WO2018049017A1

WO2018049017A1 - Block copolymer systems for local administration of toll-like receptor agonists

Info

Publication number: WO2018049017A1
Application number: PCT/US2017/050459
Authority: WO
Inventors: Amir FAKHARI; Alexander Schwarz; Janardhanan Anand Subramony
Original assignee: Medimmune, Llc
Priority date: 2016-09-08
Filing date: 2017-09-07
Publication date: 2018-03-15
Also published as: TW201822767A; AR109629A1

Abstract

Provided are compositions comprising a reverse thermosensitive polymer, an immune response modifier (IRM), and ethanol. In certain aspects, the reverse thermosensitive polymer is polyoxamer 407 or PLGA-PEG-PLGA. The IRM can be a Toll-like receptor (TLR) agonist, in particular, a TLR7, TLR8, or TLR7/8 agonist.

Description

BLOCK COPOLYMER SYSTEMS FOR LOCAL ADMINISTRATION

OF TOLL-LIKE RECEPTOR AGONISTS

BACKGROUND

[0001] The most effective treatment for localized solid cancers is to remove the tumor by surgery followed by post-operative chemotherapy or radiation treatment. However, this approach is not suitable for many cancers since many patients are not candidates for surgical procedure due to tumor size, location of the tumor, and/or stage of the cancer. In some cases, even after surgery, the overall survival rates for some patients are not promising. Therefore, therapies including chemotherapy and cancer immunotherapy are additional options for cancer treatment.

[0002] Toll-like receptor (TLR) agonists are small nucleoside analogues that have shown efficacy towards a variety of tumors. TLR agonists are used for cancer immunotherapy to stimulate the immune system locally against cancer cells. Systemic administration of TLR agonists results in the stimulation of the immune system of the entire body, which can have highly undesirable side effects, such as patient discomfort, while delivering just a small portion of the entire administrated dose to the tumor. Therefore, local delivery of TLR agonists is a preferred approach for administration, such as in dermal applications.

[0003] Thus, TLR agonists are used as a potent modulator for the topical treatment for genital warts and superficial basal cell carcinomas. They are also introduced as a treatment of malignant skin lesions including melanoma and basal cell carcinoma. TLR agonists induce pro-inflammatory cytokines and chemokines in-vitro and in-vivo that attract immune cells to the site of administration. Immune cells then treat cancer cells at the site resulting in elimination of cancer cells.

[0004] Studies have shown that direct injection of TLR agonists into the tumor site as an immune-stimulant generated anti-tumor CD8 T cell response for the treatment of low-grade lymphoma with low-dose radiotherapy. Preliminary results indicated the requirement of a controlled delivery system for these agonists at the tumor site. Accordingly, there is a need for new drug delivery approaches, including intra-tumoral delivery of TLR agonists, to limit systemic side effects and create a local immune response.

[0005] Thermogels can be made from biodegradable, biocompatible, thermo-sensitive polymers, which are a solution at room temperature. An active agent, such as a TLR agonist, can be incorporated into the thermogel by mixing the drug (dissolved in an organic solvent) with the polymer solution. Upon injection (at body temperature), they self-entangle or gel, resulting in depot formation, thereby securing the drug at the injection site. Drug loading, sol-to-gel transition, mechanical properties of the formed gel, and release of the drug from the gel can be manipulated by changing the polymer concentration and through the addition of excipients.

SUMMARY OF THE INVENTION

[0006] Some of the main aspects of the present invention are summarized below.

Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0007] The disclosure provides the application of block co-polymers for delivery, such as intra- tumoral delivery, of TLR agonists. The formulation can be secured at the site of the injection (e.g., the tumor) using gelation via in-situ phase separations (thermo-sensitive gels). Drug incorporation may have a negative impact on the sol-to-gel properties of thermogels, resulting in no gel formation at body temperature. Therefore, addition of a TLR agonist into the thermo-gelling system required us to develop a novel approach to achieve the desired gelation properties, the desired release of TLR agonist at the injection site, and greatly diminished systemic side effects. Several excipients have been used to optimize the gelation. Our approach uses particular excipients and processing to incorporate TLR agonist(s) into the thermogel. In particular, we surprisingly discovered that ethanol can be used to incorporate TLR 7/8 agonists into the thermogel with minimum or no impact on sol-to-gel properties of thermogel.

[0008] In one aspect, the invention provides a composition comprising:

(a) 10%-25% (w/v) of a reverse thermosensitive polymer;

(b) an immune response modifier (IRM) of Formula I:

wherein Ri has the formula alkylene-L-Ri-i, alkenylene-L-Ri-i, or alkynylene-L-Ri-i, wherein: the alkylene, alkenylene, and alkynylene groups, are optionally interrupted or terminated by one or more -O- groups; L is a bond or a functional linking group selected from the group consisting of -NH-S(0)₂- -NH-C(O)-, -NH-C(S)-, -NH-S(0)₂-NR₃- -NH-C(0)-NR₃- -NH-C(S)- NR₃-, -NH-C(0)-0-, -0-, -S-, and -S(0)₂-; and Ri-i is a linear or branched aliphatic group, optionally including one or more unsaturated carbon-carbon bonds;

R is selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkenyl, haloalkyl, alkoxy, alkylthio, and -N(R₃)₂; n is 0 to 4;

R₂ is selected from the group consisting of: hydrogen; alkyl; alkenyl; aryl; heteroaryl;

heterocyclyl; alkylene-Y-alkyl; alkylene-Y-alkenyl; alkylene-Y-aryl; and alkyl or alkenyl substituted by one or more substituents selected from the group consisting of: -OH; halogen; - N(R₄)₂; -C(0)-Ci-ioalkyl; -C(0)-0-Ci-ioalkyl; -N₃; aryl; heteroaryl; heterocyclyl; -C(0)-aryl; and -C(0)-heteroaryl;

Y is -O- or -S(0)o-2-;

each R₄ is independently selected from the group consisting of hydrogen, Ci-io alkyl, and C2-10 alkenyl; and

R₃ is selected from the group consisting of hydrogen and alkyl; with the proviso that when L is - NH-S(0₂)-, and n is 0, R1-1 is a linear or branched aliphatic group having at least 16 carbon atoms, optionally including one or more unsaturated carbon-carbon bonds; or a pharmaceutically acceptable salt thereof; and (c) up to 20% (v/v) ethanol.

[0009] In some embodiments, the compositions of the invention comprise an IRM of

Formula II:

wherein:

X is alkylene having up to 8 carbon atoms optionally interrupted or terminated by -0-;

R₂ is hydrogen, alkyl, alkoxyalkylenyl, alkylaminoalkylenyl, or hydroxyalkylenyl; Y is -C(O)- or -S(0)₂-;

Ri is a linear or branched aliphatic group having 1-23 carbon atoms, preferably 11-23 carbon atoms, optionally including one or more unsaturated carbon-carbon bonds; and

R is hydrogen, halogen, or hydroxyl.

[0010] In some embodiments, the IRM is a Toll-like receptor 7 (TLR7) agonist, a Tolllike receptor 8 (TLR8) agonist, or a Toll-like receptor 7/8 (TLR7/8) agonist.

[0011] In another aspect, the invention provides a composition comprising:

(a) 12%-20% (w/v) of a reverse thermosensitive polymer selected from the group consisting of (i) poloxamer 407 and (ii) poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b- poly(lactic-co-glycolic acid) copolymer (PLGA-PEG-PLGA), wherein the PLGA-PEG- PLGA number-average molecular weight (Mn) is about 1600: 1500: 1600 daltons, and wherein the PLGA ratio of lactic acid to glycolic acid is about 3: 1;

(b) an immune response modifier (IRM) having the structure:

or a pharmaceutically acceptable salt thereof; and

(c) up to 20% (v/v) ethanol.

[0012] In a further aspect of the invention the immune response modifier compound comprises N-(4-{ [4-amino-2-butyl-lH-imidazo[4,5-c]quinolin-l- yl]oxy}butyl)octadecanamide, or a pharmaceutically acceptable salt thereof. In a still further aspect of the invention the immune response modifier compound comprises N-(4-{ [4-amino- 2-butyl-lH-imidazo[4,5-c]quinolin-l-yl]oxy}butyl) methylamide.

[0013] In a further aspect the invention provides a composition comprising:

(b) an immune response modifier (IRM) having the structure:

or a pharmaceutically acceptable salt thereof; and

(c) up to 20% (v/v) ethanol.

[0014] In some embodiments, the composition comprises 5% (v/v) ethanol.

[0015] In certain embodiments, the composition comprises 15%-20% (w/v) reverse

thermosensitive polymer. In one embodiment, the reverse thermosensitive polymer is poloxamer 407. In some embodiments, the poloxamer 407 is purified. In another embodiment, the reverse thermosensitive polymer is PLGA-PEG-PLGA.

[0016] In some instances, the composition can comprise 0.05 to 1.3 mg/mL IRM. For example, the concentration of IRM can be selected from the group consisting of 0.08 mg/mL, 0.4 mg/mL, and 1 mg/mL. The compositions of the invention can comprise a second active agent, in addition to the IRM.

[0017] In particular embodiments, the sol-to-gel transition temperature (T_soi-gei) of the composition is 20-37 °C.

