WO2011084725A1

WO2011084725A1 - 4-AMINO-2-(ETHOXYMETHYL)-1-(2-HYDROXY-2-METHYLPROPYL)-1H-IMIDAZO[4,5-c]QUINOLIN-5-IUM 1-HYDROXYNAPHTHALENE-2-CARBOXYLATE COMPOSITIONS AND METHODS

Info

Publication number: WO2011084725A1
Application number: PCT/US2010/061194
Authority: WO
Inventors: Zheng Zhi Wu; Ronnie Ortiz; Lester I. Harrison; Joseph M. Beaurline
Original assignee: 3M Innovative Properties Company
Priority date: 2009-12-21
Filing date: 2010-12-20
Publication date: 2011-07-14

Abstract

A compound of formula:[ I ] Pharmaceutical compositions containing the compound, and methods of inducing cytokine biosynthesis in an animal, treating a viral infection and/or treating a neoplastic disease in an animal by administering an effective amount of the compound to the animal are also disclosed.

Description

4-AMINO-2-(ETHOXYMETHYL)-l-(2-HYDROXY-2-METHYLPROPYL)-lH- IMIDAZO [4,5-c] QUINOLIN-5-IUM l-HYDROXYNAPHTHALENE-2- CARBOXYLATE COMPOSITIONS AND METHODS

Cross-Reference to Related Application

This application claims the benefit of U. S. Provisional Application No.

61/288,598, filed December 21, 2009, the disclosure of which is incorporated by reference herein in its entirety.

Background

There has been an effort in recent years, with significant success, to discover new drug compounds that act by stimulating certain key aspects of the immune system, as well as by suppressing certain other aspects (see, e.g., U.S. Pat. Nos. 6,039,969 (Tomai et al.) and 6,200,592 (Tomai et al). These compounds, referred to herein as immune response modifiers (IRMs), appear to act through basic immune system mechanisms known as Tolllike receptors (TLRs) to induce selected cytokine biosynthesis, induction of co-stimulatory molecules, and increased antigen-presenting capacity.

Many IRMs may be useful for treating a wide variety of diseases and conditions. For example, certain IRMs may be useful for treating viral diseases (e.g., human papilloma virus, hepatitis, herpes), neoplasias (e.g., basal cell carcinoma, squamous cell carcinoma, actinic keratosis, melanoma), T_R2 -mediated diseases (e.g., asthma, allergic rhinitis, atopic dermatitis), and auto-immune diseases. Certain IRMs may also be useful, for example, as vaccine adjuvants.

Many known IRMs are imidazoquinoline amine derivatives (see, e.g., U.S. Pat. No. 4,689,338 (Gerster)), but other compound classes are known as well (see, e.g., U.S. Pat. Nos. 5,446,153 (Lindstrom et al); 6,194,425 (Gerster et al); and 6,110,929 (Gerster et al.); and International Publication Number WO2005/079195 (Hays et al.)) while more are still being discovered.

In view of the great therapeutic potential for IRMs in the treatment of a wide variety of diseases and conditions, and despite the important work that has already been done, new compounds that can modulate the immune response, by induction of cytokine biosynthesis or other mechanisms, are still needed. Summary

The present invention provides, in one aspect, a new compound useful for inducing cytokine biosynthesis in animals. The compound (i.e., 4-amino-2-(ethoxymethyl)-l-(2- hydroxy-2-methylpropyl)-lH-imidazo[4,5-c]quinolin-5-ium l-hydroxynaphthalene-2- carboxylate) has the following formula (I):

The compound of Formula I has unexpectedly different pharmacological properties than l-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]-2-methylpropan-2-ol (i.e., Resiquimod). For example, the compound of Formula I is unexpectedly effective at inducing the biosynthesis of tumor necrosis factor (TNF-a). Furthermore, the compound of Formula I is unexpectedly more effective at inducing the biosynthesis of TNF-a when delivered from an oil-based carrier than when delivered from an aqueous carrier. Also, among imidazoquinoline amine compounds, the compound of Formula I has unique solid state physicochemical properties. For example, it has low solubility in both aqueous and oil-based carriers and a high melting point relative to many other imidazoquinoline amine compounds.

The combination of beneficial solid state physicochemical properties and remarkable pharmacological properties may render the compound of Formula I suitable for a variety of applications. For example, the compound of Formula I may be useful as a vaccine adjuvant or for delivery into a localized tissue region of a subject (e.g., into a tumor).

The ability to induce cytokine biosynthesis in animals makes the compound of Formula I useful for treating a variety of conditions such as viral diseases and tumors that are responsive to such changes in the immune response. Accordingly, the present invention further provides methods of inducing cytokine biosynthesis in an animal, treating a viral infection and/or treating a neoplastic disease in an animal by administering an effective amount of a compound of Formula I to the animal. The present invention further provides a method of vaccinating an animal comprising administering an effective amount of a compound of Formula I to the animal as a vaccine adjuvant.

The invention further provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of Formula I. In some embodiments, the pharmaceutical composition further comprises an antigen (e.g., a vaccine). In some embodiments of the pharmaceutical composition, the compound of Formula I is dispersed (e.g., in particulate form) in the pharmaceutically acceptable carrier.

The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

As used herein, "a", "an", "the", "at least one", and "one or more" are used interchangeably.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

"Ameliorate" refers to any reduction in the extent, severity, frequency, and/or likelihood of a symptom or clinical sign characteristic of a particular condition.

