TW201822767A - Block copolymer systems for local administration of toll-like receptor agonists - Google Patents

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 system for local administration of toll-like receptor agonists   

The most effective treatment for local solid cancer is to remove the tumor by surgery, followed by postoperative chemotherapy or radiation therapy. However, due to the size of the tumor, the location of the tumor, and / or the stage of the cancer, many patients are not suitable candidates for surgery, so this method is not suitable for many cancers. In some cases, even after surgery, the overall survival rate of some patients is not optimistic. Therefore, treatments including chemotherapy and cancer immunotherapy are additional options for cancer treatment.

Toll-like receptor (TLR) agonist systems have been shown to be small nucleoside analogs that have efficacy against various tumors. TLR agonists are used in cancer immunotherapy to locally stimulate the immune system against cancer cells. Systemic administration of TLR agonists leads to stimulation of the entire body's immune system, which may have very undesirable side effects, such as patient discomfort, while only delivering a small portion of the total dose administered to the tumor. Therefore, local delivery of TLR agonists is the preferred method of administration, such as in dermal administration.

Therefore, TLR agonists are used as effective regulators for local treatment of genital warts and superficial basal cell carcinoma. They are also introduced as treatments for malignant skin lesions, including melanoma and basal cell carcinoma. TLR agonists induce proinflammatory cytokines and chemokines that attract immune cells to the site of administration in vitro and in vivo. Immune cells then treat cancer cells at that site, leading to the elimination of cancer cells.

Studies have shown that injecting TLR agonists as immune stimulants directly into tumor sites produces anti-tumor CD8 T cell responses and is used to treat low-grade lymphoma with low-dose radiation therapy. Preliminary results indicate that a controlled delivery system for these agonists is needed at the tumor site. Therefore, there is a need for new drug delivery methods that include intratumoral delivery of TLR agonists to limit systemic side effects and produce local immune responses.

Thermogels can be made from biodegradable, biocompatible, thermosensitive polymers, which are solutions at room temperature. The active agent (such as TLR agonist) can be incorporated into the thermal gel by mixing the drug (dissolved in an organic solvent) with the polymer solution. During injection (at body temperature), they entangle or gel themselves, leading to the formation of long-acting agents, which fix the drug at the injection site. Drug loading, solution-to-gel conversion, mechanical properties of the gel formed, and drug release from the gel can be manipulated by changing the polymer concentration and by adding excipients.

Some main aspects of the invention are summarized below. Additional aspects are described in the detailed description, examples, drawings, and patent application section disclosed herein. The description in each section of this disclosure is intended to be read together with the other sections. Furthermore, the various embodiments described in each part 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.

The present disclosure provides the administration of block copolymers for delivery (eg, intratumoral delivery) of TLR agonists. The formulation can be fixed at the injection site (eg, tumor) by in situ phase separation (thermosensitive gel) using gelation. Drug incorporation may have a negative effect on the solution-to-gel properties of the hot gel, resulting in no gel formation at body temperature. Therefore, the addition of TLR agonists to thermal gel systems requires us to develop novel methods to achieve the desired gelation properties, the desired release of TLR agonists at the injection site, and greatly reduced systemic side effects. Several excipients have been used to optimize gelation. Our method uses specific excipients and processing to incorporate one or more TLR agonists into the thermal gel. Specifically, we were surprised to find that ethanol can be used to incorporate TLR 7/8 agonists into the thermal gel with minimal or no effect on the solution-to-gel properties of the thermal gel.

In one aspect, the present invention provides a composition comprising: (a) 10% -25% (w / v) reverse thermosensitive polymer; (b) an immune response modifier having the following chemical formula I (IRM): Where R ₁ has the chemical formula alkylene-LR _1-1 , alkenyl-LR _1-1 , or alkynyl-LR _1-1 , where the alkylene, alkenyl, and alkynyl groups are as appropriate Spaced or blocked by one or more -O- groups; L is a bond or functional linking group selected from the group consisting of -NH-S (O) _2- , -NH -C (O)-, -NH-C (S)-, -NH-S (O) ₂ -NR _3- , -NH-C (O) -NR _3- , -NH-C (S) -NR _3- , -NH-C (O) -O-, -O-, -S-, and -S (O) _2- ; and R _1-1 is a linear or branched aliphatic group, the aliphatic The group optionally includes 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 (R ₃ ) ₂ ; n is 0 to 4; R _{2 is} selected from the group consisting of: hydrogen; alkyl; alkenyl; aryl; heteroaryl; heterocyclic; alkylene- Y-alkyl; alkylene-Y-alkenyl; alkylene-Y-aryl; and alkyl or alkenyl substituted with one or more substituents selected from the group consisting of The group consists of the following: -OH; halogen; -N (R ₄ ) ₂ ; -C (O) -C _1-10 alkyl; -C (O) -OC _1-10 alkyl; -N ₃ ; aryl; heteroaryl; heterocyclyl; -C (O) -aryl; and -C (O)- Heteroaryl; Y is -O- or -S (O) _0-2- ; each R _{4 is} independently selected from the group consisting of hydrogen, C _1-10 alkyl, and C _2-10 Alkenyl; and R _{3 is} selected from the group consisting of hydrogen and alkyl; the condition is that when L is -NH-S (O ₂ )-and n is 0, R _1-1 is straight or branched A chain 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) of ethanol.

In some embodiments, the compositions of the present invention include IRM of formula II:

Among them: X is an alkylene group with up to 8 carbon atoms, and these carbon atoms are optionally -O- spaced or blocked; R ₂ is hydrogen, alkyl, alkoxyalkylene (alkylenyl), alkyl Aminoalkylene group or hydroxyalkylene group; Y series -C (O)-or -S (O) _2- ; R ₁ series has 1-23 carbon atoms (preferably 11-23 carbon atoms ) Straight-chain or branched-chain aliphatic group, optionally including one or more unsaturated carbon-carbon bonds; and R is hydrogen, halogen, or hydroxyl.

In some embodiments, 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.

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 Composition: (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 number average molecular weight (Mn) of the PLGA-PEG-PLGA is about 1600: 1500: 1600 Dalton, and wherein the PLGA ratio of lactic acid to glycolic acid is about 3: 1; Immune response modifiers (IRM) of the following structure:

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

In another aspect of the invention, the immune response modifier compound includes N- (4-{[4-amino-2-butyl-1H-imidazo [4,5-c] quinolin-1-yl] oxy Yl} butyl) octadecamide, or a pharmaceutically acceptable salt thereof. In still another aspect of the invention, the immune response modifier compound includes N- (4-{[4-amino-2-butyl-1H-imidazo [4,5-c] quinolin-1-yl] Oxy} butyl) formamide.

In another aspect, the present invention provides a composition comprising: (a) 12% -20% (w / v) of a reverse thermosensitive polymer selected from the group consisting of Composition: (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 number average molecular weight (Mn) of the PLGA-PEG-PLGA is about 1600: 1500: 1600 Dalton, and wherein the PLGA ratio of lactic acid to glycolic acid is about 3: 1; (b) has the following structure Immune response modifier (IRM):

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

In some embodiments, the composition includes 5% (v / v) ethanol.

In certain embodiments, the composition includes 15% -20% (w / v) reverse thermosensitive polymer. In one embodiment, the reverse thermosensitive polymer is molybdenum 407. In some embodiments, the poloxamer 407 is purified. In another embodiment, the reverse thermosensitive polymer is PLGA-PEG-PLGA.

In some cases, the composition may include an IRM of 0.05 to 1.3 mg / mL. 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 present invention may include a second active agent in addition to IRM.

In a specific embodiment, the solution-to-gel transition temperature (T _{solution_coagulate} ) of the _composition is 20 ° C-37 ° C.

The present invention provides a method of delivering a long-acting pharmaceutical formulation including an immune response modifier (IRM) to a subject, the method comprising injecting an effective amount of the composition of the present invention into the subject. The invention also provides a method of stimulating a local immune response in a subject, the method comprising injecting an effective amount of the composition of the invention into the subject. In some aspects, the composition is administered in liquid form, and wherein the composition forms a gel when injected into a subject.

In some embodiments, the subject has a tumor (such as a cancerous tumor) and the composition is injected at the tumor site. In a specific embodiment, the tumor is breast tumor, stomach tumor, lung tumor, head or neck tumor, colorectal tumor, renal cell carcinoma tumor, pancreas tumor, basal cell carcinoma tumor, cervical tumor, melanoma Tumor, prostate tumor, ovarian tumor, liver tumor, or bladder tumor.

In some embodiments, the subject has a disease or condition of the dermis, and the composition is injected at the site of the disease or condition. In a specific embodiment, the disease or condition 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 include administration of a second active agent. In some embodiments, the second active agent is a chemotherapeutic agent.

Also provided is the use of a composition of the present invention to deliver a long-acting pharmaceutical formulation including an immune response modifier (IRM) to a subject. Also provided is the use of the composition of the invention to stimulate a local immune response in a subject. In some embodiments, the subject has a tumor. In a specific embodiment, the tumor is breast tumor, stomach tumor, lung tumor, head or neck tumor, colorectal tumor, renal cell carcinoma tumor, pancreas tumor, basal cell carcinoma tumor, cervical tumor, melanoma Tumor, prostate tumor, ovarian tumor, liver tumor, or bladder tumor.