[0018] The invention provides a method of delivering a depot formulation comprising an immune response modifier (IRM) to a subject, the method comprising injecting into the subject an effective amount of a composition of the invention. The invention also provides a method of stimulating a local immune response in a subject, the method comprising injecting into the subject an effective amount of a composition of the invention. In certain aspects, the composition is administered in liquid form and wherein the composition forms a gel upon injection into the subject.

[0019] In some embodiments, the subject has a tumor, such as a cancerous tumor, and the composition is injected at the site of the tumor. In particular embodiments, the tumor is a breast tumor, a stomach tumor, a lung tumor, a head or neck tumor, a colorectal tumor, a renal cell carcinoma tumor, a pancreatic tumor, a basal cell carcinoma tumor, a cervical tumor, a melanoma tumor, a prostate tumor, an ovarian tumor, a liver tumor, or a bladder tumor.

[0020] In some embodiments, the subject has a disease or disorder of the dermis, and the composition is injected at the site of the disease or disorder. In particular embodiments, the disease or disorder of the dermis is selected from the group consisting of: basal cell carcinoma, melanoma, and genital warts. In certain embodiments, the methods of the invention comprise administering a second active agent. In some embodiments, the second active agent is a chemotherapeutic agent.

[0021] Also provided is a use of a composition of the invention to deliver a depot

formulation comprising an immune response modifier (IRM) to a subject. Further provided is a use of a composition of the invention to stimulate a local immune response in a subject. In some embodiments, the subject has a tumor. In particular embodiments, the tumor is a breast tumor, a stomach tumor, a lung tumor, a head or neck tumor, a colorectal tumor, a renal cell carcinoma tumor, a pancreatic tumor, a basal cell carcinoma tumor, a cervical tumor, a melanoma tumor, a prostate tumor, an ovarian tumor, a liver tumor, or a bladder tumor.

[0022] In some embodiments, the subject has a disease or disorder of the dermis. In particular embodiments, the disease or disorder of the dermis is selected from the group consisting of: basal cell carcinoma, melanoma, and genital warts.

[0023] The uses of the invention can comprise a second active agent, for example, a chemotherapeutic agent.

[0024] The invention includes a method of making a composition of the invention, the method comprising (a) dissolving the reverse thermosensitive polymer in an aqueous medium to prepare an excipient solution; (b) dissolving the IRM in ethanol to prepare a drug solution; and (c) adding the drug solution to the excipient solution to prepare a composition comprising ethanol in an amount of up to 20% (v/v).

[0025] Also provided is a kit comprising a composition of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 shows the dependence of the sol-to-gel transition temperature in thermo- sensitive gels on polymer concentration.

[0027] FIG. 2A shows the structure of the TLR 7/8 agonist S-36878 (non-lipidated).

FIG. 2B shows the structure of the TLR 7/8 agonist S-36862 (lapidated).

[0028] FIG. 3 shows a plot of HPLC-ELSD poloxamer 407 purification.

[0029] FIG. 4 shows the impact of purification on gel formation.

[0030] FIG. 5 shows the T_soi-gei and maximum storage modulus of three lots of purified poloxamer 407.

[0031] FIG. 6 shows the impact of purification on viscosity of poloxamer 407 solution at

10 °C.

[0032] FIG. 7 shows the viscosity of three lots of purified poloxamer 407 measured at

10°C over a shear rate sweep.

[0033] FIG. 8 shows the impact of poloxamer 407 concentration on gel formation.

[0034] FIG. 9A-9B show the influence of poloxamer 407 concentration on T_soi-gei

transition temperature (FIG. 9A) and maximum recorded storage modulus (FIG. 9B).

[0035] FIG. 10 shows the impact of poloxamer 407 concentration on viscosity over a shear rate sweep at 10 °C.

[0036] FIG. 11 shows the impact of poloxamer 407 concentration on viscosity over a shear rate sweep at 23 °C.

[0037] FIG. 12A-12B show the influence of poloxamer 407 concentration on viscosity at

10 °C (FIG. 12A) and 23 °C (FIG. 12B).

[0038] FIG. 13 shows T_soi-gei and maximum storage modulus of poloxamer 407 dissolved in IX PBS, distilled water, and IX Tris buffer.

[0039] FIG. 14 shows the viscosity of poloxamer 407 dissolved in IX PBS, distilled water, and IX Tris buffer measured at 10 °C.

[0040] FIG. 15 shows the impact of ethanol concentration on gel formation.

[0041] FIG. 16A-16B show the influence of ethanol concentration on T_soi-gei (FIG. 16A) and maximum recorded storage modulus (FIG. 16B).

[0042] FIG. 17 shows the impact of ethanol concentration on viscosity over a shear rate sweep at 10 °C. [0043] FIG. 18 shows the influence of ethanol concentration on viscosity of poloxamer

407 solution at 10 °C.

[0044] FIG. 19 shows the formulation preparation and mixing steps.

[0045] FIG. 20 shows the impact of drug addition to poloxamer 407 solution containing

5% (v/v) ethanol on gel formation.

[0046] FIG. 21 shows the impact of drug addition to poloxamer 407 solution containing

5% (v/v) ethanol on viscosity.

[0047] FIG. 22A-22B show the impact of drug addition to PLGA-PEG-PLGA (FIG.

22A) and unpurified poloxamer 407 (FIG. 22B) on gel formation.

[0048] FIG. 23A-23B show viscosity vs. shear rate sweep for PLGA-PEG-PLGA (FIG.

23A) and unpurified poloxamer 407 (FIG. 23B) formulations. Viscosity measurements were conducted at 23 °C.

[0049] FIG. 24A-24B show the results of injectability testing for PLGA-PEG-PLGA

(FIG. 24A) and poloxamer 407 (FIG. 24B) formulations at room temperature.

[0050] FIG. 25A-25B show in vitro release of S-36878 (FIG. 25A) and S-36862 (FIG.

25B) from PLGA-PEG-PLGA and poloxamer 407 formulations.

[0051] FIG. 26A-26B show tumor drug levels (FIG. 26A: μg drug/tumor and FIG. 26B:

% of to) for injected PLGA-PEG-PLGA and poloxamer 407 formulations in the B 16-OVA tumor model.

[0052] FIG. 27 shows serum drug level for injected PLGA-PEG-PLGA and poloxamer

407 formulations in the B 16-OVA tumor model.

[0053] FIG. 28 shows measured tumor volume for the animals dosed with poloxamer

407 formulation in study one (n=9 mice/group for PBS).

[0054] FIG. 29 shows percent survival results for the animals dosed with poloxamer 407 formulation in study one (n=9 mice/group for PBS).

[0055] FIG. 30 shows measured tumor volume for the animals dosed with poloxamer

407 formulation in study two.

[0056] FIG. 31 shows percent survival results for the animals dosed with poloxamer 407 formulation in study two.

[0057] FIG. 32A-32E show measured serum cytokines: keratinocyte-derived cytokine

(FIG. 32A), chemokine C-X-C motif) ligand 10 (FIG. 32B), interleukin 10 (FIG. 32C), monocyte chemoattractant protein (FIG. 32D), and interleukin 6 (FIG. 32E), from blood samples collected 6 hours post-dose from 3 mice in each group in study two.

DETAILED DESCRIPTION OF THE INVENTION

[0058] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, protein chemistry, cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Handbook of Pharmaceutical Excipients (7th ed., Rowe et al. eds., 2012); Martin's Physical Pharmacy and Pharmaceutical Sciences (6th ed., Sinko, 2010); Remington: The Science and Practice of Pharmacy (21st ed., Univ. Sci. Philadelphia ed., 2005); Current Protocols in Molecular Biology (Ausubel et al. eds., 2016); Molecular Cloning: A Laboratory Manual (4th ed., Green and Sambrook eds., 2012); Lewin's Genes XI (1 lth ed., Krebs et al. eds., 2012); DNA Cloning: A Practical Approach, Volumes I and II (2d ed., Glover and Hames eds., 1995); Protein Engineering: A Practical Approach (1st ed., Rees et al. eds. 1993); Culture Of Animal Cells (6th ed. Freshney, 2010); Antibodies: A Laboratory Manual (2nd ed., Greenfield ed., 2013); Antibody Engineering (2d ed.,

Borrebaeck ed., 1995).

[0059] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. For example, Dictionary of Pharmaceutical Medicine (3rd ed. Nahler and Mollet eds., 2013); The Dictionary of Cell and Molecular Biology (5th ed. J.M. Lackie ed., 2013), Oxford Dictionary of Biochemistry and Molecular Biology (2d ed. R. Cammack et al. eds., 2008), and The Concise Dictionary of Biomedicine and Molecular Biology (2d ed. P-S. Juo, 2002) can provide one of skill with general definitions of some terms used herein.

[0060] Any headings provided herein are not limitations of the various aspects or

embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. [0061] All of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers' instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.

I. Definitions

[0062] As used in this specification and the appended claims, the singular forms "a,"

"an," and "the" include plural referents, unless the context clearly dictates otherwise. The terms "a" (or "an") as well as the terms "one or more" and "at least one" can be used interchangeably.

[0063] Furthermore, "and/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0064] Wherever embodiments are described with the language "comprising," otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are included.

