QUINAZOLINE DERIVATIVE USEFUL AS TOLL-LIKE RECEPTOR ANTAGONIST

FIELD OF THE INVENTION

The invention relates to generally to the field of immunology. More particularly, the invention relates to compositions and methods for altering immune function. More specifically, the invention relates to compositions and methods for reducing immune stimulation mediated through certain Toll-like receptor (TLR) molecules.

BACKGROUND OF THE INVENTION

Stimulation of the immune system, which includes stimulation of either or both innate immunity and adaptive immunity, is a complex phenomenon that can result in either protective or adverse physiologic outcomes for the host. In recent years there has been increased interest in the mechanisms underlying innate immunity, which is believed to initiate and support adaptive immunity. This interest has been fueled in part by the recent discovery of a family of highly conserved pattern recognition receptor proteins known as Toll-like receptors (TLRs) believed to be involved in innate immunity as receptors for pathogen-associated molecular patterns (PAMPs). Compositions and methods useful for modulating innate immunity are therefore of great interest, as they may affect therapeutic approaches to conditions involving autoimmunity, inflammation, allergy, asthma, graft rejection, graft-versus-host disease (GvHD), infection, cancer, and immunodeficiency.

Recently there have been a number of reports describing the immunostimulatory effect of certain types of nucleic acid molecules, including CpG nucleic acids and double-stranded RNA. Of note, it was recently reported that Toll-like receptor 9 (TLR9) recognizes bacterial DNA and CpG DNA. Hemmi H et al. (2000) Nature 408:740-5; Bauer S et al. (2001 ) Proc Natl Acad Sci U SA 98:9237-42. It was also recently reported that immune complexes containing IgG and nucleic acid can stimulate TLR9 and participate in B-cell activation in certain autoimmune diseases. Leadbetter EA et al. (2002) Nature 416:595-8.

Chloroquines have been recognized as useful not only as anti-malarial agents but also as anti-inflammatory agents. Although its mechanism of action is not well understood, chloroquine has been used effectively in the treatment of various autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). For a review, see Wallace DJ (1996) Lupus 5 Suppl 1 :S59-64. i

Recently Macfarlane and colleagues described a number of small molecule analogs and derivatives of chloroquine (4-aminoquinoline) and quinacrine (9-aminoacridine) which reportedly inhibit stimulation of the immune system. U.S. Pat. No. 6,221 ,882; U.S. Pat. No. 6,479,504; U.S. Pat. No. 6,521 ,637; PCT published application PCT/USOO/16723 (WO 00/76982); and PCT published application PCT/US98/13820 (WO 99/01154). Macfarlane and colleagues reported these small molecule inhibitors of the immune response, even when used at nanomolar concentrations, can block the action of immunostimulatory DNA. U.S. Pat. No. 6,221,882 B1. Macfarlane and coworkers studied a large number of compounds by varying substituents on a limited number of 4- aminoquinoline and 9-aminoacridine core structures related to chloroquine and quinacrine.

SUMMARY OF THE INVENTION

The invention provides a TLR antagonist, compositions including the TLR antagonist, and methods of use of the TLR antagonist and compositions of the invention. The TLR antagonist of the invention, referred to herein as CPG 52364, has a structural formula provided as formula I.

In an aspect the invention is a compound of formula I

and pharmaceutically acceptable salts thereof. MeO in Formula I represents a methoxy group, CH ₃ O-.

In an aspect the invention is a composition including a therapeutically effective amount of the compound of formula I and a pharmaceutically acceptable carrier. The composition in one embodiment includes the free base form of CPG 52364, shown as formula I. The composition in one embodiment includes a pharmaceutically acceptable salt of CPG 52364. In one embodiment the composition is formulated for oral administration.

In an aspect the invention is a method of inhibiting an immune response in a subject. The method according to this aspect includes the step of administering to the subject an effective amount of a compound of formula I

or a pharmaceutically acceptable salt thereof.

In an aspect the invention is a method of treating a subject having an autoimmune disease. The method according to this aspect includes the step of administering to the subject an effective amount of a compound of formula I

or a pharmaceutically acceptable salt thereof, to treat the autoimmune disease. In an embodiment the autoimmune disease is selected from systemic lupus erythematosus, insulin-dependent diabetes mellitus, rheumatoid arthritis, multiple sclerosis, atherosclerosis, inflammatory bowel disease, ankylosing spondylitis, autoimmune hemolytic anemia, Behcet's syndrome, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic thrombocytopenia, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polymyositis/dermatomyositis, primary biliary sclerosis, sarcoidosis, sclerosing cholangitis, Sjogren's syndrome, systemic sclerosis (scleroderma and CREST syndrome), Takayasu's arteritis, temporal arteritis, and Wegener's granulomatosis. In a

particular embodiment the autoimmune disease is systemic lupus erythematosus. In a particular embodiment the autoimmune disease is inflammatory bowel disease. In an embodiment the subject is a human.

In an aspect the invention is a method of treating a subject having or at risk of having transplant rejection. The method according to this aspect includes the step of administering to the subject an effective amount of a compound of formula I

or a pharmaceutically acceptable salt thereof, to treat the transplant rejection. In an embodiment the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures are illustrative only and are not required for enablement of the invention disclosed herein.

FIG. 1 is a bar graph depicting mean anti-dsDNA antibody levels in MRL Ipr/lpr mice treated with CPG 52364 for 12 weeks. The Y axis represents intensity of kinetoplast staining in a Crithidia luciliae assay at a serum dilution of 1 :100.

FIG. 2 is a graph depicting pharmacokinetics of CPG 52364 in blood after single dose administration to rats. Circles: 8.2 mg/kg IV. Triangles: 41.0 mg/kg oral.

FIG. 3 is a graph depicting pharmacokinetics of CPG 52364 in blood after single dose administration to cynomolgus monkeys. Circles: 8.2 mg/kg IV. Triangles: 41.0 mg/kg oral.

FIG. 4 is a graph depicting pharmacokinetics of CPG 52364 in blood after single dose oral administration (50 mg/kg) to cynomolgus monkeys.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a particular 2-aminoaryl-4-(ω-aminoalkyl)amino- quinazoline compound having a structure provided as formula I

i.e., 2-[4-(4-methyl-1-piperazinyl)phenyl]-N-[2-(4-morpholinyl)eth yl]-4-(6,7-dimethoxy- quinazolinamine), referred to herein as CPG 52364, pharmaceutically acceptable salts of CPG 52364, compositions containing CPG 52364, and methods of use of CPG 52364 as a therapeutic immunosuppressive agent. It has been discovered according to the invention that CPG 52364 is a Toll-like receptor (TLR) antagonist that selectively inhibits TLR7, TLR8, and TLR9, a subfamily of TLRs that recognize certain types of nucleic acids, notably CpG DNA and single-stranded RNA. Accordingly, CPG 52364 can be used to inhibit signaling by any one or combination of TLR7, TLR8, and TLR9. Inhibition of signaling by any one or combination of these TLRs can reduce undesirable immune activity. Accordingly, CPG 52364 can be used, either alone or in combination with other agents or treatment modalities, to reduce undesirable immune activity, for example, to treat a subject having a condition characterized by undesirable immune activity. Such conditions include, without limitation, inflammation, autoimmune diseases, acute and chronic transplant rejection, acute and chronic graft-versus-host disease (GvHD), allergy, asthma, and sepsis.

The invention in one aspect provides a compound having the structure provided in formula I. In one embodiment the invention provides CPG 52364 as the free base having the structure provided in formula I. A method of preparing CPG 52364 is disclosed in Example 1 below. CPG 52364 has a molecular weight of 492.6 Daltons and has the appearance of an off-white to tan solid. The solubilty of the free base in aqueous solution is dependent on pH and exceeds 350 mg/mL between a pH of 1.22 and 5.90. At a pH above 6.90, the solubilty in aqueous solution is less than 1.0 mg/mL. CPG 52364 is soluble in chloroform, methylene chloride, and dimethylformamide.

In one embodiment the invention provides a pharmaceutically acceptable salt of CPG 52364. The phrase "pharmaceutically acceptable salt(s)", as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the compounds of the present invention. Accordingly, in one embodiment acids that may be used to prepare pharmaceutically acceptable acid addition salts of CPG 52364 are those that form nontoxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate [, 1 ,1'-methylene- bis-(2-hydroxy-3-naphthoate)] salts. At least the following acid addition salts have been prepared: hydrochloride, sulfate, bisulfate, phosphate, tartrate, fumarate, and p- toluenesulfonate. Preparation of the bisulfate salt is described in Example 1 below.

The invention also relates to base addition salts of CPG 52364. The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of CPG 52364 are those that form non-toxic base salts , i.e., salts containing pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium, magnesium, and lithium). This invention also encompasses pharmaceutical compositions containing prodrugs of a compound of formula I. Compounds of formula I having a free amino or amido group can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues, are covalently joined through peptide bonds to a free amino group of a compound of formula I. The amino acid residues include the 20 naturally occurring amino acids commonly designated by three-letter or single-letter symbols and also include, 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone. Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters that are covalently bonded to the above substituents of formula 1 through the carbonyl carbon prodrug sidechain.

The invention further contemplates pharmaceutically acceptable salts of prodrugs of CPG 52364.

The invention also contemplates hydrates of pharmaceutically acceptable salts of CPG 52364, hydrates of prodrugs of CPG 52364, and hydrates of pharmaceutically acceptable salts of prodrugs of CPG 52364. In various individual embodiments hydrates may include various whole integer or whole-plus-one-half integer number of water molecules (for example, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 water molecules) per molecule of CPG 52364 salt or CPG 52364 prodrug.

This invention also encompasses compounds of formula I containing protective groups. One skilled in the art will also appreciate that compounds of the invention can also be prepared with certain protecting groups that are useful for purification or storage and can be removed before administration to a patient. The protection and deprotection of functional groups is described in "Protective Groups in Organic Chemistry", edited by J. W. F. McOmie, Plenum Press (1973) and "Protective Groups in Organic Synthesis", 3rd edition, T. W. Greene and P. G. M. Wuts, Wiley-lnterscience (1999).