"Antigen" refers to any substance that may be bound by an antibody in a manner that is immunospecific to some degree.

"Induce" and variations thereof refer to any measurable increase in cellular activity. For example, induction of an immune response may include, for example, an increase in the production of a cytokine, activation, proliferation, or maturation of a population of immune cells, and/or other indicator of increased immune function.

"Symptom" refers to any subjective evidence of disease or of a patient's condition.

"Therapeutic" and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition.

"Treat" or variations thereof refer to reducing, limiting progression, ameliorating, preventing, or resolving, to any extent, the symptoms or signs related to a condition.

The term "solvate" refers to an amorphous or crystalline material having one or more molecules of associated solvent, for example, a solvate may be a crystalline material having the associated solvent within the crystal lattice. The associated solvent may be water, and a "hydrate" may be formed. The term "solvate" as used herein encompasses "hydrate".

The compound 4-amino-2-(ethoxymethyl)- 1 -(2-hydroxy-2-methylpropyl)- 1H- imidazo[4,5-c]quinolin-5-ium l-hydroxynaphthalene-2-carboxylate described herein may be in any of its pharmaceutically acceptable forms including amorphous solid, crystalline solid, semi-solid, solvate (e.g., hydrate), wholly or partially dissolved (e.g., in a pharmaceutical composition), or dispersed in a pharmaceutically acceptable carrier. It should be understood that the term "compound" includes any or all such forms, whether explicitly stated or not; at times, "solvate" is explicitly stated.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

Detailed Description

The present invention provides, for example, a compound of Formula I. In some embodiments, the compound of Formula I is in solid form. In some embodiments, the compound of Formula I is in particulate form. In some embodiments, the compound of Formula I is in amorphous form. In some embodiments, the compound of Formula I is in crystalline form. In some embodiments, the compound of Formula I is in solvate (e.g., hydrate) form. In some embodiments, the compound of Formula I is in at least partially dissolved form (e.g., partially or completely dissolved). Pharmaceutical compositions disclosed herein may include the compound of Formula I in any of these forms or any combination of these forms.

The compound of Formula I can conveniently be prepared by combining, for example, 1 -[4-amino-2-(ethoxymethyl)- lH-imidazo[4,5-c]quinolin- 1 -yl]-2-methylpropan- 2-ol with l-hydroxynaphthalene-2-carboxylic acid (i.e., l-hydroxy-2-naphthoic acid) in a suitable solvent (e.g., a lower alcohol such as methanol, water, or a combination thereof). Each of 1 -[4-amino-2-(ethoxymethyl)- lH-imidazo[4,5-c]quinolin- 1 -yl]-2-methylpropan- 2-ol and l-hydroxynaphthalene-2-carboxylic acid may be dissolved in the suitable solvent before combining the respective solutions or the compounds can be added neat to the suitable solvent. The mixture of l-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin-l-yl]-2-methylpropan-2-ol and l-hydroxynaphthalene-2-carboxylic acid is typically heated at an elevated temperature (e.g., up to the boiling point of the suitable solvent) and optionally filtered to form a solution. Upon cooling the solution, typically, 4- amino-2-(ethoxymethyl)-l-(2-hydroxy-2-methylpropyl)-lH-imidazo[4,5-c]quinolin-5-ium l-hydroxynaphthalene-2-carboxylate will precipitate from solution and can be isolated using conventional methods (e.g., filtration or decanting).

The preparation of l-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]- 2-methylpropan-2-ol can be carried out, for example, according to the methods described in Example 99 of U. S. Pat. No. 5,389,640 (Gerster et al). Other routes to 1H- imidazo[4,5-c]quinolin-4-amine compounds are known and can be useful for making l-[4- amino-2-(ethoxymethyl)- lH-imidazo[4,5-c]quinolin- 1 -yl]-2-methylpropan-2-ol using the appropriate reagents and intermediates; see, e.g., U. S. Pat. Nos. 4,988,815 (Andre et al), 5,367,076 (Gerster), and 5,395,937 (Nikolaides et al.) and International App. Pub. No.

WO2007/035935 (Krepski et al.). l-Hydroxynaphthalene-2-carboxylic acid is available, for example, from a variety of chemical suppliers. Salts of l-[4-amino-2-(ethoxymethyl)- lH-imidazo[4,5-c]quinolin- 1 -yl]-2-methylpropan-2-ol and 1 -hydroxynaphthalene-2- carboxylic acid may also be useful for the preparation of the compound of Formula I.

In some embodiments of the pharmaceutical compositions and methods disclosed herein, the compound of Formula I is administered in the form of solid particles. The solid particles of the compound of Formula I may be amorphous, crystalline, or a combination thereof, and in the form of, for example, fine powders or liquid suspensions such as colloidal suspensions, liquid crystal suspensions, or may be included within gels, creams, or other semi-solid forms. Solid particles comprising the compound of Formula I may be essentially pure or may include carriers or fillers.