[0065] Units, prefixes, and symbols are denoted in their Systeme International de Unites

(SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth.

[0066] A "polymer" is a series of cross-linked repeating units or "monomers." The

monomeric units can be the same or different ("co-polymer"). A polymer can comprise two or more different cross-linked polymers. Polymers can be combined in varying ratios, to provide compositions with differing properties. Those of skill in the art of polymer chemistry will be familiar with the different properties of polymeric compounds. [0067] The compositions of the invention comprise a "reverse thermosensitive polymer," which is liquid at low temperatures and rapidly transitions to gel at physiological

temperature. This transition may occur very quickly, for example, over one-half of a degree Celsius. The transition temperature can be altered with various polymer compositions, concentrations, and buffer solutions. The aqueous, biocompatible polymer is reversible back to a liquid via cooling, and is dissolvable.

[0068] Thermo-gelation is a reversible phenomenon which is defined by a sol-gel

transition temperature (T_soi-gei). Below T_soi-gei, materials remain fluid and above this temperature, the materials turn to a semi-solid (gel). Here, the T_soi-gei was defined as the temperature at which the storage modulus (G') is half way between the values for the storage modulus for the flowable material and the gel.

[0069] An "isolated" molecule is one that is in a form not found in nature, including those which have been purified. In some embodiments, an isolated molecule is substantially pure. As used herein, the term "substantially pure" refers to purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.

[0070] A "label" is a detectable compound that can be conjugated directly or indirectly to a molecule, so as to generate a "labeled" molecule. The label can be detectable on its own (e.g., radioisotope labels or fluorescent labels) or can catalyze chemical alteration of a substrate compound or composition that is detectable (e.g., an enzymatic label).

[0071] The terms "inhibit," "block," and "suppress" are used interchangeably and refer to any statistically significant decrease in occurrence or activity, including full blocking of the occurrence or activity. For example, "inhibition" can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence.

[0072] An "active agent" is an ingredient that is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the human body. The active agent can be in association with one or more other ingredients, and can be, but is not necessarily, in a finished dosage form. The terms "active agent" and "drug substance" are used

interchangeably herein. [0073] An "effective amount" of an active agent is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.

[0074] The term "pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile and can comprise a pharmaceutically acceptable carrier, such as physiological saline. The form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), a stabilizing agent (e.g. human albumin), a preservative (e.g. sodium benzoate), an absorption promoter to enhance bioavailability and/or other conventional solubilizing or dispersing agents.

[0075] A "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and laboratory animals including, e.g., humans, non-human primates, canines, felines, porcines, bovines, equines, rodents, including rats and mice, rabbits, etc.

[0076] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to

alleviate" refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder. In certain embodiments, a subject is successfully "treated" for a disease or disorder of the eye according to the methods provided herein if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder.

[0077] "Prevent" or "prevention" refer to prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of prevention include those prone to have or susceptible to the disorder. In certain embodiments, a disease or disorder of the eye is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder, than a patient who has not been subject to the methods of the invention.

II. Reverse Thermosensitive Polymers

[0078] To control intra-tumoral delivery of TLR agonists, several configurations, such as engineered in-situ depot-forming hydrogels, particulate systems, wafers, and rods can be used to minimize systemic side effects. In most cases, these systems are made from biodegradable polymeric materials such as natural polymers including polysaccharides and polypeptides, and synthetic polymers such as PLA and PLGA. These biopolymers are biocompatible in-vivo and applicable as in-situ depot-forming systems for localized intra- tumoral drug delivery.

[0079] Among these systems, in-situ depot-forming hydrogels are three-dimensional networks of polymers with the capacity to hold the TLR agonists, and thus can be used for the intra-tumoral delivery of these cancer immunotherapy agents. Injectable biodegradable in-situ forming depots have been shown to be less invasive and to have less pain upon injection as compared to pre-formed implants, making them desirable systems for local administration of anticancer drugs. Injectable bio materials are suitable for development as delivery systems to localize the drug molecules at the tumor site. According to the mechanism of depot formation, engineered in-situ gelling depots can be classified into two categories: (1) platforms based on in-situ cross-linking, and (2) platforms based on in-situ phase separation. In-situ phase separation is a strategy to deliver drugs to the tumor site. Phase separation can be induced by changing the solubility of the polymer with respect to changes in pH, temperature, or by elimination of a solvent.

[0080] A key requirement of in-situ depot-forming systems for local delivery and, more specifically, intra-tumoral delivery, is the injectability using standard gauge needles in either a vial/syringe or a pre-filled syringe configuration. The injection should be easy to administer and should also provide minimal discomfort to the patient. Intra-tumoral injections based on in-situ gelling polymers are solutions that have low viscosity and can easily flow during administration but rapidly form gel networks once injected. Among phase separation platforms, thermogel-based platforms undergo sol-to-gel transformation with increasing temperature (FIG. 1). Since thermogels do not require the use of organic solvents, polymerization agents, or any externally applied triggers for in-situ depot- formation, they are especially attractive for the delivery of small molecules and biological molecules. Temperature-dependent phase transitions are governed by interactions between molecules, including hydrogen bonding or hydrophobic responses. Water-polymer hydrogen bonding tends to be undesirable as compared to polymer-polymer interactions at the lower critical solution temperature (LCST). In this state, the solvated macromolecules lose the water of hydration, and polymer-polymer interaction increases, resulting in formation of polymeric network structure with an increase in viscosity of the system. For intra-tumoral drug delivery, the ideal requirement would be an aqueous polymer solution that easily flows at room temperature, followed by formation of gel at physiological temperature. For this approach, both synthetic and natural polymeric materials can be used.

[0081] The compositions provided herein comprise a reverse thermosensitive polymer.

Such polymers can be composed of, for example, tri-block polymers with two hydrophilic chains connected by a hydrophobic chain. A rapid viscosity transition occurs in response to heat, which causes the polymer chains to deform and the hydrophilic chains to align, leading to the formation of micelles and a subsequent phase change to a viscous gel. The resulting gel is dissolvable, and is also reversible back to a liquid with cooling.

[0082] The transition temperature (Tsoi-gei) of a reverse thermosensitive polymer can be modified in a number of ways. For example, the transition temperature can be modified through the addition of an additive, such as fatty acid excipients, including sodium caprate, sodium laurate, or sodium oleate; humectants, such as glycerol; glycols; emulsifiers, solubilizers; paraffins; triglycerides; lipophilic substances, such as isopropyl myristate; and various solvents. The use of a modified polymer can also affect the transition temperature. Furthermore, the addition of other polymers to form polymer mixtures can influence the transition temperature. One of skill in the art can adjust the T_soi-gei of the compositions of the invention to any desired temperature. Preferably, compositions of the invention have a T_soi-gei of about 10 °C to about 40 °C, or about 20 °C to about 37 °C, or about 25°C to about 37°C.

[0083] The polymers used in the invention can be a flexible or flowable material.

"Flowable" means the ability to assume, over time, the shape of the space containing it. This characteristic includes, for example, liquid compositions or highly viscous, gel-like materials. Accordingly, the polymers can be administered in a liquid or gel form. In certain embodiments, the polymer is in an aqueous solution.

[0084] Preferably, the reverse thermosensitive polymers have a number-average

molecular weight (M_n) of about 2,000 Da to about 100,000 Da, more particularly at least about 10,000 Da, or at least about 25,000 Da, or at least about 40,000. In some

embodiments, the polymers have a M_n of about 5,000 Da to about 90,000 Da, or about 10,000 Da to about 80,000 Da, or about 20,000 Da to about 70,000 Da, or about 30,000 Da to about 60,000 Da, or about 5,000 Da to about 50,000 Da.

[0085] Poloxamers are one example of reverse thermosensitive polymers suitable for use in the invention. Poloxamers are a class of triblock polyalkyleneoxide co-polymers, typically composed of a core block of poly(propylene oxide) capped at each terminus with a block of poly(ethylene oxide). Poloxamers having a higher proportion of ethylene oxide tend to exhibit reverse gelling. Poloxamers are most commonly unbranched. Examples of particular poloxamers include poloxamer 118, poloxamer 188, poloxamer 338, poloxamer 407. Trade names for poloxamers are Pluronic® and Tetronic®.

[0086] Poloxamines, in which amine groups replace oxygens in the backbone or termini, are another example of reverse thermosensitive polymers suitable for use in the invention. Examples of particular poloxamines include poloxamine 1107 and poloxamine 1307.

[0087] Suitable polymers also include combinations, co-polymers, and derivatives of the following: poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co- glycolide), poly(ethylene glycol)-block-poly(lactide-co-glycolide)-block-poly(ethylene glycol), copolymerized materials of poly(ethylene oxide)-poly(propylene oxide)- poly(ethylene oxide) recognized as poloxamer- (Pluronic^®), poly(N-isopropyl acrylamide), poly(ethylene glycol )-block-poly(capro!actone)-block-poly(ethyleiie glycol),

poly(organophosphazene), methylcellulose, hydroxypropyl methylcellulose, chitosan solution with β-glycerophosphate, and collagen.

