Covalent anesthetic-polymer conjugates for long-lasting local anesthesia

Abstract

Anesthetics covalently conjugated to biodegradable and biocompatible hydrophilic polymers via hydrolytic linkages provide controlled release of effective amounts of local anesthetic for nerve blockade in vivo with reduced toxicity compared to unconjugated anesthetics. The anesthetic release rate can be adjusted by modifying the hydrophilicity of the polymer. Exemplary formulations of poly(glycerol sebacate) (PGS) conjugated to tetrodotoxin (TTX), optionally containing a polyethylene glycol (PEG) polymer (PGS-PEG-TTX and PGS-TTX), and methods of use thereof, are provided. Nerve blockade with PGS-PEG-TTX and PGS-TTX was associated with minimal systemic toxicity and local toxicity to muscle and peripheral nerves. A TDP-TTX conjugate homogeneously dispersed in PEG200 is also described. PEG200 served not only as a vehicle but also as a chemical penetration enhancer (CPE) to enhance the efficacy of TTX.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and claims priority to U.S. Patent Application No. 62 / 593,784, filed December 1, 2017, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION This is generally in the field of low toxicity, long duration nerve blocks and local anesthesia and local analgesia, and specifically in the field of formulations of tetrodotoxin covalently linked to biocompatible polymers for controlled release.

[0003] government support This invention was made with government support under Grant Nos. 5R01GM073626-12 and 1R01GM116920-01 awarded by the National Institute of General Medical Sciences. The U.S. Government has certain rights in this invention. [Background technology]

[0004] 2. Background of the Invention Long-lasting local anesthesia after a single injection has been the focus of clinical and scientific research for the past 20 years. However, there are many limitations associated with the conventional amino-ester and amino-amide local anesthetic compounds currently used in clinical practice. Although these compounds provide effective peripheral nerve blockade, their relatively short duration of action is often insufficient, especially in the management of chronic and neuropathic pain. These compounds also cause side effects, such as local toxicity to muscles and peripheral nerves, which increase with higher concentrations and longer durations of exposure. Unfortunately, these side effects are exacerbated when these compounds are delivered using sustained release vehicles (even if the toxicity of the delivery vehicle itself is minimized), and can cause inflammatory responses in nerves that sometimes last significantly longer than the duration of nerve blockade (Padera et al., Anesthesiology 108, 921-928, doi:10.1097 / ALN.0b013e31816c8a48 (2008); Kohane et al., Pain 104, 415-421, doi:http: / / dx.doi.org / 10.1016 / S0304-3959(03)00049-6 (2003); Kohane et al., Journal of Biomedical Materials Research 59, 450-459, doi:10.1002 / jbm.1261 (2002)). In particular, the low specificity of many reagents for peripheral nerve voltage-gated sodium channels can result in severe systemic side effects, primarily cardiovascular (e.g., life-threatening arrhythmias) and neurological (e.g., seizures) side effects.

[0005] In light of these limitations, it has been a long-standing goal to develop local anesthetic formulations that could enhance or extend nerve blockade while minimizing systemic and local side effects. Tetrodotoxin (TTX), a naturally occurring site 1 sodium channel blocker (S1SCB), has been investigated for the past 20 years as an alternative to traditional local anesthetics (Lahaye et al., Anesthesiology 123, 741-742 (2015)). Although TTX has extremely potent local anesthetic properties (Kohane et al., Regional Anesthesia and Pain Medicine 25, 52-59, doi:http: / / dx.doi.org / 10.1016 / S1098-7339(00)80011-5 (2000)), the doses of TTX required to achieve significant peripheral nerve blockade can cause hypotension and generalized muscle weakness, including diaphragmatic paralysis and respiratory failure. Despite the narrow therapeutic window of TTX, attempts to introduce it into clinical practice are ongoing (Lahaye et al., Anesthesiology 123, 741-742 (2015); PERE et al., Regional Anesthesia and Pain Medicine 18, 304-307 (1993); Lobo et al., Anesthesiology 123, 873-885 (2015)), and approaches to extend the duration of TTX nerve blockade for clinical use are desirable.

[0006] Long drug duration can be achieved by encapsulating drugs in sustained delivery systems, but TTX is highly hydrophilic, preventing effective encapsulation (Rwei et al., Proceedings of the National Academy of Sciences 112, 15719-15724, doi:10.1073 / pnas.1518791112 (2015); Shankarappa et al., Proceedings of the National Academy of Sciences 109, 17555-17560, doi:10.1073 / pnas.1214634109 (2012)). Liposomes have previously been reported as efficient carriers for the controlled release of TTX, with encapsulation efficiencies up to 50% (Rwei et al., Proceedings of the National Academy of Sciences 112, 15719-15724, doi:10.1073 / pnas.1518791112 (2015); Epstein-Barash, H. et al., Proceedings of the National Academy of Sciences 106, 7125-7130, doi:10.1073 / pnas.0900598106 (2009); Zhan, C. et al., Nano Letters 16, 177-181, doi:10.1021 / acs.nanolett.5b03440 (2016)). These liposomes achieved peripheral nerve blockade lasting up to 13.5 hours in a rat sciatic nerve model with minimal local or systemic toxicity. However, the initial burst release of TTX prevented further increases in the injected dose, thereby preventing further extension of the nerve blockade. Ideally, an injectable anesthetic for postoperative or chronic pain management could last from a few days up to a few weeks. Furthermore, the time-consuming and cumbersome preparation protocol makes the liposomal formulation inconvenient for clinical use.

[0007] It is therefore an object of the present invention to provide a biocompatible platform for the delivery of anesthetic agents over extended periods of time after a single administration with low toxicity.

[0008] It is also an object of the present invention to provide biocompatible formulations of specific Site 1 sodium channel blockers (S1SCBs) that extend the duration of anesthesia derived from S1SCBs while minimizing local or systemic toxicity. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Padera et al., Anesthesiology 108,921-928,doi:10.1097 / ALN.0b013e31816c8a48(2008) [Non-Patent Document 2] Kohane et al., Pain 104,415-421,doi:http: / / dx.doi.org / 10.1016 / S0304-3959(03)00049-6(2003) [Non-Patent Document 3] Kohane et al., Journal of Biomedical Materials Research 59,450-459,doi:10.1002 / jbm.1261(2002)) [Non-Patent Document 4] Kohane et al., Regional Anesthesia and Pain Medicine 25,52-59,doi:http: / / dx.doi.org / 10.1016 / S1098-7339(00)80011-5(2000) [Non-Patent Document 5] Lahaye et al., Anesthesiology 123,741-742(2015); PERE et al., Regional Anesthesia and Pain Medicine 18,304-307(1993) [Non-Patent Document 6] Lobo et al., Anesthesiology 123, 873-885 (2015) [Non-Patent Document 7] Rwei et al., Proceedings of the National Academy of Sciences 112,15719-15724,doi:10.1073 / pnas.1518791112(2015) [Non-Patent Document 8] Shankarappa et al., Proceedings of the National Academy of Sciences 109,17555-17560,doi:10.1073 / pnas.1214634109(2012) [Non-Patent Document 9] Epstein-Barash, H. et al., Proceedings of the National Academy of Sciences 106,7125-7130,doi:10.1073 / pnas.0900598106(2009) [Non-Patent Document 10] Zhan, C. et al., Nano Letters 16,177-181,doi:10.1021 / acs.nanolett.5b03440(2016) Summary of the Invention [Means for solving the problem]

[0010] Summary of the Invention Conjugates containing anesthetic agents, particularly site I sodium channel blockers such as tetrodotoxin, covalently attached to a biodegradable and biocompatible polymer backbone via hydrolyzable ester bonds provide long-lasting local anesthesia with significantly lower toxicity. The covalent bond is stable enough to prevent an initial "burst release" of the anesthetic agent, and slow hydrolysis of the ester bond achieves sustained release of an effective amount of the anesthetic agent, thereby blocking pain in vivo for up to or more than 72 hours and up to one month after a single dose. The anesthetic agent is released in its native form. The hydrolysis rate of the ester bond in the conjugate is adapted to the needs of the recipient by modifying the hydrophilicity of the polymer backbone.

[0011] PEG has also been demonstrated to function as a chemical permeation enhancer that enhances the efficacy of the anesthetic within the conjugate. Thus, formulations of anesthetic-polymer conjugates that are uniformly dispersed in PEG 200 are described. In a preferred embodiment, the formulations of anesthetic-polymer conjugates are dispersed in PEG 200 as a formulation that is injectable by syringe. Methods of making the covalent anesthetic-polymer formulations and their methods of use are also described.

[0012] In certain embodiments, the anesthetic agent is a site 1 sodium channel blocker (S1SCB). Exemplary S1SCBs include tetrodotoxin (TTX), saxitoxin (STX), decarbamoyl saxitoxin, neosaxitoxin, and gonyautoxin. In certain exemplary embodiments, the S1SCB is tetrodotoxin (TTX). In some embodiments, the conjugate comprises an anesthetic agent that is a vanilloid receptor subtype 1 (TRPV1) agonist. Exemplary TRPV1 agonists include trans8-methyl-N-vanillyl-6-nonenamide (capsaicin), dihydro-capsaicin, nordihydrocapsaicin, homodihydrocapsaicin, homocapsaicin, and resiniferatoxin.

[0013] The conjugates include an anesthetic covalently bonded to a biodegradable and biocompatible polymer. The hydrophilicity of the polymer can be modified in a controlled manner, for example, by adding a hydrophilic polymer. Thus, the conjugates allow for tunable anesthetic release in vivo by varying the concentration and relative ratio of hydrophilic and hydrophobic polymers and monomers in the conjugate. Preferred biocompatible polymer backbones include polyanhydrides. An exemplary polyanhydride polymer is poly(glycerol sebacate) (PGS). Preferred hydrophilic polymers include polyethylene oxide polymers and polyethylene oxide copolymers, such as poly(ethylene glycol) (PEG). Exemplary PEGs include PEG100, PEG200, PEG300, PEG400, PEG500, PEG600, PEG700, PEG800, PEG900, PEG1000, and PEG2000. Preferred anesthetics include phycotoxins and vanilloids.

[0014] One skilled in the art will appreciate that the amount of anesthetic agent in the formulation can be adapted to the needs of the recipient. In some embodiments, the formulation contains anesthetic agent in a dosage of 0.1 μg to 200 μg (inclusive), preferably 1 μg to 100 μg (inclusive) per dose.

[0015] The duration of effective nerve block resulting from administration of a formulation of the conjugate in vivo can be adjusted by changing the amount and ratio of hydrophilic polymers in the conjugate. Thus, in some embodiments, the amount and ratio of hydrophilic polymers in the conjugate is sufficient to provide effective nerve block for a period of time ranging from 1 hour to up to 1 month depending on the needs of the recipient. For example, in some embodiments, the formulation delivers an amount of anesthetic agent that provides effective nerve block in vivo for up to 72 hours. In other embodiments, the formulation delivers an amount of anesthetic agent that provides effective nerve block in vivo for at least 72 hours, up to 2 weeks or more than 2 weeks, for example, up to 1 month. The formulation provides effective nerve block with minimal systemic toxicity and substantially no local toxicity to muscle or peripheral nerves. In some embodiments, the conjugate includes one or more additional therapeutic, prophylactic or diagnostic agents covalently linked to the amphiphilic polymer. In other embodiments, the conjugate is formulated in liposomes or other secondary delivery vehicles, optionally including one or more additional therapeutic, preventive or diagnostic agents.In other embodiments, one or more additional therapeutic, preventive or diagnostic agents are included for injection with the conjugate by mixing into the infusion.The additional active agent can be covalently linked to the polymer backbone via a hydrolyzable ester bond.In some embodiments, the additional active agent is a glucocorticoid, such as dexamethasone.

[0016] Provided is a method for providing effective nerve blockade in a subject in need of effective nerve blockade with no or significantly reduced local toxicity after a single administration for a duration of up to one month.The method comprises administering to the subject an effective amount of a formulation comprising an anesthetic covalently conjugated to PGS and optionally modified by the addition of PEG in an amount effective to provide nerve blockade at the administration site for up to three days and up to one month after administration.In some embodiments, the method is effective for treating or preventing neuropathic pain in the subject. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. An anesthetic-polymer covalent conjugate for long-lasting local anesthesia, said conjugate comprising: (a) an anesthetic agent; and (b) Polymer backbone Including, the polymer backbone comprises one or more hydrophilic or hydrophobic polymers and optionally one or more polyethylene oxide polymers; A conjugate, wherein the anesthetic agent is covalently conjugated to the polymer backbone via a hydrolyzable linker in an amount effective to induce effective local nerve blockade with reduced toxicity compared to the unconjugated anesthetic agent following administration to a subject at a site requiring local nerve blockade. (Item 2) 2. The conjugate according to item 1, wherein the anesthetic agent is a site 1 sodium channel blocker (S1SCB). (Item 3) 3. The conjugate according to item 2, wherein the S1SCB is selected from the group consisting of tetrodotoxin (TTX), saxitoxin (STX), decarbamoyl saxitoxin, neosaxitoxin and gonyautoxin. (Item 4) 2. The conjugate according to item 1, wherein the anesthetic agent is a vanilloid receptor subtype 1 (TRPV1) agonist. (Item 5) 2. The conjugate of claim 1, wherein the polymer backbone comprises poly(glycerol sebacate) (PGS). (Item 6) 6. The conjugate according to item 1 or item 5, wherein the polymer backbone further comprises one or more hydrophilic polymers having a molecular weight of about 100 Da to about 200,000 Da (inclusive), preferably about 200 Da to about 2,000 Da (inclusive). (Item 7) 7. The conjugate of claim 6, wherein the polymer backbone comprises polyethylene glycol (PEG). (Item 8) 8. The conjugate of claim 7, wherein the PEG is one or more PEGs selected from the group consisting of PEG monomethyl ether (PEGMME), PEG100, PEG200, PEG300, PEG400, PEG500, PEG600, PEG700, PEG800, PEG900, PEG1000 and PEG2000. The formulation of claim 1, wherein the PGS is covalently attached to the hydrophilic polymer via a hydrolyzable ester bond. (Item 9) 2. The conjugate according to item 1, wherein the conjugate releases the anesthetic agent over a period of about 24 hours to 72 hours, 72 hours to 1 week, or 1 week to 1 month after administration in vivo. (Item 10) 10. The conjugate according to any one of items 1 to 9, further comprising one or more active agents covalently conjugated to the backbone polymer. (Item 11) 11. The conjugate according to item 10, further comprising one or more covalently attached glucocorticoids. (Item 12) 12. The conjugate according to item 11, wherein the glucocorticoid is selected from the group consisting of dexamethasone, cortisone, hydrocortisone, prednisone, beclomethasone, betamethasone, flunisolide, methylprednisone, paramethasone, prednisolone, triamcinolone, alclometasone, amcinonide, clobetasol, fludrocortisone, diflurosone diacetate, fluocinolone acetonide, fluoromethalone, flurandrenolide, halcinonide, medrysone and mometasone, and pharma- ceutically acceptable salts and mixtures thereof. (Item 13) 11. The conjugate according to item 10, wherein the conjugate further comprises covalently bound dexamethasone. (Item 14) A formulation for in vivo administration comprising the conjugate according to any one of items 1 to 13. (Item 15) 15. The formulation of item 14, wherein the formulation comprises PEG as an excipient. (Item 16) 16. The formulation of item 15, wherein the PEG is PEG200. (Item 17) 17. A dosage unit comprising the formulation according to any one of items 14 to 16, comprising an anesthetic agent in an amount of 0.1 μg to 200 μg (inclusive), preferably 1 μg to 100 μg (inclusive). (Item 18) 18. The dosage unit according to item 17, wherein the amount of anesthetic is an amount effective to induce effective local nerve blockade for up to one month after administration to a subject in need thereof. (Item 19) 18. A method for providing neuroblockade in a subject in need thereof, comprising administering to said subject an effective amount of the formulation according to any one of items 14 to 16 or the dosage unit according to item 17, in an amount effective to provide a nerve block at the administration site. (Item 20) 20. The method of claim 19, wherein the formulation is present in an amount effective to delay the onset of neuropathic pain in the subject. (Item 21) 20. The method of claim 19, wherein the subject is a human. (Item 22) 20. The method of claim 19, wherein the method provides pain relief for at least 3 days and up to 1 week, 2 weeks, 3 weeks, or 1 month after administration. (Item 23) 23. The method according to any one of items 19 to 22, wherein the formulation is a formulation injectable by syringe. (Item 24) 23. The method according to any one of items 19 to 22, wherein the formulation is administered via injection or infiltration at or near the painful site. (Item 25) 14. A method for making a conjugate according to any one of items 1 to 13, comprising covalently coupling one or more anesthetic agents to one or more polymer backbones. (Item 26) 26. The method of claim 25, wherein the covalent coupling comprises a Steglich esterification reaction. (Item 27) 26. The method of claim 25, wherein the covalent coupling is performed under conditions that do not denature or otherwise reduce the biological activity of the anesthetic agent. (Item 28) 17. A method for preparing the formulation according to any one of items 14 to 16, comprising dispersing the conjugate in an excipient suitable for in vivo administration. (Item 29) 1. A method of treating or preventing pain at a site in a subject in need thereof, the method comprising: (a) an anesthetic-polymer covalent conjugate, wherein the conjugate comprises an anesthetic agent covalently conjugated to a polymer backbone via a hydrolyzable linker; an anesthetic-polymer covalent conjugate, wherein the polymer backbone comprises one or more hydrophilic or hydrophobic polymers and optionally one or more polyethylene oxide (PEG) polymers; and (b) Aqueous solution of PEG administering to the subject a formulation comprising wherein the anesthetic-polymer covalent conjugate is dispersed within an aqueous solution of the PEG; The method, wherein the formulation is in an amount effective to provide effective nerve blockade at the site of need for a period of at least 72 hours and up to one month after administration. [Brief description of the drawings]

[0017] [Figure 1A] 1A and 1B are schematic diagrams of the stepwise synthesis of covalent conjugates of an anesthetic (tetrodotoxin; TTX) with a dicarboxylic acid / triol polymer (TD) to form a TD-TXX conjugate (FIG. 1A) or with PEG (P) to form a TDP-TXX conjugate (FIG. 1B), respectively. FIG. 1C is a schematic diagram of a molecular model of a "lattice" of a PGS-PEG-TTX conjugate. TTX is represented as (●) and PGS-PEG is represented as (-). [Figure 1B] 1A and 1B are schematic diagrams of the stepwise synthesis of covalent conjugates of an anesthetic (tetrodotoxin; TTX) with a dicarboxylic acid / triol polymer (TD) to form a TD-TXX conjugate (FIG. 1A) or with PEG (P) to form a TDP-TXX conjugate (FIG. 1B), respectively. FIG. 1C is a schematic diagram of a molecular model of a "lattice" of a PGS-PEG-TTX conjugate. TTX is represented as (●) and PGS-PEG is represented as (-). [Figure 1C] 1A and 1B are schematic diagrams of the stepwise synthesis of covalent conjugates of an anesthetic (tetrodotoxin; TTX) with a dicarboxylic acid / triol polymer (TD) to form a TD-TXX conjugate (FIG. 1A) or with PEG (P) to form a TDP-TXX conjugate (FIG. 1B), respectively. FIG. 1C is a schematic diagram of a molecular model of a "lattice" of a PGS-PEG-TTX conjugate. TTX is represented as (●) and PGS-PEG is represented as (-).

