 , chondroitin-4-sulphate, and proteinase 3 (see, for example, Hartwig JH, Thelen M, Rosen A, Janmey PA, Nairn AC, Aderem A.
- MPO myeloperoxidase
- BPI bactericidal permeability-increasing protein
- MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature 1992; 356:618-622); secondary (specific) granules contain lysozyme, lactoferrin, collagenase, complement activator, phospholipase A 2 , complement receptors, e.g., CR3, CR4, N- formylmethionyl-leucyl-phenyl alanine (FMLP) receptors, laminin receptors, cytochrome bsss, monocyte-chemotactic factor, histaminase, and vitamin Bl 2 binding protein; and small storage granules contain gelatinase, plasminogen activator, cathepsin B, cathepsin D, beta-D- gluc ⁇ ronidase, alpha-mannosidase, and cytochrome bs 5 g.
- Neutrophil granules contain antimicrobial or cytotoxic substances, neutral proteinases, acid hydrolases and a pool of cytoplasmic membrane receptors.
- MPO myeloperoxidase
- Defensins which constitute 30 to 50% of azurophilic granule protein, are small (molecule weight ⁇ 4000) potent antimicrobial peptides that are cytotoxic to a broad range of bacteria, fungi and some viruses. Their toxicity may be due to membrane permeabilization of the target cell which is similar to other channel-forming proteins (perforins).
- Bacterial permeability-increasing (BPI) protein is a member of perforins. It is highly toxic to gram-negative bacteria but not to gram-positive bacteria or fungi and can also neutralize endotoxin, the toxic lipopolysaccharide component of gram-negative bacterial cell envelope.
- Serine proteases such as elastase and cathepsin G hydrolyze proteins in bacterial cell envelopes.
- Substrates of granulocyte elastase include collagen cross-linkages and proteoglycans, as well as elastin components of blood vessels, ligaments, and cartilage.
- Cathepsin D cleaves cartilage proteoglycans, whereas granulocyte collagenases are active in cleaving type I and, to a lesser degree, type III collagen from bone, cartilage, and tendon.
- Collagen breakdown products have chemotactic activity for neutrophils, monocytes, and fibroblasts.
- protease inhibitors such as alpha2-macroglobulin and alphal -antiprotease. These antiproteases are present in serum and synovial fluids. They may function by binding to and covering the active sites of proteases. Protease-antiprotease imbalance can be important in the pathogenesis of emphysema.
- Azurophil granules function predominantly in the intracellular milieu (in the phagolysosomal vacuole), where they are involved in the killing and degradation of microorganisms.
- Neutrophil specific granules are susceptible to release their contents extracellularly and have an important role in initiating inflammation.
- Specific granules represent an intracellular reservoir of various plasma membrane components including cytochrome b (component of NADPH oxidase, an enzyme responsible for the production of superoxide), receptors for complement fragment iC3b (CR3, CR4), for laminin, and formylmethionyl-peptide chemoattractants.
- cytochrome b component of NADPH oxidase, an enzyme responsible for the production of superoxide
- receptors for complement fragment iC3b CR3, CR4
- laminin for laminin
- formylmethionyl-peptide chemoattractants formylmethionyl-peptid
- SNARE Soluble N-ethylmaleimide attachment protein receptor proteins
- SNARE motif alpha-helical coiled-coil domain
- SNAREs are localized to distinct membrane compartments of the secretory and endocytic trafficking pathways, and contribute to the specificity of intracellular membrane fusion processes.
- the t- SNARE domain consists of a 4-helical bundle with a coiled-coil twist.
- the SNARE motif contributes to the fusion of two membranes.
- SNARE motifs fall into four classes: homologues of syntaxin Ia (t-SNARE), VAMP-2 (v-SNARE), and the N- and C-terminal SNARE motifs of SNAP-25. One member from each class may interact to form a SNARE complex.
- the SNARE motif is found in the N-terminal domains of certain syntaxin family members such as syntaxin Ia, which is required for neurotransmitter release (Lerman et al., Biochemistry 39: 8470-8479 (2000)), and syntaxin 6, which is found in endosomal transport vesicles (Misura et al.-, Proc. Natl. Acad. Sci. U.S.A. 99: 9184-9189 (2002)).
- SNAP-25 neuropeptide-associated protein 25 IcDa proteins are components of SNARE complexes, which may account for the specificity of membrane fusion and to directly execute fusion by forming a tight complex (the SNARE or core complex) that brings the synaptic vesicle and plasma membranes together.
- the SNAREs constitute a large family of proteins that are characterized by 60-residue sequences known as SNARE motifs, which have a high propensity to form coiled coils and often precede carboxy-terminal transmembrane regions.
- the synaptic core complex is formed by four SNARE motifs (two from SNAP-25 and one each from synaptobrevin and syntaxin 1) that are unstructured in isolation but form a parallel four-helix bundle on assembly.
- the crystal structure of the core complex has revealed that the helix bundle is highly twisted and contains several salt bridges on the surface, as well as layers of interior hydrophobic residues.
- a polar layer in the centre of the complex is formed by three glutamines (two from SNAP-25 and one from syntaxin 1) and one arginine (from synaptobrevin) (Rizo et al., Nat Rev Neurosci 3: 641-653 (2002)).
- Members of the SNAP-25 family contain a cluster of cysteine residues that can be palmitoylated for membrane attachment (Risinger et al., J. Biol. Chem. 268: 24408-24414 (1993)).
- neutrophils The major role of neutrophils is to phagocytose and destroy infectious agents. They also limit the growth of some microbes, prior to onset of adaptive (specific) immunological responses. Although neutrophils are essential to host defense, they have also been implicated in the pathology of many chronic inflammatory conditions and in ischemia- reperfusion injury. Hydrolytic enzymes of neutrophil origin and oxidatively inactivated protease inhibitors can be detected in fluid isolated from inflammatory sites. Under normal conditions, neutrophils can migrate to sites of infection without damage to host tissues. However, undesirable damage to a host tissue can sometimes occur. This damage may occur through several independent mechanisms.
- Ischemia-reperfusion injury is associated with an influx of neutrophils into the affected tissue and subsequent activation. This may be triggered by substances released from damaged host cells or as a consequence of superoxide generation through xantine oxidase.
- blood may contain a mixture of normal, primed, activated and spent neutrophils.
- Activated neutrophils have enhanced production of reactive oxygen intermediates (ROI).
- ROI reactive oxygen intermediates
- a subpopulation of neutrophils with the enhanced respiratory burst has been detected in the blood of people with an acute bacterial infection and patients with the adult respiratory distress syndrome (ARDS).
- ARDS adult respiratory distress syndrome
- Neutrophils have been implicated in the pathology of this condition because of the large influx of these cells into the lung and the associated tissue damage caused by oxidants and hydrolytic enzymes released from activated neutrophils.
- the impairment of neutrophil microbicidal activity that occurs as the ARDS worsens may be a protective response on the part of the host, which is induced locally by inflammatory products.
- the acute phase of thermal injury is also associated with neutrophil activation, and this is followed by a general impairment in various neutrophil functions.
- Activation of neutrophils by immune complexes in synovial fluid contributes to the pathology of rheumatoid arthritis.
- Chronic activation of neutrophils may also initiate tumor development because some ROI generated by neutrophils damage DNA and proteases promote tumor cell migration.
- ROI generated by neutrophils damage DNA and proteases promote tumor cell migration.
- a correlation has been established between the onset of bacterial infection and reduction in the proportion and absolute numbers of neutrophils positive for antibody and complement receptors.
- Oxidants of neutrophil origin have also been shown to oxidize low-density lipoproteins (LDL), which are then more effectively bound to the plasma membrane of macrophages through specific scavenger receptors. Uptake of these oxidized LDL by macrophages may initiate atherosclerosis.
- primed neutrophils have been found in people with essential hypertension, Hodgkin's disease, inflammatory bowel disease, psoriasis, sarcoidosis, and septicemia, where priming correlates with high concentrations of circulating TNF-alpha (cachectin).
- Chlorinated oxidants and hydrogen peroxide can inactivate antiproteases such as alphal -protease inhibitor and alpha2-macroglobulin, which are endogenous inhibitors of elastase, but simultaneously activate latent metalloproteases such as collagenases and gelatinase, which contribute to the further inactivation of antiproteases.
- antiproteases such as alphal -protease inhibitor and alpha2-macroglobulin, which are endogenous inhibitors of elastase, but simultaneously activate latent metalloproteases such as collagenases and gelatinase, which contribute to the further inactivation of antiproteases.
- Cytoplasmic constituents of neutrophils may also be a cause of formation of specific anti-neutrophil cytoplasmic antibodies (ANCA), which are closely related to the development of systemic vasculitis and glomerulonephritis.
- ANCA are antibodies directed against enzymes that are found mainly within the azurophil or primary granules of neutrophils.