The compound of Formula I in solid form can be manipulated into a desired particle size for particulate delivery. The average size of the particles may be less than 1 μιη, or from about 1 to 20 or 1 to 100 μιη, or larger than 100 μιη, depending on the desired formulation and condition to be treated. Methods for controlling the particle size include milling, sieving, recrystallization, reprecipitation (e.g., using various surfactants and/or other surface tension-modifying components), spray drying, and electrostatic techniques. For example, particles of the compound of Formula I can be reduced in size from 10 micrometers (μιη) to 1 μιη using reprecipitation by dissolving the 10 μιη particles in 1- methyl-2-pyrrolidone (NMP) and then adding a solution 0.05% of a surfactant (e.g., available from Uniqema under the trade designation "TWEEN 80") in 0.03 molar Citrate Buffered Saline, pH 5. In another example, a colloidal suspension may be formed using the compound of Formula I dissolved in a water-miscible organic injectable solvent (e.g. N-methyl pyrrolidone or NMP) in a first package compartment. A second package compartment may contain, for example, an aqueous solution containing surfactants (e.g. surfactant "TWEEN 80") and optionally an antigen. Prior to administration to a localized tissue region, such as subcutaneous injection, colloidal particles of the compound of

Formula I may be formed by mixing the contents of the first and second compartments, causing precipitation of the compound of Formula I into fine particles. Where

appropriate, a colloidal suspension of a compound of Formula I can be made prior to packaging, with instructions for shaking or vortexing prior to administration. In a further example, a suspension may be formed using the compound of Formula I dissolved in an aqueous and/or organic solvent at a suitable pH and then adding the solution to a solvent in which the compound of Formula I is less soluble using high shear stirring to cause precipitation of small particles (e.g., up to 100 μιη). Particle size and/or the quality of a dispersion of the compound of Formula I can also be altered, for example, in a

pharmaceutical composition by homogenization, for example, with a rotor and stator or with a high pressure check valve (e.g., available from Avestin, Inc., Ontario, Canada).

In some embodiments of the methods disclosed herein, the compound of Formula I (e.g., in a pharmaceutical composition disclosed herein) is administered to a localized tissue region. In some of these embodiments, the compound of Formula I is administered in particulate form. Particles of the compound of Formula I may have a limited solubility, for example, in a localized tissue region so that once delivered to the region they dissolve slowly over an extended period, providing extended residence times in a localized area. In other embodiments, solid particle suspensions of the compound of Formula I may dissolve relatively quickly upon delivery (e.g., within an hour). The rate at which the particles dissolve may depend, for example, on particle size and the physicochemical properties or the environments that any carriers or fillers may provide, if used. The compound of Formula I may induce cytokines in a localized tissue region with or without dissolving or dissociating. A "localized tissue region" will generally be a relatively small portion of the body, e.g., less than 10 percent by volume, and often less than 1 percent by volume. For example, depending on the size of, e.g., a solid tumor or cancerous organ, the localized tissue region will typically be on the order of no more than about 500 cubic centimeters (cm³), often less than about 100 cm³, and in many instances 10 cm³ or less. For some applications the localized tissue region will be 1 cm³ or less (e.g., for small tumor nodules, viral lesions, or vaccination sites). However, in certain instances the localized tissue region may be a particularly large region, up to several liters, for example, to treat metastasized cancer within the entire peritoneal cavity. The localized tissue region may be, for example, a cancer, a viral infected lesion, or organ, or vaccination site. It may be, for example, a solid tumor, lymph tissue, reticuloendothelial system, bone marrow, mucosal tissue, etc. The localized tissue region may be, e.g., a breast cancer tumor, stomach cancer tumor, lung cancer tumor, head or neck cancer tumor, colorectal cancer tumor, renal cell carcinoma tumor, pancreatic cancer tumor, basal cell carcinoma tumor, cervical cancer tumor, melanoma cancer tumor, prostate cancer tumor, ovarian cancer tumor, or bladder cancer tumor. Delivery of the compound of Formula I to a localized tissue region may be in conjunction with image guiding techniques using, for example, ultrasound, MRI, and real-time X-ray (fluoroscopy).

In some embodiments of the pharmaceutical compositions and methods disclosed herein, the pharmaceutical composition further comprises an antigen in an amount effective to generate an immune response against the antigen. In some embodiments, the antigen is a vaccine. Vaccines include any material administered to raise either humoral and/or cell mediated immune response, such as live or attenuated viral and bacterial immunogens and inactivated viral, tumor-derived, protozoal, organism-derived, fungal, and bacterial immunogens, toxoids, toxins, polysaccharides, proteins, glycoproteins, peptides, cellular vaccines (e.g., using dendritic cells), DNA vaccines, recombinant proteins, glycoproteins, and peptides. Exemplary vaccines include vaccines for cancer, BCG, cholera, plague, typhoid, hepatitis A, B, and C, influenza A and B, parainfluenza, polio, rabies, measles, mumps, rubella, yellow fever, tetanus, diphtheria, hemophilus influenza b, tuberculosis, meningococcal and pneumococcal vaccines, adenovirus, HIV, chicken pox, cytomegalovirus, dengue, feline leukemia, fowl plague, HSV-1 and HSV-2, hog cholera, Japanese encephalitis, respiratory syncytial virus, rotavirus, papilloma virus, severe acute respiratory syndrome (SARS), anthrax, and yellow fever. See also, e.g., vaccines disclosed in International Publication No. WO 02/24225 (Thomsen et al.).

Antigens can be co-delivered with a compound of Formula I, for example, in admixture in a pharmaceutical composition according to the present invention. Such pharmaceutical compositions may include the compound in Formula I in particulate form. Particles of the compound of Formula I may dissolve slowly over an extended period or may be processed in the solid state, providing extended residence times at the vaccination site. This may allow the compound of Formula I to reach, for example, antigen presenting cells at or around the same time as the antigen. In other embodiments, the compound of Formula I and the antigen may be administered separately at or about the same time. Co- delivering a vaccine adjuvant (e.g., an IRM compound such as a compound of Formula I) and an antigen to an immune cell can increase the immune response to the antigen and improve antigen-specific immunological memory. Optimal delivery may occur, for example, when the adjuvant and the antigen are processed within an antigen presenting cell at the same time.

