WO2004082688A1

WO2004082688A1 - Phosphatidylglycerol (pg) receptor agonists and antagonists

Info

Publication number: WO2004082688A1
Application number: PCT/CA2004/000415
Authority: WO
Inventors: Anthony E. Bolton; Arkady Mandel
Original assignee: Vasogen Ireland Limited
Priority date: 2003-03-20
Filing date: 2004-03-19
Publication date: 2004-09-30

Abstract

This invention relates to phosphatidylglycerol (PG) agonists and antagonists, pharmaceutical compositions comprising PG agonists and antagonists as well as methods for ascertaining agonist/antagonist properties of putative ligands for the PG receptor.

Description

PHOSPHATIDYLGLYCEROL (PG) RECEPTOR AGONISTS AND ANTAGONISTS

BACKGROUND OF THE INVENTION

Field of the Invention

State of the Art

Pharmaceutically acceptable phosphatidylglycerol (PG) bodies are three dimensional synthetic and semi-synthetic compositions having a pronounced anti-inflammatory effect when administered in effective amounts in vivo. These bodies are believed to interact with the mammalian immune system apparently by binding to at least one receptor, which initiates an anti-inflammatory immune response. This receptor is termed the "PG receptor".

While administration of pharmaceutically acceptable PG bodies to a mammal is an effective method to therapeutically treat or prophylactically inhibit inflammation, the identification of other agonists and antagonists to the PG receptor would be beneficial in order to permit modulation of the PG receptor activity by the attending clinician.

SUMMARY OF THE INVENTION

This invention is directed, from one aspect, to methods for screening putative ligands for the PG receptor in order to ascertain which ligands exhibit agonist and antagonist properties thereto, and to such ligands themselves. The putative ligands tested can be a single compound or libraries of compounds which may be generated or obtained by any means including, by way of example, combinatorial chemistry techniques or from fermentation broths, plant extracts, cellular extracts and the like. Additional advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.

In one of its composition aspects, this invention is directed to PG receptor agonist and antagonists having a binding affinity to the PG receptor of at least 30% as compared to the binding affinity of phosphatidylglycerol to said receptor and modulates the anti-inflammatory response in a mammal.

In another of its composition aspects, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount Of a PG receptor agonist or antagonists so as to modulate an anti-inflammatory response in a mammal wherein said agonist or antagonist has a binding affinity to the PG receptor of at least 30% as compared to the binding affinity of phosphatidylglycerol to said receptor.

Preferably, ligands for the PG receptor have a binding affinity of at least about 70% relative to the binding affinity of phosphatidylglycerol. Most preferably, ligands for the PG receptor have a binding affinity of at least about 100% relative to the binding affinity of phosphatidylglycerol.

In one embodiment, the methods of this invention employ competitive assay techniques to screen compounds that exhibit for agonist/antagonist properties of interest, h an alternative embodiment, the methods of this invention employ frontal affinity chromatography (FC) to screen a compound or a library of compounds to rank those members of the library that bind to the PG receptor in a manner that an anti-inflammatory response is modulated in vivo.

In a competitive assay, an excess of ligand (phosphatidylglycerol) in labelled form and putative ligand(s) is combined with the cells carrying the PG receptor (e.g., macrophages, antigen presenting cells, etc.) in a suitable aqueous solution and the mixture is incubated under conditions which effect binding to the receptor. By using a fixed and constant concentration of labelled PG and a fixed and constant concentration of PG receptor, or a constant number of cells expressing such receptor and varying the concentration of unlabelled PG or the putative ligand , a series of inhibition curves can be obtained from which the relative binding affinity of the putative ligand compared to PG may be derived using methods known in the art, for example Scathard Analysis (Robb, RJ et al, (1981) J. Exp Med Novl; 154(5): 1455-74). The binding activity of the putative ligand relative to phosphatidylglycerol dictates the level of putative ligand bound to the receptor.

i frontal affinity chromatography, the PG receptor or cells carrying the PG receptor is typically immobilized on a suitable solid support material and packed in a column. In this embodiment phosphatidylglycerol is labeled to allow for constant detection. A mixture containing putative ligands and phosphatidylglycerol is then continuously infused through the column. Ligands having an affinity for the PG receptor bind to the column, but eventually the capacity of the column for each ligand is exceeded and the ligands elute or Dbreak throughQ at their infusion concentration. Once a ligand begins eluting from the column, it is continually present in the effluent. Compounds having little or no affinity for the PG receptor break through earlier in the effluent compared to phosphatidylglycerol.

Accordingly, in one of its method aspects, this invention is directed to a method for determining the binding affinity of a putative ligand for the PG receptor which method comprises:

(a) selecting a putative ligand or mixture of putative ligands for the PG receptor;

(b) contacting said putative ligand or mixture of said putative ligands with the PG receptor under conditions wherein the binding affinity of one or more of said putative ligands to the PG receptor can be determined; and

(c) measuring the binding affinity of said putative ligand(s) .

In a preferred embodiment, the methods of this invention employ phosphatidylglycerol as an internal standard and the binding affinity of the putative ligand is measured relative to phosphatidylglycerol, wherein said binding modulates the anti-inflammatory response. In a particularly preferred embodiment, ligands for the PG receptor are identified by having a binding affinity of about 30% relative to the binding affinity of phosphatidylglycerol to the PG receptor. Still more preferably, ligands for the PG receptor are identified by having a binding affinity of at least about 70% relative to the binding affinity of phosphatidylglycerol to the PG receptor, wherein said binding of said ligand modulates the anti-inflammatory response. Most preferably, ligands for the PG receptor are identified by having a binding affinity of at least about 100% relative to the binding affinity of phosphatidylglycerol to the PG receptor.

