Patent ID: 12186402

DEFINITIONS

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

Unless otherwise defined, 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 belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a block copolymer” is a reference to one or more block copolymers and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of” and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of” and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.

As used herein, the term “short-chain fatty acid” (“SCFA”) refers to a carboxylic acid attached to an aliphatic chain, which is either saturated or unsaturated, the aliphatic chain being 12 carbons or less in length.

As used herein the term “fatty acid derivative” (specifically “SCFA derivative”) refers to a small molecular compounds that are obtained by making simple modifications (e.g., amidation, methylation, halogenation, etc.) to fatty acid molecules (e.g., SCFA molecules). For example, butyramide, D- or L-amino-n-butyric acid, alpha- or beta-amino-n-butyric acid, arginine butyrate, butyrin, phenyl butyrates (e.g., 4-, 3-, 2-), dimethylbutyrate, 4-halobutyrates (e.g., fluoro-, chloro-, bromo-, iodo-), 3-halobutyrates (e.g., fluoro-, chloro-, bromo-, iodo-), 2-halobutyrates (e.g., fluoro-, chloro-, bromo-, iodo-), oxybutyrate, and methylbutyrates are exemplary butyrate derivatives. Other butyrate derivatives and similar derivatives of other SCFAs are within the scope of the SCFA derivatives described herein.

As used herein, the term “copolymer” refers to a polymer formed from two or more different monomer subunits. Exemplary copolymers include alternating copolymers, random copolymers, block copolymers, etc.

As used herein, the term “block copolymer” refers to copolymers wherein the repeating subunits are polymeric blocks, i.e. a polymer of polymers. In a copolymer of blocks A and B, A and B each represent polymeric entities themselves, obtained by the polymerization of monomers. Exemplary configurations of such block copolymers include branched, star, di-block, tri-block and so on.

As used herein, the term “supramolecular” (e.g., “supramolecular assembly”) refers to the non-covalent interactions between molecules and/or solution (e.g., polymers, macromolecules, etc.) and the multicomponent assemblies, complexes, systems, and/or fibers that form as a result.

As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, excipient, or carrier conventional in the art for use with a therapeutic agent for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. A “pharmaceutical composition” typically comprises at least one active agent (e.g., the copolymers described herein) and a pharmaceutically acceptable carrier.

As used herein, the term “effective amount” refers to the amount of a composition (e.g., pharmaceutical composition) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

As used herein, the term “administration” refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., pharmaceutical compositions of the present invention) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through the eyes (e.g., intraocularly, intravitreally, periocularly, ophthalmic, etc.), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

As used herein, the terms “co-administration” and “co-administer” refer to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent (e.g., in the same or separate formulations). In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s).

As used herein, the term “nanoparticles” refers to particles having mean dimensions (e.g., diameter, width, length, etc.) of less than 1 μm (e.g., <500 nm (“sub-500-nm nanoparticles”), <100 nm (“sub-100-nm nanoparticles”), <50 nm (“sub-50-nm nanoparticles”).

As used herein, the term “biocompatible” refers to materials, compounds, or compositions means that do not cause or elicit significant adverse effects when administered to a subject. Examples of possible adverse effects that limit biocompatibility include, but are not limited to, excessive inflammation, excessive or adverse immune response, and toxicity.

As used herein, the term “biostable” refers to compositions or materials that do not readily break-down or degrade in a physiological or similar aqueous environment. Conversely, the term “biodegradeable” refers herein to compositions or materials that readily decompose (e.g., depolymerize, hydrolyze, are enzymatically degraded, disassociate, etc.) in a physiological or other environment.

As used herein, the term “substituted” refers to a group (e.g., alkyl, etc.) that is modified with one or more additional group(s). Non-limiting examples of substituents include, for example: halogen, hydroxy, oxo (═O), thioxo (═S), cyano (—CN), nitro (—NO2), imino (═N—H), oximo (═N—OH), hydrazino (═N—NH2), NH2)—Rb—ORa, —Rb—OC(O)—Ra, —Rb—OC(O)—ORa, —Rb—OC(O)—N(Ra)2, —Rb—N(Ra)2, —Rb—C(O)Ra, —Rb—C(O)ORa, —Rb—C(O)N(Ra)2, —Rb—O—Rc—C(O)N(Ra)2, —Rb—N(Ra)C(O)ORa, —Rb—N(Ra)C(O)Ra, —Rb—N(Ra)S(O)tRa(where t is 1 or 2), —Rb—S(O)tRa(where t is 1 or 2), —Rb—S(O)tORa(where t is 1 or 2), and —Rb—S(O)tN(Ra)2(where t is 1 or 2); and alkyl, alkenyl, alkynyl, each of which may be optionally substituted by halogen, oxo (═O), thioxo (═S), cyano (—CN), nitro (—NO2), imino (═N—H), oximo (═N—OH), hydrazine (═N—NH2), —Rb—ORa, —Rb—OC(O)—Ra, —Rb—OC(O)—ORa, —Rb—OC(O)—N(Ra)2, —Rb—N(Ra)2, —Rb—C(O)Ra, —Rb—C(O)ORa, —Rb—C(O)N(Ra)2, —Rb—O—Rc—C(O)N(Ra)2, —Rb—N(Ra)C(O)ORa, —Rb—N(Ra)C(O)Ra, —Rb—N(Ra)S(O)tRa(where t is 1 or 2), —Rb—S(O)tRa(where t is 1 or 2), —Rb—S(O)tORa(where t is 1 or 2), —Rb—S(O)tN(Ra)2(where t is 1 or 2), carbocycle and heterocycle; wherein each Rais independently selected from hydrogen, alkyl, alkenyl, alkynyl, carbocycle and heterocycle, wherein each Ra, valence permitting, may be optionally substituted with halogen, oxo (═O), thioxo (═S), cyano (—CN), nitro (—NO2), imino (═N—H), oximo (═N—OH), hydrazine (═N—NH2), —Rb—ORa, —Rb—OC(O)—Ra, —Rb—OC(O)—ORa, —Rb—OC(O)—N(Ra)2, —Rb—N(Ra)2, —Rb—C(O)Ra, —Rb—C(O)ORa, —Rb—C(O)N(Ra)2, —Rb—O—Rc—C(O)N(Ra)2, —Rb—N(Ra)C(O)ORa, —Rb—N(Ra)C(O)Ra, —Rb—N(Ra)S(O)tRa(where t is 1 or 2), —Rb—S(O)tRa(where t is 1 or 2), —Rb—S(O)tORa(where t is 1 or 2) and —Rb—S(O)tN(Ra)2(where t is 1 or 2); and wherein each Rbis independently selected from a direct bond or a straight or branched alkylene, alkenylene, or alkynylene chain, and each Rcis a straight or branched alkylene, alkenylene or alkynylene chain. Substituent groups may be selected from, but are not limited to: alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxyl, alkoxy, mercaptyl, cyano, halo, carbonyl, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl, perfluoroalkyl, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof. A “substituted alkyl” encompasses alkynes and alkenes, in addition to alkanes displaying substituent moieties.