[0088] Preferred reverse thermosensitive polymers include poly(lactide-co-glycolide)- block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) triblock copolymer-based thermogels, and poloxamer- (Pluronic^®) based thermogels. In one embodiment, the polymer is PLGA-PEG-PLGA having an M_n of about 1600: 1500: 1600 Da and a PLGA ratio of lactic acid to glycolic acid of about 3: 1. In another embodiment, the polymer is poloxamer 407. III. Immune Response Modifiers

[0089] Compositions of the present invention comprise an immune response modifier

(IRM). IRMs have been shown to induce the production of certain cytokines, such as interferon alpha (IFN-a), tumor necrosis factor alpha (TNF-a), and certain interleukins, indicating that these compounds can inhibit tumor cell growth and virus production. The ability to modulate the immune response by inducing cytokine biosynthesis also makes IRMs useful as a vaccine adjuvant. Preferably, the IRM is a Toll-like receptor (TLR) agonist, more preferably, a Toll-like receptor 7 (TLR7) agonist, a Toll-like receptor 8 (TLR8) agonist, or a Toll-like receptor 7/8 (TLR7/8) agonist.

[0090] Compositions provided herein can comprise an IRM of Formula I:

R is selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkenyl, haloalkyl, alkoxy, alkylthio, and -N(R₃)₂; n is 0 to 4; R₂ is selected from the group consisting of: hydrogen; alkyl; alkenyl; aryl; heteroaryl; heterocyclyl; alkylene-Y-alkyl; alkylene-Y-alkenyl; alkylene-Y-aryl; and alkyl or alkenyl substituted by one or more substituents selected from the group consisting of: -OH; halogen; - N(R₄)₂; -C(0)-C i-io alkyl; -C(0)-0-CMO alkyl; -N₃; aryl; heteroaryl; heterocyclyl; -C(0)-aryl; and -C(0)-heteroaryl;

Y is -O- or -S(0)o-2-;

R₃ is selected from the group consisting of hydrogen and alkyl; with the proviso that when L is - NH-S(0₂)-, and n is 0, R1-1 is a linear or branched aliphatic group having at least 16 carbon atoms, optionally including one or more unsaturated carbon-carbon bonds.

[0091] In some embodiments, R1-1 is a linear or branched aliphatic group having 11-20 carbon atoms (preferably, 12-20 carbon atoms), optionally including one or more unsaturated carbon-carbon bonds. In some embodiments, RM is a linear (i.e., straight chain) alkyl group having 11-20 carbon atoms (preferably, 12-20 carbon atoms). These RM substituents described herein are desirable because they provide lipid-like characteristics to the IRM. This is advantageous because these lipid moieties can aid in the sequestering of the IRM at the site of application. That is, the lipid moiety can assist in preventing the rapid diffusion of an IRM away from the site of administration. This sequestering can result in enhanced adjuvancy of an IRM, which could be manifest by enhanced recruitment and activation of antigen-presenting cells at a desired site. Furthermore, this sequestering can result in less systemic distribution of an IRM, and the ability to use lesser amounts of the IRM.

[0092] Some aspects of the invention include non-lipidated versions of the IRM, wherein

Ri-i is a linear or branched aliphatic group having 1-10 carbon atoms. In some embodiments, RM is CH₃.

[0093] In some embodiments, the compositions of the invention comprise an IRM of

Formula II:

wherein:

X is alkylene having up to 8 carbon atoms optionally interrupted or terminated by -0-; R₂ is hydrogen, alkyl, alkoxyalkylenyl, alkylaminoalkylenyl, or hydroxyalkylenyl; Y is -C(O)- or -S(0)₂-;

R is hydrogen, halogen, or hydroxyl.

[0094] The term "aliphatic" group means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example.

[0095] As used herein, the terms "alkyl," "alkenyl," "alkynyl" and the prefix "alk-" are inclusive of both straight chain and branched chain groups and of cyclic groups, e.g., cycloalkyl and cycloalkenyl. Unless otherwise specified, these groups contain from 1 to 23 carbon atoms, with alkenyl groups containing from 2 to 23 carbon atoms, and alkynyl groups containing from 2 to 23 carbon atoms. In some embodiments, these groups have a total of up to 20 carbon atoms, up to 18 carbon atoms, up to 16 carbon atoms, up to 10 carbon atoms, up to 8 carbon atoms, up to 7 carbon atoms, up to 6 carbon atoms, or up to 4 carbon atoms. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 10 ring carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopropylmethyl, cyclopentyl, cyclohexyl, adamantyl, and substituted and unsubstituted bornyl, norbornyl, and norbornenyl.

[0096] Unless otherwise specified, "alkylene," "alkenylene," and "alkynylene" are the divalent forms of the "alkyl," "alkenyl," and "alkynyl" groups defined above. Likewise, "alkylenyl," "alkenylenyl," and "alkynylenyl" are the divalent forms of the "alkyl,"

"alkenyl," and "alkynyl" groups defined above. For example, an arylalkylenyl group comprises an alkylene moiety to which an aryl group is attached.

[0097] An alkylene group with carbon atoms optionally "interrupted" by -O- refers to having carbon atoms on either side of the -0-. An example is -CH2-CH2-O-CH2-CH2-.

[0098] An alkylene group with carbon atoms optionally "terminated" by -O- refers to having the -O- on either or both ends of the alkylene group or chain of carbon atoms.

Examples include -O-CH2-CH2-CH2-CH2- and -CH2-CH2-CH2-CH2-O-. In some embodiments, when X is alkylene having up to 8 carbon atoms terminated by -0-, the -CD- may be connected to either the nitrogen of the imidazole ring or the nitrogen of the amide (Y is -C(O)-) or sulfonamide (Y is -S(0)₂-) group.

[0099] The term "haloalkyl" is inclusive of groups that are substituted by one or more halogen atoms, including perfluorinated groups. This is also true of other groups that include the prefix "halo-". Examples of suitable haloalkyl groups are chloromethyl, trifluoromethyl, and the like.

[00100] The term "aryl" as used herein includes carbocyclic aromatic rings or ring

systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl and indenyl.

[00101] The term "heteroaryl" includes aromatic rings or ring systems that contain at least one ring heteroatom (e.g., O, S, N). Suitable heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl, pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl, pyrazinyl, 1-oxidopyridyl, and so on.

[00102] The term "heterocyclyl" includes non-aromatic rings or ring systems that contain at least one ring heteroatom (e.g., O, S, N) and includes all of the fully saturated and partially unsaturated derivatives of the above mentioned heteroaryl groups. Exemplary heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, thiazolidinyl, imidazolidinyl, isothiazolidinyl, tetrahydropyranyl, quinuclidinyl, homopiperidinyl, and the like.

[00103] The terms "arylene," "heteroarylene," and "heterocyclylene" are the divalent forms of the "aryl," "heteroaryl," and "heterocyclyl" groups defined above. Likewise, "arylenyl," "heteroarylenyl," and "heterocyclylenyl" are the divalent forms of the "aryl," "heteroaryl," and "heterocyclyl" groups defined above. For example, an alkylarylenyl group comprises an arylene moiety to which an alkyl group is attached.

[00104] Any pharmaceutically acceptable forms of the IRMs of Formula I and Formula II and their salts can be used, including isomers (e.g., diastereomers and enantiomers), solvates, polymorphs, and the like. In particular, if a compound is optically active, the invention specifically includes each of the compound's enantiomers as well as racemic mixtures of the enantiomers. In some embodiments, the IRM compound is not imiquimod.

[00105] For any of the compounds presented herein, each one of the variables (e.g., R, Ri,

Ri-i, L, X, and so on) in any of its embodiments can be combined with any one or more of the other variables in any of their embodiments, as would be understood by one of skill in the art. Each of the resulting combinations of variables is included in the embodiments of the present invention.

[00106] Preferred IRMs for use in the compositions and methods of the invention include

S-36862 (FIG. 2A) and S-36878 (FIG. 2B). Additional preferred IRMs for us in the compositions and methods of the invention include N-(4-{ [4-amino-2-butyl- lH-imidazo[4,5- c]quinolin-l-yl]oxy}butyl)octadecanamide and N-(4-{ [4-amino-2-butyl- lH-imidazo[4,5- c]quinolin-l-yl]oxy}butyl) methylamide.

[00107] The IRMs can be unpurified or purified using standard methods in the art.

Purification methods include, for example, chromatography, such as high pressure liquid chromatography (HPLC), solvent extraction, and precipitation.

[00108] Descriptions of suitable IRMs, and methods of preparing and using them can be found, for example, in U.S. Patent No. 7,799,800, U.S. Patent No. 9,242,980, and

International Publication No. WO 2015/069535. IV. Compositions and Methods

[00109] Compositions of the invention comprise a reverse thermosensitive polymer, an

IRM, and ethanol. Typical compositions of the invention comprise about 5% to about 30% (w/v) reverse thermosensitive polymer, or about 10% to about 25% (w/v), or about 12% to about 20% (w/v), or about 15% to about 20% (w/v) or about 17% to about 18% (w/v) reverse thermosensitive polymer.

[00110] In some embodiments, a composition of the invention comprises about 0.05 to about 1.5 mg/mL IRM, or about 0.1 to about 1.0 mg/mL. For example, the composition can comprise about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, or 1.5 mg/mL. The amount of IRM in the compositions of the invention will vary according to the IRM, the subject treated, and the intended indication. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy. It is within the purview of the ordinarily skilled artisan to determine a therapeutically effective amount of the IRM to be included in the composition.

