[go: up one dir, main page]

EP4422691A1 - Alginates modifiés et procédés de fabrication et d'utilisation associés - Google Patents

Alginates modifiés et procédés de fabrication et d'utilisation associés

Info

Publication number
EP4422691A1
EP4422691A1 EP22888557.0A EP22888557A EP4422691A1 EP 4422691 A1 EP4422691 A1 EP 4422691A1 EP 22888557 A EP22888557 A EP 22888557A EP 4422691 A1 EP4422691 A1 EP 4422691A1
Authority
EP
European Patent Office
Prior art keywords
alginate
modified
group
substituted
linker
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22888557.0A
Other languages
German (de)
English (en)
Other versions
EP4422691A4 (fr
Inventor
Yevgeny Brudno
Christopher Moody
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
North Carolina State University
Original Assignee
North Carolina State University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by North Carolina State University filed Critical North Carolina State University
Publication of EP4422691A1 publication Critical patent/EP4422691A1/fr
Publication of EP4422691A4 publication Critical patent/EP4422691A4/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/734—Alginic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/04—Alginic acid; Derivatives thereof

Definitions

  • Hydrogel biomaterials offer utility in biomedical applications due to their stability, tunable mechanics and degradation profiles, as well as biocompatibility with surrounding tissues.
  • Clinical and preclinical applications of hydrogel biomaterials include tissue engineered constructs, depots for drug and cell delivery, and cellular scaffolds used in the study of biological processes.
  • alginate is rapidly gaining attention because it gels under neutral, physiological conditions, exhibits good biocompatibility at tissue sites, and has wide chemical versatility.
  • Alginate gelation utilizes ionic cross-links between the carboxyl groups on alginate and divalent cations.
  • the calcium cross-linked hydrogels are injectable and self-healing, enabling facile injection into tissues.
  • calcium cross-linked alginate hydrogels elicits low levels of foreign body responses, very low toxicity and limited immunogenicity.
  • alginate is generally recognized as safe (GRAS) by the FDA, which has motivated alginates preclinical testing to deliver drugs, biologicals, viruses and cells and clinically as a dietary supplement, material for wound dressings, sealant agent and as an injectable implant.
  • GRAS safe
  • alginate The chemical versatility of alginate is of particular interest to researchers trying to take advantage of alginate’s biocompatibility.
  • Chemical modification of alginate has been used to decorate alginate polymers with small molecules for controlled and tunable drug delivery and with peptides and proteins to mediate cell attachment and signaling. More recently, alginate polymers have been conjugated to bioorthogonal “click” chemical motifs in order to enhance or enable alginate cross-linking, to expedite polymer modification, and to create targetable drug depots.
  • alginate is straightforward to chemically modify, modification carries undesired complications. Most frequently, alginate polymers are modified through carbodiimide coupling between the carboxyl group on alginate and nucleophiles (alcohols, amines, and others). Unfortunately, this chemical modification decreases polymer viscosity and can inhibit gelation. Indeed, alginate hydrogels modified to a high degree of substitution (DS) suffer from poor or nonexistent calcium cross-linking. What is needed are new methods of modifying alginate that do not suffer from the drawbacks currently experienced when modifying alginate with a high DS.
  • alginate strands with a high degree of substitution Disclosed are alginate strands with a high degree of substitution and methods of making the same.
  • substituted alginate strands comprising alginate strands coupled to a functional moiety (e.g., a click motif, such as, for example, an azide) via a linker, wherein the linker comprises a nucleophilic terminus (such as, for example, amino, alcohol, thiol) and additionally a carboxyl group attached to the linker; and wherein the nucleophilic terminus is coupled to a carboxyl group on the alginate strand.
  • the substituted alginate strands can form polymer strands.
  • an alginate strand to an functional moiety (e.g., a click motif, such as, for example, an azide) via carbodiimide coupling said method comprising conjugating the functional moiety to a linker comprising a nucleophilic terminus and a carboxyl group of the nucleophilic terminus of the linker (such as, amine, alcohol, thiol); wherein the functional moiety is chemically coupled to the linker at the opposing end to the nucleophilic terminus; and coupling the nucleophilic terminus of the linker to the alginate strand via carbodiimide coupling; wherein the modification allows for synthesis of highly substituted alginate; wherein the substitution does not disrupt the integrity of the gel.
  • an functional moiety e.g., a click motif, such as, for example, an azide
  • nucleophilic terminus of the linker comprise a protecting group (such as, for example, methyl, ethyl, benzyl, benzyloxy carbonyl, s-Butyl, 2-Alkyl-l,3-oxazoline, OBO, silyl, photo-sensitive group propyl, tert-butyl, and NVOC); and wherein the nucleophilic terminus is deprotected prior to being coupled to a sugar on the alginate strand.
  • a protecting group such as, for example, methyl, ethyl, benzyl, benzyloxy carbonyl, s-Butyl, 2-Alkyl-l,3-oxazoline, OBO, silyl, photo-sensitive group propyl, tert-butyl, and NVOC
  • a protecting group such as, for example, methyl, ethyl, benzyl, benzyloxy carbonyl, s-Butyl, 2-Alkyl-l,3- oxazoline, OBO, silyl, photo-sensitive group propyl, tert-butyl, and NVOC
  • modified alginates comprising one or more covalently modified monomers defined by Formula I wherein:
  • X is O, S, or NRs
  • Rs is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; Li and L2 are each independently absent or represent a linking group;
  • A represents a functional moiety (e.g., a click motif or an active agent); and Y 1 and Y2 independently are hydrogen or — PO(OR)2, or Y2 is absent, and Y 1, together with the two oxygen atoms to which Y 1 and Y2 are attached form a cyclic structure as shown below wherein wherein R.4 and Rs are, independently, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, aroxy, alkylthio, carbonyl, carboxyl, amino, or amido; or R4 and Rs, together with the carbon atom to which they are attached, form a 3- to 8-membered unsubstituted or substituted carbocyclic or heterocyclic ring.
  • R.4 and Rs are, independently, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclo
  • hydrogel matrixes or scaffolds comprising polymer strands of the substituted alginate strand of any preceding aspect or the modified alginates of any preceding aspect, wherein the polymer strands are cross-linked to other alginate polymer stands via a divalent metal cation (e.g., via calcium crosslinking).
  • Figure 1A shows an overview of depletive and restorative alginate modifications.
  • Alginate can be modified with chemical groups (blue) through the carboxyl groups (red), which depletes the carboxyl groups, and destroys calcium cross-linking.
  • carboxyl groups red
  • the modifications restore carboxyl groups
  • alginate gels maintain calcium cross-linking and gelation.
  • Figure IB shows a representation of cross-linking that occurs in traditional unmodified alginate gels.
  • Figure 1C shows the effect of modification on alginate cross-linking.
  • Figure ID shows the present solution of introducing new carboxylic acid groups and the effect of said groups on cross-linking.
  • FIG. 1 shows the synthetic approach to (S)-2-ammonio-6-(2-azidoacetamido)hexanoate (S4).
  • Figure 3 shows the coupling of azide-amine (depletive modification) and azide-lysine (restorative modification) to alginate.
  • Figures 4A, 4B, 4C, and 4D show restorative modifications at high degrees of substitution maintain alginate gel mechanics.
  • Figure 4A shows photos of calcium cross-linked alginate gels using depletive or restorative modifications at low- and high-DS azide.
  • N 3 for each gel as well as carboxyl-depleted and unmodified hydrogel can be found in Figure 5. ⁇ 5 % tolerance range of deviation was used to select the plateau value of the linearity limit.
  • Figure 4C shows representative frequency sweeps [1-100 Hz] within the LVE region showing the gel-like behavior and structural stability of calcium cross-linked gels modified with high-DS restored azide-alginate and high-DS depleted azide-alginate.
  • Storage modulus (G’) and loss modulus (G”) measurements are shown.
  • N 3 for each gel and low-DS gel mechanics can be found in Figure 6.
  • Figures 6A and 6B show frequency Sweep [1-100 Hz] of calcium cross-linked gels showing gel-like status and structural stability. Storage modulus (G’) and loss modulus (G”) measurements are shown.
  • Figure 6A shows one sample from each gel group including low-DS gels and unmodified.