[0018] [Diagram 2]Figures 2A-2C show contact angle (degrees) versus fphil (%) (Figure 2A); T mass loss (time) versus fphil (%) (Figure 2B); and TgD8

number

number

number

[0019] [Diagram 3] 3A and 3B are bar graphs showing % cell viability for C2C12 cells (FIG. 3A) or PC12 cells (FIG. 3B) incubated for 24 hours with each of TgD8; TgD8P200; TgD8P1000; and TgD8P2000, each of which was at a dose of 0.001 mg / ml (left bar), 0.01 mg / ml (middle bar), and 0.1 mg / ml (right bar), respectively. Data are means ± SD, n=4.

[0020] [Figure 4] 4A-4B show the T mass loss (time) versus fphil (%) (FIG. 4A) and the TgD8

number

number

number

[0021] [Figure 5A] 5A and 5B are schematic diagrams of the stepwise synthesis of covalent conjugates of an anesthetic (tetrodotoxin; TTX) with a dicarboxylic acid / triol polymer (TD) with one or more additional "linking" polymers to form a TD polymer-TXX conjugate (FIG. 5A) or with dexamethasone (Dex) to form a TD-Dex-TXX conjugate (FIG. 5B), respectively. [Figure 5B] 5A and 5B are schematic diagrams of the stepwise synthesis of covalent conjugates of an anesthetic (tetrodotoxin; TTX) with a dicarboxylic acid / triol polymer (TD) with one or more additional "linking" polymers to form a TD polymer-TXX conjugate (FIG. 5A) or with dexamethasone (Dex) to form a TD-Dex-TXX conjugate (FIG. 5B), respectively.

[0022] [Figure 6A] 6A to 6E are line graphs.

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

[0023] [Figure 7] FIG. 7 is a schematic diagram of the solvent evaporation process to prepare an injectable formulation of PGS-PEG-TTX conjugates in PEG200.

[0024] [Figure 8] 8A to 8C are line graphs.

number

number

number

number

number

number

[0025] [Figure 9A] 9A to 9D are respectively

number

number

number

number

number

number

number

number

[0026] [Figure 10A] 10A-10E are graphs. FIG. 10A shows the results of the respective injected legs.

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

[0027] [Figure 11] FIG. 11 shows the conjugated TTX / PEG200

number

number

[0028] [Figure 12A] Figures 12A-12D are line graphs. Figures 12A and 12B show % mortality (Figure 12A) and duration of block (hours) (Figure 12B) versus fphil (%) for 10 μg TTX conjugate, respectively. Figures 12C and 12D show % block success (Figure 12C) and duration of block (hours) (Figure 12D) versus fphil (%) for 1 μg TTX conjugate, respectively. [Figure 12B] Figures 12A-12D are line graphs. Figures 12A and 12B show % mortality (Figure 12A) and duration of block (hours) (Figure 12B) versus fphil (%) for 10 μg TTX conjugate, respectively. Figures 12C and 12D show % block success (Figure 12C) and duration of block (hours) (Figure 12D) versus fphil (%) for 1 μg TTX conjugate, respectively. [Figure 12C] Figures 12A-12D are line graphs. Figures 12A and 12B show % mortality (Figure 12A) and duration of block (hours) (Figure 12B) versus fphil (%) for 10 μg TTX conjugate, respectively. Figures 12C and 12D show % block success (Figure 12C) and duration of block (hours) (Figure 12D) versus fphil (%) for 1 μg TTX conjugate, respectively. [Figure 12D]Figures 12A-12D are line graphs. Figures 12A and 12B show % mortality (Figure 12A) and duration of block (hours) (Figure 12B) versus fphil (%) for 10 μg TTX conjugate, respectively. Figures 12C and 12D show % block success (Figure 12C) and duration of block (hours) (Figure 12D) versus fphil (%) for 1 μg TTX conjugate, respectively.

[0029] [Figure 13] FIG. 13 is a line graph showing relative fluorescence (%) versus time post-injection (hours).

[0030] [Figure 14] Figure 14 shows the PGS-Cap

number

number

number

[0031] Detailed Description of the Invention I. Definition The term "Site 1 sodium channel blocker" or "S1SCB" refers to a molecule that binds to the outer opening of a sodium channel at a location called "Site 1." In a preferred embodiment, the Site 1 sodium channel blocker is a naturally occurring toxin or a derivative thereof. The term "derivative thereof" includes any derivative of a Site 1 sodium channel blocker (e.g., biological and / or pharmacological, i.e., that effectively blocks sodium channels) that has substantially the same functional properties as the underivatized Site 1 sodium channel blocker.

[0032] The term "anesthesia" refers to loss of sensation (localized without loss of consciousness; generalized with loss of consciousness), usually without loss of vital functions. The terms "anesthetic" and "anesthetic agent" refer to an agent that induces anesthesia in a subject.

[0033] The term "vasoconstrictor" refers to an agent that causes narrowing of the lumen of a blood vessel, especially as a result of vasomotion.

[0034] The term "infiltration" refers to injection into multiple layers or regions of tissue.

[0035] The term "injection" refers to injection into a single point in a tissue or cavity.

[0036] The term "nerve block" refers to local anesthesia produced by the interruption of the flow of impulses along a nerve trunk.

[0037] The term "minimum effective concentration" (MEC) is the lowest local concentration of one or more drugs at a given location that is sufficient to provide pain relief.

[0038] The terms "individual," "individual," "subject," and "patient" are used interchangeably and refer to mammals, including but not limited to humans, rodents (e.g., mice and rats), and other laboratory animals.

[0039] The term "biocompatible" refers to one or more materials that are not themselves toxic to a host (e.g., an animal or human) and do not degrade (if the polymer degrades) at a rate that produces toxic concentrations of monomeric or oligomeric subunits or other by-products in the host.

[0040] The term "backbone polymer" or "backbone" refers to the chemical moiety or polymer contained within the anesthetic-polymer covalent conjugate to which the anesthetic is covalently attached. The backbone polymer does not form part of the excipient. Exemplary backbone polymers include dicarboxylic acid / triol derivatives. A preferred backbone polymer is poly(glycerol sebacate) (PGS)-PEG copolymer (PGS-PEG).

[0041] The term "hydrolyzable linker" refers to any chemical bond or chemical moiety that can be hydrolytically degraded, such as an ester bond.

[0042] The term "burst release" refers to the rapid release or delivery of an agent, e.g., the rapid delivery of an active agent from a delivery vehicle or delivery structure. For example, the rapid release or "burst" release of a drug from a drug delivery system in vivo can occur due to a portion of the drug being adsorbed or weakly bound to the delivery system, as compared to slower drug release due to diffusion or degradation of the delivery system.

[0043] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in therapeutic preparations is contemplated. Supplementary active compounds can also be incorporated into the preparation.

[0044] II. Composition Formulations containing local anesthetics covalently conjugated to biodegradable and biocompatible polymers such as PGS and PGS-PEG backbones have been demonstrated to provide tunable anesthetic release in vivo. These formulations allow for localized delivery of high concentrations of anesthetics with low systemic and local toxicity. The rate and amount of anesthetic released at the site of administration can be controlled by varying the amount of anesthetic, the molecular weight, composition and concentration of the polymer, and the type of covalent linker used to conjugate the molecule.

[0045] Exemplary anesthetic agents include site 1 sodium channel blockers and vanilloids. The anesthetic agent is conjugated to at least one topical amphiphilic polymer. Exemplary amphiphilic polymers include poly(glycerol sebacate) (PGS). In some embodiments, the PGS conjugate includes one or more hydrophilic polymers. Exemplary hydrophilic polymers include polyethylene-glycol (PEG). Each of these components is discussed in detail below. Formulations of anesthetic-polymer covalent conjugates may also include additional active agents (e.g., therapeutic or diagnostic agents) as well as excipients and preservatives.

[0046] A. Anesthetics The anesthetic is included in the anesthetic-polymer covalent conjugate. Prior to conjugation, the anesthetic comprises one or more free hydroxyl or carboxyl groups. In some embodiments, the anesthetic is modified to comprise one or more free hydroxyl or carboxyl groups prior to conjugation. In some embodiments, the anesthetic is a local anesthetic. Exemplary classes of anesthetic include site 1 sodium channel blocking agents (S1SCB) and vanilloid receptor subtype 1 (TRPV1) agonists. A preferred anesthetic is tetrodotoxin (TTX).

[0047] 1. Site 1 sodium channel blockers (S1SCBs) Site 1 blockers are a family of molecules long recognized for their potent and specific blockade of voltage-gated sodium channels. Site I sodium channel blockers (S1SCBs) include the phycotoxins (saxitoxin (STX), decarbamoylsaxitoxin, neosaxitoxin (Neo) and gonyautoxin), tetrodotoxin (TTX) and several of the conotoxins.

[0048] i. Tetrodotoxin (TTX) Tetrodotoxin (TTX) is a highly potent neurotoxin that blocks fast Na+ currents in human myocytes (muscle contractile cells), thereby inhibiting their contraction. Chemically, it is an amino polhydroquinoline (see Pharmacological Reviews, Vol. 18 No. 2, pp. 997-1049).

[0049] Tetrodotoxin alone is too toxic to be used as an anesthetic. In rats, the combination of tetrodotoxin with bupivacaine produced a longer duration of sciatic nerve blockade without significant systemic toxicity compared to tetrodotoxin alone (Kohane et al., Anesthesiology, 1998:119-131). Although the potent inhibition of voltage-gated Na+ channels makes TTX too harmful to be used alone as a drug, blocking such channels in a controlled manner may be desirable in the treatment of conditions such as Parkinson's disease and chronic pain in terminal cancer patients. The formation of conjugates makes this highly toxic toxin a useful and safe anesthetic. [ka]

[0050] A. The origin of tetrodotoxin Tetrodotoxin has been isolated from animals such as the blue-ringed octopus and is also produced by bacteria. The most common bacteria associated with TTX production are from the Vibrio genus, with Vibrio alginolyticus being the most common species. Pufferfish, chaetognaths and nemerteans have been shown to contain Vibrio alginolyticus and TTX, but the relevance of these facts to the production of TTX in animals has not been firmly established, and there remains much debate in the literature as to whether these bacteria are indeed the source of TTX in animals. Tetrodotoxin is perhaps the most widely known site 1 toxin, but it is expensive for clinical use because it must be derived from pufferfish. The endosymbiotic bacteria that produce TTX have reduced production of TTX when grown ex vivo. Tetrodotoxin is available from the ovaries and eggs of some species of pufferfish and certain species of California newts. Numerous schemes for the total chemical synthesis of tetrodotoxin have been reported, including the synthesis of TTX by Diels-Alder reaction or from carbohydrates and homologues (Ohyabu et al., J Am Chem Soc. Jul 23;125(29):pp8798-805(2003)); Nishikawa et al., Angew. Chem. Int. Ed., 43,4782.DOI:10.1002 / anie.200460293(2004); reviewed in Chau and Ciufolini, Mar Drugs, 9(10):2046-2074(2011)). The synthesis features rapid construction of cyclohexene by Diels-Alder cycloaddition using an enantiomerically pure dienophile, early introduction of an aminated quaternary center, and use of that center to direct the relative configuration of further functionalization around the ring.

[0051] ii. Phycotoxins Phycotoxins act as specific blockers of voltage-dependent sodium channels present in excitable cells (Kao, CY, Pharm. Rev., 18:997-1049 (1966)). Due to the inhibition of sodium channels, the transmission of nerve impulses is blocked and the release of neurotransmitters is prevented at the level of the neuromotor junction, thereby preventing muscle contraction. Due to these physiological effects, these compounds are potentially useful in pharmacology when applied locally in injectable form and when used as muscle activity inhibitors in pathologies associated with muscle overactivity (e.g. muscle spasms and focal dystonia). Furthermore, since the blockade of nerve impulses at the transmission level is brought about when these compounds are applied as local infiltrations, they can not only block efferent neurotransmission pathways, but also afferent pathways, can cause inhibition of sensory pathways, and can also produce an anesthetic effect when these compounds are injected locally. This is a surprising effect since both effects occur simultaneously, as described in US Pat. No. 4,001,413.

[0052] The chemical structures of these phycotoxins have the general structure of Formula II. [ka]

[0053] The specific chemical structure of this structure is defined by the substituents R1-R5 set forth in Table 1.1. [Table 1-1]

[0054] Saxitoxin Saxitoxin (STX) was first extracted from the Alaskan buttercrumb, Saxidomus gigantcus, and is present in algae of the genus Gonyaulax. Its reported chemical formula is C10H15N7O3.2HCl. Saxitoxin is freely soluble in water and methanol, and the toxin is believed to have a perhydropurine nucleus incorporating two guanidinium moieties. STX is involved in paralytic shellfish poisoning. STX is one of the most toxic non-protein compounds known, with a reported toxicity of 8 μg / Kg in mice (approximately 0.2-1.0 mg would prove lethal to humans), and is widely considered too toxic to be used alone as a local anesthetic.

[0055] b. Neosaxitoxin and decarbamoylsaxitoxin Neosaxitoxin and decarbamoyl saxitoxin are potentially more potent and may have advantages over saxitoxin in formulations.

[0056] Neosaxitoxin ("NeoSTX" or "Neo") is in clinical development as a long-lasting local anesthetic (Rodriguez-Navarro et al., Anesthesiology, 2007;106:339-45; Rodriguez-Navarro et al., Neurotox. Res., 2009;16:408-15; Rodriguez-Navarro et al., Reg. Anesth. Pain Med., 2011;36:103-9). A phase I study of subcutaneous infiltration in human volunteers showed that NeoSTX produced effective cutaneous hypoesthesia (Rodriguez-Navarro et al., Anesthesiology, 2007;106:339-45), and a second phase I study showed that combination with bupivacaine produced longer lasting analgesia compared with NeoSTX or bupivacaine alone (Rodriguez-Navarro et al., Neurotox. Res., 2009;16:408-15). [ka]

[0057] (i) Origin of phycotoxins Saxitoxin (STX) and its derivatives can be produced in algae-derived bioreactors. The phycotoxins neosaxitoxin, saxitoxin and gonyautoxin are active compounds produced by harmful algal blooms such as Alexandrium species, Piridinium species and Gimnodinium species (Lagos, N. Biol. Res., 31:375-386 1998). In the past 15 years, it has been demonstrated that these phycotoxins, in addition to being produced by marine dinoflagellates, can also be produced by freshwater cyanobacteria such as photosynthetic blue-green algae.

[0058] Only four genera of cyanobacteria capable of producing paralytic phycotoxins have been identified, each of which produces a phycotoxin mixture that differs in both the amount and type of phycotoxins produced, i.e., they produce different profiles of paralytic phycotoxins (Lagos et al., 1999, TOXICON, 37:1359-1373 (1999). Pereira et al., TOXICON, 38:1689-1702 (2000).

[0059] STX can also be produced by chemical synthesis according to at least three different methods (Kishi et al., J. Am. Chem. Soc., 98, 2818 (1977)); Jacobi et al., J. Am. Chem. Soc., 106(19), pp5594-5598 (1984) Fleming et al., J. Am. Chem. Soc., 3926 (2006)).

[0060] Two saxitoxin derivatives, neosaxitoxin (NeoSTX) and decarbamoyl saxitoxin, have advantages in terms of production process and potency. One study examined sciatic nerve blockade in rats using several members of the saxitoxin series, including NeoSTX (Kohane et al., Reg. Anesth. Pain Med., 25:52-9 (2000)). Saxitoxin and all of these two derivatives, when combined with bupivacaine or epinephrine, produce a markedly synergistic blockade and prolonged block (1-2 days in the rat sciatic nerve in vivo).

[0061] 2. Vanilloid receptor subtype 1 (TRPV1) agonists Capsaicin and its derivatives Capsaicin is a member of the capsaicinoid group of compounds, which are characterized as containing a 3-hydroxy-4-methoxy-benzylamide (vanilloid ring) pharmacophore and a hydrophobic alkyl side chain. Capsaicinoids are the compounds responsible for the pungency of chili pepper fruits and their products.

[0062] Capsaicin was first isolated in 1876, and its experimental structure was determined for the first time in 1919. 18 H 27 Capsaicin has been characterized as NO3 (8-methyl-N-vanillyl-trans-6-nonenamide). Capsaicin has a molecular weight of 305 daltons and a vanilloid ring pharmacophore with an 11-carbon hydrophobic alkyl side chain, as shown in formula IV. The double bond structure in the hydrophobic alkyl side chain prevents intramolecular rotation, so the molecule exhibits cis / trans isomerism. However, capsaicin exists naturally as the trans isomer, since the cis isomer is the less stable configuration. [ka]

[0063] Capsaicin has very low volatility and is completely odorless. Purified capsaicin is a waxy, colorless substance at room temperature that is insoluble in cold water, but is freely soluble in alcohol, fats, and oils. The long hydrocarbon tail allows for incorporation of capsaicin into lipid-rich cell membranes, and capsaicin is known to be effectively absorbed locally through the skin and mucous membranes. The pharmacokinetic half-life of capsaicin has been found to be approximately 24 hours.

[0064] Capsaicin is FDA approved as a topical drug for chronic pain management. Capsaicin binds to transient receptor potential vanilloid (TRPV1) (Caterina et al., Nature, 389:816-824 (1997)), which mediates nociceptive stimulation in sensory neurons. Capsaicin has been shown to be safe in animal studies (Park et al., Anticancer Res., 18:4201-4205 (1998)).

[0065] Capsaicinoids are naturally occurring compounds that are the active components of chili peppers, well known for their use in culinary applications worldwide. Capsaicinoids are produced within the fruits of plants belonging to the Capsicum genus of the Solanaceae family. The fruits of the "fragrant" chili pepper plant (e.g., commonly known as jalapeno or habanero) are a rich natural source of capsaicin. Capsaicin is located between the seeds and the ribs of the chili pepper fruit and is retained within the dried and / or crushed chili pepper fruit. Examples of Capsicum species known for their high content of capsaicinoids include C. annum (oleoresin pepper), C. frutescens (jalapeno pepper) and C. chinense (habanero pepper), which have been found to contain 0.22-20 mg of total capsaicinoids / g dry weight. Capsaicin is the most abundant capsaicinoid in the chili pepper plant, accounting for about 71% of the total capsaicinoids in most hot varieties (see de Lourdes Reyes-Escogido et al., Molecules, 16:1253-1270 (2011)). The biosynthesis of capsaicin requires the condensation of vanillylamine with 8-methylnonenoic acid, which occurs via the enzyme capsaicin synthase (CS).

