(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`Alba. A
`
`0 OO
`
`EOC Et
`
`(43) International Publication Date
`31 January 2008 (31.01.2008)
`
`(10) International Publication Number
`WO 2008/014414 A2
`
`
`
`(51) International Patent Classification:
`AG1K 38/17 (2006.01)
`
`(21) International Application Number:
`PCT/US2007/074514
`
`(22) International Filing Date:
`
`26 July 2007 (26.07.2007)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`60/833,239
`
`English
`
`English
`
`26 July 2006 (26.07.2006)
`
`US
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH,
`CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG,
`ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL,
`IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
`LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
`MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL,
`PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY,
`TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZIM, ZW.
`
`US):
`except
`States
`designated
`all
`(for
`(71) Applicant
`BIOMARCK PHARMACEUTICALS, LTD. [US/US];
`7200 Falls of Neuse Road, Suite 202, Raleigh, North
`Carolina 27615 (US).
`
`(72) Inventor; and
`PARIKH, Indu
`(75) Inventor/Applicant (for US only):
`[US/US]; 2558 Booker Creek Road, Chapel Hill, North
`Carolina 27514 (US).
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL, PL,
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`(74) Agents: HULEATT, Jayme A. et al.; Cooley Godward
`Kronish LLP, Attn: Patent Group, 1200 19th Street, N.W.,
`Suite 500, Washington, District Of Columbia 20036 (US).
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`
`[Continued on next page]
`
`
`
`(54) Title: METHODS FOR ATTENUATING RELEASE OF INFLAMMATORY MEDIATORS AND PEPTIDES USEFUL
`THEREIN
`
`MPO Secretion from LPS-Primed Human Neutrophils
`
`0.354
`
`0.30
`
`0.254
`
`0.20
`
`0.15
`
`0.104
`
`0.05
`
` 0.00
`
`
`
`Absorbance 450am
`
`10 uM 8-Br-cGMP
`+ PMA (nM)
`Treatments
`
`087014414 A2 | IN IV 0000 OO OO
`
`=
`
`(57) Abstract: The present invention includes methods of inhibiting or suppressing cellular secretory processes. More specifically
`& the present invention relates to inhibiting or reducing the release of inflammatory mediators from inflammatory cells by inhibiting
`the mechanism associated with the release of inflammatory mediators from granules in inflammatory cells. In this regard, the present
`invention discloses an intracellular signaling mechanism that illustrates several novel intracellular targets for pharmacological in-
`tervention in disorders involving secretion of inflammatory mediators from vesicles in inflammatory cells. Peptide fragments and
`variants thereof of MANS peptide as disclosed in the present invention are useful in such methods.
`
`
`
`
`MYLAN - EXHIBIT 1030
`
`

`

`
`
`WO 2008/014414 A 2
`
`{NII DA 000 000000 0000 0
`
`—
`
`with sequence listing part of description published sepa-
`rately in electronic form and available upon request from
`the International Bureau
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes and Abbreviations" appearing at the begin-
`ning of each regular issue of the PCT Gazette.
`
`

`

`WO 2008/014414
`
`PCT/US2007/074514
`
`METHODS FOR ATTENUATING RELEASE OF INFLAMMATORY
`
`MEDIATORS AND PEPTIDES USEFUL THEREIN
`
`Cross Reference to Related Application
`
`[0001]
`
`The present application claims priority to U.S. Serial No.: 60/833,239 filed on July 26,
`
`20006, which is incorporated in its entirety by reference.
`
`Field of Invention
`
`[0002]
`
`The present invention relates to peptides or peptide compositions and methods of their
`
`use to attenuate (or inhibit or reduce) the stimulated release of mediators of inflammation from
`
`inflammatory cells during inflammation.
`
`The present invention also relates to use of these
`
`peptides or peptide compositions to modulate an intracellular signaling mechanism regulating the
`
`secretion of inflammatory mediators from inflammatory cells.
`
`Background of the Invention
`
`[0003]
`
`Inflammatory leukocytes synthesize a number of inflammatory mediators that
`
`are
`
`isolated intracellularly and stored in cytoplasmic membrane-bound granules. Examples of such
`
`mediators include, but are not limited to, myeloperoxidase [MPO] in neutrophils (see,
`
`for
`
`example, 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 (see,
`
`for example, Gleich G J. Mechanisms of eosinophil-associated
`
`inflammation.
`
`J
`
`Allergy ~~ Clin
`
`Immunol ~~ 2000;
`
` 105:651-663),
`
`lysozyme
`
`in
`
`monocytes/macrophages (see, for example, 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;
`
`and
`
`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), and granzyme in natural killer (NK) cells and cytotoxic lymphocytes (see, for
`
`example, Bochan MR, Goebel WS, Brahmi Z.
`
`Stably transfected antisense granzyme B and
`
`perforin constructs inhibit human granule-mediated lytic ability. Cell Immunol 1995;164:234-
`
`239; 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;
`
`
`
`

