WO1993011784A1 - Methods for inhibition or stimulation of the inflammatory response - Google Patents

Abstract

The present invention relates to products and methods for modulation of the inflammatory response mediated by one or more chemoattractant receptors. Therapeutic methods and compositions are also provided, for the treatment of disorders where an inhibition, or alternatively, stimulation, of the inflammatory response is desired. In one emdobiment, methods and products for inhibition of the inflammatory response are provided. Such anti-inflammatory compounds include but are not limited to mastoparan analogs and chemoattractant receptors (cytoplasmic loop) analogs. In another embodiment, methods and products are provided for stimulation of the inflammatory response, by inhibition of chemoattractant receptor desensitization. In a particular embodiment, such a pro-inflammatory compound is a chemoattractant receptor cytoplasmic tail analog which blocks desensitizing phosphorylation of a chemoattractant receptor. In a preferred, non-limiting aspect, inhibition of the inflammatory response is chemoattractant receptor class-specific. Methods for identifying and obtaining anti-inflammatory or pro-inflammatory compounds of the invention are provided.

Description

METHODS FOR INHIBITION OR STIMULATION

OF THE INFLAMMATORY RESPONSE

This invention was made in part with

government support under grants DE-03738, HL-36162 and CA-29589 awarded by the National Institutes of Health. The Government has certain rights in the invention.

1. INTRODUCTION

The present invention relates to compounds and methods for inhibition, or alternatively,

stimulation of the inflammatory response mediated by one or more chemoattractant receptors. Therapeutic methods based on such inhibition or stimulation are also provided.

2. BACKGROUND OF THE INVENTION The G proteins (GTP-binding proteins) are a family of signal transducing proteins that couple activated membrane receptors (e.g., β-adrenergic receptor, chemoattractant receptors) with their intracellular effector enzymes and ion channels (e.g., adenylate cyclase, phospholipase C, potassium

channel). The G proteins consist of three subunits: an a chain which binds guanyl nucleotides (e.g., GDP, GTP), a β chain, and a γ chain. G proteins cycle between an inactive state and an active state. The inactive state occurs when GDP is bound to the α chain which associates in a trimer G_αβγ complex. The active state occurs when the α chain (G_α) binds GTP, and then dissociates from the β and γ chains. When a G

protein-coupled receptor in the membrane is activated by binding to its ligand, the receptor catalyzes activation of the G protein by increasing the rate of exchange of GTP for bound GDP. Released G_α-GTP then affects the activity of its target effector enzyme or channel. The a chains of certain G proteins are targets of bacterial toxins, which can specifically ADP-ribosylate the α chains. Stimulatory G proteins (G_s) and inhibitory G proteins (G_i) have been described. Another G protein, denoted G_o (for G-other) has also been described (id.). (See generally, Stryer and Bourne, 1986, Ann. Rev. Cell Biol. 2:391-419.)

G protein-coupled receptors are a diverse family which contain seven putative membrane spanning domains and transduce ligand-mediated signals through interactions with G proteins. Chemoattractant

receptors form one group of G protein-coupled

receptors.

Chemoattractants receptors mediate proinflammatory and chemotactic actions. These

receptors, which function as specific receptors for chemotactic factors, are present on phagocytic

leukocytes such as

neutrophils/granulocytes/polymorphonuclear leukocytes, monocytes, macrophages, and possibly eosinophils.

Phagocytes accumulate at sites of inflammation by migrating along gradients of chemoattractants produced by immune responses (Snyderman, R., and Uhing, R.J., 1988, in Inflammation: Basic Principles and Clinical Correlates, eds., Gallin, J.I. Snyderman, R. and

Goldstein, I.M., Raven Press Ltd., New York, pp. 309- 323). At higher concentrations, chemoattractants stimulate the cells' cytotoxic responses (activation of the respiratory burst, exocytosis of lysosomal enzymes). The actions mediated by chemoattractant receptors include stimulation of granule-enzyme

release and superoxide anion production, upregulation of expression and activity of the cell adhesion

molecule Mac-1 (CDIIb, CD18), increased expression of CR1, a decrease in cell surface glycoprotein 100MEL-14 on neutrophils (Gerard and Gerard, 1991, Nature 349:6- 14), and inhibition of neutrophil adherence to

activated endothelial cells (Holmes et al., 1991, Science 253:1278). In vivo, these receptors may participate in anaphylactoid and septic shock (Gerard and Gerard, supra).

The chemoattractant receptors initiate signal transduction via a pertussis toxin-sensitive GTP-binding protein (G protein) to activate

phospholipase C resulting in the formation of

diacylglycerol and phosphoinositides (Snyderman et al., 1990, in ADP-Ribosylatinσ Toxins and G Proteins: Insights Into Signal Transduction, Moss, J., ed., American Society for Microbiology, Washington, D.C., pp. 295-323; Allen et al., 1988, Hemat. Oncol. Clinics of North America 2:33-59). One of the earliest events in the pathway of cellular activation induced by occupancy of chemoattractant receptors is a transient change in cytosolic free calcium concentration ([Ca²⁺]_i) involving a rapid initial rise, a subsequent slower increase and then decline to baseline levels (Pozzan et al., 1983, Science 221:1413, Korchak et al., 1984, J. Biol. Chem. 259:4076; Prentki et al., 1984, J.

Biol. Chem. 259:13777). The best characterized chemoattractant receptor is the one which binds formylpeptides. cDNAs encoding receptors for three chemoattractants, formylpeptide [e.g. fMet-Leu-Phe (fMLP)] (Boulay et al., 1990, Biochem. Biophys. Res. Commun. 168:1103-1109; Boulay et al., 1990,

Biochemistry 29:11123-11133), C5a (Gerard and Gerard, 1991, Nature 349:614-617) and platelet activating factor [PAF, (Honda et al., 1991, Nature 349:342-346)] have been cloned. These receptors are 20-34%

identical in their amino acid sequences and have the characteristic structure of G protein-coupled receptors (Dohlman et al., 1987, Biochemistry 26:2657- 2664). The interleukin-8 (IL-8) receptor, another chemoattractant receptor, has also been cloned (Holmes et al., 1991, Science 253:1278-1280).

Attenuation of signalling, i.e., desensitization, has been most extensively studied in j8-adrenergic receptors (jSARs) and rhodopsin. Two types of rapid desensitization of βARs, homologous and heterologous, have been identified and result from phosphorylation of the agonist occupied receptor which uncouples it from the stimulatory G protein G,

(Lefkowitz et al., 1990, Trends Pharmacol. Sci.

11:190-194). Heterologous desensitization (i.e., attenuation of adenylate cyclase responsiveness to other hormones) occurs in response to low doses (e.g., 10 nM) of nonspecific agonists for receptors coupled to activation of adenylate cyclase, which results in this class of receptors being phophorylated by cAMP- dependent protein kinase (PKA) (Hausdorff et al., 1989, J. Biol. Chem. 264:12657-12665; Clark et al., 1988, Proc. Natl. Acad. Sci. USA 85:1442-1446).

Homologous desensitization occurs with higher doses of specific agonist (e.g., 2 μM). Occupancy by the specific agonist stimulates a βAR-specific kinase

(βARK) to phosphorylate specific sites on the βAR

(Benovic et al., 1986, Proc. Natl. Acad. Sci. USA

83:2797-2801; Lohse et al., 1990, Science 248:1547- 1550; Bouvier et al., 1987, J. Biol. Chem. 262:3106- 3113); the phosphorylated receptor then associates with β-arrestin (Lefkowitz et al., 1990, Trends

Pharmacol. Sci. 11:190-194) to further impair

receptor/G protein coupling (Bouvier, et al., 1987, J. Biol. Chem. 262:3106-3113). Phosphorylation of βAR by βARK occurs at sites different from those

phosphorylated by PKA. While this homologous desensitization is mediated primarily by βARK, PKA- mediated phosphorylation may also occur in concert.

In contrast, the mechanisms involved in the desensitization of chemoattractant receptors are not yet well defined. The formylpeptide chemoattractant receptor, which transduces signals through a pertussis toxin sensitive G_i-like G protein (Smith et al., 1985, J. Biol. Chem. 260:5875-5878; Spiegel, 1987, Mol.

Cell. Endocrinol. 49:1-16), can be desensitized in the presence of agonist (e.g., fMLP) (McLeish et al., 1989, Mol. Pharmacol. 36:384-390; Simchowitz et al., 1980, J. Clin. Invest. 66:736-747). Formylpeptide receptors are desensitized by exposure to

formylpeptides as well as by elevations in

intracellular cAMP (Simchowitz et al., 1980, J.

Immunol. 124:1482-1491; Rivkin et al., 1975, J.

Immunol. 115:1126-1134; Gallin et al., 1978, J.

Immunol. 120:492-496) which inhibit calcium influx (Takenawa et al., 1986, J. Biol. Chem. 261:1092-1098), by phorbol 12-myristate 13-acetate (PMA) (Naccache, et al., 1985, J. Biol. Chem. 260:2125-2131) which

disrupts coupling of the activated G protein to phospholipase C (Smith et al., 1987, J. Biol Chem.

262:6121-6127) or by pertussis toxin (Smith et al., 1985, J. Biol. Chem. 260:5875-5878; Verghese et al., 1985, Biochem. Biophys. Res. Commun. 127:450-457;

Brandt et al., 1985, Proc. Natl. Acad. Sci. U.S.A.

82:3277-3280; Volpi et al., 1985, Proc. Natl. Acad. Sci. USA 82:2708-2712; Okajima et al., 1985, J. Biol. Chem. 260:6761-6768) which disrupts receptor coupling to its G protein (Smith et al., 1987, J. Biol Chem. 262:6121-6127). McLeish et al. (1989, Mol. Pharmacol. 36:384-390) have demonstrated that formylpeptide receptors on differentiated HL-60 cells are

homologously desensitized and suggest two potential mechanisms, loss of membrane receptors and functional alteration in receptor G protein interaction.

Phosphorylation of Giα2 has been associated with loss of its function and in receptor desensitization in hepatocytes (Bushfield et al., 1990, Biochem. J.

268:449-457; Bushfield et al., 1990, Biochem. J.

271:365-372). Ligand occupancy of the cAMP

chemoattractant receptor in Dictyostelium also results in phosphorylation of its transducing Gα2 protein

(Gundersen and Devreotes, 1990, Science 248:591-593). Exposure to agonist also leads to formylpeptide receptor internalization (Jesaitis et al., 1983, J. Biol. Chem. 258:1968-1977; Sklar et al., 1984, J.