[0066] Purified capsaicin is commercially available (Sigma Aldrich # M2808, CAS#404-86-4). Capsaicin and its analogs are industrially produced using fatty acid chlorides and amines at temperatures of 140-170°C under moderate pressure (see Kaga et al., J. Org. Chem., 54:3477-3478, 1989 and Kaga et al., Tetrahedron 1996, 52, 8451-8470). However, the application of large-scale chemical synthesis of capsaicin is limited by the toxicity of the required reagents, a drawback that makes enzymatic synthesis an attractive alternative to conventional chemical synthesis. Thus, several methods are known for the in vitro chemical synthesis of capsaicinoids from substrate molecules using enzyme-catalyzed reactions (see, for example, Kobata et al., Biotechnol. Lett., 21, 547-550 (1999)). The enzymatic formation of capsaicin in vitro has been demonstrated using cells and tissues from Capsicum annum plants grown in liquid medium (see Johnson et al., Plant Sci. 70:223-229 (1990)). Cells and placenta tissue from fruits grown in vitro and immobilized in calcium alginate produced capsaicin in the medium. A greater potential for capsaicin synthesis was observed in immobilized placenta tissue than in immobilized cells. After 30 days of culture, a maximum yield of 2,400 μg capsaicin / g immobilized placenta was observed.

[0067] B. Capsaicin analogues i. Capsaicinoids In some embodiments, capsaicin is a capsaicinoid analog of capsaicin. Capsaicinoid analogs of capsaicin are known in the art. For example, see Reilly and Yost, Drug Metab.Disp.,33:550-536(2005). Reilly and Yost report five naturally occurring capsaicinoid analogs of capsaicin. These compounds have the same 3-hydroxy-4-methoxy-benzylamide (vanilloid ring) pharmacophore, but have differences in hydrophobic alkyl side chain portion (e.g., saturation at C15-16 (ω-2,3 position), loss of methyl group at C17 (loss of tertiary carbon) and change in length of hydrocarbon chain). Naturally occurring capsaicinoid analogs of capsaicin include, but are not limited to, homocapsaicin, nordihydrocapsaicin, dihydrocapsaicin, homodihydrocapsaicin, n-vanillyl octanamide, nonivamide, and n-vanillyl decanamide, as in Formula V. [ka]

[0068] In some embodiments, capsaicin is the capsinoid analog of capsaicin.The capsinoid group of compounds, including capsiate, dihydrocapsiate and nordihydrocapsoate, are structurally similar to capsaicinoid, but have different central bonds (amide moiety in capsaicinoid, ester moiety in capsinoid), as in formula VI.Capsinoid is isolated from a few varieties of non-pungent red pepper plants (e.g., cultivar CH19sweet).The biological activity of capsiate is similar to that of capsaicin, but capsinoid does not show pungency or sensory stimulation. [ka]

[0069] In some embodiments, capsaicin is a synthetic capsaicinoid or a capsinoid analog of capsaicin. Several synthetic (non-naturally occurring) analogs of capsaicin are known in the art (see, for example, Satoh et al., Biochim. Biophys. Acta., 1273:21-30 (1996)). Hot and non-hot analogs can be synthesized with different acyl chain lengths and / or chemical substitutions on the aromatic ring. In particular, both capsaicinoids and capsinoids can be synthetically modified by altering the substitution pattern and / or the number of methoxy groups on the benzene ring, which may be stacked on the quinone ring of ubiquinone. Furthermore, capsaicin can be modified by changing the position of the amphoteric amide bond unit in the molecule and / or other chemical modifications of this unit. An example of chemical modification is shown in Formula VII. [ka]

[0070] B. Polymer Conjugates for the controlled release of an anesthetic in vivo include one or more polymers to which an anesthetic is covalently conjugated, for example, by an ester bond. The polymer to which the anesthetic is attached may also be referred to as the "backbone" polymer.

[0071] Covalent conjugates of anesthetics and polymers require a "backbone" formed from a suitable polymer (e.g., a dicarboxylic acid / triol (TD) polymer) to which the anesthetic can be attached. Representative schemes for the generation and conjugation of such polymers are provided in Figures 1A and 1B, respectively.

[0072] In some embodiments, the conjugate comprises a polyester polymer. In some embodiments, the conjugate comprises a blend of more than one polyester polymer. A preferred polyester polymer is poly(glycerol sebacate) (PGS).

[0073] In some embodiments, PGS is blended with one or more additional polyester polymers to change one or more material properties of the conjugate. Suitable polyester polymers for inclusion in the conjugate include bioabsorbable synthetic polyesters (e.g., poly(lactide-co-caprolactone), poly(caprolactone) (PCL), polydioxanone (PDO), poly(ortho)esters, poly(glycolic acid) (PGA), polymers of lactic and glycolic acid, poly(lactic acid) (PLA), polybutyrate), poly(lactide-co-glycolide), poly(hydroxybutyrate), and their copolmyers, blends, and chemical derivatives (substitution, addition, hydroxylation, oxidation of chemical groups, e.g., alkyl, alkylene, and other modifications routinely performed by those skilled in the art). Any natural or synthetic polymer with -COOH or -OH groups can be used to conjugate the anesthetic agent. Thus, natural and synthetic polymers (e.g., hyaluronic acid, tannic acid, poly-acrylic acid, and other compounds containing carboxyl-terminated or side-chain moieties) can be included in the anesthetic conjugate. Generally, the polymers included in the covalent conjugate of the anesthetic are biodegradable and biocompatible. However, in some embodiments, one or more non-biodegradable polymers are included in the covalent conjugate of the anesthetic. When the covalent conjugate of the anesthetic comprises a blend of one or more biodegradable polymers and one or more non-biodegradable polymers, the ratio of the biodegradable polymer to the non-biodegradable polymer can be altered to adjust, for example, the amount and / or delivery rate of the anesthetic agent according to the needs of the recipient.

[0074] 1. Poly(glycerol sebacate) (PGS) In some embodiments, other bioabsorbable polyesters are used in combination with or in place of poly(glycerol sebacate) (PGS) for use in anesthetic-polymer covalent conjugates. Anesthetic conjugates covalently bound to polymers for controlled release of anesthetics in vivo may include poly(glycerol sebacate) (PGS). PGS is a biodegradable polyester polymer used in a variety of biomedical applications. PGS is typically prepared by polycondensation of glycerol and sebacic acid. PGS is biocompatible and biodegradable, and the mechanical properties and degradation kinetics of PGS can be tailored by controlling factors such as curing time, curing temperature, reactant concentration, and, for acrylated PGS, the degree of acrylation (Rai et al., Progress in Polymer Science V. 37, (8), pp. 1051-1078 (2012)).

[0075] Glycerol is the basic building block of lipids, while sebacic acid is a natural metabolic intermediate in the ω-oxidation of medium- to long-chain fatty acids. Furthermore, copolymers containing glycerol and sebacic acid have been approved for medical applications by the U.S. Food and Drug Administration (FDA). PGS has been proposed for tissue engineering applications due to its desirable mechanical properties, biocompatibility and controlled degradation (Jia, Y. et al., Polymer Chemistry 7, 2553-2564, doi:10.1039 / c5py01993a (2016); Rai et al., Progress in Polymer Science 37, 1051-1078 (2012); Wang et al., Journal of Biomedical Materials Research Part A 66A, 192-197, (2003); Loh et al., Journal of Materials Chemistry B 3, 7641-7652 (2015); Wang et al., Nat Biotech 20, 602-606 (2002)). PGS is terminated with hydroxyl and carboxyl groups and can be easily covalently conjugated to peptides, proteins and drugs. TTX, as depicted in Figure 1, has a molecular structure rich in hydroxyl groups and can be covalently conjugated to PGS via ester bonds and released by hydrolysis of these bonds. Furthermore, the surface-eroding nature of PGS and PGS-based copolymers, which induces consistent release of TTX over an extended period of time following a nearly zero-order profile, makes them unique and preferable over other polyesters for controlled drug delivery applications (Wang et al., Journal of Biomedical Materials Research Part A 66A, 192-197 (2003)). The incorporation of PEG allows for tuning of the hydrophilicity of the resulting conjugate, and therefore the release rate of TTX. These features endow the conjugate with the potential for long-lasting local anesthesia while minimizing systemic and local toxicity.

[0076] 2. Polymers for tunable hydrophilicity The conjugate of an anesthetic covalently bonded to poly(glycerol sebacate) (PGS) optionally includes one or more additional hydrophilic polymers. The hydrophilic polymers extend the PGS backbone and change the hydrophilicity of the conjugate. Increasing the relative amount of hydrophilic polymer to PGS increases the hydrophilicity of the conjugate. The hydrophilicity of the conjugate is related to the hydrolysis rate of the ester bond in vivo. The hydrolysis rate of the ester bond in the PGS-anesthetic conjugate is related to the release of the anesthetic from the conjugate in vivo. Thus, the release rate of the covalently bonded anesthetic from the polyester conjugate in vivo is controlled by the amount and size of the hydrophilic polymer attached or associated with the PGS-anesthetic conjugate.

[0077] In a preferred embodiment, the one or more hydrophilic polymer components comprise a poly(alkylene glycol) chain. The poly(alkylene glycol) chain may comprise 1 to 500 repeating units, more preferably 40 to 500 repeating units. Suitable poly(alkylene glycols) include polyethylene glycol, polypropylene 1,2-glycol, poly(propylene oxide), polypropylene 1,3-glycol, and copolymers thereof.

[0078] A wide variety of hydrophilic polymers can be included, including polyβ-aminoesters and 1,2-aminoalcohol lipids. In some embodiments, the polymers are alkyl-modified polymers, such as alkyl-modified poly(ethylene glycol). Other exemplary polymers include poly(alkylene glycols), polysaccharides, poly(vinyl alcohols), polypyrrolidones, polyoxyethylene block copolymers (e.g., PLURONIC®), polyethylene glycols (PEGs) and copolymers thereof. Preferred hydrophilic polymers are biocompatible (i.e., do not induce significant inflammatory or immune responses) and non-toxic. Examples of suitable hydrophilic polymers include, but are not limited to, poly(alkylene glycols), such as polyethylene glycol (PEG), poly(propylene glycol) (PPG), and copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), polyvinylpyrrolidones), poly(hydroxyalkyl methacrylates), poly(hydroxyalkyl methacrylamides), poly(saccharides), poly(amino acids), poly(vinyl alcohols), and copolymers, terpolymers, and mixtures thereof.

[0079] In some embodiments, the one or more hydrophilic polymer components are copolymers that include one or more blocks of polyethylene oxide (PEO) with one or more blocks composed of other biocompatible polymers (e.g., poly(lactide), poly(glycolide), poly(lactide-co-glycolide), or polycaprolactone). The one or more hydrophilic polymer segments can be copolymers that include one or more blocks of PEO with one or more blocks comprising polypropylene oxide (PPO). Specific examples include triblock copolymers of PEO-PPO-PEO (e.g., POLOXAMERS™ and PLURONICS®).

[0080] a. Poly(ethylene glycol) (PEG) Conjugates of anesthetic agents covalently attached to one or more optionally containing PEGs (PGS) are described.

[0081] PEG is a commonly used hydrophilic polymeric agent. The size, relative amount and distribution of amphiphilic PEG incorporated into modified dendrimer-based nanoparticles (MDNPs) can affect the biophysical characteristics (e.g., structural features and charge density) of the resulting MDNPs.

[0082] The physical properties of MDNPs are directly related to the size, relative amount and distribution of amphiphilic PEG (i.e., the degree of PEGylation).Exemplary properties that can be modified include the effectiveness of MDNP uptake by one or more types of eukaryotic cells, the speed and effectiveness of intracellular delivery of therapeutic, prophylactic and diagnostic agents, and the immunogenicity and cytotoxicity of MDNPs.In certain embodiments, PEGylation results in charge neutralization of MDNPs.

[0083] Typically, amphiphilic PEGs include short chain oligo-ethylene glycols. Exemplary oligo-ethylene glycols include di-ethylene glycol, tri-ethylene glycol, tetra-ethylene glycol, penta-ethylene glycol, hexa-ethylene glycol, and the like. [ka]

[0084] In some embodiments, the amphiphilic polymer is a phospholipid conjugated to monomethoxypolyethylene glycol (mPEG). In certain embodiments, the PEG or mPEG associated with the lipid is a branched or "multi-armed" PEG. The MDNP may include a multi-armed polyethylene glycol having at least two branches with sulfhydryl or thiopyridine end groups. However, PEG polymers with other end groups, such as succinimidyl or maleimide end groups, may also be used.

[0085] The anesthetic-polymer covalent conjugate may include polyethylene glycol polymers having various molecular weights. For example, the PEG may have a molecular weight of approximately 100 Da (i.e., PEG 100 Da) to approximately 12,000 kDa (i.e., PEG 12 KDa), inclusive. The anesthetic-polymer covalent conjugate may be formed using a single type of amphipathic PEG, or may be formed from two or more different types of amphipathic PEG. For example, the anesthetic-polymer covalent conjugate may be formed using multiple different types of PEG with various molecular weights.

[0086] Anesthetic-polymer covalent conjugates can be formed using a single amphiphilic polymer species or a mixture of multiple different amphiphilic polymer species. Amphiphilic polymers can be modified with adducts. For example, amphiphilic polymers can be modified with one or more different adducts, the same or different. Thus, anesthetic-polymer covalent conjugates can be formed using PGS and one or more PEG and other polymers (including mixtures of the same or different adducts, as appropriate).

[0087] Exemplary PEGs are PEGs having molecular weights such as PEG(100);PEG(200);PEG(300);PEG(400);PEG(500);PEG(600);PEG(750);PEG(800);PEG(900);PEG(1,000);PEG(2,000);PEG(3,000);PEG(5,000);PEG(6,000);PEG(7,000);PEG(8,000);PEG(9,000);PEG(10,000);PEG(12,000); and PEGs having molecular weights greater than 12,000, such as PEG(20,000). The lipid component may contain saturated or unsaturated fatty acid moieties.

[0088] In some embodiments, the anesthetic-polymer covalent conjugate comprises one or more species of PEG or [methoxy(polyethylene glycol)](mPEG) molecules. When more than a single species of PEG and / or mPEG is used, they may be present in the same or different molar ratios. Exemplary PEG or mPEG that may be incorporated into the anesthetic-polymer covalent conjugate include PEG or mPEG moieties having molecular weights of 100 Da to 80,000 Da, e.g., 100 Da to 10,000 Da, more preferably, 200 Da to 5,000 Da, and most preferably, 200 to 2,000 Da.

[0089] PEG and / or mPEG can be included in a polyester to PEG molar ratio of 1:01 to 1:1,000. For example, in some embodiments, PEG is present in the PGS-PEG-TTX conjugate at a molar ratio of 1:1 PGS:PEG or at a molar ratio of more than 1:1 (e.g., 1:2, 1:3, 1:4; 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 or more). In some embodiments, PEG is present in the PGS-PEG-TTX conjugate at a molar ratio of 1:1 PEG:PGS or at a molar ratio of more than 1:1 (e.g., 1:2, 1:3, 1:4; 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 or more). The amount and size of PEG (or other hydrophilic polymer) included in the conjugate can be varied depending on the desired physiological properties of the conjugate in vivo.

[0090] C. Covalent linker moiety The anesthetic conjugates covalently bonded to PGS or PGS-PEG or other polymer conjugates are linked to one or more anesthetic agents via one or more types of covalent bonds. In a preferred embodiment, the covalent bonds are biodegradable bonds that degrade or are otherwise cleaved in vivo to release the anesthetic agent at or near the site of degradation. An exemplary biodegradable covalent bond is a hydrolyzable bond, such as an ester bond.

[0091] In some embodiments, the anesthetic-polymer covalent conjugate comprises an anesthetic and / or additional active agent directly conjugated or attached to the PGS or PGS-PEG or other polymer backbone, for example, via an ester bond. Optionally, the anesthetic or compound is conjugated to the PGS or PGS-PEG or other polymer backbone via one or more spacers / linkers via various bonds (e.g., disulfide, carbonate, carbamate, thioester, hydrazine, hydrazide and amide bonds). In some embodiments, the attachment occurs via a suitable spacer that provides a disulfide bridge between the agent and the PGS or PGS-PEG or other polymer backbone. In this case, the anesthetic-polymer covalent conjugate can rapidly release the agent in vivo by thiol exchange reaction under reducing conditions found in the body.

[0092] 1. Spacer In some embodiments, the anesthetic agent is coupled to the polymer backbone via one or more intermediate "spacer" or "linker" molecules. The spacer or linker can increase the hydrophilicity of the molecule, thereby increasing the rate of hydrolysis or degradation of the anesthetic / polymer bond, resulting in increased release of the anesthetic agent in vivo. Thus, a spacer can be included to modify the release rate of the anesthetic agent and / or additional active agents from the polymer in vivo.

[0093] The term "spacer" includes compounds used to link active agents to PGS or PGS-PEG or other polymer backbones. The spacer can be a single chemical entity or two or more chemical entities that are linked together to crosslink the polymer and the anesthetic agent. The spacer can include any small molecule chemical entity, peptide or polymer with sulfhydryl, thiopyridine, succinimidyl, maleimide, vinylsulfone and carbonate termini.

[0094] The spacer may be selected from sulfhydryl, thiopyridine, succinimidyl, maleimide, vinylsulfone and carbonate terminated compounds. The spacer may include thiopyridine terminated compounds such as dithiodipyridine, N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate (LC-SPDP) or sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate (sulfo-LC-SPDP). Spacers can also include peptides, where the peptides are linear or cyclic, essentially having a sulfhydryl group (e.g., glutathione, homocysteine, cysteine ​​and its derivatives, arg-gly-asp-cys (RGDC), cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC)), cyclo(Arg-Gly-Asp-D-Tyr-Cys), cyclo(Arg-Ala-Asp-d-Tyr-Cys)). Spacers can be mercapto acid derivatives (e.g., 3-mercaptopropionic acid, mercaptoacetic acid, 4-mercaptobutyric acid, thiolan-2-one, 6-mercaptohexanoic acid, 5-mercaptovaleric acid, and other mercapto derivatives, such as 2-mercaptoethanol and 2-mercaptoethylamine).