In some embodiments, the subject has a disease or disorder of the dermis. In a specific embodiment, the disease or condition of the dermis is selected from the group consisting of basal cell carcinoma, melanoma, and genital warts.

Such uses of the invention may include a second active agent, such as a chemotherapeutic agent.

The present invention includes a method of preparing the composition of the present 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 pharmaceutical solution ; And (c) adding the drug solution to the excipient solution to prepare a composition including ethanol in an amount of up to 20% (v / v).

Kits including the composition of the present invention are also provided.

    【Details of the invention】    

Unless otherwise indicated, the practice of the present invention will employ conventional techniques of pharmacy, formulation science, protein chemistry, cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, etc. Technology is within the skill of the field. Such techniques are fully explained in the literature. See, for example, Handbook of Pharmaceutical Excipients [ Handbook of Pharmaceutical Excipients ] (7th edition, edited by Rowe et al., 2012); Martin's Physical Pharmacy and Pharmaceutical Sciences (6th edition, Sinko ), 2010); Remington: The Science and Practice of Pharmacy ] Remington: The Science and Practice of Pharmacy ] (21st Edition, Edited by Philadelphia University of Science, 2005); Current Protocols in Molecular Biology [Ausubel et al. Human Editor, 2016); Molecular Cloning: A Laboratory Manual [Molecular Transplantation: Laboratory Manual] (4th edition, edited by Green and Sambrook, 2012); Lewin's Genes XI [ Lewin's Gene XI] (11th edition, Krebs Editors et al., 2012); DNA Cloning: A Practical Approach, Volumes I and II [DNA Transplantation: Practical Methods , Volumes I and II ] (2nd edition, edited by Glover and Hames, 1995); Protein Engineering: A Practical Approach [Protein Engineering: A Practical Method] (1st edition, edited by Rees et al., 1993); Culture Of Animal Cells (6th edition, Freshney, 2010); Antibodies: A Laboratory Manual (Antibody: Laboratory Manual) (2nd edition, Greenfield editor, 2013); Antibody Engineering [antibody engineering] (2nd edition, Borrebaeck editor, 1995).

In order to make the present invention easier to understand, certain terms are first defined. Additional definitions are listed throughout the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. For example, Dictionary of Pharmaceutical Medicine [Pharmacy Dictionary] (3rd edition, edited by Nahler and Mollet, 2013); The Dictionary of Cell and Molecular Biology (5th edition, edited by JMLackie, 2013), Oxford Dictionary of Biochemistry and Molecular Biology [Oxford Dictionary of Biochemistry and Molecular Biology ] (2nd edition, edited by R. Cammack et al., 2008), and The Concise Dictionary of Biomedicine and Molecular Biology [ Concise Dictionary of Biomedicine and Molecular Biology ] (Second Edition, PS.Juo, 2002) can provide technical personnel with a general definition of some terms used in this article.

Any subheadings provided herein are not limitations of different aspects or embodiments of the invention, which can be obtained by referring to this specification as a whole. Therefore, by referring to the specification in its entirety, the terms defined immediately below are more completely defined.

All such references cited in this disclosure are incorporated by reference in their entirety. In addition, any manufacturer's instructions or catalogs for any products cited or mentioned herein are incorporated by reference. The documents incorporated herein by reference or any teachings therein may be used in the practice of the present invention. Documents incorporated by reference into this document are not admitted to be prior art.

I. Definition

As used in this specification and the scope of the accompanying patent applications, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. The terms "a / a / a" (or "an"), and the terms "one or more" and "at least one / at least one" one) "can be used interchangeably.

Furthermore, "and / or" is understood to be a specific disclosure of each of these two specified features or components with or without the other. Therefore, the term "and / or" as used in phrases 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 phrases 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).

When the language "comprising" is used to describe an embodiment, other similar embodiments described with respect to "consisting of" and / or "consisting essentially of" are included.

Units, initial codes, and symbols are expressed in the form accepted by their Système International de Unites (SI). Numeric ranges include the numbers that define the range, and any single value provided herein can be used as the endpoint of a range including other single values provided herein. For example, a set of values, such as 1, 2, 3, 8, 9, and 10, are also revealed from the numeric range of 1-10, 1-8, 3-9, and so on.

"Polymer" is a series of cross-linked repeating units or "monomers". The monomer units can be the same or different ("copolymer"). The polymer may include two or more different crosslinked polymers. The polymers can be combined in different ratios to provide compositions with different properties. Those skilled in the field of polymer chemistry will be familiar with the different properties of polymer compounds.

The composition of the present invention includes a "reverse thermosensitive polymer", which is liquid at low temperatures and rapidly converts to a gel at physiological temperatures. This conversion may occur very quickly, for example after half a degree Celsius. Various polymer compositions, concentrations, and buffer solutions can be used to change the conversion temperature. Aqueous, biocompatible polymers reversibly return to liquid by cooling, and are soluble.

Thermal gelation is a reversible phenomenon defined by the solution-gel transition temperature (T _{solution_gel} ). Under the T _{solution_gel} , the materials remain liquid, and above this temperature, the materials become semi-solid (gel). Here, T _{solution_gel is} defined as a _temperature at which the storage modulus (G ') is half of the value between the flowable material and the storage modulus of the gel.

"Isolated" molecules are molecules that exist in forms not found in nature, including those that have been purified. In some embodiments, the isolated molecules are substantially pure. As used herein, the term "substantially pure" refers to a purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.

A "labeled" is a detectable compound that can be directly or indirectly conjugated to a molecule in order to produce a "labeled" molecule. The label may be detectable in itself (eg, radioisotope label or fluorescent label), or it may catalyze a chemical change in a detectable substrate compound or composition (eg, enzymatic label).

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

"Active agent" is an ingredient intended to provide pharmacological activity or other direct effects, or to affect the structure or any function of the human body in the diagnosis, cure, alleviation, treatment, or prevention of disease. The active agent may be combined with one or more other ingredients, and may, but not necessarily be the finished dosage form. The terms "active agent" and "drug substance" are used interchangeably herein.

An "effective amount" of active agent is an amount sufficient to achieve a clearly defined purpose. Regarding the prescribed purpose, the "effective amount" can be determined empirically and in a conventional manner.

The term "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional components that have unacceptable toxicity to the subject to be administered. Such a composition may be sterile and may include a pharmaceutically acceptable carrier, such as physiological saline. The form and characteristics of a pharmaceutically acceptable carrier or diluent can be determined by the amount of active ingredient to be combined, the route of administration, and other well-known variables. Suitable pharmaceutical compositions may include one or more buffers (e.g. acetate, phosphate or citrate buffers), surfactants (e.g. polysorbate), stabilizers (e.g. human albumin), preservatives ( (Eg sodium benzoate), absorption enhancers to enhance bioavailability and / or other conventional solubilizers or dispersants.

"Subject" or "individual" or "animal" or "patient" or "mammal," means any subject whose diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammal subjects include humans, domestic animals, agricultural animals, sports animals, and laboratory animals, including, for example, humans, non-human primates, large animals, felines, pigs, cattle, horses, rodents (including large animals) Rats and mice, rabbits) and so on.

Terms such as "treating or treatment or to treat" or "alleviating or to alleviate" refer to curing, slowing the diagnosed pathological condition or disorder, and alleviating the symptoms of the diagnosed pathological condition or disorder, and / or Or stop the therapeutic measures of the progression of the diagnosed pathological condition or condition. Therefore, patients in need of treatment include those already suffering from the condition. In certain embodiments, if the patient shows, for example, full, partial, or transient relief or elimination of symptoms associated with the disease or disorder, the subject successfully "treated" the eye disease or disorder according to the methods provided herein.

"Prevent or prevention" refers to defensive or preventive measures that prevent and / or slow down the development of target pathological conditions or conditions. Therefore, patients in need of prevention include those susceptible to or susceptible to the condition. In certain embodiments, if the patient transiently or permanently exhibits, for example, fewer or less severe symptoms related to the disease or condition, or is associated with the disease or condition than the patient who has not been subjected to the method of the invention The later onset of related symptoms successfully prevented eye diseases or conditions according to the methods provided herein.

II. Reverse thermosensitive polymer

To control the intratumoral delivery of TLR agonists, several configurations can be used, such as hydrogels, particle systems, wafers, and rods formed by engineered in situ long-acting agents to minimize systemic side effects. In most cases, these systems are made of biodegradable polymeric materials such as natural polymers (including polysaccharides and polypeptides), and synthetic polymers such as PLA and PLGA. These biopolymers are biocompatible in the body and are suitable as a system for in situ long-acting drug delivery for local intratumoral drug delivery.

In such systems, hydrogels formed by long-acting agents in situ are three-dimensional networks of polymers that maintain the ability of TLR agonists, and thus can be used to deliver such cancer immunotherapeutics intratumorally. Injectable, biodegradable, in situ formed long-acting agents have been shown to be less invasive than pre-formed implants, and have less pain when injected, making it a topical anticancer agent The ideal system for medicines. Injectable biomaterials are suitable for development as delivery systems for drug molecules that locate tumor sites. According to the mechanism of the formation of long-acting agents, engineered in-situ gelling long-acting agents can be divided into two categories: (1) platforms based on in-situ crosslinking, and (2) platforms based on in-situ phase separation. In situ phase separation is a strategy for delivering drugs to the tumor site. Phase separation can be induced by changing the solubility of the polymer (relative to changes in pH, temperature) or by eliminating the solvent.