As used herein, the term “pseudo-random” refers to sequences or structures generated by processes in which no steps or measures have been taken to control the order of addition of monomers or components.

As used herein, the term “display” refers to the presentation of solvent-exposed functional group by a molecule, monomer, polymer, nanostructure or other chemical entity.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are polymer materials that find use in, for example, delivery of short-chain fatty acids. In particular, polymers are provided that form stable nanoscale structures and release their payload, for example, by cleavage of a covalent bond (e.g., via hydrolysis or enzymatic cleavage). The polymers are useful, for example, for delivery of payloads (e.g., SCFAs) to the intestine for applications in health and treatment of disease, and have broad applicability in diseases linked to changes in the human microbiota including inflammatory, autoimmune, allergic, metabolic, and central nervous system diseases, among others.

In some embodiments, provided herein are copolymers (e.g., block or random) of: hydrophilic monomers (or a block thereof) pendant-displaying methacrylamide or methacrylate monomers (or a block thereof). In some embodiments, methods are provided for the assembly of these copolymers into nanoparticles, micelles, or other delivery systems. In some embodiments, methods are provided for the administration of the copolymers, and delivery systems comprising such copolymers, for the treatment or prevention of various diseases and conditions. In particular, polymers are functionalized to deliver a pharmaceutically-relevant small molecule moiety (e.g., SCFA) relevant for treating human disease with a covalent bond that is broken (e.g., by hydrolysis or enzyme activity). In some embodiments, copolymers are obtained using reversible addition-fragmentation chain-transfer (“RAFT”) polymerization of an appropriate monomer with an initiator.

In some embodiments, polymers are random copolymers comprising N-hydroxypropyl methacrylamide (HPMA) monomers. Other synthetic natural molecules or polymers may be used as hydrophilic monomers.

In some embodiments, an HPMA-(N-hydroxyethyl methacrylamide) copolymer is a copolymer (e.g., random copolymer) of HPMA monomers and N-hydroxyethyl methacrylamide monomers. A free HPMA terminus (e.g., not connected to the N-hydroxyethyl acrylamide block) may be one of a number of chemical groups, including but not limited to hydroxyl, methoxy, thiol, amine, maleimide, halogen, polymer chain transfer agents, protecting groups, drug, biomolecule, or tissue targeting moiety. Some or all N-hydroxyethyl methacrylamide monomers display a pharmaceutically-relevant small molecule covalently attached to the hydroxyethyl functional group. A preferred embodiment of the pharmaceutically-relevant small molecule is short- and medium-chain fatty acids (“SCFA” s) and their derivatives containing up to 12 carbon atoms in the chain, for example, between 3 and 10 carbon atoms in the chain. The chain may be linear or branched. Example SCFAs include, but are not limited to, acetate, propionate, iso-propionate, butyrate, iso-butyrate, and other SCFAs described herein, as well as derivatives thereof. A free SCFA terminus may be one of a number of chemical groups including but not limited to methyl, hydroxyl, methoxy, thiol, amine, N-alkyl amine, and others.

In some embodiments, an HPMA-(N-hydroxyethyl methacrylate) copolymer is a copolymer (e.g., random copolymer) of HPMA monomers and N-hydroxyethyl methacrylate monomers. A free HPMA terminus may be one of a number of chemical groups, including but not limited to hydroxyl, methoxy, thiol, amine, maleimide, halogen, polymer chain transfer agents, protecting groups, drug, biomolecule, or tissue targeting moiety. Some or all N-hydroxyethyl methacrylate monomers display a pharmaceutically-relevant small molecule covalently attached to the hydroxyethyl functional group. A preferred embodiment of the pharmaceutically-relevant small molecule is short- and medium-chain fatty acids (“SCFA” s) and their derivatives containing, for example, between 3 and 12 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or ranges therebetween) carbon atoms in the chain. The chain may be linear or branched. Example SCFAs include, but are not limited to, acetate, propionate, iso-propionate, butyrate, iso-butyrate, and other SCFAs described herein, as well as derivatives thereof. A free SCFA terminus may be one of a number of chemical groups including but not limited to methyl, hydroxyl, methoxy, thiol, amine, N-alkyl amine, and others.

In some embodiments, an HPMA-(N-(4-hydroxybenzoyloxy)alkyl methacrylate) copolymer is a copolymer (e.g., random copolymer) of HPMA monomers and N-(4-hydroxybenzoyloxy)alkyl methacrylate monomers. A free HPMA terminus may be one of a number of chemical groups, including but not limited to hydroxyl, methoxy, thiol, amine, maleimide, halogen, polymer chain transfer agents, protecting groups, drug, biomolecule, or tissue targeting moiety. Some or all N-(4-hydroxybenzoyloxy)alkyl methacrylate monomers display a pharmaceutically-relevant small molecule covalently attached to the hydroxyethyl functional group. A preferred embodiment of the pharmaceutically-relevant small molecule is short- and medium-chain fatty acids (“SCFA” s) and their derivatives containing, for example, between 3 and 12 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or ranges therebetween) carbon atoms in the chain. The chain may be linear or branched. Example SCFAs include, but are not limited to, acetate, propionate, iso-propionate, butyrate, iso-butyrate, and other SCFAs described herein, as well as derivatives thereof. A free SCFA terminus may be one of a number of chemical groups including but not limited to methyl, hydroxyl, methoxy, thiol, amine, N-alkyl amine, and others.

In some embodiments, an HPMA-(N-(4-hydroxybenzoyloxy)alkyl methacrylamide) copolymer is copolymer (e.g., random copolymer) of HPMA monomers and N-(4-hydroxybenzoyloxy)alkyl methacrylamide monomers. A free HPMA terminus may be one of a number of chemical groups, including but not limited to hydroxyl, methoxy, thiol, amine, maleimide, halogen, polymer chain transfer agents, protecting groups, drug, biomolecule, or tissue targeting moiety. Some or all N-(4-hydroxybenzoyloxy)alkyl methacrylamide monomers display a pharmaceutically-relevant small molecule covalently attached to the hydroxyethyl functional group. A preferred embodiment of the pharmaceutically-relevant small molecule is short- and medium-chain fatty acids (“SCFA” s) and their derivatives containing, for example, between 3 and 12 (e.g., 3, 4, 5, 6, 7, 8, 9 10, 11, 12, or ranges therebetween)carbon atoms in the chain. The chain may be linear or branched. Example SCFAs include, but are not limited to, acetate, propionate, iso-propionate, butyrate, iso-butyrate, and other SCFAs described herein, as well as derivatives thereof. A free SCFA terminus may be one of a number of chemical groups including but not limited to methyl, hydroxyl, methoxy, thiol, amine, N-alkyl amine, and others.