In addition to the delivery methods mentioned specifically above, a compound of Formula I (e.g., in a pharmaceutical composition disclosed herein) may be administered in any other suitable manner (e.g., non-parenterally or parenterally). As used herein, non- parenterally refers to administration through the digestive tract, including by oral ingestion. Parenterally refers to administration other than through the digestive tract which would include nasal (e.g., transmucosally by inhalation), topical, ophthalmic, and buccal adminstration, but in practice usually refers to injection (e.g., intravenous, intramuscular, subcutaneous, intratumoral, or transdermal) using, for example, conventional needle injection, injection using a microneedle array, or any other known method of injection.

The compound of Formula I may be provided in any pharmaceutical composition suitable for administration to a subject and may be present in the pharmaceutical composition in any suitable form (e.g., a solution, a suspension, an emulsion, or any form of mixture). The pharmaceutical composition may be formulated with any

pharmaceutically acceptable excipient, carrier, or vehicle. In some embodiments, the pharmaceutically acceptable carrier comprises water (e.g., phosphate or citrate buffered saline). In some embodiments, the pharmaceutically acceptable carrier comprises an oil (e.g., corn, sesame, squalene, cottonseed, soybean, or safflower oil). The pharmaceutical composition may further include one or more additives including skin penetration enhancers, colorants, fragrances, flavorings, moisturizers, thickeners, suspending agents, surfactants, and dispersing agents.

In addition to antigens specifically described above, the pharmaceutical compositions and methods of the present disclosure can include other additional active agents, e.g., in admixture or administered separately. Such additional agents can include a chemotherapeutic agent, a cytotoxoid agent, an antibody, an antiviral agent, a cytokine, a tumor necrosis factor receptor (TNFR) agonist, or an additional immune response modifier. TNFR agonists that may be delivered in conjunction with the compound of Formula I include CD40 receptor agonists, such as disclosed in copending application U.S. Patent Publication 2004/0141950 (Noelle et al). Other active ingredients for use in combination with an IRM preparation of the present invention include those disclosed in, e.g., U.S. Patent Publication No. 2003/0139364 (Krieg et al).

In some embodiments, a pharmaceutical composition according to the present invention may be a conventional topical dosage formulation (e.g., a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, or a lotion). Suitable types of

formulations are described, for example, in U.S. Pat. No. 5,238,944 (Wick et al); U.S. Pat. No. 5,939,090 (Beaurline et al); U.S. Pat. No. 6,245,776 (Skwierczynski et al); European Patent No. EP 0394026 (Schultz); and U.S. Patent Publication No. 2003/0199538

(Skwierczynski et al).

The compound of Formula I has been shown to induce the production of TNF-a in rats as described in the Examples below. The ability to induce TNF production indicates that the compound of Formula I is useful as an immune response modifier that can modulate the immune response in a number of different ways, rendering it useful in the treatment of a variety of disorders. Other cytokines whose production may be induced by the administration of the compound of Formula I generally include Type I interferons (e.g., INF- a), IL-1, IL-6, IL-8, IL-10, IL-12, MIP-1, MCP-1, and a variety of other cytokines. Among other effects, these and other cytokines inhibit virus production and tumor cell growth, making the compound of Formula I useful in the treatment of viral diseases and neoplastic diseases. Accordingly, the invention provides a method of inducing cytokine biosynthesis in an animal comprising administering an effective amount of the compound of Formula I (e.g., in a pharmaceutical composition) to the animal. The animal to which the compound of Formula I is administered for induction of cytokine biosynthesis may have a disease (e.g., a viral or neoplastic disease), and administration of the compound may provide therapeutic treatment. Also, the compound of Formula I may be administered to the animal before the animal acquires the disease so that administration of the compound of Formula I may provide a prophylactic treatment.

In addition to the ability to induce the production of cytokines, the compound of Formula I may affect other aspects of the innate immune response. For example, natural killer cell activity may be stimulated, an effect that may be due to cytokine induction. IRM activity of the compound of Formula I also may include activating macrophages, which in turn stimulate secretion of nitric oxide and the production of additional cytokines. IRM activity of the compound of Formula I also may include inducing cytokine production by T cells, activating T cells specific to an antigen, and/or activating dendritic cells. Further, IRM activity of the compound of Formula I may include proliferation and differentiation of B-lymphocytes. IRM activity of the compound of

Formula I also may affect the acquired immune response. For example, IRM activity can include inducing the production of the T helper type 1 (T_H1) cytokine IFN-γ and/or inhibiting the production of the T helper type 2 (T_R2) cytokines IL-4, IL-5 and/or IL-13.