For local delivery, and more specifically, the key requirement of an in situ long-acting agent formation system for intratumoral delivery is the injectability of using standard metered injection needles in vial / syringe or pre-filled syringe configurations. The injection should be easy to administer and should also cause minimal discomfort to the patient. Intratumoral injection systems based on in situ gelling polymers have a low viscosity and can flow easily during administration, but upon injection, a gel network is quickly formed. In the phase separation platform, as the temperature increases, the thermal gel-based platform undergoes solution-to-gel conversion ( Figure 1 ). Since thermogels do not require the use of organic solvents, polymerization agents, or any externally applied initiators for in situ long-acting agent formation, they are particularly attractive for delivery systems of small molecules and biomolecules. Temperature-dependent phase transitions are controlled by molecular interactions, including hydrogen bonding or hydrophobic responses. Compared with polymer-polymer interaction at low critical solution temperature (LCST), water-polymer hydrogen bonding is often undesirable. In this state, the solvated macromolecule loses water of hydration, and the polymer-polymer interaction increases. As the viscosity of the system increases, a polymer network structure is formed. For intratumoral drug delivery, the ideal requirement is an aqueous polymer solution that flows easily at room temperature and then forms a gel at physiological temperature. For this method, synthetic and natural polymer materials can be used.

Such compositions provided herein include reverse thermosensitive polymers. Such a polymer may be composed of, for example, a triblock polymer having two hydrophilic chains connected by a hydrophobic chain. Rapid viscosity conversion occurs in response to heat, which causes the polymer chains to deform and align the hydrophilic chains, resulting in the formation of micelles and subsequent phases into viscous gels. The resulting gel is soluble and can also return to liquid under cooling.

The conversion temperature (T _{solution_gel} ) of the reverse thermosensitive polymer can be changed in various ways. For example, the conversion temperature can be changed by adding additives, such as fatty acid excipients, including sodium caprate, sodium laurate, or sodium oleate; wetting agents, such as glycerin; ethylene glycol; emulsifiers, solubilizers; chains Alkanes; triglycerides; lipophilic substances such as isopropyl myristate; and various solvents. The use of modified polymers can also affect the conversion temperature. In addition, the addition of other polymers to form a polymer mixture can affect the conversion temperature. Those skilled in the art can adjust the T _{solution_gel of the} composition of the present invention to any desired temperature. Preferably, the composition of the present invention has a T _{solution_gel of} about 10 ° C to about 40 ° C, or about 20 ° C to about 37 ° C, or about 25 ° C to about 37 ° C.

The polymer used in the present invention may be an elastic or flowable material. "Flowable" means the ability to maintain the shape of the space it is in over time. This feature includes, for example, a liquid composition or a high-viscosity gel-like material. Therefore, the polymers can be administered in liquid or gel form. In some embodiments, the polymer is in an aqueous solution.

Preferably, such a reverse thermosensitive polymer number average molecular weight (M _n) of from about 2,000Da to about 100,000Da, more specifically at least about 10,000Da, or at least about 25,000Da, or at least about 40,000Da. In some embodiments, M _n of these polymers is from about 5,000Da to about 90,000Da, or from about 10,000Da to about 80,000Da, or from about 20,000Da to about 70,000Da, or from about 30,000Da to about 60,000Da, or About 5,000Da to about 50,000Da.

Poloxamer is an example of a reverse thermosensitive polymer suitable for the present invention. Poloxamers are a class of triblock polyalkylene oxide copolymers, usually composed of a core block of poly (propylene oxide), which covers each block with poly (ethylene oxide) blocks On the end. Poloxamers with a higher proportion of ethylene oxide tend to exhibit reverse gelation. Poloxamer is most commonly unbranched. Examples of Poloxamers include Poloxamer 118, Poloxamer 188, Poloxamer 338, Poloxamer 407. Poloxamer's trade names are Pluronic® and Tetronic®.

Poloxamine, in which an amine group replaces oxygen in the main chain or terminal, is another example of a reverse thermosensitive polymer suitable for the present invention. Specific examples of poloxamide include poloxamine 1107 and poloxamine 1307.

Suitable polymers also include combinations, copolymers 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), considered to be molybdenum- ( Pluronic®) poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) copolymerization material, poly (N-isopropylacrylamide), poly (ethylene glycol) -block -Poly (caprolactone) -block-poly (ethylene glycol), poly (organophosphazene), methyl cellulose, hydroxypropyl methyl cellulose, chitosan solution with β-glycerophosphate, And collagen.

Preferred reverse thermosensitive polymers include poly (lactide-co-glycolide) -block-poly (ethylene glycol) -block-poly (lactide-co-glycolide) Thermogels of block copolymers, and poloxamer-based (Pluronic®) thermogels. In one embodiment, the polymer-based PLGA-PEG-PLGA, which is M _n of about 1600: 1500: 1600Da, and the ratio of lactic acid and glycolic acid PLGA of 3: 1. In another embodiment, the polymer is molybdenum 407.

III. Immune response modifiers

The composition of the present invention includes an immune response modifier (IRM). IRM has been shown to induce the production of certain cytokines such as interferon alpha (IFN-α), tumor necrosis factor alpha (TNF-α), and certain interleukins, indicating that these compounds can inhibit tumor cell growth and viruses produce. The ability to modulate the immune response by inducing cytokine biosynthesis also makes IRM 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.

The composition provided herein may include an IRM having the following chemical formula I: Where R ₁ has the chemical formula alkylene-LR _1-1 , alkenyl-LR _1-1 , or alkynyl-LR _1-1 , where the alkylene, alkenyl, and alkynyl groups are as appropriate Spaced or blocked by one or more -O- groups; L is a bond or functional linking group selected from the group consisting of -NH-S (O) _2- , -NH -C (O)-, -NH-C (S)-, -NH-S (O) ₂ -NR _3- , -NH-C (O) -NR _3- , -NH-C (S) -NR _3- , -NH-C (O) -O-, -O-, -S-, and -S (O) _2- ; and R _1-1 is a linear or branched aliphatic group, the aliphatic The group optionally includes 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 (R ₃ ) ₂ ; n is 0 to 4; R _{2 is} selected from the group consisting of: hydrogen; alkyl; alkenyl; aryl; heteroaryl; heterocyclic; alkylene- Y-alkyl; alkylene-Y-alkenyl; alkylene-Y-aryl; and alkyl or alkenyl substituted with one or more substituents selected from the group consisting of The group consists of the following: -OH; halogen; -N (R ₄ ) ₂ ; -C (O) -C _1-10 alkyl; -C (O) -OC _1-10 alkyl; -N ₃ ; aryl; heteroaryl; heterocyclyl; -C (O) -aryl; and -C (O)- Heteroaryl; Y is -O- or -S (O) _0-2- ; each R _{4 is} independently selected from the group consisting of hydrogen, C _1-10 alkyl, and C _2-10 Alkenyl; and R _{3 is} selected from the group consisting of hydrogen and alkyl; the condition is that when L is -NH-S (O ₂ )-and n is 0, R _1-1 is straight or branched A chain aliphatic group having at least 16 carbon atoms, optionally including one or more unsaturated carbon-carbon bonds.

In some embodiments, R _1-1 is a linear or branched aliphatic group having 11-20 carbon atoms (preferably 12-20 carbon atoms), optionally including one or more unsaturations Carbon-carbon bond. In some embodiments, R _1-1 is a linear (ie, linear) alkyl group having 11-20 carbon atoms (preferably 12-20 carbon atoms). The R _1-1 substituents described herein are desirable because they provide lipid-like characteristics to the IRM. This is advantageous because the lipid moieties can contribute to chelation of the IRM at the application site. That is, the lipid portion may explain the prevention of rapid diffusion of the IRM away from the administration site. This chelation can lead to IRM-enhancing adjuvants, which can be manifested by enhanced recruitment and activation of antigen-presenting cells at the desired site. In addition, this chelation can result in less systemic distribution of IRM, and the ability to use lesser amounts of IRM.

Some aspects of the invention include a non-lipidated form of IRM, wherein R _1-1 is a straight or branched chain aliphatic group having 1-10 carbon atoms. In some embodiments, R _1-1 is CH ₃ .

In some embodiments, the compositions of the present invention include IRM of formula II:

Among them: X is an alkylene group with up to 8 carbon atoms, and these carbon atoms are optionally -O- spaced or blocked; R ₂ is hydrogen, alkyl, alkoxyalkylene, alkylaminoalkyl Propylene group, or hydroxyalkylene group; Y series -C (O)-or -S (O) _2- ; R ₁ series having 1-23 carbon atoms (preferably 11-23 carbon atoms) straight A chain or branched aliphatic group optionally includes one or more unsaturated carbon-carbon bonds; and R is hydrogen, halogen, or hydroxyl.

The term "aliphatic" group refers to a saturated or unsaturated linear or branched hydrocarbon group. The term is used to cover, for example, alkyl, alkenyl, and alkynyl groups.

