Patent Application
20250114436
Hydrogels are a common class of biomaterials, with their network structure offering a surrogate of the natural extracellular matrix and their highly hydrated porosity enabling controlled release of encapsulated macromolecules. The polymers used in composing hydrogels are typically hydrophilic, and once crosslinked afford a material that can imbue water in an amount many times the dry weight of the polymer itself. Hydrogels can be characterized by their mode of crosslinking; chemical crosslinking entails the permanent formation of covalent crosslinks between polymer chains, while physical crosslinking arises from transient and reversible interactions or entanglements. The mechanical properties of the bulk hydrogel materials usually follow directly from their mode of crosslinking. Covalent crosslinks commonly yield materials with higher modulus that do not flow or permanently deform under moderate strain but exhibit permanent loss of mechanical character under high strain. Conversely, physical crosslinking typically gives rise to materials with more dynamic viscoelastic behavior, enabling flow under applied strain and exhibiting self-healing character.
Dynamic-covalent chemistry encompasses a number of equilibrium-governed covalent bonds, including many classical organic reaction mechanisms. Recently, dynamic-covalent crosslinking has gained attention for its use in the preparation of hydrogels. When used in the context of hydrogel crosslinking, this approach enables covalent bonding interactions with dynamic exchange and finite average lifetime. Accordingly, this mode of crosslinking, in principle, affords aspects of both chemical and physical crosslinking in yielding dynamic viscoelastic materials with well-defined crosslinking interactions and excellent mechanical properties while also undergoing equilibrium-governed bond exchange that enables network restructuring and self-healing. Certain of these dynamic-covalent interactions are further modulated by competition from naturally occurring analytes, enabling their equilibrium-governed bond exchange to be integrated into stimuli-responsive platforms. One such chemistry that has been explored in this regard is dynamic-covalent bonding between phenylboronic acids (PBAs) and cis-1,2 or cis-1,3 diols. In the context of drug delivery for diabetes, PBA-diol chemistry is susceptible to competition from glucose (a cis-1,2 diol), which in turn affords hydrogels where the extent of network crosslinking is rendered glucose-dependent.
Prior reports have described hydrogel materials crosslinked using PBA-diol interactions and explored glucose-responsive release of encapsulated macromolecules from these networks. Rich phenomena in polymer physics have also been elucidated from ideal network platforms prepared using this chemistry. At the same time, PBA chemistry presents two key drawbacks in its application for use in glucose-responsive materials. First, common diol chemistries used for polymer crosslinking have affinity for PBA significantly higher than that of glucose, which itself does not typically bind PBA with affinity sufficient for optimal function under physiological glucose concentrations. This challenge, in turn, limits glucose-responsive function of the material. Second, the non-specific nature of the PBA-glucose interaction means these linkages are subject to interference from binding of common analytes such as fructose and lactate, which actually bind with higher affinity than glucose to most PBA chemistries. Accordingly, limited glucose-responsiveness and sensitivity to non-glucose analytes present in the body act contrary to the envisioned application of these materials for stimuli-responsive release of insulin to control blood glucose levels in diabetes.
What is needed are boronic acid motifs that bind glucose with high affinity and improved specificity and that can be used to prepare dynamic injectable materials with improved function in encapsulation and glucose-triggered release of insulin in vitro and in vivo.
One embodiment described herein is hydrogel, the hydrogel comprising:
wherein:
wherein:
is a linking moiety; and
is a first moiety comprising a hydrophilic polymer, a hyperbranched macromolecule, or a combination thereof; and
wherein:
is a second moiety comprising a hydrophilic polymer, a hyperbranched macromolecule, or a combination thereof;
wherein the diboronate compound of formula (I) and the diol compound of formula (II) form crosslinks by dynamic-covalent bonds.
In another aspect,
In another aspect,
In another aspect, X⊖ is Br−, Cl−, NO3−, H2PO4−, H2PO3−, HSO4−, HSO3−, H3C−, SO3−, HCO3−, HCO2−, H3C—CO2−, HC2O4−, or TsO−. In another aspect, X⊖ is Br− or Cl−. In another aspect,
another aspect,
In another aspect,
has an equilibrium constant (Keq) for binding glucose of at least 350 M−1. In another aspect,
has an equilibrium constant (Keq) for binding glucose of at least 1000 M−1. In another aspect,
has an equilibrium constant (Keq) for binding glucose that is at least 20 times greater than equilibrium constant of
for binding lactate. In another aspect,
comprises:
In another aspect,
comprises:
In another aspect,
comprises
In another aspect,
comprises a branched polymer. In another aspect, the branched polymer comprises a polyalkylene glycol. In another aspect, the branched polymer is a four-armed polymer. In another aspect, the diboronate compound of formula (I) comprises:
wherein n is 2 to 250. In another aspect,
comprises a dendrimer. In another aspect, the dendrimer is a polyamidoamine dendrimer. In another aspect,
comprises a linear polymer. In another aspect, the linear polymer comprises a polysaccharide. In another aspect, the polysaccharide comprises hyaluronic acid. In another aspect, the molar ratio of the diboronate compound of formula (I) to the diol compound of formula (II) is 1:1. In another aspect,
comprises a branched polymer. In another aspect, the branched polymer is a four-armed or an eight-armed polymer. In another aspect, the branched polymer comprises polyethylene glycol. In another aspect, the diol compound of formula (II) comprises:
wherein n is 2 to 250. In another aspect,
comprises a dendrimer. In another aspect, the dendrimer is a polyamidoamine dendrimer. In another aspect,
comprises a linear polymer.
Another embodiment described herein is a pharmaceutical composition comprising insulin encapsulated within a hydrogel.
Another embodiment described herein is a method of delivering insulin to a subject in need thereof, the method comprising: administering a pharmaceutical composition to a subject in need thereof, the pharmaceutical composition comprising insulin encapsulated within a hydrogel, the hydrogel comprising:
wherein:
wherein:
a linking moiety; and
is a first moiety comprising a hydrophilic polymer, a hyperbranched macromolecule, or a combination thereof; and
wherein:
is a second moiety comprising a hydrophilic polymer, a
hyperbranched macromolecule, or a combination thereof; wherein the diboronate compound of formula (I) and the diol compound of formula (II) form crosslinks by dynamic-covalent bonds. In another aspect, the subject in need thereof has diabetes. In another aspect, the insulin is administered to the subject at 0.05-10 international units (IU)/kg. In another aspect, following administration of the pharmaceutical composition, the subject has blood glucose levels of about 60-110 mg/dL.
Another embodiment described herein is the use of a hydrogel, a pharmaceutical composition, or a method for delivering insulin to a subject in need thereof.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.
As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.
As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.
As used herein, the term “or” can be conjunctive or disjunctive.
As used herein, the term “substantially” means to a great or significant extent, but not completely.
As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ±10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol “-” means “about” or “approximately.”
All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.
As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.
As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.
As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein.
As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.
As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject's age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.
As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non-human primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.
As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.
As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.
As used herein, “treating or preventing a disease or disorder” includes alleviating and mitigating a disease or disorder, and improving symptoms, and also includes lowering the probability of getting a disease or disorder.
As used herein, the terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
As used herein, the terms “salt” or “salts” refers to an acid addition or base addition salt of a compound of the invention. “Salts” include in particular “pharmaceutical acceptable salts.” The term “pharmaceutically acceptable salts” refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable. In many cases, the compounds described herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein.
Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.
The term “alkoxy,” as used herein, refers to a group —O-alkyl. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy and tert-butoxy.
The term “alkyl,” as used herein, means a straight or branched, saturated hydrocarbon chain. The term “lower alkyl” or “C1-6alkyl” means a straight or branched chain hydrocarbon containing from 1 to 6 carbon atoms. The term “C4alkyl” means a straight or branched chain hydrocarbon containing from 1 to 4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.
The term “alkenyl,” as used herein, means a straight or branched, hydrocarbon chain containing at least one carbon-carbon double bond.
The term “alkoxyalkyl,” as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
The term “alkoxyfluoroalkyl,” as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through a fluoroalkyl group, as defined herein.
The term “alkylene,” as used herein, refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 10 carbon atoms, for example, of 2 to 5 carbon atoms. Representative examples of alkylene include, but are not limited to, —CH2—, —CD2—, —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, and —CH2CH2CH2CH2CH2—.
The term “alkylamino,” as used herein, means at least one alkyl group, as defined herein, is appended to the parent molecular moiety through an amino group, as defined herein.
The term “amide,” as used herein, means —C(O)NR— or —NRC(O)—, wherein R may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl.
The term “aminoalkyl,” as used herein, means at least one amino group, as defined herein, is appended to the parent molecular moiety through an alkylene group, as defined herein.
The term “amino,” as used herein, means —NRxRy, wherein Rx and Ry may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl. In the case of an aminoalkyl group or any other moiety where amino appends together two other moieties, amino may be —NRx−, wherein Rx may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl.
The term “aryl,” as used herein, refers to a phenyl or a phenyl appended to the parent molecular moiety and fused to a cycloalkane group (e.g., the aryl may be indan-4-yl), fused to a 6-membered arene group (i.e., the aryl is naphthyl), or fused to a non-aromatic heterocycle (e.g., the aryl may be benzo[d][1,3]dioxol-5-yl). The term “phenyl” is used when referring to a substituent and the term 6-membered arene is used when referring to a fused ring. The 6-membered arene is monocyclic (e.g., benzene or benzo). The aryl may be monocyclic (phenyl) or bicyclic (e.g., a 9- to 12-membered fused bicyclic system).
The term “cyanoalkyl,” as used herein, means at least one —CN group, is appended to the parent molecular moiety through an alkylene group, as defined herein.
The term “cyanofluoroalkyl,” as used herein, means at least one —CN group, is appended to the parent molecular moiety through a fluoroalkyl group, as defined herein.
The term “cycloalkoxy,” as used herein, refers to a cycloalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
The term “cycloalkyl” or “cycloalkane,” as used herein, refers to a saturated ring system containing all carbon atoms as ring members and zero double bonds. The term “cycloalkyl” is used herein to refer to a cycloalkane when present as a substituent. A cycloalkyl may be a monocyclic cycloalkyl (e.g., cyclopropyl), a fused bicyclic cycloalkyl (e.g., decahydronaphthalenyl), or a bridged cycloalkyl in which two non-adjacent atoms of a ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms (e.g., bicyclo[2.2.1]heptanyl).
Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, and bicyclo[1.1.1]pentanyl.
The term “cycloalkenyl” or “cycloalkene,” as used herein, means a non-aromatic monocyclic or multicyclic ring system containing all carbon atoms as ring members and at least one carbon-carbon double bond and preferably having from 5-10 carbon atoms per ring. The term “cycloalkenyl” is used herein to refer to a cycloalkene when present as a substituent. A cycloalkenyl may be a monocyclic cycloalkenyl (e.g., cyclopentenyl), a fused bicyclic cycloalkenyl (e.g., octahydronaphthalenyl), or a bridged cycloalkenyl in which two non-adjacent atoms of a ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms (e.g., bicyclo[2.2.1]heptenyl).
Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl or cycloheptenyl. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl or cycloheptenyl.
The term “carbocyclyl” means a “cycloalkyl” or a “cycloalkenyl.” The term “carbocycle” means a “cycloalkane” or a “cycloalkene.” The term “carbocyclyl” refers to a “carbocycle” when present as a substituent.