[0095] In some embodiments, the spacer is thiosalicylic acid and / or its derivatives, (4-succinimidyloxycarbonyl-methyl-alpha-2-pyridylthio)toluene, (3-[2-pyridylthio]propionylhydrazide. In some embodiments, the spacer has a maleimide terminus, where the spacer comprises a polymer or small molecule chemical entity (e.g., bis-maleimidodiethylene glycol and bis-maleimidotriethylene glycol, bis-maleimidoethane, bismaleimidohexane). In some embodiments, the spacer comprises a vinyl sulfone, e.g., 1,6-hexane-bis-vinyl sulfone. In some embodiments, the spacer comprises a thioglycoside, e.g., thioglucose. In some embodiments, the spacer is formed from one or more reduced proteins (e.g., bovine serum albumin and human serum albumin), any thiol-terminated compound capable of forming a disulfide bond. Spacers may also include maleimide, succinimidyl, and thiol-terminated polyethylene glycols.

[0096] In some embodiments, the spacer / linker is a gamma-aminobutyric acid (GABA) linker, an allyl linker, a propargyl linker, an ethanethiol linker, a pyridine disulfide linker, In some embodiments, the spacer / linker is conjugated to the PGS or PGS-PEG or other polymer backbone via one or more of an ether, thioester, carbamate, carbonate, hydrazine or amide bond, for example to improve stability under physiological conditions compared to an ester bond.

[0097] In other embodiments, various linkers, e.g., allyl, propargyl, etc., are attached to the PGS or PGS-PEG or other polymer backbone surface via various bonds, e.g., ether, ester, carbamate, carbonate, etc., that can participate in click chemistry for conjugation of active agents such as NAC.

[0098] In further embodiments, the PGS or PGS-PEG or other polymer backbone is conjugated to a first anesthetic or active agent via one linker while being conjugated to a second anesthetic or active agent via a different linker.

[0099] D. Penetration-Enhancing Excipients Polyethylene oxide has been demonstrated to function as a chemical penetration enhancer (CPE) for anesthetic-polymer conjugates.

[0100] The data in the examples demonstrate that when an anesthetic-polymer conjugate is dispersed in a solution of PEG200, PEG200 functions as a CPE. A solution of an anesthetic-polymer conjugate dispersed in PEG can increase drug influx into the nerve, which may be due to the amphiphilic nature of PEG200. CPE may allow or facilitate the anesthetic to cross the perineurial barrier and increase drug influx into the nerve.

[0101] As described in the Examples below, a dose of 1-3 μg of unconjugated anesthetic (e.g., TTX) in 0.5 mL of PEG200 resulted in complete nerve blockade in 100% of the tested animals, with the duration of block being up to 5.3 ± 0.3 hours. This is three times longer than the effect of 0.5% bupivacaine, an anesthetic commonly used in clinical practice. PEG200 is a suitable delivery vehicle for local anesthesia in clinical practice due to its low toxicity and already widely used in various pharmaceutical formulations.

[0102] Thus, in some embodiments, the formulation of the anesthetic-polymer conjugate includes an excipient that includes PEG. A preferred excipient is PEG 200. The excipient enhances the efficacy of the anesthetic-polymer conjugate.

[0103] Due to their good miscibility, the TDP-TTX conjugates were easily dispersed in PEG200 to form homogeneous TDP-TTX / PEG200 formulations that could be injected with a syringe. All prepared TDP-TTX / PEG200 formulations behaved as liquids with viscosities below 10 Pa s, allowing their administration to patients by syringe injection.

[0104] In some embodiments, the anesthetic-polymer conjugate and the CPE are integrated into one system. In an exemplary embodiment, the inclusion of PEG200 in the formulation of PGS-PEG-TTX provides a broader therapeutic index than an equivalent amount of PGS-PEG-TTX without the PEG200 excipient.

[0105] Thus, in some embodiments, conjugates of anesthetic covalently attached to PGS or PGS-PEG are dispersed in PEG, such as PEG200, to enhance the effectiveness of the injectable formulation.

[0106] In a preferred embodiment, the anesthetic covalent conjugate is formulated as a liquid for administration directly to or near the painful site via injection or infiltration. Preferred excipients include polyethylene glycols (PEGs) having molecular weights between 1 Da and 1,000 Da, e.g., PEG 200.

[0107] 1. Preparations that can be injected by syringe The choice of solvent for syringe-injectable formulations of PGS-TTX and PGS-PEG-TTX must take into account the desired specific flow properties required for proper administration to the affected area by the patient (Mastropietro et al., J Dev Drugs 2,108(2013)). For example, PGS is a thermosetting polymer, and the elastic modulus of PGS can be easily tuned by controlling various parameters (e.g., reaction time, reaction temperature, and curing time).

[0108] In some embodiments, the anesthetic-polymer covalent conjugate comprises a sufficient amount of hydrophilic polymer to allow the conjugate to be dispersed in an aqueous medium such as phosphate buffered saline (PBS). For example, PGS-PEG2000-TTX and PGS-PEG1000-TTX conjugates, which contain a high percentage of PEG in the backbone, can be uniformly suspended in PBS. However, some PGS-PEG-TTX and PGS-TTX conjugates cannot be easily uniformly suspended in PBS. Thus, in some embodiments, a solubilizer is added to increase the hydrophilicity of the conjugate, making it applicable to injection formulations. Exemplary solubilizers include PEG. A preferred PEG is PEG200. PEG200 has been demonstrated to function not only as a solubilizing medium for the conjugate, but also as a penetration enhancer that enhances the effectiveness of the conjugate as a delivery vehicle for anesthetic agents.

[0109] i.PEG In some embodiments, the covalent conjugate of an anesthetic is formulated as a syringe injectable form by homogenous suspension in PEG.

[0110] The term "injectate" refers to a material that is injected. In some embodiments, the injectate includes one or more excipient materials that allow or enhance the dispersion of the covalent conjugate of an anesthetic as a fluid medium, making it applicable for administration via injection or infiltration. Thus, in some embodiments, the covalent conjugate of an anesthetic is formulated as an injectable solution by dispersion of PEG in an aqueous solution.

[0111] Usually, PEG is added to the formulation to increase the hydrophilicity of PGS-PEG-TTX conjugate. When PEG concentration is increased, copolymer dissolves in PBS. For example, in some embodiments, PGS-TTX conjugate is uniformly suspended in PEG100, PEG-monomethyl-ether (mPEG), PEG200, PEG250, PEG300, PEG400 or PEG500.

[0112] In a preferred embodiment, the covalent conjugate of an anesthetic is formulated into a form injectable by syringe by homogeneous suspension in PEG200. PEG200 is a colorless, transparent, viscous liquid. PEG200 is widely used in various pharmaceutical formulations, in part due to its low toxicity. To make a high-quality formulation injectable by syringe, PGS-PEG-TTX and PGS-TTX conjugates are formulated into PEG200 via a solvent evaporation process, as shown in the diagram of FIG. 4.

[0113] The viscoelastic properties of the synthesized formulations were investigated by rheology. For the PGS-PEG200-TTX / PEG200 (50 mg / ml) formulations, the loss modulus (G") was higher than the storage modulus (G'), suggesting that the viscous component of the complex modulus governs the behavior of the material (Figure 5A). Furthermore, although the viscosity increases as the PEG concentration in the conjugate decreases, all PGS-PEG-TTX / PEG200 formulations (50 mg / ml) had viscosities below 10 Pa s at the angular frequencies tested (Figure 5B). Given the viscous behavior but low viscosity, all PGS-PEG-TTX / PEG200 formulations were injectable by syringe.

[0114] E. Anesthetic-Polymer Covalent Conjugates and Dosage Units Conjugates of an anesthetic covalently attached to a hydrophilic polymer (e.g., PGS or PGS-PEG) comprise: (i) an effective amount of a local anesthetic to provide nerve blockade; (ii) a hydrophilic polymer backbone (preferably, PGS); and (iii) optionally, one or more PEGs attached to the polymer backbone in relative ratios necessary to provide the desired functional parameters, such as: (a) the total amount of anesthetic loaded per unit dose of the formulation; and (b) the rate of release of the anesthetic from the polymer backbone in vivo.

[0115] The release rate and dose of anesthetic per unit time (i.e., dose / hour, dose / day, etc.) can be modified depending on the total amount administered, as well as the ratio of hydrophilic polymers to each other (e.g., the ratio of the amount of PGS to the amount of PEG). For example, in some embodiments, the addition of PEG to the polymer-anesthetic conjugate determines the overall hydrophilicity of the conjugate. The overall hydrophilicity of the conjugate determines the hydrolysis rate of the ester bonds in vivo. The hydrolysis rate of the ester bonds in vivo determines the release rate of the anesthetic in vivo. Thus, the overall hydrophilicity of the conjugate can be adjusted by adding more or less PEG to shorten or extend the period during which hydrolysis of the ester bonds in the conjugate occurs in vivo.

[0116] In some embodiments, the release rate of anesthetic in vivo is adjusted by adjusting the percentage of PEG segments in the polymer-anesthetic conjugate to provide active agent release for up to one month after exposure to physiological conditions in vivo.For example, the release rate of TTX in vivo can be adjusted by adjusting the percentage of PEG segments in the PGS-TTX conjugate.In some embodiments, the release rate of TTX increases in proportion to the molecular weight of PEG incorporated into the PGS-PEG backbone.Thus, TTX-PGS-PEG1000 releases TTX in vivo at a much faster rate than an equimolar amount of TTX-PGS-PEG100.

[0117] The conjugates can be formulated to have an in vivo release rate of approximately 50%, 60%, 70%, 80%, 90% or more than 90% of the total anesthetic released over a period of 1-30 days. In an exemplary embodiment, after 28 days, 99.7% of the TTX was released from PGS-PEG2000-TTX, whereas PGS-PEG1000-TTX, PGS-PEG200-TTX and PGS-TTX showed 89.5, 62.5 and 43.7% TTX release, respectively, over the same period. For PGS-PEG200-TTX and PGS-TTX conjugates, TTX is released according to a zero-order release profile, which is due to the degradation of the PGS-based copolymer under a surface erosion mechanism. The increase in TTX release with increasing PEG concentration is due to the increased hydrophilicity of the copolymer backbone. Such increased hydrophilicity increases water uptake, which accelerates the hydrolysis of the ester bonds. The results presented in the Examples confirm that drug release occurs due to hydrolysis of the ester bond. The tunable sustained release of the compound of interest over a period of weeks helps the conjugate provide long-lasting local anesthesia.

[0118] In one exemplary embodiment, the PEG-PGS conjugate contains an excess molar ratio of PGS to PEG. For example, the molar ratio of -COOH groups to -OH for PGS-PEG copolymer synthesis is usually 8:7. The remaining -COOH groups are conjugated to anesthetics and, if necessary, additional active agents (see Tables 1 and 2). The total amount of PEG incorporated into PGS can be calculated by using the ratio of free "-COOH" groups to free "-OH" groups in the PGS / polymer mixture. Usually, the design of the conjugate allows for the incorporation of at least one anesthetic and, if necessary, additional active agents.

[0119] F. Further Agents The formulation of the anesthetic-polymer covalent conjugate may include one or more additional pharma- ceutically active agents. The active agents (e.g., therapeutic, prophylactic and / or diagnostic agents) may be associated with the anesthetic-polymer covalent conjugate. In some embodiments, the active agents are covalently or non-covalently associated with the anesthetic-polymer covalent conjugate. For example, the additional active agents may be covalently attached to the polymer backbone via an ester bond. In other embodiments, the additional active agents are attached to the polymer backbone via a covalent non-ester linker, e.g., via one or more spacers. When the active agent is covalently associated with the polymer backbone, the agent may be released from the backbone in vivo at a time that coincides with or differs from the timing of the release of the covalently attached anesthetic in vivo. Thus, in some embodiments, the formulation comprising the anesthetic-polymer covalent conjugate delivers both the anesthetic and one or more additional active agents at the same or different rates throughout the same or different times in vivo. In some embodiments, the additional active agent is non-covalently associated with the anesthetic-polymer conjugate. For example, the formulation of the anesthetic-polymer covalent conjugate can include one or more additional active agents by mixing. Exemplary additional active agents include additional local anesthetics, vasoconstrictors, hormones, anti-inflammatory agents and anti-infective agents.

[0120] 1. Local anesthetics The formulation of the anesthetic-polymer covalent conjugate may include one or more additional local anesthetic agents. The term "local anesthetic agent" refers to a drug that provides localized numbness or pain relief. Classes of local anesthetic agents that may be utilized include aminoacylanilide compounds (e.g., lidocaine, prilocaine, bupivacaine, mepivacaine, and related local anesthetic compounds having various substituents on the ring system or on the amine nitrogen); aminoalkylbenzoate compounds (e.g., procaine, chloroprocaine, propoxycaine, hexylcaine, tetracaine, cyclomethycaine, benoxinate, butacaine, proparacaine, and related local anesthetic compounds); cocaine and related local anesthetic compounds; aminocarbonate compounds (e.g., diperodone and related local anesthetic compounds); N-phenylamidine compounds (e.g., phenacaine and related anesthetic compounds); N-aminoalkylamide compounds (e.g., dibucaine and related local anesthetic compounds); aminoketone compounds (e.g., falicaine, dyclonine, and related local anesthetic compounds); and aminoether compounds (e.g., pramoxine, dimethisoquien, and related local anesthetic compounds). Preferred local anesthetics are the amino-amides and amino esters, most preferably bupivacaine, the levoenantiomer of bupivacaine being preferred when vasoconstrictor activity of the local anesthetic is desired, as well as tetracaine and ropivacaine, which are slightly more sensory selective.

[0121] When administered at various sites and for various types of surgery, the pain relief of these drugs lasts an average of 6-10 hours. For many types of surgery, it would be preferable for the duration of pain relief to last 2 or 3 days. The preferred local anesthetics for use in combination with NeoSTX are bupivacaine, ropivacaine, tetracaine, and levobupivacaine. Bupivacaine is a particularly potent long-acting local anesthetic. Other advantages of bupivacaine include its wide availability, with partial motor blockade as well as adequate sensory anesthesia.

[0122] 2. Vasoconstrictors The formulation of anesthetic-polymer covalent conjugate may contain one or more vasoconstrictors.Useful vasoconstrictors act locally to restrict blood flow, thereby retaining the injected drugs in the area where they are administered.This has the effect of substantially reducing systemic toxicity.

[0123] Preferred vasoconstrictors are those that act on alpha adrenergic receptors (e.g., epinephrine and phenylepinephrine). Other drugs and dyes (e.g., bupivacaine and levobupivacaine) have vasoconstriction as a side effect.

[0124] In some embodiments, the conjugates of anesthetics covalently attached to PGS or PGS-PEG or other polymer conjugates are formulated with epinephrine. For example, epinephrine can be included in an amount effective to increase or prolong the nerve blockade resulting from one or more anesthetic agents in the conjugate.

[0125] 3. Glucocorticoids Formulations of the anesthetic-polymer covalent conjugates may include one or more glucocorticoids. Exemplary glucocorticoids include dexamethasone, cortisone, hydrocortisone, prednisone, beclomethasone, betamethasone, flunisolide, methylprednisone, paramethasone, prednisolone, triamcinolome, alclometasone, amcinonide, clobetasol, fludrocortisone, diflurosone diacetate, fluocinolone acetonide, fluoromethalone, flurandrenolide, halcinonide, medrysone and mometasone, and pharma- ceutically acceptable salts and mixtures thereof. Anti-inflammatory agents.

[0126] In some embodiments, conjugates of anesthetics covalently bound to PGS or PGS-PEG or other polymer conjugates are formulated with dexamethasone. Dexamethasone has a hydroxyl group that allows it to be covalently conjugated to PGS-PEG via the same ester bond used in the conjugation of TTX. As described in the Examples, the drug release profiles for TTX and dexamethasone were consistent for all PGS-PEG-drug conjugates. The mass loss followed a similar trend to that of the drug release of PGS-PEG-TTX conjugates (Figures 3A-3D). After 28 days, the mass loss of PGS-TTX was 27.3%, whereas the mass losses of PGS-PEG200-TTX, PGS-PEG1000-TTX and PGS-PEG2000-TTX were 30.4, 70.4 and 94.7%, respectively.

[0127] 4. Other Medication In some embodiments, conjugates of anesthetics covalently attached to PGS or PGS-PEG or other polymer conjugates are formulated with additional active agents, such as anti-inflammatory agents, anti-infective agents, anti-proloferative agents, etc. Exemplary anti-inflammatory agents include non-steroidal drugs, such as indomethacin, aspirin, acetaminophen, diclofenac sodium, and ibuprofen; and steroidal anti-inflammatory agents, such as dexamethasone.

[0128] G. Other Excipients and Preservatives The conjugate of the anesthetic covalently bonded to PGS or PGS-PEG or other polymers can be formulated to include one or more excipients or preservatives for administration. Usually, the composition is formulated to include an excipient for administration via parenteral route. Exemplary pharmaceutical compositions of the covalent conjugate of the anesthetic are formulated for administration via intramuscular, intraperitoneal, intravenous (iv) or subcutaneous injection, transdermal administration (passive administration, or administration using iontophoresis or electroporation).

[0129] The formulation of the anesthetic covalently bonded to PGS or PGS-PEG includes one or more additional pharma- ceutically acceptable excipients. Exemplary additional excipients include preservatives, pH adjusters, antioxidants, and isotonicity agents. In some embodiments, the covalent conjugate of the anesthetic is formulated in saline or acidic buffer, optionally including a preservative, for example, for administration via parenteral routes. The pharmaceutical composition of the covalent conjugate of the anesthetic can be formulated for administration via parenteral (intramuscular, intraperitoneal, intravenous (iv) or subcutaneous injection), transdermal (passive administration, or administration using iontophoresis or electroporation), transmucosal (nasal, vaginal, rectal or sublingual) routes of administration, or administration using biodegradable inserts, and can be formulated into a unit dosage form appropriate for each administration route.

[0130] In some embodiments, the anesthetic-polymer covalent conjugate is formulated in an aqueous solution for administration by parenteral injection. The formulation may be in the form of a suspension or emulsion. In general, a pharmaceutical composition is provided that includes an effective amount of the anesthetic-polymer covalent conjugate, which optionally includes a pharma- ceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such compositions include diluents (e.g., sterile water, buffered saline of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength); and, optionally, additives (e.g., surfactants and solubilizers (e.g., TWEEN® 20, TWEEN® 80, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite) and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol).

[0131] Examples of non-aqueous solvent or vehicle are propylene glycol, polyethylene glycol, vegetable oil (e.g., olive oil and corn oil), gelatin and injectable organic ester (e.g., ethyl oleate).The above-mentioned preparation can be lyophilized and redissolved / resuspended immediately before use.The above-mentioned preparation can be sterilized, for example, by filtering through a bacteria-retaining filter, incorporating a sterilizing agent into the preparation, irradiating the preparation, or heating the preparation.

[0132] In some embodiments, the anesthetic-polymer covalent conjugate is formulated in a solution that includes one or more proteins or polypeptides.In an exemplary embodiment, the anesthetic-polymer covalent conjugate is formulated in a solution that includes albumin protein.For example, an albumin protein such as bovine albumin can be added to the mixture of the anesthetic-polymer covalent conjugate to enable or increase the solubilization or dispersion of the conjugate in aqueous solution.