As used herein, the terms "alkyl", "alkenyl", "alkynyl" and the first "alkyl" include straight and branched chain groups and cyclic groups, such as cycloalkyl and cycloalkenyl . Unless otherwise stated, these groups contain from 1 to 23 carbon atoms, of which alkenyl groups contain from 2 to 23 carbon atoms and alkynyl groups contain from 2 to 23 carbon atoms. In some embodiments, the 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. The cyclic group may be monocyclic or polycyclic and preferably has from 3 to 10 ring carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopropylmethyl, cyclopentyl, cyclohexyl, adamantyl, and substituted and unsubstituted bornyl, norbornyl, and norbornyl Norbornenyl.

Unless otherwise stated, "alkylene", "alkenyl", and "alkynyl" are the divalent forms of the "alkyl", "alkenyl", and "alkynyl" groups defined above. Similarly, "alkylene", "alkenyl", and "alkynyl" are the divalent forms of "alkyl", "alkenyl", and "alkynyl" as defined above. For example, an arylalkylene group includes an alkylene moiety to which the aryl group is attached.

An alkylene group having carbon atoms optionally "spaced" by -O- means having carbon atoms on either side of -O-. An example is -CH ₂ -CH ₂ -O-CH ₂ -CH _2- .

An alkylene group having a carbon atom that is optionally "end-capped" by -O- means having -O- at either or both ends of the alkylene group or carbon atom chain. Examples include -O-CH ₂ -CH ₂ -CH ₂ -CH ₂ -and -CH ₂ -CH ₂ -CH ₂ -CH ₂ -O-. In some embodiments, when X is an alkylene group having up to 8 carbon atoms and the carbon atoms are terminated with -O-, the -O- can be attached to the nitrogen of the imidazole ring or amide (Y -C (O)-) or sulfonamide (Y series -S (O) 2-) group nitrogen.

The term "haloalkyl" includes groups substituted with one or more halogen atoms, including perfluorinated groups. The same is true for other groups that include the initial "halo". Examples of suitable haloalkyl groups are chloromethyl, trifluoromethyl and the like.

The term "aryl" as used herein includes carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, bisphenyl, stilbyl and indenyl.

The term "heteroaryl" includes aromatic rings or ring systems containing at least one ring heteroatom (eg, O, S, N). Suitable heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyridine Oxazolyl, Oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzo Oxazolyl, pyrimidinyl, benzimidazolyl, quino Porphyrinyl, benzothiazolyl, naphthyridyl, iso Oxazolyl, isothiazolyl, purinyl, quinazolinyl, pyridine Group, 1-oxypyridyl group, etc.

The term "heterocyclic group" includes non-aromatic rings or ring systems containing at least one ring heteroatom (eg, O, S, N), and includes all fully saturated and partially unsaturated derivatives of the aforementioned heteroaryl groups. Exemplary heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl, Porphyrinyl, thio Porphyrinyl, piperidinyl, piper Group, tetrahydrothiazolyl, imidazolidinyl, isothiazolidinyl, tetrahydropiperanyl, Pyridyl, homopiperidyl, etc.

The terms "arylene", "heteroaryl", and "heterocyclylene" are the divalent forms of "aryl", "heteroaryl", and "heterocyclyl" as defined above. Similarly, "arylene", "heteroarylene", and "heterocyclic" are the divalent forms of "aryl", "heteroaryl", and "heterocyclic" defined above. For example, an alkylarylene group includes an arylene moiety to which the alkyl group is attached.

Any pharmaceutically acceptable form of the IRM and its salts of Formula I and Formula II can be used, including isomers (eg, diastereomers and mirror isomers), solvates, polymorphs, and the like. In particular, if the compound is optically active, the present invention specifically includes each of the mirror images of the compound, as well as the racemic mixture of mirror images. In some embodiments, the IRM compound is not imiquimod.

As those skilled in the art will understand, for any compound proposed herein, each variable in any of its embodiments (eg, R, R ₁ , R _1-1 , L, X, etc.) can be combined with any of its embodiments Any one or more of the other variables in the combination. Each of the resulting combinations of variables is included in the embodiments of the present invention.

Preferred IRMs used in the compositions and methods of the present invention include S-36862 ( FIG. 2A ) and S-36878 ( FIG. 2B ). Additional preferred IRMs used in the compositions and methods of the present invention include N- (4-{[4-amino-2-butyl-1H-imidazo [4,5-c] quinoline-1 -Yl] oxy} butyl) stearic acid amide and N- (4-{[4-amino-2-butyl-1H-imidazo [4,5-c] quinolin-1-yl] Oxy} butyl) formamide.

The IRM 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 Shendian.

A description of suitable IRMs, and methods of making and using them can be found in, for example, US Patent No. 7,799,800, US Patent No. 9,242,980, and International Publication No. WO 2015/069535.

IV. Composition and methods

The composition of the present invention includes a reverse thermosensitive polymer, IRM, and ethanol. Typical compositions of the present invention include 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.

In some embodiments, the composition of the present invention includes an IRM of about 0.05 to about 1.5 mg / mL, or an IRM of about 0.1 to about 1.0 mg / mL. For example, the composition may include 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 of IRM. The amount of IRM in the composition of the invention will vary depending on the IRM, the subject being treated and the intended indication. The therapeutic dose can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy. Determining the therapeutically effective amount of IRM included in the composition is within the ordinary skill of the art.

The invention also includes the use of any hydrophobic drugs or active pharmaceutical ingredients in the thermosensitive polymer composition used to develop a local drug delivery system, such as risperidone, paclitaxel, topotecan , Doxorubicin or docetaxel. Therefore, the present invention provides a composition including a reverse thermosensitive polymer, a hydrophobic drug, and up to 20% (v / v) ethanol.

The composition of the present invention includes 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 specific embodiment, the composition includes about 5% (v / v) ethanol.

The pH of the composition administered to the subject is generally about 5.5 to about 8.5, preferably about 6.0 to about 7.8, which is a suitable pH level for injection into mammals. The pH of the composition can be adjusted by any suitable acid or base (such as hydrochloric acid or sodium hydroxide).

Preferably, the composition of the present invention is a pharmaceutical composition. The pharmaceutical composition according to the present invention may include pharmaceutically acceptable, non-toxic, sterile carriers, such as physiological saline, non-toxic buffers (eg acetate, phosphate, citrate), surfactants (eg polysorbate) ), Stabilizers (such as human albumin), and / or salts (such as acid addition salts, base addition salts), etc. The form and characteristics of the pharmaceutically acceptable carrier or diluent can be determined by the amount of active ingredient to be combined and other well-known variables.

These compositions may also contain preservatives, wetting agents, emulsifying agents, and dispersing agents. The prevention of the emergence of microorganisms can be ensured by a sterilization procedure and by including both different antibacterial and antifungal agents (eg, parabens, trichlorobutanol, sorbic acid phenol, etc.). It is also possible to add isotonic agents (such as sugar, sodium chloride, etc.) to these compositions. In addition, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin).

The pharmaceutical compositions provided herein may also include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc .; (2) oil-soluble antioxidants , Such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc .; and (3) metal chelating agents , Such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

Due to their reverse thermosensitive properties, the compositions of the present invention form a gel when injected into a subject's body. Therefore, the composition of the present invention can be used in a method of delivering a long-acting pharmaceutical formulation including IRM to a subject, the method comprising injecting an effective amount of the composition into the subject. The invention also provides a method of stimulating a local immune response in a subject, the method comprising injecting an effective amount of the composition of the invention into the subject.

Subjects in need of treatment may have tumors, such as breast tumors, stomach tumors, lung tumors, head or neck tumors, colorectal tumors, renal cell carcinoma tumors, pancreatic tumors, basal cell carcinoma tumors, cervical tumors, Melanoma tumor, prostate tumor, ovarian tumor, liver tumor, or bladder tumor. The tumor may be in organs, lymphoid tissue, reticular endothelial tissue, bone marrow, mucosal tissue, etc. The tumor may be a solid tumor and may be malignant. In one embodiment, the composition of the present invention is injected into a tumor.

Subjects in need of treatment may have a disease or condition of the dermis, such as basal cell carcinoma, melanoma, or genital warts. In one embodiment, the composition of the present invention is injected into the site of a disease or disorder.

The composition can be administered in a single dose or multiple doses. The composition can be administered as many times as necessary to achieve the target endpoint. The injection interval may be different. For example, the composition can be administered every 1, 2, 3, or 4 weeks, or every 1, 2, 3, 4, 5, or 6 months. The dosage regimen can be adjusted to provide the best desired response.

The composition may also be administered in combination therapy and / or in combination with other agents. Specifically, the method of the present invention may include administration of a second active agent in addition to IRM. For example, the second active agent can be a second IRM, an antigen, an antigen binding molecule, a chemotherapeutic agent, a cytotoxic agent, an antiviral agent, an interleukin, a tumor necrosis factor receptor agonist, or a labeling or imaging agent. In a specific embodiment, the second active agent is a chemotherapeutic agent. The composition of the present invention may include a second active agent, or the second active agent may be administered separately.

The composition of the present invention can be prepared by dissolving the reverse thermosensitive polymer in an aqueous medium to prepare an excipient solution; dissolving a hydrophobic drug such as IRM in ethanol to prepare a drug solution; The solution is added to the excipient solution to prepare a composition including ethanol in an amount of up to 20% (v / v). Combining the excipient solution and the drug solution results in the precipitation of drug particles because hydrophobic drugs such as IRM are water-insoluble. Unexpectedly, ethanol acts as a surfactant, stabilizing drug particles so that they do not aggregate. Therefore, the present invention provides a method for preparing a stable thermal gel.