[00111] The invention also includes the use of any hydrophobic drug or active

pharmaceutical ingredient, for example, risperidone, paclitaxel, topotecan, doxorubicin, or docetaxel, in the thermo- sensitive polymer compositions for development of local drug delivery systems. Accordingly, the invention provides a composition comprising a reverse thermosensitive polymer, a hydrophobic drug, and up to 20% (v/v) ethanol.

[00112] The compositions of the invention comprise up to about 20% (v/v) ethanol,

preferably about 0.5% to about 20% (v/v) ethanol, about 2% to about 15% (v/v) ethanol, about 2.5% to about 10% (v/v) ethanol, about 1% to about 6% (v/v) ethanol, or about 5% to about 7.5% (v/v) ethanol. In a particular embodiment, the composition comprises about 5% (v/v) ethanol.

[00113] The pH of the composition administered to a subject is, generally, about 5.5 to about 8.5, preferably about 6.0 to about 7.8, which are suitable pH levels for injection into a mammal. The pH of the composition can be adjusted by any suitable acid or base, such as hydrochloric acid or sodium hydroxide.

[00114] Preferably, the compositions of the invention are pharmaceutical compositions.

Pharmaceutical compositions in accordance with the present invention can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers (e.g. acetate, phosphate, citrate), surfactants (e.g. polysorbate), stabilizing agents (e.g. human albumin), and/or salts (e.g., acid addition salts, base addition salts) etc. The form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined and other well-known variables.

[00115] These compositions can also contain preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of presence of microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like, can also be added into the

compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

[00116] A pharmaceutical composition provided herein can also include a

pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil- soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[00117] Due to their reverse thermosensitive property, the compositions of the invention form a gel upon injection into the body of a subject. Accordingly, a composition of the invention can be used in a method of delivering a depot formulation comprising an IRM to a subject, the method comprising injecting into the subject an effective amount of the composition. The invention also provides a method of stimulating a local immune response in a subject, the method comprising injecting into the subject an effective amount of a composition of the invention.

[00118] A subject in need of treatment may have a tumor, such as a breast tumor, a

stomach tumor, a lung tumor, a head or neck tumor, a colorectal tumor, a renal cell carcinoma tumor, a pancreatic tumor, a basal cell carcinoma tumor, a cervical tumor, a melanoma tumor, a prostate tumor, an ovarian tumor, a liver tumor, or a bladder tumor. The tumor may be in an organ, in lymph tissue, in the reticuloendothelium, in bone marrow, in mucosal tissue, etc. The tumor may be a solid tumor, and may be malignant. In one embodiment, a composition of the invention is injected into the tumor.

[00119] A subject in need of treatment may have a disease or disorder of the dermis, such as a basal cell carcinoma, a melanoma, or genital warts. In one embodiment, a composition of the invention is injected at the site of the disease or disorder.

[00120] The composition can be administered as a single dose or multiple doses. The composition can be administered as many times as needed to achieve a targeted endpoint. Injection intervals may vary. For example, the composition can be administered every 1, 2, 3, or 4 weeks, or every 1, 2, 3, 4, 5, or 6 months. Dosage regimens can be adjusted to provide the optimum desired response.

[00121] Compositions can also be administered in combination therapy and/or combined with other agents. In particular, the methods of the invention can comprise administering a second active agent, in addition to the IRM. For instance, the second active agent can be a second IRM, an antigen, an antigen-binding molecule, a chemotherapeutic agent, a cytotoxic agent, an antiviral agent, a cytokine, a tumor necrosis factor receptor agonist, or a label or imaging agent. In a particular embodiment, the second active agent is a chemotherapeutic agent. A composition of the invention can comprise the second active agent, or the second active agent can be administered separately.

[00122] Compositions of the invention can be made by dissolving the reverse

thermosensitive polymer in an aqueous medium to prepare an excipient solution; dissolving the hydrophobic drug, e.g., the IRM, in ethanol to prepare a drug solution; and adding the drug solution to the excipient solution to prepare a composition comprising ethanol in an amount of up to 20% (v/v). Combining the excipient solution and the drug solution results in precipitation of drug particles, as the hydrophobic drug, e.g., the IRM, is water-insoluble. Unexpectedly, ethanol acts as a surfactant, stabilizing the drug particles such that they do not aggregate. Accordingly, the invention provides a method of preparing a stable thermogel.

V. Kits

[00123] Also within the scope of the disclosure are kits comprising compositions as

provided herein and instructions for use. The kit can further contain at least one additional reagent, or one or more additional compositions. Kits typically include a label indicating the intended use of the contents of the kit. The term "label" includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[00124] This disclosure further provides kits that comprise one or more compositions of the invention, which can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one composition comprising a reverse thermosensitive polymer, an IRM, and up to 20% ethanol, in one or more containers. In some embodiments, the kits contain all of the components necessary and/or sufficient to perform the methods of the invention. One skilled in the art will readily recognize that the disclosed compositions can be readily incorporated into one of the established kit formats which are well known in the art.

EXAMPLES

[00125] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain

compositions of the present disclosure and methods for making and using compositions of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.

Example 1. Materials

[00126] Poloxamer 407, Ammonium sulfate ((NH₄)2S04), and dimethyl sulfoxide

(DMSO) were purchased from Sigma Aldrich (St. Louis, MO). Undenatured USP ethanol (200 proof), and dichloromethane (DCM) were purchased from Spectrum Chemicals (New Brunswick, NJ). Phosphate Buffered Saline (PBS IX) pH 7.2 was obtained from Life Technologies (Carlsbad, CA). Poly(lactic-co-glycolic acid)-b-Poly(ethylene glycol)-b- Poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer (Mn: 1,600: 1,500: 1,600 Da, 3: 1 LA:GA) was acquired from PolySciTech (West Lafayette, IN). TLR 7/8 agonists (FIG. 2A- 2B), S-36878 (non-lipidated) and S-36862 (lipidated), were received from 3M (St. Paul, MN). Tris-Buffered Saline pH 7.2 (Tris IX) was made in-house. Example 2. Purification of Poloxamer 407

Purification Procedure

[00127] Poloxamer 407 was purified utilizing an aqueous-aqueous extraction system

disclosed in U.S. Patent No. 6,761,824. Briefly, 100 g of poloxamer was added to 900 mL of distilled water in a 2L flask and stirred for 7 hours at 5 °C until poloxamer 407 was fully dissolved. Then, a cooled 1000 mL ammonium sulfate solution (25% w/v filtered with 0.22 μιη filter) was slowly added to the solution at 5 °C while mixing, until a turbid solution was obtained. The solution was then transferred to a 2L separation funnel and kept overnight at 5°C until two distinct phases developed. The next day, the lower phase was discarded and the upper phase was transferred to a 2L glass flask. Distilled water was added to the solution while mixing, until the volume of the solution increased to 1L and the solution cooled to 5°C. Next, another cooled 800 mL ammonium sulfate solution (25% w/v filtered with 0.22 μιη filter) was slowly added to the poloxamer 407 solution until a turbid solution was observed. The turbid solution was transferred to the separation funnel and kept overnight at 5 °C until two distinct phases were obtained. The lower phase was again discarded and the upper phase was transferred to a 2L glass flask. The volume of the solution was increased to 1L by adding distilled water while stirring and cooled to 5 °C. Another cooled 800 mL ammonium sulfate solution (25% w/v filtered with 0.22 μιη filter) was slowly added to the solution until a turbid solution was achieved. Same as previously done, the lower phase was discarded and the upper phase was transferred to a glass flask. 500 mL dichloromethane (DCM) was then added to the flask and stirred well. The mixture was transferred to the separation funnel and kept overnight until two distinct phases were achieved. The lower phase was collected and another 500 mL of DCM was added to the separation funnel and mixed well. The mixture was kept overnight until two separated phases were obtained. This cycle was repeated one more time. After this step, DCM was removed via Rotavap until a concentrated poloxamer 407 solution was obtained. Finally, the purified poloxamer 407 was completely dried in a vacuum oven overnight at 30 °C.

Purification Confirmation by HPLC-ELSD

[00128] Poloxamer 407 was characterized using an Agilent 1100 HPLC system with a

Sedex 75 evaporative light scattering detector (ELSD) using a Luna C8 column (5 μιη 100 A, 4.6x150 mm) (Phenomenex, Torrance, CA). Sample (50μ1) was separated using a step- gradient of Mobile Phase A (50 mM ammonium acetate, pH 4) and Mobile Phase B

(methanol) that was at 30% B for 2 min; 100% B for 10 min; and then 30% B for 6 min at a flow rate of 1 mL/minute and a column temperature of 28°C. The ELSD was run at 50 °C drift tube temperature, 3.5 bar pressure and a gain of 3. FIG. 3 shows that the 5 minute shoulder is minimized with the subsequent poloxamer 407 purification.

Purification Confirmation by Compendial Testing

[00129] Three lots of the purified poloxamer 407 were characterized using USP methods as described below, and compared to unpurified poloxamer 407:

(A) Identification A: IR <197> indicates the IR absorption spectrum exhibiting a maxima only at the same wavelengths a corresponding USP reference standard.