The compounds, salts, and prodrugs of the present invention can exist in several tautomeric forms, including the enol and imine form, and the keto and enamine form and geometric isomers and mixtures thereof. All such tautomeric forms are included within the scope of the present invention. Tautomers exist as mixtures of a tautomeric set in solution. In solid form, usually one tautomer predominates. Even though one tautomer may be described, the present invention includes all tautomers of the present compounds.

The present invention also includes atropisomers of the present invention. Atropisomers refer to compounds of formula I that can be separated into rotationally restricted isomers.

The present invention also includes isotopically-labeled compounds, which are identical to those recited in formula I, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, and oxygen, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, and ¹⁷ O, respectively. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes such as ³ H and ¹⁴ C are incorporated, are

useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., ³ H, and carbon- 14, i.e ^r .; ¹⁴ C ₁ isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., ² H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically-labeled compounds of formula I of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the Examples below, by substituting a readily available isotopically-labeled reagent for a non-isotopically-labeled reagent. The invention in one aspect provides a composition including a therapeutically effective amount of CPG 52364, either as the free base or as a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The composition of the invention can optionally further include at least one additional therapeutic agent. In one embodiment an additional therapeutic agent is another TLR antagonist. The invention further provides a method of making a pharmaceutical composition. The method includes the step of placing a therapeutically effective amount of CPG 52364, either as the free base or as a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

As used herein, "effective amount" refers to any amount that is necessary or sufficient to realize a desired biological effect. In some instances an effective amount is a therapeutically effective amount. A "therapeutically effective amount" is any amount that is necessary or sufficient to realize a desired biological effect in a subject. In one embodiment an effective amount is an amount sufficient to treat an autoimmune disease. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular agent being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular agent without necessitating undue experimentation. Multiple doses per day may be contemplated to achieve appropriate systemic levels of compounds. Appropriate systemic levels can be

determined by, for example, measurement of the patient's peak or sustained plasma level of the compound.

As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, dilutants or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency. Various formulations are contemplated by the invention, including those suitable for oral and parenteral administration. These and other formulations contemplated by the invention are described in more detail below.

As noted above, CPG 52364 is an antagonist of certain TLRs. Mammalian TLRs are orthologs of a family of highly conserved transmembrane proteins, first discovered in Drosophila, that have intracellular domains that are homologous to the receptors for interleukin (IL)-I and IL-18 (Krieg AM. (2002) Ann Rev Immunol. 20:709-760; Takeda K et al. (2005) lnt Immunol. 17(1 ):1-14). In mammals TLRs are pattern recognition receptors which recognize pathogen-associated molecular patterns (PAMPs) and act as key signaling elements in innate immunity. Eleven mammalian TLRs have been identified, of which only ten are active in humans (TLR1 to TLR10) and only nine are active in mice (TLR1 to TLR7, TLR9, and TLR11 ). Stop codons in the mouse TIr 10 gene and the human Tlr11 gene have rendered these nonfunctional (Takeda K et al. (2005) lnt Immunol. 17(1 ):1-14). Subsets of the TLRs serve different roles in the innate immune system.

TLR1 , TLR2, TLR5, and TLR6 are expressed on the cell surface and recognize microbial structural components other than nucleic acids. In contrast, TLR3, TLR7, TLR8, and TLR9 are located in intracellular compartments and recognize nucleic acids from viruses or bacteria: dsRNA by TLR3, ssRNA by TLR7 and TLR8 (except in mice, where TLR8 appears to be nonfunctional [Hemmi H et al. (2002) Nature Immunol.

3(2): 196-200; Jurk M et al. (2002) Nature Immunol. 3(6):499]), and nonmethylated CpG motifs by TLR9 (Heil F et al. (2004) Science 303(5663):1526-1529; Means TK et al. (2005) Ann NY Acad Sci. 1062:242-251 ; Subramanian S et al. (2006) Proc Natl Acad Sci USA. 103(26):9970-9975). The natural ligand for TLR10, which is not expressed in

rodents, is not known (Hasan U et al. (2005) J. Immunol. 174(5):2942-2950). After binding of nucleic acids to TLR7, TLR8, or TLR9, all of which are located in endosomes, intracellular signaling is similar to the activated IL-1 receptor and depends on recruitment of intracellular adapter and signaling molecules (Hemmi H et al. (2002) Nature Immunol. 3(2): 196-200; Leadbetter EA et al. (2002) Nature. 416(6881 ):603-607).

TLR7 and TLR8 also signal in response to certain synthetic small molecules, including the imidazoquinoline compounds R-837 (imiquimod; Aldara™ (3M Pharmaceuticals, St. Paul, MN); 1-isobutyl-1/-/-imidazo[4,5-c]quinolin-4-amine) and R-848 (resiquimod; 4-amino-D , D-dimethyl-2-ethoxymethyl-IH-imidazo[4,5-c]quinoline-1 - ethanol), as well as various derivatives of imidazoquinoline compounds, including certain substituted imidazoquinoline amines. See, for example, Hemmi H et al. (2002) Nature Immunol. 3(2):196-200; Jurk M et al. (2002) Nature Immunol. 3(6):499; U.S. Pat. No. 6,331 ,539.

TLR9 signals in response to CpG DNA. As used herein, the term "CpG DNA" refers to an immunostimulatory deoxyribonucleic acid which contains a cytosine- guanine (CG) dinucleotide, the C residue of which is unmethylated. The entire immunostimulatory nucleic acid can be unmethylated or portions may be unmethylated but at least the C of the 5'-CG-3' must be unmethylated. Examples of CpG DNA and the effects of CpG nucleic acids on immune modulation have been described extensively in U.S. patents such as U.S. Pat. Nos. 6,194,388; 6,207,646; 6,239,116; and 6,218,371 , and published international patent applications, such as WO98/37919, WO98/40100, WO98/52581 , and WO99/56755.

CpG DNA includes both naturally occurring immunostimulatory nucleic acids, as found in bacterial DNA and plasmids, as well as synthetic oligodeoxynucleotides (ODN). In one embodiment CpG DNA is a CpG ODN. In one embodiment the CpG ODN has a base sequence provided by 5'-TCGTCG I I I I GTCG M M GTCGTT-3" (ODN 2006; SEQ ID NO:1 ).

In one aspect the invention provides a method of inhibiting an immune response in a subject. The method includes the step of administering to the subject an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. As used herein, "subject" refers to a living mammal. In various non-limiting individual embodiments a subject is a mouse, rat, guinea pig, rabbit, cat, dog, goat, sheep, horse, cow, or non-human primate. In one embodiment the subject is a human.

In one embodiment the immune response is an antigen-specific immune response. This type of immune response is also known as an adaptive immune response. An antigen-specific immune response is an immune response that involves T lymphocytes or B lymphocytes that are activated in response to contact with a particular antigen. An antigen-specific immune response may be characterized by the development of memory for a particular antigen, and thus an antigen-specific immune response includes both primary and recall antigen-specific immune responses. The immune response in this embodiment can be a cellular immune response, a humoral immune response, or a combination of a cellular immune response and a humoral response. Antigen-specific immune responses may include elaboration of certain cytokines, chemokines, and antibodies. Cytokines associated with antigen-specific immune responses include, without limitation, interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-5 (IL-5). Interferon gamma (IFN-D) is a cytokine associated with both antigen-specific and antigen-nonspecific immune responses. Chemokines associated with antigen-specific immune responses include, without limitation, interferon-gamma- inducible protein-10 (IP-10). Antibodies include antibodies of various classes or isotypes, including IgG, IgM, IgA, IgE, and IgD.

In one embodiment the immune response is an antigen-nonspecific immune response. This type of immune response is also known as an innate immune response. An antigen-nonspecific immune response is an immune response that involves phagocytic cells (macrophages and neutrophils) and natural killer (NK) cells that are activated in response to contact with certain molecular patterns that signal danger or non-self, without specificity for any particular antigen and without the development of memory. In one embodiment an antigen-nonspecific immune response is an inflammatory response. Antigen-nonspecific immune responses may include elaboration of certain cytokines and chemokines, such as interleukin-1 (IL-1), interleukin-12 (IL-12), interferon alpha (IFN-D), and tumor necrosis factor alpha (TNF-D), but not antibodies. Chemokines associated with antigen-nonspecific immune responses include, without limitation, interleukin-8 (IL-8). Inhibiting an immune response means reducing at least one measurable aspect of an immune response. The inhibition can be measured using any suitable technique, including, for example, enzyme-linked immunosorbent assay (ELISA) specific for a particular cytokine or chemokine, flow cytometry to quantify cell surface markers indicative of immune cell activation, mixed lymphocyte reaction, and cytolytic assay.

These methods are well known to those skilled in the art, and reagents used in the practice of these methods are generally readily available from commercial sources. An immune response is deemed to be inhibited if at least one measurable aspect of the immune response is reduced by at least 5 percent compared to a control immune response measured under similar conditions. In various embodiments the inhibition corresponds to at least: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99 percent reduction of a control immune response measured under similar conditions.

Plasmacytoid dendritic cells and B cells express TLR7 and TLR9 and are poised to initiate the immunologic response to microbial nucleic acids. Although the intracellular location of these receptors and their preferred repertoire of ligands might suggest that the TLR receptors in these cells would be involved in the innate response to microbial nucleic acids, indirect evidence suggests that these same receptors may help to initiate the abnormal response to self-antigens that characterizes autoimmune diseases including SLE. As disclosed in the examples below, it has been shown that CPG 52364, at concentrations of less than 10 nM, effectively antagonizes TLR signaling in vitro in human cells that have been transfected with TLR9 or TLR8 expression vectors. CPG 52364 was found to be more than an order of magnitude more potent than hydroxychloroquine as an inhibitor of TLR9 signaling, and closer to two orders of magnitude more potent than hydroxychloroquine as an inhibitor of TLR8 signaling.