V. Set

Kits including the compositions and instructions for use provided herein are also within the scope of this disclosure. The kit may further contain at least one additional reagent, or one or more additional components. The kit usually includes a mark indicating the intended use of the contents of the kit. The term "marking" includes any written or recorded material provided on or with the kit, or any other written or recorded material that accompanies the kit.

The present disclosure further provides kits comprising one or more compositions of the present invention, which can be used to perform the methods described herein. In some embodiments, the kit includes at least one composition including a reverse thermosensitive polymer, IRM, and up to 20% ethanol in one or more containers. In some embodiments, the kits contain all elements necessary and / or sufficient to perform the methods of the present invention. Those skilled in the art will readily recognize that the disclosed composition can be easily incorporated into one of the established kit forms well known in the art.

    【Example】    

Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail the preparation of certain compositions of the present disclosure and methods of making and using the disclosed compositions. Those skilled in the art should understand that many modifications can be made to both materials and methods without departing from the scope of this disclosure.

Example 1. Materials

Poloxamer 407, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (St. Louis, Missouri). Undenatured USP ethanol (200 proof) and dichloromethane (DCM) were purchased from Spectrum Chemicals (New Brunswick, New Jersey). Phosphate buffered saline (PBS 1X) pH 7.2 was obtained from Life Technologies (Carlsbad, California). Poly (lactic acid-co-glycolic acid) -b-poly (ethylene glycol) -b-poly (lactic acid-co-glycolic acid) (PLGA-PEG-PLGA) copolymer (Mn: 1600: 1500: 1600Da, LA: GA: 3: 1) Obtained from PolySciTech (West Lafayette, Indiana). TLR 7/8 agonists ( Figures 2A-2B ), S-36878 (non-lipidated) and S-36862 (lipidated) were received from 3M Company (St. Paul, Minnesota). Tris buffered saline pH 7.2 (Tris 1X) was prepared internally.

Example 2. Purification of Poloxamer 407

Purification process

Poloxamer 407 was purified using the aqueous-aqueous extraction system disclosed in US Patent No. 6,761,824. Briefly, 100 g of poloxamer was added to 900 mL of distilled water in a 2L flask, and stirred at 5 ° C for 7 hours until poloxamer 407 was completely dissolved. Then, cooled 1000 mL of ammonium sulfate solution (25% w / v filtered with 0.22 μm filter) was slowly added to the solution at 5 ° C. while mixing until a cloudy solution was obtained. The solution was then transferred to a 2L separatory funnel and kept at 5 ° C overnight until two different phases were formed. 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 1 L, and the solution was cooled to 5 ° C. Next, another cooled 800 mL of ammonium sulfate solution (25% w / v filtered with a 0.22 μm filter) was slowly added to the poloxamer 407 solution until a cloudy solution was observed. The turbid solution was transferred to a separatory funnel and kept at 5 ° C overnight until two different phases were obtained. The lower phase was discarded again, and the upper phase was transferred to a 2L glass flask. Distilled water was added to increase the volume of the solution to 1 L while stirring, and cooled to 5 ° C. Another cooled 800 mL of ammonium sulfate solution (25% w / v filtered with a 0.22 μm filter) was slowly added to this solution until a cloudy solution was obtained. As before, the lower phase was discarded and the upper phase was transferred to a glass flask. Then 500 mL of dichloromethane (DCM) was added to the flask and stirred well. The mixture was transferred to a separatory funnel and kept overnight until two different phases were obtained. Collect the lower phase and add another 500 mL of DCM to the separatory funnel and mix well. The mixture was kept overnight until two separate phases were obtained. Repeat this cycle again. After this step, DCM was removed by Rotavap until a concentrated poloxamer 407 solution was obtained. Finally, the purified poloxamer 407 was completely dried in a vacuum oven at 30 ° C overnight.

Confirmation of purification by HPLC-ELSD

Using a Luna C8 column (5 μm 100Å, 4.6 x 150mm) (Philomenex, Torrance, CA), using an Agilent 1100 HPLC with a Sedex 75 evaporative light scattering detector (ELSD) The system characterizes Poloxamer 407. A sample (50 μl) was separated using a step gradient of mobile phase A (50 mM ammonium acetate, pH 4) and mobile phase B (methanol), with a flow rate of 1 mL / min at 30% B for 2 min; 100% B for 10 min; and Then 30% B lasted 6 min, and the column temperature was 28 ° C. The ELSD was operated at a drift tube temperature of 50 ° C, a pressure of 3.5 bar and a gain of 3. Figure 3 shows that in the case of subsequent Poloxamer 407 purification, the shoulder peak at 5 minutes was minimized.

Confirmation of purification by pharmacopoeial testing

Three batches of purified poloxamer 407 were characterized using the USP method described below and compared with unpurified poloxamer 407:

(A) Identification A: IR <197> indicates that the IR absorption spectrum shows the maximum value only at the same wavelength as the corresponding USP reference standard.

(B) USP average molecular weight by titration.

(C) USP unsaturation (specification: 0.048 +/- 0.017mEq / g).

(D) USP grade 2 residual solvent <467> by GC-procedure C-methylene chloride NMT 600 ppm.

As shown in Table 1 , the USP characterization of the three batches of purified poloxamer 407 showed an increase in average molecular weight, indicating that lower molecular weight impurities were removed during the purification process, and because the purification also reduced the level of unsaturation, confirming A successful purification method for eliminating low molecular weight impurities. In addition, due to the purification process, IR results showed no change, indicating that the purification process does not affect the chemical structure of poloxamer 407. The purification results of Poloxamer 407 are still within the scope of the USP Pharmacopoeia. These similarities between the purified polymer and the unpurified starting material are translated into similar rheological properties, as described below.

Purification confirmation by rheological evaluation

Using a cone-plate geometry (radius 49.9mm, 1 ° angle) and a sample volume of 800 μL from The solution-to-gel transition temperature (T _solution-gel ) of the unpurified and purified poloxamer 407 solution, the maximum recorded storage modulus, and viscosity measurements were performed. Before setting the final measurement gap in the geometry, prepare samples without visible air bubbles by careful pipetting and visual inspection of the limited exposed surface. Cover the cone-plate geometry with a cover to reduce evaporation.

A temperature scan (10 ° C-40 ° C) was performed during the gelation process to record the energy storage (G ') and loss (G ") modulus of the Poloxamer 407 solution. T _{solution_gel} is defined as the energy storage The modulus (G ') is half the temperature between the storage modulus values of the solution and the gel. Based on the preliminary strain scan test results, a temperature sweep test is performed using a strain amplitude of 0.1% to apply sufficient torque and linear viscosity Elastic behavior: Record the maximum storage modulus of each sample as an indicator of the strength of the gel formed.

The viscosity of these samples was measured under a shear rate scan of 1-1000s ^-1 . The test was conducted at 10 ° C, and the viscosity at a shear rate of 1000 s ^-1 was reported to compare the samples.

Three batches of purified poloxamer 407 and a batch of starting, unpurified poloxamer 407 were used for rheological evaluation. For sample preparation, 17.9% (w / v) poloxamer 407 was dissolved in phosphate buffered saline (PBS) at 5 ° C overnight while stirring. Then, a temperature scan and a shear rate scan were performed on each prepared sample using a rheometer to measure the T _{solution_gel} , maximum recorded storage modulus, and viscosity of the Poloxamer 407 sample.

Figure 4 and Table 2 show the T _{solution_gel of} unpurified and purified poloxamer 407 and the maximum recorded storage modulus (G '). Purification reduced the T _{solution_gel} from 23.4 ° C to 20.9 ° C, and increased the maximum recorded storage modulus from 12.9 kPa to 22.3 kPa. In addition, the viscosity results shown in FIG. 6 and Table 2 indicate that the viscosity of purified poloxamer 407 is greater than that of unpurified poloxamer 407. These results confirm that the purification successfully removed impurities from the starting poloxamer 407. Purified poloxamer 407 undergoes solution-gel conversion at a lower temperature, and the gel formed has a higher maximum recorded storage modulus than unpurified poloxamer 407.

For batch-to-batch comparison, three purified poloxamer 407 batches were prepared and tested for batch-to-batch variation. Figure 5 , Figure 7 , and Table 3 present the rheological results and comparison of the three purified batches. The average T _{solution_gel} is 20.03 ℃ and the coefficient of variation is 2.5%. In addition, the average maximum recorded storage modulus of the three purified batches was 22.5 kPa, and the coefficient of variation was 3.0%. Finally, the viscosity recorded at 1000s ^-1 and 10 ° C was 29.1 mPa.s, and the coefficient of variation was 1.4%. The results showed that the purification process was repeatable, and the rheological properties of the three batches of purified poloxamer 407 were comparable.

Example 3. Preparation of thermal gel formulation

Effect of Poloxamer 407 concentration on gel formation

To develop a formulation based on poloxamer 407, the effect of poloxamer 407 concentration on gel formation was first evaluated. Purified poloxamer 407 in PBS at a concentration of 10.5%, 11.6%, 12.6%, 13.7%, 14.7%, 15.8%, 16.8%, 17.9%, and 18.9% (w / v) at 5 ° C Dissolve overnight. Then, a rheometer was used to evaluate the effect of concentration on gel formation and viscosity.