The terms cycloalkylene and heterocyclylene refer to divalent groups derived from the base ring, i.e., cycloalkane, heterocycle. For purposes of illustration, examples of cycloalkylene and heterocyclylene include, respectively,
Cycloalkylene and heterocyclylene include a geminal divalent groups such as 1,1-C3-6cycloalkylene
A further example is 1,1-cyclopropylene
The term “fluoroalkyl,” as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by fluorine.
Representative examples of fluoroalkyl include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl such as 3,3,3-trifluoropropyl.
The term “fluoroalkylene,” as used herein, means an alkylene group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by fluorine.
Representative examples of fluoroalkyl include, but are not limited to —CF2—, —CH2CF2—, 1,2-difluoroethylene, 1,1,2,2-tetrafluoroethylene, 1,3,3,3-tetrafluoropropylene, 1,1,2,3,3-pentafluoropropylene, and perfluoropropylene such as 1,1,2,2,3,3-hexafluoropropylene.
The term “halogen” or “halo,” as used herein, means Cl, Br, I, or F.
The term “haloalkyl,” as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by a halogen.
The term “haloalkoxy,” as used herein, means at least one haloalkyl group, as defined herein, is appended to the parent molecular moiety through an oxygen atom.
The term “halocycloalkyl,” as used herein, means a cycloalkyl group, as defined herein, in which one or more hydrogen atoms are replaced by a halogen.
The term “heteroalkyl,” as used herein, means an alkyl group, as defined herein, in which one or more of the carbon atoms has been replaced by a heteroatom selected from S, O, P and N. Representative examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, amides, and alkyl sulfides.
The term “heteroaryl,” as used herein, refers to an aromatic monocyclic heteroatom-containing ring (monocyclic heteroaryl) or a bicyclic ring system containing at least one monocyclic heteroaromatic ring (bicyclic heteroaryl). The term “heteroaryl” is used herein to refer to a heteroarene when present as a substituent. The monocyclic heteroaryl are five or six membered rings containing at least one heteroatom independently selected from the group consisting of N, O and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). The five membered aromatic monocyclic rings have two double bonds, and the six membered aromatic monocyclic rings have three double bonds. The bicyclic heteroaryl is an 8- to 12-membered ring system and includes a fused bicyclic heteroaromatic ring system (i.e., 10π electron system) such as a monocyclic heteroaryl ring fused to a 6-membered arene (e.g., quinolin-4-yl, indol-1-yl), a monocyclic heteroaryl ring fused to a monocyclic heteroarene (e.g., naphthyridinyl), and a phenyl fused to a monocyclic heteroarene (e.g., quinolin-5-yl, indol-4-yl).
A bicyclic heteroaryl/heteroarene group includes a 9-membered fused bicyclic heteroaromatic ring system having four double bonds and at least one heteroatom contributing a lone electron pair to a fully aromatic 107l electron system, such as ring systems with a nitrogen atom at the ring junction (e.g., imidazopyridine) or a benzoxadiazolyl. A bicyclic heteroaryl also includes a fused bicyclic ring system composed of one heteroaromatic ring and one non-aromatic ring such as a monocyclic heteroaryl ring fused to a monocyclic carbocyclic ring (e.g., 6,7-dihydro-5H-cyclopenta[b]pyridinyl), or a monocyclic heteroaryl ring fused to a monocyclic heterocycle (e.g., 2,3-dihydrofuro[3,2-b]pyridinyl). The bicyclic heteroaryl is attached to the parent molecular moiety at an aromatic ring atom. Other representative examples of heteroaryl include, but are not limited to, indolyl (e.g., indol-1-yl, indol-2-yl, indol-4-yl), pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl (e.g., pyrazol-4-yl), pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl (e.g., triazol-4-yl), 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl (e.g., thiazol-4-yl), isothiazolyl, thienyl, benzimidazolyl (e.g., benzimidazol-5-yl), benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, benzofuranyl, isobenzofuranyl, furanyl, oxazolyl, isoxazolyl, purinyl, isoindolyl, quinoxalinyl, indazolyl (e.g., indazol-4-yl, indazol-5-yl), quinazolinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, isoquinolinyl, quinolinyl, imidazo[1,2-a]pyridinyl (e.g., imidazo[1,2-a]pyridin-6-yl), naphthyridinyl, pyridoimidazolyl, thiazolo[5,4-b]pyridin-2-yl, and thiazolo[5,4-d]pyrimidin-2-yl.
The term “heterocycle” or “heterocyclic,” as used herein, means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. The term “heterocyclyl” is used herein to refer to a heterocycle when present as a substituent. The monocyclic heterocycle is a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. The three- or four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of O, N, and S. The five-membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The six-membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. The seven- and eight-membered rings contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocyclyls include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, 2-oxo-3-piperidinyl, 2-oxoazepan-3-yl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, oxepanyl, oxocanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to a 6-membered arene, or a monocyclic heterocycle fused to a monocyclic cycloalkane, or a monocyclic heterocycle fused to a monocyclic cycloalkene, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a monocyclic heterocycle fused to a monocyclic heteroarene, or a spiro heterocycle group, or a bridged monocyclic heterocycle ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. The bicyclic heterocyclyl is attached to the parent molecular moiety at a non-aromatic ring atom (e.g., indolin-1-yl). Representative examples of bicyclic heterocyclyls include, but are not limited to, chroman-4-yl, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzothien-2-yl, 1,2,3,4-tetrahydroisoquinolin-2-yl, 2-azaspiro[3.3]heptan-2-yl, 2-oxa-6-azaspiro[3.3]heptan-6-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), azabicyclo[3.1.0]hexanyl (including 3-azabicyclo[3.1.0]hexan-3-yl), 2,3-dihydro-1 H-indol-1-yl, isoindolin-2-yl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, tetrahydroisoquinolinyl, 7-oxabicyclo[2.2.1]heptanyl, hexahydro-2H-cyclopenta[b]furanyl, 2-oxaspiro[3.3]heptanyl, 3-oxaspiro[5.5]undecanyl, 6-oxaspiro[2.5]octan-1-yl, and 3-oxabicyclo[3.1.0]hexan-6-yl. Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a 6-membered arene, or a bicyclic heterocycle fused to a monocyclic cycloalkane, or a bicyclic heterocycle fused to a monocyclic cycloalkene, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.13,7]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.13,7]decane). The monocyclic, bicyclic, and tricyclic heterocyclyls are connected to the parent molecular moiety at a non-aromatic ring atom.
The term “hydroxyl” or “hydroxy,” as used herein, means an —OH group.
The term “hydroxyalkyl,” as used herein, means at least one —OH group, is appended to the parent molecular moiety through an alkylene group, as defined herein.
The term “hydroxyfluoroalkyl,” as used herein, means at least one —OH group, is appended to the parent molecular moiety through a fluoroalkyl group, as defined herein.
Terms such as “alkyl,” “cycloalkyl,” “alkylene,” etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance (e.g., “C1-4alkyl,” “C3-6cycloalkyl,” “C1-4alkylene”). These designations are used as generally understood by those skilled in the art. For example, the representation “C” followed by a subscripted number indicates the number of carbon atoms present in the group that follows. Thus, “C3alkyl” is an alkyl group with three carbon atoms (i.e., n-propyl, isopropyl). Where a range is given, as in “C1-4,” the members of the group that follows may have any number of carbon atoms falling within the recited range. A “C1-4alkyl,” for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).
The term “substituted” refers to a group that may be further substituted with one or more non-hydrogen substituent groups. Substituent groups include, but are not limited to, halogen, ═O (oxo), ═S (thioxo), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, and acyl.
For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
Dynamic-covalent chemistry is valuable for hydrogel crosslinking, leveraging equilibrium-governed reversible interactions to realize viscoelastic materials with dynamic properties and self-healing character. The bonding between aryl boronates and diols is one particular dynamic-covalent chemistry of interest. The extent of network crosslinking using this motif can be subject to competition from ambient diols such as glucose, offering a strategy for glucose-directed release of insulin to control diabetes. However, the majority of work to—date uses phenylboronic acids (PBAs) that suffer from low-affinity glucose binding relative to their binding of the synthetic diols used in crosslinking, limiting material responsiveness. Moreover, PBA chemistry is also subject to competition from binding non-glucose analytes such as fructose and lactate, limiting the specificity of sensing. Here, dynamic-covalent hydrogels are prepared that, for the first time, leverage a diboronate motif with enhanced glucose binding and improved glucose specificity.
This crosslinking yields hydrogels that, when compared to traditional PBA crosslinking, offer more glucose-responsive insulin release that is minimally impacted by non-glucose analytes.
A dynamic-covalent crosslinking chemistry is disclosed that leverages high-affinity and glucose-specific interactions from di-phenylboronic acid (DiPBA) motifs (
In one aspect, the invention provides hydrogels. Exemplary hydrogels of the present invention comprise a diboronate compound (e.g., a diboronate compound of formula (I)) and a diol compound (e.g., a diol compound of formula (II)). In various instances, the diboronate compound and the diol compound form crosslinks by dynamic-covalent bonds. In various instances, the molar ratio of the diboronate compound to the diol compound is 1:1.
Diboronate Compounds of Formula (1) In one aspect, the invention provides diboronate compounds of formula (I):
wherein:
are as defined herein.
In various instances,
wherein:
In various instances,
is a linking moiety.
In various instances,
is a first moiety comprising a hydrophilic polymer, a hyperbranched macromolecule, or a combination thereof.
In some instances,
may be
In some instances
may be
In various instances, X⊖ may be Br−, Cl−, NO3−, H2PO4−, H2PO3−, HSO4−, HSO3−, H3C—SO3−, HCO3, HCO2−, H3C—CO2−, HC2O4−, or TsO−.
In some instances, X⊖ may be Br− or Cl−.
In various instances,
may be
In some instances,
may be
In various instances,
may have an equilibrium constant (Keq) for binding glucose of at least 350 M−1.
In some instances,
may have an equilibrium constant (Keq) for binding glucose of at least 1000 M−1.
In some instances,
may have an equilibrium constant (Keq) for binding glucose that is at least 20 times greater than equilibrium constant of
for binding lactate.
In various instances,
may comprise:
In some instances,
may comprise:
In some instances,
may comprise:
In some instances,
may comprise a branched polymer. The branched polymer may comprise a polyalkylene glycol. The polyalkylene glycol may comprise polyethylene glycol. The branched polymer may be a four-armed polymer.
In some instances, the diboronate compound of formula (I) may comprise:
wherein n is 2 to 250.
In some instances,
may comprise a dendrimer. The dendrimer may be a polyamidoamine dendrimer.
In some instances,
may comprise a linear polymer. The linear polymer may comprise a polysaccharide. The polysaccharide may comprise hyaluronic acid.
In another aspect, the invention provides diols of formula (II):
wherein
is as defined
In various instances
is a second moiety comprising a hydrophilic polymer, a hyperbranched macromolecule, or a combination thereof.
In various instances,
comprises a branched polymer. The branched polymer may comprise polyethylene glycol.
In some instances, the diol compound of formula (II) may comprise:
wherein n is 2 to 250.
In some instances,
may comprise a dendrimer. The dendrimer may be a polyamidoamine dendrimer.
In some instances,
may comprise a linear polymer.
Compound names can be assigned by using Struct=Name naming algorithm as part of CHEMDRAW® ULTRA.
