WO2019084548A1 - Coacervats complexes liquides de solidification in situ pour l'administration locale d'agents anti-angiogéniques ou d'agents chimiothérapeutiques - Google Patents

Coacervats complexes liquides de solidification in situ pour l'administration locale d'agents anti-angiogéniques ou d'agents chimiothérapeutiques

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Publication number
WO2019084548A1
WO2019084548A1 PCT/US2018/058015 US2018058015W WO2019084548A1 WO 2019084548 A1 WO2019084548 A1 WO 2019084548A1 US 2018058015 W US2018058015 W US 2018058015W WO 2019084548 A1 WO2019084548 A1 WO 2019084548A1
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WO
WIPO (PCT)
Prior art keywords
agent
salt
liquid complex
solidifying liquid
coacervate
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PCT/US2018/058015
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English (en)
Inventor
Russell Stewart
Joshua Preston JONES
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University Of Utah Research Foundation
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Publication of WO2019084548A1 publication Critical patent/WO2019084548A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/416—Anti-neoplastic or anti-proliferative or anti-restenosis or anti-angiogenic agents, e.g. paclitaxel, sirolimus

Definitions

  • Transarterial embolization is a procedure in which a microcatheter is inserted into and guided through a peripheral artery to a target tissue for localized delivery of an embolic agent to selectively block blood flow.
  • TAE is used to treat abnormal vasculature, such as aneurysms and arteriovenous malformations (AVMs), to control gastrointestinal bleeding, and to treat tumors of the head, neck, liver, kidney, and colon.
  • AFMs arteriovenous malformations
  • TACE tumor transarterial chemoembolization
  • TACE tumor transarterial chemoembolization
  • Both TAE and TACE treatment of un-resectable tumors have repeatedly demonstrated survival benefit.
  • both of these embolization methods result in tumor hypoxia, which stimulates the release of vascular endothelial growth factors (VEGFs), often leading to re-vascularization and rebound of the tumor through angiogenesis.
  • VEGFs vascular endothelial growth factors
  • embolized AVMs can re-vascularize through angiogenesis.
  • Angiogenesis is the sprouting of new blood vessels from pre-existing vessels, as opposed to de novo vasculogenesis.
  • Angiogenesis is initiated when endothelial cells are activated in response to a variety of angiogenic signals. Activated endothelial cells detach from perivascular smooth muscle cells, sprout along concentration gradients of angiogenic growth factors, coalesce to form endothelial tubes, and recruit perivascular cells to form vessels. Remodeling and pruning complete the maturation process.
  • Angiogenesis is a cancer therapeutic target, as it has been demonstrated that angiogenesis is necessary for tumor development. In response to hypoxia and nutrient deprivation, an "angiogenic switch" leads tumors to progress into a proangiogenic phenotype. Growing evidence suggests this is a significant factor in poor clinical outcomes of cancer treatments.
  • hypoxia inducible factor 1 a transcription factor that functions as a master regulator of several genes involved in angiogenesis including VEGFs and their receptors (VEGFR-1 and VEGFR-2).
  • VEGFs and their transmembrane tyrosine kinase receptors play a prominent role in promoting angiogenesis.
  • the VEGFRs are expressed primarily on vascular endothelial cells. VEGF binding to the receptors stimulates endothelial cell mitosis and promotes vascular permeability, which leads to extravasation of plasma proteins and the formation of a provisional extracellular matrix (ECM), along which endothelial cells can migrate.
  • ECM provisional extracellular matrix
  • hypoxia secondary to embolization of hypervascularized tumors has been shown to stimulate angiogenesis.
  • TACE therapy show significantly higher VEGF expression after TACE therapy.
  • overexpression of VEGF after TACE is associated with development of metastases.
  • decreased VEGF levels are associated with longer patient survival.
  • Tyrosine kinase inhibitors are hydrophobic drugs that cross the cell membrane and bind to the kinase domain of RTKs. TKIs inhibit RTKs by one of three mechanisms: Type I TKIs are ATP analogs, Type II are allosteric inhibitors, and Type III are suicide substrates that covalently inactivate the kinase. Type I inhibitors have the widest range of activity because of the conservation of the ATP binding region. Type II inhibitors are more selective, while covalent inhibitors, targeting a single amino acid, are the most selective.
  • TKIs for the treatment of hypervascularized tumors include sunitinib malate (SUTENT®; SUN), pazopanib hydrochloride (VOTRIENT®; VAN), sorafenib tosylate (NEXAAR®; SOR) and vandetanib (CAPRELSA®; VAN).
  • SUN, PAZ, and SOR all target multiple RTKs, including VEGFR-2 and PDGF- ⁇ .
  • VAN is more selective, targeting VEGFR-2 and EGFR. All of these TKIs are administered orally and have broad systemic side effects.
  • systemic antiangiogenic therapy can have serious side effects including gastrointestinal perforation, cardiac impairment, hypothyroidism, leukoencephalopathy, impaired wound healing, hemorrhage, thrombosis, hypertension, and proteinuria.
  • sorafenib, sunitinib, and bevacizumab in conjunction with TACE have been stopped early because of severe adverse effects.
  • liquid complex coacervates composed of one or more anti-angiogenic agents or chemotherapeutic agents that solidify in situ in blood vessels.
  • Oppositely charged polyelectrolytes were designed to form liquid complex coacervates at high ionic strengths but undergo aqueous aqueous phase inversion to produce a microporous solid at physiological ionic strengths.
  • the anti-angiogenic agent or chemotherapeutic is subsequently released locally at a prolonged rate from the microporous solid.
  • Figure 1 shows a schematic of the embolic coacervate setting mechanism. Ionic shielding limits strong interpolymer interactions; dynamic interactions create a liquid form. Diffusion of ions out of the coacervate in physiological conditions allows stronger, less dynamic interactions between polyions, which creates a microporous solid form.
  • Figure 2 A shows SS injection pressures for PG-MP (+Ta) in 135 cm catheters, 0.026" ID vs. NaCl concentration.
  • FIGS 3A-D show EC injected through a 135 cm, 0.026" ID model catheter into balanced salt solution (BSS).
  • Figures 4A-4B show the in vitro cumulative release profile for drug-releasing ECs in balanced salt solution (BSS) for sunitinib malate (Sun)(8A) and doxorubicin hydrochloride (8B). Dashed lines represent fits to zero-order release model out to depletion.
  • Optional or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • the phrase “optionally comprises a contrast agent” means that the contrast agent can or cannot be included in the compositions and that the description includes both compositions including the contrast agent and excluding the contrast agent.
  • X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
  • a weight percent of a component is based on the total weight of the formulation or composition in which the component is included.
  • alkyl group as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 25 carbon atoms, such as methyl, ethyl, ⁇ -propyl, isopropyl, w-butyl, isobutyl, i-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like.
  • longer chain alkyl groups include, but are not limited to, a palmitate group.
  • a "lower alkyl” group is an alkyl group containing from one to six carbon atoms.
  • cycloalkyl group is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
  • the term “treat” as used herein is defined as maintaining or reducing the symptoms of a pre-existing condition when compared to the same symptoms in the absence of the complex coacervate.
  • prevent as used herein is the ability of the in situ complex coacervates described herein to completely eliminate the activity or reduce the activity when compared to the same activity in the absence of the complex coacervate.