  • Figures 8A, 8B, and 8C show the self-healing behavior of calcium cross-linked alginate gels demonstrated by the recovery after repeated deformation of high strain [500%] at 10 Hz followed by low strain [0.2%].
  • Figures 9A, 9B, 9C, 9D, and 9E show calcium cross-linked alginate gels with restorative azide modification reduce off-target gel migration.
  • Figure 9A shows representative images (same BLI scale) and figure 9B shows quantitation of fluorescence signal after intramuscular injection of alginate hydrogels fluorescently conjugated to carboxyl-depletive or -restorative modifications over 2 weeks. Unmodified alginates with unconjugated fluorophore served as negative control.
  • Figure 9C shows representative locations for quantified off-target migration. Quantitation of fluorescence signal in non-injected contralateral limb (9D) and ankle (9E) over 2 weeks. Region of interest (ROI) values were quantified as total radiance efficiency in equivalent sized regions.
  • ROI Region of interest
  • Figures 11A and 11B show restorative azide modification improves on-target capture of circulating DBCO fluorophores.
  • Figure 11A shows representative images (same BLI scale) and 11B shows quantitation of fluorescence at intramuscular sites from i.m. -injected alginate hydrogels cross-linked with calcium.
  • DBCO-Cy7 was administered i.v. one week following gel injection.
  • mice were imaged to compare the capture of the fluorescent signal at the injected site.
  • ROI values were quantified as total radiance efficiency in equivalent sized regions.
  • Samples show mean ⁇ SEM.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “abouf another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.
  • An “increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • reduce or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g, tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.
  • prevent or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.
  • the term “subject” refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline.
  • the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • terapéuticaally effective refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • Biocompatible generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.
  • compositions, methods, etc. include the recited elements, but do not exclude others.
  • Consisting essentially of' when used to define compositions and methods shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.
  • control is an alternative subject or sample used in an experiment for comparison purposes.
  • a control can be "positive” or “negative.”
  • Effective amount of an agent refers to a sufficient amount of an agent to provide a desired effect.
  • the amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained.
  • the term When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
  • “Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use.
  • carrier or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
  • carrier encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.
  • “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
  • “Therapeutic agent” refers to any composition that has a beneficial biological effect.
  • Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer).
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • therapeutic agent when used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
  • “Therapeutically effective amount” or “therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result.
  • a desired therapeutic result is the control of type I diabetes.
  • a desired therapeutic result is the control of obesity.
  • Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject.
  • the term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief
  • a desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
  • a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
  • Alginates are versatile polysaccharide based polymers that may be formulated for specific applications by controlling the molecular weight, rate of degradation and method of scaffold formation.
  • Alginate molecules are comprised of (l-4)-linked P-D-mannuronic acid (M units) and a L-guluronic acid (G units) monomers, which can vary in proportion and sequential distribution along the polymer chain.
  • M units P-D-mannuronic acid
  • G units L-guluronic acid
  • Alginate is a collective term used to refer to linear polysaccharides formed from -D-mannuronate and L-guluronate in any M/G ratio, as well as salts and derivatives thereof.
  • alginate encompasses any polymer having the structure shown below, as well as salts
  • Mannuronate and “Mannuronate Monomer”, as used herein, refer to mannuronic acid monomers as well as salts thereof.
  • Guluronate and “Guluronate Monomer”, as used herein, refer to guhironic acid monomers as well as salts thereof.
  • “Chemically Modified Alginate' or “Modified Alginate” are used herein interchangeably, and refer to alginate polymers which contain one or more covalently modified monomers.
  • “Covalently Modified Monomer', as used herein, refers to a monomer which is an analog or derivative of a mannuronate and/or guluronate monomer obtained from a mannuronate and/or guluronate monomer via a chemical process.
  • “Singularly Modified Alginate Polymer” refers to modified alginates that contain one or more covalently modified monomers, wherein substantially all of the covalently modified monomers possess the same covalent modification (i.e., the polymer contains one type or species of covalently modified monomer).
  • Singularly modified alginate polymers include, for example, modified alginate polymers wherein substantially all of the monomers in the modified alginate polymer are represented by mannuronate monomers, guluronate monomers, and a covalently modified monomer defined by Formula I described below. Not all of the monomers are necessarily covalently modified.
  • Multiply Modified Alginate Polymer refers to modified alginates that contain covalently modified monomers, wherein substantially all of the covalently modified monomers do not possess the same covalent modification (i.e., the polymer contains two or more different ‘types’ or species of covalently modified monomers).
  • Multiply modified alginate polymers include, for example, modified alginate polymers wherein substantially all of the monomers in the modified alginate polymer are represented by mannuronate monomers, guluronate monomers, and two or more different types of covalently modified monomers defined by Formula I.
  • a ‘type’ or ‘species’ of covalently modified monomer refers to a covalent monomer defined by Formula I, wherein all possible variable positions are chemically defined. Not all the monomers need be covalently modified.
  • singularly modified alginates are defined using formulae illustrating the structure of the covalently modified monomers incorporated in the backbone and omitting the mannuronate and guluronate monomers.
  • “Substituted,” as used herein, refers to all permissible substituents of the compounds or functional groups described herein.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats.
  • substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl
  • Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e. a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • Aryl refers to C5-C 10-membered aromatic, heterocyclic, fused aromatic, fused heterocyclic, biaromatic, or bihetereocyclic ring systems.
  • aryl includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics.”
  • the aromatic ring can be substituted at one or more ring positions with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (or quatemized amino), nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, — CFs, — CN; and combinations thereof.
  • Aryl further encompasses polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (i. e. , “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles.
  • heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, IH-indazolyl, indolenyl, indolinyl, indolizinyl
  • Alkyl refers to the radical of saturated or unsaturated aliphatic groups, including straight-chain alkyl, alkenyl, or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups, cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted alkyl, alkenyl, or alkynyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), preferably 20 or fewer, more preferably 10 or fewer, most preferably 6 or fewer.
  • the alkyl chain generally has from 2-30 carbons in the chain, preferably from 2-20 carbons in the chain, more preferably from 2-10 carbons in the chain.
  • preferred cycloalkyls have from 3-20 carbon atoms in their ring structure, preferably from 3-10 carbons atoms in their ring structure, most preferably 5, 6 or 7 carbons in the ring structure.