The compound may exist as a stereoisomer wherein asymmetric or chiral centers are present. The stereoisomer is “R” or “S” depending on the configuration of substituents around the chiral carbon atom. The terms “R” and “S” used herein are configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The disclosure contemplates various stereoisomers and mixtures thereof and these are specifically included within the scope of this invention. Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers. Individual stereoisomers of the compounds may be prepared synthetically from commercially available starting materials, which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by methods of resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and optional liberation of the optically pure product from the auxiliary as described in Furniss, Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical Organic Chemistry”, 5th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns or (3) fractional recrystallization methods.
It should be understood that the compound may possess tautomeric forms, as well as geometric isomers, and that these also constitute an aspect of the invention.
In the compounds of formula (I), formula (II), and any subformulas, any “hydrogen” or “H,” whether explicitly recited or implicit in the structure, encompasses hydrogen isotopes 1H (protium) and 2H (deuterium).
The present disclosure also includes isotopically-labeled compounds (e.g., deuterium labeled), where an atom in the isotopically-labeled compound is specified as a particular isotope of the atom. Examples of isotopes suitable for inclusion in the compounds of the invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to 2H, 3H, 13C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F, and 36Cl, respectively.
Isotopically-enriched forms of compounds of formula (I), or any subformulas, may generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using an appropriate isotopically-enriched reagent in place of a non-isotopically-enriched reagent. The extent of isotopic enrichment can be characterized as a percent incorporation of a particular isotope at an isotopically-labeled atom (e.g., % deuterium incorporation at a deuterium label).
The disclosed compounds may exist as pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to salts or zwitterions of the compounds which are water or oil-soluble or dispersible, suitable for treatment of disorders without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit/risk ratio and effective for their intended use. The salts may be prepared during the final isolation and purification of the compounds or separately by reacting an amino group of the compounds with a suitable acid. For example, a compound may be dissolved in a suitable solvent, such as but not limited to methanol and water and treated with at least one equivalent of an acid, like hydrochloric acid. The resulting salt may precipitate out and be isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide a salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, thrichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric and the like. The amino groups of the compounds may also be quaternized with alkyl chlorides, bromides, and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like.
Basic addition salts may be prepared during the final isolation and purification of the disclosed compounds by reaction of a carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine and N,N′-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.
Optimum reaction conditions and reaction times for each individual step can vary depending on the particular reactants employed and substituents present in the reactants used.
Specific procedures are provided in the Examples section. Reactions can be worked up in the conventional manner, e.g., by eliminating the solvent from the residue and further purified according to methodologies generally known in the art such as, but not limited to, crystallization, distillation, extraction, trituration, and chromatography. Unless otherwise described, the starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature.
Starting materials, if not commercially available, can be prepared by procedures selected from standard organic chemical techniques, techniques that are analogous to the synthesis of known, structurally similar compounds, or techniques that are analogous to the above-described schemes or the procedures described in the synthetic examples section.
Routine experimentations, including appropriate manipulation of the reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that cannot be compatible with the reaction conditions, and deprotection at a suitable point in the reaction sequence of the method are included in the scope of the invention. Suitable protecting groups and the methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which can be found in PGM Wuts and TW Greene, in Greene's book titled Protective Groups in Organic Synthesis (4th ed.), John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety. Synthesis of the compounds of the invention can be accomplished by methods analogous to those described in the synthetic schemes described hereinabove and in specific examples.
When an optically active form of a disclosed compound is required, it can be obtained by carrying out one of the procedures described herein using an optically active starting material (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a mixture of the stereoisomers of the compound or intermediates using a standard procedure (such as chromatographic separation, recrystallization, or enzymatic resolution).
Similarly, when a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the above procedures using a pure geometric isomer as a starting material, or by resolution of a mixture of the geometric isomers of the compound or intermediates using a standard procedure such as chromatographic separation.
It can be appreciated that the synthetic schemes and specific examples as described are illustrative and are not to be read as limiting the scope of the invention as it is defined in the appended claims. All alternatives, modifications, and equivalents of the synthetic methods and specific examples are included within the scope of the claims.
Pharmaceutical compositions of the present invention comprise insulin encapsulated within the hydrogels disclosed herein (i.e., “hydrogel-encapsulated insulin”).
Hydrogel-encapsulated insulin may be incorporated into pharmaceutical compositions suitable for administration to a subject (such as a patient, which may be a human or non-human). The pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of the active agent (insulin). A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A “therapeutically effective amount” is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
The pharmaceutical compositions may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
Thus, the hydrogels and their physiologically acceptable salts and solvates may be formulated for administration by, for example, solid dosing, eyedrop, in a topical oil-based formulation, injection, inhalation (either through the mouth or the nose), implants, or oral, buccal, parenteral, or rectal administration. Techniques and formulations may generally be found in “Remington's Pharmaceutical Sciences,” (Meade Publishing Co., Easton, Pa.). Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage.
The route by which the hydrogel-encapsulated insulin is administered, and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).
Carriers for systemic administration typically include at least one of diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, and others. All carriers are optional in the compositions. Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. The amount of diluent(s) in a systemic or topical composition is typically about 50 to about 90%.
Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10%.
Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in a systemic composition is typically about 5 to about 50%.
Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmelose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in a systemic or topical composition is typically about 0.1 to about 10%. Suitable colorants include a colorant such as an FD&C dye. When used, the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1%. Suitable flavors include menthol, peppermint, and fruit flavors. The amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%.
Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s) in a systemic or topical composition is typically about 0.001 to about 1%. Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5%. Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%. Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.
Suitable solvents include water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%. Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, PA) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%. Suitable surfactants include lecithin, Polysorbate 80, and sodium lauryl sulfate, and the TWEENS from Atlas Powder Company of Wilmington, Delaware. Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 1992, pp. 587-592; Remington's Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337; and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5%.
Although the amounts of components in the systemic compositions may vary depending on the type of systemic composition prepared, in general, systemic compositions include 0.01% to 50% of actives and 50% to 99.99% of one or more carriers. Compositions for parenteral administration typically include 0.1% to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent.
Compositions for oral administration can have various dosage forms. For example, solid forms include tablets, capsules, granules, and bulk powders. These oral dosage forms include a safe and effective amount, usually at least about 5%, and more particularly from about 25% to about 50% of actives. The oral dosage compositions include about 50% to about 95% of carriers, and more particularly, from about 50% to about 75%.
Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically include an active component, and a carrier comprising ingredients selected from diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, glidants, and combinations thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and croscarmelose. Specific lubricants include magnesium stearate, stearic acid, and talc. Specific colorants are the FD&C dyes, which can be added for appearance. Chewable tablets preferably contain sweeteners such as aspartame and saccharin, or flavors such as menthol, peppermint, fruit flavors, or a combination thereof.
Capsules (including implants, time release and sustained release formulations) typically include an active and a carrier including one or more diluents disclosed above in a capsule comprising gelatin. Granules typically comprise an active, and preferably glidants such as silicon dioxide to improve flow characteristics. Implants can be of the biodegradable or the non-biodegradable type.
The selection of ingredients in the carrier for oral compositions depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this invention. Solid compositions may be coated by conventional methods, typically with pH or time-dependent coatings, such that the hydrogel-encapsulated insulin is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action. The coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT coatings (available from Rohm & Haas G.M.B.H. of Darmstadt, Germany), waxes and shellac.
Compositions for oral administration can have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non-effervescent granules, suspensions reconstituted from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like. Liquid orally administered compositions typically include the hydrogel-encapsulated insulin and a carrier, namely, a carrier selected from diluents, colorants, flavors, sweeteners, preservatives, solvents, suspending agents, and surfactants. Peroral liquid compositions preferably include one or more ingredients selected from colorants, flavors, and sweeteners.
Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically include one or more of soluble filler substances such as diluents including sucrose, sorbitol, and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Such compositions may further include lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.
The disclosed compositions can be topically administered. Topical compositions that can be applied locally to the skin may be in any form including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers, sprays, skin patches, and the like. Topical compositions include: a disclosed hydrogel and a carrier. The carrier of the topical composition preferably aids penetration of the hydrogels into the skin. The carrier may further include one or more optional components.
The amount of the carrier employed in conjunction with the hydrogel-encapsulated insulin is sufficient to provide a practical quantity of composition for administration per unit dose of the medicament. Techniques and compositions for making dosage forms useful in the methods of this invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd ed., (1976).
A carrier may include a single ingredient or a combination of two or more ingredients. In the topical compositions, the carrier includes a topical carrier. Suitable topical carriers include one or more ingredients selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, combinations thereof, and the like. More particularly, carriers for skin applications include propylene glycol, dimethyl isosorbide, and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.
The carrier of a topical composition may further include one or more ingredients selected from emollients, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, all of which are optional.
Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and combinations thereof. Specific emollients for skin include stearyl alcohol and polydimethylsiloxane. The amount of emollient(s) in a skin-based topical composition is typically about 5% to about 95%.
Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof. The amount of propellant(s) in a topical composition is typically about 0% to about 95%.
Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinations thereof.
Specific solvents include ethyl alcohol and homotopic alcohols. The amount of solvent(s) in a topical composition is typically about 0% to about 95%.
Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. Specific humectants include glycerin. The amount of humectant(s) in a topical composition is typically 0% to 95%. The amount of thickener(s) in a topical composition is typically about 0% to about 95%. Suitable powders include beta-cyclodextrins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetra alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically-modified Montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof. The amount of powder(s) in a topical composition is typically 0% to 95%. The amount of fragrance in a topical composition is typically about 0% to about 0.5%, particularly, about 0.001% to about 0.1%. Suitable pH adjusting additives include HCl or NaOH in amounts sufficient to adjust the pH of a topical pharmaceutical composition.
The disclosed insulin-encapsulated hydrogels may be used to deliver insulin to a subject. The methods of treatment may comprise administering to a subject in need of insulin a pharmaceutical composition comprising insulin encapsulated within a hydrogel, as described herein. In various instances, subject in need thereof may have diabetes (e.g., Type 1 diabetes).
In various instances, insulin may be administered to the subject at 0.05-10 international units (IU)/kg. In various instances, insulin may be administered to the subject at 0.10-10 IU/kg; 1-10 IU/kg; 1-9 IU/kg; 2-8 IU/kg; 2-7 IU/kg; 3-7 IU/kg; 3-6 IU/kg; or 4-6 IU/kg. In various instances, insulin may be administered to the subject at no greater than 10 IU/kg; no greater than 9 IU/kg; no greater than 8 IU/kg; no greater than 7 IU/kg; no greater than 6 IU/kg; no greater than 5 IU/kg; no greater than 4 IU/kg; no greater than 3 IU/kg; no greater than 2 IU/kg; no greater than 1 IU/kg; no greater than 0.50 IU/kg; no greater than 0.10 IU/kg; or no greater than 0.05 IU/kg. In various instances, insulin may be administered to the subject at no less than 0.05 IU/kg; no less than 0.10 IU/kg; no less than 0.50 IU/kg; no less than 1 IU/kg; no less than 2 IU/kg; no less than 3 IU/kg; no less than 4 IU/kg; no less than 5 IU/kg; no less than 6 IU/kg; no less than 7 IU/kg; no less than 8 IU/kg; no less than 9 IU/kg; or no less than 10 IU/kg.
In various instances, following administration of the pharmaceutical composition, the subject has blood glucose levels of about 60-110 mg/dL. In various instances, following administration of the pharmaceutical composition, the subject has blood glucose levels of about 65-105 mg/dL; about 70-100 mg/dL; about 75-95 mg/dL; or about 80-90 mg/dL. In various instances, following administration of the pharmaceutical composition, the subject has blood glucose levels of no greater than about 110 mg/dL; no greater than about 100 mg/dL; no greater than about 90 mg/dL; no greater than about 80 mg/dL; no greater than about 70 mg/dL; or no greater than about 60 mg/dL.