  • inhibitor refers to the ability of the complex coacervate to slow down or prevent a process such as, for example, angiogenesis.
  • Subject refers to mammals including, but not limited to, humans, non- human primates, sheep, dogs, rodents (e.g., mouse, rat, guinea pig, etc.), cats, rabbits, cows, and non-mammals including chickens, amphibians, and reptiles.
  • rodents e.g., mouse, rat, guinea pig, etc.
  • cats rabbits, cows, and non-mammals including chickens, amphibians, and reptiles.
  • physiological conditions refers to conditions such as pH, temperature, etc. within the subject.
  • physiological pH and temperature of a human are 7.2 and 37 °C, respectively.
  • each of the combinations A + E, A + F, B + D, B + E, B + F, C + D, C + E, and C + F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination of A + D.
  • any subset or combination of these is also specifically contemplated and disclosed.
  • the sub-group of A + E, B + F, and C + E is specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination of A + D.
  • This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of making and using the disclosed compositions.
  • steps in methods of making and using the disclosed compositions are examples of steps in methods of making and using the disclosed compositions.
  • each such combination is specifically contemplated and should be considered disclosed.
  • Described herein are the preparation and use of in situ liquid complex coacervates for the local delivery of anti-angiogenic agents and chemotherapeutic agents.
  • the in situ liquid complex coacervates solidify upon administration of the liquid complex coacervate to the subject.
  • PEs polyelectrolytes
  • the polymers associate and undergo liquid liquid phase separation (LLPS).
  • the dense and concentrated PE phase is called a complex coacervate.
  • Fig. 1 shows a schematic of the embolic coacervate setting mechanism.
  • Ionic shielding limits strong interpolymer interactions; dynamic interactions create a liquid form. Diffusion of ions out of the coacervate in physiological conditions allows stronger, less dynamic interactions between polyions, which creates a solid in situ.
  • the solid produced in situ is a cohesive, non-fluid, water insoluble material.
  • the solid can be a gel such as, for example, a microporous gel-like solid or water insoluble ionic hydrogel.
  • gel is defined herein as a non-fluid colloidal network or polymer network that is expanded throughout its whole volume by a fluid.
  • the liquid complex coacervates described herein are liquids.
  • the liquid complex coacervates described herein have a completely different morphology compared to corresponding solids produced in situ despite the fact that the poly cation and polyanion composition in the liquid complex coacervate and the solid are identical.
  • the elastic modulus of the solid formed in situ is at least 1,000 times greater than the corresponding in situ liquid complex coacervate.
  • the elastic modulus of the solid is at least 5,000 times greater than, 10,000 times greater than, 15,000 times greater than, or 20,000 times greater than the corresponding in situ liquid complex coacervate.
  • the in situ formed solid is in the form of a microporous microparticle or depot, which makes the solids produced by the in situ solidifying liquid complex coacervates effective as a device for the local delivery of the anti-angiogenic agent.
  • compositions and methods for locally delivering an anti-angiogenic agent into a subject where the method involves introducing into the subject a in situ solidifying liquid complex coacervate with at least one polyguanidinyl copolymer, a polyphosphate, an anti-angiogenic agent, and a monovalent salt that produces monovalent ions in water, where the concentration of the monovalent ions in the complex coacervate is greater than the concentration of the monovalent ions in the subject, wherein upon introduction of the in situ solidifying liquid complex coacervate into the subject, a solid is produced in situ.
  • compositions and methods for locally delivering a chemotherapeutic agent into a subject where the method involves introducing into the subject a in situ solidifying liquid complex coacervate with at least one polyguanidinyl copolymer, a polyphosphate, a chemotherapeutic agent, and a monovalent salt that produces monovalent ions in water, where the concentration of the monovalent ions in the complex coacervate is greater than the concentration of the monovalent ions in the subject, wherein upon introduction of the in situ solidifying liquid complex coacervate into the subject, a solid is produced in situ.
  • the in situ solidifying liquid complex coacervate when introduced into a vessel in the subject, a solid is produced from the liquid complex coacervate. Further in this aspect, the adhesive solid creates an artificial embolus within the vessel. Still further in this aspect, the artificial embolus selectively reduces or inhibits blood flow to the target tissue.
  • Polyelectrolytes with opposite net charges in aqueous solution can associate into several higher order morphologies depending on the solution conditions and charge ratios. They can form stable colloidal suspensions of polyelectrolyte complex with net surface charges. Repulsion between like surface charges stabilizes the suspension from further association.
  • the initial complex can further coalesce and settle out into a dense fluid phase in which the opposite macroion charges are approximately equal. This process is referred to as complex coacervation and the dense fluid morphology as a complex coacervate.
  • the process is an associative macrophase separation of an aqueous solution of two oppositely charged polyelectrolytes into two liquid phases, a dense concentrated polyelectrolyte phase in equilibrium with a polyelectrolyte depleted phase.
  • the aqueous coacervate phase can be dispersed into the aqueous depleted phase but quickly settles back out, like oil droplets in water.
  • the spontaneous demixing of paired polyelectrolytes into complex coacervates occurs when the attractive forces between polyelectrolyte pairs are stronger than repulsive forces.
  • rheology experiments can identify copolymers that produce a liquid coacervate with the desired properties for any given situation.
  • having the lowest initial velocity is desirable to ensure easy injection through the smallest bore transarterial catheters.
  • rapid in situ setting in response to the ionic strength differential between the liquid coacervate and tissue is desirable.
  • transition to a solid (high elastic modulus, G') final form is desirable in order to ensure robust and stable occlusion in large diameter blood vessels.
  • the in situ solidifying liquid complex coacervate is introduced into a subject, wherein upon introduction of the liquid complex coacervate into the subject, a solid is produced in situ.
  • the in situ solidifying liquid complex coacervates undergo aqueous aqueous phase inversion to produce a microporous solid at physiological ionic strengths.
  • no organic solvents e.g., DMSO, N-methyl-2-pyrrolidone
  • the aqueous aqueous phase inversion of the liquid complex coacervates described herein can produce microporous solids with varying properties and features.
  • the in situ solidifying liquid complex coacervates described herein are liquids at ionic strengths higher than the ionic strength of the application site, but
  • microporous solids at the ionic strength of the application site.
  • the complex coacervates forms a solid in situ phase inversion at the application site as the salt concentration in the complex coacervate equilibrates to the application site salt concentration.
  • the polyguanidinyl copolymer is composed of a polymer backbone with a plurality of guanidinyl groups.
  • the guanidinyl groups are pendant to the polymer backbone.
  • the number of guanidinium groups present on the poly cation ultimately determines the charge density of the poly cation.
  • the guanidinium group can be derived from a residue of arginine attached to a polymer.
  • Any anionic counterions can be used in association with the cationic polymers.
  • the counterions should be physically and chemically compatible with the essential components of the composition and do not otherwise unduly impair product performance, stability, or aesthetics.
  • Non-limiting examples of such counterions include halides (e.g., chloride, fluoride, bromide, or iodide), sulfate, sulfate, methylsulfate, acetate, and other monovalent carboxylic acids.
  • the poly guanidinyl copolymer is a synthetic compound prepared by the free radical polymerization between a monomer such as an acrylate, a methacrylate, an acrylamide, a methacrylamide, or any combination thereof, and a guanidinyl monomer of formula I
  • R 1 is a hydrogen or an alkyl group
  • X is oxygen or NR 5 , where R 5 is a hydrogen or an alkyl group
  • m is from 1 to 10, or the pharmaceutically acceptable salt thereof.