Biol. Chem. 259:5661-5669) and inhibiting receptor internalization attenuates desensitization (Jesaitis et al., 1986, J. Biol. Chem. 261:13662-13669). In sum, the specific mechanism of chemoattractant

receptor desensitization, and whether phosphorylation of receptor is involved, have not been determined by the prior art.

Mastoparan is a wasp venom peptide toxin, that causes activation of human polymorphonuclear leukocytes through two independent mechanisms. One mechanism is similar to that of chemoattractant receptors in that it involves the activation of phospholipase C via a pertussis toxin-sensitive G_i protein (Perianin and Snyderman, 1989, J. Immunol.

143:1669-1673; Joyce-Brady et al., 1991, J. Biol.

Chem. 266:6589-6865).

3. SUMMARY

The present invention relates to products and methods for modulation of the inflammatory

response mediated by one or more chemoattractant receptors. Therapeutic methods and compositions are also provided, for the treatment of disorders where an inhibition, or alternatively, stimulation, of the inflammatory response is desired. In one embodiment, methods and products for inhibition of the

inflammatory response are provided. Such anti- inflammatory compounds include but are not limited to mastoparan analogs and chemoattractant receptor

(cytoplasmic loop) analogs. In another embodiment, methods and products are provided for stimulation of the inflammatory response, by inhibition of

chemoattractant receptor desensitization. In a particular embodiment, such a pro-inflammatory

compound is a chemoattractant receptor cytoplasmic tail analog which blocks desensitizing phosphorylation of a chemoattractant receptor. In a preferred, non- limiting aspect, inhibition of the inflammatory response is chemoattractant receptor class-specific. Methods for identifying and obtaining anti- inflammatory or pro-inflammatory compounds of the invention are provided.

4. DESCRIPTION OF THE FIGURES

Figure 1. Desensitization of formylpeptide receptors. (A) Intracellular calcium elevation in formylpeptide receptor transfected 293 cells. Indo-1 loaded formylpeptide receptor transfected 293 cells were exposed to varying doses of fMLP followed 5 min later by a second 10 nM dose and [Ca²⁺]_i was measured as described in Section 6.1. (B) Intracellular calcium elevation in formylpeptide receptor transfected 293 cells. As in (A) using αl agonist norepinephrine (Norep.). Shown are representative tracings of three experiments. Intracellular calcium levels were measured using the following equation: [Ca²⁺]i = K_d(F- F_min) / (F_max-F) (Cobbold and Rink, 1987, Biochem. J. 248: 313-328), which results in a nonlinear y-axis.

Figure 2. Cross desensitization of

formylpeptide and C5a receptors co-expressed in transfected TSA cells. TSA cells co-transfected with formylpeptide and C5a receptor cDNAs were exposed to an initial dose of either 10 nM fMLP (Panel A), 10 nM C5a (Panel B), 10 μM norepinephrine (Panel C) (Nor.) or 10 μM PMA (Panel D) and [Ca²⁺]_i measured. The secondary response was calculated as a percent of the primary response e by the same ligand. Each point (bar) is the mean of duplicate determinations, the maximum variability was + 4%. The average degree of inhibition (%) of the response to a second dose of ligand is indicated in parentheses.

Figure 3. Cross desensitization of formylpeptide and C5a receptors in human neutrophils. Human neutrophils were loaded with the calcium

indicator Indo-1 and exposed to the indicated

nanomolar concentrations of fMLP (closed circles), C5a (closed squares), and micromolar concentrations of ATP (closed triangles) (pretreatment dose), followed 5 min later by a second dose of agonist (Panel A, 1 nM fMLP; Panel B, 1 nM C5a; Panel C, 1 μM ATP) and the maximum [Ca²⁺]_i measured. The response to the second dose of agonist is expressed as a percent of the maximum calcium response in the absence of agonist

pretreatment. Each point is the mean of two to six determinations. N.D., not determined.

5. DETAILED DESCRIPTION OF THE INVENTION Chemoattractant receptors on leukocytes initiate a number of pro-inflammatory functions including the accumulation of leukocytes at sites of inflammation and their release of cytotoxic molecules such as oxygen radicals or proteolytic enzymes. The development of therapeutic agents which alter the chemotactic or cytotoxic activities of phagocytic cells has been a goal of the pharmaceutical industry for several decades.

Other than corticosteroids and non-steroidal anti-inflammatory agents (NSAIDS), there are no effective marketed anti-inflammatory agents.

Moreover, neither corticosteroids nor NSAIDS

specifically alter the response of inflammatory cells to chemoattractants. There exists a tremendous clinical need for effective, specific anti- inflammatory agents; such agents are provided by the present invention. Similarly, a great need exists for specific pro-inflammatory agents in cases of

immunosuppression and other disorders; such agents are provided by the present invention.

The present invention provides methods and products for modulation of the inflammatory response mediated by one or more chemoattractant receptors.

Thus, therapeutic methods and compositions are also provided, for the treatment of disorders where an inhibition, or alternatively, stimulation, of the inflammatory response is desired. In one embodiment, methods and products for inhibition of the

inflammatory response are provided. Specific

embodiments of the invention providing for inhibition of the inflammatory response include (1) mastoparan analogs which are chemoattractant receptor

antagonists; and (2) chemoattractant receptor

(cytoplasmic loop) analogs which are antagonists. In another embodiment, methods and products for

stimulation of the inflammatory response are provided. Specific embodiments of the invention providing for stimulation of the inflammatory response include chemoattractant receptor analogs which block

desensitizing phosphorylation of a chemoattractant receptor. In a preferred embodiment, the compounds of the invention which inhibit or stimulate the

inflammatory response are peptides having a sequence of 5-50 amino acids.

The chemoattractant receptors which mediate inflammatory effects subject to modulation according to the present invention include but are not limited to the receptors for formylpeptide (Boulay et al., 1990, Biochem. Biophys. Res. Commun. 168:1103-1109), C5a (Gerard and Gerard, 1991, Nature 349:614-617), IL- 8 (Holmes et al., 1991, Science 253:1278-1280), platelet activating factor (PAF; Kunz et al., 1991, manuscript submitted); leukotriene B₄ (LTB₄), and any other chemoattractant receptors which are homologous to the foregoing or appear to use the same or similar G protein to initiate their biological activities.

In a preferred aspect, the chemoattractant receptors as a class are modulated, although this is not required, since the analogs of the invention include those inducing homologous or heterologous desensitization or activation as well as class- specific modulation. Evidence for the availability of class-specific modulation is presented by way of example in Section 6, infra.

The invention is described in greater detail in the following subsections.

5.1. IDENTITY OF COMPOUNDS FOR INHIBITION

OF THE INFLAMMATORY RESPONSE

The compounds of the invention which are able to inhibit the inflammatory response are targeted to the G protein(s) that chemoattractant receptors couple to and activate. Such compounds are capable of interfering with chemoattractant receptor-G protein interactions or with the ability of the receptor to activate G protein. Chemoattractant receptors on leukocytes initiate the biological activities of phagocytes through interactions with a G protein termed Gi₂. There are currently five well-defined chemoattractant receptors (formylpeptide, C5a, IL-8, PAF, LTB₄), all of which appear to utilize the same or similar G protein to initiate their biological activities. It is likely that the relevant G protein is activated throu the binding of a specific region of the chemoattractant receptor with a specific region on the G protein. Blocking this interaction

specifically should have a selective effect on

inhibiting the activities of chemoattractant receptors as a class.

5.1.1. MASTOPARAN ANALOGS

In a specific embodiment, the invention provides analogs of the wasp venom peptide toxin mastoparan which have the ability to inhibit the inflammatory response (i.e., are anti-inflammatories). Such anti-inflammatory compounds bind to G proteins (e.g., G_i2), as does mastaparan, but inhibit rather than activate the G proteins. In a preferred aspect, such peptides inhibit activation of the class of chemoattractant receptors. Such a class-specific activity is supported by the ability of the

chemoattractant receptors to cross desensitize each other (see Section 6, infra).

Mastoparan, a peptide toxin having the sequence INLKALAALAKKIL-NH₂ (SEQ ID NO:1), activates G proteins by promoting GDP/GTP exchange in a manner nearly identical to that of ligand-occupied G protein- coupled receptors. Mastoparan competes with the receptors for binding to G proteins with a preference for G_i type G proteins. The mastoparan

tetradecapeptide is a positively charged amphiphilic α helix which is believed to mimic the G protein-binding domain on receptors.

The anti-inflammatory compounds of the invention are obtained and identified by using

mastoparan as the archetype on which to base the design of peptides which are then tested for G protein activation activity; a peptide that retains activity is then specifically altered and tested for its inhibitory (anti-inflammatory) activity. The

identification of anti-inflammatory mastoparan analogs is described by way of example in Section 7, infra.

5.1.2. CHEMOATTRACTANT RECEPTOR SECOND OR

THIRD CYTOPLASMIC LOOP ANALOGS

In another embodiment, the invention

provides chemoattractant receptor analogs which have the ability to inhibit the inflammatory response.

Such analogs are based on the sequence of positively charged regions of the second and third cytoplasmic loops of chemoattractant receptors. These regions are believed to interact with G proteins. Based on our results of manipulations of regions of the

chemoattractant receptors by chimeric receptor

construction and analysis, we have found that at least the third cytoplasmic loop is important in G protein coupling. According to the present invention,

cytoplasmic loop analog peptides based on regions of high homology among chemoattractant receptors are preferred, in particular, those based on regions of high homology among the formylpeptide, C5a and IL-8 receptors. In a specific embodiment, such inhibitory peptides comprise a sequence shown in Table II or

Table III (see Section 8, infra). In particular embodiments, the inhibitory peptide which is a second cytoplasmic loop analog is one having one of the following sequences or a portion thereof:

D R C V C V L H P V W T Q N H R T V S (SEQ ID

NO: 2);

D R F L L V F K P I W C Q N F R G A G (SEQ ID

NO: 3);

D R Y L A I V H A T R T L T Q K R H (SEQ ID

NO: 4); or

N R F Q A V T R P I K T A Q A N T R K (SEQ ID

NO:5).

In other particular embodiments, the inhibitory peptide which is a third cytoplasmic loop analog is one having one of the following sequences or a portion thereof :

K I H K Q G L I K S S R P L R (SEQ ID NO: 6);

R T W S R R A T R S T K T L K (SEQ ID NO: 7);

T L F K A H M G Q K H R A M R (SEQ ID NO: 8); or

T L L M G P V Q Q Q R N A E K R R A L W (SEQ ID NO:9).