Ligands, having at least 30% of the binding affinity of phosphatidylglycerol to the PG receptor, correspond to compounds which have either agonist or antagonist properties when administered in vivo. In one embodiment, such ligands stimulate the receptor to produce an anti-inflammatory response when administered in vivo and, accordingly, are PG receptor agonists. In another embodiment, the ligand binds to the PG receptor but either reduces or prevents an anti-inflammatory response when the receptor is exposed to phosphatidylglycerol. In such cases, the ligand exhibits antagonist behavior and thus is an antagonist.

Another embodiment of the present invention relates to a method to reduce the production of inflammatory cytokines by cells in a subject. This method includes the step of contacting the cells that express a phosphatidylglycerol receptor with an agonist of a phosphatidylglycerol receptor, wherein the agonist increases the activity of a phosphatidylglycerol receptor.

Yet another embodiment of the present invention relates to a method to promote survival of a transplanted cell or graft, comprising administering to a transplant recipient an agonist of a phosphatidylglycerol receptor, wherein the agonist increases the activity of a phosphatidylglycerol receptor.

Another embodiment of the present invention is a method for treating a T-cell function-mediated disorder, comprising administering to a mammalian patient an effective amount of pharmaceutically acceptable agonist of a phosphatidylglycerol receptor wherein the agonist increases the activity of the phosphatidylglycerol receptor to inhibit and/or reduce the progression of the T-cell function-mediated disorder.

Another embodiment of the present invention is a method for treating an inflammatory disorder, comprising administering to a mammalian patient an effective amount of pharmaceutically acceptable agonist of a phosphatidylglycerol receptor wherein the agonist increases the activity of the phosphatidylglycerol receptor to inhibit and/or reduce the progression of the inflammatory disorder.

Another embodiment of the present invention is a method for treating an endothelial function disorder, comprising administering to a mammalian patient an effective amount of pharmaceutically acceptable agonist of a phosphatidylglycerol receptor wherein the agonist increases the activity of the phosphatidylglycerol receptor to inhibit and/or reduce the progression of the endothelial function disorder.

Another embodiment of the present invention is a method for treating an immune disorder, comprising administering to a mammalian patient an effective amount of pharmaceutically acceptable agonist of a phosphatidylglycerol receptor wherein the agonist increases the activity of the phosphatidylglycerol receptor to inhibit and/or reduce the progression of the immune disorder.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is relates to phosphatidylglycerol (PG) agonists and antagonists, pharmaceutical compositions comprising PG agonists and antagonists as well as methods for ascertaining agonist/antagonist properties of putative ligands for the PG receptor. When describing the compositions and methods of this invention, the following terms have the following meanings, unless otherwise indicated. All terms not defined herein have their conventional art-recognized meaning.

Definitions

Before the present compounds, compositions, and methods are disclosed and described, it is to understood that, unless otherwise indicated, this invention is not limited to specific support structures, reagents, methods of preparation, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes one or more supports and the like.

The term "PG receptor" or "phosphatidylglycerol receptor" refers to a protein or peptide located on a cell involved in an anti-inflammatory response, such as on a macrophage, an endothelial cell or a nerve cell, which interacts with phosphatidylglycerol in a manner that an anti-inflammatory response is initiated. The term "PG" or "phosphatidylglycerol" refers to phospholipids preferably carrying a phosphate-glycerol group with a wide range of two fatty acid chains. Preferably, such PG groups are represented by the formula:

where R and R¹ are independently selected from C_I0 - C₂₄ hydrocarbon chains, saturated or unsaturated, straight chain or containing a limited amount of branching. Preferably when employed in an aqueous solution, phosphatidylglycerol forms a liposome, wherein the lipid chains R and R¹ form the structural component of the liposomes, rather than the active component. Accordingly, these can be varied to include two or one such lipid chains, the same or different, provided they fulfill the structural function. Preferably, the lipid chains may be saturated, mono-unsaturated or polyunsaturated, straight-chain or with a limited amount of branching. Laurate (C12), myristate (C14), palmitate (C16), stearate (C18), aracmdate (C20), behenate (C22) and lignocerate (C24) are examples of useful saturated lipid chains for the PG for use in the present invention. Palmitoleate (C16), oleate (C18) are examples of suitable mono-unsaturated lipid chains. Linoleate (C18), linolenate (C18) and arichidonate (C20) are examples of suitable poly-unsaturated lipid chains for use in PG in the liposomes of the present invention. Phospholipids with a single such lipid chain, also useful in the present invention, are known as lysophospholipids. The present invention also extends to cover use of liposomes in which the active component is the dimeric form of PG, namely cardiolipin, but other dimers of Formula I are also suitable. Preferably, such dimers are not synthetically cross-linked with a synthetic cross-linking agent, such as maleimide but rather are cross-linked by removal of a glycerol unit as described by Lehniger, Biochemistry, p. 525 (1970) and depicted in the reaction below:

-co- -CH, CH₂ 0- -CO-

-co- -CH O 0 CH O- -CO-

C IH₂ O I PI 0'CH₂CH(0H)CH₂0 — P I -CH, o- σ

cardiolipiπ

+

HOCH₂CH(OH)CH₂OH

where each R and R¹ are independently as defined above.

What is meant by "agonist" of a PG receptor is any compound (ligand) which is capable of specifically associating with (e.g. binding to) a receptor in a manner that the biological activity of the receptor is stimulated or increased. The term "PG receptor agonist compound" or "PG receptor agonist" refers to any compound, including, but not limited to, an antibody that specifically binds to and activates or increases the activity of the PG receptor. Agonists of PG receptors of the present invention are particularly useful in methods for regulating' inflammation, and particularly, in methods for reducing inflammation and treating diseases or conditions in which it is desirable to reduce inflammation (e. g., transplant rejection, autoimmune disease), and particularly, to reduce the production of pro-inflammatory cytokines. Agonists of the PG receptor of this invention do not include PG liposomes as described above.

The phrase "PG receptor antagonist compound" or "PG receptor antagonist" refers to any compound (ligand) that is capable of specifically associating with (e.g., binding to) a PG receptor in a manner that the biological activity of the receptor is decreased (e.g., reduced, inhibited, blocked).