Also as disclosed in the examples below, it has been shown that CPG 52364, at nanomolar concentrations, effectively antagonizes TLR signaling in vitro in human peripheral blood mononuclear cells. Results were similar for TLR7, TLR8, and TLR9. In addition to demonstrated effects in vitro, the examples below also show that CPG 52364 effectively inhibits TLR9-stimulated chemokine production in vivo. In mice it was found, for example, that an oral dose of less than 1 mg/kg body weight produced a 50 percent decrease in TLR9-stimulated IP-10 production. This represents a greater than 37-fold greater potency than chloroquine and more than 200-fold greater potency than hydroxychloroquine in this system. Significantly, Example 5 below shows that CPG 52364 effectively reduces anti- dsDNA titers in a mouse model of lupus. Anti-dsDNA titers have been described in the literature to serve as a marker of clinical activity as well as of clinical severity of systemic lupus erythematosus, an autoimmune disease with a relapsing and remitting pattern of clinical disease. MRL Ipr/lpr mice, which develop spontaneous lupus, were

treated with CPG 52364 or negative control (phosphate-buffered saline, PBS) for twelve weeks and then assessed for anti-dsDNA antibody titers. Mice treated with CPG 52364 had titers that were only about one quarter to one third of corresponding titers in mice treated with PBS. In one aspect the invention provides a method of treating a subject having an autoimmune disease. Generally, the term "treat" as used herein means to obtain a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a particular condition or disease or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for a condition or disease and/or adverse effect attributable to the condition or disease. "Treat" as used herein covers any treatment (e.g. complete or partial) of, or prevention of, a condition or disease in a non-human, such as a mammal, or more particularly a human, and includes: (a) preventing the condition disease from occurring in a subject that may be at risk of developing or predisposed to having a condition or disease but has not yet been diagnosed as having it; (b) inhibiting the condition or disease, i.e., arresting its development; or (c) relieving or ameliorating the condition or disease, i.e., cause regression of the condition or disease.

In one embodiment the term "treat" refers to reducing at least one objective manifestation of a disease or condition in a subject having the disease or condition. In one embodiment treat refers to eliminating a disease or condition in a subject having the disease or condition.

In one embodiment the term "treat" refers to preventing a disease or condition from developing in a subject at risk of developing the disease or condition. A subject may be at risk of having a disease or condition due to a genetic predisposition for developing the disease or condition. Alternatively or in addition, a subject may be at risk of having a disease or condition due to an identified or expected exposure of the subject to, or contact of the subject with, an agent associated with developing the disease or condition.

As used herein a "subject having a disease or condition" refers to a subject having at least one objective manifestation of the disease or condition. For example, a subject having an autoimmune disease refers to a subject having at least one objective manifestation of the autoimmune disease.

As used herein a "subject at risk of having a disease or condition" refers to a subject without at least one objective manifestation of the disease or condition but that

is nonetheless at risk of harboring or developing the disease or condition. For example, a subject at risk of having transplant rejection refers to a subject without at least one objective manifestation of transplant rejection but that is nonetheless at risk of developing transplant rejection. Such a subject in one embodiment is a subject that is expected to receive a tissue or organ transplant. In another embodiment such a subject is a subject that has received a tissue or organ transplant.

As used herein, "autoimmune disease" refers to immunologically mediated acute or chronic injury to a tissue or organ of host origin. The term encompasses both cellular and antibody-mediated autoimmune phenomena, as well as organ-specific and organ- nonspecific autoimmunity. Autoimmune diseases specifically include, without limitation, systemic lupus erythematosus (also known simply as lupus; also referred to herein as SLE), insulin-dependent diabetes mellitus, rheumatoid arthritis, multiple sclerosis, atherosclerosis, inflammatory bowel disease, ankylosing spondylitis, autoimmune hemolytic anemia, Behcet's syndrome, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic thrombocytopenia, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polymyositis/dermatomyositis, primary biliary sclerosis, sarcoidosis, sclerosing cholangitis, Sjogren's syndrome, systemic sclerosis (scleroderma and CREST syndrome), Takayasu's arteritis, temporal arteritis, and Wegener's granulomatosis. Inflammatory bowel disease specifically includes Crohn's disease and ulcerative colitis. Autoimmune diseases also include certain immune complex-associated diseases.

In one embodiment the invention provides a method of treating a subject having SLE. Systemic lupus erythematosus (SLE) is a complex, multifactorial, chronic, remitting and relapsing autoimmune disease with a heterogeneous clinical presentation. Although SLE patients with manifestations of SLE may be young (as young as 2 years old) or elderly (80 years of age or older), SLE most commonly affects women of childbearing age. In this age range, the female to male ratio has been estimated to be approximately 12:1 to 10:1 (Danchenko N et al. (2006) Lupus. 15(5):308-315; Manson JJ et al. (2006) Orphanet J Rare Dis. 1(1 ):6). An accurate overall incidence or prevalence rate of SLE is difficult to obtain due to differences in methods, patient ethnicity and demographics coupled with the complexity of the etiopathogenesis of this disease, which is influenced by genetics and environmental triggers, such as sunlight, hormonal influences, drugs, and infections (especially infections with Epstein-Barr virus) (Manson JJ et al. (2006) Orphanet J Rare Dis. 1(1 ):6). Nonetheless, estimates of the

incidence rates (per 100,000 people per year) of SLE have ranged from 0.3 to 0.9 among Caucasian men; 0.7 to 2.5 among African-American men; 2.5 to 3.9 among Caucasian women; and 8.1 to 11.4 among African-American women (Manzi S. (2001 ) Am J Manag Care. 16 Suppl:S474-S479). Estimates for the prevalence (per 100,000 5 people) of SLE have ranged from 3 to 19 among Caucasian men; 3 to 53 among African-American men; 17 to 71 among Caucasian women; and 56 to 283 among African-American women (Manzi S. (2001 ) Am J Manag Care. 16 Suppl:S474-S479).

SLE is characterized by loss of self-tolerance and the development of B cell autoreactivity to certain self-antigens leading to the production of autoantibodies to io DNA, RNA, and associated nuclear proteins, and tissue and organ damage from the resultant inflammation. The clinical features of SLE are diverse with a constellation of potential clinical presentations that can include constitutional symptoms, dermatologic or musculoskeletal signs, and/or hematologic, cardiovascular, renal, and sometimes neurologic (CNS) findings. Despite the disparity in the spectrum of clinical signs and

/5 severity of disease among patients with SLE, two findings are consistent among patients: elevation in serum concentration of interferon-alpha (IFN-α) and circulating autoantibodies (Means TK et al. (2005) Ann NY Acad ScL 1062:242-251 ; Baechler EC et al. (2004) Curr Opin Immunol. 16:801-807; Croker JA et al. (2005) TRENDS Immunol. 26(11 ):580-586). In both humans and murine models of lupus, the primary

20 source of the IFN-α is the plasmacytoid dendritic cells (pDC), which are an integral part of the innate immune system designed to identify components of microbial invaders and initiate an immune response (Baechler EC et al. (2004) Curr Opin Immunol. 16:801- 807).

A role for IFN-α in the pathogenesis and development of SLE has been

25 supported by a series of observations in patients with SLE, sequelae of IFN-α therapy in patients with malignancies or hepatitis, and microarray analyses of gene transcription in peripheral blood mononuclear cells (PBMCs) from patients with SLE. Elevated concentrations of IFN-α in the serum of patients with SLE appear to correlate with disease activity and severity, especially early in a patient's disease (Rόnnblom L et al.

30 (2006) Arthritis Rheum. 54(2):408-420). Other evidence for a role of IFN-α came from studies of sequelae experienced by patients with carcinoid tumors, pancreatic tumors, or hepatitis and treated with IFN-α. Approximately 22% of these patients developed serologic abnormalities (elevations in serum titers for anti-dsDNA or ANA) and

approximately 19% developed some form of autoimmune disease. Approximately 0.7% developed SLE (Baechler EC et al. (2004) Curr Opin Immunol. 16:801-807).

An intriguing recent finding that supports a role of IFN-α in SLE has come from microarray analyses of RNA isolated from PBMCs collected from SLE patients. Several investigators have demonstrated upregulation of a pattern of genes induced by IFN-α - known as the IFN-α signature - in a subset of patients with SLE. In a study of PBMC RNA from 48 patients, approximately half had the IFN-α signature. However, the study included patients who were in remission or had a mild form of disease and would not have been expected to have either elevated serum concentrations of IFN-α or, perhaps, an IFN-α signature. Nonetheless, the IFN-α signature was associated with patients with severe manifestations (i.e., CNS or renal involvement) or hematologic findings (especially leukopenia, lymphopenia, or thrombocytopenia) (Baechler EC et al. (2004) Curr Opin Immunol. 16:801-807; Rόnnblom L et al. (2006) Arthritis Rheum. 54(2):408- 420). Inappropriate production of IFN-α in SLE is by pDCs. These cells normally produce IFN-α in response to detection of microbial nucleic acids (Krieg AM. (2002) Ann Rev Immunol. 20:709-760; Baechler EC et al. (2004) Curr Opin Immunol. 16:801-807; Croker JA et al. (2005) TRENDS Immunol. 26(11 ):580-586). In SLE, these cells produce IFN-α in response to chromatin-DNA or RNA-containing immune complexes, which can also activate autoreactive B cells (Leadbetter EA et al. (2002) Nature.

416(6881 ):603-607; Lau CM et al. (2005) J Exp Med. 202(9):1171-1177; Barrat RJ et al.

(2005) J Exp Med. 202(8): 1131 -1139; Vollmer et al. (2005) J Exp Med. 202(11 ):1575- 1585; Pisitkun P et al. (2006) Science. 312(5780): 1669-1672; Subramanian S et al.

(2006) Proc Natl Acad Sci USA. 103(26):9970-9975; Savarese E et al. (2006) Blood. 107(8):3229-3234). The microbial nucleic acids and the autoantigen immune complexes are recognized in these cells by TLR7 and TLR8 (RNA and associated proteins) and TLR9 (DNA and associated proteins), which are potential therapeutic targets for SLE.