  • alkenyl and alkynyl refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • Alkyl includes one or more substitutions at one or more carbon atoms of the hydrocarbon radical as well as heteroalkyls. Suitable substituents include, but are not limited to, halogens, such as fluorine, chlorine, bromine, or iodine; hydroxyl; — NR1R2, wherein Ri and R2 are independently hydrogen, alkyl, or aryl, and wherein the nitrogen atom is optionally quatemized; — SR, wherein R is hydrogen, alkyl, or aryl; — CN; — NO2; — COOH; carboxylate; — COR, — COOR, or — CONR2, wherein R is hydrogen, alkyl, or aryl; azide, aralkyl, alkoxyl, imino, phosphonate, phosphinate, silyl, ether, sulfonyl, sulfonamido, heterocyclyl, aromatic or heteroaromatic moieties, —
  • Amino and “Amine,” as used herein, are art-recognized and refer to both substituted and unsubstituted amines, e.g., a moiety that can be represented by the general formula: wherein, R, R', and R" each independently represent a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, — (CH2)m — R'", or R and R' taken together with the N atom to which they are attached complete a heterocycle having from 3 to 14 atoms in the ring structure; R'" represents a hydroxy group, substituted or unsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle, or a poly cycle; and m is zero or an integer ranging from 1 to
  • R and R' can be a carbonyl, e.g., R and R' together with the nitrogen do not form an imide.
  • R and R' (and optionally R") each independently represent a hydrogen atom, substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, or — (CH2)m — R'".
  • alkylamine refers to an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto (i.e. at least one of R, R', or R" is an alkyl group).
  • Carbonyl is art-recognized and includes such moieties as can be represented by the general formula: wherein X is a bond, or represents an oxygen or a sulfur, and R represents a hydrogen, a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, — (CH2)m — R", or a pharmaceutical acceptable salt, R' represents a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or — (CH2)m — R"; R" represents a hydroxy group, substituted or unsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle, or a poly cycle; and m is zero or an integer ranging from 1
  • Heteroalkyl refers to straight or branched chain, or cyclic carbon- containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quatemized.
  • saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n- octyl.
  • unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3- propynyl, and 3-butynyl.
  • Alkoxy alkylamino
  • alkylthio alkylthio
  • Alkylaryl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or hetero aromatic group).
  • Heterocycle refers to a cyclic radical attached via a ring carbon or nitrogen of a monocyclic or bicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ring atoms, consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, Ci-Cio) alkyl, phenyl or benzyl, and optionally containing 1-3 double bonds and optionally substituted with one or more substituents.
  • heterocyclic ring examples include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, IH-indazolyl, indolenyl, indolinyl, ind
  • Heterocyclic groups can optionally be substituted with one or more substituents as defined above for alkyl and aryl.
  • We and others have demonstrated that poor calcium cross-linking directly caused by high DS causes loss of gel stiffness, migration of implants away from desired injection sites, and enhanced calcium leaching, leading to an increased foreign body response to the gel.
  • One approach to loss of calcium cross-linking at high DS is to use alternative cross-linkers, but this has several drawbacks.
  • the addition of new chemical cross-linkers add unnecessary complication to the regulatory pathway for clinical use and can have unexpected physiological toxicity.
  • alternative cross-links are often covalent, these cross-linkers eliminate two advantages of using alginate in the first place - alginate’s ability to self-heal and its shearthinning characteristic.
  • substituted alginate strands comprising alginate strand coupled to an functional moiety (such as, for example, azide) via a linker with a nucleophilic terminus (such as, for example, carbamic acid, carbamate ester, glycine, arginine, or lysine), wherein the linker comprises a nucleophilic terminus and a carboxyl group of the nucleophilic terminus; and wherein the nucleophilic terminus is coupled to a carboxyl group on the alginate strand.
  • the substituted alginate strands can form polymer strands.
  • Alginate polysaccharides are polyelectrolyte systems which have a strong affinity for divalent cations (e.g., Ca +2 , Mg +2 , Ba +2 ) and form stable hydrogels when exposed to these molecules. See Martinsen A., et al., Biotech. & Bioeng., 33 (1989) 79-89.)
  • calcium cross-linked alginate hydrogels are useful for the methods described herein.
  • the polymers, e.g., alginates, of the hydrogel are 0-100% crosslinked, e.g., at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, crosslinked.
  • the polymers, e.g., alginates, of the hydrogel are not crosslinked.
  • the polymers, e.g., alginates, of the hydrogel contain less than 50%, e.g., less than 50%, 40%, 30%, 20%, 10%, 50%, 2%, 1%, or less, crosslinking.
  • hydrogel matrixes or scaffolds comprising polymer strands of the substituted alginate strand disclosed herein, wherein the polymer strands are crosslinked to other alginate polymer stands via calcium crosslinking.
  • Alginate may be chemically modified to yield new properties. For example, alginate may be oxidized to increase the rate of biodegradation. Alternatively, alginate may be reduced for improved biocompatibility. Alginate can also be chemically modified to change their crosslinking behavior. For instance, alginate can be modified with bioorthogonal click groups to allow click crosslinking. In another example, alginate can be modified with acrylic groups to allow radical polymerization crosslinking. As another example, alginates can be modified with host-guest chemistries to allow host-guest crosslinking.
  • Coupling reactions can be used to covalently attach bioactive epitopes, such as the cell adhesion sequence RGD to the polymer backbone.
  • bioactive epitopes such as the cell adhesion sequence RGD
  • Chemical functionalization with small molecules regulates immune and foreign body response functionalization with peptides mediates cellular and tissue responses, and modification with reactive chemical groups enables new modes of drug delivery.
  • Alginate polymers conjugated to bioorthogonal “click” chemical motifs (as functional moieties) enhance cross-linking and expedite polymer modification. Additionally, alginates modified with click motifs have been used as targetable drug depots, capable of repeatedly capturing and releasing drugs.
  • Click chemistry refers to a class chemical reaction between two click groups that exhibit good yields, wide functional group tolerance, and are highly selective even in the presence of a complex mixture of biological molecules. These characteristics allow the click reactions to proceed even in vivo.
  • Example click motif pairs used as the first click motif and the second click motif include, but not limited to, azide with phosphine; azide with cyclooctyne; nitrone with cyclooctyne; nitrile oxide with norbomene; oxanorbomadiene with azide; trans-cyclooctene with s-tetrazine; quadricyclane with bis(dithiobenzil)nickel(II).
  • the second click motif comprises an alkene, e.g., a cyclooctene, e.g., a transcyclooctene (TCO) or norbomene (NOR), and the first click motif comprises a tetrazine (Tz).
  • the second click motif comprises an alkyne, e.g., a cyclooctyne such as dibenzocyclooctyne (DBCO), and the first click motif comprises an azide (Az).
  • the second click motif comprises a Tz
  • the first click motif comprises an alkene such as transcyclooctene (TCO) or norbomene (NOR).
  • the first click motif comprises an Az
  • the second click motif comprises a cyclooctyne such as dibenzocyclooctyne (DBCO).
  • TCO reacts specifically in a click chemistry reaction with a tetrazine (Tz) moiety.
  • DBCO reacts specifically in a click chemistry reaction with an azide (Az) moiety.
  • Norbomene reacts specifically in a click chemistry reaction with a tetrazine (Tz) moiety.
  • CuAAC copper(I)-catalyzed Azide- Alkyne Cycloaddition
  • Cu Copper
  • the Azide-Alkyne Cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2,3-triazole.
  • Staudinger ligation is a reaction that is based on the classic Staudinger reaction of azides with triarylphosphines. It launched the field of bioorthogonal chemistry as the first reaction with completely abiotic functional.
  • the azide acts as a soft electrophile that prefers soft nucleophiles such as phosphines. This is in contrast to most biological nucleophiles which are typically hard nucleophiles.
  • the reaction proceeds selectively under water-tolerant conditions to produce a stable product.
  • Phosphines are completely absent from living systems and do not reduce disulfide bonds despite mild reduction potential. Azides had been shown to be biocompatible in FDA-approved drugs such as azidothymidine and through other uses as cross linkers. Additionally, their small size allows them to be easily incorporated into biomolecules through cellular metabolic pathways.