Other inhibitory cytoplasmic loop analogs are identified as described by way of example in

Section 8, infra. 5.2. IDENTITY OF COMPOUNDS FOR STIMULATION

OF THE INFLAMMATORY RESPONSE

In another embodiment, the invention

provides compounds for stimulation of the inflammatory response. Such pro-inflammatory compounds block chemoattractant receptor desensitization by inhibiting desensitizing phosphorylation of the receptor

cytoplasmic tail. In a preferred aspect, such pro- inflammatory compounds are analogs corresponding to potential phosphorylation sites (Ser/Thr) in the tails of chemoattractant receptors that are highly

conserved. In a specific embodiment, the compound inhibiting desensitization is a peptide corresponding to the sequence ERALTEDSTQTSDTATNSTLPSAEV (see SEQ ID NO: 10) in the formylpeptide receptor; in this 25-mer are clustered 10 of the 11 potential phosphorylation sites in the cytoplasmic tail of the formylpeptide receptor. Other specific embodiments relate to peptides consisting of 50 amino acids or less and comprising the foregoing sequence, and peptides consisting of at least 5 amino acids which correspond to a portion of such sequence. Other pro-inflammatory peptides are identified as described by way of example in Section 9, infra. The calcium mobilization assay described in Section 5.4.2 infra is the preferred in vitro assay of desensitization.

5.3. SYNTHESIS OF THE ANTI- AND PRO-INFLAMMATORY

COMPOUNDS OF THE INVENTION

The compounds of the invention, in a

preferred aspect, are polypeptides, comprising a sequence of amino acids. Such amino acids can be naturally occurring amino acids. The most common naturally-occurring amino acids are listed in Table V.

However, the compounds of the invention are not limited to the 20 natural amino acids. In other embodiments, the compounds can comprise non-classical amino acids or cyclic peptides or peptidomimetics (chemical peptide analogs), as long as the compound has the appropriate activity when tested (see Section 5.4, infra). Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be the D (dextrarotary) or L (levorotary) amino acid.

The compounds of the invention can be prepared by any method known in the art. For example, polypeptides can be chemically synthesized or produced by recombinant DNA technology using a recombinant expression system. As an example of chemical

synthesis, solid phase peptide synthesis can be used, which fconsists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids. The protecting groups may be any known in the art. Before each new amino acid is added to the growing chain, the

protecting group of the previous amino acid added to the chain is removed. The coupling of amino acids to appropriate resins is described by Rivier et al., U.S. Patent No. 4,244,946. Such solid phase syntheses have been described, for example, by Merrifield, 1964, J. Am. Chem. Soc. 85:2149; Vale et al., 1981, Science 213:1394-1397; Marki et al., 1981, J. Am. Chem. Soc. 103:3178; and in U.S. Patent Nos. 4,305,872 and

4,316,891.

5.4. DEMONSTRATION OF ANTI-INFLAMMATORY

OR PRO-INFLAMMATORY ACTIVITY

The anti-inflammatory compounds of the invention are those compounds whose inhibitory

activity can be demonstrated in one or more of the appropriate assays described below, or other assay known in the art. The pro-inflammatory compounds of the invention are those compounds whose stimulatory activity can be demonstrated in one or more of the appropriate assays described below, or other assay known in the art.

5.4.1. IN VITRO ASSAYS WITH CELL MEMBRANES

The following assays are assays of the chemoattractant receptor-G protein interaction. They are implemented using human neutrophil membranes for testing of both insoluble and soluble compounds,

a. Chemoattractant Stimulated GTPase

Activity. This is the preferred in vitro cell membrane assay. The assay can be carried out as described in Okajima et al. (1985, J. Biol. Chem.

260:6761-6768). A measure of chemoattractant receptor activation of its G protein is the stimulation of GTPase activity by chemoattractant which can be inhibited by pertussis toxin. This assay measures the chemoattractant-stimulated release of ³²Pi liberated from [γ-³²P]GTP in neutrophil membranes as described (Okajima et al., supra). Compounds are tested in this system for their ability to block or stimulate this activity, which ability is an indication of their respective anti-inflammatory or pro-inflammatory activity.

b. GTP-Dependent Receptor Affinity Conversion. Nonhydrolyzable analogs of GTP (e.g., GTP7S) directly bind to and activate G proteins.

Thus, GTP7S can mimic receptor-mediated G protein activation. Chemoattractant receptors interconvert between high and low affinity states, which

interconversion is dependent on association or

nonassociation with G protein. Incubation with GTP7S results in a receptor affinity shift to low affinity (i.e., G protein is activated and dissociates from receptor). This assay (Koo et al., 1982, Biochem. Biophys. Res. Comm. 106:442-449; Koo et al., 1983, J. Clin. Invest. 72:748-753; Snyderman et al., 1984, J. Cell Biol. 98:444-448) examines radioligand binding to membranes at two doses, 1 nM for high affinity and 100 nM for low affinity (e.g., with [³H]fMLP). Compounds are tested in this system to identify candidates which prevent GTP7S elicited receptor affinity

interconversion; such candidates can potentially be pro-inflammatory or anti-inflammatory, which is determined by further assay methods.

5.4.2. IN VITRO ASSAYS WITH INTACT CELLS The following assays are implemented using human neutrophils for testing of compounds which are soluble. In some instances, where the compound in question is incapable of entry via pinocytosis, saponin or electropermeabilized cells are used.

a. Intracellular Calcium Mobilization. A consequence of chemoattractant receptor activation of its G protein is the rapid mobilization of

intracellular calcium from intracellular stores which occurs via G protein activation of phospholipase C. The resulting IP3 (inositol 1,4,5 trisphosphate) formed causes the intracellular calcium release. In this assay (Perianin and Snyderman, 1989, J. Immunol. 143:1669-1673), neutrophils are loaded with the

calcium indicator Indo-1, and chemoattractant

stimulated fluorescence changes reflecting calcium mobilization are monitored spectrophotometrically.

Compounds are assayed for their ability to inhibit or to stimulate this function, which ability is an

indication of their respective anti-inflammatory or pro-inflammatory activity. b. Suoeroxide Anion Generation (Respiratory Burst Activation). To measure effects of compounds on neutrophil cytotoxic function, one can assay for chemoattractant-stimulated superoxide anion

generation. This assay (McPhail and Snyderman, 1983, J. Clin. Invest. 72:192-200) measures O₂- production by continuous monitoring of superoxide dismutase- inhibitable reduction of ferricytochrome C. Compounds are assayed for their ability to inhibit or to

stimulate reduction of ferricytochrome C, which ability is an indication of their respective anti- inflammatory or pro-inflammatory activity.

c. Assay of Phospholipase C and D Activation. Phospholipase C and D assays can be used to demonstrate that a compound is a selective

inhibitor or stimulator of chemoattractant receptor-G protein interactions. Phospholipase C activation appears to be a prerequisite for elicitation of chemotactic activity, and phospholipase C and D activation a prerequisite for cytotoxic activity. The phospholipase C assay can be carried out as described in Dillon et al., 1987, J. Biol. Chem. 262:11546. The phospholipase D assay can be carried out as described in Murray et al., 1990, Biochem. J. 270:63.

d. Recombinant Cell Assay. Cells such as human kidney 293 cells (ATCC Accession No. CRL 1573) can be transfected with one or more chemoattractant cDNAs, such that these receptors are expressed by the cells. These cells then form a system for assaying responses to ligand binding by their recombinant chemoattractant receptors. For example, calcium elevation can be determined in these recombinant cells exposed to analogs of the invention, as a measure of analog activity (see Section 6, infra). In a

preferred aspect, the cells are stable transfectants. As assays for demonstrating pro-inflammatory (inhibition of desensitization) activity of analogs, desensitization assays (e.g., such as described in Section 6, infra, by detecting calcium mobilization in neutrophils or in recombinant cells expressing

chemoattractant receptors, after repeated exposure of the cells to a ligand of the appropriate class such as fMLP or C5a) can be used. A decrease in

desensitization in samples exposed to the analog relative to analogous, control samples not exposed to the analog indicates pro-inflammatory activity of the analog.

5.4.3. IN VIVO ASSAYS

Compounds demonstrated to have the desired activity in the assays described above can then be tested in vivo for the desired anti- or pro- inflammatory activity, as the case may be. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc. Suitable model systems are also used to demonstrate therapeutic utility (see Section 5.5.1, infra).

5.5. UTILITY OF THE INVENTION

The compounds of the invention have use therapeutically in diseases or disorders involving inflammation and/or complement activity. By

inflammatory response or inflammation, as used herein, is meant the response mediated by one or more

chemoattractant receptors, including but not limited to chemotaxis of cells carrying a chemoattractant receptor, increase in cytotoxic/microbicidal

activities of such cells (e.g., secretion of hydrolytic enzymes), activation of the respiratory burst (oxidative metabolism leading to free radical production), complement activity effects, etc., as well as the clinical correlates of the foregoing. (See generally, Sandborg and Smolen, 1988, Lab. Invest. 59:300-320; Snyderman and Uhing, 1988, in

Inflammation: Basic Principles and Clinical

Correlates, Gallin et al., eds., Raven Press, Ltd., New York, pp. 309-323; and Section 2, supra.) The invention provides methods of reducing inflammation, and of treating or preventing disorders associated therewith, by administration to a subject of an effective amount of the anti-inflammatory compounds of the invention. In an alternative embodiment, the invention provides methods of stimulating the

inflammatory response, and treating or preventing disorders associated with a deficit in the desired inflammatory response, by administration to a subject of an effective amount of the pro-inflammatory

compounds of the invention. The subject is preferably a mammal, including but not limited to animals such as cows, pigs, chickens, etc., and is most preferably human.

Disease and disorders which can be treated by administration of a therapeutically effective amount of the anti-inflammatory compounds of the invention include but are not limited to the

following:

Inflammatory arthritis - e.g., rheumatoid arthritis, seronegative spondeloarthritites (Behcets disease, Reiter's syndrome, etc.), juvenile rheumatoid arthritis, vasculitis, psoriatic arthritis,

polydermatomyositis.

Systemic lupus ervthematosus (SLE).

Asthma. Inflammatory dermatoses - e.g., psoriasis, dermatitis herpetiformis, eczema, necrotizing and cutaneous vasculitis, bullous diseases.

Reperfusion injury.

Septic Shock (Sepsis). It has been

suggested that activation of complement is involved in fatal complications of sepsis; see Hack et al., 1989, Am. J. Med. 86:20-26 regarding complement activation in sepsis.