The terms "polypeptide," "protein" and "peptide" are used interchangeably and to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues of a corresponding naturally-occurring amino acid.

The term "break through time" refers to the period of time between elution of the void volume and the front corresponding to the elution of a particular compound during frontal chromatography.

The term "compound library" refers to a mixture or collection of one or more putative ligands generated or obtained in any manner. Preferably, the library contains more than one putative ligand or member.

The term "effluent" refers to the solvent or solution emerging or exiting from the frontal chromatography column.

The term "frontal chromatography conditions" refers to chromatography conditions in which a solution of putative ligands is applied or infused continuously at constant concentration through a column containing a target receptor such that the target receptor is continuously contacted with the putative ligands during the chromatography.

The term "indicator compound" refers to a. compound having a known affinity or specificity for the target receptor and a measurable break through time under frontal chromatography conditions. In the present invention, indicator compound would be phosphatidylglycerol and modified versions of phosphate-glycerol group that bind to the PG receptor. The indicator compound is labeled, i.e., a fluorescent label, to allow for constant detection.

The term "ligand" refers to a molecule or group of molecules that specifically binds to one or more specific sites of the PG receptor in a manner that modulates the anti-inflammatory response.

The term "putative ligand" refers to a ligand whose affinity or specificity for a target receptor, if any, has not been determined. A putative ligand may either be a PG antagonist or agonist, dependent on how the anti-inflammatory response is modulated. The term "binding affinity" as used herein is meant to mean that when there is a binding affinity, said binding modulates the anti-inflammatory response. It is postulated that when the PG receptor specifically binds to PG, it is specifically bound to one or more sites. Further, said binding allows for identification of the receptor and thus begins the cascade of anti-inflammatory response. Binding may be measured by any standard assay, either cell-based or non-cell-based (e.g., an immunoassay). Effective conditions of a cell-based assay include, but are not limited to, appropriate media, temperature, pH and oxygen conditions that permit cell growth.

The term "solid support" or "solid phase support" refers to an inert material or molecule to which a target receptor may be bound or coupled, either directly or through a linking arm.

The term "immobilized" is meant to mean entrapped or bound to the support.

The term "PG receptor active site" refers to the binding site of interest on a particular PG receptor.

The methods of this invention include competitive assay and frontal affinity chromatography. The compounds and libraries of compounds employed in this invention may be prepared or obtained by any means including, but not limited to, combinatorial chemistry techniques, fermentation methods, plant and cellular extraction procedures and the like. Methods for making combinatorial libraries are well-known in the art. See, for example, E. R. Felder, Chimia 1994, 48, 512-541; Gallop et al., J. Med. Chem. 1994, 37, 1233-1251; R. A. Houghten, Trends Genet. 1993, 9, 235-239; Houghten et al, Nature 1991, 354, 84-86; Lam et al, Nature 1991, 354, 82-84; Carell et al, Chem. Biol. 1995, 3, 171-183; Madden et al, Perspectives in Drug Discovery and Design 2, 269-282; Cwirla et al., Biochemistry 1990, 87, 6378-6382; Brenner et al., Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383; Gordon et al., J. Med. Chem. 1994, 37, 1385-1401; Lebl et al, Biopolymers 1995, 37 177-198; and references cited therein. Each of these references is incorporated herein by reference in its entirety.

Any type of molecule that is capable of binding to the PG receptor may be used herein. For example, compound libraries screened using this invention may contain naturally-occurring molecules, such as carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, polynucleotides, including DNA and DNA fragments, RNA and RNA fragments and the like, lipids, phospholipids, retinoids, steroids, glycopeptides, glycoproteins, proteoglycans and the like; or analogs or derivatives of naturally-occurring molecules, such as peptidomimetics and the like; and non-naturally occurring molecules, such as polymeric compounds including polymer of ethylene glycol such as polyethylene glycol PEG, such as "small molecule" organic compounds generated, for example, using combinatorial chemistry techniques; and mixtures thereof. The term "small molecule organic compound" refers to organic compounds generally having a molecular weight less than about 1000, preferably less than about 500. The molecule of interest may be attached to synthetic, semi-synthetic or natural three-dimensional particles in a way so as to maximize its interaction with the PG receptor.

The compound libraries employed in this invention will preferably contain a plurality of putative ligands. The compound library will preferably contain less than about 50,000 putative ligands, more preferably, the compound library will contain less than about 10,000 putative ligands. More preferably, the compound library will contain less than about 10,000 putative ligands; even more preferably, from 1 to about 1,000 putative ligands; and still more preferably, from about 5 to about 100 putative ligands.

It is understood that the methods, while compatible with libraries of two or more putative ligands, are not limited to use thereof, but could be practiced using a single putative ligand.

When employing the methods of this invention, the PG receptor is optionally bound or coupled to a solid support. However, in some cases, such as when whole cells or organisms are employed, the cells or organisms may be contained within the column by using, for example, a porous frit at the outflow end of the column. Supports for receptors are well- known in the art and many are commercially available. Any such conventional support may be used in this invention. Representative supports include, by way of illustration, resin beads, glass beads, silica chips and capillaries, agarose, and the like. When silica capillaries are used as the solid support, the PG receptor is bound directly to the walls of the column.