It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.
Various embodiments and aspects of the inventions described herein are summarized by the following clauses:
wherein:
wherein:
is a linking moiety; and
is a first moiety comprising a hydrophilic polymer, a hyperbranched macromolecule, or a combination thereof; and
wherein:
is a second moiety comprising a hydrophilic polymer, a hyperbranched macromolecule, or a combination thereof;
Clause 7. The hydrogel of clause 1 or 6, wherein
has an equilibrium constant (Keq) for binding glucose of at least 350 M−1.
has an equilibrium constant (Keq) for binding glucose of at least 1000 M−1.
has an equilibrium constant (Keq) for binding glucose that is at least 20 times greater than equilibrium constant of
for binding lactate.
comprises:
comprises:
comprises:
comprises a branched polymer.
wherein n is 2 to 250.
comprises a dendrimer.
comprises a linear polymer.
comprises a branched polymer.
wherein n is 2 to 250.
comprises a dendrimer.
comprises a linear polymer.
wherein:
wherein:
is a linking moiety; and
is a first moiety comprising a hydrophilic polymer, a hyperbranched macromolecule, or a combination thereof; and
wherein:
is a second moiety comprising a hydrophilic polymer, a hyperbranched macromolecule, or a combination thereof;
All purchased chemicals were used directly as received unless otherwise stated. All reactions were performed under an inert atmosphere with dry solvents in anhydrous conditions.
Dry tetrahydrofuran (THF), chloroform, N,N′-dimethylformamide (DMF), and methylene chloride (DCM) were purchased from VWR in sure-seal bottles. N-bromosuccinimide (NBS); Oxalyl chloride, 2,5-dimethylbenzoic acid, N-(2-hydroxyethyl)maleimide, 3-pyridylboronic acid, 2-(bromomethyl)benzoic acid, 3-(bromomethyl)benzoic acid, hydroxybenzotriazole monohydrate (HOBt) were purchased from VWR. 4-Arm-PEG-NH2 (4aPEG-NH2) was purchased from Laysan Bio, Inc. 4-Arm-PEG-SH (4aPEG-SH) was purchased from Biopharma PEG. 4-carboxy-3-fluorophenylboronic acid (FPBA) was purchased from Synthonix. Tetramethyluroniumhexaflurophosphate (HBTU) was purchased from Chem-Impex. Benzoyl peroxide was purchased from Alfa Aesar. Phosphate Buffered Saline (PBS) was purchased from VWR. Regenerated cellulose dialysis tubing (molecular weight cutoff (MWCO) of 3.5 kDa) was purchased from Spectrum Labs. D-(−)-fructose (C6H12O6) was purchased from Sigma-Aldrich. D-Glucose (Dextrose) Anhydrous (C6H12O6) was purchased from VWR. Fluorescein isothiocyanate isomer I (90%, pure) was purchased from Sigma-Aldrich. Recombinant Human Insulin AOF (Lot No. 1987626) from Saccharomyces cerevisiae was purchased from ThermoFisher. Sodium L-lactate (C3H5NaO3) was purchased from Sigma-Aldrich. Streptozotocin (STZ, batch 0610596-17) was purchased from Cayman Chemical Company.
1H NMR Spectroscopy
Spectra were recorded on Bruker 400 MHz instrument and calibrated using residue undeuterated solvent ((CDCl3: δH=7.26 ppm; D2O: δH=4.79 ppm; d4-MeOD: δH=3.34 ppm).
A mixture of 2,5-dimethylbenzoic acid (3 g, 20 mmol), NBS (8.9 g, 50 mmol), and benzoyl peroxide (0.24 g, 1 mmol) were charged in an oven-dried 250 mL round-bottom flask and suspended with 90 mL chloroform. The mixture was heated to reflux for 4 h until a clear solution was observed. Then the mixture was cooled to ambient temperature and solvent was removed under reduced pressure. The residue was treated with 90 mL Et2O, and the undissolved solids were filtered. The filtrate was then transferred to a separation funnel and washed with 90 mL water. The aqueous layer was then washed with 30 mL Et2O. The organic layers were combined and washed with an aqueous solution of saturated NaCl (90 mL). The organic layer was then dried with Na2SO4, filtered, and the solvent was removed under reduced pressure. The residue was then recrystallized with hexaness and ethyl acetate at −20° C. After two days, the mixture was filtered, and the undissolved solids were then recrystallized with DCM and acetone at −20° C. The product was then filtered as a white solid with yields of 2 g (6 mmol, 30%). 1H NMR (400 MHz, Chloroform-d) δ 8.10 (d, J=2.0 Hz, 1H), 7.56 (dd, J=7.9, 2.1 Hz, 1H), 7.47 (d, J=7.9 Hz, 1H), 4.96 (d, J=2.2 Hz, 2H), 4.47 (s, 2H).
An oven-dried 100 mL round-bottom flask was charged with compound 1 (1 g, 3.25 mmol), DMF (20 μL), and dissolved with 15 mL mixed solvent of THF:DCM (1:4). The mixture was then stirred at 0° C. for 10 min before the dropwise addition of oxalyl chloride (1.3 mL, 16.2 mmol).
After the addition, the mixture was allowed to stir at ambient temperature for 90 min and the solvent was then removed under reduced pressure. The residue was diluted with 20 mL DCM, transferred to an addition funnel, and added to a mixture of N-(2-hydroxyethyl)maleimide (0.56 g, 4 mmol), triethylamine (0.58 mL, 4 mmol) and DCM (20 mL) at 0° C. After addition, the mixture was stirred at 0° C. for another 5 min before warming to ambient temperature. After 2 h, the mixture was transferred to a separation funnel and washed with 1 N HCl (25 mL). The aqueous layer was washed with DCM (20 mL). The organic layer was then combined and washed with water (25 mL) and an aqueous solution of saturated NaCl (25 mL) and dried over Na2SO4. The mixture was filtered, and the solvent was removed under reduced pressure. Then the residue was loaded on the column eluting with hexaness and ethyl acetate to obtain the product as white solids with yields of 0.86 g (2 mmol, 62%). 1H NMR (400 MHz, Chloroform-d) δ 7.96 (s, 1H), 7.57-7.41 (m, 2H), 6.74 (d, J=0.8 Hz, 2H), 5.00 (d, J=2.9 Hz, 1H), 4.91 (s, 1H), 4.55-4.42 (m, 4H), 3.98 (dt, J=5.3, 2.6 Hz, 2H).
A mixture of compound 2 (0.86 g, 2 mmol) and 3-pyridylboronic acid (0.52 g, 4.2 mmol) was charged to a 100 mL oven-dried round-bottom flask and diluted with 40 mL DMF. The mixture was stirred at 70° C. for 24 hours. Then the mixture was filtered, and the filtered solids was washed with THF and filtered to recover a white solid as the product with the yield of 1 g (1.5 mmol, 75%). 1H NMR (400 MHz, Methanol-d4) δ 8.95 (d, J=6.2 Hz, 1H), 8.91 (s, 1H), 8.86 (s, 1H), 8.79 (d, J=8.6 Hz, 3H), 8.67 (dd, J=16.1, 7.7 Hz, 2H), 8.21 (s, 1H), 8.05 (q, J=6.3 Hz, 2H), 8.00-7.93 (m, 1H), 7.86 (d, J=7.9 Hz, 1H), 7.57 (d, J=7.9 Hz, 1H), 6.84 (s, 2H), 6.17 (s, 2H), 5.94 (s, 2H), 4.47-4.33 (m, 2H), 3.89 (t, J=4.9 Hz, 2H).
An oven-dried 100 mL round bottom flask was charged with 2,5-dimethylbenzoic acid (1.5 g, 10 mmol), DMF (20 μL), and dissolved with 15 mL mixed solvent of THF:DCM (1:4). The mixture was then stirred at 0° C. for 10 min before the dropwise addition of oxalyl chloride (4 mL, 50 mmol). After the addition, the mixture was allowed to stir at ambient temperature for 90 min and the solvent was then removed under reduced pressure. The residue was treated with MeOH (40 mL) and triethylamine (1.7 mL, 12 mmol) and stirred at 0° C. for another 5 min before warming to ambient temperature. After 16 h, the solvent was removed under reduced pressure and the residue was diluted with 40 mL DCM. The mixture was transferred to a separation funnel and washed with 1 N HCl (25 mL). The aqueous layer was washed with DCM (20 mL). The organic layer was then combined and washed with water (25 mL) and an aqueous solution of saturated NaCl (25 mL) and dried over Na2SO4. The mixture was then filtered, and the solvent was removed under reduced pressure. Then the residue was loaded on the column eluting with hexaness and ethyl acetate to obtain the product as a transparent oil with yields of 1.6 g (10 mmol, 100%). 1H NMR (400 MHz, Chloroform-d) δ 7.72 (d, J=1.9 Hz, 1H), 7.21 (dd, J=7.7, 1.9 Hz, 1H), 7.13 (d, J=7.7 Hz, 1H), 3.88 (s, 3H), 2.55 (s, 3H), 2.34 (s, 3H).
A mixture of compound 4 (1.6 g, 10 mmol), NBS (4.45 g, 20 mmol), and benzoyl peroxide (0.12 g, 0.5 mmol) was charged into an oven-dried 250 mL round-bottom flask and suspended with 45 mL chloroform. The mixture was heated to reflux for 4 h until a clear solution was observed. Then the mixture was cooled to ambient temperature and the solvent was removed under reduced pressure. The residue was treated with 90 mL Et2O, and the undissolved solids were filtered. The filtrate was then transferred to a separation funnel and washed with the aqueous solution of saturated NaHCO3 (45 mL). The aqueous layer was then washed with 15 mL Et2O. The organic layers were combined and washed with an aqueous solution of saturated NaCl (45 mL). The organic layer was then dried with Na2SO4, filtered, and the solvent was removed under reduced pressure. The residue was then loaded on the column, eluting with hexaness and ethyl acetate to obtain the target product as white solids with a yield of 1.48 g (4.6 mmol, 46%). 1H NMR (400 MHz, Chloroform-d) δ 8.00 (d, J=2.0 Hz, 1H), 7.53 (dd, J=7.9, 2.0 Hz, 1H), 7.45 (d, J=7.9 Hz, 1H), 4.94 (s, 2H), 4.48 (s, 2H), 3.96 (s, 3H).
A mixture of compound 5 (0.24 g, 0.74 mmol) and 3-pyridylboronic acid (0.19 g, 1.5 mmol) was charged to a 50 mL oven-dried round-bottom flask and diluted with 10 mL DMF. The mixture was then stirred at 70° C. for 24 h. Then the mixture was filtered and the filtered solid was washed with THF to recover a white solid as the product with the yield of 0.3 g (0.53 mmol, 70%). 1H NMR (400 MHz, Methanol-d4) δ 8.96 (d, J=8.7 Hz, 2H), 8.93-8.78 (m, 3H), 8.70 (t, J=7.8 Hz, 2H), 8.29 (d, J=2.0 Hz, 1H), 8.01 (dt, J=20.8, 7.0 Hz, 2H), 7.84 (dd, J=8.0, 2.1 Hz, 1H), 7.61 (d, J=7.9 Hz, 1H), 6.18 (d, J=3.2 Hz, 2H), 5.96 (d, J=4.6 Hz, 2H), 3.89 (s, 3H).