`

`WO 2008/014414
`
`PCT/US2007/074514
`
`8:652-658; 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; and Takayama H, Trenn G,
`
`Sitkovsky MV. A novel cytotoxic T lymphocyte activation
`
`assay. J Immunol Methods 1987; 104:183-190).
`
`Such mediators are released at sites of injury
`
`and contribute to inflammation and tissue repair such as in the lung and elsewhere.
`
`It is known
`
`that leukocytes release these granules via an exocytotic mechanism (see, for example, Burgoyne
`
`RD, Morgan A. Secretory granule exocytosis. Physiol Rev 2003; 83:581-632; and Logan MR,
`
`Odemuyiwa SO, Mogbel R. Understanding exocytosis in immune and inflammatory cells: the
`
`molecular basis
`
`of mediator secretion. J Allergy Clin Immunol 2003;
`
`111: 923-932), but
`
`regulatory molecules and specific pathways involved in the exocytotic process have not been
`
`fully described.
`
`[0004]
`
`Several exogenous stimuli can provoke degranulation of leukocytes via a pathway that
`
`involves activation of protein kinase C and subsequent phosphorylation events (see, for example,
`
`Burgoyne RD, Morgan A. Secretory granule exocytosis. Physiol Rev 2003; 83:581-632; Logan
`
`MR, Odemuyiwa SO, Mogbel R. Understanding exocytosis in immune and inflammatory cells:
`
`the molecular basis of mediator secretion. J Allergy Clin Immunol 2003; 111: 923-932; Smolen
`
`JE, Sandborg RR. Ca2+-induced secretion by electropermeabilized human neutrophils: the roles
`
`of Ca2+, nucleotides and protein kinase C. Biochim Biophys Acta 1990; 1052:133-142; Niessen
`
`HW,
`
`Verhoeven
`
`AJ.
`
`Role
`
`of
`
`protein
`
`phosphorylation
`
`in
`
`the
`
`degranulation
`
`of
`
`electropermeabilized human neutrophils. Biochim. Biophys. Acta
`
`1994; 1223:267-273; and
`
`Naucler C, Grinstein S, Sundler R., Tapper H. Signaling to localized degranulation in neutrophils
`
`adherent to immune complexes. J Leukoc Biol 2002; 71:701-710).
`
`[0005]
`
`MARCKS protein (where MARCKS as used herein means “Myristoylated Alanine-
`
`Rich C Kinase Substrate™), is a ubiquitous phosphorylation target of protein kinase C (PKC), and
`
`is highly expressed in leukocytes (see, for example, Aderem AA, Albert KA, Keum MM, Wang
`
`JK, Greengard P, Cohn ZA. Stimulus-dependent myristoylation of a major substrate for protein
`
`kinase C. Nature 1988; 332:362-364; Thelen M, Rosen A, Nairn AC, Aderem A. Regulation by
`
`phosphorylation of reversible association of a myristoylated protein kinase C substrate with the
`
`plasma membrane. Nature 1991; 351:320-322; and Hartwig JH, Thelen M, Rosen A, Janmey
`
`PA, Nairn AC, Aderem A. MARCKS is an actin filament crosslinking protein regulated by
`
`
`
`