The PG receptor, or cells carrying the PG receptor, can be bound or coupled to the support using any art-recognized procedure. For example, the PG receptor can be bound using direct immobilization techniques (i.e., covalent binding via a sulfhydryl, amino or carboxyl group and the like), covalent binding through a linking or spacer arm, biotin-avidin binding, biotin- streptavidin binding, antibody binding, GST-glutathione binding, ion exchange absorption, hydrophobic interaction, expression of the PG receptor as a recombinant protein fused to maltose binding protein, fusion of the PG receptor with a peptide which binds selectively to an affinity column, and the like. Such methods are well-known in the art and kits for practicing many of these methods are commercially available. See, for example, Stammers et al.., FEBSLett. 1991, 283, 298-302; Herman et al.., Anal. Biochemistry 1986, 156, 48; Smith et al., FEBSLett. 1987, 215, 305; Kilmartin et al, J.Cell. Biol.1982, 93, 576-582; Skinner et al, J. Biol. Chem. 1991, 266, 14163-14166; Hopp et al, Bio/Technology 1988, 6, 1204-1210; H.M. Sassenfeld, TIBTECH 1990, 8, 88-93; Hanke et al., J General Virology 1992, 73, 654-660; Ellison et al, J. Biol. Chem. 1991, 267, 21150-21157; U. K. Pati, Gene 1992, 114, 285-288; Wadzinski et al., J. Biol Chem. 1992, 267, 16883-16888; Field et al, Mol. Cell. Biol. 1988, 8, 2159-2165; Gerard et al., Biochemistry 1990, 29, 9274-9281; Ausselbergs et al, Fibrinolysis 1993, 7, 1-13; Hopp et al., Biotechnology 1988, 6, 1205-1210; Blanar et al., Science 1992, 256, 1014-1018; Lin et al., J. Org. Chem. 1991, 56, 6850-6856; Zastrow et al, J. Biol. Chem. 1992, 267, 3530-3538; Goldstein et al, EMBO Jml. 1992, 11, 0000-0000; Lim et al., J Infectious Disease 1990, 162, 1263-1269; Goldstein et al., Virology 1992, 190, 889-893; and the articles mlBIFLAG Epitope Vol. 1: No. 1, Sept. 1992; and references cited therein. Each of these references is incorporated herein by reference in its entirety.

Competitive Assay

The relative binding activity of putative ligands compared to phosphatidylglycerol may be computed using a competitive assay. When employing a competitive assay in the present invention, the PG receptor, or cells carrying the PG receptor, may be immobilized either on a solid substrate such as a column or on a surface of a microtiter plate or used in an insoluble form as intact cells or cell membrane preparations. A label (e.g. a fluorescent label or radiolabel) is attached to the phosphatidylglycerol. In a competitive assay, an excess of ligand (phosphatidylglycerol) in labelled form and putative ligand(s) is combined with the cells carrying the PG receptor (e.g., macrophages, antigen presenting cells, etc.) in a suitable aqueous solution and the mixture is incubated under conditions which effect binding to the receptor. By using a fixed and constant concentration of labelled PG and a fixed and constant concentration of PG receptor, or a constant number of cells expressing such receptor and varying the concentration of unlabelled PG or the putative ligand , a series of inhibition curves can be obtained from which the relative binding affinity of the putative ligand compared to PG may be derived using methods known in the art. Phosphatidylglycerol and a compound that acts either as an agonist or antagonist is applied to the column in excess compared to the amount of PG receptor that is immobilized in the column. Any such conventional support may be used in this invention. Representative supports include, by way of illustration, resin beads, glass beads, silica chips and capillaries, agarose, and the like. When silica capillaries are used as the solid support, the target receptor is bound directly to the walls of the column.

The column is then allowed to incubate under sufficient binding conditions. After incubation, the surface of the column is then washed with a suitable solvent in order to wash away the unbound putative ligands and phosphatidylglycerol. Detection of the amount of bound PG compared to bound agonist/antagonist is completed by a measurement of the labeled PG.

In an alternative embodiment the competitive assay system is preconditioned to block the phosphatidylserine receptor that may be present with the PG receptor.

It will be appreciated in the art that the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the ligands or PG used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, radiographic, biochemical, immunochemical, electrical, optical or chemical means.

The label may be coupled directly or indirectly to the PG of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the PG. The ligand then binds to another molecule's (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. PG can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3- dihydrophthalazinediones, e.g., luminol.

Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting . the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultiphers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple calorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

Frontal Affinity Chromatography

Ranking of binding affinity of the putative ligands to receptor is determined by frontal affinity chromatography (FC). In an alternative embodiment, a mass spectrometer (MS) is utilized in conjunction with FC. Such methods are particularly amenable to libraries of putative ligands. FC-MS is known in the art and described by U.S. Patent 6,355,163, which is incorporated by reference in its entirety.

In a preferred embodiment, the PG receptor or cells carrying the PG receptor is bound or coupled to the solid support using biotin-avidin, biotin-streptavidin or a related-type binding. Procedures for biotinylating biomolecules are well-known in the art and various biotin reagents are commercially available. In this procedure, the PG receptor is typically biotinylated with a biotin reagent containing a spacer arm. The biotinylated PG receptor is then contacted with an avidin-containing solid support. The resulting biotin-avidin complex binds the PG receptor to the solid support. The PG receptor may be bound or coupled to the solid support either prior to or after introducing the solid support material into a column. For example, the biotinylated PG receptor may be contacted or incubated with the avidin- or srreptavidin-containing solid support and the resulting solid support containing the PG receptor subsequently introduced into a column. Alternatively, the avidin- or streptavidin-containing solid support can be first introduced into the column and the biotinylated PG receptor then cycled through the column to form the solid support containing the PG receptor in the column. Either of these methods may also be used with any of the other previously mentioned procedures for coupling the PG receptor to the solid support.

The solid support material may be introduced into the column using any conventional procedure. Typically, the solid support is slurried in a suitable diluent and the resulting slurry is pressure packed or pumped into the column. Suitable diluents include, by way of example, buffers such as phosphate buffered saline (PBS) solutions, preferably containing a preservative such as sodium azide, and the like.

Typically, the column employed in this invention will have an internal diameter (i.d.) ranging from about 10 μm to about 4.6 mm. Preferably, the internal diameter of the column will be in the range of from about 100 μm to about 250 μm. The column will typically range in length from about 1 cm to about 30 cm, preferably from about 2 cm to about 20 cm. Preferably, the column will have from about 1 pmol to about 10 nmol of PG receptor active sites per column; more preferably, from about 10 pmol to about 250 pmol of PG receptor active sites per column.