Evidence for involvement of B cell TLRs in the autoimmune response to self antigens has come from investigations in lupus-prone mouse strains. These investigations suggest a model in which nucleosomal DNA or RNA immune complexes recognized by the Ig portion of B cell receptors on the surface of autoreactive B cells are internalized and delivered to endosomes where the nucleic acids bind to TLRs: ssRNA to TLR7 and unmethylated DNA t^ TLR9. Although single-stranded nucleic

acids would be recognized by human TLR8, the murine TLR8 is defective. Binding of the nucleic acids to these TLR receptors stimulates both antibody secretion and proliferation of the B cells.

Investigations of lupus-prone mice, which share many of the features and disease heterogeneity experienced by SLE patients, have yielded important insights into the pathogenesis of lupus. These murine models (MRL Ipr/lpr, NZW x NZB F1 , and BXSB mice) share many of the serologic, and clinical features of human lupus including hyperactivity of B cells, circulating auto-antibodies (anti-dsDNA antibodies and ANA), circulating immune complexes, and development of glomerulonephritis (Andrews et al., 1978). The mouse models are not identical and heterogeneity has permitted the investigation of different aspects of lupus. Differences among the mouse models include the type or quantity of specific autoantibodies (RF and anti-Sm in MRL Ipr/lpr mice), marked lymphoproliferation in MRL Ipr/lpr mice, and gender incidence (male predominance in BXSB mice, but female predominance in NZB x NZW F1 mice). Investigations with these mouse models have indicated that TLR9 and TLR7 have an important mechanistic role in the pathogenesis of SLE.

Evidence for TLR9 involvement has come from crossbreeding experiments that generated MRL Ipr/lpr mice that express RF-specific autoreactive B cells (Leadbetter et al., 2002). Exposure of these mice to the specific RF antigen elicits an expected response: increased proliferation and autoantibody secretion by the autoreactive B cells. However, the cellular responses were not seen in vitro in the presence of pharmacologic inhibitors of TLR9 and were not seen in similar mice that also had a defect in TLR9 signaling. These results demonstrated that two T cell-independent receptors are essential for the B cell responses: the B cell receptor specific for the autoantigen and TLR9 (Leadbetter EA et al. (2002) Nature. 416(6881 ):603-607; Krieg AM. (2002) Nature Immunol. 3(5):423-424).

A role of TLR7 has been demonstrated in the BXSB mouse model in which the lupus-like syndrome develops only in males and is associated with stimulatory and inhibitory genetic loci. The Y-linked autoimmune accelerator (yaa) locus has been shown recently to be an X to Y chromosomal translocation that results in duplication of the Tlr7 gene and 2-fold over-expression of TLR7 (Subramanian S et al. (2006) Proc Natl Acad Sci USA. 103(26):9970-9975; Pisitkun P et al. (2006) Science. 312(5780):1669-1672). The overexpression of TLR7 was associated with acceleration of lupus only in lupus-prone mice.

The evidence from the genetic studies and in vitro investigations with mutant mice evokes a model in which activation of TLR7 and TLR9, and possibly TLR8 in humans, has a role in initiation of immunologic responses to self-antigens, which is central to the pathogenesis of SLE. A role for these receptors may help to explain the serendipitous discovery made decades ago that antimalarials, such as chloroquine and hydroxychloroquine, ameliorated autoimmune symptoms in patients with rheumatoid arthritis or SLE. Recent studies have revealed that these drugs are TLR antagonists, at clinically relevant concentrations, suggesting that this may be their therapeutic mechanism of action (Vollmer et al. (2005) J Exp Med. 202(11 ):1575-1585). An unanswered question is why TLR9, which normally has a ligand repertoire restricted to unmethylated DNA that is characteristic of microbial and not mammalian DNA, would instead be activated by self DNA. Indirect and circumstantial evidence that changes in DNA methylation could lead to recognition of self DNA by TLR9 comes from two observations: DNA from lupus patients is often hypomethylated and drugs that inhibit DNA methylation (procainamide and hydralazine) can induce lupus in predisposed individuals (Lu Q et al. (2005) J Immunol. 174 (10):6212-6219). In addition, the presence of the DNA in immune complexes, which are taken up by pDC through Fc receptor (FcR) ₁ seems to change the properties of the relatively low level of unmethylated CpG dinucleotides in self DNA from nonstimulatory to immune stimulatory (Boule MW et al. (2004) J Exp Med. 199(12):1631-40) TLR9-dependent and TLR9- independent dendritic cell activation by chromatin-immunoglobulin G complexes.

The current treatment goal for patients with SLE is to manage acute episodes or exacerbations of symptoms or sequelae, minimize disease flares, and control or manage chronic disease symptoms. Standard therapy has included hydroxychloroquine and low-dose glucocorticoids for mild disease and high-dose glucocorticoids coupled with more potent immunosuppressives such as cyclophosphamide or azathioprine for patients with more severe disease or symptoms (Manson JJ et al. (2006) Orphanet J Rare Dis. 1 (1 ):6). Early phase 1 trials have been instituted to evaluate the therapeutic safety and efficacy of B cell depletion in lupus patients by administration of an anti-CD20 monoclonal antibody (rituximab), which is currently used in anti-lymphoma therapy and has been approved for treatment of rheumatoid arthritis (Eisenberg R. (2003) Arthritis Res Ther. 5(4):157-159; Browning J. (2006) Nat Rev Drug Discov. 5(7):564-576). However, none of these investigational

therapies selectively inhibit multiple potential molecular targets of SLE ₁ such as TLR7, TLR8, and TLR9.

The invention in one aspect provides a method of treating a subject having or at risk of having transplant rejection. The method according to this aspect of the invention includes the step of administering to the subject an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof to treat the transplant rejection.

As used herein, "transplant rejection" refers to acute or chronic immune-mediated damage to cells, tissues, or organs transplanted from one individual to another individual, i.e., from a donor to a recipient. The rejection can be diagnosed by any suitable method, including but not limited to physical examination; noting the presence of at least one clinical sign or symptom consistent with decreased function of the transplanted cells, tissues, or organ; medical imaging such as computed tomography (CT) or magnetic resonance imaging (MRI); and biopsy of the transplanted tissue or organ. Transplants specifically include, without limitation, kidney, heart, liver, pancreas, pancreatic islets, lung, small intestine, bladder, bone, bone marrow, artery, vein, muscle, limb, and any combination thereof.

CPG 52364 can be used, alone or in combination with at least one other therapeutic agent, to treat a subject having other conditions characterized by undesirable immune activity, including inflammatory conditions, allergy, asthma, and sepsis.

Inflammatory conditions include, but are not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjuctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, meningitis, metritis, mucitis, myocarditis, myositis, myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis media, pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis, prostatitis, pulpritis, retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis.

An "allergy" refers to acquired hypersensitivity to a substance (allergen). An allergen refers to a substance (antigen) that can induce an allergic or asthmatic response in a susceptible subject. The list of allergens is enormous and can include pollens, insect venoms, animal dander dust, fungal spores and drugs (e.g. penicillin).

Examples of natural, animal and plant allergens include but are not limited to proteins specific to the following genuses: Agropyron (e.g. Agropyron repens); Agrostis (e.g.

Agrostis alba); Alder, Alnus (Alnus gultinoasa); Alternaria (Alternaria alternata);

Ambrosia (Ambrosia artemiisfolia; Anthoxanthum (e.g. Anthoxanthum odoratum); Apis (e.g. Apis multiflorum); Arrhenatherum (e.g. Arrhenatherum elatius); Artemisia

(Artemisia vulgaris); Avena (e.g. Avena sativa); Betula (Betula verrucosa); Blattella (e.g.

Blattella germanica); Bromus (e.g. Bromus inermis); Canine (Canis familiaris);

Chamaecyparis (e.g. Chamaecyparis obtusa); Cryptomeria (Cryptomeria japonica);

Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Dactylis (e.g. Dactylis glomerata); Dermatophagoides (e.g.

Dermatophagoides farinae); Felis (Felis domesticus); Festuca (e.g. Festuca elatior);

Holcus (e.g. Holcus lanatus); Juniperus (e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Lolium (e.g. Lolium perenne or

Lolium multiflorum); Olea (Olea europa); Parietaria (e.g. Parietaria officinalis or Parietaria judaica); Paspalum (e.g. Paspalum notatum); Periplaneta (e.g. Periplaneta americana); Phalaris (e.g. Phalaris arundinacea); Phleum (e.g. Phleum pratense);

Plantago (e.g. Plantago lanceolata); Poa (e.g. Poa pratensis or Poa compressa);

Quercus (Quercus alba); Secale (e.g. Secale cereale); Sorghum (e.g. Sorghum halepensis); Thuya (e.g. Thuya orientalis); and Triticum (e.g. Thticum aestivum). The term "asthma" as used herein refers to a disease of the airways that is characterized by inflammation, narrowing of the airways, and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively, associated with atopic or allergic symptoms. In one embodiment asthma is allergic asthma.

Asthma is typically an episodic disease with symptom-free periods between acute exacerbations.

The term "sepsis" refers to a systemic inflammatory response syndrome in association with infection. Sepsis is characterized by two or more features of fever or hypothermia, tachypnea, tachycardia, elevated or depressed leukocyte counts, and, in advanced cases, cardiovascular collapse (shock), organ dysfunction or failure, and disseminated intravascular coagulation. Both microbial signals and host immune response are believed to contribute to sepsis.

It is believed that CPG 52364 can be used, alone or in combination with at least one antimalarial agent, to treat malaria. Antimalarial agents include, without limitation,

various quinoline-based agents including chloroquine, hydroxychloroquine, primaquine, quinine, amodiaquine, mefloquine.

CPG 52364 can be combined with other therapeutic agents. CPG 52364 and other therapeutic agent may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time. The other therapeutic agents are administered sequentially with one another and with CPG 52364, when the administration of the other therapeutic agents and CPG 52364 is temporally separated. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer. Other therapeutic agents include but are not limited to anti-inflammatory drugs, glucocorticoids, other immunosuppressive agents, cytokines, antibodies, antimicrobial agents, etc.