  • Copper-free click chemistry is a bioorthogonal reaction first developed by Carolyn Bertozzi as an activated variant of an azide alkyne cycloaddition. Unlike CuAAC, Cu-free click chemistry has been modified to be bioorthogonal by eliminating a cytotoxic copper catalyst, allowing reaction to proceed quickly and without live cell toxicity. Instead of copper, the reaction is a strain-promoted alkyne-azide cycloaddition (SPAAC). It was developed as a faster alternative to the Staudinger ligation, with the first generations reacting over sixty times faster. The enormous bioorthogonality of the reaction has allowed the Cu-free click reaction to be applied within cultured cells, live zebrafish, and mice.
  • SPAAC strain-promoted alkyne-azide cycloaddition
  • Cyclooctynes were selected as the smallest stable alkyne ring which increases reactivity through ring strain which has calculated to be 19.9 kcal/mol. Copper-free click chemistry also includes nitrone dipole cycloaddition. Copper-free click chemistry has been adapted to use nitrones as the 1,3-dipole rather than azides and has been used in the modification of peptides.
  • click chemistry includes norbomene cycloaddition.
  • 1,3 dipolar cycloadditions have been developed as a bioorthogonal reaction using a nitrile oxide as a 1,3- dipole and a norbomene as a dipolarophile. Its primary use has been in labeling DNA and RNA in automated oligonucleotide synthesizers.
  • Norbomenes were selected as dipolarophiles due to their balance between strain- promoted reactivity and stability.
  • the drawbacks of this reaction include the cross-reactivity of the nitrile oxide due to strong electrophilicity and slow reaction kinetics.
  • click chemistry includes oxanorbomadiene cycloaddition.
  • the oxanorbomadiene cycloaddition is a 1,3-dipolar cycloaddition followed by a retro-Diels Alder reaction to generate a triazole-linked conjugate with the elimination of a furan molecule. This reaction is useful in peptide labeling experiments, and it has also been used in the generation of SPECT imaging compounds.
  • Ring strain and electron deficiency in the oxanorbomadiene increase reactivity towards the cycloaddition rate-limiting step.
  • the retro-Diels Alder reaction occurs quickly afterwards to form the stable 1,2,3 triazole. Limitations of this reaction include poor tolerance for substituents which may change electronics of the oxanorbomadiene and low rates (second order rate constants on the order of 10 4 ).
  • the tetrazine ligation is the reaction of a trans-cyclooctene and an s-tetrazine in an inverse-demand Diels Alder reaction followed by a retro-Diels Alder reaction to eliminate nitrogen gas.
  • the reaction is extremely rapid with a second order rate constant of 2000 M '-s 1 (in 9: 1 methanol/water) allowing modifications of biomolecules at extremely low concentrations.
  • the highly strained trans-cyclooctene is used as a reactive dienophile.
  • the diene is a 3,6- diaryl-s-tetrazine which has been substituted in order to resist immediate reaction with water.
  • the reaction proceeds through an initial cycloaddition followed by a reverse Diels Alder to eliminate N2 and prevent reversibility of the reaction. Not only is the reaction tolerant of water, but it has been found that the rate increases in aqueous media. Reactions have also been performed using norbomenes as dienophiles at second order rates on the order of 1 M 1 -s 1 in aqueous media. The reaction has been applied in labeling live cells and polymer coupling.
  • click chemistry includes is [4+1] cycloaddition.
  • This isocyanide click reaction is a [4+1] cycloaddition followed by a retro-Diels Alder elimination of N2.
  • reaction proceeds with an initial [4+1] cycloaddition followed by a reversion to eliminate a thermodynamic sink and prevent reversibility.
  • This product is stable if a tertiary amine or isocyanopropanoate is used. If a secondary or primary isocyanide is used, the produce will form an imine which is quickly hydrolyzed.
  • Isocyanide is a favored chemical reporter due to its small size, stability, non-toxicity, and absence in mammalian systems. However, the reaction is slow, with second order rate constants on the order of 10 2 M 1 • s 1 .
  • quadricyclane ligation utilizes a highly strained quadricyclane to undergo [2+2+2] cycloaddition with 71 systems.
  • Quadricyclane is abiotic, unreactive with biomolecules (due to complete saturation), relatively small, and highly strained ('80 kcal/mol). However, it is highly stable at room temperature and in aqueous conditions at physiological pH. It is selectively able to react with electron-poor 71 systems but not simple alkenes, alkynes, or cyclooctynes.
  • Bis(dithiobenzil)nickel(II) was chosen as a reaction partner out of a candidate screen based on reactivity.
  • di ethyldithiocarbamate is added to chelate the nickel in the product.
  • the exemplary click chemistry reactions have high specificity, efficient kinetics, and occur in vivo under physiological conditions. See, e.g., Baskin et al. Proc. Natl. Acad. Sci. USA 104(2007): 16793; Oneto et al. Acta biomaterilia (2014); Neves et al. Bioconjugate chemistry 24(2013):934; Koo et al. Angewandte Chemie 51(2012): 11836; and Rossin et al. Angewandte Chemie 49(2010):3375.
  • Alginate polymers are formed into a variety of hydrogel types.
  • Alginate hydrogels can be formed from low molecular weight (MW) alginate or from high MW alginate. Differences in hydrogel formulation control the kinetics of hydrogel degradation. Release rates of pharmaceutical compositions, e.g., small molecules, morphogens, or other bioactive substances, from alginate hydrogels is controlled by hydrogel formulation to present the pharmaceutical compositions in a spatially and temporally controlled manner. This controlled release eliminates systemic side effects and the need for multiple injections.
  • MW molecular weight
  • Differences in hydrogel formulation control the kinetics of hydrogel degradation. Release rates of pharmaceutical compositions, e.g., small molecules, morphogens, or other bioactive substances, from alginate hydrogels is controlled by hydrogel formulation to present the pharmaceutical compositions in a spatially and temporally controlled manner. This controlled release eliminates systemic side effects and the need for multiple injections.
  • Mannuronate and guluronate monomers contain a carboxylic acid moiety which can serve as a point of covalent modification.
  • the carboxylic acid moiety present on one or more mannuronate and/or guluronate residues can be covalently modified via amidation.
  • amidation of alginate typically causes loss of carboxyl residues which in turn results in destructive effects on the ability of alginate polymers to crosslink via Calcium crosslinking. The result is a loss of many of the advantageous properties of the alginate polymers. Accordingly, disclosed herein are substituted alginate strands that do not suffer from these problems and methods for achieving said modified alginate strands.
  • a functional moiety e.g., a click motif discussed above, such as, for example, an azide
  • a linker comprising an nucleophilic terminus and a carboxyl group of the nucleophilic terminus of the linker (such as, for example, carbamic acid, carbamate ester, glycine, arginine, or lysine); wherein the functional moiety is chemically coupled to the linker at the opposing end to the nucleophilic terminus; and coupling the nucleophilic terminus of the linker to the alginate strand via carbodiimide coupling; wherein the modification allows for synthesis of highly substituted alginate; wherein the substitution does not disrupt the integrity of the gel.
  • a linker comprising an nucleophilic terminus and a carboxyl group of the nucleophilic terminus of the linker (such as, for example, carbamic acid, carbamate ester, glycine, arginine, or lysine)
  • the carboxyl group on the linker can be blocked with a protecting group.
  • Any protecting group known and used in the art can be used for this purpose, including, but not limited to methyl, ethyl, benzyl, benzyloxy carbonyl, s-Butyl, 2- Alkyl-l,3-oxazoline, OBO, silyl, photo-sensitive group propyl, tert-butyl, and NVOC.
  • nucleophilic terminus of the linker comprises a protecting group (such as, for example, /c/v-butyloxy carbonyl, a-boc, -methoxybenz l carbonyl, carbobenzyloxy, acetyl, benzoyl, benzyl, carbamate, -methoyxybenzyl. 3,4-dimethoxybenyl, p-methoxyphyl, trichloroethyl chloroformate, or tosyl); and wherein the nucleophilic terminus is deprotected prior to being coupled to the alginate strand.
  • a protecting group such as, for example, /c/v-butyloxy carbonyl, a-boc, -methoxybenz l carbonyl, carbobenzyloxy, acetyl, benzoyl, benzyl, carbamate, -methoyxybenzyl. 3,4-dimethoxybenyl,
  • modified alginates include alginate monomers that have been covalently modified to facilitate crosslinking while without sacrificing calcium cross-linking.