In some embodiments, polymers are block copolymers comprising poly(N-hydroxypropyl methacrylamide) as a hydrophilic block. Other synthetic natural molecules or polymers may be used as hydrophilic blocks.

In some embodiments, an HPMA-(N-hydroxyethyl methacrylamide) copolymer is composed of the hydrophilic polyHPMA block covalently attached to the N-hydroxyethyl methacrylamide block. The free HPMA terminus not connected to the N-hydroxyethyl methacrylamide block may be one of a number of chemical groups, including but not limited to hydroxyl, methoxy, thiol, amine, maleimide, halogen, polymer chain transfer agents, protecting groups, drug, biomolecule, or tissue targeting moiety. The N-hydroxyethyl methacrylamide block displays a pharmaceutically-relevant small molecule covalently attached to the hydroxyethyl functional group. A preferred embodiment of the pharmaceutically-relevant small molecule is short- and medium-chain fatty acids (“SCFA” s) and their derivatives containing, for example, between 3 and 12 (e.g., 3, 4, 5, 6, 7, 8, 9 10, 11, 12, or ranges therebetween) carbon atoms in the chain. The chain may be linear or branched. Example SCFAs include, but are not limited to, acetate, propionate, iso-propionate, butyrate, iso-butyrate, and other SCFAs described herein, as well as derivatives thereof. The free SCFA terminus not connect to the N-hydroxyethyl methacrylamide block may be one of a number of chemical groups including but not limited to methyl, hydroxyl, methoxy, thiol, amine, N-alkyl amine, and others.

In some embodiments, an HPMA-(N-hydroxyethyl methacrylate) copolymer is composed of the hydrophilic polyHPMA block covalently attached to the N-hydroxyethyl methacrylate block. The free HPMA terminus not connected to the N-hydroxyethyl methacrylate block may be one of a number of chemical groups, including but not limited to hydroxyl, methoxy, thiol, amine, maleimide, halogen, polymer chain transfer agents, protecting groups, drug, biomolecule, or tissue targeting moiety. The N-hydroxyethyl methacrylate block displays a pharmaceutically-relevant small molecule covalently attached to the hydroxyethyl functional group. A preferred embodiment of the pharmaceutically-relevant small molecule is short- and medium-chain fatty acids (“SCFA” s) and their derivatives containing, for example, between 3 and 12 (e.g., 3, 4, 5, 6, 7, 8, 9 10, 11, 12, or ranges therebetween)carbon atoms in the chain. The chain may be linear or branched. Example SCFAs include, but are not limited to, acetate, propionate, iso-propionate, butyrate, iso-butyrate, and other SCFAs described herein, as well as derivatives thereof. The free SCFA terminus not connect to the N-hydroxyethyl methacrylate block may be one of a number of chemical groups including but not limited to methyl, hydroxyl, methoxy, thiol, amine, N-alkyl amine, and others.

In some embodiments, an HPMA-(N-(4-hydroxybenzoyloxy)alkyl methacrylate) copolymer is composed of the hydrophilic polyHPMA block covalently attached to the N-(4-hydroxybenzoyloxy)alkyl methacrylate block. The free HPMA terminus not connected to the N-(4-hydroxybenzoyloxy)alkyl methacrylate block may be one of a number of chemical groups, including but not limited to hydroxyl, methoxy, thiol, amine, maleimide, halogen, polymer chain transfer agents, protecting groups, drug, biomolecule, or tissue targeting moiety. The N-(4-hydroxybenzoyloxy)alkyl methacrylate block displays a pharmaceutically-relevant small molecule covalently attached to the hydroxyethyl functional group. A preferred embodiment of the pharmaceutically-relevant small molecule is short- and medium-chain fatty acids (“SCFA” s) and their derivatives containing, for example, between 3 and 12 (e.g., 3, 4, 5, 6, 7, 8, 9 10, 11, 12, or ranges therebetween)carbon atoms in the chain. The chain may be linear or branched. Example SCFAs include, but are not limited to, acetate, propionate, iso-propionate, butyrate, iso-butyrate, and other SCFAs described herein, as well as derivatives thereof. The free SCFA terminus not connect to the N-(4-hydroxybenzoyloxy)alkyl methacrylate block may be one of a number of chemical groups including but not limited to methyl, hydroxyl, methoxy, thiol, amine, N-alkyl amine, and others.

In some embodiments, an HPMA-(N-(4-hydroxybenzoyloxy)alkyl methacrylamide) copolymer is composed of the hydrophilic polyHPMA block covalently attached to the N-(4-hydroxybenzoyloxy)alkyl methacrylamide block. The free HPMA terminus not connected to the N-(4-hydroxybenzoyloxy)alkyl methacrylamide block may be one of a number of chemical groups, including but not limited to hydroxyl, methoxy, thiol, amine, maleimide, halogen, polymer chain transfer agents, protecting groups, drug, biomolecule, or tissue targeting moiety. The N-(4-hydroxybenzoyloxy)alkyl methacrylamide block displays a pharmaceutically-relevant small molecule covalently attached to the hydroxyethyl functional group. A preferred embodiment of the pharmaceutically-relevant small molecule is short- and medium-chain fatty acids (“SCFA”s) and their derivatives containing, for example, between 3 and 12 (e.g., 3, 4, 5, 6, 7, 8, 9 10, 11, 12, or ranges therebetween)carbon atoms in the chain. The chain may be linear or branched. Example SCFAs include, but are not limited to, acetate, propionate, iso-propionate, butyrate, iso-butyrate, and other SCFAs described herein, as well as derivatives thereof. The free SCFA terminus not connect to the N-(4-hydroxybenzoyloxy)alkyl methacrylamide block may be one of a number of chemical groups including but not limited to methyl, hydroxyl, methoxy, thiol, amine, N-alkyl amine, and others.