`

`WO 2008/014414
`
`PCT/US2007/074514
`
`protein kinase C and calcium-calmodulin. Nature 1992; 356:618-622. MARCKS protein is
`
`mechanistically involved in a process of exocytotic secretion of mucin by goblet cells that line
`
`respiratory airways (see, for example, Li et al., J Biol Chem 2001; 276:40982-40990; and Singer
`
`et al.,, Nat Med 2004; 10:193-196). MARCKS is myristoylated via an amide bond at the N-
`
`terminal amino acid in the MARCKS protein’s amino acid sequence at the alpha-amine position
`
`of the glycine which resides at the N-terminus (i.e.,
`
`at position 1) of amino acid sequence. In
`
`airway epithelial cells, the myristoylated N-terminal region of MARCKS appears to be integral
`
`to the secretory process.
`
`By the N-terminus of the MARCKS protein is meant the MANS
`
`peptide which contains Myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1),
`
`which are L-amino acids.
`
`Additionally, the peptide fragments of the MANS peptide disclosed
`
`herein, also preferably are composed of L-amino acids.
`
`The mechanism appears to involve
`
`binding of MARCKS, a myristoylated protein, to membranes of intracellular granules.
`
`[0006]
`
`An N-terminal myristoylated peptide from the N-terminus of MARCKS has been
`
`shown to block both mucin secretion and binding of MARCKS to mucin granule membranes in
`
`goblet cells (see, for example, Singer et al., Nat Med 2004; 10:193-196). This peptide contains
`
`24 amino acids of the MARCKS protein beginning with the N-terminal glycine of the MARCKS
`
`protein which is myristoylated via an amide bond and is known as myristoylated alpha-N-
`
`terminal sequence (MANS); i.e., Myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID
`
`NO: 1). Also Vergeres et al., J. Biochem. 1998, 330; 5-11, discloses that the N-terminal glycine
`
`residue of MARCKS proteins is myristoylated via a reaction catalyzed by myristoyl CoA:protein
`
`N-myristoyl transferase (NMT).
`
`[0007]
`
`In inflammatory diseases, 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, arthritis or auto-immune diseases, inflammatory cells are
`
`usually found in or migrate to areas of injury or infection associated with inflammatory disease
`
`states, especially in or to respiratory passages or airways of patients suffering from such diseases.
`
`These inflammatory cells can contribute greatly to the pathology of diseases via tissue damage
`
`done by inflammatory mediators released from these cells. One example of such tissue damage
`
`or destruction via this chronic inflammation occurs in cystic fibrosis patients where mediators
`
`
`
`

`

`WO 2008/014414
`
`PCT/US2007/074514
`
`released from neutrophils (e.g., myeloperoxidase [MPO]) induce the desquamation of the airway
`
`epithelial tissue.
`
`[0008]
`
`MARCKS, a protein of approximately 82 kD, 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 (or PSD), and a multiple homology 2 (MH2)
`
`domain. Human MARCKS cDNA and protein is known and reported by Harlan et al.,
`
`J. Biol.
`
`Chem. 1991, 266:14399 (GenBank Accession No. M68956) and also by Sakai er al., Genomics
`
`1992, 14: 175. These sequences are also provided in a WO 00/50062, which is incoporated in its
`
`entirety by reference. The N-terminus, an alpha-amino acid sequence comprising 24 amino acid
`
`residues with a myristic acid moiety attached via an amide bond to the N-terminal glycine
`
`residue is involved in binding of MARCKS to membranes in cells (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.
`
`Summary of the Invention
`
`[0009]
`
`Involvement of 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. However, 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., Nut Med
`
`2004; 10:193-196). While mucus production and secretion can be provoked by a number of
`
`factors, including mediators released by inflammatory cells, there is no known direct link
`
`whereby excess mucus causes inflammation.
`
`[00010] In one aspect of this invention, the MANS peptide can play a role in the reducing the
`
`rate and/or amount of release of inflammatory mediators granules or vesicles in inflammatory
`
`leukocytes.
`
`[00011]
`
`In another aspect, peptides derived from the MARCKS N-terminus, especially from the
`
`24 amino acid N-terminal sequence, i.e., active contiguous peptide fragments derived from
`
`
`
`