  • the modified alginates can comprise one or more covalently modified monomers defined by Formula I below wherein:
  • X is O, S, or NRs
  • Rs is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl;
  • Li and L2 are each independently absent or represent a linking group
  • A represents a functional moiety; and Y i and Y2 independently are hydrogen or — PO(OR)2, or Y2 is absent, and Y 1, together with the two oxygen atoms to which Y 1 and Y2 are attached form a cyclic structure as shown below wherein wherein R4 and Rs are, independently, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, aroxy, alkylthio, carbonyl, carboxyl, amino, or amido; or R4 and Rs, together with the carbon atom to which they are attached, form a 3- to 8-membered unsubstituted or substituted carbocyclic or heterocyclic ring.
  • Y 1 and Y2 are both H.
  • X is NH
  • A can comprise a click motif. Examples of suitable click motifs are described above.
  • A can comprise an azide, a phosphine, a cyclooctene (e.g., a transcyclooctene (TCO)), anorbomene (NOR), a tetrazine (Tz), an alkyne, a cyclooctyne such as dibenzocyclooctyne (DBCO), or a quadri cyclane.
  • the functional moiety comprises an active agent, as described below.
  • both Li and L2 are present. In some embodiments, Li is absent and L2 is present. In some embodiments, Li is present and L2 is absent. In some embodiments, both Li and L2 are absent.
  • the linking group can be any suitable group or moiety which is at minimum bivalent, and connects the two radical moieties to which the linking group is attached in the monomers of Formula I.
  • the linking group can be composed of any assembly of atoms, including oligomeric and polymeric chains.
  • the total number of atoms in the linking group can be from 3 to 200 atoms (e.g., from 3 to 150 atoms, from 3 to 100 atoms, from 3 and 50 atoms, from 3 to 25 atoms, from 3 to 15 atoms, or from 3 to 10 atoms).
  • the linking group can be, for example, an alkyl, alkoxy, alkylaryl, alkylheteroaryl, alkylcycloalkyl, alkylheterocycloalkyl, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, dialkylamino, alkylcarbonyl, alkoxy carbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, or polyamino group.
  • the linking group can comprises one of the groups above joined to one or both of the moieties to which it is attached by a functional group.
  • suitable functional groups include, for example, secondary amides (-CONH-), tertiary amides (-CONR-), secondary carbamates (-OCONH-; -NHCOO-), tertiary carbamates (-OCONR-; -NRCOO-), ureas (-NHCONH-; -NRCONH-; -NHCONR-, or -NRCONR-), carbinols ( -CHOH-, -CROH-), ethers (-O-), and esters (-COO-, -CH2O2C-, CHRO2C-), wherein R is an alkyl group, an aryl group, or a heterocyclic group.
  • the linking group can comprise an alkyl group (e.g., a C1-C12 alkyl group, a Ci-Cs alkyl group, or a Ci-Ce alkyl group) bound to one or both of the moieties to which it is attached via an ester (-COO-, -CH2O2C-, CHRO2C-), a secondary amide (-CONH-), or a tertiary amide (-CONR-), wherein R is an alkyl group, an aryl group, or a heterocyclic group.
  • the linking group can be chosen from one of the following: where m is an integer from 1 to 12 and R 1 is, independently for each occurrence, hydrogen, an alkyl group, an aryl group, or a heterocyclic group.
  • the linker can serve to modify the solubility of the compounds described herein.
  • the linker is hydrophilic.
  • the linker can be an alkyl group, an alkylaryl group, an oligo- or polyalkylene oxide chain (e.g., an oligo- or polyethylene glycol chain), or an oligo- or poly(amino acid) chain.
  • Modified alginate polymers can be of any desired molecular weight. The weight average molecular weight of the alginates is preferably between 1,000 and 1,000,000 Daltons, more preferably between 10,000 and 500,000 Daltons as determined by gel permeation chromatography.
  • Modified alginate polymers can contain any ratio of mannuronate monomers, guluronate monomers, and covalently modified monomers. In some embodiments, greater than 2.5%, 5%, 7.5%, 10%, 12%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 26%, 28%, 30%, 32.5%, 35%, 37.5%, 40%, 45%, 50%, 55%, or 60% of the monomers in the modified alginate polymer are covalently modified monomers. Preferably greater than 10%, more preferably greater than 20%, and most preferably greater than 30% of the monomers in the modified alginate polymer are covalently modified monomers.
  • Active Agent refers to a physiologically or pharmacologically active substance that acts locally and/or systemically in the body.
  • An active agent is a substance that is administered to a patient for the treatment (e.g., therapeutic agent), prevention (e.g, prophylactic agent), or diagnosis (e.g, diagnostic agent) of a disease or disorder.
  • the active agent can be a small molecule, or a biologic.
  • a biologic is a medicinal product manufactured in, extracted from, or semi-synthesized from biological sources which is different from chemically synthesized pharmaceuticals.
  • biologies used as the active agent can include, for example, antibodies, blood components, allergenics, gene therapies, and recombinant therapeutic proteins.
  • Biologies can comprise, for example, sugars, proteins, or nucleic acids, and they can be isolated from natural sources such as human, animal, or microorganism.
  • the active agent can comprise an anti-cancer drug, a drug that promotes wound healing, a drug that treats or prevents infection, or a drug that promotes vascularization.
  • the active agent can comprise an anti-cancer drug, such as a chemotherapeutic or a cancer vaccine.
  • the anti-cancer drug can include a small molecule, a peptide or polypeptide, a protein or fragment thereof (e.g., an antibody or fragment thereof), or a nucleic acid.
  • Exemplary anti-cancer drugs can include, but are not limited to, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado- Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate, Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Ari
  • the active agent can comprise a drug that promotes wound healing or vascularization.
  • the active agent can comprise a drug that reduces ischemia, e.g., due to peripheral artery disease (PAD) or damaged myocardial tissues due to myocardial infarction.
  • PAD peripheral artery disease
  • the drug can comprise a protein or fragment thereof, e.g., a growth factor or angiogenic factor, such as vascular endothelial growth factor (VEGF), e.g., VEGFA, VEGFB, VEGFC, or VEGFD, and/or IGF, e.g., IGF-1, fibroblast growth factor (FGF), angiopoietin (ANG) (e.g., Angl or Ang2), matrix metalloproteinase (MMP), delta-like ligand 4 (DLL4), paclitaxel, or combinations thereof.
  • VEGF vascular endothelial growth factor
  • VEGFA vascular endothelial growth factor
  • VEGFB vascular endothelial growth factor
  • VEGFC vascular endothelial growth factor
  • IGF e.g., IGF-1, fibroblast growth factor (FGF), angiopoietin (ANG) (e.g., Angl or Ang2),
  • the active agent can comprise an anti-proliferative drug, e.g., my cophenolate mofetil (MMF), azathioprine, sirolimus, tacrolimus, paclitaxel, biolimus A9, novolimus, myolimus, zotarolimus, everolimus, or tranilast.
  • MMF my cophenolate mofetil
  • azathioprine sirolimus, tacrolimus, paclitaxel
  • biolimus A9 biolimus A9
  • novolimus myolimus
  • zotarolimus everolimus
  • tranilast tranilast
  • the active agent can comprise an anti-inflammatory drug, e.g., corticosteroid anti-inflammatory drugs (e.g., beclomethasone, beclometasone, budesonide, flunisolide, fluticasone propionate, triamcinolone, methylprednisolone, prednisolone, or prednisone); or non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., acetylsalicylic acid, diflunisal, salsalate, choline magnesium trisalicylate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, fluribiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, na
  • the active agent can comprise a drug that prevents or reduces transplant rejection, e.g., an immunosuppressant.
  • immunosuppressants include calcineurin inhibitors (e.g., cyclosporine, Tacrolimus (FK506)); mammalian target of rapamycin (mTOR) inhibitors (e.g., rapamycin, also known as Sirolimus); antiproliferative agents (e.g., azathioprine, my cophenolate mofetil, my cophenolate sodium); antibodies (e.g., basiliximab, daclizumab, muromonab); corticosteroids (e.g., prednisone).
  • calcineurin inhibitors e.g., cyclosporine, Tacrolimus (FK506)
  • mTOR mammalian target of rapamycin
  • antiproliferative agents e.g., azathioprine, my cophenolate mofetil, my cophenolate
  • the active agent can comprise an anti -thrombotic drug, e.g., an anti-platelet drug, an anticoagulant drug, or a thrombolytic drug.
  • an anti -thrombotic drug e.g., an anti-platelet drug, an anticoagulant drug, or a thrombolytic drug.
  • Exemplary anti-platelet drugs include an irreversible cyclooxygenase inhibitor (e.g., aspirin or triflusal); an adenosine diphosphate (ADP) receptor inhibitor (e.g., ticlopidine, clopidogrel, prasugrel, or tricagrelor); a phosphodiesterase inhibitor (e.g., cilostazol); a glycoprotein IIB/IIIA inhibitor (e.g., abciximab, eptifibatide, or tirofiban); an adenosine reuptake inhibitor (e.g., dipyridamole); or a thromboxane inhibitor (e.g., thromboxane synthase inhibitor, a thromboxane receptor inhibitor, such as terutroban).