A mixture of 1,3-bis(bromomethyl)benzene (0.5 g, 1.9 mmol) and 3-pyridylboronic acid (0.48 g, 3.9 mmol) was charged to a 50 mL oven-dried round-bottom flask and diluted with 20 mL DMF. The mixture was stirred at 70° C. for 24 hours. Then the mixture was filtered, and the filtered solids was washed with Et2O and filtered to recover a white solid as the product. 1H NMR (400 MHz, Methanol-d4) δ 8.90 (t, J=5.4 Hz, 4H), 8.78 (d, J=5.8 Hz, 2H), 8.71 (d, J=7.6 Hz, 2H), 8.00 (q, J=8.8, 8.0 Hz, 3H), 7.67-7.63 (m, 1H), 7.57 (d, J=1.6 Hz, 3H), 5.85 (s, 4H).
In a 50 mL round bottom flask, 2,5-dimethylaniline (1.21 g, 0.01 mol) was charged and dissolved with 10 mL 6 M HCl. The mixture was kept stirred at 0° C. for 5 min before the dropwise addition of the NaNO3 solution in water (1 g, 0.012 mol). After the addition, the mixture was kept at the same temperature and stirred for 30 mins. Then the reaction was treated with NaN3 (0.04 mmol in 50 mL water) solution dropwise and was kept stirring for another hour. The mixture was then washed with ethyl acetate three times and the organic layer was combined, washed with water, and dried over Na2SO4. The mixture was filtered, concentrated, and loaded on the column eluting with hexaness to get the final product as light yellow oil with yield of 33% (0.49 g, 3.33 mmol). 1H NMR (400 MHz, DMSO-d6) δ 7.11 (d, J=7.6 Hz, 1H), 7.04 (s, 1H), 6.92-6.88 (m, 1H), 2.30 (s, 3H), 2.11 (s, 3H).
A mixture of 2-azido-1,4-dimethylbenzene (0.49 g, 3.33 mmol), NBS (1.27 g, 7.16 mmol), and azobisisobutyronitrile (AIBN, 0.1 g, 0.62 mmol) were charged in an oven-dried 250 mL round-bottom flask and suspended with 80 mL chloroform. The mixture was heated to reflux for 12 h and the solvent was removed. The residue was suspended in hexanes and the resulting solids were filtered. The hexanes solution was washed with sat. NaHCO3, water, sat. NaCl, and dried over Na2SO4. The mixture was filtered, concentrated, and loaded on the column eluting with hexanes to provide the target product as a white solid with a 31% yield (0.18 g, 0.59 mmol). 1H NMR (400 MHz, Chloroform-d) δ 7.35 (d, J=7.8 Hz, 1H), 7.18 (s, 1H), 7.14 (d, J=8.0 Hz, 1H), 4.46 (s, 2H), 4.45 (s, 2H).
A mixture of 2-azido-1,4-bis(bromomethyl)benzene (0.18 g, 0.59 mmol) and 3-pyridylboronic acid (0.15 g, 1.2 mmol) was charged to a 50 mL oven-dried round-bottom flask and diluted with 20 mL DMF. The mixture was stirred at 70° C. for 24 hours. Then the mixture was filtered. The filtered solids were then washed with THF and filtered to recover the product as a white solid. 1H NMR (400 MHz, Methanol-d4) δ 8.89 (td, J=12.6, 5.5 Hz, 4H), 8.81-8.74 (m, 2H), 8.68 (t, J=8.9 Hz, 2H), 8.05-7.94 (m, 3H), 7.69 (d, J=7.9 Hz, 1H), 7.57 (d, J=1.7 Hz, 1H), 7.40-7.33 (m, 1H), 5.87 (s, 2H), 5.77 (s, 2H).
A mixture of 1,4-phenylenedimethanamine (0.28 g, 2.06 mmol) and (2-formylphenyl)boronic acid (0.64 g, 4.22 mmol) was charged to a 100 mL oven-dried round-bottom flask and diluted with 20 mL MeOH. The mixture was stirred overnight. Then the mixture was treated with NaBH4 (0.12 g, 3.15 mmol) and stirred for another hour. The precipitate was filtered, washed with deionized (DI) water, and recovered as the target product. 1H NMR (400 MHz, Deuterium Oxide/Deuterium Chloride) 6 7.73 (dd, J=7.3, 1.7 Hz, 2H), 7.52-7.35 (m, 10H), 4.31 (d, J=8.2 Hz, 8H).
An oven-dried 100 mL round-bottom flask was charged with 3-(bromomethyl)benzoic acid (1 g, 4.6 mmol) and DMF (20 μL), and dissolved with 20 mL THF. The mixture was then stirred at 0° C. for 10 min before the dropwise addition of oxalyl chloride (1.9 mL, 23 mmol). After the addition, the mixture was stirred at ambient temperature for 90 min and the solvent was then removed under reduced pressure. The residue was then diluted with 20 mL DCM and transferred to an addition funnel and added to a mixture of N-(2-hydroxyethyl)maleimide (0.78 g, 5.5 mmol), triethylamine (0.79 mL, 5.5 mmol) and DCM (20 mL) at 0° C. After the addition, the mixture was stirred at 0° C. for another 5 min before being warmed to ambient temperature. After 2 h, the mixture was transferred to a separation funnel and washed with 1 N HCl (25 mL). The aqueous layer was washed with DCM (20 mL). The organic layer was then combined and washed with water (25 mL) and an aqueous solution of saturated NaCl (25 mL) and dried over Na2SO4. The mixture was filtered, and the solvent was removed under reduced pressure. Then the residue was loaded to a silica column and eluted with hexanes and ethyl acetate to obtain the product as a white solid with yield of 1 g (2.9 mmol, 64%). 1H NMR (400 MHz, Chloroform-d) δ 8.02 (t, J=1.8 Hz, 1H, Ar H), 7.93 (ddt, J=10.6, 7.7, 1.4 Hz, 1H, Ar H), 7.60 (dq, J=7.7, 1.6 Hz, 1H, Ar H), 7.43 (q, J=7.7 Hz, 1H, Ar H), 6.74 (d, J=0.7 Hz, 2H, CH═CH), 4.62 (s, 1H, Ar—CH2—), 4.52 (s, 1H, Ar—CH2—), 4.49-4.42 (m, 2H, —O—CH2—), 4.00-3.92 (m, 2H, —CH2—N—).
A mixture of compound 7 (1.04 g, 3.08 mmol) and 3-pyridylboronic acid (0.79 g, 6.46 mmol) was charged to a 100 mL oven-dried round-bottom flask and diluted with 40 mL DMF. The mixture was stirred at 70° C. for 24 h. Then the mixture was concentrated to a small volume and treated with 80 mL THF. The undissolved solids were filtered and washed with THF and recovered as product with a yield of 1 g (2.3 mmol, 75%). 1H NMR (400 MHz, D2O) δ 8.87 (s, 1H), 8.79 (s, 1H), 8.77-8.69 (m, 2H), 8.66 (d, J=7.3 Hz, 1H), 8.02-7.89 (m, 3H), 7.69 (d, J=7.8 Hz, 1H), 7.56 (t, J=7.8 Hz, 1H), 6.80 (s, 2H), 5.82 (s, 2H), 4.46 (t, J=5.0 Hz, 2H), 3.89 (t, J=5.0 Hz, 2H).
An oven-dried 100 mL round bottom flask was charged with (0.5 g, 2.32 mmol) and DMF (20 μL) and dissolved with 15 mL mixed solvent of THF:DCM (1:4). The mixture was then stirred at 0° C. for 10 min before dropwise addition of oxalyl chloride (5 eq). After the addition, the mixture was allowed to stir at ambient temperature for 90 min and the solvent was then removed under reduced pressure. The residue was treated with MeOH (80 eq) and triethylamine (1.2 eq) and stirred at 0° C. for another 5 min before warming to ambient temperature. After 16 h, the solvent was removed under reduced pressure and the residue was diluted with 40 mL DCM. The mixture was then transferred to a separation funnel and washed with 1 N HCl (25 mL). The aqueous layer was washed with DCM (20 mL). The organic layer was then combined and washed with water (25 mL) and an aqueous solution of saturated NaCl (25 mL) and dried over Na2SO4. The mixture was then filtered, and the solvent was removed under reduced pressure and used directly for the next step. Methyl 3-(bromomethyl)benzoate was then mixed with 3-pyridylboronic acid (0.3 g, 2.44 mmol) in a 100 mL oven-dried round-bottom flask diluted with 20 mL DMF. The mixture was then stirred at 70° C. for 24 h. Then the mixture was concentrated to a small volume and treated with 40 mL THF. The undissolved solid was filtered and washed with THF and recovered as the product with a yield of 0.56 g (1.6 mmol, 70%). 1H NMR (400 MHz, Methanol-d4) δ 8.96 (s, 1H), 8.93 (dd, J=6.1, 1.6 Hz, 1H), 8.88 (s, 1H), 8.79 (d, J=5.8 Hz, 1H), 8.74 (d, J=7.7 Hz, 1H), 8.69 (d, J=7.6 Hz, 1H), 8.16-8.09 (m, 2H), 8.01 (ddd, J=10.3, 7.7, 6.1 Hz, 2H), 7.76 (dt, J=7.9, 1.4 Hz, 1H), 7.62 (t, J=7.7 Hz, 1H), 5.91 (s, 2H), 3.94 (s, 3H). 1H NMR (400 MHz, Methanol-d4) δ 8.96-8.86 (m, 2H), 8.77 (dd, J=19.3, 6.8 Hz, 1H), 8.69 (d, J=7.6 Hz, 1H), 8.16-8.09 (m, 2H), 8.01 (ddd, J=10.3, 7.7, 6.1 Hz, 2H), 7.76 (dt, J=7.9, 1.4 Hz, 1H), 7.62 (t, J=7.7 Hz, 1H), 5.91 (s, 2H), 3.94 (s, 3H).
Benzoyl amine (1.02 mL, 9.3 mmol) and D-gluconolactone (1.51 g, 8.8 mmol) were charged to a 50 mL oven-dried round-bottom flask and diluted with 10 mL MeOH. The mixture was then heated to reflux and stirred for 3 h. The solvent was removed under reduced pressure and the residue was recrystallized with EtOH to obtain the target product as a white solid with the yield of 2 g (7 mmol, 80%). 1H NMR (400 MHz, D2O) δ 7.32-7.19 (m, 5H), 4.34 (d, J=1.7 Hz, 2H), 4.24 (d, J=3.7 Hz, 1H), 3.98 (t, J=3.2 Hz, 1H), 3.68 (dd, J=11.5, 2.3 Hz, 1H), 3.62 (dd, J=5.6, 2.5 Hz, 2H), 3.55-3.47 (m, 1H).
In a 50 mL oven-dried round-bottom flask, compound 3 (162 mg, 0.24 mmol) was diluted with 10 mL DI water and stirred at 0° C. A mixture of 4aPEG-thiol (0.4 g, 0.04 mmol) and DI water (10 mL) was prepared in an addition funnel and added dropwise to the solution of compound 3.