Exemplary conditions that may be treated by administering the compound of Formula I include:

(a) viral diseases such as diseases resulting from infection by an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), a poxvirus (e.g., an orthopoxvirus such as variola or vaccinia, or molluscum contagiosum), a picornavirus (e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g., influenzavirus), a paramyxovirus (e.g.,

parainfluenzavirus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), a coronavirus (e.g., SARS), a papovavirus (e.g., papillomaviruses, such as those that cause genital warts, common warts, or plantar warts), a hepadnavirus (e.g., hepatitis B virus), a flavivirus (e.g., hepatitis C virus or Dengue virus), or a retrovirus (e.g., a lentivirus such as HIV);

(b) bacterial diseases such as diseases resulting from infection by bacteria of, for example, the genus Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus,

Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, or Bordetella;

(c) other infectious diseases such as chlamydia, fungal diseases (e.g., candidiasis, aspergillosis, histoplasmosis, or cryptococcal meningitis), or parasitic diseases (e.g., malaria, Pneumocystis carnii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosome infection);

(d) neoplastic diseases such as intraepithelial neoplasias, cervical dysplasia, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, Kaposi's sarcoma, melanoma, leukemias (e.g., myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, B- cell lymphoma, and hairy cell leukemia), breast cancer, lung cancer, prostate cancer, colon cancer, and other cancers;

(e) T_H2 -mediated, atopic diseases such as atopic dermatitis or eczema,

eosinophilia, asthma, allergy, allergic rhinitis, and Ommen's syndrome;

(f) certain autoimmune diseases such as systemic lupus erythematosus, essential thrombocythaemia, multiple sclerosis, discoid lupus, and alopecia areata; and

(g) diseases associated with wound repair such as inhibition of keloid formation and other types of scarring (e.g., enhancing wound healing, including chronic wounds).

The mechanism for the antiviral and antitumor activity of the compound of

Formula I may be due in substantial part to enhancement of the immune response by induction of various important cytokines (e.g., at least one of tumor necrosis factor, interferons, or interleukins). Such compounds have been shown to stimulate a rapid release of certain monocyte/macrophage-derived cytokines and are also capable of stimulating B cells to secrete antibodies which play an important role in these IRM compounds' antiviral and antitumor activities.

It will be understood that in the treatment of the diseases mentioned above, for example, the compound of Formula I can be used in combination with other therapies such as the active agents mentioned above and other procedures (e.g., chemoablation, laser ablation, cryotherapy, and surgical excision).

An amount of a compound effective to induce cytokine biosynthesis is an amount sufficient to cause one or more cell types, such as monocytes, macrophages, dendritic cells and B-cells to produce an amount of one or more cytokines such as, for example, IFN- a, TNF- a, IL-1, IL-6, IL-10 and IL-12 that is increased over a background level of such cytokines. The precise amount will vary according to factors known in the art but is expected to be a dose of about 100 nanograms per kilograms (ng/kg) to about 50 milligrams per kilogram (mg/kg), in some embodiments about 10 micrograms per kilogram ^g/kg) to about 5 mg/kg. The invention also provides a method of treating a viral infection in an animal and a method of treating a neoplastic disease in an animal comprising administering an effective amount of a compound or pharmaceutical composition of the invention to the animal. An amount effective to treat or inhibit a viral infection is an amount that will cause a reduction in one or more of the manifestations of viral infection, such as viral lesions, viral load, rate of virus production, and mortality as compared to untreated control animals. The precise amount that is effective for such treatment will vary according to factors known in the art but is expected to be a dose of about 100 ng/kg to about 50 mg/kg, in some embodiments about 10 μg/kg to about 5 nig/kg. An amount of a compound or pharmaceutical composition effective to treat a neoplastic condition is an amount that will cause a reduction in tumor size or in the number of tumor foci. Again, the precise amount will vary according to factors known in the art but is expected to be a dose of about 100 ng/kg to about 50 mg/kg, in some embodiments about 10 μg/kg to about 5 mg/kg. The methods of the present invention may be performed on any suitable subject. Suitable subjects include animals such as humans, non-human primates, rodents, dogs, cats, horses, pigs, sheep, goats, or cows.

The composition of a formulation suitable for practicing the invention, the precise amount of a compound of Formula I effective for methods according to the present invention, and the dosing regimen, for example, will vary according to factors known in the art including the nature of the carrier, the state of the subject's immune system (e.g., suppressed, compromised, stimulated), the method of administering the compound of Formula I, and the species to which the formulation is being administered. Accordingly, it is not practical to set forth generally the composition of a formulation that includes a compound of Formula I, an amount of a compound of Formula I that constitutes an effective amount, or a dosing regimen that is effective for all possible applications. Those of ordinary skill in the art, however, can readily determine appropriate formulations, amounts of the compound of Formula I, and dosing regimen with due consideration of such factors.

In some embodiments, the methods of the present invention include administering a compound of Formula I to a subject in a formulation, for example, having a

concentration of the compound from about 0.0001% to about 20% (unless otherwise indicated, all percentages provided herein are weight/weight with respect to the total formulation), although in some embodiments the compound of Formula I may be administered using a formulation that provides the compound in a concentration outside of this range. In some embodiments, the method includes administering to a subject a formulation that includes from about 0.01% to about 1% of the compound of Formula I, for example, a formulation that includes about 0.1 % to about 0.5% compound of Formula I.

In some embodiments, the methods of the present invention include administering sufficient compound to provide a dose of, for example, from about 100 ng/kg to about 50 nig/kg to the subject, although in some embodiments the methods may be performed by administering compound in a dose outside this range. In some of these embodiments, the method includes administering sufficient compound to provide a dose of from about 10 μg/kg to about 5 mg/kg to the subject, for example, a dose of from about 100 μg/kg to about 1 mg/kg. In some embodiments, the methods of the present invention may include administering sufficient compound to provide a dose of, for example, from about 0.01 mg/m² to about 10 mg/m². Alternatively, the dose may be calculated using actual body weight obtained just prior to the beginning of a treatment course. For the dosages calculated in this way, body surface area (m²) is calculated prior to the beginning of the treatment course using the Dubois method: m 2 = (wt kg 0 ^' 425 x height cm 0 ^' 725 ) x 0.007184.