The body of the column employed in this invention may be comprised of any conventional column body material including, by way of illustration, poly(ether ether ketone) (PEEK), fused silica, silicon microchips, stainless steel, nylon, polyethylene, polytetrafluoroethylene (Teflon) and the like. Preferably, the column body is comprised of poly(ether ether ketone).

After the solid support containing the PG receptor is introduced or formed in the column, the column is typically flushed with a suitable diluent to remove any unbound PG receptor or impurities. Suitable diluents for flushing the column include, for example, phosphate buffered saline, TRIS buffers and the like. If desired, a detergent may also be included in the buffer to facilitate removal of unbound PG receptor or impurities. After the column is flushed, the column is typically equilibrated with a buffer suitable for frontal chromatography and compatible with mass spectrometry. Volatile buffers are generally preferred for use with mass spectrometry. For frontal chromatography, a buffer is typically selected to promote receptor-ligand interaction. Suitable buffers for use in FC-MS include, byway of example, ammonium acetate, ammonium formate and the like.

In the methods of this invention, phosphatidylglycerol is added to the column at the same time as the compound library. Further, phosphatidylglycerol is labeled in a manner as described above to allow for constant detection.

Following equilibration of the column, the compound library is then continuously applied to the column under frontal chromatography conditions. Typically, when applied to the column, the compound library comprises a solution of the library members or putative ligands in a suitable diluent. Typically, the diluent is the buffer solution used to equilibrate the column. Generally, the concentration of the library members in the diluent will range from about 0.01 μM to about 50 μM. Preferably, the concentration of library members ranges from about 0.1 μM to about 10 μM.

Procedures for conducting frontal chromatography are well-known in the art. See, for example, K.-I. Kasai et al., Journal of Chromatography 1986, 376, 3-A1; D. S. Hage et al., Journal of Chromatography B, 1997, 669, 449-525 and references cited therein. The disclosures of these references are incorporated herein by reference in their entirety. Typically, the compound library is continuously applied or infused into the column containing the PG receptor. Under these conditions, the PG receptor is continuously contacted or challenged with each of the members of the compound library. The column is driven to dynamic equilibrium by continuously applying the compound library to the column. Library members having different binding constants to the PG receptor display different break through times or hold-up volumes on the column, i.e., those members having a higher affinity for the target ligand have a longer break through time on the column or a larger holdup volume until they begin to elute from or break-though the column at their initial infusion concentration. Unlike zonal chromatographic methods, no physical separation'of the library members is achieved using frontal chromatography. Any putative ligands that break-through prior to the labeled phosphatidylglycerol have little or no binding affinity. During the frontal chromatography, the column is typically at a temperature in range from about 0°C to about 90°C; preferably from about 4°C to about 60°C; more preferably from about 20°C to about 40°C.

When a ligand has a very high affinity for the PG receptor, it may be desirable to pre-

_* equilibrate the column with the compound library before conducting the FC-MS analysis. The column can be pre-equilibrated by either by infusing the compound library through the column for a period sufficient to allow the column to reach equilibrium, i.e., for about 0.25 to 24 hours, or by infusing the compound library into the column, stopping the flow, and allowing the system to come to equilibrium for a period of up to one day before conducting the analysis. If desired, a sequence of stop-flow cycles may also be conducted.

Preferably, a mass spectrometer is coupled to the column to analyze the effluent. Mass spectrometry is particularly useful in the present invention since it allows for both detection and identification of the library members present in the effluent. In this regard, mass spectrometry allows the eluting members of the library to be identified based on their mass/charge ratio.

Prior to analyzing the effluent from the column by mass spectrometry, the effluent is optionally diluted with a supplemental diluent or "make-up flow" and the combined flow is directed into, for example, the electrospray mass spectrometer.. Typically, the supplemental diluent comprises a major amount of an organic solvent and a minor amount of an aqueous buffer. The organic solvent is selected so as to promote a stable and efficient electrospray. Representative organic solvents suitable for use in the supplemental diluent include, by way of example, acetonitrile, methanol, isopropanol and the like. A preferred organic solvent is acetonitrile. Typically, the amount of supplemental diluent employed is adjusted so that the combined flow rate of the effluent and the supplemental diluent is less than about 100 μL/min. Preferably, the combined flow rate entering the mass spectrometer ranges from about 100 nL/min to about 20 μL/min.

Methods for analyzing effluents using mass spectrometry are well-known in the art. Any type of mass spectrometry which is capable of directly or indirectly analyzing the components present in a solution may be employed in this invention including, for example, electrospray mass spectrometry (ES-MS), atmospheric pressure chemical ionization (APCI), membrane introduction mass spectrometry (MLMS), continuous flow fast atom bombardment (cf-FAB), thermospray techniques, particle beam, moving belt interfaces and the like. Electrospray mass spectrometry is particularly preferred. Apparatus and techniques for conducting electrospray mass spectrometric analysis are described, for example, in S. J. Gaskell, '''Electrospray: Principles and Practice" , J. Mass. Spectrom. 1997, 32, 677-688 and reference cited therein. The mass spectrometer may be of any type (i.e., scanning or dynamic) including, byway of illustration, quadrupole, time of flight, ion trap, FTICR and the like.

Typically, the mass spectrometer parameters are set to provide the highest sensitivity for the eluting compounds. Generally, when an electrospray mass spectrometer is employed, such adjustments will involve optimization of, for example, nebulizer pressure, drying gas flow rate, ion transmission and electrospray needle position. For example, the nebulizer pressure will typically range from about 0 psi to about 60 psi; and the drying gas flow rate will range from about 0 L/min to about 50 L/min. A total ion chromatogram is typically measured and monitored in real-time. The size of the column, the concentration of the compound library and the flow rate will generally determine the run-time. Typical run times range from about 1 min to about 60 min.

Upon completion of the frontal chromatography, the column is typically regenerated by washing with a large volume of the binding buffer, with or without a competitive ligand. In this regard, a particular advantage of the present method is that denaturing of the PG receptor is not required at any point in the procedure. Accordingly, columns may be re-used many times generally with no observable loss of activity or leaching of the PG receptor.