  • R 1 is methyl
  • X is NH
  • m is 3.
  • the monomer is methacrylamide.
  • the mole ratio of the guanidinyl monomer of formula I to the monomer is from 1 : 1 to 10: 1, or is 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1, where any ratio can be a lower and upper end-point of a range (e.g., 2: 1 to 5: 1, etc.). In one aspect, the mole ratio of the guanidinyl monomer of formula I to the monomer is from 3: 1 to 4: 1.
  • the poly guanidinyl copolymer can be synthesized by using polymerization techniques known in the literature such as, for example, RAFT polymerization (i.e., reversible addition-fragmentation chain-transfer polymerization) or other methods such as free radical polymerization.
  • the polymerization reaction can be carried out in an aqueous environment.
  • the molecular weight distribution of the polyguanidinyl copolymer is distributed around an average molecular weight between 5 kDa to 100 kDa, or can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 kDa, where any value can be a lower and upper end-point of a range (e.g., 10 to 30 kDa, etc.).
  • multiple copolymers with controlled M m and narrow polydispersity indices can be synthesized by RAFT polymerization.
  • the copolymer has an average molar weight (M m ) from about 2 kg/mol to about 80 kg/mol, or can be about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 kg/mol, where any value can be a lower and upper end-point of a range (e.g., 10 to 25 kg/mol, etc.).
  • the polyguanidinyl copolymer is a multimodal
  • multimodal polyguanidinyl copolymer is polyguanidinyl copolymer with a distribution curve being the sum of at least two or more molecular weight unimodal distribution curves.
  • polyguanidinyl copolymer has a multimodal distribution of polyguanidinyl copolymer molecular weights with modes between 5 and 100 kDa, or can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 kDa, where any value can be a lower and upper end-point of a range (e.g., 10 to 30 kDa, etc.).
  • the number of guanidinyl side groups can vary from about 50 to about 100 mol %, or can be about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mol %, where any value can be a lower and upper end-point of a range (e.g., 60 to 90 mol %, etc.).
  • the guanidinyl side groups are from about 70 to about 80 mol % of the polyguanidinyl copolymer.
  • comonomer concentration can vary from about 50 to about 0 mol %, or can be about 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 0 mol %, where any value can be a lower and upper end-point of a range (e.g., 10 to 40 mol %, etc.).
  • the M n , PDI, and structures of the copolymers can be verified by size exclusion chromatography (SEC), X H NMR, and 1 C NMR or other common techniques. Exemplary procedures for preparing and characterizing co polymers useful herein are provided in the Examples below.
  • the in situ solidifying liquid complex coacervates described herein include a polyphosphate, which functions as a polyanion.
  • the polyphosphate can be a synthetic polymer or naturally-occurring having a plurality of phosphate groups (PO 3 ).
  • PO 3 phosphate groups
  • the polyphosphate has from 5 to 100 phosphate groups, or can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 groups, where any value can be a lower and upper end-point of a range (e.g., 5 to 50, 5 to 20, 10 to 30, etc.).
  • the polyphosphate can be an inorganic polyphosphate including a cyclic inorganic polyphosphate having the formula (P n 0 3n ) n" or a linear inorganic polyphosphate having the formula (P n 03n+i) n+2" .
  • the polyanion is an inorganic polyphosphate possessing a plurality of phosphate groups (e.g., NaP0 3 ) n , where n is 3 to 10 or is 3, 4, 5, 6, 7, 8, 9, or 10).
  • examples of inorganic phosphate include, but are not limited to, Graham salts, hexametaphosphate salts, and triphosphate salts.
  • the counterions of these salts can be monovalent cations such as, for example, Na + , K + , NH 4 + , or a combination thereof.
  • the polyanion is a phosphorylated sugar.
  • the sugar can be a hexose or pentose sugar. Additionally, the sugar can be partially or fully
  • the phosphorylated sugar is inositol hexaphosphate (IP6).
  • Salts that Produce Ions Any salt that produces an ion in water can be used in the complex coacervates described herein.
  • concentration and identity of the salt can be varied depending upon the application and conditions (e.g., pH, valency of the salt, etc.) of which the in situ liquid complex coacervate is used.
  • the salt is a monovalent salt.
  • the monovalent salt can be any salt that produces monovalent ions in water.
  • the monovalent salt can be a biocompatible salt such as, for example, sodium chloride, sodium acetate, sodium carbonate, or any combination thereof.
  • the salt produces zwitterions in water.
  • the salt can produce a zwitterion by varying the pH of the solution.
  • an amino acid salt can produce zwitterions in the complex coacervates described herein.
  • the compound can be a multivalent salt that produces ions having a plus or minus charge of greater than or equal to 2.
  • the multivalent salt can be a salt of a compound possessing two or more carboxylic acid groups (e.g., a tartrate salt, a citrate salt, etc.).
  • the multivalent salt can be a phosphate salt (e.g., sodium phosphate).
  • the salt that produces ions is present at a concentration of from 1.5 to 10 times greater than the concentration of the ions at the administration site of the subject.
  • concentration of the ions present in the subject can vary in the subject; thus, the concentration of the salt that produces ions in the complex coacervate can be tailored to specific applications.
  • concentration in the complex coacervate is 1.5, 2, 2.5, 3, 3.4, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times greater than the concentration of the ions at the administration site of the subject, where any value can be a lower and upper end-point of a range (e.g., 2.5 to 7.5, etc.).
  • the salt concentration can be from about 150 to about 1500 mM, or can be about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1150, 1200, 1250, 1300, 1400, or 1500 mM, where any value can be a lower and upper end-point of a range (e.g., 1,000 to 1,400 mM, etc.).
  • the salt is NaCl and the concentration is about 1200 mM (1.2 M).
  • the in situ solidifying liquid complex coacervate can be formulated in hypertonic saline solutions that can be used for parenteral or intravenous administration or by injection to a subject.
  • the in situ solidifying liquid complex coacervate can be formulated in Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or other buffered saline solutions that can be safely administered to a subject, wherein the saline concentration has been adjusted so that it is greater than saline concentration at physiological conditions.
  • the in situ solidifying liquid complex coacervates described herein are useful in delivering anti-angiogenic agents to a subject.
  • Anti-angiogenic agents are in general hydrophobic molecules, and the in situ solidifying liquid complex coacervates permit the delivery of these molecules in aqueous environments.
  • the coacervates disclosed herein are effective vehicles for the delivery of high local concentrations of anti-angiogenic agents.
  • guanidinium itself has aromatic character, referred to as Y aromaticity, because of the derealization of the pi electrons over three N-C bonds.
  • the anti-angiogenic agent is present in an amount of from 0.1 mg/mL to 100 mg/mL of in situ solidifying liquid complex coacervate. Further in this aspect, the anti-angiogenic agent is present at about 0.001, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4., 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/mL of in situ solidifying liquid complex coacervate, , where any value can be a lower and upper end-point of a range (e.g., 0.1 to 10 mg/mL, etc.).
  • the anti-angiogenic agent is an FDA-approved anti-angiogenic agent.
  • the anti-angiogenic agent is a tyrosine kinase inhibitor (TKI).