In some embodiments of the methods disclosed herein, the compound of Formula I may be administered, for example, from a single dose to multiple doses per week, although in some embodiments the methods of the present invention may be performed by administering the compound of Formula I at a frequency outside this range. In some embodiments, the compound of Formula I may be administered from about once per month to about five times per week. In some embodiments, the compound of Formula I is administered once per week. Selected Embodiments of the Invention

In a first embodiment, the present invention provides a compound of formula:

In a second embodiment, the present invention provides the compound of the first embodiment in solid form.

In a third embodiment, the present invention provides the compound of the first or second embodiment in amorphous form.

In a fourth embodiment, the present invention provides the compound of the first, second, or third embodiment in particulate form.

In a fifth embodiment, the present invention provides the compound of the second embodiment in crystalline form.

In a sixth embodiment, the present invention provides the compound of the fifth embodiment in solvate form.

In a seventh embodiment, the present invention provides the compound of the first embodiment in at least partially dissolved form.

In an eighth embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the compound of any one of the first to seventh embodiments.

In a ninth embodiment, the present invention provides a pharmaceutical composition comprising a dispersion of a therapeutically effective amount of the compound of any one of the first to fifth embodiments in a pharmaceutically acceptable carrier.

In a tenth embodiment, the present invention provides the pharmaceutical composition according to the ninth embodiment, wherein the dispersion is homogenized.

In an eleventh embodiment, the present invention provides the pharmaceutical composition according to any one of the eighth to tenth embodiments, further comprising an antigen in an amount effective to generate an immune response against the antigen. In a twelfth embodiment, the present invention provides the pharmaceutical composition according to the eleventh embodiment wherein the antigen is a bacterial antigen.

In a thirteenth embodiment, the present invention provides the pharmaceutical composition according to the eleventh embodiment wherein the antigen is a viral antigen.

In a fourteenth embodiment, the present invention provides the pharmaceutical composition according to the eleventh embodiment wherein the antigen is a tumor antigen.

In a fifteenth embodiment, the present invention provides the pharmaceutical composition according to any one of the eighth to fourteenth embodiments, wherein the pharmaceutically acceptable carrier comprises an oil, for example, at least one of corn oil, sesame oil, squalene oil, cottonseed oil, soybean oil, or safflower oil.

In a sixteenth embodiment, the present invention provides a method of vaccinating an animal comprising administering an effective amount of the compound of any one of embodiments 1 to 7 or the pharmaceutical composition of any one of embodiments 8 to 15 to the animal as a vaccine adjuvant.

In a seventeenth embodiment, the present invention provides a method of inducing cytokine biosynthesis in an animal, the method comprising administering an effective amount of the compound of any one of embodiments 1 to 7 or the pharmaceutical composition of any one of embodiments 8 to 15 to the animal.

In an eighteenth embodiment, the present invention provides a method of treating a viral disease in an animal, the method comprising administering to the animal the compound of any one of embodiments 1 to 7 or the pharmaceutical composition of any one of embodiments 8 to 10 or 13 to the animal in an amount effective to ameliorate at least one symptom of the viral disease.

In a nineteenth embodiment, the present invention provides a method of treating a neoplastic disease in an animal, the method comprising administering to the animal the compound of any one of embodiments 1 to 7 or the pharmaceutical composition of any one of embodiments 8 to 10 or 14 to the animal in an amount effective to ameliorate at least one symptom of the neoplastic disease.

In a twentieth embodiment, the present invention provides the method of any one of embodiments 16 to 19, wherein administering comprises delivering the compound or the composition to a localized tissue region of the animal. In a twenty-first embodiment, the present invention provides the method of the twentieth embodiment, wherein the localized tissue region is a tumor.

In a twenty-second embodiment, the present invention provides the method of the twenty- first embodiment, wherein the tumor is a breast cancer tumor, a stomach cancer tumor, a lung cancer tumor, a head or neck cancer tumor, a colorectal cancer tumor, a renal cell carcinoma tumor, a pancreatic cancer tumor, a basal cell carcinoma tumor, a melanoma cancer tumor, a prostate cancer tumor, an ovarian cancer tumor, or a bladder cancer tumor. As shown in the following Examples, the compound of Formula I is unexpectedly effective at inducing the biosynthesis of tumor necrosis factor-alpha (TNF-a) in rats. While under some conditions the compound of Formula I may be a source of l-[4-amino- 2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]-2-methylpropan-2-ol, the compound of Formula I is unexpectedly different from l-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin- 1 -yl]-2-methylpropan-2-ol in inducing the biosynthesis of TNF-a.

Interestingly, the level of cytokine induction is not proportional to the amount of l-[4- amino-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]-2-methylpropan-2-ol found in a blood sample. For example, as shown in the Examples below, intramuscular injection of the compound of Formula I in corn oil into rats provides considerably lower levels of l-[4- amino-2-(ethoxymethyl)- lH-imidazo[4,5-c]quinolin- 1 -yl]-2-methylpropan-2-ol in blood serum than a comparable injection of l-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin-l-yl]-2-methylpropan-2-ol in phosphate buffered saline, but the injection of the compound of Formula I provided more than twice the level of induced TNF-a in rats than the comparable injection of l-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l- yl]-2-methylpropan-2-ol. Furthermore, l-hydroxynaphthalene-2-carboxylic acid and corn oil were found to be inactive for cytokine induction. These results demonstrate that the TNF-a response following injection of the compound of Formula I is not due to the serum levels of 1 -[4-amino-2-(ethoxymethyl)- lH-imidazo[4,5-c]quinolin- 1 -yl]-2-methylpropan- 2-ol and suggest that the compound

itself unexpectedly has pharmacological activity. Furthermore, the increased effectiveness of the compound of Formula I when delivered from an oil-based carrier rather than an aqueous carrier is also unexpected. Since the compound of Formula I provides low systemic levels of l-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]-2- methylpropan-2-ol but induces a high level of cytokines upon intramuscular injection, it may be useful for providing an enhanced local immune response while minimizing undesirable systemic side effects. Objects and advantages of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention.