  • ADP adenosine diphosphate
  • a phosphodiesterase inhibitor e.g., cilostazol
  • anti-platelet drugs are non-limiting, as the skilled artisan would be able to readily identify other anti-platelet drugs.
  • Exemplary anticoagulant drugs include coumarins (e.g., warfarin, acenocoumarol, phenprocoumon, atromentin, brodifacoum, or phenindione); heparin and derivatives thereof (e.g., heparin, low molecular weight heparin, fondaparinux, or idraparinux); factor Xa inhibitors (e.g., rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, letaxaban, or eribaxaban); thrombin inhibitors (e.g., hirudin, lepirudin, bivalirudin, argatroban, or dabigatran); antithrombin protein; batroxobin; hementin; and thrombomodulin.
  • coumarins e.
  • Exemplary thrombolytic drugs include tissue plasminogen activator (t-PA) (e.g., alteplase, reteplase, or tenecteplase); anistreplase; streptokinase; or urokinase.
  • tissue plasminogen activator e.g., alteplase, reteplase, or tenecteplase
  • anistreplase e.g., anistreplase
  • streptokinase e.g., reteplase, or tenecteplase
  • urokinase urokinase
  • the active agent can comprise a drug that prevents restenosis, e.g., an anti-proliferative drug, an anti-inflammatory drug, or an anti-thrombotic drug.
  • a drug that prevents restenosis e.g., an anti-proliferative drug, an anti-inflammatory drug, or an anti-thrombotic drug.
  • anti-proliferative drugs, anti-inflammatory drugs, and anti-thrombotic drugs are described herein.
  • the active agent can comprise a drug that treats or prevents infection, e.g., an antibiotic.
  • antibiotics include, but are not limited to, beta-lactam antibiotics (e.g., penicillins, cephalosporins, carbapenems), polymyxins, rifamycins, lipiarmycins, quinolones, sulfonamides, macrolides lincosamides, tetracyclines, aminoglycosides, cyclic lipopeptides (e.g., daptomycin), glycylcyclines (e.g., tigecycline), oxazonidinones (e.g., linezolid), and lipiarmycines (e.g., fidazomicin).
  • beta-lactam antibiotics e.g., penicillins, cephalosporins, carbapenems
  • polymyxins e.g., rifamycins, lipi
  • antibiotics include erythromycin, clindamycin, gentamycin, tetracycline, meclocycline, (sodium) sulfacetamide, benzoyl peroxide, and azelaic acid.
  • Suitable penicillins include amoxicillin, ampicillin, bacampicillin, carbenicillin, cioxacillin, dicloxacillin, flucioxacillin, mezlocillin, nafcillin, oxacillin, penicillin g, penicillin v, piperacillin, pivampicillin, pivmecillinam, and ticarcillin.
  • cephalosporins include cefacetrile, cefadroxil, cephalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil, cefuroxime, cefuzonam, cfcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefpimizole, cefpodoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime,
  • Monobactams include aztreonam.
  • Suitable carbapenems include imipenem/cilastatin, doripenem, meropenem, and ertapenem.
  • Exemplary macrolides include azithromycin, erythromycin, larithromycin, dirithromycin, roxithromycin, and telithromycin.
  • Lincosamides include clindamycin and lincomycin.
  • Exemplary streptogramins include pristinamycin and quinupristin/dalfopristin.
  • Suitable aminoglycoside antibiotics include amikacin, gentamycin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, and tobramycin.
  • Exemplary quinolones include flumequine, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, lomefloxacin, nadifloxacin, norfloxacin, ofoxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, repafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, besifloxacin, clinafoxacin, gemifloxacin, sitafloxacin, trovafloxacin, and prulifloxacin.
  • Suitable sulfonamides include sulfamethizole, sulfamethoxazole, and trimethoprim-sulfamethoxazone.
  • Exemplary tetracyclines include demeclocycline, doxycycline, minocycline, oxy tetracycline, tetracycline, and tigecycline.
  • antibiotics include chloramphenicol, metronidazole, tinidazole, nitrofurantoin, vancomycin, teicoplanin, telavancin, linezolid, cycloserine, rifampin, rifabutin, rifapentin, bacitracin, polymyxin B, viomycin, and capreomycin.
  • metronidazole metronidazole
  • tinidazole nitrofurantoin
  • vancomycin teicoplanin
  • telavancin linezolid
  • cycloserine rifampin
  • rifabutin rifapentin
  • bacitracin polymyxin B
  • viomycin viomycin
  • capreomycin capreomycin
  • the active agent can comprise a drug that reduces macular degeneration.
  • macular degeneration One common current treatment for macular degeneration involves the injection of anti-angiogenesis compounds intraocularly (Lucentis, Eylea). The repeated intraocular injections are sometimes poorly tolerated by patients, leading to low patient compliance.
  • the ability to noninvasively refill drug depots for macular degeneration significantly improves patient compliance and patient tolerance of disease, e.g., macular degeneration, treatment. Controlled, repeated release made possible by the methods described herein allows for fewer drug dosings and improved patient comfort.
  • the active agent can comprise a drug that prevents immunological rejection.
  • a drug that prevents immunological rejection Prior to the invention described herein, to prevent immunological rejection of cells, tissues or whole organs, patients required lifelong therapy of systemic anti-rejection drugs that cause significant side effects and deplete the immune system, leaving patients at greater risk for infection and other complications.
  • the ability to locally release anti-rejection drugs and to repeatedly load compound allows for more local anti-rejection therapy with fewer systemic side effects, improved tolerability and better efficacy.
  • the active agent can comprise a drug that prevents thrombosis.
  • Some vascular devices such as vascular grafts and coated stents suffer from thrombosis, in which the body mounts a thrombin-mediated response to the devices.
  • Anti-thrombotic drugs released from these devices, allows for temporary inhibition of the thrombosis process, but the devices have limited drugs and cannot prevent thrombosis once the drug supply is exhausted. Since these devices are implanted for long periods of time (potentially for the entire lifetime of the patient), temporary thrombosis inhibition is not sufficient.
  • the ability to repeatedly and locally administer anti-thrombotic drugs and release the drug significantly improves clinical outcomes and allows for long-term thrombosis inhibition.
  • the active agent can comprise a drug that treats inflammation.
  • Chronic inflammation is characterized by persistent inflammation due to non-degradable pathogens, viral infections, or autoimmune reactions and can last years and lead to tissue destruction, fibrosis, and necrosis.
  • inflammation is a local disease, but clinical interventions are almost always systemic.
  • Anti-inflammatory drugs given systemically have significant side-effects including gastrointestinal problems, cardiotoxicity, high blood pressure and kidney damage, allergic reactions, and possibly increased risk of infection.
  • the ability to repeatedly and locally release anti-inflammatory drugs such as NSAIDs and COX-2 inhibitors could reduce these side effects.
  • Suitable active agents include, for example, immunotherapeutics/ immunoadjuvants such as checkpoint inhibitors and STING agonists and agonists for toll-like receptors.
  • STING ligands e.g., natural cyclic dinucleotides, cAIMP dinucleotide, fluorine-containing cyclic dinulcoetides, phosphorothioate-containing cyclic dinucleotides, DMXAA
  • TLR2 ligands e.g., poly(EC)
  • TLR4 ligands e.g., lipopolysaccharides, monophosphoryl lipid A, CRX-527
  • TLR5 ligands e.g., gardiquimod, imiquimod, loxoribine, resiquimod, imidazoquinolines, adenine base analogs, benzoazepine analogs
  • TLR9 ligands e.g., gardiquimod, imi
  • representative active agents include doxorubicin, paclitaxel, gemcitabine, topotecan, tacrolimus, mycophenolic acid, rapamycin, tesiquimod, erlotinib, DMXAA, CdN, temozolomide, and docetaxel.
  • the active agent can comprise an integrin-binding peptide, collagen-mimetic peptide, hydrazide, aldehyde, immunomodulating factor, angiogenesispromoting factor, or cell-signaling factor.
  • Alginate polymers modified with azide groups lose the ability to cross-link in the presence of calcium.
  • azide-PEG4-amine azide-amine
  • modifying alginate polymers with modifications that contain carboxyl groups would restore calcium cross-linking and termed such modifications “restorative”.
  • Azide-lysine was prepared utilizing a modified three-step literature protocol starting from 2-azidoacetic acid ( Figure 2). Synthesis of compound S4 began with NHS derivatization of 2-azidoacetic acid SI with N-hydroxysuccinimide. The azide-NHS was coupled to a-BOC protected lysine S2 to give a-BOC-y-azido-lysine S3, which was deprotected to give compound the final azide-lysine S4.