  • TKI tyrosine kinase inhibitor
  • angiogenesis is, in large part, initiated and maintained by cell signaling through receptor tyrosine kinases (RTKs).
  • RTKs include receptors for several angiogenesis promoters, including VEGF, which stimulates vascular permeability, proliferation, and migration of endothelial cells; PDGF, which recruits pericytes and smooth muscle cells that support the budding endothelium; and FGF, which stimulates proliferation of endothelial cells, smooth muscle cells, and fibroblasts.
  • the anti-angiogenic agent is a TKI such as sunitinib malate (SUN), pazopanib hydrochloride (PAZ), sorafenib tosylate (SOR), vandetanib (VAN), cabozantinib, or any combination thereof.
  • SUN sunitinib malate
  • PAZ pazopanib hydrochloride
  • SOR sorafenib tosylate
  • VAN vandetanib
  • cabozantinib vandetanib
  • inclusion of an anti-angiogenic agent such as a TKI does not affect the material properties or setting reaction of the in situ solidifying liquid complex coacervate.
  • the effective plasma concentration of a TKI when administered orally is as low as 50 ng/mL.
  • the coacervates described herein allow for delivery of high concentrations of TKIs directly into hypervascular tumors while simultaneously cutting off blood supply to the tumors.
  • humanized anti-VEGF and anti-VEGFR Fab' fragments can be incorporated into the in situ solidifying liquid complex coacervates.
  • electrostatic interactions can control release kinetics.
  • the native charge of the Fab' fragment is sufficient to interact with the poly electrolyte components of the coacervate. In another aspect, the native charge of the Fab' fragment is insufficient to interact with the polyelectrolyte components of the coacervate and the Fab' fragment is modified to increase charge density by attaching a short polyelectrolyte to reactive sulfhydryl groups using maleamide conjugation chemistries.
  • the anti-angiogenic agent is an anti-VEGF antibody.
  • the anti-VEGF antibody is bevacizumab or is a biosimilar anti-VEGF antibody, or is an anti-VEGF antibody derivative such as, for example, ranibizumab.
  • the in situ solidifying liquid complex coacervates described herein are useful in delivering chemotherapeutic agents to a subject.
  • the coacervates disclosed herein are effective vehicles for the delivery of high local concentrations of water-soluble chemotherapeutic agents such as, for example, doxorubicin.
  • the chemotherapeutic agent is present in an amount of from 0.001 mg/mL to 100 mg/mL of in situ solidifying liquid complex coacervate.
  • the anti-angiogenic agent is present at about 0.001, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4., 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/mL of in situ solidifying liquid complex coacervate, where any value can be a lower and upper end-point of a range (e.g., 0.1 to 10 mg/mL, etc.).
  • the in situ solidifying liquid complex coacervates disclosed herein are formulated with a contrast agent.
  • the contrast agent is a radiographic contrast agent.
  • the radiographic contrast agent can be tantalum metal particles (Ta) or gold particles.
  • Ta tantalum metal particles
  • gold particles up to 30 % (w/w) of Ta can be included in the formulations.
  • inclusion of Ta can be beneficial to interventional radiologists in the operating room.
  • the contrast agent can be a fluoroscopic contrast agent.
  • the fluoroscopic contrast agent can be tantalum oxide (TaCh, Ta 2 0s) particles.
  • the contrast agent can be an iodinated compound.
  • the iodinated compound can be an iodide salt (e.g., sodium iodide).
  • the iodinated compound can be an ionic compound such as diatrizoate (HYPAQUE 50®), metrizoate (CORONAR 370®), and ioxaglate (HEXABRIX®), or nonionic compound such as iopamidol (ISOVUE-370®), iohexol (OMNIPAQUE 350®), and iodixanol (VISIPAQUE 320®).
  • the in situ solidifying liquid complex coacervate also includes a reinforcing component.
  • the term "reinforcing component” is defined herein as any component that enhances or modifies one or more properties of the in situ solidifying liquid complex coacervates described herein (e.g., cohesiveness, fracture toughness, elastic modulus, dimensional stability after curing, etc.).
  • the mode in which the reinforcing component can enhance the mechanical properties of the coacervate can vary and will depend on the selection of the components used to prepare the complex coacervate and reinforcing component.
  • the reinforcing component can occupy a space or "phase" in the coacervate, which ultimately increases the mechanical properties of the coacervate. Examples of reinforcing component useful herein are provided below.
  • the reinforcing component is a coil or fiber.
  • the coil or fiber can be platinum, plastic, nylon, another natural or synthetic fiber, a polymerizable monomer, a nanostructure, a micelle, a liposome, a water-insoluble filler, or any combination thereof.
  • the coil or fiber is administered concurrently with the coacervate. In another aspect, the coil or fiber is administered sequentially either before or after the coacervate.
  • the reinforcing component can be a water-insoluble filler.
  • the filler can have a variety of different sizes and shapes, ranging from particles (micro and nano) to fibrous materials. The selection of the filler can vary depending upon the application of the in situ solidifying liquid complex coacervate.
  • the fillers useful herein can be composed of organic and/or inorganic materials.
  • the nanostructures can be composed of organic materials like carbon or inorganic materials including, but not limited to, boron, molybdenum, tungsten, silicon, titanium, copper, bismuth, tungsten carbide, aluminum oxide, titanium dioxide, molybdenum disulphide, silicon carbide, titanium diboride, boron nitride, dysprosium oxide, iron (III) oxide-hydroxide, iron oxide, manganese oxide, titanium dioxide, boron carbide, aluminum nitride, or any combination thereof.
  • the filler comprises a metal oxide, a ceramic particle, or a water insoluble inorganic salt.
  • fillers useful herein include those manufactured by Sky Spring Nanomaterials, Inc., which is listed below.
  • Ni coated with carbon 99.9%, 20 nm
  • Fe 2 0 3 gamma 99%, 20-40 nm Fe 3 0 4 , 98+%, 20-30 nm
  • Si0 2 99%, 10-30 nm, treated with Silane Coupling Agents S1O2, 99%, 10-30 nm, treated with Hexamethyldisilazane
  • the filler is nanosilica.
  • Nanosilica is commercially available from multiple sources in a broad size range.
  • aqueous Nexsil colloidal silica is available in diameters from 6-85 nm from Nvacol Nanotechnologies, Inc.
  • Amino-modified nanosilica is also commercially available, from Sigma Aldrich for example, but in a narrower range of diameters than unmodified silica.
  • Nanosilica does not contribute to the opacity of the coacervate, which is an important attribute of the adhesives and glues produced therefrom.
  • the filler can be composed of calcium phosphate.
  • the filler can be hydroxyapatite, which has the formula CasiPO ⁇ OH.
  • the filler can be a substituted hydroxyapatite.
  • a substituted hydroxyapatite is hydroxyapatite with one or more atoms substituted with another atom.
  • the substituted hydroxyapatite is depicted by the formula M 5 X 3 Y, where M is Ca, Mg, Na; X is P0 4 or C0 3 ; and Y is OH, F, CI, or C0 3 .
  • the calcium phosphate comprises a calcium orthophosphate.