Examples

Example 1

l-Hydroxy-2 -naphthoic acid (3.1 g) and l-[4-amino-2-(ethoxymethyl)-lH- imidazo[4,5-c]quinolin-l-yl]-2-methylpropan-2-ol (5.2 g) were added with stirring to methanol (100 mL). The resulting mixture was heated until it became a solution. The solution was filtered while hot and the filtrate was cooled to room temperature. The crystals that formed in the filtrate were recovered by vacuum filtration, washed with three portions of methanol (5 mL each), and dried overnight at 50 °C. The solid was cooled to room temperature to yield 6.2 g of 4-amino-2-(ethoxymethyl)-l-(2-hydroxy-2- methylpropyl)- lH-imidazo[4,5-c]quinolin-5-ium 1 -hydroxynaphthalene-2-carboxylate as an off-white crystalline solid, mp 181-183 °C.

1H NMR (700 MHz, DMSO-d₆) δ 15.0 (broad s, 2H), 9.28 (broad s, 2H), 8.50 (d, J = 8.5 Hz, 1H), 8.27 (d, J= 8.2 Hz, 1H), 7.91 (d, J= 8.5 Hz, 1H), 7.86 (d, J= 8.2 Hz, 1H), 7.82 (d, J= 7.8 Hz, 1H), 7.70 (t, J= 7.7 Hz, 1H), 7.56 (t, J= 7.5 Hz, 1H), 7.49 (t, J= 7.5 Hz, 1H), 7.48 (t, J= 7.5 Hz, 1H), 7.23 (d, J= 8.9 Hz, 1H), 5.19 (broad s, 2H), 4.99 (broad s, 1H), 4.76 (broad s, 2H), 3.57 (q, J= 6.8 Hz, 2H), 1.21 (broad s, 6H), 1.17 (t, J= 7.0 Hz, 3H).

Examples 2 and 3

A sample of 4-amino-2-(ethoxymethyl)-l-(2-hydroxy-2-methylpropyl)-lH- imidazo[4,5-c]quinolin-5-ium l-hydroxynaphthalene-2-carboxylate was dispersed in corn oil at the content levels shown in Table 1, below, for each Example. The content attributable only to l-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]-2- methylpropan-2-ol (i.e., Resiquimod, Compound A) is also shown in Table 1, below.

Table 1

Injection into Rats and Analysis

Within 5 minutes after preparation, Examples 2 and 3 were shaken and then injected into CD male rats (200-250 grams) using the following protocol. Three animals, non-randomized, were used per Example tested. Each animal was anesthetized or restrained, and the injection site in the upper thigh was shaved. Example 2 or 3 (0.3 mL) was then administered by intramuscular injection into the upper thigh. After about 1 hour (± 15 minutes) after injection, each animal was anesthetized or restrained, and a 0.2 mL blood sample was collected from the tail vein. After about 2 hours (± 15 minutes) after injection, each animal was anesthetized or restrained, and a 0.2 mL blood sample was collected from the tail vein. After about 3 hours (± 15 minutes) after injection, each animal was anesthetized or restrained, and a 0.2 mL blood sample was collected from the tail vein. After about 4 hours (± 15 minutes) after injection, each animal was anesthetized, and a 3.0 mL blood sample was collected from the heart. The animal was then euthanized. After each blood collection, the blood was allowed to clot for about 30 minutes and then centrifuged to allow separation and collection of serum. The 4.0-hour sample was divided into two aliquots.

Analyses for the concentrations of l-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin-l-yl]-2-methylpropan-2-ol (Compound A) in the rat serum were done by LC/MS/MS using an instrument obtained from Applied Biosystems, Foster City, California, under the trade designation "Sciex API-3000" or by HPLC with fluorescence detection using an Agilent 1100 instrument obtained from Agilent Technologies, Santa Clara, California. The results are shown in Table 2, below.

Table 2

Analyses for the tumor necrosis factor-alpha (TNF-a) levels in the rat serum were carried out using an enzyme-linked immunosorbent assay (ELISA) obtained from

Pierce/Thermo Scientific, Rockford, IL, under the trade designation "ERTNFA2". TNF-a levels were determined using a calibration curve from 0 to 2500 pg/mL. The results are shown in Table 3, below.

Table 3

Comparative Examples A and B

The injection protocol for Examples 2 and 3 was repeated using a 0.3 mL injection of a solution of l-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]-2- methylpropan-2-ol (Compound A) in phosphate buffered saline. The phosphate buffered saline contained sodium chloride, potassium chloride, and sodium phosphate dibasic, and water, had a pH of 7.3 to 7.5, and was obtained from EMD Chemicals, Gibbstown, NJ. For Comparative Example A, a 1.0 mg/mL solution was used, and for Comparative Example B a 0.1 mg/mL solution was used. Analyses for the concentrations of l-[4- amino-2-(ethoxymethyl)- lH-imidazo[4,5-c]quinolin- 1 -yl]-2-methylpropan-2-ol

(Compound A) in the rat serum and for TNF-a levels in rat serum were carried out using the techniques described above. The results are shown in Tables 2 and 3, above.

