2'-6'-pipecoloxylidide as a sensory-selective anesthetic

Abstract

A method of inducing local anesthesia in a subject includes administering to the subject in need thereof 2',6'-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce local anesthesia in the subject. An active molecule that is metabolized to PPX in the subject is not administered to the subject with the PPX.

Description

Attorney Docket No.701039-000139WOPT 2’-6’-PIPECOLOXYLIDIDE AS A SENSORY-SELECTIVE ANESTHETIC CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.63 / 553,346 filed on February 14, 2024 and U.S. Provisional Application No. 63 / 574,736 filed on April 4, 2024, the contents of all of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0002] This disclosure relates generally to pharmaceutical compositions and methods pertaining to 2’-6’-pipecoloxylidide (PPX). BACKGROUND

[0003] Acute and chronic pain are frequently managed with opioids, local anesthetics, or a combination thereof. However, opioids are associated with nausea, constipation, sedation, and more seriously, misuse, abuse, diversion, addiction, and death from overdose (see References 1, 2), highlighting the need for safe and effective pharmacologic alternatives. Conventional local anesthetics (CLAs) are highly effective analgesics but cause motor nerve block in addition to sensory nerve block. This can be problematic in many settings and has limited their widespread use. For example, in labor anesthesia there is a trade-off between keeping the patient from feeling pain (sensory block) and inhibiting her ability to push (motor block). Consequently, pursuit of analgesia can reduce ambulation, associated with increased rates of instrumental vaginal delivery, and lead to arrest of labor (see References 3, 4). In the management of acute post-surgical pain, a concurrent motor block prevents the patient from mobilizing and participating in physical therapy, elements critical to overall recovery and prevention of serious complications. Use of continuous neuraxial or regional local anesthetics for chronic pain is similarly limited by resultant paralysis of involved body parts. Further, CLAs can be associated with local tissue injury and lethal systemic toxicity related to cardiac arrythmias, irrespective of the route of administration (see References 5, 6). Consequently, the identification and development of safe sensory-selective local anesthetics has received considerable attention, however with little success.

[0004] Previous attempts to achieve clinically relevant local anesthetic formulations with sensory selectivity have largely been limited by local and systemic toxicity. In peripheral nerve block models, co-administration of the quaternary lidocaine derivative QX-314 with the vanilloid receptor agonist capsaicin resulted in sensory selective local anesthesia throughAttorney Docket No.701039-000139WOPT preferential entry of QX-314 to sensory cells only (see Reference 7). Similar approaches used combinations of CLAs with capsaicin (see References 8, 9), CLAs with QX-314 (see Reference 10), and chemical permeation enhancers with QX-314 and QX-222 (see Reference 11). However, quaternary lidocaine derivatives lead to severe myotoxicity when administered peripherally (see Reference 12) and to irritation and death when administered intrathecally (see Reference 13). Selective blockade of specific voltage gated sodium channels has also been proposed as a potential target sensory-selective anesthesia (i.e., pain relief) due to selective expression of Nav 1.7, Nav1.8, and Nav1.9 in peripheral nociceptive neurons of the dorsal-root ganglia (see References 14, 15). However, investigation of selective sodium channel blockers has largely focused on oral and intraperitoneal agents, including VX-548, A-803467, PF- 01247324, and VX-150 (see References 15–18).

[0005] Therefore, a solution is needed that: produces sensory selective local and neuraxial anesthesia when administered at low doses for peripheral nerve block as well as neuraxial (i.e., intrathecal) anesthesia; selectively inhibits Nav1.8 channels; and exhibits less systemic toxicity and similar tissue effects as clinically used CLAs. SUMMARY

[0006] Described herein are pharmaceutical compositions and methods pertaining to 2’-6’- pipecoloxylidide (PPX).

[0007] According to some embodiments, a method of inducing anesthesia e.g., local anesthesia or systemic anesthesia in a subject includes administering to the subject in need thereof 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia e.g., local anesthesia or systemic anesthesia in the subject. An active molecule that is metabolized to PPX in the subject is not administered to the subject with the PPX.