Figure 8 shows the effect of poloxamer 407 concentration on gel formation. The results showed that no gels were formed at 11.6% w / v and 10.5% w / v poloxamer 407 concentrations. 9A-9B show that increasing the concentration of poloxamer 407 reduces the T _{solution_gel} and increases the maximum recorded storage modulus. Higher poloxamer 407 concentration results in gel formation at lower temperatures. In addition, the increase in poloxamer 407 concentration increased the maximum recorded storage modulus. This indicates that a stronger gel is formed at higher poloxamer 407 concentration.

Figures 10-12 show the effect of poloxamer 407 concentration on viscosity at 10 ° C and 23 ° C. These results indicate that an increase in the concentration of poloxamer 407 increases the viscosity at 10 ° C and 23 ° C.

Figure 13, Figure 14 , and Table 4 show the rheological properties of Poloxamer 407 (17.9% w / v) in PBS, distilled water, and tris buffer. The addition of buffer (1X PBS or 1X tris) reduced the T _{solution_gel} , but had no effect on the maximum recorded storage modulus and viscosity. The presence of salt in poloxamer 407 solution leads to the formation of micelles (and eventually gel) at lower temperatures, but it does not affect the poloxamer 407 polymerization interaction, which causes Variety. As shown in Table 4, by adding salt does not affect the viscosity, and for the test sample, at 1000 (s ^-1 ) and 10 ℃, the viscosity remains constant at about 29mPa. s.

Effect of ethanol addition on Poloxamer 407

TLR 7/8 agonists (S-36878 and S-36862) are not soluble in aqueous systems, but soluble in ethanol. In order to introduce TLR 7/8 agonist into the thermogel formulation, first ethanol was added in 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, and 25% (v / v) The concentration was added to the poloxamer 407 solution to evaluate its effect on the gel formation of the poloxamer 407 solution, T _{solution_gel} , the strength and viscosity of the formed gel. After the addition of ethanol, the final concentration of poloxamer 407 was 17%. The prepared samples were characterized by rheometer.

Figures 15 and 16A-16B suggest the effect of increasing ethanol concentration on gel formation. Increasing ethanol from 0% v / v to 20% v / v reduced the T _{solution_gel} , but had no effect on the maximum recorded storage modulus. Increase the ethanol concentration up to 25% to prevent gel formation.

Figures 17 and 18 show the effect of ethanol concentration on the viscosity of poloxamer 407 at 10 ° C. As shown in FIG. 18, the ethanol concentration increased by increasing the viscosity of the poloxamer 407 solution.

Add drug to Poloxamer 407 formulation

In two separate experiments, the drug was added to the Poloxamer 407 formulation. The purified poloxamer 407 was used in the first experiment to prepare the formulation with S-36862, and the unpurified poloxamer 407 was used in the second experiment to prepare the S-36878 and S-36862 Of the formulation.

In the first experiment, the purified poloxamer 407 was dissolved in PBS at 5 ° C overnight while mixing at 500 rpm (excipient solution). The TLR 7/8 agonist (S-36862) was then dissolved in ethanol (drug solution). Finally, the drug solution was added to the excipient solution and mixed well 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 were formed in situ within the poloxamer 407 solution ( Figure 19 ).

The rheological properties of the prepared formulations were tested and compared with drug-free formulations of 17% w / v poloxamer 407 and 5% v / v ethanol to evaluate the addition of drugs to the poloxamer 407 formulation Effect. Figure 20 , Figure 21 , and Table 5 show the effect of adding drugs on gel formation and viscosity. As shown in Table 5 , the addition of this drug does not affect the T _{solution_gel} and the maximum recorded storage modulus. In addition, the addition of drugs did not affect the viscosity of the Poloxamer 407 formulation. Therefore, the results confirmed that the addition of S-36862 did not affect the rheological properties of the formulation with purified poloxamer 407.

In the second set of experiments, the 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, LA: GA: 3: 1; solution-gel conversion at around 37 ° C) and poloxamer 407 (unpurified) were used for formulation development. These formulations were prepared using TLR 7/8 agonists (S-36878 and S-36862).

Similar to the first experiment, PLGA-PEG-PLGA and poloxamer 407 (unpurified) were dissolved in PBS and stirred at 500 rpm (excipient solution) at 5 ° C. TLR 7/8 agonist, S-36878 and S-36862 were dissolved in ethanol (drug solution), respectively. Finally, add the drug solution to the excipient solution and mix thoroughly to achieve 17% w / v PLGA-PEG-PLGA or poloxamer 407, 5% v / v ethanol, and 0.4 mg / in the formulation mL S-36862 or S-36878 ( Table 6 ). By mixing the drug solution with the excipient solution, the drug particles are formed in situ within the excipient solution ( Figure 19 ). Prepare a blank sample without adding any drug solution as a control.

Figures 22A-22B and Table 6 show the effect of added drugs (S-36878 or S-36862) on the gel formation of PLGA-PEG-PLGA and poloxamer 407 (unpurified). The results showed that the addition of S-36878 or S-36862 to the PLGA-PEG-PLGA or Poloxamer 407 formulation did not affect the T _{solution_gel} and the maximum recorded storage modulus. In addition, Figures 23A-23B and Table 6 show that the addition of drugs to these formulations does not affect the viscosity.

[Table 6]: Effects of drugs on polymer solutions containing 5% (v / v) ethanol

In the next set of experiments, the injectability of these formulations was evaluated. The formulation (150 μL) was carefully pipetted into a 1 mL long BD glass syringe to measure the sliding force when the injection rate was set to a constant 260 mm / min. In short, the Instron 5542 (Norwood, Massachusetts) was calibrated with a 50N load cell, which was carefully installed and tightened in place. Next, first load the filled glass syringe at the loading station, and use the navigation buttons to attach the crosshead to the syringe head ( Figures 24A-24B ). Bluehill software (Instron, Norwood, Massachusetts) was used to measure the maximum glide force, average glide force, and release force. The average sliding force value is reported in Table 7 .

Similar to gel formation and viscosity results, the injectability of these formulations is not affected by the addition of drugs. Compared with the PLGA-PEG-PLGA formulation, the recorded average slip force of the Poloxamer 407 formulation with S-36878 and S-36862 was higher, indicating that the Poloxamer 407 formulation required more force to inject ( Table 7 ).

Example 4. Quantification of drug content

An Agilent 1100 HPLC was used to measure the TLR 7/8 agonist in the formulated thermogel at 246 nm. The sample was diluted 10-fold in ethanol and injected (10 μL) into a Zorbax Bonus-RP column (4.6 × 150 mm, 5 mm column; temperature 50 ° C.). This separation method uses a gradient of 1 mL / min consisting of mobile phase A (50 mM ammonium acetate, pH 4) and mobile phase B (methanol). The gradient program was increased from 25% to 100% B between 0-15 minutes; maintained at 100% B from 15-20 minutes; and then slowly decreased to 25% B from 20 to 25 minutes. Linear algorithms for peak area fitting using several concentrations (100, 50, 20, 10, and 2 μg / mL) of TLR 7/8 agonists (S-36862 and S-36878) were used to interpolate TLR 7/8 The concentration of agonist in several thermal gel formulations. Table 8 shows the TLR 7/8 antagonist content in the formulated samples. The TLR 7/8 antagonist was measured within the target concentration

Example 5. In vitro release studies

The PLGA-PEG-PLGA and poloxamer 407 (unpurified) formulations were prepared according to the protocol described in Example 3. After preparing the thermogel formulation, 0.5 mL of drug-loaded thermogel was pipetted into a scintillation vial and incubated at 37 ° C for 10 minutes until a gel was formed. Once gelled, 3 mL of PBS buffer was added to the hot gel, and 0.3 mL was immediately removed from the vial and stored as t = 0. Refill the vial with fresh 0.3 mL of buffer. At specific time points, 0.3 mL of culture medium was carefully removed for drug release analysis by RP-HPLC. Fresh PBS buffer is replaced at any time, and samples are taken for measurement.

In vitro release studies have shown that in PLGA-PEG-PLGA and poloxamer 407 formulations, lipidated drug molecules (S-36862) have a slower release rate than non-lipidated drug molecules (S-36878) ( Figure 25A-25B ).

Example 6. In vivo evaluation of prepared formulations

Two sets of animal studies were conducted to evaluate the pharmacokinetics and pharmacodynamics of the formulation in the B16-OVA tumor model.

Mice, tumor cell lines, and tumor implants

C57BL / 6J-TyrC-2J female mice (C57BL / 6J Albino, strain B6 (Cg) -TyrC-2J / J, order number 000058) were obtained from Jackson Laboratories (Bar Harbor, Maine). When received at 3M's veterinary services, the mice were 14-20 grams. Prior to tumor implantation, mice were acclimated to 7-14 days.

The melanoma cell line B16-OVA was obtained from Dr. Wynette Dietz, University of Minnesota. The tumor strain was cultured in Du's modified Eagle's medium with 10% heat-inactivated fetal bovine serum and 1 mg / mL G-418 (Life Technologies, Carlsbad, California).