  • Examples of calcium orthophosphates include, but are not limited to, monocalcium phosphate anhydrate, monocalcium phosphate monohydrate, dicalcium phosphate dihydrate, dicalcium phosphate anhydrous, octacalcium phosphate, beta tricalcium phosphate, alpha tricalcium phosphate, super alpha tricalcium phosphate, tetracalcium phosphate, amorphous tricalcium phosphate, or any combination thereof.
  • the calcium phosphate can also include calcium-deficient hydroxyapatite, which can preferentially adsorb bone matrix proteins.
  • the filler can be functionalized with one or more amino or activated ester groups.
  • the filler can be covalently attached to the poly cation or polyanion.
  • aminated silica can be reacted with the polyanion possessing activated ester groups to form new covalent bonds.
  • the filler can be modified to produce charged groups such that the filler can form electrostatic bonds with the coacervates.
  • aminated silica can be added to a solution and the pH adjusted so that the amino groups are protonated and available for electrostatic bonding.
  • the reinforcing component can be micelles or liposomes.
  • the micelles and liposomes used in this aspect are different from the micelles or liposomes used as poly cations and polyanions for preparing the coacervate.
  • the micelles and liposomes can be prepared from the nonionic, cationic, or anionic surfactants described above.
  • the charge of the micelles and liposomes can vary depending upon the selection of the poly cation or polyanion as well as the intended use of the coacervate.
  • the micelles and liposomes can be used to solubilize hydrophobic compounds such pharmaceutical compounds.
  • the adhesive complex coacervates described herein can be effective as a bioactive delivery device.
  • the synthesis of the in situ solidifying liquid complex coacervates described herein can be performed using a number of techniques and procedures. Exemplary techniques for producing the coacervates are provided in the Examples.
  • the polyguanidinyl copolymer (i.e., polycation) and polyphosphate (i.e., polyanion) are mixed as dilute solutions. Upon mixing, when the co polymer (i.e., polycation) and polyphosphate associate they condense into a fluid/liquid phase at the bottom of a mixing chamber (e.g., a tube) to produce a condensed phase.
  • a mixing chamber e.g., a tube
  • the condensed phase i.e., liquid complex coacervate
  • the condensed phase i.e., liquid complex coacervate
  • the polyphosphate is added to a solution of the salt that produces ions followed by the addition of the polyguanidinyl copolymer to produce the in situ solidifying liquid complex coacervate.
  • the copolymer is added to a solution of the salt that produces ions followed by the addition of the polyphosphate to produce the in situ solidifying liquid complex coacervate.
  • the anti-angiogenic agent and additional agents can be added after the in situ solidifying liquid complex coacervate (i.e., composition composed of polyguanidinyl copolymer, polyphosphate, monovalent salt) has been formed.
  • the anti-angiogenic agent and additional agents e.g., contrast agent
  • properties of the in situ solidifying liquid complex coacervates can be fine-tuned by varying the number of guanidinyl groups in the polyguanidinyl copolymer relative to the number of phosphate groups present in the polyphosphate.
  • the charge ratio between the guanidinyl groups (positive) and phosphate groups (negative) is from 1 :2 to 2: 1, 0.5: 1 to 1.5: 1 or is 0.5: 1, 0.75: 1, 1 : 1, 1.25: 1, or is about 1 : 1 at a specified pH, temperature, and monovalent salt concentration.
  • the in situ solidifying liquid complex coacervate is composed of (1) a polyguanidinyl copolymer produced by the polymerization between the monomer of formula I, where R ⁇ s methyl, X is NH, and m is 3 (referred to herein as GPMA) in the amount of 50 to about 100 mol %, and methacrylamide in the amount of 0 to about 50 mol %, where the polymer has an average molar weight (M m ) from about 2 kg/mol to about 80 kg/mol; (2) the polyphosphate is sodium
  • the charge ratio between the guanidinyl groups (positive) and phosphate groups (negative) is from 0.5: 1 to 1.5: 1, preferably about 1 : 1;
  • the pH of the in situ solidifying liquid complex coacervate has a pH of from 7 to 7.5, or of about 7, 7.1, 7.2, 7.3, 7.4, or 7.5;
  • the concentration of the salt that produces ions is from 500 mM to 1,500 mM;
  • chemotherapeutic agent (7) and is a liquid complex coacervate prior to administration to a subject.
  • kits for making the in situ solidifying liquid complex coacervates and adhesives described herein comprising (1) a co polymer, (2) a polyphosphate, wherein the positive/negative charge ratio of the poly cation to the polyanion is from 0.5 to 1.5, preferably about 1 : 1, (3) an aqueous solution comprising a salt that produces ions at a concentration from 0.5 M to 2.0 M; and (4) an anti-angiogenic agent or chemotherapeutic agent.
  • the kits can also include additional components as described herein (e.g., reinforcing components, bioactive agents, contrast agents, etc.).
  • water can be added to the copolymer and/or polyphosphate to produce the coacervate.
  • the pH of the polyguanidinyl copolymer and polyphosphate can be adjusted such that when they are admixed in water the desired pH is produced without the addition of acid or base.
  • the in situ solidifying liquid complex coacervate can be loaded in a syringe for future. Due to the stability of the in situ solidifying liquid complex coacervate, a sterilized solution of the complex coacervate can be stored in the syringe for extended periods of time and used as needed.
  • the in situ solidifying liquid complex coacervates described herein can locally deliver an anti-angiogenic agent or chemotherapeutic agent over a prolonged of time.
  • the localized delivery of the anti-angiogenic agent prevents systemic toxicity of the anti-angiogenic agent while reducing or preventing angiogenesis at or near a tumor or vascular malformation.
  • the in situ solidifying liquid complex coacervate is introduced into a subject, wherein upon introduction of the in situ solidifying liquid complex coacervate into the subject, a solid is produced in situ via an aqueous aqueous phase inversion mechanism.
  • the in situ solidifying liquid complex coacervates described herein are liquids at ionic strengths higher than the ionic strength of the application site, but solids at the ionic strength of the application site.
  • the complex coacervates forms a solid in situ at the application site as the salt concentration in the complex coacervate equilibrates to the application site salt concentration.
  • the anti-angiogenic agent or chemotherapeutic agent is released from the microporous solid produced in situ solidifying liquid complex coacervate at constant rate (i.e., zero order kinetics).
  • the anti-angiogenic agent or the chemotherapeutic agent is released from the solid over a period of 1 day, 5 days, 10 day, 15 days, 20 days, 25 days, or 60 days, where any value can be a lower and upper end-point (e.g., 1 days to 30 days, 10 days to 25 days, etc.).
  • the amount of anti-angiogenic agent or the chemotherapeutic agent that is incorporated in the in situ solidifying liquid complex coacervate can vary, which will affect the duration of release of the anti-angiogenic agent or the chemotherapeutic agent from the solid.
  • the in situ solidifying liquid complex coacervates are fluids with low viscosity and are readily injectable via a narrow gauge device, syringe, catheter, needle, cannula, or tubing.
  • the in situ solidifying liquid complex coacervates are water-borne eliminating the need for potentially toxic solvents.
  • the in situ solidifying liquid complex coacervates can form solids in situ under physiological conditions.
  • the complex coacervates can administered or applied to any tissue in the subject (e.g., organs, blood vessels, muscle tissue, connective tissue) and bone.
  • the physiological ionic strength is approximately 300 mOsm/L.