[0008] In some embodiments, the active molecule that is metabolized to PPX in the subject is ropivacaine, bupivacaine, levobupivacaine, mepivacaine, or a pharmaceutically acceptable salt, or an analog or derivative thereof. Accordingly, in some embodiments, the method of inducing anesthesia e.g., local anesthesia or systemic anesthesia in a subject comprises administering to the subject in need thereof PPX or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia e.g., local anesthesia or systemic anesthesia in the subject, and wherein an active molecule that is metabolized to 3’-hydroxy-2’,6’- pipecoloxylidide in the subject is not administered to the subject with the PPX.Attorney Docket No.701039-000139WOPT

[0009] In some embodiments, the method of inducing anesthesia e.g., local anesthesia or systemic anesthesia in a subject comprises administering to the subject in need thereof PPX or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia e.g., local anesthesia or systemic anesthesia in the subject, and wherein ropivacaine, bupivacaine, levobupivacaine, mepivacaine, or a pharmaceutically acceptable salt, or an analog or derivative thereof is not administered to the subject with the PPX. As used herein, derivative of ropivacaine, bupivacaine, levobupivacaine, mepivacaine or analog of thereof does not include PPX.

[0010] In some embodiments, the method of inducing anesthesia e.g., local anesthesia or systemic anesthesia in a subject includes administering to the subject in need thereof an effective amount of a pharmaceutical composition. The pharmaceutical composition includes: (i) 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof; and (ii) an active molecule capable of inducing anesthesia in the subject and being metabolized to PPX in the subject. The pharmaceutical composition includes the PPX in an amount that is at least about 5% or more of the amount of the active molecule in the composition.

[0011] In some embodiments, the effective amount of PPX is a concentration of 15 mM or higher, e.g., at least about 17.5 mM, at least about 20 mM, at least about 22.5 mM, at least about 25 mM, at least about 27.5 mM, at least 30 mM or higher. For example, the effective amount of PPX is a concentration of from higher than 15 mM to about 250 mM, e.g., from about 17.5 mM to about 200 mM, from about 20 mM to about 150 mM, from about 25 mm to about 100 mM, or from about 22.5 mM to about 42.5 mM.

[0012] In some embodiments, the PPX or a pharmaceutical composition comprising the PPX is administered locally, and wherein the therapeutic effect (e.g., mitigation of pain, alteration of consciousness, sedation, or anesthesia) is local or systemic. In some other embodiments, the PPX or a pharmaceutical composition comprising the PPX is administered systemically, and wherein the therapeutic effect (e.g., mitigation of pain, alteration of consciousness, sedation, or anesthesia) is local or systemic.

[0013] In some embodiments, the pharmaceutical composition capable of inducing anesthesia, e.g., local anesthesia or systemic anesthesia in a subject includes 2’,6’- pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia e.g., local anesthesia or systemic anesthesia in a subject; and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition is substantially free an active molecule that is metabolized to PPX in the subject, e.g., theAttorney Docket No.701039-000139WOPT pharmaceutical composition does not include an active molecule that is metabolized to PPX in the subject.

[0014] In some embodiments, a pharmaceutical composition capable of inducing anesthesia, e.g., local anesthesia or systemic anesthesia in a subject, includes 2’,6’- pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia, e.g., local anesthesia or systemic anesthesia in a subject; an active molecule capable of inducing anesthesia in the subject and being metabolized to PPX in the subject; and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition includes PPX in an amount that is at least about 5% or more of the amount of the active molecule in the composition.

[0015] In some embodiments, the pharmaceutical composition capable of inducing anesthesia, e.g., local anesthesia or systemic anesthesia in a subject comprises PPX or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia e.g., local anesthesia or systemic anesthesia in a subject; and a pharmaceutically acceptable carrier or excipient, and wherein the composition is substantially free of an active molecule that is metabolized to 3’-hydroxy-2’,6’-pipecoloxylidide in the subject.

[0016] In some embodiments, the pharmaceutical composition capable of inducing anesthesia, e.g., local anesthesia or systemic anesthesia in a subject comprises PPX or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia e.g., local anesthesia or systemic anesthesia in a subject; and a pharmaceutically acceptable carrier or excipient, and wherein the composition is substantially free of ropivacaine, bupivacaine, levobupivacaine, mepivacaine, or a pharmaceutically acceptable salt, or an analog or derivative thereof.

[0017] In some embodiments, the pharmaceutical composition capable of inducing anesthesia, e.g., local anesthesia or systemic anesthesia in a subject, comprises PPX or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia, e.g., local anesthesia or systemic anesthesia in a subject; an active molecule capable of inducing anesthesia in the subject and being metabolized to PPX in the subject; and a pharmaceutically acceptable carrier or excipient, wherein the pharmaceutical composition includes PPX in an amount that is at least about 5% or more of the amount of the active molecule in the composition, and wherein the active molecule is ropivacaine, bupivacaine, levobupivacaine, mepivacaine, or a pharmaceutically acceptable salt, or an analog or derivative thereof.