All animal studies were approved by the 3M Institutional Animal Care and Use Committee and conducted according to the 3M Institutional Animal Care and Use Committee. Before tumor implantation, the tumor cells were washed in Hanks Balanced Salt Solution (HBSS) (pH 7.4) and diluted to 4-5 × 106 cells / mL in HBSS. Load the cells into a 0.5cc allergy syringe (26 gauge × ½ inch, Becton, Dickinson and Co, Franklin Lakes, New Jersey), and mix 0.1 mL (4-5 × 105 Cells) were injected into the subcutaneous space on the right flank of the anesthetized mouse's shaved hair.

Pharmacokinetics in B16-OVA tumor model

Dosing started approximately 15 days after tumor implantation (tumor diameter 7-10 mm): mice were first anesthetized (n = 5), and then frozen formulations were administered by intratumoral (IT) injection ( Table 9 ). Using a 0.5 mL allergy syringe (26 gauge × ½ inch, Becton, Dickinson and Co., Franklin Lake, New Jersey), a single injection with a total volume of 0.05 mL (50 μg) was given The center of each tumor.

Blood and tumors were collected immediately after administration (about 20 minutes after IT administration), and blood and tumors were collected 6 hours, 3 days, and 14 days after administration. Blood was collected from anesthetized mice by terminal cardiac puncture (Monoject syringe, 22 gauge x ¾ inch). According to the manufacturer's instructions (product number 365967, Becton, Dickinson and Co, Franklin Lakes, New Jersey), use BD Microtainer tubes to process serum from the blood and store the serum in -70 ° C until used for serum cytokines analysis. Tumors were collected from euthanized mice by excision, carefully removing any fat and connective tissue, and stored at -70 ° C in a 20 mL glass vial with screw cap until the drug level was analyzed. Note that these glass vials are weighed in advance in order to accurately calculate the tumor weight.

Tumor drug levels were measured by HPLC-UV. At a concentration of 225 mg tumor / mL or lower, 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, Soren ( Solon), Ohio) digested tumor samples. Tumor samples were digested in a shaking water bath at 55 ° C for 5 hours. The internal standard was added and the samples were cooled to room temperature. 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 nitrogen flow at 55 ° C for 15 minutes and reconstituted in 0.150 mL of solution. Using Zorbax Bonus RP, 150 x 4.6 mm, 3.5 μm column, analysis was performed by HPLC-UV detection at 247 nm. Using a gradient program of 0.1% formic acid in water and methanol, linear from 15% -40% methanol in 2.5 minutes; and then linear in a gradient of 40% -95% methanol from 2.5 to 17.5 minutes. Quantification was performed with calibration standards at 10 mcg / mL.

Serum drug levels were measured by the LC-MS / MS method of Will Research Laboratories (LLC) (Ashland, OH). Figures 26A-26B and Figure 27 show the tumor drug levels and serum drug levels of PLGA-PEG-PLGA and poloxamer 407 formulations injected in the B16-OVA tumor model. Compared with poloxamer 407 formulation, PLGA-PEG-PLGA showed higher levels of tumor drugs. An initial burst release was also observed in the serum drug level of the PLGA-PEG-PLGA formulation. For pharmacodynamic evaluation, only Poloxamer 407 formulation is considered.

Pharmacodynamics in B16-OVA tumor model

Tumor growth inhibition (TGI), survival, and serum cytokines were measured in B16-OVA mice administered IT. The tumor was implanted in mice as described above. Prior to administration, mice were randomly grouped according to tumor diameter. Mice were given one or two IT injections of frozen poloxamer 407 formulation or vehicle (n = 10). PBS injection was used as a control (n = 9). Two studies were conducted to evaluate the pharmacodynamics of the poloxamer 407 formulation in the B16-OVA tumor model: Study 1, in which the two IT injections were separated by 7 days (average tumor diameter approximately 5 mm) at a dose of 50 μg / 50 μL ( Table 10 ); and Study II, in which one IT injection (with an average tumor diameter of about 10 mm) was given at 0.4 μg, 4 μg, and 20 μg / 50 μL ( Table 11 ).

After administration, the tumor was measured twice a week with a calibrated digital caliper (product number 62379-531, VWR International, Radner, Pennsylvania), and the mice were euthanized when the tumor diameter was> 20 mm . The result is expressed as the tumor volume calculated using the following formula: V = p / 6 * 1.58 (L * W) 1.5

In addition, in Study 2, using multiple array method (Bio-Plex 200 system), serum interleukins were measured from blood samples collected from 3 mice per group 6 hours after administration. Multiple measurements are performed according to the manufacturer's instructions. 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 (Minnesota) Apollis (Minneapolis, Minnesota). Data analysis was performed using Bio-Plex Manager software (version 6.1) (Bio-Rad Life Science Research (Bio-Rad Life Science Research), Heracles, CA).

Repeated measurement two-way ANOVA analysis and Tukey's multiple comparison test were used to determine differences in tumor volume. A log-rank (Mantel-Cox) test was used to determine differences in survival curves.

Figures 28 and 29 show that the Poloxamer 407 formulation performs better in terms of TGI and survival rate than PBS. Figures 30 and 31 show that increasing the dose enhances the performance of the Poloxamer 407 formulation, inhibits tumor growth and leads to a higher survival rate. Figures 32A-32E show that increasing the dose lowers serum interleukin levels, which may indicate reduced drug diffusion and escape from the tumor site.

***

The above description of the detailed description will fully reveal the general nature of the present invention, so that others can easily target by applying knowledge in the art without undue experimentation without departing from the general concept of the present invention. Various applications of such specific embodiments are modified and / or adapted. Therefore, based on the teaching content and guidance presented herein, such adaptations and modifications are intended to be within the meaning and equivalent scope of the disclosed embodiments. It should be understood that the phrases or terms herein are for the purpose of description rather than limitation, so that the terms or phrases of this specification can be understood by the skilled person based on such teaching content and guidance. The present invention is further described by the following patent applications.

[ Figure 1 ] shows the dependence of the solution-to-gel transition temperature on the polymer concentration in the thermosensitive gel.

[ Figure 2A ] shows the structure of the TLR 7/8 agonist S-36878 (non-lipidated). Figure 2B shows the structure of the TLR 7/8 agonist S-36862 (lipidated).

[ Figure 3 ] shows the purification diagram of HPLC-ELSD poloxamer 407.

[ Figure 4 ] shows the effect of purification on gel formation.

[ Figure 5 ] shows three batches of purified poloxamer 407 T _solution-gel and maximum storage modulus.

[ Figure 6 ] shows the effect of purification at 10 ° C on the viscosity of poloxamer 407 solution.

[ Figure 7 ] shows the viscosity of three batches of purified poloxamer 407 measured at a shear rate scan at 10 ° C.

[ Figure 8 ] shows the effect of poloxamer 407 concentration on gel formation.

[ Figures 9A-9B ] shows the effect of poloxamer 407 concentration on the T _solution-gel transition temperature ( Figure 9A ) and the maximum recorded storage modulus ( Figure 9B ).

[ Figure 10 ] shows the effect of poloxamer 407 concentration on the viscosity when scanned at a shear rate of 10 ° C.

[ Figure 11 ] shows the effect of poloxamer 407 concentration on the viscosity when scanned at a shear rate of 23 ° C.

[ Figures 12A-12B ] shows the effect of poloxamer 407 concentration on the viscosity at 10 ° C ( Figure 12A ) and the effect on the viscosity at 23 ° C ( Figure 12B ).

[ Figure 13 ] shows the T _{solution_gel} and maximum storage modulus of poloxamer 407 dissolved in 1X PBS, distilled water and 1X Tris buffer.

[ Figure 14 ] shows the viscosity of poloxamer 407 dissolved in 1X PBS, distilled water, and 1X Tris buffer measured at 10 ° C.

[ Figure 15 ] shows the effect of ethanol concentration on gel formation.

[ Figure 16A-16B ] shows the effect of ethanol concentration on the T _{solution_gel} ( Figure 16A ) and the effect on the maximum recorded storage modulus ( Figure 16B ).

[ Figure 17 ] shows the effect of ethanol concentration on the viscosity when scanned at a shear rate of 10 ° C.

[ Figure 18 ] shows the effect of ethanol concentration on the viscosity of poloxamer 407 solution at 10 ° C.

[ Figure 19 ] shows the formulation preparation and mixing steps.

[ Figure 20 ] shows the effect of adding a drug to a poloxamer 407 solution containing 5% (v / v) ethanol on gel formation.

[ Figure 21 ] shows the effect of adding a drug to a poloxamer 407 solution containing 5% (v / v) ethanol on viscosity.

[FIGS. 22A-22B] shows drug was added to the PLGA-PEG-PLGA affect the formation of the gel (FIG. 22A) and the effect on gel formation (407 in FIG drug was added to the unpurified poloxamer 22B ).

[ Figures 23A-23B ] shows the viscosity and shear rate scan of the PLGA-PEG-PLGA formulation ( Figure 23A ) and the viscosity and shear rate scan of the unpurified poloxamer 407 formulation ( Figure 23B ). The viscosity measurement is performed at 23 ° C.