  • in situ solidifying liquid complex coacervates having an ionic strength greater than 300 mOsm/L are introduced to a subject (e.g., injected into a mammal), the liquid complex coacervate is converted to a solid at the site of application as the internal ionic strength equilibrates to the ionic strength of the physiological environment.
  • the in situ solidifying liquid complex coacervate upon solidifying can perform two functions.
  • the microporous solid produced by the liquid complex coacervate can release the anti- angiogenic agent at a controlled rate.
  • the composition of the in situ solidifying liquid complex coacervate e.g., molar mass of the polyguanidinyl copolymer
  • selection of the anti-angiogenic agent it is possible fine-tune the release partem of the anti-angiogenic agent from the solid produced from the in situ solidifying liquid complex coacervate.
  • the hydrophobic properties of the anti-angiogenic agent can be used to evaluate the release rate of the agent.
  • a less hydrophobic TKI like SUN can be released faster when compared to a more hydrophobic TKI such as, for example, SOR or VAN.
  • two or more anti-angiogenic agents of varying hydrophobicity can be used herein.
  • the anti-angiogenic agent is released from the solid in a time ranging from about 1 day to about 10 days, or in about 1, 2, 3 4, 5, 6, 7, 8, 9, or 10 days.
  • the solid produced in the subject also creates an artificial embolus within the vessel.
  • the in situ solidifying liquid complex coacervates described herein can reduce or prevent angiogenesis via two different mechanisms.
  • the in situ solidifying liquid complex coacervates can be used as synthetic embolic agents.
  • the in situ solidifying liquid complex coacervate described herein can include one or more additional embolic agents.
  • Embolic agents commercially-available are microparticles used for embolization of blood vessels. The size and shape of the microparticles can vary.
  • the microparticles can be composed of polymeric materials. An example of this is BearinTM nsPVA particles manufactured by Merit Medical Systems, Inc., which are composed of polyvinyl alcohol ranging in size from 45 ⁇ to 1,180 ⁇ .
  • the embolic agent can be a microsphere composed of a polymeric material.
  • embolic agents examples include Embosphere ® Microspheres, which are made from trisacryl cross linked with gelatin ranging in size from 40 ⁇ to 1,200 ⁇ ; HepaSphereTM Microspheres (spherical, hydrophilic microspheres made from vinyl acetate and methyl aery late) ranging in size from 30 ⁇ to 200 ⁇ ; and QuadraSphere ® Microspheres (spherical, hydrophilic microspheres made from vinyl acetate and methyl acrylate) ranging in size from 30 ⁇ to 200 ⁇ , all of which are manufactured by Merit Medical Systems, Inc.
  • the microsphere can be impregnated with one or more metals that can be used as a contrast agent.
  • EmboGold ® Microspheres manufactured by Merit Medical Systems, Inc., which are made from trisacryl cross linked with gelatin impregnated with 2% elemental gold ranging in size from 40 ⁇ to 1,200 ⁇ .
  • the embolic agent can be a coil or fiber.
  • the in situ solidifying liquid complex coacervates can include one or more contrast agents.
  • the physician can monitor precisely the position of the solid that is produced in situ.
  • Contrast agents known in the art can be used herein.
  • the contrast agent can be admixed with the components used to prepare the in situ solidifying liquid complex coacervates.
  • metal particles such as tantalum powder or gold can be used.
  • soluble iodinated compounds can be used as the contrast agent.
  • the contrast agent can be detected using techniques known in the art including X-ray, NMR imaging, ultrasound, and fluoroscopes.
  • the contrast agent can be tantalum particles having a particle size from 0.5 ⁇ to 50 ⁇ , 1 ⁇ to 25 ⁇ , 1 ⁇ to 10 ⁇ , or 1 ⁇ to 5 ⁇ .
  • contrast agent is tantalum particles in the amount of 10% to 60%, 20% to 50%, or 20% to 40%.
  • the solids produced from the in situ solidifying liquid complex coacervates described herein can encapsulate, scaffold, seal, or hold one or more bioactive agents in addition to the anti-angiogenic agent and chemotherapeutic agent.
  • the bioactive agents can be any drug including, but not limited to, antibiotics, an anti-inflammatory agent, growth factors, enzyme inhibitors, hormones, messenger molecules, cell signaling molecules, receptor agonists, oncolytic agents (e.g., oncolytic viruses), chemotherapy agents, receptor antagonists, MAB fragments, or monoclonal antibodies.
  • the agent may also be autologous or homologous (allogeneic) cells, platelet rich plasma (PRP), or other like tissue.
  • water soluble bioactive agents can be incorporated into the liquid complex coacervates described herein.
  • the in situ solidifying liquid complex coacervates described herein can include an anti-angiogenic agent in combination with a chemotherapeutic agent such as doxorubicin or an antibiotic such as tobramycin.
  • the in situ solidifying liquid complex coacervates described herein can include an anti-angiogenic agent or a chemotherapeutic agent in combination with an anti-inflammatory agent in order to reduce or prevent inflammation at the site where the complex coacervate is administered to the subject.
  • the antiinflammatory agent is an NSAIDs including, but are not limited to, acetaminophen, aspirin, ibuprofen, naproxen sodium, naproxen, indomethacin, flurbiprofen, ketoprofen, lornoxicam, meloxicam, piroxicam, oxaprozin, etodolac, ketorolac, nabumetone, or other nonselective nonsteroidal anti-inflammatory drugs (NSAIDs).
  • NSAIDs nonselective nonsteroidal anti-inflammatory drugs
  • the anti-inflammatory agent is a COX-2 inhibitors including, but not limited to, celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib, CS-502, JTE-522, L-745,337,and NS398).
  • the anti-inflammatory agent is a COX-2 inhibitors including, but not limited to, celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib, CS-502, JTE-522, L-745,337,and NS398).
  • the anti-inflammatory agent is a COX-2 inhibitors including, but not limited to, celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib, CS-502, JTE-522, L-745,337,and NS398).
  • the anti-inflammatory agent is a COX-2 inhibitors including, but not limited to, celecoxib,
  • corticosteroid including, but not limited to, Cortisol, cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fludrocortisones acetate, and deoxycorticosterone acetate.
  • the bioactive agent can be a nucleic acid.
  • the nucleic acid can be an oligonucleotide, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or peptide nucleic acid (PNA).
  • the nucleic acid of interest can be nucleic acid from any source, such as a nucleic acid obtained from cells in which it occurs in nature, recombinantly produced nucleic acid, or chemically synthesized nucleic acid.
  • the nucleic acid can be cDNA or genomic DNA or DNA synthesized to have the nucleotide sequence corresponding to that of naturally-occurring DNA.
  • the nucleic acid can also be a mutated or altered form of nucleic acid (e.g., DNA that differs from a naturally occurring DNA by an alteration, deletion, substitution or addition of at least one nucleic acid residue) or nucleic acid that does not occur in nature.
  • a mutated or altered form of nucleic acid e.g., DNA that differs from a naturally occurring DNA by an alteration, deletion, substitution or addition of at least one nucleic acid residue
  • nucleic acid that does not occur in nature e.g., DNA that differs from a naturally occurring DNA by an alteration, deletion, substitution or addition of at least one nucleic acid residue
  • the in situ solidifying liquid complex coacervates described herein are useful in any application where it is desirable to reduce or prevent angiogenesis as well as inhibit blood flow in a subject.