After the addition, the mixture was warmed to ambient temperature and stirred for 6 h. The mixture was then dialyzed against DI water (MWCO of 3,500) for 8 h, and then lyophilized. 1H NMR (500 MHz, D2O) δ 8.65 (s, 1H), 8.56 (d, J=6.3 Hz, 1H), 8.52 (s, 1H), 8.48 (dd, J=15.3, 7.6 Hz, 2H), 8.40 (d, J=6.3 Hz, 1H), 8.02 (s, 1H), 7.83-7.78 (m, 1H), 7.76-7.72 (m, 1H), 7.64 (d, J=7.9 Hz, 1H), 7.32 (d, J=7.9 Hz, 1H), 5.96 (d, J=3.1 Hz, 2H), 5.73 (s, 2H), 4.41 (d, J=9.2 Hz, 1H), 4.31 (d, J=11.8 Hz, 1H), 4.05 (s, 2H), 3.87 (d, J=8.7 Hz, 2H), 3.59 (s, 219H), 3.15 (dd, J=19.1, 9.0 Hz, 1H), 3.01 (dd, J=13.5, 6.8 Hz, 2H), 2.61-2.54 (m, 1H), 2.49 (s, 1H), 2.38 (s, 1H).
In a 50 mL oven-dried round-bottom flask, compound 8 (111 mg, 0.24 mmol) was diluted with 10 mL DI water and stirred at 0° C. A mixture of 4aPEG-thiol (0.4 g, 0.04 mmol) and DI water (10 mL) was prepared in an addition funnel and added dropwise to the solution of compound 8. After addition, the mixture was warmed to ambient temperature and stirred for 6 h. The mixture was then dialyzed against DI water (MWCO of 3,500) for 8 h, and then lyophilized. 1H NMR (400 MHz, D2O) δ 8.75 (s, 1H), 8.58 (dd, J=29.4, 7.0 Hz, 2H), 7.91 (d, J=9.1 Hz, 2H), 7.84 (t, J=6.8 Hz, 1H), 7.64 (d, J=7.9 Hz, 1H), 7.50 (t, J=7.7 Hz, 1H), 5.72 (s, 2H), 4.46 (s, 1H), 4.36 (d, J=11.9 Hz, 1H), 4.04 (s, 2H), 3.90 (dd, J=9.1, 3.8 Hz, 2H), 3.59 (s, 192H), 3.18 (dd, J=19.1, 9.0 Hz, 1H), 3.01 (d, J=5.7 Hz, 2H), 2.64-2.54 (m, 1H), 2.46-2.37 (m, 1H).
A mixture of 4aPEG-NH2 (10 kDa, 3 g, 3 mmol), 4-formylphenylboronic acid (FPBA, 0.33 g, 1.8 mmol), hydroxybenzotriazole (HOBt, 0.24 g, 1.8 mmol), and triethylamine (0.26 mL, 1.8 mmol) was added to a 100 mL oven-dried round-bottom flask and diluted with 25 mL DMF. The mixture was stirred for 5 min before the addition of (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 0.68 g, 1.8 mmol). After the addition, the mixture was then stirred for 2 d and the solvent was removed under reduced pressure. The residue was then diluted with 20 mL MeOH and the mixture was dialyzed against MeCOH for 24 h, followed by further dialysis against DI water for 2 d, and then lyophilized. 1H NMR (400 MHz, Chloroform-d) δ 8.00 (t, J=7.6 Hz, 1H), 7.71 (d, J=7.8 Hz, 1H), 7.58 (d, J=12.6 Hz, 1H), 6.66 (s, 1H), 3.65 (s, 211H).
A mixture of 4aPEG-NH2 (10 kDa, 3.0 g, 0.3 mmol), D-gluconolactone (0.3 g, 4.8 m ol), and triethylamine (0.7 mL, 4.8 mmol) was added to a 100 mL oven-dried round-bottom flask and diluted with 50 mL MeOH. The mixture was allowed to stir at ambient temperature for 3 days before being transferred to a dialysis tube (MWCO of 3,500) and dialyzed against MeOH for 24 hours, followed by further dialyzed against DI water for 24 hours, and lyophilized. 1H NMR (400 MHz, 020) δ 4.29 (d, J=3.7 Hz, 1H), 4.05 (t, J=3.2 Hz, 1H), 3.67 (s, 193H), 3.51-3.38 (in, 5H).
In a 50 mL oven-dried round-bottom flask, compound 3 (81 mg, 0.12 mmol) was diluted with 10 mL DI water and stirred at 0° C. A mixture of mPEG-thiol (5k Da, 0.5 g, 0.1 mmol) and DI water (10 mL) was prepared in an addition funnel and added dropwise to the solution of compound 3. After the addition, the mixture was warmed to ambient temperature and stirred for 6 h. The mixture was then dialyzed against DI water (MWCO of 3,500) for 8 h, and then lyophilized. 1H NMR (400 MHz, D2O) δ 8.71 (s, 1H), 8.67-8.48 (m, 4H), 8.45 (d, J=6.2 Hz, 1H), 8.11 (d, J=2.0 Hz, 1H), 7.91-7.84 (m, 1H), 7.84-7.78 (m, 1H), 7.76-7.68 (m, 1H), 7.39 (d, J=8.0 Hz, 1H), 6.04 (s, 2H), 5.81 (s, 2H), 3.67 (s, 405H), 3.35 (s, 3H), 2.82 (dt, J=12.3, 6.0 Hz, 1H), 2.62 (ddd, J=18.0, 12.2, 4.9 Hz, 2H).
A mixture of mPEG-NH2 (5 kDa, 0.5 g, 0.1 mmol), d-gluconolactone (71.2 mg, 0.4 mmol), and triethylamine (58 μL, 0.4 mmol) was added to a 50 mL oven-dried round-bottom flask and diluted with 10 mL MeOH. The mixture was allowed to stir at ambient temperature for 3 days before being transferred to a dialysis tube (MWCO of 3,500) and dialyzed against MeOH for 24 hours, followed by further dialyzed against DI water for 24 hours, and lyophilized. 1H NMR (400 MHz, D2O) δ 4.29 (d, J=3.6 Hz, 1H), 4.05 (s, 1H), 3.67 (s, 382H), 3.35 (s, 3H).
Hyaluronic acid (HA, 60 kDa, 0.23 g, 0.004 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM, 0.14 g, 0.5 mmol) were diluted with 20 mL 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH=˜4-5) and stirred at ambient temperature until fully dissolved. Then 2-aminoethane-1-thiol (38 mg, 0.5 mmol) was added, and the mixture was kept stirring for 2 days before being transferred to a dialysis tube (MWCO of 10,000) and dialyzed against 10 wt % NaCl for 1 day, followed by further dialysis in water for 2 days, and lyophilized. 1H NMR (400 MHz, D2O) δ 4.45-4.40 (m, 2H), 3.87-3.11 (m, 14H), 1.89 (s, 3H).
HA-thiol (60 kDa, 0.3 g) and compound 3 (0.34 g, 0.5 mmol) were diluted with 40 mL DI water and stirred at ambient temperature 1 day before being transferred to a dialysis tube (MWCO of 10,000), dialyzed against water for 24 hrs, and lyophilized. 1H NMR (400 MHz, Deuterium Oxide) 0 8.74 (d, J=23.8 Hz, 2H), 8.56 (s, 3H), 7.90 (s, 1H), 7.81 (s, 1H), 7.64 (s, 2H), 7.40 (s, 2H), 5.94 (s, 2H), 5.75 (s, 2H), 4.38 (s, 2H), 3.93-3.09 (m, 20H), 1.79 (s, 3H). This protocol was suitable for HA MWs of 60 kDa, 500 kDa, and 700 kDa. The % of modification can be tuned ranging from 5%-25%. The subscript “m” may range from 130-1500.
An oven-dried 100 mL round-bottom flask was charged with 2,5-bis(bromomethyl)benzoic acid (1 g, 3.25 mmol), DMF (20 μL), and dissolved with 15 mL mixed solvent of THF:DCM (1:4). The mixture was then stirred at 0° C. for 10 min before the dropwise addition of oxalyl chloride (1.3 mL, 16.2 mmol). After the addition, the mixture was allowed to stir for 90 min. The solvent was then removed under reduced pressure. The residue was diluted with 20 mL DCM, transferred to an addition funnel, and added to a mixture of 1-hydroxypyrrolidine-2,5-dione (0.46 g, 4 mmol), triethylamine (0.58 mL, 4 mmol) and DCM (20 mL) at 0° C. After addition, the mixture was stirred at 0° C. for another 5 min before warming to ambient temperature. After 2 h, the mixture was transferred to a separation funnel and washed with washed with water (25 mL) and sat. NaCl (25 mL) solution and dried over Na2SO4. The mixture was filtered, and the solvent was removed under reduced pressure and the residue was used directly for the next step.
An oven-dried 100 mL round-bottom flask was charged with 2,5-dioxopyrrolidin-1-yl 2,5-bis(bromomethyl)benzoate (1.1 g, 2.8 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (1.1 g, 5.5 mmol) and 40 mL DMF. The mixture was stirred at 70° C. for 16 hr before the mixture was cooled down to the ambient temperature. The mixture was then treated with tert-butyl (2-aminoethyl)carbamate (0.43 g, 2.8 mmol), and stirred for another 24 hr, and then the solvent was removed under vacuum. After that, 20 mL TFA was added to the residue and stirred for 2 hrs. TFA was then removed, and the residue was added to excess TFA. The precipitate was collected, washed with acetone and Et2O to provide the target product. 1H NMR (400 MHz, D2O) δ 8.83 (s, 1H), 8.76-8.68 (m, 2H), 8.65-8.57 (m, 3H), 7.88 (dt, J=16.9, 7.1 Hz, 2H), 7.65 (s, 1H), 7.57 (d, J=8.8 Hz, 1H), 7.50 (d, J=8.0 Hz, 1H), 5.84 (d, J=5.6 Hz, 2H), 5.77 (s, 2H), 3.44 (dt, J=12.3, 6.3 Hz, 2H), 3.05 (dt, J=12.5, 6.2 Hz, 2H).
Hyaluronic acid (HA) (60 kDa, 0.23 g, 0.004 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM, 0.14 g, 0.5 mmol) were diluted with 20 mL 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH=˜4-5) and stirred at ambient temperature until fully dissolved. Then DiPBA-NH2 (35 mg, 0.05 mmol) was added, and the mixture was kept stirring for 5 days before being transferred to a dialysis tube (MWCO of 10,000) and dialyzed against 10 wt % NaCl for 1 day, followed by further dialysis in water for 2 days, and lyophilized. This protocol was also suitable for HA MWs of 500 kDa or 700 kDa. The % of modification can be tuned ranging from 5%-25%. The subscript “m” may range from 130-1500.
2-Amino-5-((tert-butoxycarbonyl)amino)pentanoic acid (2.46 g, 10 mmol) was suspended in 38 mL of dry THF in a oven-dried round bottom flask and stirred at 45° C. before the treatment of triphosgene (1.48 g, 5 mmol) solution in THF (10 mL). The mixture was kept stirring at 45° C. for 45 min and centrifuged. The supernatant was collected and concentrated and the resulting solids were recrystallized from THF/hexanes at −20° C. to obtain the target product as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 6.88 (t, J=5.8 Hz, 1H), 4.44 (td, J=5.5, 2.8 Hz, 1H), 2.92 (q, J=6.5 Hz, 2H), 1.77-1.68 (m, 1H), 1.65-1.56 (m, 1H), 1.43 (dt, J=17.6, 6.4 Hz, 2H), 1.37 (s, 9H).