[ Figure 24A-24B ] shows the injectability test results of the PLGA-PEG-PLGA formulation at room temperature ( Figure 24A ) and the injectability test results of the poloxamer 407 formulation ( Figure 24B ).

[ Figures 25A-25B ] shows the in vitro release of S-36878 ( Figure 25A ) and the in vitro release of S-36862 ( Figure 25B ) from PLGA-PEG-PLGA and poloxamer 407 formulations.

[ Figs. 26A-26B ] shows the levels of tumor drugs for PLGA-PEG-PLGA and poloxamer 407 formulations injected in the B16-OVA tumor model ( Figure 26A : μg drug / tumor and Figure 26B :% of t ₀ ).

[ Figure 27 ] shows the serum drug levels for the PLGA-PEG-PLGA and poloxamer 407 formulations injected in the B16-OVA tumor model.

[ FIG. 28 ] shows the measured tumor volume for animals administered with Poloxamer 407 formulation in Study One (n = 9 mice / PBS group).

[ FIG. 29 ] shows the results of the survival percentage of the animals given the Poloxamer 407 formulation in Study One (n = 9 mice / PBS group).

[ Figure 30 ] shows the measured tumor volume for animals administered with Poloxamer 407 formulation in Study 2.

Figure 31 ] shows the results of the percent survival for animals administered Poloxamer 407 formulation in Study 2.

[ Figures 32A-32E ] shows the serum cytokines measured from blood samples collected from 3 mice in each group in Study 2 6 hours after administration: keratinocyte-derived cytokines ( Figure 32A ) , Chemokine (CXC motif) ligand 10 ( Figure 32B ), interleukin 10 ( Figure 32C ), monocyte chemoattractant protein ( Figure 32D ) and interleukin 6 ( Figure 32E ).

Claims (32)

A composition comprising: a. 10% -25% (w / v) reverse thermosensitive polymer; b. An immune response modifier (IRM) having the following chemical formula I: Where R ₁ has the chemical formula alkylene-LR _1-1 , alkenyl-LR _1-1 , or alkynyl-LR _1-1 , where the alkylene, alkenyl, and alkynyl groups are as appropriate Spaced or blocked by one or more -O- groups; L is a bond or functional linking group selected from the group consisting of -NH-S (O) _2- , -NH -C (O)-, -NH-C (S)-, -NH-S (O) ₂ -NR _3- , -NH-C (O) -NR _3- , -NH-C (S) -NR _3- , -NH-C (O) -O-, -O-, -S-, and -S (O) _2- ; and R _1-1 is a linear or branched aliphatic group, the aliphatic The group optionally includes 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 (R ₃ ) ₂ ; n is 0 to 4; R _{2 is} selected from the group consisting of: hydrogen; alkyl; alkenyl; aryl; heteroaryl; heterocyclic; alkylene- Y-alkyl; alkylene-Y-alkenyl; alkylene-Y-aryl; and alkyl or alkenyl substituted with one or more substituents selected from the group consisting of The group consists of the following: -OH; halogen; -N (R ₄ ) ₂ ; -C (O) -C _1-10 alkyl; -C (O) -OC _1-10 alkyl; -N ₃ ; aryl; heteroaryl; heterocyclyl; -C (O) -aryl; and -C (O)- Heteroaryl; Y is -O- or -S (O) _0-2- ; each R _{4 is} independently selected from the group consisting of hydrogen, C _1-10 alkyl, and C _2-10 Alkenyl; and R _{3 is} selected from the group consisting of hydrogen and alkyl; the condition is that when L is -NH-S (O ₂ )-and n is 0, R _1-1 is straight or branched A chain 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. A composition comprising: a. 10% -25% (w / v) reverse thermosensitive polymer; b. Immune response modifier (IRM) having the following chemical formula II: Among them: X is an alkylene group with up to 8 carbon atoms, and these carbon atoms are optionally -O- spaced or blocked; R ₂ is hydrogen, alkyl, alkoxyalkylene, alkylaminoalkyl Propylene, or hydroxyalkylene; Y-C (O)-or -S (O) _2- ; R ₁ is a linear or branched aliphatic group having _{1 to 23} carbon atoms, as the case may include One or 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.     The composition as described in item 1 of the patent application scope or item 2 of the patent application scope, 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.     A composition comprising: a. 12% -20% (w / v) reverse thermosensitive polymer selected from the group consisting of (i) poloxamer 407 and (ii) ) Poly (lactic acid-co-glycolic acid) -b -poly (ethylene glycol) -b -poly (lactic acid-co-glycolic acid) copolymer (PLGA-PEG-PLGA), in which the average number of PLGA-PEG-PLGA The molecular weight (Mn) is about 1600: 1500: 1600 Dalton, and the PLGA ratio of lactic acid and glycolic acid is about 3: 1; b. Immune response modifier (IRM) with the following structure: Or a pharmaceutically acceptable salt thereof; and c. Up to 20% (v / v) ethanol. A composition comprising: a. 12% -20% (w / v) reverse thermosensitive polymer selected from the group consisting of (i) poloxamer 407 and (ii) ) Poly (lactic acid-co-glycolic acid) -b -poly (ethylene glycol) -b -poly (lactic acid-co-glycolic acid) copolymer (PLGA-PEG-PLGA), wherein the average number of the PLGA-PEG-PLGA The molecular weight (Mn) is about 1600: 1500: 1600 Dalton, and the PLGA ratio of lactic acid and glycolic acid is about 3: 1; b. Immune response modifier (IRM) with the following structure: Or a pharmaceutically acceptable salt thereof; and c. Up to 20% (v / v) ethanol.     The composition as described in any of the aforementioned patent applications includes 5% (v / v) ethanol.         The composition as described in any of the aforementioned patent applications includes 15% -20% (w / v) reverse thermosensitive polymer.         The composition as described in any one of claims 1 to 7, wherein the reverse thermosensitive polymer is poloxamer 407.         The composition as described in item 8 of the patent application scope, wherein the poloxamer 407 is purified.         The composition as described in any one of claims 1 to 7, wherein the polymer is PLGA-PEG-PLGA.         Compositions as described in any of the aforementioned patent applications, including 0.05 to 1.3 mg / mL IRM.         A composition as described in the scope of any preceding patent application, including the concentration of IRM selected from the group consisting of 0.08 mg / mL, 0.4 mg / mL, and 1 mg / mL.     The composition as described in any of the aforementioned patent applications has a solution-to-gel transition temperature (T _{solution_gel} ) of 20 ° C-37 ° C.     The composition as described in any of the aforementioned patent applications further includes a second active agent.         A method of delivering a long-acting pharmaceutical formulation including an immune response modifier (IRM) to a subject, the method comprising injecting an effective amount of the subject into any one of patent application items 1 to 14 The composition mentioned.         A method for stimulating a local immune response in a subject, the method comprising injecting into the subject an effective amount of the composition as described in any one of patent application items 1 to 14.         The method according to item 15 or 16 of the patent application scope, wherein the subject has a tumor, and wherein the composition is injected into the tumor site.         The method according to item 17 of the patent application scope, wherein the tumor is breast tumor, stomach tumor, lung tumor, head or neck tumor, colorectal tumor, renal cell carcinoma tumor, pancreas tumor, basal cell carcinoma tumor, Cervical tumors, melanoma tumors, prostate tumors, ovarian tumors, liver tumors, or bladder tumors.         The method of any one of claims 15 to 18, wherein the subject has a disease or condition of the dermis, and wherein the composition is injected at the site of the disease or condition.         The method according to item 19 of the patent application scope, wherein the disease or condition of the dermis is selected from the group consisting of basal cell carcinoma, melanoma, and genital warts.         The method of any one of claims 15 to 20 of the patent application, further comprising administering a second active agent.         The method according to item 21 of the patent application scope, wherein the second active agent is a chemotherapeutic agent.         Use of the composition as described in any one of patent application items 1 to 14 for delivering a long-acting pharmaceutical formulation containing an immune response modifier (IRM) to a subject.         Use of the composition as described in any one of patent application items 1 to 14 for stimulating a local immune response in a subject.         The use as described in item 23 or 24 of the patent application scope, wherein the subject has a tumor.         The use as described in item 25 of the patent application scope, wherein the tumor is breast tumor, stomach tumor, lung tumor, head or neck tumor, colorectal tumor, renal cell carcinoma tumor, pancreas tumor, basal cell carcinoma tumor, Cervical tumors, melanoma tumors, prostate tumors, ovarian tumors, liver tumors, or bladder tumors.         The use according to any one of claims 23 to 26, wherein the subject has a dermal disease or disorder.         The use as described in item 27 of the patent application scope, wherein the disease or condition of the dermis is selected from the group consisting of basal cell carcinoma, melanoma, and genital warts.         The use as described in any of items 23 to 28 of the patent application scope further includes a second active agent.         The use as described in item 29 of the patent application scope, wherein the second active agent is a chemotherapeutic agent.         A method for preparing a composition as described in any one of claims 1 to 14 of the patent application, the method comprising: a. Dissolving the reverse thermosensitive polymer in an aqueous medium to prepare an excipient solution; b . Dissolve the IRM in ethanol to prepare a drug solution; and c. Add the drug solution to the excipient solution to prepare a composition containing ethanol in an amount of up to 20% (v / v).         A kit containing the composition as described in any one of items 1 to 14 of the patent application.