[0018] In some embodiments, the pharmaceutical composition comprises PPX at a concentration of 15 mM or higher, e.g., at least about 17.5 mM, at least about 20 mM, at leastAttorney Docket No.701039-000139WOPT about 22.5 mM, at least about 25 mM, at least about 27.5 mM, at least 30 mM or higher. For example, the pharmaceutical composition comprises the PPX at a concentration of from higher than 15 mM to about 250 mM, e.g., from about 17.5 mM to about 200 mM, from about 20 mM to about 150 mM, from about 25 mm to about 100 mM, or from about 22.5 mM to about 42.5 mM.

[0019] It is noted that the effect of administering PPX can be altered by second agent co- administered to the subject. For example, an effect of the effective amount of PPX can be altered by second agent co-administered to the subject. Some exemplary agents that can alter the effect of an amount of PPX administered to the subject include but are not limited to vasoconstrictors, glucocorticoid receptor agonists, and chemical permeation enhancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] Disclosed herein are embodiments of pharmaceutical compositions and methods pertaining to 2’-6’-pipecoloxylidide (PPX). This description includes drawings, wherein:

[0022] FIG.1 is a skeletal structure diagram of 2’-6’-pipecoloxylidide (PPX).

[0023] FIG. 2 is a dose-response curve for PPX in a rodent peripheral nerve blockade model. The curve is a demonstration that concentrations of 22.5 – 30 mM of PPX at the sciatic nerve in rats leads to a sensory block, but not a motor block. At higher concentrations, both sensory and motor block can occur. (** indicates p < 0.01, *** indicates p< 0.001 comparing the sensory block durations at the indicated concentrations)

[0024] FIG. 3 is a comparison of intrathecal administration of PPX to commercially / clinically used Ropivacaine (Rop) in a rodent intrathecal model. The comparison demonstrates that 0.75% PPX produces a sensory block when administered intrathecally but does not produce a motor block. In contrast, 0.5% ropivacaine produces a sensory and motor block of equivalent durations.

[0025] FIG.4 is a graph of sensory latency in seconds as a function of time in minutes for repeat intrathecal administrations of PPX in one animal, followed by intrathecal administration of Ropivacaine. Repeat doses of PPX can be given to produce recurrent periods of sensory anesthesia. However, no motor block is observed. This is in contrast to administration of ropivacaine which produces a sensory and motor blockade.Attorney Docket No.701039-000139WOPT

[0026] FIG.5 is a gross tissue examination at 4 and 30 days following injection of 30 mM PPX at the sciatic nerve in a rodent model.

[0027] FIG. 6 is a graph of dissection scores, subcutaneous injury scores, and muscle injury scores at 4 and 30 days following injection of 30 mM PPX at the sciatic nerve in a rodent model. The graph shows that there is no gross subcutaneous or muscle tissue injury at dissection.

[0028] FIG.7 is a microscopic tissue examination at 4 and 30 days following injection of 30 mM PPX at the sciatic nerve in a rodent model.

[0029] FIG. 8 is a graph of inflammation and myotoxicity scores at 4 and 30 days following injection of 30 mM PPX at the sciatic nerve in a rodent model. There is minimal inflammation and myotoxicity 4 and 30 days following PPX administration.

[0030] FIG.9 is a skeletal diagram showing the synthesis of PPX.

[0031] FIG.10 is a1H NMR graph of PPX.

[0032] FIG.11 is a13C NMR graph of PPX.

[0033] FIG.12 is a mass spectrometry (HRMS) characterization of PPX.

[0034] FIG.13 is a graph of duration of sciatic nerve blockade in minutes as a function of concentration in mM with Rop (15-90mM) and PPX. Data are means with standard deviations, n = 4 except n = 6 for 15mM Rop and n = 8 for 30mM, 45mM, and 60mM PPX. *p < 0.05; ****p < 0.0001.

[0035] FIG.14 is a graph of sensory to motor block duration ratio (S:M ratio) as a function of concentration for PPX versus Rop. Data are means with standard deviations, n = 4 except n = 6 for 15mM Rop and n = 8 for 30mM, 45mM, and 60mM PPX. Comparison of sensory and motor blocks by paired t-test.

[0036] FIG. 15 is a schematic of multi-channel single-fiber recordings from split sciatic nerve filaments. Action potentials were evoked from the proximal end and recorded at the distal end by four Pt-Ir wire electrodes (white arrow). Scale bar: 3 mm.

[0037] FIG. 16 is representative single-fiber recordings before (as control), immediately after (drug), and 60 min after (wash) local application of chemicals to the sciatic nerve trunk. Action potentials (AP) from fast-conducting A-fibers were labeled by blue arrows, while those from slow-conducting C-fibers by red arrows. The blocked action potential was labeled by an open arrowhead.