The break through time for the phosphatidylglycerol is first determined by applying the phosphatidylglycerol to the column containing the PG receptor under frontal chromatography conditions. The column is then typically equilibrated with the compound library to be screened. Generally, the compound library is applied or infused into the column for a time sufficient to allow all of the library members to break through the column. In some cases, such as when very strong binding ligands are present, not all members of the library will achieve equilibrium. The effluent during this period may be presented to the mass spectrometer for analysis or may be collected for recycling or disposal. Once the column has been equilibrated (or partially equilibrated) with the compound library, a mixture comprising the compound library and the phosphatidylglycerol is applied to or infused into the column using the frontal chromatography procedures described herein. Preferably, the phosphatidylglycerol will be present in the mixture in an amount ranging from about 1 nM to about 10 μM, more preferably from about 10 nM to about 100 nM. The effluent from the column is analyzed to determine the break through time for the phosphatidylglycerol on the compound library-equilibrated column and this time period is compared to the predetermined break through time for the phosphatidylglycerol to ascertain whether the compound library has a higher affinity for the PG receptor relative to the phosphatidylglycerol_.

Alternatively, the phosphatidylglycerol alone can be applied or infused into the column after equilibration of the column with the compound library. This technique would allow very strongly bound ligands or those with slow off rates to be detected.

In addition to detecting the phosphatidylglycerol using mass spectrometry, it is also contemplated that other methods of detection may be employed. For example, an indicator compound can be detected in the effluent from the column using, by way of example, fluorescence, infra-red absorption, UV- visible absorption, nuclear magnetic resonance (NMR), atomic spectroscopy (i.e., AAS, ICP-OES, etc.), flow cytometry and the like.

It will be appreciated by one of skill in the art that multiple columns may be employed.

Methods of Treatment

Phosphatidylglycerol (PG) agonists of the present invention are indicated for use in prophylaxis and/or treatment of a wide variety of mammalian disorders where T-cell function, inflammation, endothelial dysfunction and inappropriate cytokine expression are involved. A patient having or suspected of having such a disorder may be selected for treatment. DTreatmentD refers to a reduction of symptoms, such as, but not limited to, a decrease in the severity or number of symptoms of the particular disease or a limit on the further progression of symptoms.

With respect to T-cell function (T-cell mediated) disorders, these disorders include any and all disorders mediated at least in part by T-cells and include for example, ulcers, wounds, and autoimmune disorders including, but not limited to diabetes, scleroderma, psoriasis and rheumatoid arthritis.

The PG agonists of the invention are indicated for use with inflammatory allergic reactions, organ and cell transplantation reaction disorders, and microbial infections giving rise to inflammatory reactions. It is also indicated for use in prophylaxis against oxidative stress and/or ischemia reperfusion injury, ingestion of poisons, exposure to toxic chemicals, radiation damage, and exposure to airborne and water-borne irritant substances, etc., which cause damaging inflammation. It is also indicated for inflammatory, allergic and T-cell- mediated disorders of internal organs such as kidney, liver, heart, etc.

With respect to disorders involving inappropriate cytokine expression for which the present invention is indicated, these include any and all disorders involving inappropriate cytokine expression and include, for example, neurodegenerative diseases. Neurodegenerative diseases, including Down's syndrome, Alzheimer's disease and Parkinson's disease, are associated with increased levels of certain cytokines, including interleukin-1 (IL-1) (see Griffin WST et al. (1989); Mogi M. et al. (1996)). It has also been shown that IL-la inhibits long-term potentiation in the hippocampus (Murray, C. A. et al. (1998)). Long-term potentiation in the hippocampus is a form of synaptic plasticity and is generally considered to be an appropriate model for memory and learning (Bliss, T.V.P. et al. (1993)). Thus, inappropriate cytokine expression in the brain is currently believed to be involved in the development and progression of neurodegenerative diseases and neuroinflammatory disorders.

Thus, the PG agonists of the invention are indicated for the treatment and prophylaxis of a wide variety of mammalian neurodegenerative and other neurological disorders, including Downs syndrome, Alzheimer's disease, Parkinson's disease, senile dementia, depression, Huntingdon's disease, peripheral neuropathies, Guillain Barr syndrome, spinal cord diseases, neuropathic joint diseases, chronic inflammatory demyelinating disease, neuropathies including mononeuropathy, polyneuropathy, symmetrical distal sensory neuropathy, neuromuscular junction disorders, myasthenias and amyotrophic lateral sclerosis (ALS). Treatment and prophylaxis of these neurodegenerative diseases represents a particularly preferred embodiment of the invention, with treatment of AlzheimerOs disease, ParkinsonDs disease and ALS particularly preferred.

Regarding disorders involving endothelial dysfunction, the PG agonists of the present invention are indicated for the treatment and prophylaxis of a wide variety of such mammalian disorders including, any and all disorders mediated at least in part by endothelial dysfunction and include, for example, cardiovascular diseases, such as atherosclerosis, peripheral arterial or arterial occlusive disease, congestive heart failure, cerebrovascular disease (stroke), myocardial infarction, angina, hypertension, etc., vasospastic disorders such as Raynaud's disease, cardiac syndrome X, migraine etc., and the damage resulting from ischemia (ischemic injury or ischemia-reperfusion injury). In summary, it can be substantially any disorder the pathology of which involves an inappropriately functioning endothelium.

Further indications for the compositions and processes of the present invention include the treatment of patients to accelerate their rate of wound healing and ulcer healing, and treatment of patients prior to surgical operations, to accelerate their rate of recovery from surgery including their rate of healing of surgical wounds and incisions.

The following examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this invention. Unless otherwise stated, all temperatures are in degrees Celsius.

EXAMPLES

In the examples below, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.