- Diffuse fine granular cytoplasmic fluorescence is typically found in Wegener's granulomatosis, in some cases of microscopic polyarteritis and Churg Strauss syndrome, and in some cases of crescentic and segmental necrotizing glomerulonephritis.
- the target antigen is usually proteinase 3.
- Perinuclear fluorescence pANCA is found in many cases of microscopic polyarteritis and glomerulonephritis. These antibodies are often directed against myeloperoxidase but other targets include elastase, cathepsin G, lactoferrin, lysozyme and beta-D-glucuronidase.
- the third group designated "atypical" ANCA includes neutrophil nuclear fluorescence and some unusual cytoplasmic patterns and while a few of the target antigens are shared with pANCA, the others have not been identified yet.
- pANCA are also found in a third of patients with Crohn's disease. The reported incidence of ANCA in rheumatoid arthritis and SLE varies considerably but the patterns are predominantly pANCA and atypical ANCA.
- the eosinophil is a terminally differentiated, end-stage leukocyte that resides predominantly in submucosal tissue and is recruited to sites of specific immune reactions, including allergic diseases.
- the eosinophil cytoplasm contains large ellipsoid granules with an electron-dense crystalline nucleus and partially permeable matrix. In addition to these large primary crystalloid granules, there is another granule type that is smaller (small granule) and lacks the crystalline nucleus.
- the large specific granules of eosinophils contain at least four distinct cationic proteins, which exert a range of biological effects on host cells and microbial targets: major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN), and eosinophil peroxidase (EPO).
- MBP major basic protein
- ECP eosinophil cationic protein
- EDN eosinophil derived neurotoxin
- EPO eosinophil peroxidase
- Basophils contain about one fourth as much major basic protein as eosinophils together with detectable amounts of EDN, ECP and EPO. Small amounts of EDN and ECP are also found in neutrophils (Gleich G J. Mechanisms of eosinophil-associated inflammation. J Allergy Clin Immunol 2000; 105:651- 663).
- MBP appears to lack enzymatic activity but is a highly cationic polypeptide which may exert its toxic activities by interactions with lipid membranes leading to their derangement.
- Both MBP and EPO can act as selective allosteric inhibitors of agonist binding to M2 muscarinic receptors. These proteins may contribute to M2 receptor dysfunction and enhance vagally mediated bronchoconstriction in asthma.
- EDN can specifically damage the myelin coat of neurons. Histaminase and a variety of hydrolytic lysosomal enzymes are also present in the large specific granules of eosinophils.
- Eosinophils can elaborate cytokines which include those with potential autocrine growth-factor activities for eosinophils and those with potential roles in acute and chronic inflammatory responses.
- cytokines have growth-factor activities for eosinophils: granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3 and IL-5.
- cytokines produced by human eosinophils that may have activities in acute and chronic inflammatory responses include IL-I -alpha, IL-6, IL-S, TNF-alpha and both transforming growth factors, TGF-alpha and TGF-beta.
- Eosinophils contain crystalloid granules that contain MBP, eosinophil cationic protein, EPO, and eosinophil-derived neurotoxin (Gleich, J Allergy Clin Immunol 2000; 105:651-663).
- the human promyelocytic cell line HL-60 clone 15 can be used to examine secretion of EPO.
- This cell line was established from a clone of HL-60 that had been grown at an elevated pH for two months (Fischkoff, Leuk Res 1988; 12:679-686) and then treated with butyric acid to allow the cells to differentiate so as to exhibit many of the characteristics of peripheral blood eosinophils, including expression of eosinophil-specific granule proteins (Rosenberg et al., J Exp Med 1989; 170:163-176; Tiffany et al., J Leukoc Biol 1995; 58:49- 54; Badewa et al., Exp Biol Med 2002; 227:645-651).
- Eosinophils can participate in hypersensitivity reactions, especially through two lipid inflammatory mediators, leukotriene C 4 (LTC 4 ) and platelet activating factor (PAF). Both mediators contract airway smooth muscle, promote the secretion of mucus, alter vascular permeability and elicit eosinophil and neutrophil infiltration.
- LTC 4 leukotriene C 4
- PAF platelet activating factor
- MBP can stimulate the release of histamine from basophils and mast cells
- MBP can stimulate the release of EPO from mast cells.
- Eosinophils can serve as a local source of specific lipid mediators as well as induce the release of mediators from mast cells and basophils.
- Eosinophil granule content is released following similar stimuli to neutrophil granules, e.g. during phagocytosis of opsonized particles and by chemotactic factors.
- Neutrophil lysosomal enzymes act primarily on material engulfed in phagolysosomes, while the eosinophil granule contents act mainly on extracellular target structure such as parasites and inflammatory mediators.
- Monocyte and macrophage development takes place in the bone marrow and passes through the following steps: stem cell; committed stem cell; monoblast; promonocyte; monocyte in bone marrow; monocyte in peripheral blood; and macrophage in tissues.
- Monocyte differentiation in the bone marrow proceeds rapidly (1.5 to 3 days).
- granules are formed in monocyte cytoplasm and these can be divided as in neutrophils into at least two types. However, they are fewer and smaller than their neutrophil counterparts (azurophil and specific granules). Their enzyme content is similar.
- Granule-bound enzymes of monocytes/macrophages include lysozyme, acid phosphatase, and beta-glucuronidase. As a model for in vivo studies, lysozyme secretion from U937 cells was used.
- This cell line is derived from a human histiocytic lymphoma and has been used as a monocytic cell line that can be activated by a variety of agonists, such as PMA (Hoff et al., J Leukoc Biol 1992; 52:173-182; Balboa et al., J Immunol 2003; 170:5276- 5280; Sundstrom et al., lnt J Cancer 1976; 17:565-577).
- PMA Haoff et al., J Leukoc Biol 1992; 52:173-182
- Balboa et al. J Immunol 2003; 170:5276- 5280
- Sundstrom et al. lnt J Cancer 1976; 17:565-577.
- Natural killer (NK) cells and cytotoxic lymphocytes contain potent cytotoxic granules including perforin, a pore-forming protein, and granzymes, lymphocyte-specific serine proteases.
- the NK-92 cell line is an IL-2-dependent human line established from a patient with rapidly progressive non-Hodgkin's lymphoma (Gong JH., Maki G, Klingemann HG. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 1994; 8:652-658).
- NK-92 cells express high levels of molecules involved in the perforin-granzyme cytolytic pathway that targets a wide range of malignant cells (Gong et al, vide infra, and Maki G, Klingemann HG, Martinson JA, Tam YK. Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. J Hematother Stem Cell Res 2001 ; 10:369-383).
- Granzymes are exogenous serine proteases that are released by cytoplasmic granules within cytotoxic T cells and natural killer cells. Granzymes can induce apoptosis within virus-infected cells, thus destroying them.
- Extracellular release of a mediator of inflammation (inflammatory mediator) from a granulocyte (or leukocyte), and extracellular release of more than one mediator of inflammation (inflammatory mediator) from a granulocyte (or leukocyte) is sometimes referred to herein as degranulation.
- the release of a mediator of inflammation comprises release of said mediator from a granule located in the interior of a granulocyte or leukocyte.
- the release of inflammatory mediator is preferably the release of an inflammatory mediator from these granules.
- MARCKS neutrophils and macrophages
- MARCKS lipopolysaccharide
- MARCKS Tumor necrosis factor alpha modifies agonist-dependent responses in human neutrophils by inducing the synthesis and myristoylation of a specific protein kinase C substrate. Proc Natl Acad Sci USA 1990; 87:5603-5607).
- MARCKS can thus have an important role in subsequent release of inflammatory mediators when granule-containing cells, such as neutrophils and macrophages, are stimulated by agonists, especially those that work by activating PKC (Burgoyne et al., Physiol Rev 2003; 83:581-632; Logan et al. J Allergy Clin Immunol 2003; 111 : 923-932; Smolen et al., Biochim Biophys Acta 1990; 1052:133-142; Niessen et al., Biochim. Biophys. Acta 1994; 1223:267-273 ; Naucler et al., J Leukoc Biol 2002; 71 :701- 710).
- administering can reduce the amount of a mediator of inflammation released from infiltrating leukocytes at the site of inflammation, where the leukocytes are preferably granulocytes.
- the administration of the MANS peptide and/or at least one active fragment thereof can reduce the amount of a mediator of inflammation released from leukocytes such as granulocytes infiltrating into the site of inflammation.
- the degranulation-inhibiting amount of MANS peptide, or the degranulation-inhibiting amount of an active fragment thereof is sufficient to reduce or inhibit the exocytotic release of inflammatory mediators from granules contained within the inflammatory cells infiltrating into the site.
- Degranulation-inhibiting efficacy is measured at a time after administration of the MANS peptide or the fragment thereof by comparison of the percent of inhibition (i.e., percent of reduction) of the release of mediators of inflammation from said cells (leukocytes or granulocytes or other inflammatory cells) relative to the level or amount or concentration of said mediators of inflammation released or produced at approximately the same time in the absence of MANS peptide and/or in the absence of the active fragment thereof.