  • the in situ solidifying liquid complex coacervates reduce or prevent angiogenesis in a tumor.
  • Complete shutdown of the blood supply to a tumor induces hypoxia and activation of HIF-1 on a time scale of minutes, which ultimately stimulates angiogenesis in the tumor.
  • the in situ solidifying liquid complex coacervates not only can reduce or prevent blood flow to the tumor, they can also reduce or prevent angiogenesis from occurring. This two- prong approach makes the in situ solidifying liquid complex coacervates described herein very useful in cancer treatment. These unique attributes can combine to significantly improve outcomes of TAE and TACE as a minimally invasive treatment for hypervascularized tumors and AVMs of all types.
  • the in situ solidifying liquid complex coacervates described herein can perform as embolic agents to prevent or reduce blood flow in a subject.
  • the in situ solidifying liquid complex coacervates can be used in a number of applications where it is desirable to reduce or inhibit blood flow.
  • the in situ solidifying liquid complex coacervates can reduce or inhibit blood flow to an aneurysm, a varicose vein, an arteriovenous malformation, or an open or bleeding wound.
  • N-(3-aminopropyl) methacrylamide hydrochloride was obtained from Polysciences, Inc. (cat# 21200).
  • lH-Pyrazole-l-Carboxamidine hydrochloride was purchased from Chem-Ipex International (cat# 21678).
  • Methacrylamide (MA; L15013) and glacial acetic acid (cat# 36289) were obtained from Alfa Aesar.
  • 4- methoxyphenol was purchased from TCI chemicals (cat #M0123).
  • 4,4'-Azobis(4- cyanovaleric acid) V501; cat# 11590
  • azobisisobutyronitrile ⁇ ; cat# 441090
  • Tantalum metal powder (1-5 micron particle size) was purchased from Atlantic Equipment Engineers, Inc. (cat# TA-101).
  • Doxorubicin hydrochloride was obtained from Meiji Seika Pharma (cat# 25316-40-9). Sunitinib malate (SUN) was obtained from Selleckchem (cat#S1042). All solvents were ACS grade or better. Solutions were made in ultrapure double deionized water.
  • APMA N-(3-aminopropyl) methacrylamide hydrochloride
  • DMF 112 mL
  • TEA 18.7 mL; 134 mmol
  • /H-pyrazole-l-carboxamidine hydrochloride (16.4 g; 112 mmol) was added. The mixture was reacted at 20°C under N 2 .
  • RAFT polymerization was employed using 4-cyano-4- (thiobenzoylthio)pentanoic acid as the chain transfer agent (CTA) and V-501 as the initiator at a 5:1 molar ratio.
  • CTA chain transfer agent
  • V-501 V-501 as the initiator at a 5:1 molar ratio.
  • a fixed molar ratio of 80:20 (GPMA:MA) and molecular weight of 20 kD were targeted.
  • GPMA (9.12 g, 41 mmol)
  • MA (0.88 g, 10 mmol)
  • 4-cyano-4-(thiobenzoylthio)pentanoic acid (112 mg, 0.400 mmol)
  • V-501 (22.4 mg; 0.080 mmol) were dissolved in 1 M (pH 5.3) acetate buffer (52 mL).
  • the resulting solution was degassed by bubbling for 2 hours with N 2 before being septum sealed. The reaction was kept under N 2 while it proceeded at 70°C for 16 hours. The resulting polymer was cooled, exposed to air, and precipitated in acetone. For end group modification, the polymer was redissolved in methanol ( ⁇ 100 mL), and AIBN (1.3 g, 8 mmol) was added. The solution was degassed and reacted under N 2 for 4 hours at 60°C. The product was precipitated in acetone, collected by filtration, and dried under vacuum. A Millipore ultrafiltration system equipped with a Pellicon 2 Minicassette (Biomax ® 5 kDa) was used to purify the final product.
  • the polymer was characterized by aqueous size exclusion chromatography (SEC) on an Aglient HPLC 1260 Infinity equipped with refractive index detector and a Wyatt miniDAWN TREOS light scattering detector.
  • SEC aqueous size exclusion chromatography
  • the dn/dc value for p(GPMA-co-MA) was determined by injecting known stock solutions of PG ranging from 0.25-2 mg/mL at 1 mL/min using a syringe pump (PHD Ultra, Harvard
  • Coacervates of p(GPMA-co-MA) (PG) and sodium hexametaphosphate (MP) were prepared with 1-5 micron Ta powder added as a radiocontrast agent (30 wt% of final coacervate), unless otherwise noted.
  • Aqueous stock solutions of PG and MP were made at 100 mg/mL and 200 mg/mL, respectively. The pH of both solutions was adjusted to 7.2.
  • Coacervation was achieved by sequential addition of DI water, 5M NaCl, MP solution, Ta powder, and PG solution, while mixing with an overhead mixer. In this final mixture, PG concentration was fixed at 50 mg/mL; MP concentration was 42 mg/mL based upon calculated charge densities and a 1 :2 charge ratio.
  • Amounts of DI water and 5 M salt were adjusted to form a NaCl concentration of 800 mM. Phase separation occurred immediately upon addition of PG, and the coacervate was allowed to settle for 12 hr. Afterwards, the supernatant was removed and 5 M NaCl was mixed into the condensed phase using trituration to bring the overall NaCl concentration in the coacervate to its final concentration (1400 mM, unless otherwise noted).
  • a stock solution of 4 mg/mL SUN solution in 4 M NaCl was produced by dissolving SUN in 1 part DI water at 20 mg/mL and subsequently diluting it with 4 parts 5 M NaCl. After removal of the PG-MP supernatant, the SUN+NaCl stock was added to raise the salt to 1400 mM and load SUN at 750 ⁇ g/mL, forming the antiangiogenic embolic coacervate (EC-Sun).
  • the Dox-EC was loaded in the same manner, with a 40 mg/mL Dox + 4 M NaCl solution.
  • the drug loading was experimentally determined to be 33 mg/mL (absorbance at 487 nm). Both brightly colored drugs appeared soluble and remained well-dispersed in the high-salt liquid coacervate.
  • DL-EC Drug-loaded embolic coacervates
  • Doxorubicin concentration was determined using absorbance at 487 nm. Cumulative release at each timepoint is reported and is normalized by the surface area of the AA- EC exposed to BSS in the cuvette (0.4 cm 2 ).
  • AP — ⁇ r where P is pressure, r is the radius of the tube, L is the length of the tube, Q is the volumetric flow rate, and ⁇ is viscosity. This linear conversion was also verified experimentally in our model. Poiseuille's law was also used to compare measured injection pressures with rheological flow curves.
  • ECs were injected into filtration tubes (Supelco, Inc.; cat #57240-U) to a height of 1 cm (-300 of EC).
  • a cut section (-10 cm in length) of 3 F catheter (Renegade HI-FLO, Boston Scientific Inc.) was placed in the center of each EC-filled tube.
  • the filtration tubes were completely submerged in a dish containing BSS and allowed to incubate.
  • the force required to remove the catheter from the solidified EC was measured on an Instron 3342 materials tester (Instron, Inc.) equipped with a 10 N load cell and controlled with Bluehill 3 software.
  • the catheter was removed in extension mode with a strain rate of 600 mm- min "1 .