CPG 52364 may be administered to a subject with an anti-microbial agent. An anti-microbial agent, as used herein, refers to a naturally-occurring or synthetic compound which is capable of killing or inhibiting infectious microorganisms. The type of anti-microbial agent useful according to the invention will depend upon the type of microorganism with which the subject is infected or at risk of becoming infected. Antimicrobial agents include but are not limited to anti-bacterial agents, anti-viral agents, anti-fungal agents and anti-parasitic agents. Phrases such as "anti-infective agent", "anti-bacterial agent", "anti-viral agent", "anti-fungal agent", "anti-parasitic agent" and "parasiticide" have well-established meanings to those of ordinary skill in the art and are defined in standard medical texts. Briefly, anti-bacterial agents kill or inhibit bacteria, and include antibiotics as well as other synthetic or natural compounds having similar functions. Antibiotics are low molecular weight molecules which are produced as secondary metabolites by cells, such as microorganisms. In general, antibiotics interfere with one or more bacterial functions or structures which are specific for the microorganism and which are not present in host cells. Anti-viral agents can be isolated from natural sources or synthesized and are useful for killing or inhibiting viruses. Antifungal agents are used to treat superficial fungal infections as well as opportunistic and primary systemic fungal infections. Anti-parasite agents kill or inhibit parasites.

Examples of anti-parasitic agents, also referred to as parasiticides useful for human administration include but are not limited to albendazole, amphotericin B, benznidazole, bithionol, chloroquine HCI, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine, diloxanide furoate, eflomithine, furazolidaone,

glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumine antimoniate, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethanmine- sulfonamides, pyrimethanmine-sulfadoxine, quinacrine HCI, quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium (sodium antimony gluconate), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimethroprim-sulfamethoxazole, and tryparsamide some of which are used alone or in combination with others.

Antibacterial agents kill or inhibit the growth or function of bacteria. A large class of antibacterial agents is antibiotics. Antibiotics, which are effective for killing or inhibiting a wide range of bacteria, are referred to as broad spectrum antibiotics. Other types of antibiotics are predominantly effective against the bacteria of the class gram- positive or gram-negative. These types of antibiotics are referred to as narrow spectrum antibiotics. Other antibiotics which are effective against a single organism or disease and not against other types of bacteria, are referred to as limited spectrum antibiotics. Antibacterial agents are sometimes classified based on their primary mode of action. In general, antibacterial agents are cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, nucleic acid synthesis or functional inhibitors, and competitive inhibitors. Antiviral agents are compounds which prevent infection of cells by viruses or replication of the virus within the cell. There are many fewer antiviral drugs than antibacterial drugs because the process of viral replication is so closely related to DNA replication within the host cell, that non-specific antiviral agents would often be toxic to the host. There are several stages within the process of viral infection which can be blocked or inhibited by antiviral agents. These stages include, attachment of the virus to the host cell (immunoglobulin or binding peptides), uncoating of the virus (e.g. amantadine), synthesis or translation of viral mRNA (e.g. interferon), replication of viral RNA or DNA (e.g. nucleotide analogues), maturation of new virus proteins (e.g. protease inhibitors), and budding and release of the virus. Nucleotide analogues are synthetic compounds which are similar to nucleotides, but which have an incomplete or abnormal deoxyribose or ribose group. Once the nucleotide analogues are in the cell, they are phosphorylated, producing the triphosphate formed which competes with normal nucleotides for incorporation into the viral DNA or RNA. Once the triphosphate form of the nucleotide analogue is

incorporated into the growing nucleic acid chain, it causes irreversible association with the viral polymerase and thus chain termination. Nucleotide analogues include, but are not limited to, acyclovir (used for the treatment of herpes simplex virus and varicella- zoster virus), gancyclovir (useful for the treatment of cytomegalovirus), idoxuridine, ribavirin (useful for the treatment of respiratory syncitial virus), dideoxyinosine, dideoxycytidine, zidovudine (azidothymidine), imiquimod, and resimiquimod.

The interferons are cytokines which are secreted by virus-infected cells as well as immune cells. The interferons function by binding to specific receptors on cells adjacent to the infected cells, causing the change in the cell which protects it from infection by the virus, α and β-interferon also induce the expression of Class I and Class Il MHC molecules on the surface of infected cells, resulting in increased antigen presentation for host immune cell recognition, α and β-interferons are available as recombinant forms and have been used for the treatment of chronic hepatitis B and C infection. At the dosages which are effective for anti-viral therapy, interferons have severe side effects such as fever, malaise and weight loss.

Anti-viral agents useful in the invention include but are not limited to immunoglobulins, amantadine, interferons, nucleotide analogues, and protease inhibitors. Specific examples of anti-virals include but are not limited to Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate; Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine; Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride; Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate; Viroxime; Zalcitabine; Zidovudine; and Zinviroxime. Anti-fungal agents are useful for the treatment and prevention of infective fungi.

Anti-fungal agents are sometimes classified by their mechanism of action. Some antifungal agents function as cell wall inhibitors by inhibiting glucose synthase. These include, but are not limited to, basiungin/ECB. Other anti-fungal agents function by destabilizing membrane integrity. These include, but are not limited to, immidazoles,

such as clotrimazole, sertaconzole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconacole, as well as FK 463, amphotericin B, BAY 38-9502, MK 991 , pradimicin, UK 292, butenafine, and terbinafine. Other anti-fungal agents function by breaking down chitin (e.g. chitinase) or immunosuppression (501 cream). CPG 52364 may also be administered in conjunction with an anti-cancer therapy.

Anti-cancer therapies include cancer medicaments, radiation and surgical procedures. As used herein, a "cancer medicament" refers to an agent which is administered to a subject for the purpose of treating a cancer. As used herein, "treating cancer" includes preventing the development of a cancer, reducing the symptoms of cancer, and/or inhibiting the growth of an established cancer. In other aspects, the cancer medicament is administered to a subject at risk of developing a cancer for the purpose of reducing the risk of developing the cancer. Various types of medicaments for the treatment of cancer are described herein. For the purpose of this specification, cancer medicaments are classified as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormone therapy, and biological response modifiers.

The chemotherapeutic agent may be selected from the group consisting of methotrexate, vincristine, adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RAS famesyl transferase inhibitor, famesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, lncel/VX-710, VX-853, ZD0101 , ISI641 , ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomal doxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Xeload/Capecitabine, Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, BMS-182751 /oral platinum, UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer, Campto/Levamisole, Camptosar/lrinotecan, Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU

103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide, Ifes/Mesnex/lfosamide, Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331 , Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylating agents such as melphelan and cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorambucil, Cytarabine HCI, Dactinomycin, Daunorubicin HCI, Estramustine phosphate sodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue), Lomustine (CCNU), Mechlorethamine HCI (nitrogen mustard), Mercaptopurine, Mesna, Mitotane (o.p ^' -DDD), Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCI, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), lnterleukin 2, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Pentostatin

(2'deoxycoformycin), Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate, but it is not so limited.

The immunotherapeutic agent may be selected from the group consisting of Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11 , MDX- 22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1 , CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD- 72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK- 2, MDX-260, ANA Ab, SMART 1 D10 Ab, SMART ABL 364 Ab and ImmuRAIT-CEA, but it is not so limited.

The cancer vaccine may be selected from the group consisting of EGF, Anti- idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL theratope, BLP25 (MUC-1 ), liposomal idiotypic vaccine, Melacine, peptide antigen vaccines, toxin/antigen vaccines, MVA-based vaccine, PACIS, BCG vacine, TA-HPV, TA-CIN, DISC-virus and ImmuCyst/TheraCys, but it is not so limited.

CPG 52364 may be administered to a subject with an immunosuppressive agent. As used herein, an "immunosuppressive agent" refers to an agent which is administered to a subject for the purpose of down-regulating an immune response. These agents

may be used in the treatment of graft rejection, graft-versus-host disease, and autoimmune disease. Immunosuppressive agents specifically include but are not limited to corticosteroids such as prednisone and methylprednisolone, azathioprine, cyclosporine A, tacrolimus, mycophenolate mofetil, rapamycin, polyclonal antibodies (e.g., antithymocyte globulin), and monoclonal antibodies (e.g. OKT3, basiliximab, and daclizumab).

CPG 52364 may be administered to a subject with an anti-inflammatory agent. As used herein, an "anti-inflammatory agent" refers to an agent which is administered to a subject for the purpose of down-regulating an inflammatory response. Anti- inflammatory agents are generally less potent than immunosuppressive agents, and they specifically include, without limitation, non-steroidal anti-inflammatory agents, prostaglandin inhibitors, gold, colchicine, and methotrexate.

CPG 52364 may be administered to a subject with an agent useful for the treatment of allergy. Agents useful for the treatment of allergy include, but are not limited to, anti-histamines, corticosteroids, and prostaglandin inducers. Anti-histamines are compounds which counteract histamine released by mast cells or basophils. These compounds are well known in the art and commonly used for the treatment of allergy. Anti-histamines include, but are not limited to, acrivastine, astemizole, azatadine, azelastine, betatastine, brompheniramine, buclizine, cetirizine, cetirizine analogs, chlorpheniramine, clemastine, CS 560, cyproheptadine, desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR 609, hydroxyzine, levocabastine, loratidine, methscopolamine, mizolastine, norastemizole, phenindamine, promethazine, pyrilamine, terfenadine, and tranilast.