`

`WO 2008/014414
`
`PCT/US2007/074514
`
`within the N-terminal 1-to-24 amino acid sequence of MARCKS having a glycine at position 1,
`
`as well as N-terminal amides of such fragments, such as N-terminal acetic acid amides of such
`
`fragments, and/or as well as C-terminal amides of such fragments, such as C-terminal amides of
`
`ammonia, can inhibit or reduce the rate and/or amount of release of inflammatory mediators
`
`from inflammatory leukocytes. Such inhibition or reduction in release comprises inhibition of a
`
`MARCKS-related release of inflammatory mediators from inflammatory leukocytes.
`
`[00012]
`
`In another aspect, peptides derived from the MARCKS N-terminus, especially from the
`
`1-to-24 amino acid N-terminal sequence, i.e., active contiguous peptide fragments derived from
`
`within the N-terminal 1 to 24 amino acid sequence of MARCKS having a glycine at position 1,
`
`as well as N-terminal amides of such fragments such as N-terminal acetic acid amides of such
`
`fragments, and as well as C-terminal amides of such fragments such as C-terminal amides of
`
`ammonia, can inhibit the rate of release and/or amount of release of inflammatory mediators
`
`such as those identified herein in this invention, by inhibiting the process of degranulation in
`
`inflammatory leucocytes.
`
`[00013]
`
`In another aspect, the MANS peptide and active fragments thereof, and active amides
`
`of such fragments as described herein, can compete for membrane binding in inflammatory cells
`
`with native MARCKS protein to attenuate (lessen or reduce) MARCKS-related release of
`
`mediators of inflammation from granules or vesicles containing such mediators of inflammation
`
`in such inflammatory cells.
`
`[00014] Leukocyte cell types and model cell types that secrete specific granule contents in
`
`response to phorbol ester induced activation of PKC are useful for in vitro demonstration of
`
`efficacy of peptides of this invention and of substituted peptides (e.g., alpha-N-amides, C-
`
`terminal amides and esters) of this invention.
`
`[00015] The attenuation of release of membrane-bound inflammatory mediators by compounds
`
`and compositions of this invention can be demonstrated using human leukocyte cell lines. For
`
`example, neutrophils isolated from human blood can be used to demonstrate attenuation or
`
`inhibition of release of myeloperoxidase (MPO). The human promyelocytic cell
`
`line HL-60
`
`clone 15 can be used to demonstrate attenuation of release or inhibition of release or secretion of
`
`eosinophil peroxidase (EPO) by compounds and compositions of this invention (see,
`
`for
`
`
`
`

`

`WO 2008/014414
`
`PCT/US2007/074514
`
`example, Fischkoff SA. Graded increase in probability of eosinophilic differentiation of HL-60
`
`promyelocytic leukemia cells induced by culture under alkaline conditions. Leuk Res 1988;
`
`12:679-686; Rosenberg HF, Ackerman S
`
`J, Tenen DG. Human eosinophil cationic protein:
`
`molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity. J Exp Med
`
`1989;
`
`170:163-176; Tiffany HL,
`
`Li
`
`F, Rosenberg HF. Hyperglycosylation of eosinophil
`
`ribonucleases in
`
`a promyelocytic leukemia cell
`
`line and in differentiated peripheral blood
`
`progenitor cells. J Leukoc Biol 1995; 58:49-54; and Badewa AP, Hudson CE, Heiman AS.
`
`Regulatory effects
`
`of eotaxin,
`
`eotaxin-2,
`
`and eotaxin-3
`
`on eosinophil degranulation and
`
`superoxide anion generation. Exp Biol Med 2002; 227:645-651). The monocytic leukemia cell
`
`line U937 can be used to demonstrate attenuation of release or inhibition of release or secretion
`
`of lysozyme by compounds and compositions of this invention (see,
`
`for example, 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; and Sundstrom C, Nilsson K. Establishment
`
`and characterization of a human histiocytic lymphoma cell line (U-937). Int J Cancer 1976;
`
`17:565-577). The lymphocyte natural
`
`killer
`
`cell
`
`line NK-92 can be used to demonstrate
`
`attenuation or inhibition of release of granzyme by compounds and compositions of this
`
`invention (see, for example, 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; 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; and Takayama H, Trenn G,
`
`Sitkovsky MV. A novel cytotoxic T
`
`lymphocyte activation assay. J Immunol Methods 1987; 104:183-190). In an in vitro method to
`
`inhibit or attenuate the release of a mediator of inflammation such as those described herein,
`
`each of the cell types is preincubated with a peptide compound or peptide composition of this
`
`invention over a range of concentrations followed by incubation of these cells by a stimulator of
`
`release of inflammatory mediators, such as phorbol ester. The percent of inhibition of release of
`
`a mediator of inflammation is determined as compared to the release of the mediator in the
`
`absence of the peptide compound or peptide composition, such as in a specrophotometric readout
`
`of a concentration of the mediator released.
`
`
`
`