(B) USP Average molecular weight by titration.

(C) USP Unsaturation (Specification: 0.048 +/- 0.017 mEq/g).

(D) USP Class 2 Residual Solvent <467> by GC - Procedure C - Methylene chloride NMT 600 ppm.

[00130] As indicated in Table 1, the USP characterization of the three purified poloxamer

407 lots shows that the average molecular weight increased, indicating that lower molecular weight impurities were removed during the purification process and the unsaturation levels were also decreased as a result of the purification confirming successful purification process for elimination of low molecular weight impurities. Moreover, IR results showed no change as a result of purification process indicating that the purification process did not impact on the chemical structure of the poloxamer 407. The results of the purification were still within USP compendial ranges for poloxamer 407. These similarities between the purified polymers and the unpurified starting material translated into similar rheological properties, as described below.

Table 1: Characterization of Three Lots of Purified Poloxamer 407

Identification by Average MW Class 2 Residual

Poloxamer Lot Unsaturation

USP<197> (Titration) Solvent <467> (DCM)

USP specification Meets 9,840 to 14,600 0.048 + 0.017 <600 ppm

Unpurified Meets 10,988 0.057 mEq/g < 600 ppm

Purified lot #1 Meets 12,590 0.025 mEq/g < 600 ppm Identification by Average MW Class 2 Residual

Poloxamer Lot Unsaturation

USP<197> (Titration) Solvent <467> (DCM)

Purified lot #2 Meets 11,956 0.019 mEq/g < 600 ppm

Purified lot #3 Meets 12,128 0.020 mEq/g < 600 ppm

Purification Confirmation by Rheological Evaluation

[00131] Sol-to-gel transition temperature (T_soi-gei), maximum recorded storage modulus, and viscosity measurements for unpurified and purified poloxamer 407 solutions were conducted using a cone-plate geometry (radius 49.9 mm, 1° angle) and 800 μΐ_^ sample volume with an MCR-301 torsional rheometer (Anton Paar, Graz, Austria) that detects torque (T) in the range of 0.1 mNm to 200 mNm. Samples, free of visible air bubbles, were prepared before setting the final measurement gap in the geometry by careful pipetting and visual inspection of the limited exposed surface. A hood covered the cone-plate geometry to mitigate evaporation.

[00132] A temperature sweep (10-40 °C) was conducted to record storage (G') and loss

(G") modulus of the poloxamer 407 solutions during gelation. T_soi-gei is defined as the temperature at which the storage modulus (G') is half way between the values of the storage modulus for the solution and the gel. Based on the preliminary strain sweep test results, a strain amplitude of 0.1% was used for the temperature sweep tests to apply adequate torque and linear viscoelastic behavior. The maximum storage modulus was recorded for each samples as an indication of the strength of the formed gel.

[00133] The viscosity of the samples was measured over a shear rate sweep of 1-1000 s^"1.

The testing was conducted at 10 °C and the viscosity at shear rate of 1000 s^"1 was reported to compare the samples.

[00134] Three lots of purified poloxamer 407 and one lot of starting, unpurified

poloxamer 407 were employed for rheological evaluations. For sample preparation, 17.9% (w/v) poloxamer 407 was dissolved in phosphate buffered saline (PBS) overnight at 5 °C while stirring. Then, the temperature sweep and shear rate sweep were performed for each prepared sample using the rheometer to measure T_soi-gei, maximum recorded storage modulus, and viscosity of the poloxamer 407 samples. [00135] FIG. 4 and Table 2 show the T_soi-gei and maximum recorded storage modulus (G') for unpurified and purified poloxamer 407. Purification reduced the T_soi-gei from 23.4 °C to 20.9 °C and increased the maximum recorded storage modulus from 12.9 kPa to 22.3 kPa. Moreover, the viscosity results shown in FIG. 6 and Table 2 indicate that the viscosity of purified poloxamer 407 is greater than the viscosity of unpurified poloxamer 407. These results confirmed that the purification was successful in removing impurities from the starting poloxamer 407. The purified poloxamer 407 underwent sol-to-gel transition at lower temperature and the formed gel had higher maximum recorded storage modulus as compared to unpurified poloxamer 407.

Table 2: 1 sol-gel, Max. Storage Modulus, and Viscosity of

Purified and Unpurified Poloxamer 407 Samples

[00136] For lot-to-lot comparison, three purified poloxamer 407 batches were prepared and tested for lot-to-lot variation. FIG. 5, FIG. 7, and Table 3 present the rheological results and comparison of the three purified lots. The average T_soi-gei was 20.03 °C, with a 2.5% coefficient of variation. Additionally, the average maximum recorded storage modulus for the three purified lots was 22.5 kPa, with a 3.0% coefficient of variation. Finally, the recorded viscosity at 1000 s^"1 and 10 °C was 29.1 mPa.s, with a 1.4% coefficient of variation. The results indicated that the purification process was reproducible and rheological properties of the three lots of purified poloxamer 407 were comparable.

Table 3: Tsoi-gei, Max. Storage Modulus, and Viscosity of Purified Poloxamer 407 Samples

Maximum Recorded Viscosity (mPa*s) at

Sample ID Tsol-gel (°C)

Storage Modulus (Pa) 1000 (s -¹), 10 °C

Poloxamer 407 in PBS, 19.9 21600 28.9 purified, lot #3

Average 20.03 22500 29.1

Coefficient Variation 2.5% 3.0% 1.4%

Example 3. Preparation of Thermogel Formulations

Impact of Poloxamer 407 Concentration on Gel Formation

[00137] In order to develop formulations based on poloxamer 407, first the impact of the poloxamer 407 concentration on the gel formation was evaluated. Purified poloxamer 407 at concentrations of 10.5%, 11.6%, 12.6%, 13.7%, 14.7%, 15.8%, 16.8%, 17.9%, and 18.9% (w/v) were dissolved in PBS overnight at 5 °C. Then, the impact of concentration on gel formation and viscosity were evaluated using the rheometer.

[00138] FIG. 8 shows the impact of poloxamer 407 concentration on gel formation. The results demonstrated that no gel was formed at 11.6% w/v and 10.5% w/v poloxamer 407 concentrations. FIG. 9A-9B indicate that increasing the poloxamer 407 concentration decreased the T_soi-gei and increased the maximum recorded storage modulus. Higher poloxamer 407 concentration resulted in gel formation at lower temperatures. Moreover, the increase in the poloxamer 407 concentration increased the maximum recorded storage modulus. This indicated that at higher poloxamer 407 concentrations stronger gel were formed.

[00139] FIG. 10-12 show the effect of poloxamer 407 concentration on viscosity at 10 °C and 23 °C. These resuts demonstrated that the increase in poloxamer 407 concentration increased the visocsity at both 10 °C and 23 °C.

[00140] FIG. 13, FIG. 14, and Table 4 show the rehological properties of poloxamer 407

(17.9% w/v) in PBS, distilled water, and tris buffer. Addition of buffer (IX PBS or IX tris) reduced the T_soi-gei but did not have an impact on maximum recorded storage modulus and viscosity. The presence of the salt in the poloxamer 407 solution resulted in formation of micelles (and eventually gel formation) at lower temperature however it did not influence on poloxamer 407 polymeric interaction which causes change on maximum recorded storage modulus. As shown in table 4, the viscosity was not impacted by addition of salt and it remained constant at ~ 29 mPa- s at 1000 (s ¹) and 10 °C for the tested samples.

Table 4: Tsoi-gei, Max. Storage Modulus and Viscosity of Poloxamer 407 in Different Media

Effect of Ethanol Addition to Poloxamer 407

[00141] Both TLR 7/8 agonists (S-36878 and S-36862) are not soluble in aqueous-based systems but are soluble in ethanol. To introduce the TLR 7/8 agonists into the thermogel formulations, ethanol was first added to the poloxamer 407 solution at concentrations of 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, and 25% (v/v) to evaluate its impact on gel formation, Tsoi-gei, strength of the formed gel, and the viscosity of the poloxamer 407 solution. The final concentration of poloxamer 407 after ethanol addition was 17%. Prepared samples were characterized by rheometer.

[00142] FIG. 15 and FIG. 16A-16B present the effect of increasing ethanol concentration on gel formation. Increasing ethanol from 0% v/v to 20% v/v decreased the T_soi-gei but did not have an impact on maximum recorded storage modulus. Increasing ethanol

concentration up to 25% prevented gel formation.

[00143] FIG. 17 and FIG. 18 show the impact of ethanol concentration on viscosity of poloxamer 407 at 10 °C. As shown in FIG. 18, increasing ethanol concentration increased the viscosity of poloxamer 407 solution.

Addition of Drug to Poloxamer 407 Formulation

[00144] In two separate experiments, the drug was added to the poloxamer 407

formulation. Purified poloxamer 407 was used in the first experiment to prepare the formulation with S-36862, and unpurified poloxamer 407 was used in the second experiment to prepare formulations with both S-36878 and S-36862.