Adult respiratory distress syndrome (ARDS). ARDS is a fulminant form of respiratory failure affecting many critically ill patients; for complement involvement in ARDS, see Hangen et al., 1990, J. Surg. Res. 48:196-203; Hatherill et al., 1989, J. Surg. Res. 46:195-199; Hatherill et al., 1989, J. Biol. Response Mod. 8:614-624.

Tissue damage relating to tissue

transplantation.

Other autoimmune disorders. In addition to the autoimmune disorders SLE and rheumatoid arthritis, disorders such as glomerulonephritis can be treated.

Thermal injury (burn). The main

complications due to burn are inflammatory in nature, including shock, and pulmonary edema.

Cardiopulmonary bypass. Systemic inflammation has been associated with the use of pump- oxygenator systems in cardiopulmonary bypass and hemodialysis, which can lead to organ dysfunction, termed the post-pump syndrome or post-perfusion

syndrome.

In addition, other clinical correlates of undesirable inflammatory responses can be treated with the anti-inflammatory compounds of the invention, including but not limited to that associated with hemolytic anemia, hemodialysis (in which the alternative complement pathway is activated; levels of Bb, iC3b, C3a, and C5a, but not C4d, increase;

Opperman et al., 1988, Klin. Wochenscher 66:857-864; Kojima et al., 1989, Nippon Jenzo Gakkai Shi 31:91-97; Ueda et al., 1990, Nippon Jenzo Gakkai Shi 32:19-24), blood transfusion, certain hematologic malignancies, pneumonia, post-ischemic myocardial inflammation and necrosis, and barotrauma (decompression sickness; see Ward et al., 1990, Undersea Biomed. Res. 17:51-66 regarding complement involvement).

Diseases or disorders that can be treated by the pro-inflammatory compounds of the invention include but are not limited to immunosuppression

(e.g., due to AIDS, cancer chemotherapy, radiation therapy, corticosteroid therapy, or other therapy for autoimmune disease), and congenital

immunodeficiencies. 5.5.1. DEMONSTRATION OF THERAPEUTIC UTILITY

Suitable in vitro (e.g., see Section 5.4.2) and in vivo assays are used to demonstrate therapeutic utility of the compounds of the invention. For in vivo testing, prior to administration to humans, any animal model system known in the art may be used. For example, several animal models are available to demonstrate the efficacy of anti-inflammatory

compounds of the invention in the treatment of adult respiratory distress syndrome (ARDS). These include New Zealand white rabbits infused with activated complement (Nuytinck et al., 1986, Brit. J. Exp.

Pathol. 67:537-548); cerulean-induced acute

pancreatitis in rats (Guice et al., 1988, Ann. Surg. 208:71-77); a porcine model produced by infusion of live Pseudomonas aeruginosa (Dehring et al., 1987, J. Trauma 27:615-625); cynomolgus monkeys (Macaca fascicularis) made acutely septic with infusions of E. coli. resulting in severe sepsis and ARDS (Stevens et al., 1986, J. Clin. Invest. 77:1812-1816).

Two animal models of sepsis which can be used are a rat cecal ligation and puncture model (von Allmen et al., 1990, J. Surg. Res. 48:476-480) and a sheep common bile duct contamination model (Barke et al., 1990, Arch. Surg. 125:437-440).

A rabbit model of barotrauma is known (Ward et al., 1990, Undersea Biomed. Res. 17:51-66).

For animal models of thermal, injury, see Bjornson et al., 1986, J. Infect. Dis. 153:1098-1107; Oldham et al., 1988, Surgery 104:272-279; Friedl et al., 1989, Am. J. Pathol. 135:203-217; Demling et al., 1989, Surgery 106:52-59.

An animal model system for rheumatoid arthritis is that consisting of animals of the

autoimmune MRL/1 mouse strain (Murphy, E.D. and Roths, J.B., 1978, in Genetic Control of Autoimmune Disease, Rose, N.R., et al., eds., Elsevier/North-Holland, New York, pp. 207-219), that develop a spontaneous

rheumatoid arthritis-like disease (Hang et al., 1982, J. Exp. Med. 155:1690-1701).

5.5.2. THERAPEUTIC ADMINISTRATION AND COMPOSITIONS

Various delivery systems are known and can be used to administer the compounds of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, etc. Other methods of introduction include but are not limited to intradermal, intramuscular,

intraperitoneal, intravenous, subcutaneous,

intranasal, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound of the invention, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically,

compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a

solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either

separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be

administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

The amount of the compound of the invention which will be effective in the treatment of a

particular disorder or condition will depend on the nature of the disorder or condition, and can be

determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges are generally about several hundred micrograms of active compound per subject. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.

The invention also provides a pharmaceutical pack comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.

6. EXAMPLE: RECEPTOR CLASS DESENSITIZATION

OF LEUKOCYTE CHEMOATTRACTANT RECEPTORS

The data described herein support a novel mechanism of receptor regulation, class

desensitization, which is intermediate between

homologous and heterologous desensitization. Class desensitization of chemoattractant receptors is less selective and requires higher agonist doses than does homologous desensitization, but is far more efficient and specific than heterologous desensitization.

Receptor class desensitization appears to affect functional classes of receptors.

As detailed below, to define their regulation better, formylpeptide and C5a

chemoattractant receptor cDNAs were transiently expressed with high efficiency (about 35-54%) in human kidney cells. As in neutrophils, both receptors were active in elevating intracellular calcium (ED₅₀ about 0.5-1 nM). Agonist-specific desensitization for calcium elevation was observed for both

chemoattractant receptors at doses of about 1 nM.

Heterologous desensitization of formylpeptide, C5a and αl adrenergic receptors required high doses of phorbol ester (100 nM PMA). To further study the phenomenon of desensitization, formylpeptide and C5a receptor cDNAs were co-transfected, resulting in about 80% of receptor positive cells expressing both receptors.

These cells also possessed endogenous αl adrenergic receptors. Interestingly, chemoattractant receptors were cross desensitized by pretreatment with low doses of either C5a or f-Met-Leu-Phe (10 nM) but not by the αl adrenergic agonist norepinephrine (up to 10 μM). Neither chemoattractant desensitized αl adrenergic receptors. This phenomenon was reproduced in human neutrophils.

6.1. MATERIALS AND METHODS

Materials. PMA (phorbol 12-myristate 13- acetate), norepinephrine, carbachol, and C5a from Sigma. fMet-Leu-Phe from Peninsula Laboratories.

(Burlingame, CA). Indo-1 AM from Molecular Probes Inc. (Eugene, OR). Adenosine triphosphate (ATP) from Pharmacia/LKB. Tissue culture media from GIBCO.

DNA Cloning/Plasmid Constructions. Reverse transcription polymerase chain reaction (PCR) of differentiated HL-60 cell mRNA to obtain formylpeptide receptor encoding cDNA was carried out essentially as described (Lee and Caskey, 1989, in Genetic

Engineering: principles and methods, Vol. 11, Setlow & Hollaender, eds., Plenum Press, New York, pp. 159-170) using antisense primer (5'-CTTTGCCTGTAACGCCACCTC-3' (SEQ ID NO: 11)) and amplified with antisense and sense primer (5'-ATGGAGACAAATTCCTCTCTCC-3' (SEQ ID NO: 12) ) using VENT polymerase (New England Biolabs) for 29 cycles (denaturation at 94°C, 30 sec, annealing at 55°C, 30 sec, and extension at 72°C, 30 sec). The resulting cDNA fragment was labeled by nick

translation and used to screen a λgt10 HL-60d library (Didsbury and Snyderman, 1987, FEBS Lett. 219:259-263) under low stringency. Formylpeptide receptor positive clones were confirmed by double stranded DNA

sequencing as described (Chen and Seeburg, 1985, DNA 4:165-170). The open reading frame, devoid of

nontranslated sequences, of the formylpeptide receptor cDNA from a receptor positive clone was generated by PCR to contain a unique EcoRl site immediately 5' of the start codon and a Hind III site immediately 3' to the stop codon using the cloning primers described above which had an additional 12 bases at their 5' end encoding EcoRl and Hind III sites, respectively. The EcoRl/Hind III digested PCR-generated receptor cDNA was then directionally cloned into EcoRl/Hind III cut pRK5 plasmid DNA (Eaton et al., 1986, Biochemistry 25:8343-8347). The EcoRl digested cDNA fragment encoding the C5a chemoattractant receptor (Gerard and Gerard, 1991, Nature 349:614-617) was inserted into the same pRK5 vector.

Cells/Cell Culture/Transfection. Adenovirus type 5 transformed human embryonic kidney 293 cells from American Type Culture Collection (ATCC Accession No. CRL 1573). TSA cells (a clonal variant of 29 cells stably expressing viral large T antigen) kindly provided by Dr. G. Rice, Genentech, Inc. Cells were maintained in DMEM/F12 (50:50) supplemented with 10% fetal bovine serum, 2 mM glutamine and containing penicillin/streptomycin. Calcium phosphate mediated transfection of cells was carried out as described (Gorman, 1985, in DNA Cloning: A Practical Approach. Glover, ed., IRL Press, Washington, D.C., pp.143-165) using a 8-9 μg of CsCl-purified plasmid DNA as a calcium phosphate precipitate in 0.5 ml [0.82% NaCl (w/v), 0.6% Hepes (w/v) pH 7.1, 0.02% Na₂HPO₄ (w/v), 0.25 M CaCl₂] added to 5 ml of complete medium per 60 mm plate. Cells were exposed to DNA for 18 h,

glycerol shocked by exposure to 15% glycerol (v/v) in Dulbecco's phosphate buffered saline without Ca²⁺/Mg²⁺ for 15 sec, washed with PBS, incubated in complete medium for 48 h, and analyzed. Transfection

efficiency was monitored by flow cytometry and radioligand binding. The approximate degree of formylpeptide and C5a receptor co-expression was determined by flow cytometric analysis of

fluoresceinated formylpeptide and C5a binding in conjunction with the degree of cross desensitization between fMLP and C5a elicited calcium responses in the cell population. Human neutrophils were isolated from peripheral blood of normal volunteers as described (Smith and Snyderman, 1987, Meth. Enzymol. 141:261- 271).