`

`WO 2008/014414
`
`PCT/US2007/074514
`
`[00016] To demonstrate the importance of the relative amino acid sequence positioning in the
`
`peptides of the invention, the relative ability to inhibit or reduce the amount of inflammatory
`
`mediator released by a peptide which is identical to the 24 amino acid sequence of the MARCKS
`
`protein N-terminus region (i.e., the MANS- myristoylated alpha-N-terminal sequence peptide)
`
`was compared to the ability to inhibit or reduce the amount of inflammatory mediator released
`
`by a peptide containing the same 24 amino acid residues present in MANS but which are
`
`sequenced in
`
`a random order (i.e., an RNS peptide, otherwise referred to
`
`as a “Random N-
`
`terminal sequence peptide”) with respect to the sequence order in MANS. In each of the cell
`
`types examined, the MANS peptide, but not the RNS peptide, attenuated release of inflammatory
`
`mediators in a concentration-dependent manner over a time course of 0.5-3.0 hrs. These results
`
`suggest that the relative amino acid sequence positioning in the peptides of the invention which
`
`are in the order found in the MARCKS protein, specifically its N-terminal region, and more
`
`specifically its 24 amino acid residue N-terminal region are involved in at least one intracellular
`
`pathway dealing with the inhibition of leukocyte degranulation.
`
`[00017] The invention relates to a new use for the 24 amino acid peptide sequence, and to the
`
`alpha-N-terminal acetylated peptide sequence, the myristoylated polypeptide, also known as the
`
`MANS peptide, and to active fragments thereof, which active fragments can be selected from the
`
`group of peptides having from 4 to 23 contiguous amino acid residues of the MANS peptide
`
`amino acid sequence, and which fragments may be N-terminal-myristoylated if they do not begin
`
`with the N-terminal glycine at position 1 in SEQ ID NO: 1, or which may be N-terminal-acylated
`
`with C2 to C12 acyl groups, including N-terminal-acetylated, and/or C-terminal amidated with
`
`an NH2 group.
`
`[00018] The invention also relates to
`
`a new method for blocking MARCKS-related cellular
`
`secretory processes, especially those that involve the MARCKS-related 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.
`
`[00019] 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
`
`
`
`

`

`WO 2008/014414
`
`PCT/US2007/074514
`
`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 as described herein 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.
`
`[00020] 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.
`
`[00021]
`
`More particularly, 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-
`
`GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1)) or an active fragment thereof. The
`
`active fragment is at least four and preferably at least six amino acids in length. As used herein,
`
`an "active fragment" of a MARCKS protein is one that affects (inhibits or reduces) MARCKS
`
`protein-mediated release, such as MARCKS protein-mediated release of an inflammatory
`
`mediator.
`
`An
`
`active
`
`fragment
`
`can
`
`be
`
`selected
`
`from
`
`the
`
`group
`
`consisting
`
`of
`
`GAQFSKTAAKGEAAAERPGEAAV (SEQ ID NO: 2); GAQFSKTAAKGEAAAERPGEAA
`
`(SEQ
`
`ID
`
`NO:
`
`4);
`
`GAQFSKTAAKGEAAAERPGEA
`
`(SEQ
`
`ID
`
`NO:
`
`7);
`
`GAQFSKTAAKGEAAAERPGE (SEQ ID NO: 11); GAQFSKTAAKGEAAAERPG (SEQ ID
`
`NO: 16); GAQFSKTAAKGEAAAERP (SEQ ID NO: 22); GAQFSKTAAKGEAAAER (SEQ
`
`ID NO: 29); GAQFSKTAAKGEAAAE (SEQ ID NO: 37); GAQFSKTAAKGEAAA (SEQ ID
`
`
`
`
`
`