- a sufficient degranulation-inhibiting amount is the percentage of reduction of a mediator of inflammation released from a granulocyte, at the site of inflammation, which is from about 1 % to about 99%, preferably from 5% to about 99%, more preferably from about 10% to about 99%, even more preferably from about 25% to 99%, and even more preferably from about 50% to about 99% of the amount of said mediator of inflammation released from said granulocyte in the absence of MANS peptide or an active fragment thereof tested under the same conditions.
- administering can reduce the amount of a mediator of inflammation released from a granulocyte, which granulocyte is stimulated by said inflammatory stimulant at said site of inflammatory stimulation, from about 1% to about 99%, preferably from 5% to about 99%, more preferably from about 10% to about 99%, even more preferably from about 25% to 99%, and even more preferably from about 50% to about 99% of the amount of said mediator of inflammation released from said granulocyte in the absence of MANS peptide in the presence of the identical inflammation-stimulating amount of said inflammatory stimulant.
- administering can reduce the amount of a mediator of inflammation released from a granulocyte, which granulocyte is stimulated by said inflammatory stimulant at said site of inflammatory stimulation, by 100% of the amount of said mediator of inflammation released from said granulocyte in the absence of MANS peptide in the presence of the identical inflammation-stimulating amount of said inflammatory stimulant.
- An example of an inflammatory stimulant used in in vitro examples herein is phorbol 12-myristate 13-acetate (PMA).
- PMA phorbol 12-myristate 13-acetate
- Monocyte chemoattractant protein (MCP-I) is nearly as effective as C5a, and much more potent than IL-8, in the degranulation of basophils, resulting in histamine release. Histamine release can occur after stimulation with chemokines (i.e., chemoattractant cytokines), RANTES and MIP-I.
- the degranulation-inhibiting amount of MANS peptide administered to a site of inflammatory stimulation in an animal comprises from about 1 time to about 1,000,000 times the concentration of the MARCKS peptide at said site of inflammatory stimulation, preferably from about 1 time to about 100,000 times the concentration of the MARCKS peptide at said site of inflammatory stimulation, more preferably from about 1 time to about 10,000 times the concentration of the MARCKS peptide at said site of inflammatory stimulation, even more preferably from about 1 time to about 1 ,000 times the concentration of the MARCKS peptide at said site of inflammatory stimulation, even more preferably from about 1 time to about 100 times the concentration of the MARCKS peptide at said site of inflammatory stimulation, and even more preferably from about 1 time to about 10 times the concentration of the MARCKS peptide at said site of inflammatory stimulation.
- the granulocyte resides on or in the airway of an animal, preferably a human, and the MANS peptide is administered by inhalation, such as by inhalation of a pharmaceutical composition comprising the MANS peptide, for example a pharmaceutical composition comprising the MANS peptide and an aqueous solution, which composition is administered in the form of an aerosol, or a pharmaceutical composition comprising the MANS peptide in the form of a dry powder, which composition is administered using a dry powder inhaler.
- a pharmaceutical composition comprising the MANS peptide
- a pharmaceutical composition comprising the MANS peptide
- an aerosol a pharmaceutical composition comprising the MANS peptide in the form of a dry powder
- Other methods and devices known in the art for administration of a solution or powder by inhalation such as, for example, droplets, sprays, and nebulizers, can be useful.
- the peptide of the present invention may block secretory processes that are physiologically important, including basal secretory functions.
- basal secretory mechanisms may require less MARCKS protein than stimulated secretion. Basal secretion may be preserved since all therapies to block MARCKS-mediated secretion may not eliminate all MARCKS function,.
- MARCKS nucleotide sequence refers to any nucleotide sequence derived from a gene encoding a MARCKS protein, including, for example, DNA or RNA sequence, DNA sequence of the gene, any transcribed RNA sequence, RNA sequence of the pre-mRNA or mRNA transcript, and DNA or RNA bound to protein.
- Precise delivery of the MARCKS-blocking peptide may also overcome any potential limitations of blocking important secretory processes. Delivering such agents to the respiratory tract should be readily accomplished with inhaled formulations. Since these agents may be useful in treating inflammatory bowel disease, one can envision delivery of the blocking agents into the rectum/colon/intestinal tract via enema or suppositories. Injections or transdermal delivery into inflamed joints may yield relief to patients with arthritic or autoimmune diseases by limiting the secretion from localized inflammatory cells. Injection into areas surrounding nerve endings may inhibit secretion of some types of neurotransmitters, blocking transmission of severe pain or uncontrolled muscle spasms. Delivery of the peptide for the treatment of inflammatory skin diseases should be readily accomplished using various topical formulations known in the art.
- MARCKS myristoylatcd alaninc-rich C kinase substrate
- NHBE normal human bronchial epithelial
- MARCKS PKC-dependent phosphorylation of MARCKS, which releases MARCKS from the plasma membrane into the cytoplasm, where it is in turn dephosphorylated by a protein phosphatase 2A (PP2A) that is activated by PKG.
- P2A protein phosphatase 2A
- MARCKS interacts with actin and myosin in the cytoplasm and thus may be able to tether the granules to the cellular contractile apparatus, thus, mediating subsequent granule movement and exocytosis.
- MARCKS serves as the point of convergence for coordinating actions of these two protein kinases that control secretion from membrane- bound compartments in inflammatory cells (i.e. secretion of MPO from neutrophils).
- the present invention demonstrates secretion of the inflammatory mediator MPO from canine or human neutrophils was enhanced by concurrent activation of both PKC and PKG, while activation of either kinase alone was insufficient to induce a maximal secretory response.
- An enhanced secretory response to PMA alone was documented in NHBE cells (FIG. 1, column 4) and in neutrophils (FIG. 11), although the magnitude of the response was much less than that observed by others in a rat goblet-like cell line. See, Abdullah et al, supra.
- the present invention may be used in a pharmaceutical formulation.
- the drug product is present in a solid pharmaceutical composition that may be suitable for oral administration.
- a solid composition of matter according to the present invention may be formed and may be mixed with and/or diluted by an excipient.
- the solid composition of matter also may be enclosed within a carrier, which may be, for example, in the form of a capsule, sachet, tablet, paper, or other container.
- the excipient serves as a diluent, it may be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the composition of matter.
- excipients will be understood by those skilled in the art and may be found in the National Formulary, 19: 2404-2406 (2000), the disclosure of pages 2404 to 2406 being incorporated herein in their entirety.
- suitable excipients include, but are not limited to, starches, gum arabic, calcium silicate, microcrystalline cellulose, methacrylates, shellac, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose.
- the drug product formulations additionally can include lubricating agents such as, for example, talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propyl hydroxybenzoates; sweetening agents; or flavoring agents.
- lubricating agents such as, for example, talc, magnesium stearate and mineral oil
- wetting agents such as, for example, talc, magnesium stearate and mineral oil
- emulsifying and suspending agents such as methyl- and propyl hydroxybenzoates
- sweetening agents or flavoring agents.
- Polyols, buffers, and inert fillers also may be used. Examples of polyols include, but are not limited to, mannitol, sorbitol, xylitol, sucrose, maltose, glucose, lactose, dextrose, and the like.
- Suitable buffers include, but are not limited to, phosphate,
- inert fillers that may be used include those that are known in the art and are useful in the manufacture of various dosage forms.
- the solid formulations may include other components such as bulking agents and/or granulating agents, and the like.
- the drug products of the invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.
- the composition of matter of the present invention may be made by a direct compression process.
- the active drug ingredients may be mixed with a solid, pulverant carrier such as, for example, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin, and mixtures thereof, as well as with an antifriction agent such as, for example, magnesium stearate, calcium stearate, and polyethylene glycol waxes.
- a solid, pulverant carrier such as, for example, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin, and mixtures thereof, as well as with an antifriction agent such as, for example, magnesium stearate, calcium stearate, and polyethylene glycol waxes.
- the mixture may then be pressed into tablets using a machine with the appropriate punches and dies to obtain the desired tablet size.
- tablets for oral administration may be formed by a wet granulation process.
- Active drug ingredients may be mixed with excipients and/or diluents.
- the solid substances may be ground or sieved to a desired particle size.
- a binding agent may be added to the drug.
- the binding agent may be suspended and homogenized in a suitable solvent.
- the active ingredient and auxiliary agents also may be mixed with the binding agent solution.
- the resulting dry mixture is moistened with the solution uniformly. The moistening typically causes the particles to aggregate slightly, and the resulting mass is pressed through a stainless steel sieve having a desired size.
- the mixture is then dried in controlled drying units for the determined length of time necessary to achieve a desired particle size and consistency.
- the granules of the dried mixture are sieved to remove any powder.
- disintegrating, antifriction, and/or anti-adhesive agents may be added.
- the mixture is pressed into tablets using a machine with the appropriate punches and dies to obtain the desired tablet size.
- the operating parameters of the machine may be selected by the skilled artisan.