Cytosolic Calcium Measurements. Transfected cells were removed from cell culture dishes with

Versene (GIBCO), washed once with 10 mM Hepes-buffered Hanks balanced salt solution (HHBSS) at room

temperature and resuspended in 1.2 ml of HHBSS (~5-6 X 10⁶/ml). Cells were loaded with 1 μM Indo-1 AM for 20 min at room temperature, washed twice with HHBSS, resuspended in 1.2 ml HHBSS and placed in a cuvette. The cuvette was placed into a heated (37°C) cuvette holder of a Perkin-Elmer fluorescence

spectrophotometer (Model 650-19). Calcium analyses were carried out after equilibration of the cells to 37°C (5 min) with an excitation wavelength of 355 nm and an emission wavelength of 405 nm. Maximal and minimal fluorescence were determined in the presence of 0.02% digitonin and 20 mM Tris pH 8, 5 mM EGTA, respectively. Intracellular calcium levels were measured using the following formula: [Ca²⁺]i = K_d(F-

F_min) / F_max-F) (Cobbold and Rink, 1987, Biochem. J.

248:313-328).

6.2. RESULTS

The human formylpeptide chemoattractant receptor was transiently expressed in 293 human kidney cell lines with high efficiency (40-55%). Using intracellular calcium elevation as a measure of receptor action, cells transfected with formylpeptide receptors were exposed to doses of fMLP followed 5 min later by a second 10 nM dose of fMLP (Fig. 1, Panel A). The formylpeptide receptors underwent homologous desensitization with an ED₅₀= 1-5 nM fMLP.

Norepinephrine, acting through endogenous αl

adrenergic receptors had no effect on the calcium response elicited by fMLP although αl adrenergic receptors were homologously desensitized (Fig. 1, Panel B).

To determine if chemoattractant receptors, which as a class utilize a pertussis toxin sensitive G protein to affect calcium elevation (Snyderman et al., 1990, in ADP-Ribosylating Toxins and G Proteins:

Insights into Signal Transduction, Moss, ed., American Society for Microbiology, Washington, D.C., pp. 295- 323; Allen et al., 1988, Hemat. Oncol. Clinics of North America 2:33-59) could more effectively

desensitize one another, formylpeptide and C5a

receptor (Gerard and Gerard, 1991, Nature 349:614-617) cDNAs were co-transfected in the same cell population. Expression levels of both chemoattractant receptors, determined by flow cytometric analysis taken together with the degree of cross desensitization observed (Fig. 2), indicated a maximum of about 80% of

receptor-expressing cells potentially expressing both formylpeptide and C5a receptors. Co-transfected cells were first exposed to a 10 nM dose of fMLP and

subsequently (5 min later) exposed to either 10 nM fMLP, 10 nM C5a, or 10 μM norepinephrine. As

expected, fMLP pretreatment desensitized by about 90% the secondary response to fMLP with no desensitizing effect on norepinephrine-mediated calcium

responsiveness. Interestingly, the calcium responsiveness to C5a was inhibited by 72-76% by fMLP pretreatment (Fig. 2, Panel A). Conversely, co- transfectants exposed first to 10 nM C5a desensitized by 91-93% the secondary response to C5a, also with no effect on norepinephrine-mediated calcium

responsiveness. The secondary response to fMLP however, was inhibited by 70-76% by C5a pretreatment (Fig. 2 , Panel B). Assuming that 80% of positive transfectants expressed both formylpeptide and C5a receptors, then the degree of cross desensitization would approach 90%. At these doses, norepinephrine pretreatment had no desensitizing effect on either fMLP or C5a mediated calcium responses. Heterologous desensitization was demonstrated by pretreatment with 100 nM PMA which lowered by 94-97% the calcium responsiveness to fMLP, C5a, and norepinephrine (Fig. 2, Panel D).

To determine whether the phenomenon of receptor class desensitization, observed with cloned and expressed formylpeptide and C5a receptors, could be demonstrated in human neutrophils which

endogenously contain these receptors, the ability of fMLP and C5a to cross desensitize the calcium

responsiveness of each other was examined. Human neutrophils were exposed to primary doses of fMLP ranging from 1-100 nM and followed 5 min later by a second dose of 1 nM C5a (this dose elicits a calcium response that is about 75% of maximal whereas a 10 nM dose is about 75% of maximal in transfected cells). A dose dependent desensitizing effect was observed with greater than 80% inhibition of the C5a response by pretreatment with 100 nM fMLP (Fig. 3B). Conversely, a similar dose dependent desensitizing effect of C5a on intracellular calcium elevation by 1 nM fMLP was apparent (Fig. 3A). Statistical nonpaired t-test analysis of data points at 10 and 100 nM revealed significant desensitizing dose response differences of fMLP vs. C5a on fMLP-elicited intracellular calcium elevation, p<0.01 at 10 nM, p<0.001 at 100 nM (Fig. 3A) and on C5a-elicited intracellular calcium

elevation, p<0.001 at 10 nM, p<0.02 at 100 nM (Fig. 3B). F variance analysis, comparing the homologous with receptor class desensitization curves of figures 3A and 3B from 10 to 100 nM doses indicated a

significant difference between the two with a p<0.025. To distinguish receptor class desensitization from heterologous desensitization, we measured the effect of ATP, a P2 purinergic receptor agonist, on C5a and fMLP mediated calcium elevation and vice versa.

Pretreatment with 100 μM ATP, which elicits a calcium response greater than 100 nM doses of

chemoattractants, did not inhibit the subsequent calcium response to 1 nM fMLP or C5a and actually "primed" the response to these chemoattractants (Figs. 3A & 3B). Conversely, fMLP pretreatment had no desensitizing effect on ATP-mediated calcium

responses, while C5a had a small desensitizing effect at high concentrations (100 nM) (Fig. 3C). All three agonists at these concentrations (1 nM fMLP, C5a and 1 μM ATP) gave a submaximal calcium response (about 75%, 75% and 45%, respectively). Calcium elevation via P2 purinergic receptors was equally as sensitive to inhibition by pertussis toxin treatment as were chemoattractant receptors.

6.3. DISCUSSION

The present studies define mechanisms of chemoattractant receptor regulation by analysis of cloned and expressed formylpeptide and C5a receptors. Desensitization of these receptors was analyzed using calcium elevation as a measure of receptor action. Both homologous and heterologous desensitization was observed. Homologous desensitization was seen with as little as 1 nM fMLP pretreatment. The αl adrenergic agonist norepinephrine, at doses which elicited calcium responses greater than that evoked by a 100 nM dose of fMLP, did not affect fMLP responsiveness, indicating the specificity of homologous

desensitization. These observations also indicated that homologous desensitization of formylpeptide receptors was not due to depletion of intracellular calcium stores. Unlike chemoattractant receptors, α1- adrenergic receptors couple to a pertussus toxin insensitive G protein to stimulate calcium elevation (Exton, 1988, FASEB J. 2:2670-2676; Cotecchia et al., 1990, J. Biol. Chem. 265:63-69; and data not shown). Heterologous desensitization was observed in

chemoattractant receptor transfected cells by PMA (100 nM) which desensitized nearly completely the

formylpeptide and C5a chemoattractant receptors as well as α1-adrenergic receptors.

To determine whether chemoattractant receptors as a class were regulated coordinately, C5a and formylpeptide receptors were expressed in the same cells. Unexpectedly, cells expressing both C5a and formylpeptide receptors efficiently cross desensitized each other. The desensitization was receptor class selective since neither C5a nor fMLP desensitized norepinephrine elicited calcium responses or vice versa. In contrast, PMA heterologously desensitized all three receptor types. The observation of receptor class desensitization in this receptor expression system led us to examine he phenomenon in

neutrophils. The same receptor class desensitization was observed in these cells. The activity and potency of the receptor class desensitization process was less than homologous desensitization as evidenced by the lower ability of 10 nM and 100 nM doses of each chemoattractant to desensitize the other

chemoattractant type whereas merely complete

desensitization was seen to the same chemoattractant (Fig. 3) . Statistical analysis of the dose response curves distinguishing homologous from receptor class desensitization (Fig. 3A & 3B) revealed them to be significantly different. In neutrophils, receptor class desensitization was distinguished from

heterologous desensitization by the fact that ATP, acting through P2 purinergic receptors, did not desensitize the response to fMLP or C5a or vice versa in agreement with previous observations (Walker et al., 1991, Lab. Invest. 64:105-112). At high doses of C5a (100 nM), there may have been some heterologous desensitization of P2 purinergic receptors. We noted a potentiating effect of ATP pretreatment on C5a- and fMLP-mediated calcium responses.

Thus, there appears to be at least three types of chemoattractant receptor desensitization, homologous (i.e., fMLP→ fMLP), heterologous (i.e. PMA → fMLP) and a novel receptor class desensitization

[i.e. (fMLP→ C5a) as distinct from αl adrenergic or

P2 purinergic]. Unlike homologous desensitization, which for β-adrenergic receptors results in

phosphorylation of only agonist-occupied receptors (Lefkowitz et al., 1990, Trends Pharmacol. Sci.

11:190-194), chemoattractant receptor class

desensitization does not require agonist occupancy since neither formylpeptide nor C5a compete in binding to each others receptor (Rollins et al., 1985, J.

Biol. Chem. 260:715-716; Bender et al., 1987, J. Immunol. 139:3028-3033) but do efficiently desensitize each other. Receptor class desensitization occurred with relatively low doses of chemoattractant (e.g., 10 nM) whereas even high doses of fMLP (> 100 nM) caused no heterologous desensitizing effect on other calcium elevating receptors (e.g., αl adrenergic receptors on TSA cells or P2 purinergic receptors on neutrophils).

There is no desensitizing effect of

norepinephrine or ATP on chemoattractant response even though they are equally effective for activating phospholipase C (Cotecchia et al., 1990, J. Biol.

Chem. 265:63-69; Cockcroft and Stutchfield, 1989, Biochem. J. 263:715-723). This again indicates the novelty of this mechanism for desensitization.

7. EXAMPLE: IDENTIFICATION OF ANTI-INFLAMMATORY MASTOPARAN ANALOGS

Anti-inflammatory mastoparan analogs are obtained and identified as follows:

1. The lead molecule (i.e., mastoparan) is tested for activity, which should be stimulatory for G protein activation. Peptides consisting of at least 5 amino acids and corresponding to the lead molecule shortened from each end are tested to determine the smallest molecule which retains such biological

activity. This is done in two steps. First, peptides corresponding to the lead molecule shortened from one end, e.g., the amino terminus, are tested to determine the smallest peptide with activity (the "parent

peptide"). A series of peptides corresponding to the parent peptide shortened from the other end, e.g., the carboxy terminus, are then tested to determine the smallest peptide with activity.