To an oven-dried 10 mL round bottom flask, NCA-Orni (1 g, 3.9 mmol) and DMF (8 mL) were added and stirred at ambient temperature. The solution was freeze-pump-thawed for 3 cycles, then 4aPEG-NH2 (2 kDa, 48 mg, 0.024 mmol) was added. The mixture was kept at ambient temperature for 6 hr before exposed to the air. Then, HBTU (46 mg, 0.12 mmol), HOBt (16 mg, 0.12 mmol), 3-mercaptopropanoic acid (13 mg, 0.12 mmol), and Et3N (17 μL, 0.12 mmol) were added to the mixture and stirred for another 6 hr. The solution was then transferred to a dialysis tube (MWCO of 3,500) and dialyzed against MeOH (for 24 hr) and water (for 24 hr). The undissolved solids were collected by filtration. The collected solids were treated with TFA (50 mL) for 2 hr and concentrated to small volume. The residue was then diluted with 20 mL water and transferred to a dialysis tube (MWCO of 3,500) dialyzed in water for 24 hr, then lyophilized and used directly for the next step.
4aPEG10(Orni)m-SH (0.2 g, 0.017 mmol) and compound 3 (46 mg, 0.07 mmol) were diluted with 10 mL DI water and stirred at ambient temperature 1 day before being transferred to a dialysis tube (10,000 MWCO) and dialyzed against water for 24 hr, and lyophilized. 1H NMR (400 MHz, D2O) δ 8.67 (s, 6H), 8.60 (s, 4H), 8.54 (s, 9H), 8.51 (s, 8H), 8.41 (s, 5H), 7.82 (d, J=21.3 Hz, 13H), 7.67 (d, J=17.0 Hz, 5H), 7.33 (d, J=22.4 Hz, 4H), 6.04 (s, 8H), 5.81 (s, 8H), 4.31 (s, 108H), 3.68 (s, 181H), 3.00 (s, 210H), 1.74 (s, 413H).
DiPBA-NH2 (0.5 g, 0.7 mmol), succinic anhydride (78 mg, 0.77 mmol), and Et3N (0.12 mL, 0.77 mmol) were charged to a 100 mL round bottom flask and diluted with 20 mL MeOH. The mixture was stirred at ambient temperature for 24 hr before concentrated to small column. The residue was added to large volume of Et2O, and the precipitate was collected, washed with DCM and acetone and dried under vacuum as the target product. 1H NMR (400 MHz, D2O) δ 8.80 (s, 1H), 8.71 (d, J=6.2 Hz, 1H), 8.61 (d, J=7.0 Hz, 4H), 7.91 (q, J=7.6 Hz, 2H), 7.67 (d, J=8.0 Hz, 1H), 7.58 (d, J=7.8 Hz, 1H), 7.48 (s, 1H), 5.87 (s, 2H), 5.82 (s, 2H), 3.27 (dd, J=18.7, 6.1 Hz, 4H), 2.50 (t, J=6.5 Hz, 2H), 2.41 (t, J=6.4 Hz, 2H).
Sixth generation (G6) PAMAM dendrimer (160 mg, 0.003 mmol), DiPBA-COOH (180 mg, 0.26 mmol), and DMTMM (73 mg, 0.26 mmol) were charged to a 100 mL round bottom flask and diluted with 20 mL of DI water. The mixture was stirred at ambient temperature for 4 days before being transferred to a dialysis tube (MWCO of 10,000), dialyzed against water for 24 hours, and lyophilized. 1H NMR (400 MHz, D2O) δ 8.55 (s, 65H), 8.52-8.30 (m, 314H), 7.71 (dt, J=19.3, 6.7 Hz, 138H), 7.55-7.39 (m, 203H), 5.81-5.63 (m, 262H), 3.77 (d, J=21.0 Hz, 256H), 3.47-2.98 (m, 1355H), 2.88 (s, 293H), 2.81-2.05 (m, 2400H).
A mixture of generation 6.0 (G6) PAMAM dendrimer (58 kDa, 5 g, 0.086 mmol), D-gluconolactone (4.7 g, 26.5 mmol), and triethylamine (TEA, 3 mL, 21.5 mmol) was added to a 1000 mL oven-dried round-bottom flask and diluted with 700 mL MeOH. The mixture was allowed to stir at ambient temperature for 7 days. Methanol was evaporated off and the crude mixture was dissolved in water, transferred to a regenerated cellulose dialysis tube (MWCO of 3,500), dialyzed against water for 24 hours, and lyophilized. 1H NMR (400 MHz, D2O) δ 4.3 (d, 256H), 4.1-3.6 (bs), 3.5-3.1 (bs, 1528H), 3.0-2.6 (bs, 1524H), 2.5-2.3 (bs, 1016H).
A mixture of generation 2.0 (G2) PAMAM dendrimer (3.2 kDa, 1 g, 0.31 mmol), D-gluconolactone (1.3 g, 7.37 mmol), and triethylamine (TEA, 0.7 mL, 5 mmol) was added to a 500 mL oven-dried round-bottom flask and diluted with 300 mL MeOH. The mixture was allowed to stir at ambient temperature for 5 days. Methanol was evaporated off and the crude was dissolved in water, transferred to a regenerated cellulose dialysis tube (MWCO of 3,500), dialyzed against water for 24 hours, and lyophilized. 1H NMR (400 MHz, D2O) δ 4.3 (d, 16H), 4.1-3.6 (bs), 3.5-3.1 (bs, 88H), 3.0-2.6 (bs, 84H), 2.5-2.3 (bs, 56H).
(2,2,5,5-Tetramethyltetrahydro-8aH-[1,3]dioxolo[4′,5′:4,5]furo[3,2-d][1,3]dioxin-8a-yl)methanol (1 g, 4 mmol), 4-dimethylaminopyridine (4-DMAP, 24 mg, 0.2 mmol), and Et3N (1.15 mL, 8 mmol) were charged to a 100 mL round bottom flask and diluted with 20 mL DCM. The mixture was kept stirring at ambient temperature for 5 mins before the dropwise addition of 4-methylbenzenesulfonyl chloride (1.5 g, 8 mmol) solution in DCM (20 mL). After the addition, the mixture was kept stirring for 24 hrs. After that, the solution was transferred to a separation funnel and washed with water and sat. NaCl, and dried over Na2SO4. Then the mixture was filtered and concentrated, and the residue was loaded on a column eluting with hexanes/ethyl acetate (EA) for purification. The target product was recovered as a transparent oil. 1H NMR (400 MHz, Chloroform-d) δ 7.84-7.77 (m, 2H), 7.34 (d, J=8.0 Hz, 2H), 4.43 (s, 1H), 4.30 (d, J=2.3 Hz, 1H), 4.22 (d, J=10.5 Hz, 1H), 4.14 (d, J=10.4 Hz, 1H), 4.08 (d, J=1.7 Hz, 1H), 4.02 (dd, J=13.6, 2.2 Hz, 1H), 3.93 (d, J=13.6 Hz, 1H), 2.44 (s, 3H), 1.39 (s, 3H), 1.37 (s, 3H), 1.27 (s, 3H).
Fructose-OTs (1 g, 2.4 mmol) and NaN3 (0.9 g, 7.2 mmol) were diluted with 20 mL DMF and stirred at 100° C. for 3 days. The solvent was then removed under vacuum and the residue was loaded on the column, eluting with hexanes/ethyl acetate (EA) for purification. The final target was recovered as light-yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 4.41 (s, 1H), 4.33 (d, J=2.3 Hz, 1H), 4.12 (q, J=2.1 Hz, 1H), 4.08-3.98 (m, 2H), 3.72 (d, J=13.2 Hz, 1H), 3.40 (d, J=13.2 Hz, 1H), 1.47 (s, 3H), 1.42 (s, 3H), 1.36 (s, 3H).
In a 25 mL oven-dried Schlenk flask, 8aPEG-alkyne (0.57 g), copper (II) sulfate pentahydrate (CuSO45·H2O, 2 mg, BDH, ACS grade), and N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 98%, 3.2 μL, Acros) were added and diluted with DMF (10 mL). The flask was degassed by three freeze-pump-thaw cycles. On the last cycle, the flask was opened to quickly add sodium ascorbate (20 mg) into the flask before re-capping the flask. The flask was vacuumed and backfilled with N2 for 5 cycles, then transferred to a 50° coil for reaction. After 5 days, the reaction was quenched by exposure to air, concentrated to small volume, diluted with 10 mL DCM, and passed through a short Al2O3 column. The DCM was removed, and residue was diluted with 5 mL MeOH, transferred to a dialysis tube (molecular weight cutoff (MWCO) of 10,000) and dialysis against MeOH. Then the MeOH was removed under vacuum and the residue was treated with 90% TFA in water for 24 hr. After that, the mixture was concentrated and diluted with 10 mL water and transferred to a dialysis tube (MWCO of 10,000), dialyzed against water for 24 hr, and lyophilized. This protocol was suitable for any armed-PEG macromer (4arm, 8arm etc.) of any length (e.g., n=1-100).
Recombinant Human Insulin (5.8 kDa, 50 mg) was dissolved in sodium carbonate solution (0.1 M, 5 mL), while FITC (3.2 mg) was dissolved in dimethyl sulfoxide (DMSO, 1 mL). Both insulin and FITC solutions were adjusted to pH 11, and the FITC solution was added to the insulin solution dropwise. The reaction mixture was kept in the dark for 12 h. Following this time, the reaction mixture was pH adjusted to 5.3 and the resulting cloudy solution was centrifuged (4000 rpm, 30 min, 4° C.). The pellet was resuspended and dialyzed in DI water in the dark. Finally, the product was collected and lyophilized, yielding a yellow powder. The FITC-insulin product was verified by electrospray ionization mass spectrometry (ESI-MS, Advion) to ensure successful conjugation.
A 0.01 M stock solution of the PBA of interest was prepared by dissolving 0.2 mmol of each PBA in 20 mL DI water. The solution was then titrated with 0.005 M NaOH solution under constant stirring with pH monitoring.
Hydrogel mechanical properties were evaluated with a TA Instruments HR-2 rheometer fitted with a Peltier stage set to 25° C. All measurements were performed using a 25 mm parallel plate geometry. Oscillatory strain amplitude sweep measurements were first conducted at a frequency of 20 rad/s. Oscillatory frequency sweep measurements were then conducted at 3% strain after verification that this was in the linear viscoelastic region for all materials. Several rheology studies were performed, and hydrogels were prepared according to the various parameters being assessed: (i) For studies of concentration-dependent hydrogelation, stock solutions of PBA-bearing macromers (4aPEG-DiPBA, 4aPEG-PyPBA, or 4aPEG-FPBA) and 4aPEG-diol were prepared in 1× PBS. To formulate hydrogels, appropriate volumes of each macromer stock solution (at 1:1 motif to diol by mole) and PBS were combined to yield the final desired polymer concentration. (ii) For studies of glucose-dependent hydrogelation, glucose-containing buffers were prepared by dissolving glucose with PBS to yield a desired glucose concentration (0 mg/dL, 100 mg/dL, 200 mg/dL, and 400 mg/dL). Then stock solutions of PBA-bearing macromers (4aPEG-DiPBA, 4aPEG-PyPBA, or 4aPEG-FPBA) and 4aPEG-diol were prepared in these various glucose-containing PBS solutions. To formulate hydrogels, appropriate volumes of each macromer stock solution (at 1:1 motif to diol by mole) were combined to yield a final desired polymer concentration of 2 mM. (iii) For analyte-dependent hydrogelation, lactate, fructose, and glucose were dissolved in PBS to yield their final desired concentrations (Lactate: 5 mM, Fructose: 1 mM, Glucose: 22 mM). Then stock solutions of PBA-bearing macromers (4aPEG-DiPBA, 4aPEG-PyPBA, or 4aPEG-FPBA) and 4aPEG-diol were prepared in these various PBS solutions. To formulate the hydrogels, appropriate volumes of each macromer stock solution (at 1:1 motif to diol by mole) were combined to yield a final desired polymer concentration of 2 mM in the buffer containing the desired analyte.