- the above prepared core may be coated with a concentrated solution of sugar or cellulosic polymers, which may contain gum arab ⁇ c, gelatin, talc, titanium dioxide, or with a lacquer dissolved in a volatile organic solvent or a mixture of solvents.
- a concentrated solution of sugar or cellulosic polymers which may contain gum arab ⁇ c, gelatin, talc, titanium dioxide, or with a lacquer dissolved in a volatile organic solvent or a mixture of solvents.
- various dyes may be added in order to distinguish among tablets with different active compounds or with different amounts of the active compound present.
- the active ingredient may be present in a core surrounded by one or more layers including enteric coating layers.
- Soft gelatin capsules may be prepared in which capsules contain a mixture of the active ingredient and vegetable oil.
- Hard gelatin capsules may contain granules of the active ingredient in combination with a solid, pulverulent carrier, such as, for example, lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives, and/or gelatin.
- a solid, pulverulent carrier such as, for example, lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives, and/or gelatin.
- Liquid preparations for oral administration may be prepared in the form of syrups or suspensions, e.g., solutions containing an active ingredient, sugar, and a mixture of ethanol, water, glycerol, and propylene glycol. If desired, such liquid preparations may comprise one or more of following: coloring agents, flavoring agents, and saccharin. Thickening agents such as carboxymethylcellulose also may be used. 100096 ⁇ In the event that the above pharmaceuticals are to be used for parenteral administration, such a formulation may comprise sterile aqueous injection solutions, nonaqueous injection solutions, or both, comprising the composition of matter of the present invention. When aqueous injection solutions are prepared, the composition of matter may be present as a water soluble pharmaceutically acceptable salt.
- Parenteral preparations may contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient.
- Aqueous and non-aqueous sterile suspensions may comprise suspending agents and thickening agents.
- the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampules and vials.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
- composition of matter also may be formulated such that it may be suitable for topical administration (e.g., skin cream).
- These formulations may contain various excipients known to those skilled in the art. Suitable excipients may include, but are not limited to, cetyl esters wax, cetyl alcohol, white wax, glyceryl monostearate, propylene glycol, monostearate, methyl stearate, benzyl alcohol, sodium lauryl sulfate, glycerin, mineral oil, water, carbomer, ethyl alcohol, acrylate adhesives, polyisobutylene adhesives, and silicone adhesives.
- NHBE cells were exposed to the following two specific protein kinase activators: the phorbol ester, phorbol 12-myristate 13-acetate (PMA), for activation of PKC, and the nonhydrolyzable cGMP analogue, 8-Br-cGMP, for activation of PKG.
- PMA phorbol 12-myristate 13-acetate
- 8-Br-cGMP nonhydrolyzable cGMP analogue
- UTP is a well defined pathophysiological ⁇ relevant mucin secretagogue. Lethem et al, Am. J. Respir. Cell MoI. Biol. 9, 315-322 (1993).
- the present invention further demonstrates that UTP, at various concentrations, preferably 40 to 140 ⁇ M, may induce a significant increase in mucin secretion from NHBE cells after a 2-h exposure.
- PKC and PKG were involved in regulation of mucin secretion in response to a pathophysiological stimulus
- effects of PKC/PKG inhibitors on UTP-induced mucin secretion were investigated. NHBE cells were preincubated with various inhibitors for 15 min and then exposed to UTP (100 ⁇ M) plus the inhibitor for 2 h.
- mucin secretion provoked by UTP may require both PKC and PKG activities, as the secretory response was attenuated independently by the PKC inhibitor calphostin C (500 nM), the PKG inhibitor R p -8-Br-PET-cGMP (10 ⁇ M), or the soluble guanylyl cyclase (GC-S) inhibitor LY83583 (50 ⁇ M) but likely not by the protein kinase A (PKA) inhibitor KT5720 (500 nM) (FIG. ID).
- PKC protein kinase A
- 8-Br-cGMP was utilized in these studies. Although the primary physiological effect of 8-Br-cGMP is to activate PKG, it also has been reported to act as an agonist for cGMP-gated ion channels in some cells and, at high concentrations, to cross-activate PKA.
- R p -8-Br-cGMP a unique cGMP analogue that can activate cGMP-gated ion channels similar to 8-Br-cGMP but inhibit PKG activity, was used as an agonist to distinguish the effects of PKG and cGMP- gated ion channels on mucin release.
- FIG. IA column 11
- R p -8-Br-cGMP did not enhance mucin secretion when added to the cells with PMA.
- the present invention examines potential intracellular substrates of these enzymes that could play a role in signaling events downstream of the kinase activation.
- Numerous intracellular substrates can be phosphorylated by PKC or PKG, and phosphorylation by PKC of one such substrate, MARCKS protein, seemed to be of particular interest.
- MARCKS phosphorylation has been observed to correlate with a number of cellular processes involving PKC signaling and cytoskeletal contraction, such as cell movement, mitogenesis, and neural transmitter release.
- MARCKS appeared to be a candidate for a mediator molecule connecting PKC/PKG activation and mucin granule exocytosis.
- MARCKS is a Key Molecule Linking PKC/PKG Activation to Mucin Secretion in NHBE Cells
- MARCKS protein a specific cellular substrate of PKC that might play a role in linking kinase activation to granule release was studied.
- MARCKS protein a specific cellular substrate of PKC that might play a role in linking kinase activation to granule release was studied.
- FIG. 2A MARCKS was expressed in NHBE cells, and the majority of this protein was membrane-associated under unstimulated conditions.
- FIG. 2A cells were labeled with [ H]myristic acid overnight and the membrane (lane 1) and the cytosol (lane 2) fractions were then isolated by differential centrifugation.
- a role for MARCKS as a key regulatory component of the mucin secretory pathway may be demonstrated in three different ways.
- MARCKS mucin secretion in response to stimulation by PMA+8-Br-cGMP or UTP was inhibited in a concentration- dependent manner by the MANS peptide, which had the amino acid sequence identical to the N-terminal region of MARCKS, whereas the corresponding control peptide (RNS), containing the same amino acid composition but arranged in random order, did not affect secretion.
- the N-terminal myristoylated domain of MARCKS is known to mediate the MARCKS-membrane association. As indicated in FIG.
- MARCKS may function as a molecular linker by interacting with granule membranes at its N-terminal domain and binding to actin filaments at its PSD site, thereby tethering granules to the contractile cytoskeleton for movement and exocytosis.
- FIG. 8 shows a possible mechanism depicting that mucin secretagogue interacts with airway epithelial (goblet) cells and activates two separate protein kinases, PKC and PKG.
- Activated PKC phosphorylates MARCKS, causing MARCKS translocation from the plasma membrane to the cytoplasm, whereas PKG, activated via the nitric oxide (NO) — * ⁇ GC-S — > cGMP ⁇ PKG pathway, in turn activates a cytoplasmic PP2A, which dephosphorylates MARCKS. This dephosphorylation stabilizes MARCKS attachment to the granule membranes.
- MARCKS also interacts with actin and myosin, thereby linking granules to the cellular contractile machinery for subsequent movement and exocytotic release.
- MARCKS The attachment of MARCKS to the granules after it is released into the cytoplasm may also be guided by specific targeting proteins or some other forms of protein- protein interactions in which the N-terminal domain of MARCKS is involved.
- the MANS peptide, or an active fragment thereof, comprising at least 6 amino acids would act to inhibit competitively targeting of MARCKS to the membranes of mucin granules, thereby blocking secretion.
- a second test demonstrated the inhibitory effect of a MARCKS-specific antisense oligonucleotide on mucin secretion.
- the antisense oligonucleotide down-regulated MARCKS mRNA and protein levels in NHBE cells and substantially attenuated mucin secretion induced by PKC/PKG activation.
- FIG. 3A is a Northern blot that showed a decrease of .about.15% in MARCKS mRNA compared with controls in the attached chart;
- FIG. 3 B is Western blot that showed a decrease of about 30% in MARCKS protein in the attached graph; and FIG. 3 C shows mucin hypersecretion was attenuated significantly by the antisense oligonucleotide, whereas the control oligonucleotide had no effect.
- CTO is the control oligonucleotide
- ASO is an antisense oligonucleotide.
- antisense oligonucleotides that are complementary to specific RNAs can inhibit the expression of cellular genes as proteins. See Erickson and Izant, Gene Regulation: Biology Of Antisense RNA And DNA, Vol. I 5 Raven Press, N.Y., 1992. For example, selective inhibition of a p21 gene that differed from a normal gene by a single nucleotide has been reported. Chang et al., Biochemistry (1991), 30:8283-8286.
- a third experiment indicated that transfection of HBEl cells with a PSD-deleted mutant MARCKS resulted in significant repression of mucin secretion induced by PKC/PKG activation. Deletion of the PSD would abolish the ability of MARCKS to bind to act ⁇ n. As indicated in FIG. 8, by competing with native MARCKS for binding to granule membrane, the PSD-truncated MARCKS could thereby inhibit granule release as it is unable to interact with the actin filaments. Transfection of these cells with the wild-type MARCKS cDNA did not further enhance mucin secretion.