  • GPMA 3-guanidinopropyl methacrylamide
  • APMA 3-aminopropyl methacrylamide
  • APMA 3-aminopropyl methacrylamide
  • PDI controlled molecular weight and low polydispersity index
  • the reaction was done in an aqueous acetate buffer, pH 5.3. To set polymerization parameters, the kinetics of this reaction were studied. The rate of polymerization was proportional to monomer concentration out to 16 hours, following pseudo-first order kinetics. Furthermore, a minimal induction period was observed and the reaction reached 90% monomer conversion. Next, large scale polymer synthesis (batch size 6-20 g) was performed eight times, which typically yielded around 50% after conversion to the hydrochloride salt and ultrafiltration. SEC analysis with light scattering was used to determine molecular weights and size distributions with an experimentally determined dn/dc value of 0.1782.
  • ECs were formed using the synthetic p(GPMA-co-MA) (PG) and sodium hexametaphosphate (MP).
  • MP is a mixture of inorganic phosphate oligomers, both cyclic and linear, usually containing 10-20 phosphorous atoms per chain.
  • cyclic inorganic polyphosphates In their fully ionized form, cyclic inorganic polyphosphates have the formula (P n 03n) n" , while the linear form is comprised of (P n 03 n +i) n+2" . Regardless of the whether the polyphosphate is linear or cyclic, each phosphorus atom has one strongly ionized hydrogen, with a pK a of -4.5 or less.
  • the weakly acidic end groups of linear polyphosphates are usually dissociated between pH 4.5 and 9.5. Overall this contribution to charge density is relatively small, so 1 charge per phosphorous atom was assumed at pH 7.2.
  • the strongly basic GPMA sidechains on the polymer were considered fully protonated.
  • coacervates were first formed in 800 mM NaCl. After removal of the supernatant, salt content of the coacervate was raised by mixing 5 M NaCl into the condensed phase. 30 wt% tantalum metal powder was added to the complex as a radiocontrast agent, necessary for the delivery of the embolic agent under fluoroscopic guidance.
  • liquid embolic agents require a viscosity that allows for injection through microcatheters without exceeding their rated burst pressure.
  • Clinically used embolic microcatheters have rated pressures of 5-8 MP a; thus, an injection pressure less than 4 MPa at 0.3 mL min "1 , the maximum injection rate of current liquid embolics, was set as a design specification.
  • the viscosity of the EC was lowered by increasing the amount of NaCl in the coacervate.
  • Corresponding changes in injection pressures were determined in model catheters.
  • the model catheters have the same inner surface (PTFE) and internal diameter (0.026”) as commonly used 3 F embolic catheters.
  • ECs were loaded into 1 mL syringes and a syringe pump, equipped with a force transducer, was used to measure the steady state injection force through the model catheter at 0.3 mL min "1 .
  • the measured forces were converted to pressure and extrapolated to a catheter length of 135 cm, the most common length used in the clinic.
  • injection pressure was 10.1 MPa, exceeding the limits of clinically used catheters.
  • Increasing NaCl concentration drastically reduced injection pressures (Fig. 2A), with 1400 mM NaCl being sufficient to lower injection pressures below 4 MPa (3.25 MPa).
  • a further increase in NaCl concentration resulted in even lower injection pressures.
  • a NaCl concentration of 1400 mM was chosen for further development.
  • the viscosity of PG-MP was investigated over a range of shear rates (0.1-500 s "1 ) to simulate transcatheter delivery (Fig. 2B). Without Ta, PG-MP displayed some slight shear thinning at low shear rates, but the viscosity (1.1 Pa s) remained nearly unchanged at shear rates greater than 0.3 s "1 . With PG-MP displayed significantly more shear thinning, with a low shear viscosity of about 5 Pa s at 0.1 s "1 . However, by 10 s "1 the viscosity was 1.2 Pa s, only slightly higher than those without Ta.
  • the force required to remove the catheter from solidified ECs was measured both 2 minutes and 24 hours after injection of the EC into saline. Sections of 3 F catheters were embedded into 1 cm of EC. After allowing the EC to solidify in saline, the force required to remove the catheter from the embolic plug was measured at 2 minutes and 24 hours. At 2 minutes, the force required to remove the catheter at 1 cm s "1 was 16.7 raN (+/- 5.8 mN). Even in the worst-case scenario (24 hours), the force required to remove the embolic was only 384 mN (+/- 107 mN). Additionally, no embolic agent remained adhered to the catheter upon removal.
  • DL-ECs Drug-loaded embolic coacervates
  • ECs were prepared as described previously in 800 mM NaCl.
  • 4 mg/mL Sun or 40 mg/mL Dox were suspended in 4 M NaCl.
  • the brightly colored drugs were soluble and well-dispersed in the liquid coacervate.
  • ECs were loaded with 750 ⁇ g/mL of Sun (EC-Sun) or 33 mg/mL of Dox (EC -Dox), which were experimentally verified.
  • DL-ECs exhibited sustained release of drug for 14 days (EC-Sun) and 23 days (EC-Dox) (Fig. 4A-4B).
  • the release profiles were determined by placing 50 of DL-EC into a cuvette containing 1 mL balanced salt solution (BSS) at pH 6.9. These cuvettes allowed for controlling surface area of the DL-EC exposed to the solution (0.4 cm 2 ), and thus the amount released normalized to surface area was calculated.
  • the BSS release solutions were replaced at intervals of 1-3 days to maintain sink conditions. Cumulative drug release from EC-SUN was 83% (-Ta) and 75% (+Ta). EC-Dox released 48% (+Ta) and 38% (-Ta) of total drug loaded.

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  • Engineering & Computer Science (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des coacervats complexes liquides composés d'un ou de plusieurs agents anti-angiogéniques ou agents chimiothérapeutiques qui se solidifient in situ dans des vaisseaux sanguins. Les polyélectrolytes chargés de manière opposée ont été conçus pour former des coacervats complexes liquides à des forces ioniques élevées mais subissent une inversion de phase aqueuse pour produire un solide microporeux à des forces ioniques physiologiques. L'agent anti-angiogénique ou l'agent chimiothérapeutique est ensuite libéré localement à une vitesse prolongée à partir du solide microporeux.
PCT/US2018/058015 2017-10-27 2018-10-29 Coacervats complexes liquides de solidification in situ pour l'administration locale d'agents anti-angiogéniques ou d'agents chimiothérapeutiques WO2019084548A1 (fr)

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WO2016011028A1 (fr) * 2014-07-14 2016-01-21 University Of Utah Research Foundation Coacervats complexes de solidification in situ et leurs procédés de fabrication et d'utilisation
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WO2006120557A1 (fr) * 2005-05-12 2006-11-16 Pfizer Inc. Polytherapie anticancereuse faisant intervenir du malate de sunitinib
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JOHNSTON ET AL.: "The Effect of Comb Architecture on Complex Coacervation", ORGANIC & BIOMOLECULAR CHEMISTRY, vol. 15, no. 36, 23 August 2017 (2017-08-23), pages 7630 - 7642, XP055593519, ISSN: 1477-0520, DOI: 10.1039/C7OB01314K *

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Publication number Priority date Publication date Assignee Title
WO2022140513A1 (fr) * 2020-12-22 2022-06-30 Fluidx Medical Technology, Llc Compositions injectables à solidification in situ avec agents de contraste transitoires et leurs procédés de fabrication et d'utilisation

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