  • Table 1 Select information for modified alginate polymers used in this study.
  • Alginate hydrogels formed through vigorous mixing of alginate and calcium sulfate were subjected to rheological testing.
  • Rheological testing confirmed the overall observation that while low-DS alginate with either modification form viscoelastic gels, only the carboxyl-restoring modifications permitted calcium cross-linking at high DS. Because the advantage of restorative modifications was most marked at high-DS, subsequent experiments compared high-DS carboxyl-depleted and -restored alginate gels.
  • Alginate with restorative modifications showed gel-like behavior (storage modulus (G’) > loss modulus (G”)) with a relatively strong three-dimensional (3D) network due to the appearance of the G” peak as shown in Fig. 4B).
  • the linearity limiting value of the linear viscoelastic (LVE) range for the sample with restorative modifications was 26% as compared to 5% for unmodified gels (Fig. 4B, Fig. 5) indicating the modification indeed induced a relatively high degree of cross-linking and strengthened the structural organization of the sample.
  • Frequency sweep tests were performed within the LVE region (Fig.
  • Alginate conjugated to restorative modifications demonstrates improved gel retention and systemic capture in vivo.
  • Hydrogel function requires gels to remain at injected sites. We found that poorly crosslinking hydrogels lose their ability to stay at the injection site and migrate to other parts of the body. We tested the ability of alginate carrying depletive and restorative modifications to be retained at target sites.
  • Hydrogel retention at introduced sites is central to their use as tissue engineering scaffolds and as cell and drug delivery platforms. It was initially surprising that alginate hydrogels incorporating both restorative and depletive modifications were well retained at the injected tissue sites over two weeks, especially in light of previous published results in which poorly cross-linked alginate rapidly migrated away from the injection site. Thus, despite the complete loss of gelation behavior in vitro, some hydrogel coherence must have remained. However, calcium cross-linked alginate gels containing depletive modification had significantly increased accumulation at off-target sites, indicating that the loss of cross-linking does lead to polymer shedding over time. Lack of precise control over hydrogel localization and off-target accumulation can severely impede clinical translation so the restorative modifications are highly desirable in scaffold and drug delivery platforms that require high DS.
  • Alginate hydrogels using restorative modification of carboxyl groups demonstrated improved DBCO fluorescent capture from the blood at the site of the depot as compared to highly modified, poorly gelling hydrogels.
  • highly- modified alginates lost their retention capacity after injection into tissues. This difference is very likely due to degradation of the poorly cross-linked hydrogels and their migration away from target sites.
  • Alginate hydrogels with restorative carboxyl groups improved small molecule capture at intramuscular sites.
  • the amount of DBCO-fluorophore injected is still a small percentage of the theoretical number of available azides and we only expect 1-10% of the total molecule reaching the gel at any point, but the increased chance of an interaction between an azide and a circulating DBCO molecule increases the accumulation
  • alginate modifications that restore carboxyl groups on the polymer backbone represent a significant improvement in hydrogel and depot stability for in vivo applications. Modifications that restore carboxyl groups make possible mechanically robust, highly modified alginate gels with superior calcium cross-linking behavior, mechanical properties, in vivo retention and depot capture compared to modifications that deplete the carboxyl group.
  • Calcium sulfate dihydrate C3771
  • MES M3671
  • /V,/V-Diisopropylethylamine D125806
  • Azide-PEG4-amine (1868) was purchased from Lumiprobe corporation.
  • DBCO- sulfo-amine (1227) and DBCO-Cy7 (1047) were purchased from Click Chemistry Tools.
  • Ethyl-3- [3 -dimethylaminopropyl] carbodiimide hydrochloride (EDC, 024810) was purchased from Oakwood Chemical.
  • N-hydroxysuccinimide was purchased from Chem-Impex International (00182) or Alfa Aesar (Al 0312).
  • /V,/V-Dimethylformamide (227056) was purchased from Acros Organics.
  • Trifluoroacetic Acid (04901-500), Methanol (A433P-4), Acetonitrile (A998-4) and Toluene (T290-4) were purchased from Fisher Chemical.
  • Septa was removed and l-(3-dimethylaminopropyl)-3- ethylcarbondiimide hydrochloride (5.06 g, 26.4 mmol, 1.3 equiv.) was added. Septa was replaced and the reaction was set to stirring at room temperature. Then 2-azidoacetic acid (SI) (1.52 mL, 20.3 mmol 1 equiv.) was injected into reaction.
  • SI 2-azidoacetic acid
  • Nutrition grade, high guluronic acid content, high molecular weight (MW) alginate purchased from Dupont Nutrition and Health was first dissolved in DI water and charcoal was added (0.5 g of charcoal per 1 g of alginate), filtered, and then MES buffer was added to reach the desired 0.5% weight per volume (w/v) concentration (1 g of alginate in 200 mL, 20 pM, 1 eq.) (100 mM MES, 300 mM NaCl, pH: 6.5).
  • EDC 40 mM, 2000 eq.
  • NHS 20 mM, 1000 eq.
  • the solution was dialyzed against 4 L of water with successively lower salt content, changing solution 2-3 times per day for 4-5 days. Dialyzed solutions were frozen and lyophilized under high vacuum.
  • the samples were dissolved in 0.5% w/v in IX MES buffer solution (1 g in 200 mL) and the coupling steps were repeated as described. Azide quantitation. The number of azides conjugated to the alginate was quantified by looking at the decrease in DBCO absorbance upon incubation of alginate azides with a known excess amount of DBCO. 0.1% w/v (2.5 mg in 2.5 ml) alginate solutions were made in phosphate buffer solution (PBS).
  • PBS phosphate buffer solution
  • 80 pM solutions of DBCO-amine were made and three different amounts of alginate was added (400 pmol, 800 pmol and 1.6 nmol of alginate) to separate 80 pM solutions of DBCO-amine.
  • a DBCO negative control solution was also tested along with a positive control consisting of DBCO reacted with sodium azide (1 umol, 2500 eq.).
  • Spectrophotometry was performed using a UV/Vis spectrophotometer (Thermo Scientific Nanodrop 2000c) using a cuvette with a 1 cm pathlength. Absorbance changes were observed at 308 nm wavelength. This process was repeated with replicates of the 0.1% alginate (alg) solutions and the absorbance values were averaged. Decrease in absorbance in alginate samples compared to DBCO negative control indicated the quantity of azides that reacted with DBCO.
  • Alginate was dissolved in PBS (2% w/v, 20 mg in 1 mL). 10:1 ratio of the 2% w/v alginate solution was mixed with a calcium sulfate solution (final concentration of 18.2 mM) in a two syringe system while minimizing air. The solutions were mixed by pushing the syringe barrels back and forth 10 times. This system was used to create gel sizes ranging from 400 pL to 1 mL.
  • Time sweep Two minutes of time sweep was performed at 0.2% strain and a frequency of 10 Hz and a total 150 data points at a constant rate were collected before and between each test performed.
  • Amplitude sweep test of the gel samples was performed on a logarithmic ramp ranging from 0.01 to 500% at 10 Hz with 3 seconds of conditioning and sampling time, and 10 data points per decade were recorded.
  • Frequency sweep Frequency sweep on a logarithmic ramp ranging from 0.01-100 Hz at 0.2% strain was performed with 3 seconds of sample conditioning and sampling time, and 10 data points per decade were recorded.
  • Cyclic amplitude sweep A continuous cyclic amplitude sweep test was performed in five intervals (1-5). Intervals 1, 3 and 5 were set at 0.2% strain, 10 Hz for 2 minutes, whereas intervals 2 and 4 set at 500% strain, 10 Hz for one minute.
  • Shear rate ramp Two continuous shear rate ramps from 0 to 50 s-1 and 50 to 0 s-1 for 2.5 minutes each were performed to study the continuous flow behavior of the gel. Total of 74 data points with 20 data points per decade was recorded.
  • Cy7 fluorescence was monitored over two weeks using an IVIS imager to obtain a fluorescence signal.
  • ICG/ICG excitation and emission filters were used for all IVIS images presented and no image math in the Living Image software was performed.
  • For all IVIS images only radiance efficiency values were used to normalize the data over variable exposure times.
  • Regions of Interest (ROIs) were used to sum the fluorescent signal associated with the injected calf, the injected ankle, and the contralateral calf.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Polymers & Plastics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Materials Engineering (AREA)
  • Biochemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)
  • Materials For Medical Uses (AREA)