- MARCKS serves as a Convergent Signaling Molecule Mediating Cross-talk of PKC and PKG Pathways
- MARCKS was involved integrally in the mucin secretory process.
- the present inventors addressed how MARCKS acts as a key regulatory molecule upon which PKC and PKG converge to regulate mucin secretion.
- FIG. 5 MARCKS was phosphorylated by PKC and consequently translocated from the membrane to the cytoplasm.
- PKG appeared to induce dephosphorylation of MARCKS (FIG. 5A, lane 4, and FIG. 5B).
- R p ⁇ 8-Br-PET ⁇ cGMP FIG. 5 A, lane 5
- the NHBE cells were labeled with [ 32 P]orthophosphate and then exposed to the indicated reagents.
- MARCKS phosphorylation in response to the treatments was evaluated by immunoprecipitation assay.
- 8-Br- cGMP reversed MARCKS phosphorylation induced by PMA, and this effect of 8-Br-cGMP could be blocked by R p -8-Br-PET-cGMP (PKG inhibitor) or okadaic acid (PP 1/2 A inhibitor).
- PMA-induced phosphorylation of MARCKS was reversed by subsequent exposure of cells to 8-Br-cGMP.
- Lane 1 medium alone for 8 min; lane 2, 100 nM PMA for 3 min; lane 3, 100 nM PMA for 3 min and then with 1 ⁇ M 8-Br-cGMP for 5 min; lane 4, 100 nM PMA for 8 min; lane 5, medium alone for 3 min and then 100 nM PMA+1 ⁇ M 8-Br- cGMP for 5 min.
- 8-Br-cGMP-induced MARCKS dephosphorylation was attenuated by fostriecin in a concentration-dependent manner.
- FIG. 6 helps to demonstrate that PP2A is an essential component of the mucin secretory pathway.
- NHBE cells were preincubated with the indicated concentration of fostriecin, okadaic acid (500 nM), or medium alone for 15 min and then stimulated with PMA (100 nM)+8-Br-cGMP (1 ⁇ M) for 15 min or with UTP (100 ⁇ M) for 2 h.
- FIG. 4A shows the activation of PKC results in MARCKS phosphorylation in NHBE cells.
- Cells were labeled with [ 32 P]orthophosphate for 2 h and then exposed to the stimulatory and/or inhibitory reagents.
- MARCKS phosphorylation in response to the treatments was evaluated by immunoprecipitation as described.
- FIG. 4B demonstrates phosphorylated MARCKS is translocated from the plasma membrane to the cytoplasm. 32 P-Labeled cells were exposed to PMA (100 nM) or medium alone for 5 min, and then the membrane and the cytosol fractions were isolated.
- FIG. 7 depicts a radiolabeled immunoprecipitation assay which reveals that MARCKS may associate with two other proteins (.about .200 and .about .40 kDa) in the cytoplasm.
- NHBE cells were labeled with [ 3 H]leucine and [ 3 H]proline overnight, and the membrane and the cytosol fractions were prepared as described under "Experimental Procedures.” Isolated fractions were precleared with the nonimmune control antibody (6F6).
- the cytosol was then divided equally into two fractions and used for immunoprecipitation carried out in the presence of 10 ⁇ M cytochalasin D (Biomol, Plymouth Meeting, Pa.) with the anti-M ARCKS antibody 2Fl 2 (lane 2) and the nonimmune control antibody 6F6 (lane 3), respectively.
- MARCKS protein in the membrane fraction was also assessed by immunoprecipitation using the antibody 2Fl 2 (lane 1).
- the precipitated protein complex was resolved by 8% SDS-polyacrylamide gel electrophoresis and visualized by enhanced autoradiography. MARCKS appeared to associate with two cytoplasmic proteins with molecular masses of .about 200 and .about 40 kDa, respectively.
- MARCKS-associated proteins were excised from the gel and analyzed by matrix-assisted laser desorption ionization/time of flight mass spectrometry/ ⁇ nternal sequencing (the Protein/DNA Technology Center of Rockefeller University, N.Y.). The obtained peptide mass and sequence data were used to search protein databases via Internet programs ProFound and MS-Fit. Results indicate that they are myosin (heavy chain, non-muscle type A) and actin, respectively. Matrix-assisted laser desorption ionization/time of flight mass spectrometry/internal sequence analysis indicates that these two MARCKS-associated proteins were myosin (heavy chain, non-muscle type A) and actin, respectively.
- MARCKS serves as a key mediator molecule regulating mucin granule release in human airway epithelial cells. It is believed that elicitation of airway mucin secretion requires dual activation and synergistic actions of PKC and PKG. Activated PKC phosphorylates MARCKS, resulting in translocation of MARCKS from the inner face of the plasma membrane into the cytoplasm.
- MARCKS Activation of PKG in turn activates PP2A, which dephosphorylates MARCKS in the cytoplasm. Because the membrane association ability of MARCKS is dependent on its phosphorylation state this dephosphorylation may allow MARCKS to regain its membrane-binding capability and may enable MARCKS to attach to membranes of cytoplasmic mucin granules. By also interacting with actin and myosin in the cytoplasm (FIG. 7), MARCKS may then be able to tether granules to the cellular contractile apparatus, mediating granule movement to the cell periphery and subsequent exocytotic release. The wide distribution of MARCKS suggests the possibility that this or a similar mechanism may regulate secretion of membrane-bound granules in various cell types under normal or pathological conditions.
- the invention also relates to a new method for blocking any cellular exocytotic secretory process, especially those releasing inflammatory mediators from granules contained within inflammatory cells, whose stimulatory pathways involve the protein kinase C (PKC) substrate MARCKS protein and release of contents from membrane-bound vesicles.
- PKC protein kinase C
- the inventors have shown that stimulated release of the inflammatory mediator myloperoxidase from human (FIG. 9) or canine (FIG. 10) neutrophils can be blocked in a concentration-dependent manner by the MANS peptide.
- FIG. 9 stimulated release of the inflammatory mediator myloperoxidase from human
- FIG. 10 canine neutrophils
- FIG. 9 shows isolated neutrophils that were stimulated to secrete myloperoxidase (MPO) with 100 nM PMA and 10 .mu.M 8-Br-cGMP.
- 10 ⁇ M MANS causes a slight decrease in MPO secretion.
- 10 or 100 ⁇ M of a control peptide (RNS) has no effect on MPO secretion.
- isolated neutrophils were stimulated to secrete myloperoxidase (MPO) with 100 nM PMA and 10 ⁇ M 8-Br-cGMP.
- 10 ⁇ M MANS causes a slight decrease in MPO secretion.
- 10 or 100 ⁇ M of a control peptide (RNS) has no effect on MPO secretion.
- RNS control peptide
- the peptide may be used therapeutically to block the release of mediators of inflammation secreted from infiltrating inflammatory cells in any tissues.
- inflammatory diseases i.e., respiratory diseases such as asthma, chronic bronchitis and COPD, inflammatory bowel diseases including ulcerative colitis and Crohn's disease, autoimmune diseases, skin diseases such as rosacea, eczema; and severe acne, arthritic and pain syndromes such as rheumatoid arthritis and fibromyalgia.
- This invention may be useful for treating diseases such as arthritis, chronic bronchitis, COPD and cystic fibrosis.
- This invention is accordingly useful for the treatment in both human and animal diseases, especially those affecting equines, canines, felines, and other household pets.
- FIGS. 11-15 show MPO secretion for both humans and canines.
- isolated neutrophils were stimulated with LPS at a concentration of 1 x 1 CT 6 M for 10 minutes at 37 0 C. prior to adding the stimuli as indicated in the figures.
- the LPS primes the cells so they can respond to a secretagogue.
- this invention discloses a method of regulating an inflammation in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a MANS peptide or an active fragment thereof.
- said active fragment of the MANS protein comprises at least six amino acids.
- said inflammation is caused by respiratory diseases, bowel diseases, skin diseases, autoimmune diseases and pain syndromes.
- said respiratory diseases are selected from the group consisting of asthma, chronic bronchitis, and COPD.
- said bowel diseases are selected from the group consisting of ulcerative colitis, Crohn's disease and irritable bowel syndrome.
- said skin diseases are selected from the group consisting of rosacea, eczema, psoriasis and severe acne.
- said inflammation is caused by arthritis or cystic fibrosis.
- said subject is a mammal.
- said mammal is selected from the group consisting of humans, canines, equines and felines.
- said administering step is selected from the group consisting of topical administration, parenteral administration, rectal administration, pulmonary administration, nasal administration, inhalation and oral administration.
- said pulmonary administration is selected from the group of aerosol, dry powder inhaler, metered dose inhaler, and nebulizer.
- this invention discloses a method for regulating a cellular secretory process in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition comprising at least one compound comprising a MANS peptide or an active fragment thereof, that regulates an inflammatory mediator in a subject.