[0133] H. Dosage form In a preferred embodiment, the anesthetic-polymer covalent conjugate is provided in an aqueous solution in a vial. Depending on the type of formulation, as outlined above and below, the size of the vial may range from 10 μl to 200 ml, preferably from about 100 μl to about 10 ml, and one to five vials may be used for one patient in various situations. In another embodiment, the anesthetic-polymer covalent conjugate is provided in one or more vials, optionally lyophilized, and then rehydrated and combined prior to use.

[0134] Preferably, the dosage form comprises an amount of anesthetic effective to reduce or prevent pain in a subject in need thereof without toxicity. In animal studies, 350-400 g rats were administered anesthetic-polymer covalent conjugates containing approximately 80 μg of conjugated TTX without systemic toxicity. Dosage forms for administration to humans are contemplated that comprise anesthetic-polymer covalent conjugates having up to and including 1.5 mg of conjugated TTX.

[0135] III.How to use Methods of using the anesthetic-polymer covalent conjugates to deliver anesthetic agents are provided. Methods of providing neuroblockade in a subject in need thereof by administering to the subject an anesthetic-polymer covalent conjugate are provided.

[0136] The method may include contacting tissue surrounding one or more nerves with an effective amount of an anesthetic-polymer covalent conjugate to reduce or inhibit sensory activity in the nerves relative to a control. The method may prolong blockade of Site 1 sodium channels (S1SC) while minimizing or reducing toxicity.

[0137] In some embodiments, the active agent binds to or otherwise inhibits the activity of cell surface receptors associated with the physiological process of sensory nerve activity. For example, the anesthetic agent may be effective to block ion channels, such as the site 1 sodium channel (S1SC) or TRPV1 receptor. In a preferred embodiment, the anesthetic agent covalently coupled to one or more polymers is S1SCB tetrodotoxin (TTX). A method is provided that includes contacting one or more nerves with an effective amount of TTX to reduce or inhibit sensory activity in the nerves compared to a control. The anesthetic agent is administered as a covalently attached polymer conjugate and then released in vivo at or near the site of administration by degradation of the covalent bond to the polymer backbone.

[0138] The above method can consistently release a quantity of anesthetic in a physiologically active state to achieve effective nerve blockade at the administration site for an extended period of time. The method of administering an anesthetic-polymer covalent conjugate can provide effective nerve blockade for a significantly longer period of time than would be achieved by administering an equivalent amount of anesthetic to the same site in the absence of the polymer conjugate. Thus, the method of administering Site 1 sodium channel blocker in vivo as a covalent polymer conjugate can effectively block Site 1 sodium channels for a longer period of time than an equivalent amount of Site 1 sodium channel blocker administered alone to the same site. Typically, these methods do not cause vasoconstriction.

[0139] The anesthetic-polymer covalent conjugate for use in the method can be formulated so that the anesthetic is released over a period of time, from minutes to hours, days, weeks or months. In some embodiments, the method allows for the safe administration of an amount of anesthetic that may be toxic to the recipient when administered in the absence of the covalent polymer conjugate. For example, when the anesthetic-polymer covalent conjugate is formulated to deliver anesthetic to the administration site over an extended period of time, the serum concentration of free anesthetic at any given time may be less than the serum concentration when the anesthetic is administered alone as a bolus.

[0140] Thus, in some embodiments, the method provides long-term nerve blockade with TTX with low toxicity. Usually, the anesthetic agent is released from the polymer backbone in vivo in its native form (i.e., in a physiologically active state). In some embodiments, the anesthetic agent is physiologically inactive when covalently conjugated to the polymer backbone.

[0141] A. Methods for Treating or Preventing Pain Provided is a method of providing sensory and / or motor nerve blockade in a subject, comprising administering an effective amount of one or more anesthetic agents covalently conjugated to a polymer backbone to a nerve or near the nerve of the subject to reduce or inhibit sensory and / or motor function in the nerve. In a preferred embodiment, the subject is a human. In some embodiments, the method is effective in delaying the onset of pain, such as nociceptive and / or neuropathic pain, in the subject. In some embodiments, the subject is an adult. In other embodiments, the subject is a child. All of the methods described may include a step of identifying a subject in need of pain relief.

[0142] In some embodiments, the method includes administering the anesthetic-polymer covalent conjugate in combination with one or more additional active agents (e.g., a local anesthetic agent). The term "combined" or "combined" is used to refer to the co-administration, simultaneous administration, or sequential administration of two or more different agents. Thus, the combination can be administered simultaneously (e.g., as a mixture), separately but simultaneously (e.g., by separate applications to the same subject), or sequentially (e.g., one of the compounds or agents is administered first, followed by the second). For example, the combination therapy can include co-administration of TTX-PGS-PEG and / or TTX-PGS with one or more additional anesthetic or other active agents, either separately as two different formulations, or together as the same formulation (i.e., a single pharmaceutical composition containing both active agents). When the two agents are administered as separate formulations, the co-administration can include simultaneous and / or sequential administration of the two agents. The appropriate time course for sequential administration can be selected by the physician according to factors such as the nature of the patient's disease and the patient's symptoms. In certain embodiments, sequential administration includes co-administration of two agents within a period of hours, days, or weeks of each other. For example, in some embodiments, TTX-PGS-PEG and / or TTX-PGS are administered first, followed by an alpha-2-adrenergic agonist. In other embodiments, TTX-PGS-PEG and / or TTX-PGS are administered first, followed by an additional active agent.

[0143] 1. Dosage One or more anesthetic-polymer covalent conjugates may be administered to separate painful sites in an amount effective to provide highly localized selective nerve blockade in separate focal areas involved in eliciting pain in order to reduce or eliminate pain emanating from the separate locations.

[0144] In some embodiments, the dosage is an amount effective to treat or prevent pain when administered for infiltration anesthesia of one or more tissues, hi some embodiments, the dosage is an amount effective to treat or prevent pain when administered for conduction anesthesia or nerve block anesthesia.

[0145] The above method minimizes the potential adverse effects of anesthetic agents outside the location of pain. For example, PGS-PEG and / or TTX-PGS can be administered to separate painful sites in an amount effective to provide highly localized selective nerve blockade in separate localized areas involved in pain generation for the purpose of reducing or eliminating pain emanating from separate locations in a subject for about 1 hour to about 72 hours, depending on the needs of the subject.

[0146] A pharmaceutical composition of an anesthetic-polymer covalent conjugate for attenuating pain at a site in a human or animal in need of pain attenuation may contain 1 μg to 1.5 mg of anesthetic conjugate conjugated to a polymer backbone. For example, a pharmaceutical composition of an anesthetic-polymer covalent conjugate for attenuating pain at a site in a human or animal in need of pain attenuation may contain 1 μg to 5,000 mg of the conjugate. The amount of the conjugate administered depends on the size and type of the polymer backbone and the anesthetic in the conjugate. In certain embodiments, the dose of the anesthetic in the conjugate ranges from about 1 μg to about 2000 μg, about 10 μg to about 200 μg, or about 20 μg to about 50 μg. Typically, at least 75%, at least 80% or 90%, up to and including 100% of the total amount of anesthetic is released from the polymer backbone conjugate in vivo over a desired time period after administration.

[0147] The formulations of the present disclosure for long-term local anesthesia may be administered by any means known in the art, including via injection at a separate site through the skin of a human or animal; via implantation at a separate site by implanting the dose into the skin, tissue, muscle, tendon, joint or other body part of a human or animal; by infiltration into a separate wound, tissue surface, or surgical site where a surgical wound is open.

[0148] For long term anesthesia in animals and humans, the subject's body weight can be used as a guide to determine the appropriate dosage (see Table 8 below). Different clinical situations pose different demands on the safety and efficacy of local anesthetics. Systemic safety determines the upper limit of the total dose (mg or mg / kg) of TTX or other disclosed active agents. Although there are small differences in the acceptable total dose based on the time course of uptake, vascularity, etc., every local anesthetic has its own maximum acceptable total dose.

[0149] In any localized region of the body, a sufficient local tissue concentration of a local anesthetic is necessary to block afferent transmission. The lowest local concentration of one or more drugs at a given location sufficient to provide pain relief is called the "minimum effective concentration" (MEC).

[0150] Thus, clinical situations requiring infiltration of large volumes of tissue require a greater total volume of local anesthetic at or beyond the MEC than clinical situations involving smaller volumes of tissue. If the MECs at different locations are similar, larger volumes of tissue will require a greater total dose than smaller volumes of tissue.

[0151] The dose of anesthetic in the anesthetic-polymer covalent conjugate depends on the anesthetic administered, as well as the site to which the local anesthetic is administered. Typically, dosage units are prepared for use in volumes ranging from about 0.1 ml to about 120 ml. In certain embodiments, the anesthetic in the PGS or PGS-PEG conjugate is present in a concentration range of 0.01% (weight / volume) to 5% (w / v) and the one or more alpha-2-adrenergic agonists are present in a concentration range of 0.01% (w / v) to 5% (w / v). Typically, the total systemic dose is only about 1 mg / kg body weight to about 200 mg / kg body weight in adults. For example, in embodiments in which the local anesthetic is administered via a regional block (e.g., ankle block), the dose of anesthetic ranges from about 1 ml to about 30 ml of a 0.5% (w / v) solution. In other embodiments, a 3 mg / kg dose (maximum 200 mg) of a 2% (w / v) solution may be administered by intra-articular infiltration. In other embodiments, the dose of local anesthetic may range from 0.5 ml to about 60 ml of a 0.25% to 5% (w / v) solution.

[0152] In a preferred embodiment, the anesthetic agent is TTX. TTX in the conjugate may be present at a concentration ranging from 0.01 mM to 100 mM, preferably 0.1 mM to 0.3 mM, and most preferably 0.21 mM. In some embodiments, the dose of the conjugate administered in a single application is sufficient to reduce or inhibit sensory and / or motor function for a period of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 6 months, or more than 6 months. In certain embodiments, TTX-PGS-PEG is administered as an injectable formulation in an amount sufficient to reduce pain at or around the administration site of a subject for up to 72 hours. In an exemplary method, injection of a formulation containing PGS-PEG1000-TTX and / or PGS-PEG2000-TTX containing 1.0 μg of TTX into a subject is effective to produce nerve blockade in the subject for a period of 1 to 6 hours. Amounts empirically determined based on animal data are shown in Table 1.2 below. [Table 1-2] a Mass of rat used in experiment = 0.35 kg b PGM = poly(glycerol malonate)

[0153] 2. Treated Pain Conjugates of anesthetic agents with Site 1 sodium channel blockers and / or other anesthetics may be used to treat many conditions, where the anesthetic formulation may be administered to or near the painful area, including, but not limited to, the treatment of acute or chronic pain, nociceptive and neuropathic pain, pre- and post-operative pain, cancer pain, pain associated with neurotransmitter dysregulation syndromes and orthopedic disorders, sports-related injuries, acute traumatic pain, nociceptive pain, and neurotransmitter dysregulation syndromes.

[0154] i. Surgery The formulations of the present disclosure may be used to prevent or reduce pain associated with surgical procedures. In some embodiments, the dosage units are used to treat or prevent pain associated with large, multi-layered surgical wounds for full-length open laparotomy, thoracoabdominal incision, or flank incision; Caesarean section, open hysterectomy, esophagogastrotomy, nephrectomy, or large abdominal cancer surgery (e.g., colectomy); wound infiltration for total hip replacement (hip arthroplasty) or total knee replacement (knee arthroplasty); peripheral nerve block or plexus block (perineural injection); infiltration (injection along the layers of the wound); shoulder, hand, or arm surgery, infiltration or ilioinguinal / iliohypogastric block for inguinal hernia repair, penile block for hypospadias repair, femoral block for total knee replacement or anterior cruciate ligament repair, for thoracotomy, or for thoracotomy. the administration of one or more site 1 sodium channel blockers conjugated to PGS or PGS-PEG in an amount effective to provide an intercostal nerve block for leg amputation or femoral and sciatic nerve block for leg amputation or foot and ankle surgery; a lumbar sympathetic block for complex regional pain syndrome / reflex sympathetic dystrophy or vascular insufficiency of the leg, or a celiac plexus block for pancreatitis or pancreatic cancer; a nerve block to the hip or knee for total joint replacement surgery (femoral and sciatic, lumbar plexus and sciatic nerve block); or a long duration sciatic nerve block when rapid motor recovery is not required, such as in lower limb amputation.

[0155] Also provided is a dosage unit comprising an amount of one or more Site 1 sodium channel blockers conjugated to PGS and / or PGS-PEG to provide long duration anesthesia to the eye or orbit. In other embodiments, the dosage unit may be prepared in an amount effective to prevent, reduce or inhibit sensory and / or motor function in the cornea of ​​the eye.

[0156] In some embodiments, dosage units may be prepared in an amount effective to prevent, reduce or inhibit sensory and / or motor function in one or more peripheral nerves.

[0157] In other embodiments, the dosage unit comprises a quantity of one or more anesthetic agents covalently conjugated to one or more polymers to extend the delivery or release of the anesthetic agent at or near the sciatic nerve.

[0158] ii.Chronic pain In some embodiments, the formulations of the present disclosure can be used in the treatment or prevention of pain over a long period of time.The method can include administering to a subject a formulation that includes one or more anesthetics covalently conjugated to one or more polymers for controlled or delayed release of anesthetic at the administration site over a long period of time.For example, a method is provided that includes administering to a subject a formulation that includes TTX-PEG-PGS for controlled or delayed release of TTX at or near the administration site over a long period of time. Methods are provided that include administering an anesthetic-polymer covalent conjugate comprising one or more Site 1 sodium channel blockers and one or more other anesthetics or other agents to a painful structure or near a painful structure of a subject in an amount effective to reduce or inhibit sensory and / or motor function for a period of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 6 months, or more than 6 months.

[0159] In certain embodiments, the method can be used to treat or attenuate chronic pain, for example, chronic pain associated with a disease or disorder. For example, in patients suffering from chronic pain, the dose of TTX released from TTX-PGS and / or TTX-PGS-PEG is up to 100 μg / hour, up to 10 μg / hour, up to 1 μg / hour, up to μg / day, or up to 1 μg / week, etc. The amount of anesthetic administered to the subject in the first administration can be calculated taking into account the desired hourly or daily dose. The total dosage and release rate can be calculated taking into account the serum half-life of the anesthetic so that the serum concentration does not exceed the serum concentration at which toxicity and / or side effects occur. Usually, the total dosage is administered at once to the site close to or near the site close to the disease or disorder, or to the area surrounding the painful structure.

[0160] IV. Formulations and Methods of Preparation Animal and human studies are necessary to determine effective doses and volumes for human treatment. For example, the potency rankings of site 1 sodium channel blockers obtained from in vitro physiological experiments did not predict the potency rankings of those compounds in vivo (Kohane et al., Reg. Anesth. Pain Med., 25:52-9 (2000)).

[0161] Different clinical situations impose different demands on the safety and efficacy of local anesthetics. Systemic safety determines the upper limit of the total dose (mg or mg / kg) of TTX or other local anesthetics. Although there is a small difference in the total dose that can be tolerated based on the time course of uptake, vascularity, etc., every local anesthetic has a maximum total dose that can be tolerated. As described herein, the inclusion of TTX as a covalent conjugate in the PGS or PGS-PEG polymer backbone allows for delayed release of TTX via hydrolysis of the ester bond in vivo. Delayed release allows for longer nerve blockade, higher efficacy and systemic safety, allowing for higher doses or more.

[0162] In some clinical situations, it is important not to administer large volumes of local anesthetic (i.e., more than 20 ml) to prevent the spread of numbness or weakness to other body locations where the effects of the local anesthetic are undesirable or even dangerous. The described anesthetic-polymer covalent conjugates can be administered in a desired volume, for example, from 1 ml to 500 ml, according to the dose, release rate and other parameters desired by the recipient.

[0163] As described in the Examples (below), poly(triol dicarboxylic acid)-co-poly(ethylene glycol) (TDP) polymers were synthesized by forming ester bonds between the carboxyl groups of dicarboxylid acid and the hydroxyl groups of triol and PEG via a Steglich esterification reaction. The TDP polymers were degradable by hydrolysis of the ester bonds and showed good biocompatibility with minimal cytotoxicity. Various f phil We have synthesized a family of TDP polymers with f phil determined the hydrolysis rate of the ester bonds of the polymer network. TDP polymers have multiple active end groups (hydroxyl and carboxyl groups) to which drugs can be covalently conjugated. These features suggest that TDP polymers have the potential to serve as a universal platform for the controlled release of a wide range of therapeutic drugs.

[0164] Covalent conjugation of TTX with TDP polymer was achieved by forming an ester bond between the carboxyl group of TDP polymer and the hydroxyl group of TTX via Steglich esterification reaction. Steglich esterification synthesis was performed at room temperature to avoid drug degradation. The synthesized TDP-TTX conjugate gradually decomposed into smaller polymer-TTX fragments, and finally decomposed into the native form of TTX by hydrolysis of the ester bond, achieving the controlled release of TTX. The release rate of TTX was determined by the f phil f less than 37.8% phil In this study, the TDP-TTX conjugate achieved a long-term TTX release of more than one month in vitro.

[0165] A. Methods for Making Covalent Polymer-Anesthetic Conjugates The synthesis of poly(triol dicarboxylic acid)-co-poly(ethylene glycol) (TDP) polymers is typically carried out in a three-step reaction, typically under conditions that minimize the destruction or denaturation of the anesthetic agent.

[0166] In an exemplary method, the synthesis of PGS-PEG-TTX is carried out in a three-step reaction at room temperature to avoid drug denaturation. The first step is the Steglich esterification of PEG and sebacic acid, which forms a linear pre-polymer chain without any crosslinks. A schematic of this reaction is shown in Figures 1A-1B. The hydrophilicity of the final copolymer is adjusted by the addition of PEG. An exemplary method uses PEG of various molecular weights (e.g., 200 Da, 1,000 Da, and 2,000 Da).

[0167] The second step is the addition of glycerol, which results in the block copolymer of PGS-PEG. As shown in the examples, for the synthesis of PGS-PEG copolymer, the molar ratio of -COOH group to -OH group is 8:7, and the extra -COOH group was used for drug conjugation (Tables 1 and 2).

[0168] In the third step, the PGS-PEG-TTX conjugate is obtained via Steglich esterification between the remaining -COOH groups of the PGS-PEG copolymer and the -OH groups of TTX (Figures 1A, 1B).