Abstract

La perte de capacités de réticulation dans des échafaudages d'alginates hautement substitués est un problème important affectant l'utilisation de polymères d'alginates dans des implants, l'administration de cellules et l'ingénierie tissulaire. Des échafaudages d'alginates modifiés qui conservent leurs propriétés de gélification sous une forte modification chimique sont divulgués, ainsi que des procédés de fabrication et d'utilisation associés.
EP22888557.0A 2021-10-28 2022-10-28 Alginates modifiés et procédés de fabrication et d'utilisation associés Pending EP4422691A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US202163272975P 2021-10-28 2021-10-28
US202163288906P 2021-12-13 2021-12-13
PCT/US2022/078944 WO2023077112A1 (fr) 2021-10-28 2022-10-28 Alginates modifiés et procédés de fabrication et d'utilisation associés

Publications (2)

Publication Number Publication Date
EP4422691A1 true EP4422691A1 (fr) 2024-09-04
EP4422691A4 EP4422691A4 (fr) 2025-08-27

Family

ID=86158838

Family Applications (1)

Application Number Title Priority Date Filing Date
EP22888557.0A Pending EP4422691A4 (fr) 2021-10-28 2022-10-28 Alginates modifiés et procédés de fabrication et d'utilisation associés

Country Status (4)

Country Link
EP (1) EP4422691A4 (fr)
JP (1) JP2024540178A (fr)
CA (1) CA3236482A1 (fr)
WO (1) WO2023077112A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117123277B (zh) * 2023-07-21 2025-07-22 华中农业大学 一种硫掺杂核壳状异质结构多相Fenton催化剂及其制备方法与应用
WO2025064911A1 (fr) * 2023-09-21 2025-03-27 North Carolina State University Systèmes, compositions et procédés de transduction universelle

Also Published As

Publication number Publication date
JP2024540178A (ja) 2024-10-31
WO2023077112A1 (fr) 2023-05-04
EP4422691A4 (fr) 2025-08-27
CA3236482A1 (fr) 2023-05-04

Similar Documents

Publication Publication Date Title
AU2019200810B2 (en) Modified alginates for anti-fibrotic materials and applications
Huang et al. Hydrogel scaffolds for differentiation of adipose-derived stem cells
US20200197526A1 (en) Refillable drug delivery devices and methods of use thereof
Ding et al. Weak bond-based injectable and stimuli responsive hydrogels for biomedical applications
Kempe et al. In situ forming implants—an attractive formulation principle for parenteral depot formulations
EP2455104B1 (fr) Hydrogels de PEG bio-fonctionnalisés et solubles dépendant à un stimulus
CN102573944B (zh) 原位成型水凝胶及其生物医学用途
Liu et al. Chitosan derivatives co-delivering nitric oxide and methicillin for the effective therapy to the methicillin-resistant S. aureus infection
Wei et al. Biomimetic joint paint for efficient cartilage repair by simultaneously regulating cartilage degeneration and regeneration in pigs
CA2720203A1 (fr) Composition osteogenique comprenant un complexe facteur de croissance/polymere amphiphile un sel soluble de cation et un support organique
EP2178565A1 (fr) COMPLEXES ENTRE UN POLYMÈRE AMPHIPHILE ET UNE PROTÉINE OSTÉOGÉNIQUE APPARTENANT À LA FAMILLE DES BMPs
EP4422691A1 (fr) Alginates modifiés et procédés de fabrication et d'utilisation associés
Moore et al. Impact of composite scaffold degradation rate on neural stem cell persistence in the glioblastoma surgical resection cavity
US20240409672A1 (en) Modified alginates and methods of making and using thereof
JP2009523791A (ja) １．０〜１００ｋＤａの分子量を有するデキストランを含む薬剤及びその調製方法
US20210353767A1 (en) Compositions and methods for drug delivery
CN118434448A (zh) 修饰的海藻酸盐及其制备和使用方法
ES2874930T3 (es) Dispositivos recargables de administración de fármacos y procedimientos de uso de los mismos
JP2019528837A (ja) アルブミンを用いたコンビネーション、特に軟骨欠損治療用のコンビネーション
KR102709709B1 (ko) 주사 가능한 포도당 산화 효소 고정화 젤라틴 하이드로겔의 종양 재발 억제 용도
US20230390403A1 (en) Compositions and methods for drug delivery
US20220401562A1 (en) Refillable drug delivery devices and methods of use thereof
Pandit Structure-Function Relationships of Polyester Coacervates as Hemostats and Drug Carriers
KR101986175B1 (ko) 케라틴을 포함하는 항암용 조성물
Noverraz Development of functionalized alginate-based hydrogels to reduce fibrosis in the transplantation of microencapsulated cells

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20240426

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20250725

RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 47/36 20060101AFI20250721BHEP

Ipc: A61K 31/734 20060101ALI20250721BHEP

Ipc: A61K 47/30 20060101ALI20250721BHEP

Ipc: C08B 37/00 20060101ALI20250721BHEP

Ipc: C08J 3/075 20060101ALI20250721BHEP

Ipc: C08L 5/04 20060101ALI20250721BHEP

Ipc: A61K 47/61 20170101ALI20250721BHEP

Ipc: A61K 47/69 20170101ALI20250721BHEP