- said active fragment of the MANS protein comprises at least six amino acids.
- said regulating a cellular secretory process is blocking or reducing a cellular secretory process.
- said inflammatory mediator is caused by respiratory diseases, bowel diseases, skin diseases, autoimmune diseases and pain syndromes.
- said respiratory diseases are selected from the group consisting of asthma, chronic bronchitis, and COPD.
- said bowel diseases are selected from the group consisting of ulcerative colitis, Crohn's disease and irritable bowel syndrome.
- said skin diseases are selected from the group consisting of rosacea, eczema, psoriasis and severe acne.
- said inflammatory mediator is caused by arthritis or cystic fibrosis.
- said subject is a mammal.
- said mammal is selected from the group consisting of humans, canines, equines and felines.
- said administering step is selected from the group consisting of topical administration, parenteral administration, rectal administration, pulmonary administration, nasal administration, inhalation and oral administration.
- said pulmonary administration is selected from the group of aerosol, dry powder inhaler, metered dose inhaler, and nebulizer.
- this invention discloses a method of reducing inflammation in a subject comprising administering a therapeutically effective amount of a compound that inhibits the MARCKS-related release of inflammatory mediators, whereby the release of inflammatory mediators in the subject is reduced compared to that which would occur in the absence of said treatment.
- said compound is at least one active fragment of a MARCKS protein.
- said active fragment is at least six amino acids in length.
- said compound is a MANS peptide or an active fragment thereof.
- said compound is an antisense oligonucleotide directed against the coding sequence of a MARCKS protein or an active fragment thereof.
- said active fragment is at least six amino acids in length.
- this invention discloses a method of reducing inflammation in a subject comprising administering a therapeutically effective amount of a pharmaceutically active composition comprising a compound that inhibits the MARCKS- related release of inflammatory mediators, whereby the inflammation in the subject is reduced compared to that which would occur in the absence of said treatment.
- said compound is an active fragment of a MARCKS protein.
- said active fragment is at least six amino acids in length.
- said compound is a MANS peptide or an active fragment thereof.
- said compound is an antisense oligonucleotide directed against the coding sequence of a MARCKS protein or an active fragment thereof.
- said active fragment is at least six amino acids in length.
- the present invention is intended to encompass a composition that contains one or more of the MANS peptide or its active fragments and use thereof in the treatment of inhibiting the release of inflammatory mediators from granules or vesicles of inflammatory cells.
- this invention discloses a method of regulating mucin granule release in a subject comprising administering a compound that regulates mucin granule release, whereby mucin granules are reduced as compared to that which would occur in the absence of said mucin granules.
- said compound is an active fragment of a MARCKS protein.
- said compound is a MANS peptide.
- this invention discloses a method of regulating exocytotic secretion of airway mucin granules in a subject comprising: administering a compound that regulates mucin granule release, whereby mucin granules are reduced as compared to that which would occur in the absence of said mucin granules.
- said compound is an active fragment of a MARCKS protein.
- said compound is a MANS peptide.
- this invention discloses a method of modulating mucus secretion in a subject comprising: administering a therapeutic amount of an antisense sequence that are complementary to sequences encoding a MARCKS protein or an active fragment thereof, wherein mucus secretion by said cell is inhibited compared to that which would occur in the absence of such administration.
- said sequence is at least eighteen nucleic acids in length.
- said compound is complementary to sequences encoding a MANS peptide or an active fragment thereof.
- said modulating mucus secretion is blocking or reducing mucus secretion.
- this invention discloses a method of reducing or inhibiting inflammation in a subject comprising administering a therapeutically effective amount of at least one peptide comprising MANS peptide or an active fragment thereof effective to modulate an inflammatory mediator at the inflammation site.
- said active fragment is at least six amino acids in length.
- said inflammatory mediators are produced by cells selected from the group consisting of neutrophils, basophils, eosinophils, monocytes and leukocytes.
- the cells are leukocytes, more preferably granulocytes, and even more preferably neutrophils, basophils, eosinophils or a combination thereof.
- the agent is administered orally, parenterally, cavitarily, rectally or through an air passage.
- said composition fUrther comprises a second molecule selected from the group consisting of an antibiotic, an antiviral compound, an antiparasitic compound, an anti-inflammatory compound, and an immunosuppressant.
- An active fragment of a MANS peptide can be selected from the group consisting of the myristoylated peptides of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:
- the methods disclosed in this invention can be accomplished by use of or administering of combinations of the peptides disclosed in the invention, i.e., by use of or administering of a peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4. SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
- SEQ ID NO 19 SEQ ID NO 19 and combinations thereof.
- a single peptide is used or administered in the methods disclosed herein.
- PKC protein kinase C
- degranulation in a cell selected from the group consisting of neutrophils, eosinophils, monocytes/macrophages and lymphocytes can be attenuated by pre-incubation and by co- incubation with a peptide identical to the N-terminal region of MARCKS protein, wherein the peptide is selected from the group consisting of the MANS peptide (SEQ ID NO: 1) and myristoylated N-terminal fragments thereof (SEQ ID NO: 3 to 19).
- MANS peptide SEQ ID NO: 1
- myristoylated N-terminal fragments thereof SEQ ID NO: 3 to 19
- [000129IMZS 1 E Cell Culture-Expansion, cryopreservation, and culture of NHBE cells in the air/liquid interface were performed as described previously. See, Krunkosky et ⁇ l. Briefly, NHBE cells (Clonetics, San Diego, Calif.) were seeded in vented T75 tissue culture flasks (500 cells/cm 2 ) and cultured until cells reached 75-80% confluence. Cells were then dissociated by trypsin/EDTA and frozen as passage-2.
- Air/liquid interface culture was initiated by seeding passage-2 cells (2 x 10 4 cells/cm 2 ) in TRANSWELL® clear culture inserts (Costar, Cambridge, Mass.) that were thinly coated with rat tail collagen, type I (Collaborative Biomedical, Bedford, Mass.). Cells were cultured submerged in medium in a humidified 95% air, 5% CO 2 environment for 5-7 days until nearly confluent. At that time, the air/liquid interface was created by removing the apical medium and feeding cells basal ateral Iy. Medium was renewed daily thereafter. Cells were cultured for an additional 14 days to allow for full differentiation.
- Both base line and test secretions were analyzed by ELISA using an antibody capture method as known in the art. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, pp. 570-573, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1988).
- the primary antibody for this assay was 17Q2 (Babco, Richmond, Calif.), a monoclonal antibody that reacts specifically with a carbohydrate epitope on human airway mucins.
- the ratio of test/base-line mucin is similar to a "secretory index", was used to quantify mucin secretion, allowing each culture dish to serve as its own control and thus, minimizing deviation caused by variability among culture wells. Wright et al., Am. J. Physiol. 271, L854-L861 (1996). Levels of mucin secretion were reported as percentage of the medium control.
- Radiolabeled Immunoprecipitation Assay When labeling with [ 32 P]phosphate, cells were preincubated for 2 h in phosphate-firee Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and then labeled with 0.1 mCi/ml [ 32 P] orthophosphate (9000 Ci/mmol, PerkinElmer Life Sciences) for 2 h.
- Post-nuclear supernatants were separated into membrane and cytosol fractions by ultracentrifugation at 400,000 x g for 30 min at 4 0 C.
- the membrane pellet was solubilized in the lysis buffer by sonication. lmmunoprecipitation was then carried out as described above.
- MARCKS-related Peptides Both the myristoylated N-terminal sequence (MANS) and the random N-terminal sequence (RNS) peptides were synthesized at Genemed Synthesis, Inc. (San Francisco, Calif.), then purified by high pressure liquid chromatography (>95% pure), and confirmed by mass spectroscopy with each showing one single peak with an appropriate molecular mass.
- the corresponding control peptide contained the same amino acid composition as the MANS but arranged in random order, MA-GTAPAAEGAGAEVKRASAEAKQAF (SEQ ID NO: 2).
- MA-GTAPAAEGAGAEVKRASAEAKQAF SEQ ID NO: 2.
- the presence of the hydrophobic myristate moiety in these synthetic peptides enhances their permeability to the plasma membranes, enabling the peptides to be taken up readily by cells.
- cells were preincubated with the peptides for 15 min prior to addition of secretagogues, and mucin secretion was then measured by ELISA.
- Antisense Oligonucleotides MARCKS antisense oligonucleotide and its corresponding control oligonucleotide were synthesized at Biognostik GmbH (Gottingen, Germany). NHBE cells were treated with 5 ⁇ M antisense or control oligonucleotide apically for 3 days (in the presence of 2 ⁇ g/ml lipofectin for the first 24 h). Cells were then incubated with secretagogues, and mucin secretion was measured by ELISA. Total RNA and protein were isolated from treated cells. MARCKS mRNA was assessed by Northern hybridization according to conventional procedures using human MARCKS cDNA as a probe. MARCKS protein level was determined by Western blot using purified anti-MARCKS IgGl (clone 2Fl 2) as the primary detection antibody.