Thus, initially, the following peptides (see

SEQ ID NO:l) are tested for stimulatory activity: N L K A L A A L A K K I L

L K A L A A L A K K I L K A L A A L A K K I L A L A A L A K K I L L A A L A K K I L A A L A K K I L A L A K K I L L A K K I L A K K I L

The smallest peptide from among those listed above which retains activity is identified; peptides corresponding to this peptide shortened one amino acid at a time from the carboxyl-terminal end are then tested to determine the smallest peptide with

activity. Thus, for example, assuming arcruendo that K A L A A L A K K I L (see SEQ ID NO: 1) is identified as the smallest peptide retaining activity, then the following series of peptides (see SEQ ID NO: 1) are tested to determine the smallest peptide among them which retains activity:

K A L A A L A K K I K A L A A L A K K K A L A A L A K

K A L A A L A

K A L A A L

K A L A A

2. A series of mastoparan analogs are then made, corresponding to the smallest active peptide identified in step 1 in which an alanine is

substituted at each position (or a glycine residue at positions which contain an alanine residue in the lead molecule) (initially, with each analog containing a single substitution), and tested for stimulatory activity to determine which are the critical residues for such activity.

3. The critical residues necessary for stimulatory activity are then changed with the residues listed below in Table I (e.g., if residue #2 is critical, then it is changed to Asp, Glu or Gln). Analogs are now tested for the ability to inhibit rather than stimulate G protein activation by

chemoattractants.

TABLE I

*SEQ ID NO:1

4. Non-critical residues are then replaced with the above listed replacements to increase the inhibitory activity of the optimal analog identified in step #3 above.

The peptide analog with inhibitory activity identified in step #4, above, is then, if desired, altered in a variety of ways, including but not limited to the following: The peptide can be used with the N-terminus as a free amine or chemically derivatized so as to modify either the polypeptide's activity, susceptibility to degradation or clearance from biological fluids. The C-terminus can be

modified to an amide to affect the polymer's activity, susceptibility to degradation or clearance from biological fluids. The analog may also be made as homodimers or heterodimers by a variety of chemical coupling means. Likewise, it can be prepared with an additional cysteine on either terminus to crosslink two polypeptides through disulfide bond formation. The analog can also be cyclized as monomers or polymers or coupled to a carrier molecule in order to promote activity or stability or to a carrier molecule (e.g., an antibody or fragment thereof or ligand of an in vivo receptor) to direct targeting.

8. EXAMPLE: IDENTIFICATION OF ANTI-INFLAMMATORY CHEMOATTRACTANT RECEPTOR CYTOPLASMIC LOOP ANALOGS

Anti-inflammatory chemoattractant receptor third cytoplasmic loop analogs are identified as follows:

1. The lead molecule is the peptide

corresponding to the third cytoplasmic loop of the formylpeptide chemoattractant receptor (Table II).

The lead molecule is tested for its ability to inhibit chemoattractant (fMLP) stimulated GTPase activity.

If the peptide does not have a sufficient inhibitory effect, it is structurally altered to potentiate or increase inhibitory activity. Such structural alterations include but are not limited to one or more of the following: C-terminal modification to an amide, construction of homodimers or

heterodimers by a variety of chemical coupling means

(e.g., the peptide can be prepared with an additional cysteine on either terminus to crosslink two peptides through disulfide bond formation), cyclization as monomers or polymers, and coupling to a carrier

molecule.

2. Peptides consisting of at least 5 amino acids and corresponding to the molecule shortened from each end are tested to determine the smallest molecule which retains inhibitory activity (see Section 7, step 1, for analogous procedure). First, peptides

corresponding to the lead molecule shortened from one end, e.g., the amino terminus, are tested to determine the smallest peptide with activity (the "parent peptide" ) . Thus , e . g . , the following peptides (see SEQ ID NO: 6) are initially tested:

I H K Q G L I K S S R P L R H K Q G L I K S S R P L R K Q G L I K S S R P L R Q G L I K S S R P L R G L I K S S R P L R L I K S S R P L R I K S S R P L R K S S R P L R S S R P L R S R P L R

A series of peptides corresponding to the parent peptide shortened from the other end, e.g., the carboxy terminus, are then tested to determine the smallest peptide with activity.

3. A series of analogs are then made, corresponding to the smallest active peptide

identified in step 1 in which an alanine is

substituted at each position (or a glycine residue at positions which contain an alanine residue in the lead molecule) (initially, each analog containing a single substitution), and tested to determine which are the critical residues for inhibitory activity.

4. The critical residues necessary for activity are then replaced with the residues listed below in Table II (e.g. if residue #2 is critical, then it will be changed to Leu, Met or Val). Analogs which are enhanced in their inhibitory activity are identified. 5. If desired, non-critical residues are then replaced with the replacements listed in Table II to increase the inhibitory activity of the optimal analog identified in step #3 above.

In other embodiments, the lead molecule in the identification of anti-inflammatory

chemoattractant receptor cytoplasmic loop analogs is the third cytoplasmic loop of the C5a receptor, IL-8 receptor, or PAF receptor.

In another specific embodiment, the identification of anti-inflammatory chemoattractant receptor second cytoplasmic loop analogs are

identified as described above for third cytoplasmic loop analogs, except using the lead molecule and residue replacements as set forth in Table III.

If desired, the peptide analog resulting from step #5 above is then altered in a variety of ways including but not limited to the following: The peptide analog can be used with the N-terminus as a free amine or chemically derivatized so as to modify either the polypeptide's activity, susceptibility to degradation or clearance from biological fluids. The C-terminus can be modified to an amide to affect the polymer's activity, susceptibility to degradation or clearance from biological fluids. The analog can also be made as homodimers or heterodimers by a variety of chemical coupling means. It can be prepared with an additional cysteine on either terminus to crosslink two peptides through disulfide bond formation. The analogs can be cyclized as monomers or polymers. The analogs can also be coupled to a carrier molecule in order to promote stability or activity or to a carrier molecule to direct targeting.

In the production of the chemoattractant receptor cytoplasmic loop analogs of the invention, amino acid residue numbers 1, 2, 3, 9, 16 and 17 in Tables II and III, which are amino acid residues that are highly conserved among all G protein-coupled receptors, will not initially be modified.

9. EXAMPLEIDENTIFICATION OF PRO-INFLAMMATORY ANALOGS

Pro-inflammatory analogs of the invention are identified as follows:

1. The lead peptide, corresponding to the sequence ERALTEDSTQTSDTATNSTLPSAEV (see SEQ ID NO: 10) in the formylpeptide receptor, is tested for its ability to inhibit desensitization.

If desired, the peptide is then structurally altered to potentiate or increase its inhibitory effect. Such alterations can be carried out by a variety of means, including but are not limited to C- terminal modification to an amide, construction of homodimers or heterodimers which can be accomplished by one of a variety of chemical coupling means (e.g., making an additional cysteine on either terminus to crosslink two peptides through disulfide bond

formation), cyclization as monomers or polymers, and coupling to a carrier molecule.

2. Peptides consisting of at least 5 amino acids and corresponding to the molecule shortened from each end are tested to determine the smallest molecule which retains biological activity (see Section 7, step 1, for analogous procedure). First, peptides

corresponding to the lead molecule shortened from one end, e.g., the amino terminus, are tested to determine the smallest peptide with activity (the "parent peptide"). Thus, e.g., the following peptides (see SEQ ID NO: 10) are initially tested:

R A L T E D S T Q T S D T A T N S T L P S A E V A L T E D S T Q T S D T A T N S T L P S A E V L T E D S T Q T S D T A T N S T L P S A E V T E D S T Q T S D T A T N S T L P S A E V E D S T Q T S D T A T N S T L P S A E V D S T Q T S D T A T N S T L P S A E V S T Q T S D T A T N S T L P S A E V T Q T S D T A T N S T L P S A E V Q T S D T A T N S T L P S A E V T S D T A T N S T L P S A E V S D T A T N S T L P S A E V D T A T N S T L P S A E V T A T N S T L P S A E V A T N S T L P S A E V T N S T L P S A E V N S T L P S A E V S T L P S A E V

T L P S A E V L P S A E V P S A E V

A series of peptides corresponding to the parent peptide shortened from the other end, e.g., the carboxy terminus, are then tested to determine the smallest peptide with activity.

3. A series of analogs are then made, corresponding to the smallest active peptide

identified in step 1 in which an alanine is

substituted at each position (or a glycine residue at positions which contain an alanine residue in the lead molecule) (initially, each analog containing a single substitution), and tested to determine which are the critical residues for desensitization-inhibitory activity.

4. If desired, the critical residues necessary to activity are then changed with the residues listed below in Table IV (e.g., if residue #2 is critical than it is changed to Leu, Met or Val). Positions where a serine or threonine is present are only altered to contain the other potential

phosphorylatable amino acid (e.g., Ser to Thr or Thr to Ser, for example, as listed for residue #5 below). Analogs are identified which are enhanced in their inhibitory activity.

5. If desired, non-critical residues are replaced with the replacements listed in Table IV, to increase the desensitization-inhibitory activity of the optimal analog found in step #3 above.

If desired, the candidate peptide analog is then be altered in a variety of ways including but limited to the following: It may be used with the N- terminus as a free amine or chemically derivatized so as to modify either the polypeptide's activity, susceptibility to degradation or clearance from biological fluids. The C-terminus can be modified to an amide to affect the polymer's activity,

susceptibility to degradation or clearance from biological fluids. The analog can also be made as homodimers or heterodimers by a variety of chemical coupling means. It can be prepared with an additional cysteine on either terminus to crosslink two peptides through disulfide bond formation. The peptides can also be made as cyclized monomers or polymers, or coupled to a carrier molecule in order to promote stability or activity or to a carrier molecule to direct targeting.

In other embodiments, the lead peptide for the identification of pro-inflammatory analogs is all or a portion of the chemoattractant receptor

cytoplasmic tail sequences set forth in Table IV (SEQ ID NOS:10, 13, 14, 15). The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become

apparent to those skilled in the art from the

foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Snyderman, Ralph

Didsbury, John R.

Uhing, Ronald J.

(ii) TITLE OF INVENTION: Methods For Inhibition Or Stimulation Of

The Inflammatory Response

(iii) NUMBER OF SEQUENCES: 15

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Pennie & Edmonds

(B) STREET: 1155 Avenue of the Americas

(C) CITY: New York

(D) STATE: New York

(E) COUNTRY: U.S.A.