`

`WO 2008/014414
`
`PCT/US2007/074514
`
`NO: 46); GAQFSKTAAKGEAA (SEQ ID NO: 56); GAQFSKTAAKGEA (SEQ ID NO: 67);
`
`GAQFSKTAAKGE (SEQ ID NO: 79); GAQFSKTAAKG (SEQ ID NO: 92); GAQFSKTAAK
`
`(SEQ ID NO: 106); GAQFSKTAA (SEQ ID NO: 121); GAQFSKTA (SEQ ID NO: 137);
`
`GAQFSKT (SEQ ID NO: 154); GAQFSK (SEQ ID NO: 172); GAQFS (SEQ ID NO: 191) and
`
`GAQF (SEQ ID NO: 211). These peptides, instead of containing a myristoyl moiety at the N-
`
`terminal amino acid, either contain no chemical moiety or a non-myristoyl chemical moiety at
`
`the N-terminal amino acid and/or a chemical moiety at the C-terminal amino acid, such as an N-
`
`terminal acetyl group and/or a C-terminal amide group as described herein. The presence of the
`
`hydrophobic N-terminal myristoyl moiety in the MANS peptides and N-terminal myristoylated
`
`fragments thereof 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 a myristoyl group into a membrane lipid bilayer can provide a partition coefficient or
`
`apparent association constant with lipids of up to 10* M™! 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. 32: 10436-10443) which is sufficient, at least in part, to permit a partitioning of
`
`the MANS peptide and of myristoylated MANS peptide fragments into the plasma membrane of
`
`a cell while additional functional groups and their interactions within the MANS peptide (which
`
`is myristoylated) and within myristoylated MANS peptide fragments can potentiate their relative
`
`membrane permeabilities. 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. and White, Peter D.Eds., Fmoc Solid Phase
`
`Peptide Synthesis: A Practical Approach, Oxford University Press, New York, New York
`
`(2000); and Lloyd-Williams, P.
`
`et
`
`al. Chemical Approaches to the Synthesis of Peptides and
`
`Proteins (1997)) and purified by methods known in the art, such as by high pressure liquid
`
`chromatography. Molecular weight of each peptide can be confirmed by mass spectroscopy with
`
`each showing a peak with an appropriate molecular mass. Efficacy of the individual peptides and
`
`of combinations of individual peptides (for example, combinations of 2
`
`of the peptides,
`
`combinations of 3 of the peptides, combinations of 4 of the peptides) in the methods of this
`
`disclosure can be readily determined without undue experimentation using the procedures
`
`
`
`
`
`

`

`WO 2008/014414
`
`PCT/US2007/074514
`
`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 (i.e., 1:1) to 99.99 to 0.01,
`
`which ratio can be readily determined using the procedures described in the examples disclosed
`
`herein.
`
`[00022]
`
`Preferably
`
`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 inhibiting 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 inhibits
`
`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.
`
`[00023] Administration of a composition 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 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. In one aspect, 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. In another aspect, 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. While the amount and frequency of dose can be determined by clinical evaluation and be
`
`a function of the disease or source of inflammation and the extent of tissue involved and the age
`
`and size of the patient, it
`
`is anticipated that dosing of a pharmaceutical composition can be
`
`10
`
`
`
`

`

`WO 2008/014414
`
`PCT/US2007/074514
`
`repeated after 3
`
`to
`
`§ hours, preferably after 6
`
`to
`
`8 hours after the first administration of the
`
`pharmaceutical composition.
`
`[00024] 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. As used herein "reducing" generally means a lessening of the effects
`
`of inflammation. Preferably, release of inflammatory mediators are inhibited or blocked by the
`
`methods disclosed.
`
`[00025] 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 inhibit an inflammatory mediator at
`
`the inflammation site. The term "inhibiting" means a reduction in the amount of inflammatory
`
`mediator secretion. The term “completely inhibiting” means a reduction to zero in the amount of
`
`inflammatory mediator secretion. Again, as stated above, the active fragment is at least four and
`
`preferably at least six amino acids in length. The term “exocytotic process” means exocytosis,
`
`1.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.
`
`The term “degranulation-