- PSD phosphorylation site domain
- MARCKS contains the PKC-dependent phosphorylation sites and the actin filament-binding site.
- two fragments flanking the PSD sequence (coding for 25 amino acids) were generated by polymerase chain reaction and then ligated through the Xhol site that was attached to the 5'-ends of oligonucleotide primers designed for the polymerase chain reaction.
- the resultant mutant cDNA and the wild-type MARCKS cDNA were each inserted into a mammalian expression vector pcDNA4/TO (Invitrogen, Carlsbad, Calif.). Isolated recombinant constructs were confirmed by restriction digests and DNA sequencing.
- HBEl is a papilloma virus-transformed human bronchial epithelial cell line capable of mucin secretion when cultured in air/liquid interface. Transfection of HBEl cells was carried out using the Effectene transfection reagent (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. Briefly, differentiated HBEl cells grown in air/liquid interface were dissociated by trypsin/EDTA and re-seeded in 12-well culture plates at 1 x 10 5 cells/cm 2 . After overnight incubation, cells were transfected with the wild-type MARCKS cDNA, the PSD-truncated MARCKS cDNA, or vector DNA.
- Protein Phosphatase Activity Assay PTl and PP2A activities were measured using a protein phosphatase assay system (Life Technologies, Inc.) as known in the art with slight modification. Huang et al., Adv. Exp. Med Biol. 396, 209-215 (1996). Briefly, NHBE cells were treated with 8-Br-cGMP or medium alone for 5 min.
- Cells were then scraped into a lysis buffer (50 mM Tris-HCl (pH 7.4), 0.1% .beta.-mecaptoethanol, 0.1 mM EDTA, 1 mM benaamidine, 10 ⁇ g/ml pepstatin A, 10 ⁇ g/ml Ieupeptin) and disrupted by sonication for 20 s at 4 0 C.
- Cell lysates were centrifuged and the supernatants saved for phosphatase activity assay. The assay was performed using 32 P-labeled phosphorylase A as a substrate. Released 32 Pi was counted by scintillation. The protein concentration of each sample was determined by the Bradford assay.
- PP2A activity was expressed as the sample total phosphatase activity minus the activity remaining in the presence of 1 nM okadaic acid.
- PPl activity was expressed as the difference between the activities remaining in the presence of 1 nM and 1 ⁇ M okadaic acid, respectively.
- Protein phosphatase activities were reported as nmol of Pj released per min/mg total protein.
- Cytotoxicity Assay All reagents used in treating NHBE cells were examined for cytotoxicity by measuring the total release of lactate dehydrogenase from the cells. The assay was carried out using the Promega Cytotox 96 Kit according to the manufacturer's instructions. All experiments were performed with reagents at non-cytotoxic concentrations.
- the steps involved in isolating PMN include collecting 10 ml ACD anti coagulated blood. Then layering 5 ml on 3.5 ml PMN isolation media while ensuring that the PMN isolation media (IM) was at room temperature (RI). Next, the blood was centrifuged at room temperature for 30', 550 X g at 1700 RPMs. The low lower white band was transferred into 15 ml conical centrifuge tube (CCFT). Next, 2V HESS with 10% fetal bovine serum (PBS) was added and centrifuged at room temperature for 10', 400 X g at 1400 RPMs.
- PBS fetal bovine serum
- the pellet was then resuspended in 5 ml 1-1 ESS with PBS.
- the cell suspension was added to 50 ml CCFT containing 20 ml of ice cold 0.88% NH 4 Cl and inverted two to three times.
- the resulting product was centrifuged for 10', 800 X g at 2000 RPMs, then aspirated and resuspended in 5 ml HBSS with FBS.
- the prep was examined by counting and cytospin and preferably for whole blood, the cell number should be between lO 9 -l ⁇ " cells and for PMNs, cell number should be between 2-4 x 10 7 cells. See generally, Wang et al., J. Immunol., "Neutrophil-induced changes in the biomechanical properties of endothelial cells: roles of ICAM-I and reactive oxygen species," 6487-94 (2000).
- the final concentrations in the assay mixture are: 30 mM potassium phosphate, pH 6.0, 0.05% Triton X-100, 0.1 mM hydrogen peroxide, and 0.32 mM O- Diannisidine Dihydrochloride.
- the assay mixture was incubated at room temperature for 5 minutes, and MPO enzyme activity determined spectrophoto ⁇ ietrically at 550 nanometers. Samples were assayed in duplicate.
- the monocytic leukemia cell line U937 was used to assess secretion of lysozyme (Hoff T, Spencker T, Emmendoerffer A., Goppelt-Struebe M. Effects of glucocorticoids on the TPA-induced monocytic differentiation. J Leukoc Biol 1992; 52:173-182; Balboa M A, Saez Y, Balsinde J. Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes. J Immunol 2003; 170:5276-5280; Sundstrom C, Nilsson K.
- a human histiocytic lymphoma cell line U-937. Int J Cancer 1976; 17:565-577.
- the lymphocyte natural killer cell line NK-92 used to assess release of granzyme (Gong JH., Maki G 5 Klingemann HG. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 1994; 8:652-658; Maki G, Klingemann HG, Martinson JA, Tarn YK. Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92.
- GTAPAAEGAGAEVKRASAEAKQAF (SEQ ID NO: 2)], which consists of the same 24 amino acids but arranged in random order sequence which possesses less than 13% sequence identity to the MANS peptide sequence.
- MANS but not RNS, attenuates release of inflammatory mediators in a concentration-dependent manner.
- a useful time course of observation is 0.5- 3.0 hrs. The results are consistent with the N-terminal region of the MARCKS protein being involved in intracellular pathways leading to leukocyte degranulation.
- erythrocytes in the sediment were lysed in chilled distilled water. Isolated granulocytes were washed twice with Hanks' balanced salts solution (HBSS) and resuspended in HBSS on ice.
- HBSS Hanks' balanced salts solution
- the neutrophils used for the experiments were of >98% purity with ⁇ 2% contamination by eosinophils, and the viability was >99% as determined by Trypan blue dye exclusion.
- PMA control reference was established using purified human neutrophils suspended in HBSS aliquoted at 4 x 10 6 cells/ml in 15 ml tubes and stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) in the absence of MANS or RNS peptide for the same time periods. The reaction was terminated by placing the tubes on ice and centrifiigation at 400g for 5 min at 4°C.
- TMB tetramethylbenzidine
- HL-60 clone 15 cells (ATCC CRL-1964) were maintained in medium consisting of RPMI 1640 with L-glutamine supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Invitrogen Co., Carlsbad, CA), 50 IU/ml penicillin, 50 ⁇ g/ml streptomycin, and 25 mM HEPES buffer, pH 7.8, at 37°C in an atmosphere containing 5% CO2.
- FBS heat-inactivated fetal bovine serum
- FBS heat-inactivated fetal bovine serum
- 50 IU/ml penicillin 50 ⁇ g/ml streptomycin
- 25 mM HEPES buffer pH 7.8, at 37°C in an atmosphere containing 5% CO2.
- CC chemokine receptors 1 and 3 are differentially regulated by IL-5 during maturation of eosinophilic HL-60 cells. J Immunol 1998; 160:1385-1392).
- U937 cells (ATCC CRL- 1593.2) were grown at 37°C in an atmosphere of 5% CO 2 in complete medium consisting of RPMI 1640 with L-glutamine supplemented with 10% FBS, 50 IU/ml penicillin, and 50 ⁇ g/ml streptomycin.
- NK-92 cells (ATCC CRL-2407) were maintained in alpha-MEM medium (Sigma-Aldrich Co.) supplemented with 20% FBS, 100 U/ml of interleukin-2 (IL-2) (Chemicon International, Inc., Temecula, CA) 3 5 x 10 "5 M of 2- mercaptoethanol, 50 IU/ml penicillin, and 50 ⁇ g/ml streptomycin at 37°C in an atmosphere containing 5% CO 2 .
- IL-2 interleukin-2
- HL-60 clone 15, U937, and NK-92 cells were washed and resuspended at 2.5 x 10 6 cells/ml in phenol red-free RPMI- 1640 (Cellgro; Mediatech, Inc.) for all degranulation assays. Aliquots of cells in 15 ml rubes were preincubated with indicated concentrations of MANS or RNS peptide for 10 m ⁇ n at 37°C. The cells then were stimulated with PMA for up to 2 hr.
- a control reference (PMA control reference) was established for each cell type using HL-60 clone 15, U937, and NK-92 cells, respectively, which were washed and resuspended at 2.5 x 10 6 cells/ml in phenol red-free RPMI- 1640 and stimulated with PMA but in the absence of MANS or RNS peptide for the same time periods. The reaction was terminated by placing tubes on ice and centrifuging cells at 40Og for 5 min at 4°C.