(F) ZIP: 10036-2711

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Misrock, S. Leslie

(B) REGISTRATION NUMBER: 18,872

(C) REFERENCE/DOCKET NUMBER: 7337-017

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 212 790-9090

(B) TELEFAX: 212 8698864/9741

(C) TELEX: 66141 PENNIE

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

lle Asn Leu Lys Ala Leu Ala Ala Leu Ala Lys Lys lle Leu

1 5 10

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Asp Arg Cys Val Cys Val Leu His Pro Val Trp Thr Gln Asn His Arg 1 5 10 15

Thr Val Ser

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESSr single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Asp Arg Phe Leu Leu Val Phe Lvs Pro lle Trp Cys Gln Asn Phe Arg 1 5 10 15

Gly Ala Gly

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTHi 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Asp Arg Tyr Leu Ala lle Val His Ala Thr Arg Thr Leu Thr Gln Lys 1 5 10 15

Arg His

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Asn Arg Phe Gln Ala Val Thr Arg Pro lle Lys Thr Ala Gln Ala Asn 1 5 10 15

Thr Arg Lys (2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Lys lle His Lys Gln Gly Leu lle Lys Sep. Ser Arg Pro Leu Arg 1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Arg Thr Trp Ser Arg Arg Ala Thr Arg Ser Thr Lys Thr Leu Lys 1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Thr Leu Phe Lys Ala His Met Gly Gln Lys His Arg Ala Met Arg 1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Thr Leu Leu Met Gly Pro Val Gln Gln Gln Arg Asn Ala Glu Lys Arg 1 5 10 15

Arg Ala Leu Trp

20 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 :

Gly Gln Asp Phe Arg Glu Arg Leu He His Ala Leu Pro Ala Ser Leu

1 5 10 15

Glu Arg Ala Leu Thr Glu Asp Ser Thr Gln Thr Ser Asp Thr Ala Thr

20 25 30

Asn Ser Thr Leu Pro Ser Ala Glu Val Ala Leu Gln Ala Lys

35 40 45

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

CTTTGCCTGT AACGCCACCT C 21

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

ATGGAGACAA ATTCCTCTCT CC 22

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Gly Gln Gly Phe Gln Gly Arg Leu Arg Lys Ser Leu Pro Ser Leu Leu 1 5 10 15

Arg Asn Val Leu Thr Glu Glu Ser Val Val Arg Glu Ser Lys Ser Phe

20 25 30

Thr Arg Ser Thr Val Asp Thr Met Ala Gln Lys Thr Gln Ala Val

35 40 45

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Gly Gln Asn Phe Arg His Gly Phe Leu Lys He Leu Ala Met His Gly 1 5 10 15

Leu Val Ser Lys Glu Phe Leu Ala Arg His Arg Val Thr Ser Tyr Thr

20 25 30

Ser Ser Ser Val Asn Val Ser Ser Asn Leu

35 40

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Thr Lys Lys Phe Arg Lys His Leu Thr Glu Lys Phe Tyr Ser Met Arg 1 5 10 15

Ser Ser Arg Lys Cys Ser Arg Ala Ser Ser Asp Thr Val Thr Glu Val

20 25 30

Val Val Pro Phe Asn Gln Ile Pro Gly Asn Ser Leu Lys Asn

35 40 45

Claims

WHAT IS CLAIMED IS: 1. A substantially purified peptide having a sequence of not more than fifty amino acids, and comprising the following amino acid sequence: Glu- Arg-Ala-Leu-Thr-Glu-Asp-Ser-Thr-Gln-Thr-Ser-Asp-Thr- Ala-Thr-Asn-Ser-Thr-Leu-Pro-Ser-Ala-Glu-Val (see SEQ ID NO: 10).

2. A substantially purified peptide having the following amino acid sequence: Glu-Arg-Ala-Leu- Thr-Glu-Asp-Ser-Thr-Gln-Thr-Ser-Asp-Thr-Ala-Thr-Asn- Ser-Thr-Leu-Pro-Ser-Ala-Glu-Val (see SEQ ID NO: 10).

3. A substantially purified peptide having a sequence of not more than fifty amino acids, and comprising an amino acid sequence selected from the group consisting of:

(a) Lys-Ile-His-Lys-Gln-Gly-Leu-Ile-Lys-Ser-Ser-Arg- Pro-Leu-Arg (SEQ ID NO: 6);

(b) Arg-Thr-Trp-Ser-Arg-Arg-Ala-Thr-Arg-Ser-Thr-Lys- Thr-Leu-Lys (SEQ ID NO: 7);

(c) Thr-Leu-Phe-Lys-Ala-His-Met-Gly-Gln-Lys-His-Arg- Ala-Met-Arg (SEQ ID NO: 8); and

(d) Thr-Leu-Leu-Met-Gly-Pro-Val-Gln-Gln-Gln-Arg-Asn- Ala-Glu-Lys-Arg-Arg-Ala-Leu-Trp (SEQ ID NO: 9).

4. A substantially purified peptide having an amino acid sequence selected from the group

consisting of:

(a) Lys-Ile-His-Lys-Gln-Gly-Leu-Ile-Lys-Ser-Ser-Arg- Pro-Leu-Arg (SEQ ID NO: 6);

(b) Arg-Thr-Trp-Ser-Arg-Arg-Ala-Thr-Arg-Ser-Thr-Lys- Thr-Leu-Lys (SEQ ID NO:7); (c) Thr-Leu-Phe-Lys-Ala-His-Met-Gly-Gln-Lys-His-Arg- Ala-Met-Arg (SEQ ID NO: 8); and

(d) Thr-Leu-Leu-Met-Gly-Pro-Val-Gln-Gln-Gln-Arg-Asn- Ala-Glu-Lys-Arg-Arg-Ala-Leu-Trp (SEQ ID NO: 9).

5. A substantially purified peptide having a sequence of not more than fifty amino acids, and comprising an amino acid sequence selected from the group consisting of:

(a) Asp-Arg-Cys-Val-Cys-Val-Leu-His-Pro-Val-Trp-Thr- Gln-Asn-His-Arg-Thr-Val-Ser (SEQ ID NO:2);

(b) Asp-Arg-Phe-Leu-Leu-Val-Phe-Lys-Pro-Ile-Trp-Cys- Gln-Asn-Phe-Arg-Gly-Ala-Gly (SEQ ID NO: 3);

(c) Asp-Arg-Tyr-Leu-Ala-Ile-Val-His-Ala-Thr-Arg-Thr- Leu-Thr-Gln-Lys-Arg-His (SEQ ID NO: 4); and

(d) Asn-Arg-Phe-Gln-Ala-Val-Thr-Arg-Pro-Ile-Lys-Thr- Ala-Gln-Ala-Asn-Thr-Arg-Lys (SEQ ID NO: 5).

6. A substantially purified peptide having an amino acid sequence selected from the group

consisting of:

(a) Asp-Arg-Cys-Val-Cys-Val-Leu-His-Pro-Val-Trp-Thr- Gln-Asn-His-Arg-Thr-Val-Ser (SEQ ID NO:2);

(b) Asp-Arg-Phe-Leu-Leu-Val-Phe-Lys-Pro-Ile-Trp-Cys- Gln-Asn-Phe-Arg-Gly-Ala-Gly (SEQ ID NO: 3);

(c) Asp-Arg-Tyr-Leu-Ala-Ile-Val-His-Ala-Thr-Arg-Thr- Leu-Thr-Gln-Lys-Arg-His (SEQ ID NO: 4); and

(d) Asn-Arg-Phe-Gln-Ala-Val-Thr-Arg-Pro-Ile-Lys-Thr- Ala-Gln-Ala-Asn-Thr-Arg-Lys (SEQ ID NO: 5).

7. A method for identifying a peptide with the ability to inhibit the inflammatory response mediated by one or more chemoattractant receptors comprising the following steps in the stated order: (a) testing a first series of peptides to determine which peptides have the ability to stimulate an inflammatory response mediated by one or more chemoattractant receptors, which series consists of the following peptides (see SEQ ID NO:1):

Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu, Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu,

Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu,

Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu,

Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu,

Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu,

Ala-Leu-Ala-Lys-Lys-Ile-Leu,

Leu-Ala-Lys-Lys-Ile-Leu,

Ala-Lys-Lys-Ile-Leu;

(b) identifying the smallest peptide within the first series which is determined to have the ability to stimulate the inflammatory response;

(c) testing a second series of peptides to

determine which peptides have the ability to stimulate the inflammatory response, each peptide in the second series consisting of at least five amino acids and having the sequence of the peptide identified in step (b) except with a carboxy-terminal deletion, each peptide differing in size by one amino acid from the other peptides in the second series;

(d) identifying the smallest peptide within the second series which is determined to have the ability to stimulate the inflammatory response;

(e) testing a third series of peptides to

determine which peptides lack the ability to stimulate the inflammatory response, each peptide in the third series having the sequence of the peptide identified in step (d) except that each contains a single substitution of an alanine within the sequence, or where an alanine is present, a glycine for an alanine; each peptide in the third series having a different substituted residue than the other peptides in the third series;

(f) identifying the substituted residue in the sequence of a peptide determined to lack the ability in step (e);

(g) obtaining a peptide having the sequence of the peptide determined to lack the ability in step (e), except that the residue

identified in step (f) is substituted according to the following, where the number of the residue is the number of the

corresponding residue in the sequence Ile- Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys- Ile-Leu (SEQ ID NO: 1), numbered from amino to carboxyl terminus:

If the residue is residue number 1, the

substitution is Leu, Met or Val;

If the residue is residue number 2, the

substitution is Asp, Glu, or Gin;

If the residue is residue number 3 , the

substitution is lie, Met, or Val;

If the residue is residue number 4, the

substitution is Arg or His;

If the residue is residue number 5, the

substitution is Gly, Pro, Ser, or Thr;

If the residue is residue number 6, the

substitution is lie. Met, or Val; If the residue is residue number 7, the

substitution is Gly, Pro, Ser, or Thr;

If the residue is residue number 8, the

substitution is Gly, Pro, Ser, or Thr;

If the residue is residue number 9, the

substitution is lie, Met, or Val; If the residue is residue number 10, the

substitution is Gly, Pro, Ser, or Thr;

If the residue is residue number 11, the

substitution is Arg or His;

If the residue is residue number 12, the

substitution is Arg or His;

If the residue is residue number 13, the

substitution is Leu, Met, or Val;

If the residue is residue number 14, the

substitution is lie. Met, or Val;

(h) testing the peptide obtained in step (g) to determine whether it has the ability to inhibit the inflammatory response mediated by one or more chemoattractant receptors; (i) repeating steps (f)-(h) until a peptide is obtained which is determined in step (h) to have the ability to inhibit; and (j) identifying a peptide which is determined in step (h) to have the ability to inhibit the inflammatory response.