Corticosteroids include, but are not limited to, methylprednisolone, prednisolone, prednisone, beclomethasone, budesonide, dexamethasone, flunisolide, fluticasone propionate, and triamcinolone. Although dexamethasone is a corticosteroid having antiinflammatory action, it is not regularly used for the treatment of allergy or asthma in an inhaled form because it is highly absorbed and it has long-term suppressive side effects at an effective dose. Dexamethasone, however, can be used according to the invention for treating allergy or asthma because when administered in combination with a composition of the invention it can be administered at a low dose to reduce the side effects. Some of the side effects associated with corticosteroid use include cough, dysphonia, oral thrush (candidiasis), and in higher doses, systemic effects, such as adrenal suppression, glucose intolerance, osteoporosis, aseptic necrosis of bone,

cataract formation, growth suppression, hypertension, muscle weakness, skin thinning, and easy bruising. Barnes & Peterson (1993) Am Rev Respir Dis 148:S1-S26; and Kamada AK et al. (1996) Am J Respir Crit Care Med 153:1739-48.

CPG 52364 may be administered to a subject with an agent useful for the treatment of asthma. Agents useful for the treatment of asthma are generally separated into two categories, quick-relief medications and long-term control medications. Asthma patients take the long-term control medications on a daily basis to achieve and maintain control of persistent asthma. Long-term control medications include anti-inflammatory agents such as corticosteroids, chromolyn sodium and nedocromil; long-acting bronchodilators, such as long-acting β ₂ agonists and methylxanthines; and leukotriene modifiers. The quick-relief medications include short-acting β ₂ agonists, anticholinergics, and systemic corticosteroids. There are many side effects associated with each of these drugs and none of the drugs alone or in combination is capable of preventing or completely treating asthma. Asthma medicaments include, but are not limited, PDE-4 inhibitors, bronchodilator/beta-2 agonists, K ⁺ channel openers, VLA-4 antagonists, neurokin antagonists, thromboxane A2 (TXA2) synthesis inhibitors, xanthines, arachidonic acid antagonists, 5 lipoxygenase inhibitors, TXA2 receptor antagonists, TXA2 antagonists, inhibitor of 5-lipox activation proteins, and protease inhibitors. Bronchodilator/β ₂ agonists are a class of compounds which cause bronchodilation or smooth muscle relaxation. Bronchodilator/β ₂ agonists include, but are not limited to, salmeterol, salbutamol, albuterol, terbutaline, D2522/formoterol, fenoterol, bitolterol, pirbuerol methylxanthines and orciprenaline. Long-acting β ₂ agonists and bronchodilators are compounds which are used for long-term prevention of symptoms in addition to the anti-inflammatory therapies. Long-acting β ₂ agonists include, but are not limited to, salmeterol and albuterol. These compounds are usually used in combination with corticosteroids and generally are not used without any inflammatory therapy. They have been associated with side effects such as tachycardia, skeletal muscle tremor, hypokalemia, and prolongation of QTc interval in overdose.

Methylxanthines, including for instance theophylline, have been used for long- term control and prevention of symptoms. These compounds cause bronchodilation resulting from phosphodiesterase inhibition and likely adenosine antagonism. Dose- related acute toxicities are a particular problem with these types of compounds. As a

result, routine serum concentration must be monitored in order to account for the toxicity and narrow therapeutic range arising from individual differences in metabolic clearance. Side effects include tachycardia, tachyarrhythmias, nausea and vomiting, central nervous system stimulation, headache, seizures, hematemesis, hyperglycemia and hypokalemia. Short-acting β _∑ agonists include, but are not limited to, albuterol, bitolterol, pirbuterol, and terbutaline. Some of the adverse effects associated with the administration of short-acting β ₂ agonists include tachycardia, skeletal muscle tremor, hypokalemia, increased lactic acid, headache, and hyperglycemia.

Chromolyn sodium and nedocromil are used as long-term control medications for preventing primarily asthma symptoms arising from exercise or allergic symptoms arising from allergens. These compounds are believed to block early and late reactions to allergens by interfering with chloride channel function. They also stabilize mast cell membranes and inhibit activation and release of mediators from inosineophils and epithelial cells. A four to six week period of administration is generally required to achieve a maximum benefit.

Anticholinergics are generally used for the relief of acute bronchospasm. These compounds are believed to function by competitive inhibition of muscarinic cholinergic receptors. Anticholinergics include, but are not limited to, ipratropium bromide. These compounds reverse only cholinerigically-mediated bronchospasm and do not modify any reaction to antigen. Side effects include drying of the mouth and respiratory secretions, increased wheezing in some individuals, and blurred vision if sprayed in the eyes.

Generally, daily oral doses of CPG 52364 will be from about 0.01 milligrams/kg per day to 1000 milligrams/kg per day. It is expected that oral doses in the range of 5 to 500 milligrams/kg, in one or several administrations per day, will yield the desired results. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. For example, it is expected that intravenous administration would be from an order to several orders of magnitude lower dose per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.

As disclosed in the examples below, single intravenous doses of CPG 52364 of ca. 8 mg/kg and single oral doses of 40-50 mg/kg resulted in therapeutically meaningful blood levels on the order of 500 to 1000 ng/ml.

For any compound described herein the therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for CPG 52364 which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.

The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic ingredients.

For use in therapy, an effective amount of CPG 52364, alone or formulated with at least one other therapeutic agent, can be administered to a subject by any mode that delivers CPG 52364 to the desired surface. Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Preferred routes of administration include but are not limited to oral, parenteral (including, without limitation, intravenous, intraperitoneal, intramuscular), intranasal, sublingual, intratracheal, inhalation, ocular, vaginal, and rectal.

For oral administration, the compounds (i.e., CPG 52364 and other therapeutic agents) can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,

hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers, i.e. EDTA for neutralizing internal acid conditions or may be administered without any carriers.

Also specifically contemplated are oral dosage forms of the above component or components. The component or components may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of hydrolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline. Abuchowski and Davis, 1981 , "Soluble Polymer-Enzyme Adducts" In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-lnterscience, New York, NY, pp. 367-383; Newmark, et al., 1982, J. Appl. Biochem. 4:185-189. Other polymers that could be used are poly-1,3- dioxolane and poly-1 ,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties.

For the component (or derivative) the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of CPG 52364 (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used. The therapeutic can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, CPG 52364 (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents. One may dilute or increase the volume of the therapeutic with an inert material.

These diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberiite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants. Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride. The list of potential non-ionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of CPG 52364 or derivative either alone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation

from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of CPG 52364 (or derivatives thereof). CPG 52364 (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Other reports of inhaled molecules include Adjei et al., 1990, Pharmaceutical Research, 7:565-569; Adjei et al., 1990, International Journal of Pharmaceutics, 63:135-144 (leuprolide acetate); Braquet et al., 1989, Journal of Cardiovascular Pharmacology, 13(suppl. 5):143-146 (endothelin-1); Hubbard et al., 1989, Annals of Internal Medicine, Vol. IM, pp. 206-212 (a1- antitrypsin); Smith et al., 1989, J. Clin. Invest. 84:1145-1146 (a-1 -proteinase); Oswein et al., 1990, "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J. Immunol. 140:3482-3488 (interferon-gamma and tumor necrosis factor alpha) and Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating factor). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Patent No. 5,451 ,569, issued September 19, 1995 to Wong et al.

Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acom Il nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts.

All such devices require the use of formulations suitable for the dispensing of CPG 52364 (or derivative). Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes,

microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise CPG 52364 (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active CPG 52364 per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for CPG 52364 stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the CPG 52364 caused by atomization of the solution in forming the aerosol. Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing CPG 52364 (or derivative) suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1 ,1 ,1 ,2-tetrafluoroethane, or combinations thereof.

Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing CPG 52364 (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. CPG 52364 (or derivative) should most advantageously be prepared in particulate form with an average particle size of less than 10 micrometers (Dm), most preferably 0.5 to 5 Dm, for most effective delivery to the distal lung. Nasal delivery of a pharmaceutical composition of the present invention is also contemplated. Nasal delivery allows the passage of a pharmaceutical composition of the present invention to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In one embodiment, the metered dose is delivered by drawing the pharmaceutical composition of the present invention solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed.

The chamber is compressed to administer the pharmaceutical composition of the present invention. In a specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation. Preferably, the nasal inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug. The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an

acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro )capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference. CPG 52364 and optionally other therapeutics may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004- 0.02% w/v).

The pharmaceutical compositions of the invention contain an effective amount of CPG 52364 and optionally therapeutic agents included in a pharmaceutically-acceptable carrier.

The therapeutic agent(s), including specifically but not limited to CPG 52364, may be provided in particles. Particles as used herein means nano or microparticles (or in some instances larger) which can consist in whole or in part of CPG 52364 or the other

therapeutic agent(s) as described herein. The particles may contain the therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. The therapeutic agent(s) also may be dispersed throughout the particles. The therapeutic agent(s) also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero order release, first order release, second order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles may be microcapsules which contain CPG 52364 in a solution or in a semi-solid state. The particles may be of virtually any shape.

Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic agent(s). Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, CP. Pathak and J.A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhyd rides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). The therapeutic agent(s) may be contained in controlled release systems. The term "controlled release" is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release formulations. The term "sustained release" (also referred to as "extended release") is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term "delayed release" is used in its conventional sense to refer to a

drug formulation in which there is a time delay between administration of the formulation and the release of the drug there from. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and thus may or may not be 'sustained release."

Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

EXAMPLES

Example 1 Synthesis of CPG 52364

This example describes the synthesis of CPG 52364 as the free base (Steps 1-6) and as a bisulfate salt (Step 7).

Step i

124.11 100.17 204.27

A mixture of 4-fluorobenzaldehyde (49.6 gm, 0.40 moles) and N- methylpiperazine (69.5 gm, 0.695 moles) in water (400 mL) containing sodium carbonate (63.0 gm, 0.60 moles) was stirred at reflux for 21 hours. After cooling, the mixture was poured into a separatory funnel containing water (1000 mL) and the oily product was extracted into methylene chloride (3 X 200 mL). The combined extracts were washed with water and the solvent was then stripped under vacuum to give the aldehyde as an oil which solidified upon cooling. The solid was triturated in hexane and was then isolated by filtration. After drying there was obtained 75 gm (92%) of N- methyl-N'-(4-formyl)phenyl-piperazine as a tan solid.