Blocks may vary in molecular weight and therefore size, the adjustment of which alters the ratio of inert, unfunctionalized, pharmaceutically inactive material and active, functionalized pharmaceutically-active material. Some embodiments are a linear HPMA-(N-oxyethyl methacrylamide) block copolymer whose relative block sizes are between 0.25 and 3.5 (e.g., 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, and ranges therebetween (e.g., 0.7-1.8)). Other embodiments are a linear HPMA-(2-(4-hydroxybenzoyloxy)ethyl methacrylate) block copolymer whose relative block sizes are between 0.25 and 3.5 (e.g., 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, and ranges therebetween (e.g., 0.7-1.8)). A “relative block size” of 0.25-3.5 means that for every 1 mole of HPMA weight, the N-oxyethyl methacrylamide block (or a SCFA functionalized derivative) is 0.25 mole-3.5 mole). In some embodiments, block copolymers described herein form nanoparticles or micelles of diameter 10-1000 nm (e.g., 10, 20, 50, 100, 200, 500, 1000 nm, or ranges therebetween (e.g., 50-500 nm) when dispersed (e.g. in a liquid). The nanoparticles or micelles thus formed can then be isolated as a solid (e.g. in a powder, by lyophilization, etc.) with or without stabilizers (e.g. surfactants).

Although specific embodiments herein refer to polyHPMA as the hydrophilic block, other hydrophilic polymers may be used in place of polyHPMA including poly(ethylene oxide)-co-poly(propylene oxide) random, di- or multiblock copolymers, poly(vinyl alcohol), poly ethylene-co-vinyl alcohol), poly(N-vinyl pyrrolidone), poly(acrylic acid), poly(methyloxazoline) (“PMOXA”), poly(ethyloxazoline), poly(alkylacrylates), poly(acrylamide), poly(N-alkylacrylamides), hydrophilic polypeptide, polysaccharide, poly(N,N-dialkylacrylamides), hyaluronic acid, alginate, cyclodextrin, or poly(N-acryloylmorpholine). The hydrophilic block may be present at a molecular weight of between 3000 and 50,000 Da (e.g., 3000, 4000, 5000 Da, 6000 Da, 7000 Da, 8000 Da, 9000 Da, 10000 Da, 11000 Da, 12000 Da, 13000 Da, 14000 Da, 15000 Da, 20000 Da, 25000 Da, 30000 Da, 35000 Da, 40000 Da, 45000 Da, 50000 Da, or ranges therebetween (e.g., 9000-14000 Da, 14000-30000)).

In certain embodiments, the copolymer compositions herein are administered in the form of a pharmaceutical composition, a dietary supplement, or a food or beverage. When the compositions herein are used as a food or beverage, the food or beverage can be, e.g. a health food, a functional food, a food for a specified health use, a dietary supplement, or a food for patients. The composition may be administered once or more than once. If administered more than once, it can be administered on a regular basis (e.g. two times per day, once a day, once every two days, once a week, once a month, once a year) or on as needed, or irregular basis. The frequency of administration of the composition can be determined empirically by those skilled in the art.

Release of the pharmaceutically-active small molecule (e.g., SCFA) is a necessarily important aspect of the copolymer performance for material processing or downstream biological applications. In some embodiments, the pharmaceutically-active small molecule may be cleaved from the polymer backbone under suitable biological conditions, including hydrolysis (e.g. at certain pH) and enzyme activity (e.g. an esterase). In this regard, the copolymer may be termed a prodrug. In various embodiments, the pharmaceutical composition includes about 10-80% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or ranges therebetween) of pharmaceutically-active small molecule, e.g. a SCFA or derivative thereof, by weight. Those skilled in the art of clinical pharmacology can readily arrive at dosing amounts using routine experimentation.

Release of the pharmaceutically-active small molecule necessarily has a therapeutic effect recapitulating the therapeutic effects of SCFAs, including targeting the barrier function of the intestine and the mucus layer of the gut and all diseases in which SCFAs have been implicated to have a therapeutic benefit, including increasing mucus layer thickness or barrier function are implicated may be treated. In some embodiments, the human diseases that are treatable include, but are not limited to, rheumatoid arthritis, celiac disease and other autoimmune diseases, food allergies of all types, eosinophilic esophagitis, allergic rhinitis, allergic asthma, pet allergies, drug allergies, and other allergic and atopic diseases, inflammatory bowel disease, ulcerative colitis, Crohn's diseases, and additional inflammatory conditions, infectious diseases, metabolic disorders, multiple sclerosis, Alzheimer's disease, Parkinson's disease, dementia, and other diseases of the central nervous system, thalassemia and other blood disorders, colorectal cancer, diarrhea and related diseases effecting gut motility, Type I diabetes, and autism spectrum disorders, among others. This list is not exhaustive, and those skilled in the art can readily treat additional indications that have been shown to have therapeutic effect of SCFAs.

Pharmaceutical preparations can be formulated from the composition of the invention by drug formulation methods known to those skilled in the art. Formulations are prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective, without causing undesirable biological side effects or unwanted interactions. Suitable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The composition can be adapted for the mode of administration and can be in the form of, e.g., a pill, tablet, capsule, spray, powder, or liquid. In some embodiments, the pharmaceutical composition contains one or more pharmaceutically acceptable additives suitable for the selected route and mode of administration, such as coatings, fillers, binders, lubricant, disintegrants, stabilizers, or surfactants. These compositions may be administered by, without limitation, any parenteral route, including intravenous, intra-arterial, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal, as well as topically, orally, and by mucosal routes of delivery such as intranasal, inhalation, rectal, vaginal, buccal, and sublingual. In some embodiments, the pharmaceutical compositions of the invention are prepared for administration to vertebrate (e.g. mammalian) subjects in the form of liquids, including sterile, non-pyrogenic liquids for injection, emulsions, powders, aerosols, tablets, capsules, enteric coated tablets, or suppositories.

EXAMPLES

Example 1: Preparation of N-(2-hydroxyethyl) methacrylamide monomers with pharmaceutically-active small molecules

Ethanolamine (3.70 mL, 61.4 mmol, 2.0 eq), triethylamine (4.72 mL, 33.8 mmol, 1.1 eq) and 50 mL DCM were added into a 250 mL flask. After the system was cooled down by an ice bath, (1) methacryloyl chloride (3.00 mL, 30.7 mmol, 1.0 eq) was added dropwise under the protection of nitrogen. The reaction was allowed to warm up to room temperature and reacted overnight. Then the reaction mixture was concentrated by rotary evaporation and purified on a silica column using DCM/MeOH. The product N-(2-hydroxyethyl) methacrylamide (2) was obtained as colorless oil (3.42 g, 86.3%) and analyzed by 1H-NMR (500 MHz, CDCl3) δHppm 1.93 (s, 3H, C(CH2)—CH3), 3.43 (m, 2H, NH—CH2), 3.71 (m, 2H, O—CH2), 5.32 (s, 1H, CCH2), 5.70 (s, 1H, CCH2), 6.44 (br s, 1H, NH).