[0169] B. Methods for Making a Formulation Injectable by Syringe In some embodiments, the method includes making a formulation that can be injected by syringe. In an exemplary embodiment, PGS-PEG-TTX and / or PGS-TTX conjugate are fully dissolved in DCM, and then PEG200 is added. PEG200 is fully miscible with DCM, so a homogeneous solution is obtained after vortexing the mixture. DCM is gradually removed by rotary evaporation and lyophilization. Preferably, a homogeneous solution is formed that includes PGS-PEG-TTX and / or PGS-TTX conjugate and PEG200.

[0170] The invention will be further understood with reference to the following non-limiting examples. EXAMPLES

[0171] Example 1: Conjugates of anesthetic drugs with amphiphilic polymers and PEG Amphiphilic and biodegradable conjugates of poly(glycerol sebacate) (PGS), PEG and TTX were designed, produced and assayed according to the following method.

[0172] method material Sebacic acid (99%), poly(ethylene glycol) (PEG, 200, 1000, 2000 kDa), N,N'-diisopropylcarbodiimide (DIC, 99%), 4-dimethylaminopyridine (DMAP, 99%), anhydrous N,N-dimethylformamide (DMF, 99.8%), anhydrous dimethyl sulfoxide (DMSO, 99.9%), anhydrous dichloromethane (DCM, 99.8%), glycerol (99%), dexamethasone (98%), fluorescein isothiocyanate isomer I (FITC, 90%), phosphate-buffered saline (PBS, pH 7.4, 0.15 M, 138 mM NaCl, 2.7 mM KCl), chloroform-d (100%, 99.96 atom % D), hexamethylene diisocyanate (99.0%) and dibutyltin dilaurate (95.0%) were purchased from Sigma-Aldrich Inc. (St. Louis, MO). Cyanine 5.5 carboxylic acid (Cy5.5, 95%) was purchased from Lumiprobe Corporation (Hallandale Beach, FL). Tetrodotoxin (TTX) was obtained from Abcam plc (Cambridge, MA); tetrodotoxin ELISA kit was purchased from Reagen LLC (Moorestown, NJ).

[0173] Synthesis of TDP polymer Poly(triol dicarboxylic acid)-co-poly(ethylene glycol) (TDP) polymers were synthesized via Steglich esterification using N,N'-diisopropylcarbodiimide (DIC) as the coupling reagent and 4-dimethylaminopyridine (DMAP) as the catalyst. Briefly, dry PEG (0.005 mol) and sebacic acid (2.02 g, 0.01 mol) were added to a round-bottom flask, and the flask was placed under nitrogen after the remaining water was evaporated. After the addition of anhydrous N,N-dimethylformamide (DMF, 8 mL) and anhydrous dichloromethane (DCM, 4 mL), the mixture was sonicated for a few minutes, and DIC (4.336 mL, 0.028 mol) and DMAP (0.489 g, 0.004 mol) were added. The mixture was left at room temperature for 24 hours. Glycerol (184 μl, 0.0025 mol) was added and the mixture was left at room temperature for 24 hours. After reaction, the DCM in the reaction mixture was removed by rotary evaporation, and then the residue was washed twice with 30 mL of DI water and twice with 30 mL of 10% ethanol. The polymer was precipitated from the water and centrifuged at 20,000 rpm for 5 min. After drying, the TDP polymer was obtained in 88-96% yield. The dried TDP polymer was stored in a desiccator until further use.

[0174] Synthesis of TDP-drug conjugates TDP-drug conjugates were synthesized via a similar Steglich esterification. Briefly, dry PEG (0.005 mol) and sebacic acid (2.02 g, 0.01 mol) were added to a round-bottom flask, and the remaining water was evaporated and the flask was placed under nitrogen. Anhydrous N,N-dimethylformamide (DMF, 8 mL) and anhydrous dichloromethane (DCM, 4 mL) were added, and the mixture was sonicated for a few minutes, and DIC (4.336 mL, 0.028 mol) and DMAP (0.489 g, 0.004 mol) were added. The mixture was left at room temperature for 24 hours. Glycerol (184 μl, 0.0025 mol) was added and the mixture was left at room temperature for 24 hours. A solution of TTX (1 mg, 0.003 mmol) and / or dexamethasone (10 mg, 0.026 mmol) and / or FITC (1 mg, 0.003 mmol) and / or Cy5.5 (1.6 mg, 0.003 mmol) in anhydrous DMSO (10 mL) was added and the mixture was left at room temperature for 7 days. After the reaction, the DCM in the reaction mixture was removed by rotary evaporation. To eliminate the unbound drug and to isolate the pure polymer conjugate, the residue was washed twice with 30 mL of DI water and twice with 30 mL of 10% ethanol. The TDP-drug conjugate was precipitated from the water and centrifuged at 20000 rpm for 5 min. Upon drying, the TDP-drug conjugate was obtained in 88-96% yield. The dried TDP-drug conjugate was stored in a desiccator until further use.

[0175] T g Synthesis of D8-isocyanate Typically, T of 0.240 g g D8 (Mn = 6011, 0.4 × 10 -3 mol) was dried overnight in a 100 mL flask under high vacuum. Then, 5 mL of anhydrous DMSO was added to the flask, and 1.13 g of HMDI (240 μl, 1.5 × 10 -3 mol) and 2 drops of dibutyltin dilaurate (8 × 10 -3g) was added continuously. The reaction mixture was stirred overnight under nitrogen atmosphere at 60° C. At the end of the reaction, the resulting polymer was precipitated from diethyl ether and further purified by redissolving in 1,2-dichloroethane, followed by precipitation in a mixture of methanol and diethyl ether (5 / 95, v / v) to remove the remaining dibutyltin dilaurate. Upon drying, T g D8-isocyanate was obtained in 80-95% yield.

[0176] T g Synthesis of D8-TTX urethane Typically, T of 0.325 g g D8-isocyanate was dried overnight in a 100 mL flask under high vacuum. Then, 5 mL of anhydrous DMSO was added to the flask and 1 drop of dibutyltin dilaurate (4×10 -3 g) and 0.1 mg of TTX were successively added. The reaction mixture was stirred overnight under nitrogen atmosphere at 60° C. At the end of the reaction, the resulting polymer was precipitated from diethyl ether and further purified by redissolving in 1,2-dichloroethane, followed by precipitation in a mixture of methanol and diethyl ether (5 / 95, v / v) to remove the remaining dibutyltin dilaurate. Upon drying, T g D8-TTX urethane was obtained in 90-95% yield.

[0177] Contact angle procedure Water contact angle measurements for polymer films spin-coated on silicon wafer substrates were performed using a goniometer (Rame-Hart, model 500) equipped with an automatic dispenser. The sessile drop method was applied. A volume of 1 μL of water was dispensed onto the sample surface, and the contact angle was determined based on high-resolution images.

[0178] 1 H NMR measurement The polymers and polymer-drug conjugates were analyzed by nuclear magnetic resonance ( 1The polymers were analyzed using H NMR spectroscopy (Varian 400 MHz equipped with a 5 mm AutoX OneProbe and a Varian 7600 autosampler). The polymers were dissolved in CDCl3 and spectra were recorded at 400 MHz. The chemical shifts (δ in ppm) of the peaks corresponding to the italicized hydrogens in the list of polymers below are provided. s / d / m indicates the peak shape (i.e. singlet, doublet, triplet). 1 H NMR(T g D8)(400MHz,CDCl3)δ / ppm:1.30(2H,m,-CH2-),1.62(2H,d,-CH2CH2O(CO)-),2.35(2H,m,-CH2O(CO)-),3.50-3.85(2H,m ,OHCH2CHO-),3.94(1H,m,-OCH2CHOH),4.05-4.35(2H,m,-OCH2CHO-),5.09(1H,s,OHCH2CHO-),5.26(1H,s,-OCH2CHO-). 1 H NMR(T g D8P 1k ) (400MHz, CDCl3) δ / ppm: 1.30 (2H,m,-CH2-), 1.62 (2H,d,-CH2CH2O(CO)-), 2.35 (2H,m,-CH2O(CO)-), 3.64 (2H,m,-OCH2-), 3.94 (1H,m,-OCH2CHOH), 4.05-4.35 (2H,m,-OCH2CHO-), 5.09 (1H,s,OHCH2CHO-), 5.26 (1H,s,-OCH2CHO-). 1 The 1 H NMR spectrum is shown in Figure 1a with the key structural elements assigned. Some peaks could not be assigned due to overlapping signals.

[0179] FTIR Measurement Fourier transform infrared spectroscopy (FTIR) spectra of the samples were recorded using an Alpha Bruker spectrometer. -1 An average of 48 scans at a resolution of 100 nm was collected for each sample.

[0180] Molecular weight measurement Molecular weights were measured using a Waters gel permeation chromatograph (GPC) model 440. THF was used as the mobile phase with a flow rate of 1.0 mL / min. The molecular weights and polydispersities of the macromonomers and copolymers were calibrated with polystyrene standards.

[0181] In vitro degradation of polymers Mass loss studies were performed by applying the polymer to a Slide-A-Lyzer MINI dialysis device with 10,000 MW cutoff (Thermo Scientific, Tewksbury, MA), further dialyzing against 14 mL of PBS, and incubating at 37° C. on a platform shaker (New Brunswick Innova 40, 60 rpm). At each time point, the dialysis solution was replaced with fresh pre-warmed PBS. 14 mL of the dialysis solution was frozen, lyophilized, and the mass of the retentate was weighed for mass loss analysis. All experiments were performed in triplicate.

[0182] cell culture Cell culture of C2C12 mouse myoblast cells (American Type Culture Collection (ATCC) CRL-1772) and PC12 rat adrenal pheochromocytoma cells (ATCC, CRL-1772) was performed as previously described. 2 Briefly, C2C12 cells were cultured in DMEM containing 20% ​​FBS and 1% penicillin-streptomycin (Invitrogen). Cells were seeded in 24-well plates at 50,000 cells / mL and incubated in DMEM containing 2% horse serum and 1% penicillin-streptomycin for 10–14 days to differentiate into myotubes. PC12 cells were grown in DMEM containing 12.5% ​​horse serum, 2.5% FBS and 1% penicillin-streptomycin. Cells were seeded in 24-well plates and 50 ng / mL nerve growth factor was added 24 h after seeding (Invitrogen).

[0183] cell viability cells (1×10 4 / well) were incubated with various concentrations of polymer-TTX conjugates for 24 h. After incubation, cells were washed up to 5 times with PBS to remove the polymer, and cell viability was determined by MTT. Briefly, culture supernatants from control and polymer-containing wells were collected and cells were incubated with MTT (0.5 mg / mL; 3 h). Formazan was dissolved in 200 μl of DMSO and the optical density was measured at 550 nm. The absorbance of the control wells was assumed to be 100%, and cell viability of the treated wells was measured relative to the control wells.

[0184] Equilibrium solubility method 1 Solubility studies of TTX were performed by equilibrating excess amounts of TTX in organic solvents. Assays were performed in plastic flasks with a volume of 2 mL. To each flask, 1 mL of organic solvent and 1 mg of TTX were added. This amount of TTX was sufficient to saturate each solvent, which was characterized by the precipitation of undissolved TTX. Samples were maintained at 25°C with shaking at 150 rpm using an incubator shaker for 72 hours (until the samples reached equilibrium conditions) during the study. After this period, samples were immediately filtered through disposable capsule filters with 0.45 μm pore size (Dezani et al., Brazilian Journal of Pharmaceutical Sciences 49,853-863 (2013)).

[0185] Solubility of TTX in DMSO and DMF 50 μl of the filtrate was diluted with 450 μl of PBS. The concentration of TTX in the mixture was measured using a TTX Elisa kit. For the quantitative process of TTX, a TTX ELISA kit was used at the maximum absorbance wavelength of each solvent, and the solubility value was calculated using the calibration curve obtained for each substance. The same ratio of DMSO or DMF was added to the standard solution in the TTX Elisa kit to generate the calibration curve. The concentration was considered as the saturation or equilibrium solubility of TTX.

[0186] Solubility of TTX in DCM 1 ml of the filtrate was transferred to a round-bottom flask and the DCM was removed by rotary evaporation. 0.1 ml of citrate buffer was added to the round-bottom flask to dissolve all the TTX. 50 μl of the solution was diluted with 450 μl of PBS. The TTX concentration in the mixture was measured using a TTX Elisa kit.

[0187] Measurement of the extent of drug binding. After the esterification reaction was completed, the DCM in the reaction mixture was removed by rotary evaporation, and then the reaction mixture was washed with 30 mL of DI water. The polymer-drug conjugate was centrifuged at 20000 rpm for 5 minutes, and the supernatant was collected as Supernatant #1. The polymer-drug conjugate was washed with 40 mL of DI water and centrifuged again. The supernatant was collected as Supernatant #2. The TTX concentration of the collected supernatant was measured by ELISA. The dexamethasone concentration of the collected supernatant was determined by HPLC.

[0188] The extent of drug binding was calculated as follows:

number

[0189] In vitro drug release Drug release and mass loss studies were performed by applying the polymer-drug conjugates to a Slide-A-Lyzer MINI dialysis device with 10,000 MW cutoff (Thermo Scientific, Tewksbury, MA), further dialyzing with 14 mL of PBS, and incubating at 37°C on a platform shaker (New Brunswick Innova 40, 60 rpm). At each time point, the dialysis solution was replaced with fresh pre-warmed PBS. 0.5 mL of the dialysis solution was set aside for drug analysis. The concentration of TTX was quantified by ELISA. The concentration of dexamethasone was determined by HPLC (Agilent 1260 Infinity, Agilent Co., Palo Alto, CA, USA) using a C18 column (Poroshell 120 EC-C18, 4.6 × 100 mm, id 2.7 μm, Phenomenex, Torrance, CA, USA) and a mobile phase of acetonitrile / water (70:30) and a flow rate of 0.5 mL / min. Dexamethasone was detected by UV absorbance at λ = 254 nm.

[0190] LC-MS instrumentation and conditions Analyses were performed on a Waters® Xevo™ TQ MS ACQUITY UPLC® instrument. Chromatographic separation was performed using a Kinetex Hilic column (100 × 2.1 mm, 100 Å, 2.6 μm particles; Phenomenex) with an in-line filter assembly (Waters) and 0.05% (v / v) formic acid in water as mobile phase A and 0.05% (v / v) formic acid in methanol as mobile phase B. Gradient elution with 10% to 30% B in 0-2.2 min, followed by a ramp to 95% B (2.2-2.5 min) and equilibration to 10% B (2.5-3.0 min) was used. The mobile phase flow rate was 500 μl / min, the column temperature was 25 °C, and the sample manager temperature was 10 °C. The injection volume was 5 μl; the time between injections was 4 min.

[0191] Solvent Evaporation Method To prepare the syringe-injectable TDP-TTX / PEG200 formulation, a given amount of TDP-TTX conjugate was completely dissolved in an excess amount of DCM, and then a given amount of PEG200 was added. The resulting mixture was vortexed for 1 min to obtain a homogenous solution. The DCM was evaporated by rotary evaporation, followed by vacuum at room temperature overnight.

[0192] Rheological Testing The rheological properties of the TDP-TTX / PEG200 formulations were monitored using a temperature-controlled AR2000 rheometer (TA instruments, USA). Parallel plates of 20 mm diameter were used for all the tests. The gap distance between the plates was 0.3 mm. Frequency sweeps ranging from 0.1 to 100 rad / s were performed at room temperature. A constant stress of 0.1 Pa was used.

[0193] Animal testing Animal studies were performed in accordance with the guidelines of the International Association for the Study of Pain and according to protocols approved by the Boston Children's Hospital Animal Care and Use Committee.Adult male Sprague-Dawley rats (Charles River Laboratories), weighing 350–400 g, were housed in groups on a 12-h / 12-h light / dark cycle with lights on at 6:00 AM.

[0194] After anesthesia with isoflurane-oxygen, the left sciatic nerve was injected. The animals were injected with drug / carrier using a 23G needle, which was inserted posteromedial to the greater trochanter and oriented in an anteromedial direction, and once bone was contacted, the formulation was injected into the sciatic nerve.

[0195] Nerve blockade was tested at a site distal to the cuticle innervated by the sciatic nerve (i.e., the plantar part of the left paw), with the right paw (uninjected) serving as an untreated control that could show evidence of contralateral blockade for systemic side effects. To assess sensory nerve blockade, the hind paws were exposed to a 56°C hot plate in sequence (left, then right) and thermal latency was measured. Thermal latency was defined as the time the animal was able to keep the paw on the hot plate. A thermal latency of 2 seconds indicated no nerve blockade (baseline), whereas a thermal latency of 12 seconds indicated strong nerve blockade. Successful nerve blockade was defined as a thermal latency of more than 7 seconds. The hind paw was removed from the hot plate after 12 seconds to prevent thermal injury.

[0196] Motor nerve block was assessed by weight-bearing testing to determine the motor strength of the rat's hind paw. Briefly, rats were allowed to stand on one hind paw on a digital balance and bear their own body weight. The maximum weight that the rat could bear without touching the balance with its ankle was recorded, and motor block was considered to be achieved when motor strength was less than half-maximal, as previously described.

[0197] The duration of sensory block was calculated by the time required for the thermal latency to return to 7 s, using 2 s as baseline and 12 s as complete sensory block. The duration of motor block was defined as the time required for weight bearing to return from maximal block to half of normal.

[0198] Confocal imaging Under isoflurane-oxygen anesthesia, rats were administered 0.5 mL of the test formulation (25 mg FITC-T in PEG200). gD8 conjugate, 0.25 mg sodium fluorescein in PEG200, 0.25 mg sodium fluorescein in PBS) and euthanized at designated intervals after sciatic nerve injection. Sciatic nerves were collected with surrounding tissues, embedded in OCT compound, and then frozen and stored at -20°C. Sections (10 μm) were prepared using a cryostat microtome and mounted on glass slides. Slides were then fixed in pre-cooled 4% paraformaldehyde for 20 min at room temperature and washed three times with PBS buffer (pH 7.4). Finally, slides were mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA) and coverslips. All imaging was performed using a Zeiss LSM 710 multiphoton confocal microscope.

[0199] IVIS Imaging Under isoflurane-oxygen anesthesia, rats were shaved and administered 0.5 mL of the test formulation (25 mg of Cy5.5-T in PEG200). g The rats were injected with 100 μg / mL of ...

[0200] Tissue collection and histological examination Rats were sacrificed 4 and 14 days after injection (these time points are useful in assessing both inflammation and myotoxicity), and the sciatic nerve was removed along with the surrounding tissue. The dissector was blinded to which solution was injected into each rat. The nerve and surrounding tissue were placed in 10% formalin and processed for histological examination (hematoxylin-eosin stained slides) using standard techniques. An observer (RP) blinded to the nature of the individual samples analyzed the slides. The primary morphological indicator of myotoxicity in this study was the presence of regenerating fibers (basophilic small diameter fibers with central nuclei containing prominent nucleoli), with only occasional myofibers showing ongoing degenerative changes.