Step 2

227.18 226.20

To a stirred slurry of 2-nitro-4,5-dimethoxybenzoic acid (100 gm, 0.44 moles) in methylene chloride (750 mL) was added thionyl chloride (SOCI ₂ , 52.4 gm, 0.44 moles) and dimethylformamide (DMF) (0.5 mL). The slurry was heated at 30 - 35°C until a clear solution resulted and then heating was continued an additional 30 minutes. The total time was about two hours. After cooling to room temperature, this solution was dripped into a vigorously stirred mixture of methylene chloride (200 mL) and concentrated ammonia (NH ₄ OH, 200 mL) which was cooled in a 2O ⁰ C water bath. After complete addition of the acid chloride solution, the mixture was stirred for another 30 minutes. The solid product was isolated by filtration, washed with water followed by methylene chloride and then water again and dried. The yield of 2-nitro-4,5- dimethoxybenzamide was 91 gm, 91.4% as a pale yellow solid. Optionally, the product can be recrystallized from aqueous ethanol.

The use of DMF as catalyst in this step is not required. The acid chloride can be formed without using a catalyst as follows. To a stirred slurry of 2-nitro-4,5- dimethoxybenzoic acid (11.4 gm, 0.05 moles) in toluene (100 mL) was added thionyl chloride (6.0 gm, 0.504 moles). The slurry was stirred and heated in an oil bath set at 100°C which caused the thionyl chloride to reflux. The solution was kept at reflux until a

clear solution resulted and then heating was continued an additional 30 minutes by which time most of the thionyl chloride had been consumed and reflux had slowed to a few drops per minute. The total time was about two hours. After cooling to room temperature, this solution was dripped into a vigorously stirred mixture of toluene (100 ml_) and concentrated ammonia (100 ml_) which was cooled in a 20 ⁰ C water bath. After complete addition of the acid chloride solution, the mixture was stirred for another 30 minutes. The solid product was isolated by filtration, washed with toluene and then with water and dried. The yield of 2-nitro-4,5-dimethoxybenzamide was 10.4 gm, 92% as a pale yellow solid.

Step 3

226.20 196.21

A slurry of 2-nitro-4,5-dimethoxybenzamide (91 gm, 0.40 moles) in ethanol (700 ml_) was stirred under nitrogen as 10% palladium on carbon (Pd/C, 1.0 gm) was added. This slurry was stirred and heated to reflux as a solution of ammonium formate (76.1 gm, 1.20 moles) dissolved in water (50 mL) was added in portions through a dropping funnel over a four hour period. A wide bore condenser was used since ammonium carbonate forms in the condenser as the reduction proceeds. Periodically, the condenser was removed and washed with water to remove the ammonium carbonate. After the addition was complete, the reaction was heated at reflux for an additional 30 minutes. Thin layer chromatography (TLC; silica, 10% methanol in methylene chloride) showed complete conversion to the anthranilamide. The solution was filtered free of catalyst and was then stripped under vacuum. The solid residue was recrystallized from water to give the product as an off-white solid. The yield was 66 gm (84.1%).

Step 4

196.21 204.27 380.45

4,5-dimethoxyanthranilamide (66.0 gm, 0.336 moles) and N-methyl-N'- (4-formylphenyl)piperazine (68.7 gm, 0.336 moles) were combined with sodium metabisulfite (NaHSO ₃ , 47.9 gm, 0.251 moles) and water (4.53 ml_, 0.251 moles) in dimethylacetamide (DMAC, 500 ml_). This mixture was stirred at 150 - 155°C for 90 minutes. The slurry, containing precipitated product, was cooled to 90 ⁰ C and was poured into water and ice (1000 mL) which caused the product to crystallize. The mixture was stirred on ice for 30 minutes and was then filtered to isolate the quinazolinone as a tan powder which was washed well with water. After drying there was obtained 113 gm (88.4%) of the dimethoxyquinazolinone. TLC (silica, 10% methanol in methylene chloride) indicated the product was >95% purity by estimation. This was used in the next step without further purification. The quinazolinone will recrystallize from DMF or 1 ,2-dichlorobenzene if purification is necessary

Step 5

380.44 398.88

The dimethoxyquinazolinone (113 gm, 0.297 moles) was placed in a 1000 mL round bottom flask and phosphorous oxychloride (500 mL) was added. This mixture was stirred and refluxed for 8 hours to provide an orange slurry. Alternatively, the dimethoxyquinazolinone can be added slowly to the phosphorous oxychloride as the

latter is stirred, prior to the refluxing. After cooling, the slurry was filtered and washed with diethyl ether. The orange solid chloroquinazoline salt was stirred in a mixture of methylene chloride (1000 ml_) water (500 ml_) as a 20% solution of potassium carbonate was carefully added. After stirring for 15 minutes the methylene chloride solution was isolated and filtered to remove a small amount of insoluble material. The methylene chloride solution was concentrated under vacuum to a volume of about 200 ml_ and this solution was cooled on ice. The mixture was filtered and the solid was washed with a small amount of cold methylene chloride to give the chloroquinazoline as the solid, off-white free base in a yield of. 84.7 gm (71.5%). The chloroquinazoline will recrystallize from toluene.

Step 5 (alternate)

The dimethoxyquinazolinone (64.4 gm, 0.169 moles) was placed in a 2000 mL round bottom flask along with 1 ,2-dichlorobenzene (250 ml_) and this mixture was stirred and heated to 130 ⁰ C. At this temperature phosphorous oxychloride (POCI ₃ , 57.9 gm, 0.339 moles, 31.6 mL) was added slowly through a dropping funnel. As the phosphorous oxychloride was added, the initial slurry became an orange solution. This solution was stirred and heated at 130°C for one hour to provide an orange slurry. After cooling, the slurry was diluted with dichloromethane (500 mL) and this mixture was stirred in an ice bath. To the cold mixture water (250 mL) was added via the dropping funnel. After stirring for 15 minutes a solution of sodium hydroxide (67.8 gm, 1.70 moles) dissolved in water (125 mL) was added through the dropping funnel at a rate that kept the temperature below 20 ⁰ C. As the sodium hydroxide was added, a thick slurry formed and the color changed from orange to tan. A full five moles of base are needed to form the soluble free base of the chloroquinazoline. This mixture was stirred for 15 minutes after which the methylene chloride solution was isolated. The methylene chloride solution was stripped under vacuum and the 1 ,2-dichlorobenzene slurry was diluted with hexane (250 mL). This mixture was cooled on ice. The mixture was filtered and the solid was washed with a small amount of hexane to give the chloroquinazoline as the solid, off-white free base in a yield of 52.4 gm (77.7%).

Step 6

398.88 492.62 CPG 52364

The chloroquinazoline (84.7 gm, 0.212 moles) was combined with 2- morpholinoethyl amine (55.3 gm, 0.424 moles) and n-butanol (300 ml_). This slurry was stirred and heated at reflux for one hour to provide a yellow solution. The heating was stopped and the solution was cooled below 100 ⁰ C after which water (15 ml_) was added. This hot solution was seeded with CPG 52364 to induce crystallization. After stirring and cooling at room temperature overnight, the free base of CPG 52364, formed as a fine crystalline solid, was isolated by filtration and was washed with 2-propanol and dried at 100 ⁰ C to constant weight. The yield of CPG 52364, as tan crystals, was (96.07 gm, 92.0%). The free base can be easily recrystallized from anisole.

Step 7

492.62 737.82 CPG 52364 bisulfate

CPG 52364 (59.1 gm, 0.12 moles) was stirred in boiling methanol (600 ml_) and the hot slurry was treated, rapidly, with a solution of sulfuric acid (35.3 gm, 0.36 moles)

in methanol (200 ml_) which caused the formation of a yellow solution. Once all of the acid had been added, the solution was cooled on ice which caused the bisulfate salt to crystallize. The slurry was stirred at ice temperature for 30 minutes and was then filtered to isolate the product. The yellow solid was washed with methanol and then with diethyl ether. After drying at 100 ⁰ C to constant weight there was obtained 84.5 gm (89.5% based on the free base) of the bisulfate salt as a yellow powder.

Example 2 CPG 52364 Antagonizes TLR Signaling in Transfected Human Cells In Vitro

Concentrations of hydroxychloroquine and CPG 52364 necessary to inhibit agonist-stimulated human TLR9 and TLR8 signaling were assessed in human embryonic kidney (HEK293) cells that had been cotransfected with a reporter vector (NFκB-luciferase) and either recombinant human TLR9 (hTLR9) or TLR8 (hTLR8). Cells were incubated with varying concentrations of CPG 52364 or hydroxychloroquine for 1 hour before addition of specific agonists (unmethylated CpG DNA for hTLR9; RNA for hTLR8; or IL-1 for control cells, which had been transfected with reporter vector alone). After 16 hours, cells were harvested and luciferase activity measured by a luminometer. Inhibitory concentration (50%) values were calculated from these measurements. As shown in Table 1 , results from three to 10 independent experiments demonstrated that compared with hydroxychloroquine CPG 52364 is approximately 14- fold more potent antagonist of hTLR9 and 71 -fold more potent antagonist of hTLRδ.

Table 1: Human TLR9 and TLR8 Antagonism by CPG 52364 and Hydroxychloroquine in Transfected HEK Cells

IC ₅₀ = Inhibitory concentration (50%): Concentration that produced a 50% decrease in signaling in presence of agonist alone.

Example 3

CPG 52364 Antagonizes TLR Signaling in Transfected Human Cells and in Isolated

Human PBMCs In Vitro

CPG 52364 TLR7/8/9 antagonism was also evaluated in isolated human peripheral mononuclear cells (PBMCs). Human PBMCs stimulated by TLR7, TLR8, or TLR9 agonists in vitro elaborate various cytokines into culture medium that can be detected by ELISA. Inhibition of agonist-stimulated cytokine secretion was used as another assay to compare TLR antagonists. Because of the comparability of results of TLR8 and TLR9 antagonism in both the transfected HEK cells and isolated PBMCs, results of TLR7, TLR8, and TLR9 antagonism were pooled: TLR8 and TLR9 antagonism from assays with transfected HEK293 cells or isolated PBMCs and TLR7 antagonism from human PBMCs.