N-(2-hydroxyethyl) methacrylamide (3.30 mL, 25.6 mmol, 1.0 eq), triethylamine (7.15 mL, 51.2 mmol, 2.0 eq) and 50 mL DCM were added into a 250 mL flask. After the reaction system was cooled down by an ice bath, butyric anhydride (5.00 mL, 30.7 mmol, 1.2 eq) was added dropwise under the protection of nitrogen. The system was allowed to react overnight. The reaction mixture was filtered and washed by NH4Cl solution, NaHCO3solution, and water. After dried by anhydrous MgSO4, the organic layer was concentrated by rotary evaporation and purified on a silica column using DCM/MeOH. The product N-butanoyloxyethyl methacrylamide (3) was obtained as pale yellow oil (4.56 g, 89.6%) and analyzed by 1H-NMR (500 MHz, CDCl3) δHppm 0.95 (t, 3H, CH2CH2—CH3), 1.66 (m, 2H, CH2—CH2), 1.97 (s, 3H, C(CH2)—CH3), 2.32 (t, 2H, CO—CH2), 3.59 (dt, 2H, NH—CH2), 4.23 (t, 2H, O—CH2), 5.35 (s, 1H, CCH2), 5.71 (s, 1H, CCH2), 6.19 (br s, 1H, NH). This example demonstrates the feasibility of attaching pharmaceutically-active small molecules to a monomeric unit of the block copolymer. Additional SCFAs can be attached to the monomeric unit in a similar manner.

Example 2A: Preparation of Block Copolymers of Hydrophilic Polymers with N-(2-Hydroxyethyl Methyacrylamide

pHPMA was prepared using 2-cyano-2-propyl benzodithioate as the RAFT chain transfer agent and 2,2′-Azobis(2-methylpropionitrile) (AIBN) as the initiator. Briefly, HPMA (4) (1.0 g, 7.0 mmol, 1.0 eq), 2-cyano-2-propyl benzodithioate (15 mg, 0.07 mmol, 1/100 eq), and AIBN (2.9 mg, 0.017 mmol, 1/400 eq) were dissolved in 2.0 mL MeOH in a Schlenk tube. The reaction mixture was subjected to four freeze-pump-thaw cycles. The polymerization was conducted at 70° C. for 15 h. The polymer was precipitated in petroleum ether for five times and dried in the vacuum chamber overnight. The product poly(HPMA) (5) was obtained as light pink solid (0.79 g, 79%) and analyzed by 1H-NMR (500 MHz, DMSO-d6), δHppm 0.8-1.2 (m, 6H, CH(OH)—CH3and backbone CH3), 1.5-1.8 (m, 2H, backbone CH2), 2.91 (m, 2H, NH—CH2), 3.68 (m, 1H, C(OH)—H), 4.70 (m, 1H, CH—OH), 7.18 (m, 1H, NH).

The block copolymer was prepared using pHPMA as the macro-RAFT chain transfer agent and N-butanoyloxyethyl methacrylamide (3) as the monomer of the second RAFT polymerization. Briefly, pHPMA (0.71 g, 1.0 eq), N-butanoyloxyethyl methacrylamide (2.0 g, 10.0 mmol, 100 eq), and AIBN (4.1 mg, 0.025 mmol, 0.25 eq) were dissolved in 4.0 mL MeOH in a Schlenk tube. The reaction mixture was subjected to four freeze-pump-thaw cycles. The polymerization was conducted at 70° C. for 15 h. The polymer was precipitated in petroleum ether for five times and dried in the vacuum chamber overnight. The product was obtained as light pink solid (1.54 g, 75%) and analyzed by 1H-NMR (500 MHz, DMSO-d6) δHppm 0.80-1.1 (m, 9H, CH(OH)—CH3(HPMA), CH2—CH3(BMA), and backbone CH3), 1.55 (m, 4H, CH2—CH2(BMA) and backbone CH2), 2.28 (m, 2H, CO—CH2(BMA)), 2.91 (m, 2H, NH—CH2(HPMA)), 3.16 (m, 2H, NH—CH2(BMA)), 3.67 (m, 1H, CH(OH)—H), 3.98 (m, 2H, O—CH2(BMA)), 4.71 (m, 1H, CH—OH (HPMA)), 7.19 (m, 1H, NH), 7.44 (m, 1H, NH). Extending from this example, molecular weights varying from 5 kD and 14 kD for each block respectively can be readily obtained, leading to a variety of ratios of inert and active blocks in the polymer. In this way, the range of pharmaceutically-active ingredient thereby incorporated can be about 10-80% of the total polymer weight.

Example 2B: Preparation of 2-(4-Hydroxybenzoyloxy)Ethyl Methacrylate Monomers with Pharmaceutically-Active Small Molecules

2-Bromoethanol (4.36 mL, 61.5 mmol, 1.5 eq), triethylamine (9.15 mL, 65.6 mmol, 1.6 eq) and 60 mL DCM were added into a 250 mL flask. After the system was cooled down by an ice bath, (1) methacryloyl chloride (4.00 mL, 41.0 mmol, 1.0 eq) was added dropwise under the protection of nitrogen. The reaction was allowed to warm up to room temperature and reacted overnight. Then the reaction mixture was concentrated by rotary evaporation and purified on a silica column using DCM/MeOH. The product 2-bromoethyl methacrylate (7) was obtained as colorless oil (6.62 g, 84.1%) and analyzed by 1H-NMR (500 MHz, CDCl3) δHppm 1.95 (s, 3H, C(CH2)—CH3), 3.57 (m, 2H, Br—CH2), 4.45 (m, 2H, O—CH2), 5.62 (s, 1H, CCH2), 6.18 (s, 1H, CCH2).

4-Hydroxybenzoic acid (2.00 g, 14.5 mmol, 1.2 eq), sodium bicarbonate (2.94 g, 35.0 mmol, 2.9 eq) and 20 mL DMF were added into a two-armed flask. The mixture was remained at 70° C. for 1 h. Then, 2-bromoethyl methacrylate (2.32 g, 12.1 mmol, 1.0 eq) was dissolved in 10 mL DMF and added into the flask dropwise. The reaction was conducted at 70° C. overnight. The reaction mixture was cooled, poured into 100 mL water and extracted three times with 100 mL of a 50:50 hexane/ethyl acetate mixture. The organic phases were washed twice with water (100 mL), dried over Na2SO4and purified on a silica column using DCM/MeOH. The product 2-(4-hydroxybenzoyloxy)ethyl methacrylate (8) was obtained as viscous oil (0.87 g, 58%) and analyzed by 1H-NMR (500 MHz, CDCl3) δHppm 1.95 (s, 3H, C(CH2)—CH3), 4.48 (m, 2H, O—CH2), 4.54 (m, 2H, O—CH2), 5.62 (s, 1H, CCH2), 6.14 (s, 1H, CCH2), 6.86 (d, 2H, ArH), 7.96 (d, 2H, ArH).