[00145] In the first experiment, purified poloxamer 407 was dissolved in PBS overnight at

5 °C while mixing at 500 rpm (excipient solution). TLR 7/8 agonist (S-36862) then was dissolved in ethanol (drug solution). Finally, the drug solution was added to the excipient solution and well mixed to achieve 17% w/v poloxamer 407, 5% v/v ethanol, and 0.3 mg/mL S-36862 in the formulation (Table 5). By mixing the drug solution with the excipient solution, drug particles formed within the poloxamer 407 solution in-situ (FIG. 19).

[00146] Rheological properties of the prepared formulation were tested and compared to the formulation with no drug at 17% w/v poloxamer 407 and 5% v/v ethanol to evaluate the effect of drug addition to the poloxamer 407 formulation. FIG. 20, FIG. 21, and Table 5 show the impact of the addition of the drug on the gel formation and viscosity. As shown in Table 5, addition of the drug did not affect T_soi-gei and maximum recorded storage modulus. Moreover, addition of the drug did not impact the viscosity of poloxamer 407 formulation. Therefore, the results confirmed that the rheological properties of the formulation with purified poloxamer 407 were not impacted by the addition of S-36862.

Table 5: Impact of Drug on Poloxamer 407 Solution Containing 5% (v/v) Ethanol

[00147] In the second set of experiments, poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) copolymer (PLGA-PEG-PLGA) (M_n: 1600: 1500: 1600 Da, 3: 1 LA:GA; sol-gel transition around 37 °C) and poloxamer 407 (unpurified) were used for the formulation development. Both TLR 7/8 agonists (S-36878 and S-36862) were used to prepare the formulations. [00148] Similar to the first experiments, PLGA-PEG-PLGA and poloxamer 407

(unpurified) were dissolved in PBS and stirred at 500 rpm at 5 °C (excipient solution). TLR 7/8 agonists, S-36878 and S-36862, were separately dissolved in ethanol (drug solutions). Finally, the drug solutions were added to the excipient solutions and well mixed to achieve 17% w/v PLGA-PEG-PLGA or poloxamer 407, 5% v/v ethanol, and 0.4 mg/mL S-36862 or S-36878 in the formulation (Table 6). By mixing the drug solution to the excipient solution, particles of the drug form within excipient solution in-situ (FIG. 19). Blank samples without any addition of drug solution were prepared as controls.

[00149] FIG. 22A-22B and Table 6 show the impact of addition of the drug (S-36878 or

S-36862) on the gel formation for both PLGA-PEG-PLGA and poloxamer 407 (unpurified). The results indicate that the addition of S-36878 or S-36862 to either PLGA-PEG-PLGA or poloxamer 407 formulations does not impact T_soi-gei and maximum recorded storage modulus. Moreover, FIG. 23A-23B and Table 6 show that the viscosity was not impacted by the addition of drugs to the formulations.

Table 6: Impact of Drug on Polymer Solutions Containing 5% (v/v) Ethanol

[00150] In the next set of experiments, the injectability of the formulations was evaluated.

Formulations were carefully pipetted (150

into a 1 mL, long BD glass syringe to measure the glide force when the injection rate was set at a constant 260 mm/min. Briefly, an Instron 5542 (Norwood, MA) was calibrated with a 50 N load cell carefully mounted and screwed in position. Next, the filled glass syringe was first loaded at the loading station and the crosshead was brought closely to the syringe head using the navigation buttons (FIG. 24A-24B). Using the Bluehill software (Instron, Norwood, MA), maximum glide force, average glide force, and break loose force were measured. The average glide force values are reported in Table 7.

Table 7: Recorded Average Glide Force

[00151] Similar to gel formation and viscosity results, injectability of the formulations was not impacted as a result of the addition of the drug. Recorded average glide force for the poloxamer 407 formulations with both S-36878 and S-36862 were higher as compared to PLGA-PEG-PLGA formulations, indicating that injection of poloxamer 407 formulations required more force (Table 7).

Example 4. Drug Content Quantification

[00152] The TLR 7/8 agonist in formulated thermogels was measured using an Agilent

1100 HPLC at 246 nm. Samples were diluted ten-fold in ethanol and injected (10 μί) onto a Zorbax Bonus-RP column (4.6x 150 mm, 5 mm column; temperature 50 °C). The separation method used a gradient that consisted of mobile phase A (50 mM ammonium acetate pH 4) and mobile phase B (methanol) at 1 mL/min. The gradient program increased from 25-100% B between 0-15 minutes; stayed at 100% B from 15-20 min; and then ramped down to 25% B from 20 to 25 minutes. The peak area for several concentrations (100, 50, 20, 10, and 2μg/mL) of the TLR 7/8 agonist (S-36862 and S-36878) fitted to a linear algorithm was used to interpolate the concentration of TLR 7/8 agonist in several thermogel formulations. Table 8 shows that the TLR 7/8 agonist content in the formulated samples measured the TLR 7/8 agonist within the targeted concentration. Table 8: Drug Content Quantification in Formulated Samples

Example 5. In Vitro Release Study

[00153] Both PLGA-PEG-PLGA and poloxamer 407 (unpurified) formulations were prepared according to the protocol described in Example 3. After preparing the thermogel formulations, 0.5 mL of drug-loaded thermogel was pipetted into a scintillation vial and incubated at 37 °C for 10 minutes until gel formation. Upon gelation, 3 mL of PBS buffer was added to the thermogel, and immediately, 0.3 mL was removed from the vial and stored as t=0. The vial was replenished with fresh 0.3 mL of buffer. At specific time points, 0.3 mL of the media was carefully removed for drug release analysis by RP-HPLC. Fresh PBS buffer was replaced any time samples were removed for assays.

[00154] The in-vitro release study showed that the lipidated drug molecule (S-36862) had a slower release rate as compared to the non-lipidated drug molecule (S-36878) in both PLGA-PEG-PLGA and poloxamer 407 formulations (FIG. 25A-25B).

Example 6. In Vivo Evaluation of Prepared Formulations

[00155] Two sets of animal studies were performed to evaluate pharmacokinetics and pharmacodynamics of formulations in B 16-OVA tumor model.

Mice, Tumor Cell Line, and Tumor Implantation

[00156] C57BL/6J-TyrC-2J female mice (C57BL/6J Albino, Strain B6(Cg)-TyrC-2J/J,

Order No. 000058) were obtained from Jackson Laboratories (Bar Harbor, ME). The mice were 14-20 grams when received at 3M Veterinary Services. The mice were acclimated for 7-14 days prior to tumor implantation.

[00157] The melanoma cell line B 16-OVA was obtained from Dr. Wynette Dietz,

University of Minnesota. The tumor line was cultured in Dulbecco's Modified Eagle Medium with 10% heat- inactivated fetal calf serum and 1 mg/mL G-418 (Life Technologies, Carlsbad, CA).

[00158] All animal studies were conducted following the approval of and in accordance with 3M's Institutional Animal Care and Use Committee. Prior to tumor implantation, the tumor cells were washed in Hanks Balanced Salt Solution (HBSS), pH 7.4, and diluted to 4- 5x106 cells/mL in HBSS. The cells were loaded into 0.5 cc Allergy Syringes (26 gauge x ½ inch, Becton, Dickinson and Co, Franklin Lakes, NJ), and 0.1 mL (4-5x105 cells) were injected into the subcutaneous space on the shaved right flank of anesthetized mice.

Pharmacokinetics in the B 16-QVA Tumor Model

[00159] Dosing was initiated approximately 15 days post tumor implantation (tumor diameter of 7-10 mm) by first anesthetizing the mice (n=5), and then administering chilled formulations (Table 9) via intratumoral (IT) injection. A single injection was administered into the center of each tumor in a total volume of 0.05 mL (50 μg) using a 0.5 mL Allergy Syringe (26 gauge x ½ inch, Becton, Dickinson and Co, Franklin Lakes, NJ).

Table 9: Formulated Samples Containing 5% (v/v) Ethanol for Pharmacokinetics Study

[00160] Blood and tumors were collected immediately post-dose (-20 min after IT

dosing), and 6 hours, 3 days, and 14 days postdose. Blood was collected from anesthetized mice by terminal cardiac puncture (Monoject syringe, 22 gauge x ¾ inch). Serum was processed from the blood using BD Microtainer tubes according to manufacturer's directions (Product No. 365967, Becton, Dickinson and Co, Franklin Lakes, NJ), and the serum was stored at -70°C until serum cytokine analysis. Tumors were collected by excision from euthanized mice, carefully removing any fat and connective tissue, and stored in 20 mL glass vials with screw caps at -70°C until drug level analysis. Note that the glass vials were pre- weighed in order to accurately calculate the tumor weight. [00161] Tumor drug levels were measured by HPLC-UV. Tumor samples were digested at a concentration of 225 mg tumor/mL or less, in digestion solution (100 mM Tris-HCl pH 8.5, 1 mM EDTA, 0.2% SDS, 200 mM NaCl) and proteinase K enzyme (0.1 U/mg tumor, Amresco, Solon, OH). Tumor samples were digested at 55°C for 5 hours in a shaking water bath. Internal standard was added, and the samples were cooled to room temperature. The TLR 7/8 agonist (S-36862) was isolated from 0.300 mL digested tumor by protein precipitation with 1.2 mL ethanol. After centrifugation, the supernatant was removed and dried under a stream of nitrogen at 55°C for 15 minutes, and reconstituted in 0.150 mL of solution. Analysis was by HPLC-UV detection at 247 nm, using a Zorbax Bonus RP, 150 x 4.6 mm, 3.5 μιη column. The gradient program, using 0.1% formic acid in water and methanol, was linear from 15%-40% methanol over 2.5 minutes, and then from 40%- 95% methanol in a gradient from 2.5 to 17.5 minutes. Quantitation was performed with a calibration standard at 10 mcg/mL.