[0201] Muscle samples were scored for inflammation (0-4 points) and myotoxicity (0-6 points) (Hirata, Y. Pure and Applied Chemistry Vol. 50 979 (1978)). Inflammation scores were subjective assessments of severity (0: no inflammation, 1: peripheral inflammation, 2: deep inflammation, 3: unilateral inflammation, 4: whole muscle nerve bundle inflammation). Myotoxicity scores reflected two characteristic features of local anesthetic myotoxicity: nuclear internalization and regeneration. Nuclear internalization is characterized by muscle cell size and chromicity being normal, but the nucleus being displaced from its usual location to the periphery of the cell (Gewert, B., Plassmann, MM & MacLeod, M. Pathways for degradation of plastic polymers floating in the marine environment. Environmental Science: Processes & Impacts 17, 1513-1521 (2015)). Regeneration is characterized by shrunken myocytes with basophilic cytoplasm. Scoring was as follows: 0. normal; 1. perifascicular internalization; 2. deep internalization (>5 cell layers), 3. perifascicular regeneration, 4. deep regeneration, 5. unilateral fascicular regeneration, 6. entire fascicular regeneration. The grade for the sample represents the worst area (most severe damage) present on the slide.

[0202] Statistics. Data are presented as mean ± SD (n = 4 in release kinetics, cell function and neurobehavioral studies). To account for multiple comparisons, all statistical comparisons were performed by Tukey-Kramer test using Origin software. P < 0.05 was considered to mean statistical significance.

[0203] result Synthesis of TDP polymer The synthesis of TDP polymers was carried out via Steglich esterification of triols, dicarboxylic acids and PEG at room temperature (Figure 1A). The hydrophilicity of the polymers could be modified by using hydrophilic triols (glycerol) or hydrophobic triols (PCL triol), by varying the number of carbons in the aliphatic chain of the dicarboxylic acid (1, 5 or 8), and by changing the molecular weight of the PEG. Herein, the nomenclature of the synthesized TDP polymers is T x D y P z where "x" represents the type of triol (g is glycerol and c is PCL triol), "y" represents the number of carbons in the aliphatic chain of the dicarboxylic acid, and "z" represents the molecular weight of the PEG (200, 1000, 2000 kDa) (Table 1). The presence of glycerol and PEG in the TDP polymers increases the hydrophilicity (f phil ), which is defined as the weight percentage of glycerol and PEG within the TDP polymer. [Table 1-3] a Polymer names have been abbreviated or simplified as described in the text. b The hydrophilicity of the polymer (f phil ): weight percentage of PEG and glycerol in the polymer. c Determined by GPC. d PCL triol = polycaprolactone triol.

[0204] Characterization of TDP polymers The structure of the TDP polymer is 1 The TDP polymer was investigated by H NMR spectroscopy. 1 In the H NMR, the methylene peaks of the dicarboxylic acid were detected at 1.30, 1.62, and 2.35 ppm, and the methylene peaks of the triol were detected at 4.05 to 4.35 ppm. g D8P 200 , T g D8P 1000 and T g D8P 2000 In the FTIR spectra, additional methylene peaks at 3.45–3.60 ppm were observed, suggesting the presence of PEG segments. The molecular weights of the TDP polymers were determined by gel permeation chromatography (GPC) (Table 1). The Mn of the TDP polymers was in the range of 4,000–7,000.

[0205] The hydrophilicity of TDP polymers was investigated by evaluating both surface and bulk characterization. The surface characteristics of thin polymer films were measured by optical tensiometry (geniometry). This technique measures the angle between the polymer surface and the tangent to the water droplet surface (contact angle). The contact angle correlates with hydrophilicity. The f phil The increase in f was inversely correlated with the contact angle (Figure 2A): phil As the concentration of α-dimethylformamide increased from 0 to 83.5%, the contact angle of the polymer thin film decreased from 73.9±1.3 to 31.3±2.1°. The bulk characteristics of the polymers were investigated by comparing the aqueous solubility of the polymers. phil The increase in pH induced dissolution in water.

[0206] The in vitro degradation rate of the polymer was measured under physiological conditions (PBS, pH 7.4, 37°C). phil This increased as f increased (Fig. 2B). phil The higher the f, the greater the water uptake and the faster the hydrolysis of the ester bond. phil When it is less than 37.8% (Tg D8 and T g D8P 200 ), the polymer followed an approximately linear mass loss (Figure 2C). Cytotoxicity

[0207] The cytotoxicity of TDP polymers was evaluated in myotubes derived from myoblast cell line C2C12 to assess potential myotoxicity, and in myotubes derived from pheochromocytoma cell line PC12, which is commonly used in neurotoxicity assays. TDP polymers were dispersed in PEG200 (50 mg / ml) and added to cell cultures, and cell viability was assessed after 24 hours by MTT assay (Figures 3A and 3B). There was no decrease in cell viability in either group of cell lines.

[0208] Synthesis of TDP-TTX conjugate The synthesis of TDP-TTX conjugates was performed via Steglich esterification reaction (Figure 1B), since Steglich esterification allows the reaction to proceed at room temperature, avoiding drug degradation. The difficulty in TTX conjugation is due to the low solubility of TTX in organic solvents, but TTX is very polar and only poorly soluble in acidified water. Since the Steglich esterification reaction must be carried out in a dry, non-aqueous solvent, a suitable organic solvent for TTX was required to facilitate the binding of TTX to the conjugate. Three anhydrous solvents, DMF, DCM, and DMSO, were used for the Steglich esterification reaction. DMF is a good solvent for the dicarboxylic acid, and DCM is a good solvent for the catalyst (DMAP), coupling agent (DIC), glycerol, PEG, and the resulting polymer. TTX had the highest solubility in DMSO (10 μg / mL) among the organic solvents tested, so DMSO was selected as the solvent for TTX (Table 2). In detail, 1 mg of TTX was added to the reaction mixture (see Methods). Due to its low solubility in DMSO, initially only a small amount of TTX was dissolved in DMSO and did not participate in the reaction. However, as the Steglich esterification reaction proceeded, the dissolved TTX was conjugated to the TDP polymer, which disrupted the solution equilibrium of TTX in DMSO, and subsequently more TTX was dissolved in DMSO and participated in the reaction. To increase the degree of conjugation of TTX with TDP (the mass fraction of TTX conjugated to the polymer based on the total TTX supplied), the reaction was allowed to proceed at room temperature for 7 days. After the reaction, the degree of conjugation was determined by measuring the unbound TTX in the reaction mixture by ELISA. >99.0% of TTX was conjugated to the TDP polymer (Table 3, Table 4, Table 5). [Table 2]

[0209] See Methods for a description of the equilibrium solubility method for measuring solubility. [Table 3] a Determined by ELISA. b Degree of conjugation: mass fraction of TTX conjugated to the polymer based on total TTX supplied. [Table 4] See Methods for a description of the measurement of the extent of drug binding. a After the steglich esterification reaction, the DCM in the reaction mixture was removed by rotary evaporation, and then the reaction mixture containing 10 mL of DMSO, the polymer-TTX conjugate and unbound TTX was washed with 30 mL of DI water. The polymer-TTX conjugate was centrifuged at 20000 rpm for 5 min, and the supernatant containing 30 mL of DI water, 10 mL of DMSO and unbound TTX was collected as supernatant #1. The polymer-TTX conjugate was washed with 40 mL of DI water and centrifuged again. The supernatant containing 40 mL of DI water and unbound TTX was collected as supernatant #2. b The TTX concentration of the supernatant was measured using a TTX Elisa kit. Because of the presence of organic solvents in the supernatant #1, the supernatant #1 was diluted 10-fold with PBS to avoid the influence of organic solvents on the ELISA measurement.

[0210] The extent of TTX binding was calculated as follows:

number

number

[0211] Characterization of TDP-TTX conjugates The conjugation of the TDP polymer with TTX was directly confirmed by Fourier transform infrared spectroscopy (FTIR). -1 The absorption peaks in the range of 3,000–3,600 cm are characteristic of the guanidinium groups of TTX and the hydroxyl groups of the TDP polymer. -1 In the range of 0.1 to 1.0 μm, the TDP polymer showed stronger absorption than the unmodified TDP polymer, indicating the presence of TTX.

[0212] Example 2: TTX-PGS / PEG provides sustained in vitro drug release without cytotoxicity result To assess the potential of the TDP-TTX conjugate to provide sustained neuronal blockade, the release kinetics was investigated in vitro under physiological conditions (PBS, pH 7.4, 37°C). HPLC of the release sample revealed a peak at approximately 5.0 min. Liquid chromatography-mass spectrometry (LC-MS) revealed that the molecular weight of the molecule in that fraction corresponds to the molecular weight of TTX (m / z 320.1 corresponds to [TTX+H] +It was confirmed that TTX was released from the TDP-TTX conjugates in its native form. The half-life of TTX release, which is the time it takes to release half of the loaded TTX, was examined. All TDP-TTX conjugates significantly extended the duration of TTX release compared to free TTX (Figures 4A and 4B).

[0213] f of TDP polymer phil The release rate of TTX was determined. phil The release half-life of TTX increased from 25±5 h to 723±75 h as the f phil Since the profile of the g D8-TTX and T g D8P 200 -TTX), suggesting the possibility of releasing TTX at a constant rate over a long duration of local anesthesia while minimizing systemic toxicity.

[0214] To demonstrate that TTX release was dependent on ester bond cleavage, TTX was covalently conjugated to TDP via a urethane bond (Chaffin, KA, Chen, X., McNamara, L., Bates, FS & Hillmyer, MA Polyether Urethane Hydrolytic Stability after Exposure to Deoxygenated Water. Macromolecules 47, 5220-5226, doi:10.1021 / ma500904d (2014)), which can be hydrolyzed, albeit at a much slower rate than the ester bond (Figure 5A). In vitro, after 28 days of incubation in physiological conditions (PBS, pH 7.4, 37 °C), ELISA-detectable TTX was observed in 25 mg of TDP containing 10 μg of TTX. g There was no release from the D8-TTX urethane conjugate (Figure 6).

[0215] In vitro drug release from PGS-PEG-TTX conjugates and PGS-TTX conjugates The in vitro drug release and degradation of PGS-PEG-TTX and PGS-TTX conjugates were investigated under physiological conditions (PBS, 37°C) over a period of 28 days. LC-MS analysis confirmed that TTX was released from the polymer-TTX conjugates in its native form. All polymer-TTX conjugate formulations significantly extended the duration of TTX release compared to free TTX (Figures 6B, 6C). The addition of PEG to the PGS-TTX conjugates determined the hydrophilicity of the polymer backbone, which in turn determined the hydrolysis rate of the ester bonds and the TTX release rate. Thus, the TTX release rate can be regulated by adjusting the percentage of PEG segments in the polymer-TTX conjugates. The release of TTX increased proportionally to the amount of PEG incorporated. After 28 days, PGS-PEG2000-TTX showed 99.7% TTX release, whereas PGS-PEG1000-TTX, PGS-PEG200-TTX and PGS-TTX showed 89.5, 62.5 and 43.7% TTX release, respectively. In the case of PGS-PEG200-TTX and PGS-TTX conjugates, TTX was released according to a zero-order release profile, which was attributed to the degradation of PGS-based copolymers under a surface erosion mechanism. The increase in TTX release with increasing PEG concentration can be attributed to the increased hydrophilicity of the copolymer backbone. Such increased hydrophilicity increases the uptake of water, thereby accelerating the hydrolysis of ester bonds.

[0216] A similar trend was observed for drug-dexamethasone, which has a hydroxyl group that can be covalently attached to PGS-PEG via the same ester bond used in the conjugation of TTX. The concentration of dexamethasone was measured using HPLC. The drug release profiles of TTX and dexamethasone were consistent for all PGS-PEG-drug conjugates examined (see Table 6). The mass loss followed a similar trend to that of the drug release of PGS-PEG-TTX conjugates (Figures 6A-6E). After 28 days, the mass loss of PGS-TTX was 27.3%, whereas the mass losses of PGS-PEG200-TTX, PGS-PEG1000-TTX, and PGS-PEG2000-TTX were 30.4, 70.4, and 94.7%, respectively. These results confirmed that drug release occurred due to hydrolysis of the ester bond. The tunable sustained release of the compound of interest over a period of weeks confirmed the potential of the conjugate to provide long-lasting local anesthesia.

[0217] The above data demonstrate that any drug containing a hydroxyl or carboxyl group can be covalently conjugated to the TDP polymer via a hydrolyzable ester bond, which has the potential to serve as a universal platform for the controlled release of a wide range of therapeutic drugs, such as dexamethasone (Figure 5B, Table 6, Figures 6A-6E). [Table 6] a Dex=dexamethasone. b Determined by HPLC.

[0218] Example 3: Preparation of a formulation injectable by syringe result Injectable solutions and suspensions have the potential to be injected into the body by any route of administration (Mastropietro, D., Nimroozi, R. & Omidian, H. Rheology in pharmaceutical formulations-A perspective. J Dev Drugs 2,108(2013)). However, there is a high f phil With T g D8P 2000 could be uniformly suspended in PBS to produce an injectable formulation, but phil Other TDP polymers with low affinity could not be homogenously suspended in PBS (Figures 3A and 3B). To administer the TDP-TTX conjugate to patients by injection, a homogenous TDP-TTX / PEG200 formulation was made by solvent evaporation (Figure 7). Briefly, the TDP-TTX conjugate was dissolved in DCM to generate a homogenous solution, and then PEG200, which is miscible with DCM, was added. The DCM was removed by rotary evaporation and lyophilization to obtain a homogenous suspension of TDP-TTX in PEG200.

[0219] The dynamic storage modulus (G') and loss modulus (G") and complex viscosity of the TDP-TTX / PEG200 formulations were characterized over a range of angular frequencies (Figures 8A-8C). Viscosity is important when considering syringeability and injectability. At 50 mg / mL, all TDP-TTX / PEG200 formulations had viscosities below 10 Pa s over the range of angular frequencies tested. G" was higher than G', suggesting that the viscous component of the complex modulus dominated the behavior of the materials, i.e., they behaved as liquids (Figure 8C). The viscous behavior and low viscosity of the TDP-TTX / PEG200 formulations suggest that they are injectable by syringe.

[0220] f of the above polymer philwas inversely correlated with the viscosity of the TDP-TTX / PEG200 formulation (Figure 8A). This is probably because the decrease in hydrophilicity meant a decrease in miscibility with PEG200. c The viscosity of D8-TTX / PEG200 is T g The viscosity was lower than that of D8-TTX / PEG200, which may be due to the better miscibility of PCL-triol in PEG200 than that of glycerol.

[0221] Example 4: TDP-TTX conjugate induces sciatic nerve blockade in vivo result Effect of TTX dose on nerve blockade Rats (4 per group) were injected into the left sciatic nerve with 0.5mL of PBS or PEG200 containing free TTX, or 0.5mL of PEG200 containing TDP-TTX conjugate. They then underwent neurobehavioral testing to measure the duration of functional impairment (i.e., nerve blockade) in both hind paws. The duration of impairment on the injected (left) side reflected the duration of nerve block. The impairment on the non-injected (right, contralateral) side reflected the systemic distribution of TTX.

[0222] Free TTX with and without PEG200 Groups of rats receiving sciatic nerve injections of free TTX showed a dose-dependent increase in the frequency of successful nerve blocks and the median duration of nerve blocks (Figures 9A and 9B). Low doses of TTX (1 or 2 μg; 6 or 12 μM, respectively) in PBS did not cause detectable nerve blocks or toxicity. Blocks from 4 μg (24 μM) free TTX in PBS were successful in 100% of animals and resulted in a median duration of sensory nerve block of 1.9 ± 1.0 h. This is comparable to the effect of 0.5% bupivacaine, an anesthetic agent commonly used in clinical practice. However, blocks with 4 μg TTX in PBS were accompanied by significant systemic toxicity, evidenced by sensory loss in the uninjected (contralateral) leg (Figure 9C and Figures 10A-10E). Injections of 5 μg (30 μM) free TTX in PBS caused contralateral lesions in all animals and were uniformly lethal (Figure 9D). There was no statistically significant difference between the duration of sensory and motor blockade at any dose of free TTX in PBS (FIG. 11).

[0223] Free TTX in 0.5 mL of PEG200 provided a significantly faster rate of nerve block success and a longer duration of nerve block than TTX in PBS (Figures 9A, 9B). 1 μg (6 μM) and 3 μg (18 μM) of free TTX in PEG200 provided 100% block with durations of 3.6 ± 0.3 h and 5.3 ± 0.3 h, respectively. PEG200 did not affect the incidence of systemic toxicity (Figures 9C, 9D). The improved nerve block success rate and duration with PEG200 is consistent with the effects of chemical penetration enhancers or nanoencapsulation.

[0224] T g D8-TTX / PEG200 The duration of the nerve block is T g The T was significantly prolonged by the formulation in D8-TTX / PEG200 (Figures 9A and 9B). gConjugated TTX as D8-TTX / PEG200 provided 50% block success with a median duration of 1.6±1.1 hours. The success and duration of sensory block increased with increasing doses of conjugated TTX. g Sensory nerve block with conjugated TTX as D8-TTX / PEG200 lasted for 71.5±6.9 hours (FIG. 9B) and did not result in animal mortality or contralateral lesions (FIGS. 9C, 9D, and 10A-10E). Importantly, due to limited toxicity in this animal model, it is feasible to use sustained release (Rwei, AY et al., Repeatable and adjustable on-demand sciatic nerve block with phototriggerable liposomes. Proceedings of the National Academy of Sciences 112, 15719-15724, (2015)), chemical penetration enhancers (Lahaye, LA & Butterworth, IVJF Site-1 Sodium Channel Blockers as Local Anesthesia Will Neosaxitoxin Supplant the Need for Continuous Nerve Blocks? Anesthesiology 123, 741-742 (2015)), or drugs to enhance the efficacy of S1SCB (Rai, R., Tallawi, M., Grigore, A. & Boccaccini, ARSynthesis, properties and biomedical applications of poly(glycerol sebacate) (PGS): A review. Progress in Polymer Science 37, 1051-1078, (2012)), such a long nerve block cannot be achieved with TTX. The motor block was longer than the sensory block at all doses of conjugated TTX. For example, gMotor block with 80 μg (480 μM) of conjugated TTX as D8-TTX / PEG200 lasted for 83.5±10.5 hours (FIG. 11).

[0225] T g The fact that low doses of TTX (e.g., 1-3 μg) in D8-TTX / PEG200 had a higher blocking success rate than free TTX in PBS was consistent with a CPE-like effect of the excipients in that formulation. Otherwise, in general, one would expect free drug to have a higher blocking success rate due to the higher free fraction of drug. g To determine whether D8 was involved in the CPE-like effect, 25 mg of T in 0.5 mL of PEG200 was g Injection of 3 μg (18 μM) of free TTX with (but not conjugated to) the D8 polymer into the sciatic nerve produced 100% block with a duration of 3.5±1.1 hours, which was longer than the duration of block from free TTX (p<0.05) but not longer than TTX in PEG200, indicating that T g These results suggest that PEG200, not D8, is responsible for the CPE-like effect. g The D8 polymer did not produce a nerve block (Table 7). [Table 7] Data for duration of nerve block are mean ± SD.