2-(4-hydroxybenzoyloxy)ethyl methacrylate (0.87 g, 3.48 mmol, 1.0 eq), triethylamine (1.46 mL, 10.44 mmol, 3.0 eq) and 30 mL DCM were added into a 100 mL flask. After the reaction system was cooled down by an ice bath, butyric anhydride (1.14 mL, 6.96 mmol, 2.0 eq) was added dropwise under the protection of nitrogen. The system was allowed to react overnight. The reaction mixture was filtered and washed by NH4Cl solution, NaHCO3solution, and water. After dried by anhydrous MgSO4, the organic layer was concentrated by rotary evaporation and purified on a silica column using DCM/MeOH. The product 2-(4-butanoyloxybenzoyloxy)ethyl methacrylate (9) was obtained as pale oil (1.02 g, 92%) and analyzed by 1H-NMR (500 MHz, CDCl3) δHppm 1.06 (t, 3H, CH2CH2—CH3), 1.72 (m, 2H, CH2—CH2), 1.94 (s, 3H, C(CH2)—CH3), 2.54 (t, 2H, CO—CH2), 4.48 (m, 2H, O—CH2), 4.56 (m, 2H, O—CH2), 5.59 (s, 1H, CCH2), 6.14 (s, 1H, CCH2), 7.16 (d, 2H, ArH), 8.06 (d, 2H, ArH).

Example 3: Preparation of Block Copolymers of Hydrophilic Polymers with N-(2-Hydroxyethyl Methyacrylamide

pHPMA was prepared using 2-cyano-2-propyl benzodithioate as the RAFT chain transfer agent and 2,2′-Azobis(2-methylpropionitrile) (AIBN) as the initiator. Briefly, HPMA (4) (1.0 g, 7.0 mmol, 1.0 eq), 2-cyano-2-propyl benzodithioate (15 mg, 0.07 mmol, 1/100 eq), and AIBN (2.9 mg, 0.017 mmol, 1/400 eq) were dissolved in 2.0 mL MeOH in a Schlenk tube. The reaction mixture was subjected to four freeze-pump-thaw cycles. The polymerization was conducted at 70° C. for 15 h. The polymer was precipitated in petroleum ether for five times and dried in the vacuum chamber overnight. The product poly(HPMA) (5) was obtained as light pink solid (0.79 g, 79%) and analyzed by 1H-NMR (500 MHz, DMSO-d6), δHppm 0.8-1.2 (m, 6H, CH(OH)—CH3and backbone CH3), 1.5-1.8 (m, 2H, backbone CH2), 2.91 (m, 2H, NH—CH2), 3.68 (m, 1H, C(OH)—H), 4.70 (m, 1H, CH—OH), 7.18 (m, 1H, NH)

The block copolymer (pHPMA-b-pBMA) was prepared using pHPMA as the macro-RAFT chain transfer agent and N-butanoyloxyethyl methacrylamide (3) as the monomer of the second RAFT polymerization. Briefly, pHPMA (0.71 g, 1.0 eq), N-butanoyloxyethyl methacrylamide (2.0 g, 10.0 mmol, 100 eq), and AIBN (4.1 mg, 0.025 mmol, 0.25 eq) were dissolved in 4.0 mL MeOH in a Schlenk tube. The reaction mixture was subjected to four freeze-pump-thaw cycles. The polymerization was conducted at 70° C. for 15 h. The polymer was precipitated in petroleum ether for five times and dried in the vacuum chamber overnight. The product was obtained as light pink solid (1.54 g, 75%) and analyzed by 1H-NMR (500 MHz, DMSO-d6) δHppm 0.80-1.1 (m, 9H, CH(OH)—CH3(HPMA), CH2—CH3(BMA), and backbone CH3), 1.55 (m, 4H, CH2—CH2(BMA) and backbone CH2), 2.28 (m, 2H, CO—CH2(BMA)), 2.91 (m, 2H, NH—CH2(HPMA)), 3.16 (m, 2H, NH—CH2(BMA)), 3.67 (m, 1H, CH(OH)—H), 3.98 (m, 2H, O—CH2(BMA)), 4.71 (m, 1H, CH—OH (HPMA)), 7.19 (m, 1H, NH), 7.44 (m, 1H, NH). Extending from this example, molecular weights varying from 5 kD and 14 kD for each block respectively can be readily obtained, leading to a variety of ratios of inert and active blocks in the polymer. In this way, the range of pharmaceutically-active ingredient thereby incorporated can be about 10-80% of the total polymer weight.

Example 4: Preparation of Block Copolymers of Hydrophilic Polymers with 2-(4-Hydroxybenzoyloxy)Ethyl Methacrylate

The block copolymer (pHPMA-b-pBBOMA) was prepared using pHPMA as the macro-RAFT chain transfer agent and 2-(4-butanoyloxybenzoyloxy)ethyl methacrylate (10) as the monomer of the second RAFT polymerization. Briefly, pHPMA (0.50 g, 1.0 eq), 2-(4-butanoyloxybenzoyloxy)ethyl methacrylate (1.12 g, 3.5 mmol, 50 eq), and AIBN (3.0 mg, 0.018 mmol, 0.005 eq) were dissolved in 4.0 mL MeOH in a Schlenk tube. The reaction mixture was subjected to four freeze-pump-thaw cycles. The polymerization was conducted at 70° C. for 15 h. The polymer was precipitated in petroleum ether for five times and dried in the vacuum chamber overnight. The product was obtained as light pink solid (1.34 g, 83%) and analyzed by 1H-NMR (500 MHz, DMSO-d6) δHppm 0.80-1.1 (m, 9H, CH(OH)—CH3(HPMA), CH2—CH3(BBOMA), and backbone CH3), 1.57 (m, 4H, CH2—CH2(BBOMA) and backbone CH2), 2.46 (m, 2H, CO—CH2(BBOMA)), 2.90 (m, 2H, NH—CH2(HPMA)), 3.67 (m, 1H, CH(OH)—H), 4.00-4.40 (m, 4H, O—CH2(BBOMA)), 4.71 (m, 1H, CH—OH (HPMA)), 7.17 (m, 2H, ArH (BBOMA)), 7.92 (m, 2H, ArH (BBOMA)).