The binding affinities (Keq) between different small molecule PBAs and model analytes (
A variety of studies were performed to assess the glucose-responsive and glucose-specific release of insulin from hydrogels. (i) To evaluate glucose-dependent FITC-insulin release from hydrogels, 0.1 mL hydrogels were prepared in a pH 7.4 PBS buffer at 2 mM polymer concentration (at 1:1 PBA to diol by mole) along with 20 μg FITC-insulin per hydrogel. Gels were then incubated in circular molds placed within 12-well plates and immersed in 3.5 mL of pH 7.4 release buffer containing 2.3, 5.5, 11 or 22 mM of glucose. At each time point, a 20 μL aliquot was taken and further diluted to 200 μL for fluorescence analysis (Ex: 485 nm, Em: 520 nm) on a Tecan M200 plate reader. The bulk was adjusted by addition of 20 μL of the same release buffer to maintain constant volume with each sampling. Released FITC-insulin concentrations were determined using a standard curve. After 8 h, gels were manually destroyed by treating with HCl solution to disrupt any remaining gel network and free residual FITC-insulin. The pH of this mixture was adjusted to pH 7.4 and insulin was quantified for mass balance closure. (ii) To evaluate FITC-insulin release upon a sudden increase in glucose level to mimic a hyperglycemic spike, hydrogels were prepared as before and immersed in 3.5 mL of pH 7.4 release buffer containing 2.3 mM glucose for 2 h. Subsequently, the release buffer was completely removed and replaced with 3.5 mL of pH 7.4 buffer containing 22 mM glucose and release was monitored for an additional 2 h. At each time point, a 20 μL aliquot was taken and further diluted to 200 μL for fluorescence analysis, and endpoint analysis and mass balance closure were performed, as before. (iii) To evaluate glucose-specific FITC-insulin release from hydrogels, 0.1 mL of hydrogel were prepared as before in pH 7.4 PBS at 2 mM polymer concentration and containing 20 μg FITC-insulin. Gels were then immersed in 3.5 mL of pH 7.4 PBS containing either (a) 5 mM glucose and 0.5 mM sodium lactate, (b) 5 mM glucose and 5 mM sodium lactate, (c) 10 mM glucose and 0.5 mM sodium lactate, or (d) 10 mM glucose and 5 mM sodium lactate. At each time point, 20 μL samples were collected, diluted to 200 μL, and analyzed as normal, along with replacement of 20 μL fresh buffer to the bulk. After 8 hours, gels were manually destroyed by HCl and analyzed for insulin content to ensure mass balance closure. (iv) To evaluate FITC-insulin release with a sudden increase in sodium lactate to mimic post-exercise elevation, 0.1 ml of hydrogel were prepared as before in pH 7.4 PBS at 2 mM polymer concentration and containing 20 μg FITC-insulin. Gels were then immersed in 3.5 mL of pH 7.4 buffer containing 10 mM glucose and 0.5 mM sodium lactate for 2 h. Subsequently, the release buffer was completely removed and replaced with 3.5 mL of pH 7.4 buffer containing 10 mM glucose and 5 mM sodium lactate for 2 h. At each time point, a 20 μL aliquot was taken and further diluted to 200 μL for fluorescence analysis, and endpoint analysis and mass balance closure were performed, as before.
To evaluate the performance hydrogels for blood glucose control, male C57BL6/J mice (8 weeks old, ˜25 g/mouse; Jackson Laboratory) were induced to be insulin deficient using streptozotocin (STZ). Mice were fasted for 4 h, following which a single intraperitoneal (i.p.) injection of STZ at a dose of 150 mg/kg was administered. Following an additional 30 min fast, food was returned. Seven days following STZ treatment, insulin-deficient diabetes was verified using hand-held blood glucose meters (CVS brand) with unfasted blood glucose (BG) levels ensured to be above 600 mg/dL for study inclusion. Mice were then fasted for 12 h, and those with BG >550 mg/dL were randomly divided into 4 groups (n=5-6/group). Groups were treated with one of the following: (a) 0.1 mL pH 7.4 PBS buffer, (b) 0.1 mL human recombinant insulin (4 IU/kg), (c) 0.1 mL insulin-loaded DiPBA hydrogel (1:1 molar ratio of 4aPEG-DiPBA to 4aPEG-diol, insulin dose of 7 IU/kg), or (d) 0.1 mL insulin-loaded FPBA hydrogel (1:1 molar ratio of 4aPEG-FPBA to 4aPEG-diol, insulin dose of 7 IU/kg) via subcutaneous (s.c.) injection. BG level were continuously monitored for 3 h after treatment. To examine gel response to a sudden increase in BG, a glucose tolerance test was performed by i.p. injection of glucose (1.25 g/kg glucose, 0.1 mL). BG were subsequently monitored for 3 h. A total of two IPGTT cycles were performed. Mice were fasted for the duration of the experiment with continuous access to water. All experiments followed a protocol approved by the University of Notre Dame Animal Care and Use Committee (IACUC) and adhered to all relevant Institutional, State, and Federal guidelines. Areas under the curve (AUC) were calculated using the trapezoidal rule and statistical analyses were performed to compare DiPBA and FPBA treatment groups using GraphPad Prism v9.0, with significance obtained using a Student's t-test.
To calculate overlap concentration (c*) for these networks, the following relation was used:
where M is the macromer molecular weight (10,000 g/mol), Rg is its radius of gyration (nm), and NA is Avagadro's number. See Wehrman et al., A/ChE J. 64 3168-3176 (2018). The Rg for a star PEG polymer can be estimated as follows:
where Rgarm is the radius of gyration of a single arm of the star and f is the number of arms on the macromer. See Wehrman, id. To estimate Rgarm (in nm) of PEG in a good solvent, the following relation was used:
with M for a single arm of 2500 g/mol. See Devanand and Selser, Macromolecules 24: 5943-5947 (1991). From this relationship, Rgarm was determined to be 2.06 nm, resulting in a value for Rgstar of 3.25 nm. Accordingly, c* was estimated to be 0.115 g/mL or 11.5 wt %.
Previously reported diboronate glucose sensors include architectures of two phenylboronic acids attached to an aryl core via charged ammonium linkers. In a variation on this approach, the DiPBA motif explored here has introduced adjacent charge via pyridine-based phenylboronic acid structures (
To first quantify the affinities of binding for these different PBA motifs to glucose, related analytes, and model diols, a set of small molecules (
Once small molecules were synthesized and validated for their binding, PBA-modified macromers were prepared by end-group functionalization of 10 kDa 4-arm polyethylene glycol (4aPEG,
With modified 4aPEG macromers prepared, ideal network hydrogels prepared from these macromers were next evaluated. Dynamic-covalent hydrogels were prepared over a range of macromer concentrations by combining equimolar Diol-4aPEG with each of the PBA-modified 4aPEGs for oscillatory rheology, first performing a strain sweep to verify the linear viscoelastic region and then performing a frequency sweep at constant strain of 3% (
Glucose-dependent dynamic properties were next evaluated for these hydrogels using oscillatory rheology, comparing dynamic-covalent networks prepared from DiPBA-diol and FPBA-diol crosslinking. Hydrogels were formulated by mixing PBA-bearing 4aPEG macromers with equimolar Diol-4aPEG at a total polymer concentration of 2 mM (˜2% w/v) in a pH 7.4 buffer containing various glucose concentrations (
After confirming glucose-responsive hydrogelation, controlled release of an encapsulated insulin payload was next assessed (
To improve function in blood glucose control, accelerated insulin release upon an increase in glucose level—as occurs following a meal—is a desirable property for a hydrogel depot. Accordingly, this function was assessed for hydrogels with encapsulated insulin by a sudden change in glucose concentration of the bulk release media (
After establishing and comparing the relative glucose-responsive function for these hydrogels, their interaction with non-glucose analytes was next evaluated. Hydrogels were formulated at 2 mM (˜2 wt %) macromer concentration, as before, with different amounts of competing analytes. From the initial ITC results, it was hypothesized that the DiPBA hydrogel should be less sensitive to crosslink disruption by non-glucose analytes than would the FPBA hydrogel. The concentrations of the competing analytes studied were 1 mM for fructose and 5 mM for lactate, selected to be on the upper end of their physiologically relevant range of exposure. These results were compared to the hydrogel response resulting from incubation with 22 mM glucose, also on its upper end of diabetic physiological exposure concentration. Oscillatory rheology was performed as before (
In the context of insulin therapy, interference from lactate presents an especially problematic outcome for a delivery depot; whereas fructose arises from dietary sources and typically overlaps with glucose consumption and insulin need, lactate is frequently elevated during and after periods of vigorous exercise. Lactate is also known to be elevated in diabetics with poorly managed disease. Thus, the impact of lactate was further explored for its role in triggering undesired insulin release from PBA-diol hydrogels (
As lactate levels may rise quickly with vigorous exercise, this scenario was recreated by studying the change in insulin release upon a sudden change in an environment of stable and slightly elevated glucose (10 mM) from low (0.5 mM) to high (5 mM) lactate levels (
In order to verify therapeutic function of this DiPBA-based hydrogel platform, an in vivo study in streptozotocin-induced diabetic mice was performed (
Herein, a new DiPBA motif was developed and used for the first time to prepare dynamic-covalent ideal network hydrogels. Molecular-scale binding studies using ITC demonstrated this new DiPBA to have glucose affinity that was 150 times higher than that of a traditional PBA motif. Simultaneously, this DiPBA motif showed reduced binding to fructose and lactate; interference from these non-glucose analytes presents a significant hurdle to the use of PBA-based materials due to the possibility that these physiological analytes may trigger non-specific insulin release. Rheology studies on dynamic-covalent ideal network hydrogels demonstrated DiPBA-diol crosslinking to be more glucose-sensitive than FPBA-diol crosslinking. In addition, hydrogels crosslinked by DiPBA-diol interactions were minimally impacted by non-glucose analytes like fructose and lactate; these analytes were at least as effective as glucose in disrupting crosslinking of FPBA-diol materials. In the context of glucose-responsive insulin delivery for blood glucose management in diabetes, the glucose sensing and specificity of DiPBA-diol crosslinking translated to improved glucose-responsive insulin release from the hydrogels. The improved responsiveness of DiPBA-based crosslinking was further validated in a diabetic mouse model, exhibiting more rapid blood glucose correction following multiple glucose challenges. This approach to use more sensitive and specific DiPBA-diol crosslinking thus offers a new material-centered approach with the potential to achieve the longstanding goal of glucose-responsive insulin therapy, overcoming limitations of commonly used PBA-based crosslinking chemistries.
This application claims priority to U.S. Provisional Patent Application No. 63/267,762 filed on Feb. 9, 2022, which is incorporated by reference herein in its entirety.
This invention was made with government support under grant number 1944875 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/012572 | 2/8/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63267762 | Feb 2022 | US |