B, dynamic binding capacity C = degrees Celsius cm = centimeter eq. equivalents

FAB fast atom bombardment

FC frontal chromatography

g grams κ_d dissociation constant

L liter

MALDI = matrix-assisted laser desorption/ionization meq. = milliequivalent mg milligram mL = milliliter mM millimolar mmol = millimole

MS = mass spectrometry m/z = mass charge ratio

N = normal

PBS = phosphate buffered saline

PEEK = poly(ether ether ketone) pmol = picomole

TIC = total ion chromatogram

μ = micrograms μL = microliter μm = micrometer μM= micromolar

Vo = void volume

Example 1

Screening of an Mono-chained PG Library Using FC-MS

hi this example, a compound library containing a mixture of six putative ligands are screened using frontal chromatography in combination with a mass spectrometer to determine the relative affinity of the putative ligands for the PG receptor.

The PG receptor (0.5 mg) is biotinylated with a biotin reagent containing a long-chain spacer arm (NHS-LC-biotin, Pierce). The extent of biotin incorporation is monitored by matrix- assisted laser desorption/ionization and the reaction is terminated at 14 biotins/PG receptor (average). The biotinylated receptor is then coupled to a beaded support by incubating the receptor with 25 μL of Ultralmk immobilized avidin (Pierce, Cat. No. 53119) in bicarbonate buffer (pH 8.5) for 1 hour. The beads are then thoroughly washed with the bicarbonate buffer. A UV quantitation indicates an immobilization of -45 μg receptor/25 μL beads is achieved. The beads are then slurry-packed into a 500 μm i.d. by 11.5 cm poly(ether ether ketone) (PEEK) column body (-23 μL column volume). In this experiment, a mixing tee serves a dual role as a column end-fitting and mixing chamber for the column eluent and organic make-up flow. The column is then directly connected to an electrospray mass spectrometer (Hewlett-Packard series 1100 MSD, single quadrupole).

For operation in frontal chromatography mode, the column is first flushed with ammonium acetate buffer (NH₄OAc, 2 mM, pH 6.7). After flushing, the flow is switched to a second solution containing a mixture of the six putative ligand in ammonium acetate buffer, each present at 1 μM. All solutions are infused concurrently with a multi-syringe pump (PHD 200, Harvard Apparatus) at a flow rate of 8 μL/min/syringe (1 cc syringes). A Rheodyne valve (Model 9725) is used for flow switching. The column effluent combined with the make-up flow (10% 2 mM NH OAc buffer in acetonitrile) in the tee are to provide a flow rate of 16 μL/min into the mass spectrometer.

For the analysis of this mixture, the spectrometer is scanned from m/z 100-1500. Data is collected in scan mode with positive ion detection. A total ion chromatogram (TIC) is constructed from a 50 minute run time. This represents the consumption of only 400 pmol of each putative ligand. Peaks at specific m/z values are then identified through the analysis of the mass spectra giving rise to the TIC and selected ion chromatograms for all six compounds are reconstructed from the TIC.

Putative ligands having no affinity for the PG receptor break through at the void volume (Vo), while compounds having an affinity for the PG receptor break through later, at volumes relating to their concentrations and Ka values.

Example 2

Screening of a Putative Ligand Library Using FC-MS and an Indicator Compound

In this example, the use of an indicator compound to screen a compound library is demonstrated. The PG receptor in this example is the same as that used in Example 1. The column is also essentially the same as the column in Example 1 and it was prepared and operated as described therein; however, an appropriate amount of PG is added to the putative ligand library prior to addition to the column. The indicator compound was monitored, to determine whether the representative library contained a compound with a higher affinity for the PG receptor.

Example 3

Measure of Relative Binding Affinity Using a Competitive Assay In this example, the relative binding affinity of a putative ligand compared to PG is measured.

Macrophages comprising the PG receptor are cultivated and then the macrophages are affixed to a porous fritted glass tip encased in a column (Aldrich Chemical, Milwaukee, WI). Dipalmitoyl phosphatidylglycerol is then labeled with radioactive phosphorous-32. A fivefold excess of the labeled dipalmitoyl phosphatidylglycerol and a five-fold excess of the putative ligand is added to the column. The column is allowed to incubate under sufficient binding conditions for approximately two hours. After incubating, the cells are washed with an appropriate buffered solution. The extend of radio-labeled dipalmitoyl phosphatidylglycerol is measured with a scintillation counter to determine the amount of bound PG molecule as compared with bound putative ligand.

Example 4

Measure of Relative Binding Affinity Using a Competitive Assay in Antigen Presenting Cells

U937 is a monocytic leukemia cell line that can be differentiated into macrophages by administration of a phorbol ester. The cells are cultured by growing in RPMI medium with a 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°V, 5% CO₂. They are then seeded into 6 well and differentiated into macrophages by treating with 150nM phorbol myristate acetate (PMA) for 2-3 days. The cell media is removed after macrophages have differentiated and replaced with complete media for 24 hrs prior to commencement of experiment.

Liposomes of size 100 + 20 nanometers are prepared according to standard methods known in the art and comprise the following:

(a) 75% phosphatidylglycerol (PG) and 25% phosphatidylcholine (PC) with approximately 1% phosphatidylethanolamine (PE) conjugated to a rhodamine label incorporated therein, (b) 75 % phosphatidylglycerol (PG) and 25 % phosphatidylcholine (PC)

The stock concentration of both liposome preparations are 38.9 mM lipid or 2.89 x 10¹⁴ liposomes per ml.

Treatment Protocol

The differentiated macrophages are first treated with LPS (1 Ong/nl for 18hrs) to induce maximal expression of the PG receptor. The cells are then detached from the plate, counted and resuspended at a concentration of 1 x 10⁶ cells/ml and then allowed to reattach to a new plate. lOOμM of the fluorescently-labelled liposomes is then added to the cell with increasing concentrations of the unlabelled liposomes composition (100 μM — lOmM) to compete for binding to the PG receptor.