Example 5: Preparation of Polymer Solutions

The polymer solutions were prepared using cosolvent evaporation method. 80 mg pHPMA-b-pBMA was dissolved in 1.0 mL ethanol in a beaker. Then, 1.0 mL 1× phosphate buffered saline (PBS) was added into the beaker. The solution was stirred vigorously for 6 hours to let ethanol evaporate. The polymeric solution was obtained at 80 mg/mL in 1×PBS and was stored at 4° C. The size of the polymeric formulation was measured by dynamic light scattering. This example demonstrates the assembly into particles in solution. Particles can be prepared using other solutions and additives in solutions as embodied in this example. Particle sizes from 10-1000 nm are attainable as embodied in this example.

Example 6: Administration of the Polymer Solutions Via Oral Gavage and Increase in Fecal SCFA

Mice were orally gavaged using the polymer solutions prepared. 125 μL of a 80 mg/mL solution was administered by oral gavage into the stomach of a mouse using a needle. Mouse fecal samples were obtained every 4 hours and the fecal samples processed for gas chromatography. Gas chromatography samples were prepared as adapted from Kaur, 2012 by adding 0.4 mL of phosphate buffered saline to 0.1 g of feces and homogenizing by vortex. 0.1 mL of an internal standard mixture comprising 0.5 mM 4-methylvaleric acid, 5% meta-phosphoric acid, and 1.56 mg/mL copper sulfate was added, the sample vortexed, and then centrifuged at 13000 rpm for 10 min. 10 μL of the supernatant was injected into the gas chromatograph for analysis and concentrations of fecal SCFA were determined using a calibration curve. This example demonstrates that oral administration of the block copolymer increases the fecal concentration of the SCFA attached to the block polymer

Example 7: Administration of the Polymer Solutions Via Oral Gavage and Decrease in Physiological Markers of Allergic Reaction and Anaphylaxis

An allergy to peanuts was induced in mice by sensitization via intragastric gavage with 5 mg peanut protein+10 μg cholera toxin as adjuvant (“allergic positive control” or “allergic” in this example andFIGS.13,14,15, and16) or 10 μg cholera toxin adjuvant only (“non-allergic negative control” in this example andFIGS.13,14,15, and16) at weaning and once weekly for 5 weeks total following a procedure previously reported and known to those skilled in the art. Data is reported for male mice only to control for subject weight and dosing. 80 mg/mL stock solutions of polymer were prepared, then diluted to 13.3 mg/mL (for a 2 mg daily dose of polymer) and 53.3 mg/mL (for a 8 mg daily dose of polymer) using phosphate buffered saline. 150 μL of the polymer solution was administered daily by intragastric gavage into the stomach of “allergic” mice using a needle. For “allergic” mice dosed with 2 mg daily (N=6), the 13.3 mg/mL solution was used; For “allergic” mice dosed with 8 mg daily (N=5), the 53.3 mg/mL solution was used; for “allergic” mice receiving no dose of polymer (N=6), 150 μL of 1×PBS was used as control. For “non-allergic negative control” mice (N=4), 150 μL of 1×PBS was dosed daily as control. One week after the 5th sensitization (day 35 of the experiment), the mice were given 5 mg of peanut protein via intraperitoneal injection. Core body temperature was monitored rectally for 90 minutes after injection and mouse serum was collected 90 minutes and 24 hours after injection. Serum was processed for ELISA and peanut-specific Immunoglobulin E (IgE), peanut-specific Immunoglobulin G1 (IgG1), and mouse mast cell protease-1 (mMCP-1) was determined. This example demonstrates that oral administration of the block copolymer reduces the physiological markers and symptoms of an allergic reaction when dosed daily

Example 8: Administration of Sodium Butyrate Solutions Via Oral Gavage and Inferiority of Sodium Butyrate Compared to Polymer in Decreasing Physiological Markers of Allergic Reaction and Anaphylaxis

An allergy to peanuts was induced in mice by sensitization via oral gavage with 6 mg peanut protein+10 μg cholera toxin (“allergic positive control” or “allergic”; N=19) at weaning and once weekly for 5 weeks total following a procedure previously reported and known to those skilled in the art. Mice received a dose of 2.83 mg sodium butyrate per g of mouse body weight daily by oral gavage of sodium butyrate dissolved in phosphate buffered saline into the stomach of a “allergic” mice using a needle. This dose is equivalent to the dose administered by polymer at 0.8 mg polymer per g of mouse body weight. Mice were weighed once weekly and the amount of solution adjusted based on the new weight. One week after the 5th sensitization (day 35 of the experiment), the mice were given 5 mg of peanut protein via intraperitoneal injection. Core body temperature was monitored rectally for 90 minutes after injection and mouse serum was collected 90 minutes and 24 hours after injection. Serum was processed for ELISA and peanut-specific Immunoglobulin E (IgE), peanut-specific Immunoglobulin G1 (IgG1), and mouse mast cell protease-1 (mMCP-1) was determined. These results demonstrate that oral administration of sodium butyrate at the same dose as an equivalent block copolymer is not effective at reducing the physiological markers and symptoms of an allergic reaction when dosed daily (FIG.17).

Example 9: Administration of Different Formulations of Polymer Via Oral Gavage do not Lead to Equivalent Effectiveness at Decreasing Physiological Markers of Allergic Reaction and Anaphylaxis

Two formulations of polymer were prepared using the methods described in Examples 3 and 5. One polymer was characterized and demonstrated to have a ratio of HPMA:BMA block in the copolymer of 1.01 and a particle diameter of 329 nm by dynamic light scattering (“Formulation 2”). The other polymer was characterized and demonstrated to have a ratio of HPMA:BMA block in the copolymer of 0.75 and a particle diameter of 64.6 nm by dynamic light scattering (“Formulation 1”) (FIG.18).

An allergy to peanuts was induced in mice by sensitization via oral gavage with 6 mg peanut protein+10 μg cholera toxin (“allergic positive control” or “allergic”) or 10 μg cholera toxin alone (“non-allergic negative control”; N=4) at weaning and once weekly for 5 weeks total following a procedure previously reported and known to those skilled in the art. 80 mg/mL stock solutions of Formulation 2 polymer were prepared as in Example 3, then diluted to 53.3 mg/mL. 0.8 mg of polymer per g of mouse body weight was administered daily by oral gavage into the stomach of a “allergic” mice using a needle. Another group of “allergic” mice received 2.83 mg of sodium butyrate identically to Example 7. Another group of “allergic” mice received a 0.8 mg per g of mouse body weight daily dose of a control polymer that could not release butyric acid. For “allergic” mice receiving no dose of polymer, volume-matched solution of 1×PBS was used as control. For “non-allergic negative control” mice, 0.8 mg of control polymer per g of mouse body weight was dosed daily as control. One week after the 5th sensitization (day 35 of the experiment), the mice were given 5 mg of peanut protein via intraperitoneal injection. Core body temperature was monitored rectally for 90 minutes after injection and mouse serum was collected 90 minutes and 24 hours after injection (FIG.19). Serum was processed for ELISA and peanut-specific Immunoglobulin E (IgE), peanut-specific Immunoglobulin G1 (IgG1), and mouse mast cell protease-1 (mMCP-1) was determined. These results demonstrate that increase in fecal butyrate is not sufficient to predict efficacy, and that molecular and morphological characteristics of the polymer non-obviously result in differences in therapeutic effect. Specifically, Formulation 2, which differs from Formulation 1 only by molecular weight and block ratio, does not reduce the physiological markers and symptoms of an allergic reaction when dosed daily.