8. A substantially purified peptide identified according to the method of claim 7.

9. A method for identifying a peptide with the ability to inhibit the inflammatory response mediated by one or more chemoattractant receptors comprising the following steps in the stated order: (a) testing a first series of peptides to determine which peptides have the ability to inhibit an inflammatory response mediated by one or more chemoattractant receptors, which series consists of the following peptides (see SEQ ID NO: 6):

Ile-His-Lys-Gln-Gly-Leu-Ile-Lys-Ser-Ser-Arg-Pro-Leu- Arg,

His-Lys-Gln-Gly-Leu-Ile-Lys-Ser-Ser-Arg-Pro-Leu-Arg, Lys-Gln-Gly-Leu-Ile-Lys-Ser-Ser-Arg-Pro-Leu-Arg,

Gln-Gly-Leu-Ile-Lys-Ser-Ser-Arg-Pro-Leu-Arg,

Gly-Leu-Ile-Lys-Ser-Ser-Arg-Pro-Leu-Arg,

Leu-Ile-Lys-Ser-Ser-Arg-Pro-Leu-Arg,

Ile-Lys-Ser-Ser-Arg-Pro-Leu-Arg,

Lys-Ser-Ser-Arg-Pro-Leu-Arg,

Ser-Ser-Arg-Pro-Leu-Arg,

Ser-Arg-Pro-Leu-Arg;

(b) identifying the smallest peptide within the first series which is determined to have the ability to inhibit the inflammatory response;

(c) testing a second series of peptides to

determine which peptides have the ability to inhibit the inflammatory response, each peptide in the second series consisting of at least five amino acids and having the sequence of the peptide identified in step (b) except with a carboxy-terminal deletion, each peptide differing in size by one amino acid from the other peptides in the second series; and

(d) identifying the smallest peptide within the second series which is determined to have the ability to inhibit the inflammatory response.

10. A substantially purified peptide identified according to the method of claim 9.

11. A method for identifying a peptide with the ability to inhibit the inflammatory response mediated by one or more chemoattractant receptors comprising the following steps in the stated order:

(a) testing a first series of peptides to

determine which peptides have the ability to inhibit an inflammatory response mediated by one or more chemoattractant receptors, which series consists of the following peptides (see SEQ ID NO: 2):

Arg-Cys-Val-Cys-Val-Leu-His-Pro-Val-Trp-Thr-Gln-Asn-

His-Arg-Thr-Val-Ser,

Cys-Val-Cys-Val-Leu-His-Pro-Val-Trp-Thr-Gln-Asn-

His-Arg-Thr-Val-Ser,

Val-Cys-Val-Leu-His-Pro-Val-Trp-Thr-Gln-Asn-His-Arg-

Thr-Val-Ser,

Cys-Val-Leu-His-Pro-Val-Trp-Thr-Gln-Asn-His-Arg-Thr-

Val-Ser,

Val-Leu-His-Pro-Val-Trp-Thr-Gln-Asn-His-Arg-Thr-Val-

Ser,

Leu-His-Pro-Val-Trp-Thr-Gln-Asn-His-Arg-Thr-Val-Ser, His-Pro-Val-Trp-Thr-Gln-Asn-His-Arg-Thr-Val-Ser,

Pro-Val-Trp-Thr-Gln-Asn-His-Arg-Thr-Val-Ser,

Val-Trp-Thr-Gln-Asn-His-Arg-Thr-Val-Ser,

Trp-Thr-Gln-Asn-His-Arg-Thr-Val-Ser,

Thr-Gln-Asn-His-Arg-Thr-Val-Ser,

Gln-Asn-His-Arg-Thr-Val-Ser,

Asn-His-Arg-Thr-Val-Ser,

His-Arg-Thr-Val-Ser;

(b) identifying the smallest peptide within the first series which is determined to have the ability to inhibit the inflammatory

response;

(c) testing a second series of peptides to

determine which peptides have the ability to inhibit the inflammatory response, each peptide in the second series consisting of at least five amino acids and having the sequence of the peptide identified in step (b) except with a carboxy-terminal deletion, each peptide differing in size by one amino acid from the other peptides in the second series; and

(d) identifying the smallest peptide within the second series which is determined to have the ability to inhibit the inflammatory response.

12. A substantially purified peptide identified according to the method of claim 11.

13. A method for identifying a peptide with the ability to inhibit the desensitization of one or more chemoattractant receptors comprising the

following steps in the stated order:

(a) testing a first series of peptides to

determine which peptides have the ability to inhibit the desensitization of one or more chemoattractant receptors, which series consists of the following peptides (see SEQ

ID NO: 10):

Arg-Ala-Leu-Thr-Glu-Asp-Ser-Thr-Gln-Thr-Ser-Asp-Thr-

Ala-Thr-Asn-Ser-Thr-Leu-Pro-Ser-Ala-Glu-Val,

Ala-Leu-Thr-Glu-Asp-Ser-Thr-Gln-Thr-Ser-Asp-Thr-Ala- Thr-Asn-Ser-Thr-Leu-Pro-Ser-Ala-Glu-Val, Leu-Thr-Glu-Asp-Ser-Thr-Gln-Thr-Ser-Asp-Thr-Ala-Thr-

Asn-Ser-Thr-Leu-Pro-Ser-Ala-Glu-Val,

Thr-Glu-Asp-Ser-Thr-Gln-Thr-Ser-Asp-Thr-Ala-Thr-Asn- Thr-Leu-Pro-Ser-Ala-Glu-Val,

Glu-Asp-Ser-Thr-Gln-Thr-Ser-Asp-Thr-Ala-Thr-Asn-Thr-

Leu-Pro-Ser-Ala-Glu-Val ,

Asp-Ser-Thr-Gln-Thr-Ser-Asp-Thr-Ala-Thr Asn-Thr-Leu- Pro-Ser-Ala-Glu-Val,

Ser-Thr-Gln-Thr-Ser-Asp-Thr-Ala-Thr-Asn-Thr-Leu-Pro- Ser-Ala-Glu-Val,

Thr-Gln-Thr-Ser-Asp-Thr-Ala-Thr-Asn-Thr-Leu-Pro-Ser-

Ala-Glu-Val,

Gln-Thr-Ser-Asp-Thr-Ala-Thr-Asn-Leu-Pro-Ser-Ala-Glu- Val,

Thr-Ser-Asp-Thr-Ala-Thr-Asn-Leu-Pro-Ser-Ala-Glu-Val , Ser-Asp-Thr-Ala-Thr-Asn-Leu-Pro-Ser-Ala-Glu-Val, Asp-Thr-Ala-Asn-Leu-Pro-Ser-Ala-Glu-Val,

Thr-Ala-Asn-Leu-Pro-Ser-Ala-Glu-Val,

Ala-Asn-Leu-Pro-Ser-Ala-Glu-Val,

Asn-Leu-Pro-Ser-Ala-Glu-Val,

Leu-Pro-Ser-Ala-Glu-Val,

Pro-Ser-Ala-Glu-Val;

(b) identifying the smallest peptide within the first series which is determined to have the ability to inhibit the desensitization;

(c) testing a second series of peptides to

determine which peptides have the ability to inhibit the desensitization, each peptide in the second series consisting of at least five amino acids and having the sequence of the peptide identified in step (b) except with a carboxy-terminal deletion, each peptide differing in size by one amino acid from the other peptides in the second series; and (d) identifying the smallest peptide within the second series which is determined to have the ability to inhibit the desensitization.

14. A substantially purified peptide identified according to the method of claim 13.

15. A method of inhibiting the inflammatory response mediated by one or more chemoattractant receptors comprising exposing a cell expressing a chemoattractant receptor to an effective amount of the peptide of claim 4.

16. A method of inhibiting the inflammatory response mediated by one or more chemoattractant receptors comprising exposing a cell expressing a chemoattractant receptor to an effective amount of the peptide of claim 6.

17. A method of inhibiting the inflammatory response mediated by one or more chemoattractant receptors comprising exposing a cell expressing a chemoattractant receptor to an effective amount of the peptide of claim 8.

18. A method of inhibiting the inflammatory response mediated by one or more chemoattractant receptors comprising exposing a cell expressing a chemoattractant receptor to an effective amount of the peptide of claim 10.

19. A method of inhibiting the inflammatory response mediated by one or more chemoattractant receptors comprising exposing a cell expressing a chemoattractant receptor to an effective amount of the peptide of claim 12.

20. A method of inhibiting the desensitization of one or more chemoattractant

receptors comprising exposing a cell expressing a chemoattractant receptor to an effective amount of the peptide of claim 2.

21. A method of inhibiting the

desensitization of one or more chemoattractant

receptors comprising exposing a cell expressing a chemoattractant receptor to an effective amount of the peptide of claim 14.

22. A pharmaceutical composition comprising a therapeutically effective amount of the peptide of claim 2; and a pharmaceutically acceptable carrier.

23. A pharmaceutical composition comprising a therapeutically effective amount of the peptide of claim 4; and a pharmaceutically acceptable carrier.

24. A pharmaceutical composition comprising a therapeutically effective amount of the peptide of claim 6; and a pharmaceutically acceptable carrier.

25. The method according to claim 15 in which the class of chemoattractant receptors

comprising the formyl-Met-Leu-Phe receptor and the C5a receptor is inhibited.

26. The method according to claim 16 in which the class of chemoattractant receptors comprising the formyl-Met-Leu-Phe receptor and the C5a receptor is inhibited.

27. The method according to claim 17 in which the class of chemoattractant receptors

comprising the formyl-Met-Leu-Phe receptor and the C5a receptor is inhibited.

28. The method according to claim 18 in which the class of chemoattractant receptors

comprising the formyl-Met-Leu-Phe receptor and the C5a receptor is inhibited.

29. The method according to claim 19 in which the class of chemoattractant receptors

comprising the formyl-Met-Leu-Phe receptor and the C5a receptor is inhibited.

30. The method according to claim 20 in which the desensitization of the class of

chemoattractant receptors comprising the formyl-Met- Leu-Phe receptor and the C5a receptor is inhibited.

31. The method according to claim 21 in which the desensitization of the class of

chemoattractant receptors comprising the formyl-Met- Leu-Phe receptor and the C5a receptor is inhibited.