Due to the fact that both compounds are competing for the same receptor, once the concentration of the unlabelled PG liposome reaches a sufficient level, it will compete with the labelled compound, thus reducing the amount of labelled compound binding to the PG receptor or to cells expressing the PG receptor. The initial binding of the labelled PG liposome to the cell in the absence of unlabelled PG causes the cell to become fluorescent, and this fluorescence can be monitored by fluorimetry, flow cytometry or by other means. Increasing the concentration of the unlabelled PG liposome results in a competition for binding to the PG receptor or cell expressing the PG receptor and causes a reduction in fluorescence associated with the cell; the higher the concentration of unlabelled PG the lower the proportion of the labelled PG that binds. The binding of fluorescent PG to the receptor or cells expressing the PG receptor, measured by fluorimetry, flow cytometry or by other means, in the presence of different concentrations of unlabelled PG can be expressed graphically, yielding a binding curve, the slope of which reflects the affinity of binding of PG to the PG receptor. In a similar manner a range of putative ligands can be examined and the relative binding affinity of the putative ligand compated to the PG may then be derived using standard computational methods known in the art. The binding activity of the putative ligand relative to phosphatidylglycerol dictates the level of putative ligand bound to the receptor. Example 5

Measure of Relative Binding Affinity Using a Competitive Assay in Endothelial Cells

Human umbilical vein endothelial cells (HUVECs) are a primary cell line of endothelial cells that are isolated from umbilical vein cords as follows:

T75 flasks are prepared by coating with 0.2% gelatin (5-7mls/flask) for a minimum of 15/20 minutes or overnight. The excess is then removed. The cord is sprayed with 70% ethanol prior to procedure and any placenta still remaining attached to the cord was cut away. The cord is then cut to an approximate length of 5-6 inches. The cord has two arteries which are thick walled and one vein that is bigger and thin walled. The vein is located and the serrated edge of a stopper placed into it. Approximately 20cm of string is then used to tie the. cord onto the stopper.

The cord is then washed through with phosphate buffered saline (PBS) a number of times until the PBS runs clear. Following this 15-20mls of Collagenase solution is placed into the cord; it is wrapped in tinfoil and incubated for 15 minutes at 37°C. After incubation the tied end of the cord is cut and the collagenase drained into a 50ml tube. .: Collagenase is then passed through the cord again, the cord is massaged to loosen the endothelial cells and then PBS is passed through the cord and collected into the same tube containing the collagenase solution. This is then centrifuged at 1600RPMs, the supernatant removed and the pellet resuspended in 10-12 mis of Ml 99 complete medium. Finally the medium containing the cells is added to the gelatinised flasks.

Liposome compositions and treatment protocols used are as in Example 3.

Example 6

Measure of Relative Binding Affinity Using a Competitive Assay in Neuronal Cells

Cells from the human terotocarcinoma cell line NT2 can be differentiated into nondividing cells with moφhological and biochemical characteristics of embryonal human neurons. They develop axons, dendrites and other indices of neuronal polarity and express central nervous system neuronal marker proteins (Pleasure SJ et al (1992), J. Neurosci 12, 1802-1815).

Cells are cultured as described in Pleasure SJ. Briefly cells are plated at a density of 2.3 x 10⁶ cells per T75 flask in DMEM-high glucose, 10% FCS, 100IU/ml penicillin, 1 OOμg/streptomycin, and lOμM retinoic acid for 4 weeks to allow for them to differentiate into neurons. They are then split 1 :6 and grown in the same medium without the retinoic acid. Cells are then mechanically dislodged and replated on matrigel. Treatment at this stage with mitotic inhibitors (lμM cytosine arabinoside, lOμM flurodeoxyuridine and lOμM uridine), removes undifferentiated cells, leaving differentiated neurons.

Liposome compositions and treatment protocols used are as in Example 3.

Claims

What is Claimed is :

1. A phosphatidylglycerol receptor agonist or antagonist, other than phosphatidylglycerol itself, having a binding affinity to the phosphatidylglycerol receptor of at least 30% as compared to the binding affinity of phosphatidylglycerol to said receptor and having the ability to modulate the inflammatory - anti-inflammatory response to intramuscular injection thereof in a mammal.

2. A phosphatidylglycerol receptor agonist or antagonist according to claim 1 having a binding affinity to the phosphatidylglycerol receptor of at least 70% as compared to the binding affinity of phosphatidylglycerol to said receptor.

3. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of a phosphatidylglycerol receptor agonist or antagonist as defined in claim 1 or claim 2.

4. A method for screening putative ligands for agonist or antagonist properties of the phosphatidylglycerol receptor, wherein said method comprises performing an assay of said putative ligands to determine binding affinity for said phosphatidylglycerol receptor in comparison with binding affinity of phosphatidylglycerol itself.

5. The method according to Claim 4, wherein said assay is a competitive assay.

6. The method according to Claim 4 or claim 5 wherein said assay is frontal affinity chromatography.

7. The method according to Claim 6, wherein said method comprises:

(a) selecting a putative ligand or mixture of putative ligands for the phosphatidylglycerol receptor;

(b) contacting said putative ligand or mixture of said putative ligands with the phosphatidylglycerol receptor under conditions wherein the binding affinity of one or more of said putative ligands to the phosphatidylglycerol receptor can be determined; and

(c) measuring the binding affinity of said putative ligand(s).

8. The method according to Claim 7, wherein the method comprises the further step of using labeled phosphatidylglycerol.

9. Use in the preparation of a medicament to reduce the production of inflammatory cytokines by cells in a mammalian subject, to treat an inflammatory disorder in a mammalian subject, to treat a T-cell mediated disorder in a mammalian subject, to treat an endothelial function disorder in a mammalian subject, or to treat an immune disorder in a mammalian subject, of an agonist of a phosphatidylglycerol receptor as claimed in claim 1 or claim 2.

10. Use in the preparation of a medicament to promote survival of a transplanted cell or graft, of an agonist of a phosphatidylglycerol receptor as claimed in claim 1 or claim 2.