Comparative Examples C and D

The injection protocol for Examples 2 and 3 was repeated using corn oil (Comp.

Ex. C) and a solution of 0.3 mg/mL l-hydroxynapthalene-2-carboxylic acid in corn oil (Comp. Ex. D.). No induction of TNF-a was observed for either of Comparative Examples C or D. Examples 4 and 5

Examples 4 and 5 were carried out according to the method of Examples 2 and 3 with the modification that Example 4 was a 1.6 mg/mL solution in phosphate buffered saline used in Comparative Examples A and B, and Example 5 was a 1.6 mg/mL solution in corn oil. The injection protocol used for Examples 2 and 3 was repeated for Examples 4 and 5. The results from the analyses of the blood serum for Compound A and TNF-a are shown in Tables 4 and 5, respectively, below.

Table 4

Table 5

Injection into mice tumors

Examples 2 to 5 can be injected into the tumors of 6 albino black mice (C57BL/6J- Tyr) using the following protocol. On day zero, a 0.050 mL intradermal tumor inoculation (lxlO⁵ B16-luc) can be administered to the mice. On day 1, 0.050 mL of any of Examples 2 to 5 can be injected into a tumor. Blood collection and tumor site biopsies (about 30 mg) can then be carried out 1, 2, 4, 6, 8, 12, 24, 30, and 48 hours after the injection of any of Examples 2 to 5. The serum collected from the blood at these times can be analyzed for TNF-a using an ELISA kit available from Biosource International, Camarillo, Calif, and type 1 IFN-a2 using a bioassay from Interferonsource.com, Piscataway, NJ. The tumor site biopsies collected at the indicated times can be stored at -20 °C in a preservative available from Ambion, Inc., Austin, TX, under the trade designation "RNALATER" until processing. The biopsies can then be transferred to 2-mL microcentrifuge tubes containing a single 2.3 mm stainless steel bead and 0.700 mL buffer "RLT" and 2- mercaptoethanol available from Qiagen, Inc., Valencia, CA. The samples can then be processed using the TissueLyser for 2 disruption cycles (3 minutes at 30 Hz), or more if needed, and stored frozen until they are analyzed for mRNA, TNF-a, and IFN-a2. On days 1, 2, 3, any of Examples 2 to 5 can be injected into a tumor along with controls to investigate tumor development. On days 4 to 10, the body weights and tumor

development in the mice can be observed. On day 11 , Xenogen monitoring of the tumors can be carried out using a field of view of C, an exposure time of 15 seconds, a medium bining, and click numbers 092908; 93455; 93913. The mice can be euthanized following imaging.

Based on the unexpected results found for Examples 2 to 5 one might predict

Examples 2 to 5 would generate more TNF-a and IFN-a2 under these conditions than Comparative Examples using similar content levels of l-[4-amino-2-(ethoxymethyl)-lH- imidazo[4,5-c]quinolin-l-yl]-2-methylpropan-2-ol. One might further predict that Examples 2 to 5 will inhibit tumor growth better and generate more tumor killing cells than Comparative Examples using similar content levels of l-[4-amino-2-(ethoxymethyl)- lH-imidazo[4,5-c]quinolin- 1 -yl]-2-methylpropan-2-ol. The complete disclosures of the patents, patent documents, and publications cited herein are incorporated by reference in their entirety as if each were individually incorporated. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein as follows.

Claims

What is claimed is:

1. A compound of formula:

The compound of claim 1 in solid form.

3. The compound of claim 1 or claim 2 in amorphous form.

4. The compound of any preceding claim in particulate form.

5. The compound of claim 2 in crystalline form.

6. The compound of claim 5 in solvate form.

7. The compound of claim 1 in at least partially dissolved form.

8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the compound of any preceding claim.

9. A pharmaceutical composition comprising a dispersion of a therapeutically effective amount of the compound of any one of claims 1 to 5 in a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 8 or 9, further comprising an antigen in an amount effective to generate an immune response against the antigen.

11. The pharmaceutical composition of any one of claims 8 to 10, wherein the pharmaceutically acceptable carrier comprises an oil.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutically acceptable carrier comprises corn oil.

13. A method of vaccinating an animal comprising administering an effective amount of the compound of any one of claims 1 to 7 or the pharmaceutical composition of any one of claims 8 to 12 to the animal as a vaccine adjuvant.

14. A method of inducing cytokine biosynthesis in an animal, the method comprising administering an effective amount of the compound of any one of claims 1 to 7 or the pharmaceutical composition of any one of claims 8 to 12 to the animal.

15. A method of treating a viral disease in an animal, the method comprising administering to the animal the compound of any one of claims 1 to 7 or the

pharmaceutical composition of any one of claims 8 to 12 to the animal in an amount effective to ameliorate at least one symptom of the viral disease.

16. A method of treating a neoplastic disease in an animal, the method comprising administering to the animal the compound of any one of claims 1 to 7 or the

pharmaceutical composition of any one of claims 8 to 12 to the animal in an amount effective to ameliorate at least one symptom of the neoplastic disease.

17. The method of any one of claims 13 to 16, wherein administering comprises delivering the compound or the composition to a localized tissue region of the animal.

18. The method of claim 17, wherein the localized tissue region is a tumor.