Pooled results from antagonist assays of agonist-stimulated responses of human TLR7, TLR8, and TLR9 by antimalarial reference compounds, which have been used to treat SLE, and CPG 52364 are summarized in Table 2. In addition to mean IC ₅₀ S from the pooled results, the response ratios of TLR8/TLR9 and TLR7/TLR9 antagonism have been calculated in order to compare cross-family antagonism. These ratios can be interpreted as the relative antagonism of a compound for one TLR versus another; lower ratios represent greater cross-antagonism between the receptors. Compared with hydroxychloroquine in these pooled results, CPG 52364 is a 6- fold more potent TLR9 antagonist with an IC ₅₀ of 18 nM (~ 9 ng/mL); a 52-fold more potent TLR8 antagonist with an IC ₅₀ of 35 nM (~ 17 ng/mL); and a 62-fold more potent TLR7 antagonist with an IC ₅₀ of 13 nM (~ 6 ng/mL). In contrast to hydroxychloroquine, which appears to be more specific for TLR9, CPG 52364 is a potent antagonist for all three members of the TLR7/8/9 family: TLR7, TLR8, and TLR9. Thus, CPG 52364 would appear to be an improvement over hydroxychloroquine not only in potency for TLR7/8/9 antagonism but also in cross-family antagonism.

Table 2: Antagonist Potency of CPG 52364 Relative to Reference Antimalarial Compounds (Pooled Data)

IC _5O = Inhibitory concentration (50%): Concentration that produced a 50% decrease in signaling in presence of agonist alone

Example 4 CPG 52364 Inhibits TLR9-Stimulated IP-10 Production In Vivo

Inhibitory effects of CPG 52364 and antimalarials were compared in vivo by evaluation of the inhibitory dose 50% (ID ₅₀ ) of these agents on TLR9- and ^" Unstimulated IP-10 production in mice. Mice were given various doses of CPG 52364 or antimalarial agents or PBS (vehicle controls) either by intraperitoneal (IP) injection or oral gavage before administration of a TLR9 or TLR7 agonist. Agonist activity was assessed by measurement of plasma IP-10 concentration at three hours after agonist administration.

Comparison of antagonism of TLR9-stimulated IP-10 production demonstrated that CPG-52364, regardless of whether administered by IP or oral route, is a more potent antagonist than any of the antimalarials (Table 3). After IP administration, CPG 52364 was approximately 6-fold to 43-fold more potent than the antimalarials. After oral administration, CPG 52364 was approximately 37-fold more potent than chloroquine and more than 200-fold more potent than hydroxychloroquine.

Table 3: Antagonism of TLR9-Stimulated IP-10 Production in Mice After IP or Oral Administration

Antagonist

IP Injection Oral Gavage

CPG 52364 0.93 0.6 Quinacrine 5.3 ND Chloroquine 8.7 22.4 Hydroxychloroquine 39.5 133.1

ID ₅₀ = Inhibitory dose 50%: Dose that produced a 50% decrease in TLR9-stimulated

IP-10 production, (mg/kg based on 20 g mouse). ND = Not Determined.

Example 5 CPG 52364 Reduces Anti-dsDNA Titers in Murine Autoimmune Model of SLE

The activity of CPG 52364 was compared with that of hydroxychloroquine in the well-studied mouse model of lupus, lupus-prone MRL Ipr/lpr mice that have many disease sequelae similar to human SLE. Andrews BS et al., J Exp Med. 1978; Nov 1 ; 148(5): 1198-1215. Affected mice develop spontaneous systemic autoimmunity with both molecular (IFN signature) and clinical (autoantibody production and glomerulonephritis) features similar to human SLE. The lpr (lymphoproliferative) mutation arose as a spontaneous mutation on the MRL genetic background and was found to be a null allele for Fas (CD95), a member of the tumor necrosis factor (TNF) family of receptors involved in programmed cell death. Mice develop serum autoantibody by 6 weeks of age and evidence of lymphadenopathy by 12 weeks of age. Massive lymphadenopathy observed in these animals has been shown to be due to the accumulation of an unusual population of B220+CD4-CD8- (double negative) T cells, a condition not found in human lupus. Animals develop progressive renal disease and marked proteinuria by 16 weeks of age. Mortality is approximately 50% by 20 weeks of age. Therefore, these animals have an inherent genetic mutation contributing to aggressive disease. The TLRs presumably have no direct role in the genetically driven etiology of disease but may contribute to disease exacerbation (Leadbetter EA et al.,

Nature. 2002; Apr 11 ;416(6881 ):603-607; Krieg AM, Nature Immunology. 2002; 3(5):423-424), thus allowing any potential TLR contribution to be monitored.

The ability of CPG 52364 and hydroxychloroquine to inhibit or reduce disease development was evaluated in MRL Ipr/lpr mice. Female MRL Ipr/lpr mice (n=10/group; n=20 for placebo group) were treated with daily IP injections of CPG 52364 (51 or 154 μg; approximately 2.55 or 7.7 mg/kg) or hydroxychloroquine sulfate (500 μg; approximately 25 mg/kg) for 12 weeks starting from 4 weeks of age. At the end of treatment, animals were euthanized and serum was analyzed for anti-dsDNA antibodies (Crithidia luciliae assay). Anti-dsDNA antibodies are correlated with human SLE and the reduction in anti-dsDNA antibodies is believed to be of potential clinical benefit.

Mice treated with CPG 52364 had a significant reduction in mean anti-dsDNA titers (FIG. 1). Data in FIG. 1 are presented as group mean (±SD). The scoring was performed relative to a positive control serum from a MRL-/pr mouse at 21 weeks of age given a score of 3.0 and the negative control serum from a BALB/c mouse given a score of 0. Hydroxychloroquine was ineffective at the maximum tolerated dose of 500 μg daily.

Example 6

Absorption and Pharmacokinetics of CPG 52364 in Rats Following Single Dose Oral Administration

Pharmacokinetics in whole blood and oral bioavailability of CPG 52364 was assessed in rats (3/sex/dose) after single dose administration (41 mg/kg) (FIG. 2). After oral administration, CPG 52364 was absorbed readily in blood with mean T _max of 2.3 hours in males and 3.3 hours in females and eliminated from blood slowly with mean apparent half-life of 13.1 hours in males and 11.4 hours in females. Mean oral bioavailability of CPG 52364 (normalized for dose and calculated based on AUCo-24h) was approximately 52% in males and 42% in females.

Example 7 Absorption and Pharmacokinetics of CPG 52364 in Rats Following Single Dose

Intravenous Administration

After a single IV dose (8.2 mg/kg) to rats, CPG 52364 was eliminated slowly from blood with a mean elimination half-life of 22.1 hours in males and 18.4 hours in females.

However, these values may be underestimated because samples were only collected for up to 24 hours after injection. The mean volume of distribution at steady state (V _dss ) was more than 30-fold greater than the volume of total body water in the rat (Foy JM et al., J. Physiol. 1960 Nov.;154: 169-176), which suggests extensive distribution of CPG 52364 outside the blood.

Example 8

Absorption and Pharmacokinetics of CPG 52364 in Dogs Following Single Dose

Intravenous Administration

Blood pharmacokinetic parameters were measured in beagle dogs that received

CPG 52364 as single IV dose (8.2 mg/kg). After IV administration, CPG 52364 was eliminated slowly from blood with a mean elimination half-life of approximately 22 to 24 hours. CPG 52364 appeared to be well-distributed outside of the blood compartment based on a mean volume of distribution at steady state (V _dss ) greater than 7200 ml_/kg, which exceeds the range of estimated total body water for female beagle dogs (approximately 537 to 589 mL/kg). Wamberg S et al., J Nutr. 2002; 132 (6 Suppl. 2):1711S-1713S. No gender differences were apparent.

Example 9 Absorption and Pharmacokinetics of CPG 52364 in Monkeys Following Single Dose

Oral Administration

Pharmacokinetic parameters in whole blood and oral bioavailability of CPG 52364 were characterized in cynomolgus monkeys (3/sex/dose) after single dose oral administration (41 or 50 mg/kg). After oral administration (41 mg/kg), CPG 52364 was absorbed slowly with mean T _ma χ of 7.3 hours for both males and females (FIG. 3). CPG 52364 appeared to be eliminated slowly from blood and was still present in blood at the last measured time point of 24 hours. Mean oral bioavailability of CPG 52364 (normalized for dose) was 31.0% for males and 35.8% for females, when calculated based on AUCo-24h- No noteworthy gender differences were apparent. Because an elimination phase could not be calculated accurately in this study due to insufficient time points, a second study was conducted in which 3 monkeys/sex/group were given 50 mg/kg orally and samples were collected up to 336 hours post-dose. In this study, CPG 52364 was absorbed slowly into blood with a mean T _max of 9.3 hours in males and 10.7

hours in females (FIG. 4). CPG 52364 was eliminated slowly from blood with a mean apparent half-life of 145.4 hours for males and 161.4 hours for females.

Example 10 Absorption and Pharmacokinetics of CPG 52364 in Monkeys Following Single Dose

Intravenous Administration

Pharmacokinetic parameters were assessed in the same monkeys as in Example 9 with a 7-day wash-out period between administration of 41 mg/kg orally and 8.2 mg/kg IV. As mentioned in Example 9, pharmacokinetic parameters in whole blood and intravenous bioavailability of CPG 52364 were characterized in cynomolgus monkeys (3/sex/dose) after single dose IV administration (8.2 mg/kg). After IV administration, CPG 52364 appeared to be well distributed outside of the blood compartment with a mean volume of distribution at steady state (V _dss ) of more than 2161 mL/kg, which is greater than the estimated volume of total body water in cynomolgus monkeys (approximately 558 mL/kg). Sweet AY et al., Am J Physiol. 1961 ; 201(6):975-979.

EQUIVALENTS

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.