[00162] Serum drug levels were measured by LC-MS/MS method by WIL Research

Laboratories, LLC (Ashland, OH). FIG. 26A-26B and FIG. 27 show tumor drug levels and serum drug levels for injected PLGA-PEG-PLGA and poloxamer 407 formulations in the B 16-OVA tumor model. PLGA-PEG-PLGA showed higher tumor drug level as compared to poloxamer 407 formulation. Initial burst was also observed in serum drug level for PLGA- PEG-PLGA formulation. For pharmacodynamics evaluation, only poloxamer 407 formulation was considered.

Pharmacodynamics in the B 16-OVA Tumor Model

[00163] Tumor growth inhibition (TGI), survival, and serum cytokines were measured in

IT dosed B 16-OVA mice. Mice were implanted with tumors as described above. Prior to dosing, the mice were randomized on the basis of tumor diameter. Mice were given either one or two IT injections of chilled poloxamer 407 formulation or vehicle (n=10). PBS injection was used as control (n=9). Two studies were conducted to evaluate

pharmacodynamics of poloxamer 407 formulation in the B 16-OVA tumor model: Study One, in which two IT injections were given 7-days apart (average tumor diameter ~5 mm), dosed at 50 μg/50 μΐ_^ (Table 10); and Study Two, in which one IT injection was given (average tumor diameter -10 mm), dosed at 0.4 μg, 4 μg, and 20 μg per 50 μΐ_^ (Table 11). Table 10: Formulated Samples Containing 5% (v/v) Ethanol for Pharmacodynamics Study

One

Table 11: Formulated Samples Containing 5% (v/v) Ethanol for Pharmacodynamics Study

Two

[00164] After dosing, the tumors were measured two-times per week with calibrated

digital calipers (Product No. 62379-531, VWR International, Radnor, PA), and the mice were euthanized when tumor diameter >20 mm. The results are represented as tumor volume calculated using the following formula:

V=p/6* 1.58 (L*W)1.5

[00165] In addition, in Study Two, serum cytokines were measured using the multiplex array method (Bio-Plex 200 system) in blood samples collected from 3 mice in each group at 6 hours post-dose. The multiplex assay was performed according to manufacturer's directions. Magnetic Luminex Screening Assay reagents for mouse KC (CXCL1), CXCL10 (IP- 10), MCP-5 (CCL12), IL-6, and IL-10 were obtained from R&D Systems (Minneapolis, MN). Data analysis was performed using Bio-Plex Manager Software, Version 6.1 (Bio-Rad Life Science Research, Heracles, CA).

[00166] Differences in tumor volume were determined using Repeated Measures Two- way ANOVA and Tukey's multiple comparison tests. Differences in survival curves were determined using the Log-rank (Mantel-Cox) test.

[00167] FIG. 28 and FIG. 29 show that the poloxamer 407 formulations performed better in regards to TGI and survival as compared to PBS. FIG. 30 and FIG. 31 show that increasing the dose increased the performance of the poloxamer 407 formulation, inhibiting tumor growth and resulting in higher survival rate. FIG. 32A-32E show that increasing the dose decreased the serum cytokine level, which could indicate a reduction of drug diffusion and escape from the tumor site.

[00168] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. The present invention is further described by the following claims.

Claims

1. A composition comprising:

a. 10%-25% (w/v) of a reverse thermosensitive polymer;

b. an immune response modifier (IRM) of Formula I:

Y is -O- or -S(0)o-2-;

each R₄ is independently selected from the group consisting of hydrogen, Ci-ioalkyl, and C₂- loalkenyl; and R₃ is selected from the group consisting of hydrogen and alkyl; with the proviso that when L is - NH-S(0₂)-, and n is 0, Ri-i is a linear or branched aliphatic group having at least 16 carbon atoms, optionally including one or more unsaturated carbon-carbon bonds; or a pharmaceutically acceptable salt thereof; and

c. up to 20% (v/v) ethanol.

2. A composition comprising:

a. 10%-25% (w/v) of a reverse thermosensitive polymer;

b. an immune response modifier (IRM) of Formula II:

wherein:

Ri is a linear or branched aliphatic group having 1-23 carbon atoms, optionally including more unsaturated carbon-carbon bonds; and

R is hydrogen, halogen, or hydroxyl;

or a pharmaceutically acceptable salt thereof; and

c. up to 20% (v/v) ethanol.

3. The composition of claim 1 or claim 2, wherein the IRM is a Toll-like receptor 7 (TLR7) agonist, a Toll-like receptor 8 (TLR8) agonist, or a Toll-like receptor 7/8 (TLR7/8) agonist.

4. A composition comprising:

a. 12%-20% (w/v) of a reverse thermosensitive polymer selected from the group consisting of (i) poloxamer 407 and (ii) poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) copolymer (PLGA-PEG-PLGA), wherein the PLGA- PEG-PLGA number-average molecular weight (Mn) is about 1600: 1500: 1600 daltons, and wherein the PLGA ratio of lactic acid to glycolic acid is about 3: 1;

b. an immune response modifier (IRM) having the structure:

or a pharmaceutically acceptable salt thereof; and c. up to 20% (v/v) ethanol.

5. A composition comprising:

b. an immune response modifier (IRM) having the structure:

or a pharmaceutically acceptable salt thereof; and

c. up to 20% (v/v) ethanol.

6. The composition of any preceding claim, comprising 5% (v/v) ethanol.

7. The composition of any preceding claim, comprising 15%-20% (w/v) of the reverse thermosensitive polymer.

8. The composition of any one of claims 1 to 7, wherein the reverse thermosensitive polymer is poloxamer 407.

9. The composition of claim 8, wherein the poloxamer 407 is purified.

10. The composition of any one of claims 1 to 7, wherein the polymer is PLGA-PEG-

PLGA.

11. The composition of any preceding claim, comprising 0.05 to 1.3 mg/mL IRM.

12. The composition of any preceding claim, comprising a concentration of IRM selected from the group consisting of 0.08 mg/mL, 0.4 mg/mL, and 1 mg/mL.

13. The composition of any preceding claim, having a sol-to-gel transition temperature (T_soi-gei) of 20-37 °C.

14. The composition of any preceding claim, further comprising a second active agent.

15. A method of delivering a depot formulation comprising an immune response modifier (IRM) to a subject, the method comprising injecting into the subject an effective amount of the composition of any one of claims 1 to 14.

16. A method of stimulating a local immune response in a subject, the method comprising injecting into the subject an effective amount of the composition of any one of claims 1 to 14.

17. The method of claim 15 or 16, wherein the subject has a tumor and wherein the composition is injected at the site of the tumor.

18. The method of claim 17, wherein the tumor is a breast tumor, a stomach tumor, a lung tumor, a head or neck tumor, a colorectal tumor, a renal cell carcinoma tumor, a pancreatic tumor, a basal cell carcinoma tumor, a cervical tumor, a melanoma tumor, a prostate tumor, an ovarian tumor, a liver tumor, or a bladder tumor.

19. The method of any one of claims 15 to 18, wherein the subject has a disease or disorder of the dermis and wherein the composition is injected at the site of the disease or disorder.

20. The method of claim 19, wherein the disease or disorder of the dermis is selected from the group consisting of: basal cell carcinoma, melanoma, and genital warts.

21. The method of any one of claims 15 to 20, further comprising administering a second active agent.

22. The method of claim 21, wherein the second active agent is a chemotherapeutic agent.

23. Use of the composition of any one of claims 1 to 14 to deliver a depot formulation comprising an immune response modifier (IRM) to a subject.

24. Use of the composition of any one of claims 1 to 14 to stimulate a local immune response in a subject.

25. The use of claim 23 or 24, wherein the subject has a tumor.

26. The use of claim 25, wherein the tumor is a breast tumor, a stomach tumor, a lung tumor, a head or neck tumor, a colorectal tumor, a renal cell carcinoma tumor, a pancreatic tumor, a basal cell carcinoma tumor, a cervical tumor, a melanoma tumor, a prostate tumor, an ovarian tumor, a liver tumor, or a bladder tumor.

27. The use of any one of claims 23 to 26, wherein the subject has a disease or disorder of the dermis.

28. The use of claim 27, wherein the disease or disorder of the dermis is selected from the group consisting of: basal cell carcinoma, melanoma, and genital warts.

29. The use of any one of claims 23 to 28, further comprising a second active agent.

30. The use of claim 29, wherein the second active agent is a chemotherapeutic agent.

31. A method of making the composition of any one of claims 1 to 14, the method comprising:

a. dissolving the reverse thermosensitive polymer in an aqueous medium to prepare an excipient solution;

b. dissolving the IRM in ethanol to prepare a drug solution; and c. adding the drug solution to the excipient solution to prepare a composition comprising ethanol in an amount of up to 20% (v/v).

32. A kit comprising the composition of any one of claims 1 to 14.