- [00015O]EPO activity released by HL-60 clone 15 cells was assayed using TMB according to a previously established technique (Lacy P, Mahmudi-Azer S 5 Bablitz B, Hagen SC, Velazquez JR, Man SF, Moqbel R. Rapid mobilization of intracellularly stored RANTES in response to interferon-gamma in human eosinophils. Blood 1999; 94:23-32).
- the amount of secreted EPO was expressed as percentage of total content, using the amount obtained in the same number of triton X-100-lysed cells.
- Lysozyme secreted by U937 cells was measured using a spectrophotometric assay as described previously (Balboa M A, Saez Y, Balsinde J. Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes. J Immunol 2003; 170:5276-5280) with slight modification.
- 100 ⁇ l of sample was mixed with 100 ⁇ l of a Micrococcus lysodeikticus (Sigma-Aldrich Co.) suspension (0.3 mg/ml in 0.1 M sodium phosphate buffer, pH 7.0) in a 96-wcll microplate. The decrease in absorbance at 450 nm was measured at room temperature.
- a calibration curve was constructed using chicken egg white lysozyme (EMD Biosciences, Inc.) as a standard.
- Granzyme secreted from NK-92 cells was assayed by measuring hydrolysis of Na- benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) essentially as described previously (Takayama H, Trenn G, Sitkovsky MV. A novel cytotoxic T lymphocyte activation assay. J Immunol Methods 1987; 104:183-190).
- BLT Na- benzyloxycarbonyl-L-lysine thiobenzyl ester
- MANS 100 ⁇ M
- the 50 ⁇ M MANS sample measured about 290% of control or about 15% reduction relative to the PMA control reference.
- the PMA control reference MPO activity was about 540% of control, the 50 ⁇ M MANS (measuring about 375% of control) caused an approximately 30% reduction of MPO release versus the PMA control reference; and 100 ⁇ M MANS (measuring about 295% of control) caused an approximately 45% reduction of MPO release versus the PMA control reference.
- the PMA control reference MPO activity was about 560% of control, 50 ⁇ M MANS (measuring about 375% of control) caused an approximately 33% reduction of MPO release versus the PMA control reference; 100 ⁇ M MANS (measuring about 320% of control) caused an approximately 40% reduction of MPO release versus the PMA control reference.
- the RNS peptide did not affect PMA-induced MPO release at any of the time points or concentrations tested.
- EPO activity in the supernatant of HL-60 clone 15 cells was significantly enhanced at 1 and 2 hrs after PMA stimulation.
- the PMA control reference measured at about 110%; the sample containing 10 ⁇ M MANS measured at about 95% to give about 15% reduction in EPO activity relative to the PMA control reference; the sample containing 50 ⁇ M MANS measured at about 78% to give about 30% reduction in EPO activity relative to the PMA control reference; and the sample containing 100 ⁇ M MANS measured at about 65% to give about 40% reduction in EPO activity relative to the PMA control reference.
- the PMA control reference measured at about 145%; the sample containing 10 ⁇ M MANS measured at about 130% to give about 10% reduction in EPO activity relative to the PMA control reference; the sample containing 50 ⁇ M MANS measured at about 70% to give about 50% reduction in EPO activity relative to the PMA control reference; and the sample containing 100 ⁇ M MANS measured at about 72% to give about 50% reduction in EPO activity relative to the PMA control reference.
- MANS at 50 or 100 ⁇ M significantly attenuated EPO release.
- the RNS peptide did not affect PMA- enhanced EPO release at any of the time points or concentrations tested.
- Lysozyme secretion by U937 cells was increased by PMA stimulation by 1 hr after incubation, and increased even more at 2 hrs.
- the PMA control reference measured at about 210%; the sample containing 10 ⁇ M MANS measured at about 170% to give about 20% reduction in lysozyme secretion by U937 cells relative to the PMA control reference; the sample containing 50 ⁇ M MANS measured at about 170% to give about 20% reduction in lysozyme secretion by U937 cells relative to the PMA control reference; and the sample containing 100 ⁇ M MANS measured at about 115% to give about 45% reduction in lysozyme secretion by U937 cells relative to the PMA control reference.
- the PMA control reference measured at about 240%; the sample containing 10 ⁇ M MANS measured at about 195% to give about 20% reduction in lysozyme secretion by U937 cells relative to the PMA control reference; the sample containing 50 ⁇ M MANS measured at about 185% to give about 25% reduction in lysozyme secretion by U937 cells relative to the PMA control reference; and the sample containing 100 ⁇ M MANS measured at about 140% to give about 40% reduction in lysozyme secretion by U937 cells relative to the PMA control reference.
- lysozyme secretion was significantly attenuated at both 1 and 2 hours post-stimulation by 100 ⁇ M of MANS but not as much by 50 or 10 ⁇ M of MANS.
- the RNS peptide did not affect PMA-enhanced lysozyme secretion at any of the time points or concentrations tested.
- the PMA control reference measured at about 125%; the sample containing 10 ⁇ M MANS measured at about 115% to give about 10% reduction in granzyme secretion by NK-92 cells relative to the PMA control reference; and the sample containing 100 ⁇ M MANS measured at about 85% relative to the PMA control reference to give about 30% reduction in granzyme secretion by NK-92 cells relative to the PMA control reference.
- the PMA control reference measured at about 220%; the sample containing 10 ⁇ M MANS measured at about 200% to give about 10% reduction in granzyme secretion by NK-92 cells relative to the PMA control reference; and the sample containing 100 ⁇ M MANS measured at about 80% to give about 60% reduction granzyme secretion by NK-92 cells relative to the PMA control reference.
- granzyme secretion by NK-92 cells was not significantly increased by PMA at 1 hr, but increased over two-fold at 2 hours.
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EP07752392A EP1991250A4 (en) | 2006-03-06 | 2007-03-06 | Methods for regulating inflammatory mediators and peptides useful therein |
AU2007223983A AU2007223983A1 (en) | 2006-03-06 | 2007-03-06 | Methods for regulating inflammatory mediators and peptides useful therein |
CA002645019A CA2645019A1 (en) | 2006-03-06 | 2007-03-06 | Methods for regulating inflammatory mediators and peptides useful therein |
CN2007800162729A CN101500593B (en) | 2006-03-06 | 2007-03-06 | Methods for regulating inflammatory mediators and peptides useful therein |
BRPI0708672-5A BRPI0708672A2 (en) | 2006-03-06 | 2007-03-06 | methods for regulating inflammatory mediators and useful peptides therein. |
JP2008558354A JP2009531307A (en) | 2006-03-06 | 2007-03-06 | Methods for controlling inflammation mediators and peptides useful in the methods |
MX2008011375A MX2008011375A (en) | 2006-03-06 | 2007-03-06 | Methods for regulating inflammatory mediators and peptides useful therein. |
US11/834,446 US8501911B2 (en) | 1999-02-24 | 2007-08-06 | Methods of reducing inflammation and mucus hypersecretion |
IL193846A IL193846A (en) | 2006-03-06 | 2008-09-02 | Use of myristoylated n-terminal peptide for the manufacture of a medicament for the treatment of inflammatory conditions |
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EP (1) | EP1991250A4 (en) |
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EP2053915A2 (en) * | 2006-07-26 | 2009-05-06 | Biomarck Pharmaceuticals, Ltd. | Methods for attenuating release of inflammatory mediators and peptides useful therein |
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US8501911B2 (en) | 1999-02-24 | 2013-08-06 | Biomarck Pharmaceuticals, Ltd | Methods of reducing inflammation and mucus hypersecretion |
US8563689B1 (en) | 2001-06-26 | 2013-10-22 | North Carolina State University | Methods for regulating inflammatory mediators and peptides for useful therein |
US20140302057A1 (en) * | 2013-04-05 | 2014-10-09 | North Carolina State University | Inhibitors of metastasis |
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2006
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US8501911B2 (en) | 1999-02-24 | 2013-08-06 | Biomarck Pharmaceuticals, Ltd | Methods of reducing inflammation and mucus hypersecretion |
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CN101500593B (en) | 2012-08-22 |
SG10201405436WA (en) | 2014-10-30 |
KR20090069255A (en) | 2009-06-30 |
JP2009531307A (en) | 2009-09-03 |
CN101500593A (en) | 2009-08-05 |
EP1991250A4 (en) | 2012-07-04 |
CA2645019A1 (en) | 2007-09-13 |
US7544772B2 (en) | 2009-06-09 |
BRPI0708672A2 (en) | 2012-05-29 |
IL193846A0 (en) | 2011-08-01 |
ZA200807626B (en) | 2009-11-25 |
IL193846A (en) | 2015-05-31 |
WO2007103368A3 (en) | 2009-04-16 |
US8563689B1 (en) | 2013-10-22 |
EP1991250A2 (en) | 2008-11-19 |
RU2429004C2 (en) | 2011-09-20 |
RU2008139414A (en) | 2010-04-20 |
AU2007223983A1 (en) | 2007-09-13 |
US20060217307A1 (en) | 2006-09-28 |
SG170082A1 (en) | 2011-04-29 |
MX2008011375A (en) | 2008-12-15 |
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