[0226] Both sodium fluorescein and TTX, fluorescent dyes with excitation wavelengths of 460 nm and emission wavelengths of 515 nm, are very hydrophilic, so sodium fluorescein was used as a substitute for TTX to investigate the possibility that PEG200 could act as a CPE molecule to increase TTX influx into the nerve. 0.25 mg of sodium fluorescein in 0.5 mL of PEG200 or PBS was injected into the sciatic nerve of rats. After 1 and 4 hours, the animals were euthanized and the sciatic nerve and surrounding tissues were collected. Frozen sections of those tissues were made and fluorescent images were taken. Fluorescent images of sections of the sciatic nerve and surrounding tissues were captured 1 and 4 hours after injection of 0.25 mg of sodium fluorescein in 0.5 ml of PEG or PBS.

[0227] In animals injected with sodium fluorescein in PEG200, fluorescence was observed throughout the nerve at 1 hour, but not at 4 hours after injection. No fluorescence was observed in the nerve of animals injected with PBS containing the same dose of sodium fluorescein at any time point, or in the uninjected limb. These results demonstrate that PEG200 can act as a chemical penetration enhancer to aid in the penetration of molecules into the nerve.

[0228] The critical hypothesis underlying this study was that TTX bound to the polymer may be inactive and that it may become active upon release. To test this hypothesis, we used a urethane bond to bind TTX. g 10.0 μg of conjugated TTX to D8 was injected into the sciatic nerve.No sensory nerve blockade was produced in any of the animals tested.These results suggest that TTX has no biological activity when covalently conjugated to the polymer backbone.

[0229] T diluted in PBS g D8-TTX / PEG200 To eliminate the CPE effect induced by PEG200 and to evaluate the effect of T on nerve blockg To investigate the effect of sustained release of TTX from D8-TTX itself. g The D8-TTX / PEG200 solution was diluted 4-fold with PBS containing 10 wt% bovine serum albumin (BSA). BSA acts as a surfactant to prevent the release of T cells from the solution. g Precipitation of D8-TTX was avoided. Free TTX at 1 μg (6 μM) in 0.5 ml of its diluted solution did not cause detectable nerve block or toxicity, suggesting the elimination of the CPE effect of pure PEG200 (Table 8). 25 mg of TTX containing 40 μg (240 μM) TTX in 0.5 ml of diluted solution g D8-TTX did not cause detectable nerve block or toxicity, suggesting that T g These results suggest that the slow release of TTX from D8-TTX did not achieve a therapeutic dose of TTX that resulted in nerve block. g The CPE-like function of PEG200 in the D8-TTX / PEG200 formulation was further confirmed. [Table 8] Data for duration of nerve block are mean ± SD.

[0230] Example 5:f phil Effects of Conjugated TTX on Toxicity and Efficacy f of TDP polymer phil determined the TTX release rate from TDP-TTX conjugates in vitro. phil We hypothesized that the higher f also determines the toxicity and efficacy of a particular dose of conjugated TTX in vivo. phil induced a faster release of TTX in its native form, thus increasing the toxicity of certain high doses of conjugated TTX, but on the other hand increasing the efficacy of certain low doses of conjugated TTX.

[0231] To test our hypothesis, rats were injected into the left sciatic nerve with TDP-TTX conjugates containing two doses of conjugated TTX: 10 μg (60 μM) and 1 μg (6 μM). phil The effect of f of TDP polymer was investigated. phil An increase in f enhanced the systemic toxicity of 10 μg (60 μM) of conjugated TTX, as evidenced by increased mortality (Figures 12A-12D). On the other hand, the f phil Increasing the concentration of TTX enhanced the efficacy of 1 μg (6 μM) conjugated TTX, as evidenced by increased success rate and duration of block.

[0232] Example 6: TDP polymers biodistributed to nerves in vivo result To investigate the local distribution of TDP polymers after sciatic nerve injection, TDP was covalently conjugated to fluorescein isothiocyanate (a fluorescent dye with an excitation wavelength of 488 nm and an emission wavelength of 519 nm) so that the dye could not diffuse on its own. g D8 (in 0.5 mL of PEG200) was injected into the animals. g At the indicated time points after injection, the animals were euthanized and the nerve and surrounding tissues were processed for histological examination. Confocal fluorescence imaging showed FITC fluorescence in the connective tissue between the muscle and the nerve at 24, 48 and 168 hours after injection. No FITC fluorescence was observed in the uninjected limb.

[0233] To assess the time course of localized retention of TDP in tissues, we used TDP covalently conjugated to Cy5.5, a fluorescent dye that emits a near-infrared signal ideal for fluorescence measurements in living animals. g Animals were injected with D8 (in 0.5 mL of PEG200). The polymer was Cy5.5-T g D8. Fluorescence imaging was performed using an in vivo imaging system (IVIS) to measure the Cy5.5-T expression in tissues.g The location of D8 was assessed over time. Fluorescent signal was seen in the sciatic nerve of all animals, with no detectable fluorescence elsewhere. Over a 4-week period, the fluorescent signal decreased, suggesting that TDP was gradually degraded in vivo (Figure 13). At necropsy 24 hours after injection, visible deposits were located in the sciatic nerve. These were fluorescent, confirming that they contained TDP.

[0234] Example 7: TDP-TTX conjugates do not illicit tissue reactions Results Free TTX and various f phil Animals injected with the TDP-TTX conjugates were euthanized 4 and 14 days after sciatic nerve injection. g Representative tissue responses to the D8-TTX conjugate are shown in Figure 8. At dissection, there was some residual material detected by confocal and IVIS. The tissues did not appear edematous or discolored, and had no other gross signs of tissue damage. The sciatic nerve and surrounding tissue were cut and collected for histological evaluation. Muscle tissue was processed for hematoxylin-eosin staining, and nerve tissue was processed for toluidine blue staining.

[0235] Microscopic examination revealed no significant myotoxicity or inflammation in animals injected with free TTX and TDP-TTX at 4 and 14 days after injection. Myotoxicity and inflammation were quantified using a scoring system (Table 3) (Wang, Y., Kim, YM & Langer, R. In vivo degradation characteristics of poly(glycerol sebacate). Journal of Biomedical Materials Research Part A 66A, 192-197, (2003)). The scores for free TTX and the various fractions were compared. phil There was no statistically significant difference between the scores for the TDP-TTX conjugates with

[0236] Because H&E staining is relatively insensitive for identifying nerve damage, toluidine blue-stained Epon-embedded sections of sciatic nerves from animals injected with TDP-TTX were obtained. Histological and microscopic examination of the nerves showed normal axonal distribution and myelin structure in all injected rats. Microscopic examination of the nerves at 4 and 14 days after injection revealed normal unmyelinated fibers with no evidence of injury (e.g., swelling, collapse, or darkly staining axoplasm). In all groups, myelinated fibers were occasionally seen to have deposits within the axon or circular breaks within the myelin sheath, which are common findings of normal peripheral nerve fibers. [Table 9] a 3 μg free TTX in 0.5 mL PBS, b TDP-TTX conjugates were formulated in 0.5 mL of PEG200. Data are median with 25th and 75th percentiles (n=4). Inflammation scores range from 0 to 4; myotoxicity scores range from 0 to 6 (McAlvin, JB, Reznor, G., Shankarappa, SA, Stefanescu, CF & Kohane, DS Local Toxicity from Local Anesthetic Polymeric Microparticles. Anesthesia and analgesia 116, 794-803, (2013); F Padera, R., Tse, J., Bellas, E. & S Kohane, D. Tetrodotoxin for prolonged local anesthesia with minimal myotoxicity. Vol. 34 (2006)).

[0237] Example 8: PGS-PEG-TTX conjugates provide a universal therapeutic drug platform for all alcoholic or carboxylic acid drugs result PGS-PEG copolymers have tunable hydrophilicity and therefore can serve as a universal therapeutic drug platform for all alcoholic or carboxylic acid drugs. Capsaicin, a conventional local anesthetic containing one hydroxyl group, was conjugated to PGS-PEG copolymers via an ester bond (Figure 14). To produce nerve blockade in a rat model, the minimum effective dose of capsaicin is approximately 50 μg, which is significantly higher than the minimum effective dose of TTX27-28. Therefore, to achieve the minimum effective dose of capsaicin, a higher injection dose of capsaicin and a faster release rate of capsaicin are desired than the injection dose and release rate of TTX. To prove this concept, PGS-capsaicin conjugates and PGS-PEG1000-capsaicin conjugates were synthesized. Each of them has two grafting densities: PGS-capsaicin and PGS-PEG1000-capsaicin have a grafting density of 44.4 μg / mg, and PGS-capsaicin-2 and PGS-PEG1000-capsaicin-2 have a grafting density of 182.9 μg / mg (Table 10). [Table 10]

[0238] Consistent with the release trends of TTX and dexamethasone, capsaicin was released from the PGS-PEG1000-capsaicin conjugates much faster than the PGS-capsaicin conjugates. After 1 day of incubation in PBS, 70.8 μg of capsaicin was released from 25 mg of the PGS-PEG1000-capsaicin-2 conjugate, whereas only 5.3, 21.2, and 15.2 μg of capsaicin were released from 25 mg of PGS-capsaicin, PGS-capsaicin-2, and PGS-PEG1000-capsaicin, respectively ( FIG. 10 ). In vivo, injection of 25 mg of PGS-PEG1000-capsaicin-2 produced nerve blockade in all animals tested, which lasted from 1 to 27 days. On the other hand, injection of 25 mg of PGS-PEG1000-capsaicin, PGS-capsaicin and PGS-capsaicin-2 did not produce nerve blockade in all animals tested (Table 11), suggesting that the minimum effective dose of capsaicin was not reached. These results demonstrate that PGS is the most suitable platform for TTX, but that PGS-PEG1000 is a better platform than PGS for the controlled release of capsaicin to produce nerve blockade. [Table 11] a Formulated in 0.5 ml PEG200

[0239] PGS-PEG-TTX conjugates have the potential to be applied in larger animals, such as humans. Because the median toxic dose of local anesthetics (including TTX) is directly proportional to the mass (volume of distribution) of the recipient2, the toxicity of a given amount of preparation can be significantly reduced when used in larger animals. Because the dose that blocks nerves is proportional to the body size, both the injected dose and release rate of TTX should increase in larger animals. PGS-PEG-TTX may be more suitable than PGS-TTX in larger animals because of the faster release rate of TTX.

[0240] summary The TDP polymer was synthesized by forming an ester bond between the carboxyl group of the dicarboxylic acid and the hydroxyl group of the triol and PEG via the Steglich esterification reaction. The TDP polymer was degradable by hydrolysis of the ester bond and showed good biocompatibility with minimal cytotoxicity. Various f phil We have synthesized a family of TDP polymers with f phil determined the hydrolysis rate of the ester bonds in the polymer network. TDP polymers have multiple active end groups (hydroxyl and carboxyl groups) to which drugs can be covalently conjugated. These features suggest that TDP polymers have the potential to act as a universal platform for the controlled release of a wide range of therapeutic drugs.

[0241] Covalent conjugation of TTX with TDP polymer was achieved by forming an ester bond between the carboxyl group of TDP polymer and the hydroxyl group of TTX via Steglich esterification reaction. Steglich esterification synthesis was performed at room temperature to avoid drug degradation. The synthesized TDP-TTX conjugate gradually decomposed into smaller polymer-TTX fragments, and finally decomposed into the native form of TTX by hydrolysis of the ester bond, achieving the controlled release of TTX. The release rate of TTX was determined by the f phil f less than 37.8% phil In this study, the TDP-TTX conjugate achieved a long-term TTX release of more than one month in vitro.

[0242] The CPE function of PEG200 was confirmed for the first time. PEG200 could increase drug influx into the nerve through the perineurial barrier, which could be attributed to the amphiphilic nature of PEG200. A dose of 1-3 μg of free TTX in 0.5 mL of PEG200 produced 100% blockade with a maximum duration of block of 5.3 ± 0.3 hours, which is three times longer than the effect of 0.5% bupivacaine, an anesthetic commonly used in clinical practice. Due to its low toxicity and already widespread use in various pharmaceutical formulations (D'souza, AA & Shegokar, R. Polyethylene glycol (PEG): a versatile polymer for pharmaceutical applications. Expert Opinion on Drug Delivery 13, 1257-1275, doi:10.1080 / 17425247.2016.1182485(2016)), PEG200 has great potential to serve as a TTX delivery vehicle for local anesthesia in clinical practice.

[0243] Due to their good miscibility, the TDP-TTX conjugates were easily dispersed in PEG200 to form homogeneous TDP-TTX / PEG200 formulations that could be injected with a syringe. All the prepared TDP-TTX / PEG200 formulations behaved as liquids with viscosities below 10 Pa s, suggesting the possibility of administration to patients via syringe injection.

[0244] In vivo animal studies confirmed that the TDP-TTX / PEG200 formulation could achieve sustained release of TTX and significantly expand the therapeutic index of TTX for local anesthesia. (i) TDP-TTX / PEG200 enabled injection of TTX at doses up to 80 μg without increasing its systemic toxicity. Furthermore, the TDP-TTX conjugate was retained in the epineural tissue around the nerve for a long time, which lasted up to 4 weeks, thereby maintaining therapeutic TTX concentrations for a long period of time and allowing the duration of local anesthesia to be extended. (ii) The TDP-TTX / PEG200 formulation could enhance the efficacy of TTX. The TDP-TTX / PEG200 formulation significantly enhanced the efficacy of as little as 1 μg of TTX to produce nerve blockade. Overall, in a wide range of TTX injection doses from 1.0 to 80.0 μg, the TDP-TTX / PEG200 formulation provided an adjustable duration of nerve block from several hours to 3 days.

[0245] The TDP-TTX conjugate can be used in large animals, including humans. The median toxic dose of local anesthetics (including TTX) was 1.2 mg / kg / day for recipients. 2 Since the toxicity is directly proportional to the mass (volume of distribution) of the TTX, the toxicity of a given amount of our preparation can be significantly reduced when used in larger animals. Since the dose at which the nerve is blocked is proportional to the body size, both the injected dose and the release rate of TTX should increase in larger animals. A higher f phil The TDP-TTX conjugate with the formula (I) may be more suitable in larger animals due to its faster release rate of TTX.

[0246] Aiming to introduce TTX into clinical practice, a delivery system was rationally designed to expand the therapeutic index of TTX. TTX was covalently conjugated to a biodegradable and biocompatible TDP scaffold via a hydrolyzable ester bond. The above data demonstrated that the TDP-TTX conjugate could provide tunable TTX release. TTX could be released in its native form by hydrolysis of the ester bond, and the release rate could be tunable by controlling the hydrophilicity of the TDP scaffold. PEG200 could increase drug influx into the nerve through the perineurial barrier by acting as a CPE molecule. PEG200 could significantly enhance the efficacy of TTX to produce nerve blockade. TDP-TTX / PEG200 is a syringe-injectable formulation with sustained TTX release from the TDP-TTX conjugate along with the CPE function of PEG200, and was demonstrated to significantly expand the therapeutic window of TTX. The TDP-TTX / PEG200 formulation provided nerve block of adjustable duration from several hours to 3 days at a wide range of TTX injection doses from 1.0 to 80.0 μg in in vivo experiments. Furthermore, nerve blockade was accompanied by minimal systemic toxicity and virtually no local toxicity to muscle and peripheral nerves. The TDP-TTX / PEG200 formulation provides a successful and safe approach for long-lasting and adjustable local anesthesia.

Claims

1. 1. A formulation for long-lasting local anesthesia comprising an anesthetic-polymer covalent conjugate, said conjugate comprising: (a) an anesthetic agent that is a site 1 sodium channel blocker (S1SCB) selected from the group consisting of tetrodotoxin (TTX), saxitoxin (STX), decarbamoylsaxitoxin, neosaxitoxin, and gonyautoxin; and (b) a polymer backbone comprising one or more hydrophilic or hydrophobic polymers, the polymer backbone comprising an ester bond; Including, the anesthetic agent is covalently conjugated to the polymer backbone via a hydrolyzable linker that is inactive while conjugated to the polymer backbone, the hydrolyzable linker comprising an ester bond; A formulation in which the linker is cleaved after administration to release an effective amount of Site 1 sodium channel blocker from the polymer backbone to induce local nerve blockade with less toxicity than unconjugated anesthetic agents after administration to a subject at a site requiring local nerve blockade.

2. The formulation of claim 1 , wherein the polymer backbone comprises poly(glycerol sebacate) (PGS).

3. 10. The formulation of claim 1, wherein the polymer backbone further comprises one or more hydrophilic polymers having a molecular weight of about 100 Da to about 200,000 Da, inclusive.

4. 4. The formulation of claim 3, wherein the polymer backbone comprises polyethylene glycol (PEG), methoxy(polyethylene glycol), or a combination thereof.

5. 10. The formulation of claim 1, wherein the conjugate releases the anesthetic agent over a period of about 24 hours to 72 hours, 72 hours to 1 week, or 1 week to 1 month after administration in vivo.

6. The formulation of claim 1 , wherein the conjugate further comprises one or more active agents covalently conjugated to the backbone polymer.

7. The formulation of claim 6, wherein the conjugate further comprises one or more covalently attached glucocorticoids.

8. 8. The formulation of claim 7, wherein the glucocorticoid is selected from the group consisting of dexamethasone, cortisone, hydrocortisone, prednisone, beclomethasone, betamethasone, flunisolide, methylprednisone, paramethasone, prednisolone, triamcinolone, alclometasone, amcinonide, clobetasol, fludrocortisone, diflurosone diacetate, fluocinolone acetonide, fluoromethalone, flurandrenolide, halcinonide, medrysone and mometasone, and pharma- ceutically acceptable salts and mixtures thereof.

9. The formulation of claim 6 , wherein the conjugate further comprises covalently attached dexamethasone.

10. The formulation of claim 1 for administration in vivo in a pharma- ceutically acceptable vehicle for injection.

11. The formulation of claim 10, wherein the formulation comprises a chemical penetration enhancer as an excipient.

12. 12. The formulation of claim 11, wherein the chemical penetration enhancer is PEG.

13. The formulation of claim 12, wherein the PEG is PEG 200.

14. 11. A formulation according to claim 10 in a dosage unit containing anaesthetic agent in an amount between 0.1 μg and 200 μg, both ends included.

15. 15. The formulation of claim 14, wherein the amount of anesthetic in the dosage unit is an amount effective to induce effective local nerve blockade for up to one month after administration to a subject in need thereof.

Images