Example 10: Administration of formulations of Polymer Via Oral Gavage Leads to Equivalent Changes to Fecal Butyrate Concentration, but does not Lead to Equivalent Effectiveness at Decreasing Physiological Markers of Allergic Reaction and Anaphylaxis. Equivalent Effectiveness Requires Strict Control of Certain Polymer Molecular Characteristics

Three formulations of polymer were prepared using the methods described in Examples 3 and 5 (FIG.20). One polymer was characterized and demonstrated to have a ratio of HPMA:BMA block in the copolymer of 0.75, a molecular weight of about 40,000 g/mol, and a particle diameter of 60-70 nm by dynamic light scattering (“Formulation 1”). A second polymer was characterized and demonstrated to have a ratio of HPMA:BMA block in the copolymer of 0.71, a molecular weight of about 52,000 g/mol, and a particle diameter of 120-130 nm by dynamic light scattering (“Formulation 3”). A third polymer was characterized and demonstrated to have a ratio of HPMA:BMA block in the copolymer of 0.77, a molecular weight of 39,000 g/mol, and a particle diameter of 75-82 nm by dynamic light scattering (“Formulation 4”).

Analysis of the three formulations by GPC, DLS, and NMR are depicted inFIGS.21-24.

8 mg of Formulation 1 and Formulation 3 were administered to groups of mice (N=8 per group) in a single dose oral administration via intragastric gavage and the fecal material from each mouse collected and combined over a period of 24 hours. The samples were processed for gas chromatography and the concentration of free butyrate was determined using the method described in Example 6. The ratio of free butyrate to theoretically released butyrate was determined and converted to percent release (FIG.26).

These three formulations were compared using the induced allergy model procedure described in Examples 5 and 7 (FIG.27). After the mice were given 5 mg of peanut protein via intraperitoneal injection, core body temperature was monitored rectally for 90 minutes after injection and mouse serum was collected 90 minutes and 24 hours after injection. Serum was processed for ELISA and peanut-specific Immunoglobulin E (IgE), peanut-specific Immunoglobulin G1 (IgG1), and mouse mast cell protease-1 (mMCP-1) was determined. These results demonstrate that molecular weight differences, and not block ratio differences, lead to differences in therapeutic effect. Specifically, Formulation 2, which does not differ significantly in block ratio from Formulations 1 and 3, but does differ in molecular weight and morphology does not reduce the physiological markers and symptoms of an allergic reaction when dosed daily. Formulations 1 and 3, which differ only slightly in molecular and morphological characteristics, do reduce the physiological markers and symptoms of an allergic reaction when dosed daily.

REFERENCES

The following references, some of which are cited above, are herein incorporated by reference in their entireties.Arpaia, N., Campbell, C., Fan, X., Dikiy, S., van der Veeken, J., deRoos, P., Liu, H., Cross, J. R., Pfeffer, K., Coffer, P. J., and Rudensky, A. Y. (2013). Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 504, 451-455.Berni Canani, R., Gilbert, J. A., and Nagler, C. R. (2015). The role of the commensal microbiota in the regulation of tolerance to dietary allergens. Curr Opin Allergy Clin Immunol, 15, 243-249.Berni Canani, R., Sangwan, N., Stefka, A. T., Nocerino, R., Papro, L., Aitoro, R., Calignano, A., Kahn, A. A., Gilbert, J. A., and Nagler, C. R. (2016).Lactobacillus rhamnosusGG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants. ISME J, 10, 742-750.Furusawa, Y., Obata, Y., Fukuda, S., Endo, T. A., Nakato, G., Takahashi, D., Nakanishi, Y., Uetake, C., Kato, K., Kato, T., Takahashi, M., Fukuda, N. N., Murakami, S., Miyauchi, E., Hino, S., Atarashi, K., Onawa, S., Fujimura, Y., Lockett, T., Clarke, J. M., Topping, D. L., Tomita, M., Hori, S., Ohara, O., Morita, T., Koseki, H., Kikuchi, J., Honda, K., Hase, K., and Ohno, H., (2013). Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 504, 446-450.Haghikia, A., Jörg, S., Duscha, A., Berg, J., Arndt, M., Waschbisch, A., Hammer, A., Lee, D.-H., May, C., Wilck, N., Balogh, A., Ostermann, A. I., Schebb, N. H., Akkad, D. A., Grohme, D. A., Kleinewietfeld, M., Kempa, S., Thone, J., Demir, S., Möller, D. N., Gold, R., and Linker, R. A. (2015). Dietary Fatty Acids Directly Impact Central Nervous System Autoimmunity via the Small Intestine. Immunity, 43, 817-829.Kaur, A., (2012) Modulation of gut microbiota and its environment using starch-entrapped microspheres and cereal arabinoxylans. Purdue.MacFabe, D. F. (2015) Enteric short-chain fatty acids: microbial messengers of metabolism, mitochondria, and mind: implications in autism spectrum disorders. Microb Ecol Health Dis, 26, 28177.Meijer, K. de Vos, P., and Priebe, M. G. (2010). Butyrate and other short-chain fatty acids as modulators of immunity: what relevance for health? Curr Opin Clin Nutr Metab Care. 13, 715-721.Nylund, L., Nermes, M., Isolauri, E., Salminen, S., de Vos, W. M., and Satokari, R. (2015). Severity of atopic disease inversely correlates with intestinal microbiota diversity and butyrate-producing bacteria. Allergy, 70, 241-244.Sandin, A., Bråbäck, L., Norin, E., and Bengt Björkstén, B. (2009). Faecal short chain fatty acid pattern and allergy in early childhood. Acta Paediatrica, 98, 823-827.

Smith, P. M., Howitt, M. R., Panikov, N., Michaud, M., Gallini, C. A., Bohlooly-Y, M., Glickman, J. N., and Garrett, W. S. (2013). The microbial metabolites, short chain fatty acids, regulate colonic Treg cell homeostasis. Science 341, 569-573.