[0038] FIG.17 is a series of graphs showing the effect of local chemical application to the sciatic nerve trunk on the axonal conduction velocity as assessed by single-fiber recordings. The y-axes represent current velocities (CV) in m / sec.Attorney Docket No.701039-000139WOPT

[0039] FIG.18 is a graph of the percentage of Nav1.7 block as a function of concentration in µM to show the effect of Ropivacaine (Rop) and PPX on voltage gated sodium channel currents. Data are means with standard deviations, n = 3 except n = 5 for 100µM PPX and n=7 for 100µM Rop. *p < 0.05; *** p < 0.001, ****p < 0.0001.

[0040] FIG.19 is a graph of the percentage of Nav1.8 block as a function of concentration in µM to show the effect of Ropivacaine (Rop) and PPX on voltage gated sodium channel currents. Data are means with standard deviations, n = 3 except n = 5 for 100µM PPX and n=7 for 100µM Rop. *p < 0.05; *** p < 0.001, ****p < 0.0001.

[0041] FIG.20 is a graph of the percentage of Nav1.9 block as a function of concentration in µM to show the effect of Ropivacaine (Rop) and PPX on voltage gated sodium channel currents. Data are means with standard deviations, n = 3 except n = 5 for 100µM PPX and n=7 for 100µM Rop. *p < 0.05; *** p < 0.001, ****p < 0.0001.

[0042] FIG. 21 is a graph of the mean ratio of Rop:PPX inhibition of specified Nav currents as a function of concentration in µM.

[0043] FIG.22 is a bar graph of duration of sensory and motor nerve blockade with 0.75% (w / v) Rop and PPX. Data are means with standard deviations, n = 8 except n = 6 for 1.5% PPX, n = 10 for 2.0% PPX, and n=18 for 0.75% PPX (single dose administrations). ****p < 0.0001.

[0044] FIG. 23 is a graph of duration of nerve blockade in minutes as a function of concentration of PPX (0.75%-2.0% (w / v)). Data are means with standard deviations, n = 8 except n = 6 for 1.5% PPX, n = 10 for 2.0% PPX, and n=18 for 0.75% PPX (single dose administrations). ****p < 0.0001.

[0045] FIG. 24 is a graph of sensory latency in seconds as a function of time in minutes for nerve blockade following administration of 3 repeated doses of 0.75% (w / v) PPX at 35min intervals, followed by single dose of 0.75% administered 50min after third dose of PPX. Data are means with standard deviations, n = 8 except n = 6 for 1.5% PPX, n = 10 for 2.0% PPX, and n=18 for 0.75% PPX (single dose administrations). ****p < 0.0001.

[0046] FIG.25 is a graph of % hERG block as a function of concentration in µM to show the effect of Rop versus PPX on hERG currents. Data are means with standard deviations. For current inhibition, n = 3. *p < 0.05; ****p < 0.0001.

[0047] FIG.26 is a graph of % Nav1.5 block as a function of concentration in µM to show the effect of Rop versus PPX on Nav1.5 currents. Data are means with standard deviations. For current inhibition, n = 3 except n = 4 for 100µM Rop, n = 6 for 10µM PPX, and n = 8 for 100µM PPX. ****p < 0.0001.Attorney Docket No.701039-000139WOPT

[0048] FIG.27 is a graph of mean ratio of Rop:PPX inhibition of specified currents as a function of concentration in µM based on the data values presented in FIGS.25–26.

[0049] FIG. 28 is a bar chart of the percent of animals affected by respiratory distress, contralateral latency, and seizures after sciatic injection of Rop versus PPX to evaluate acute systemic toxicities. Data are means with standard deviations. n=4.

[0050] FIG.29 is a graph of cell viability as a percentage as a function of the logarithm of the concentration of PPX in mM. Data are means with standard deviations, n = 4.

[0051] FIG.30 is a bar graph of IC50(the concentration required to inhibit growth of half of the cells) in mM for PPX and bupivacaine (Bup) in PC12 and C2C12 lines to compare the cytotoxicity of PPX and Bup. ****p < 0.0001; data are means with standard deviations, n = 4.

[0052] FIG.31 is a graph of inflammation scores as a function of days post-injection from a microscopic tissue toxicity on days 4, 30, and 60 following sciatic injection of 15 mM Rop and 30 mM PPX. Data are means with standard deviations, n = 5.

[0053] FIG.32 is a graph of mytotoxicity scores as a function of days post-injection from a microscopic tissue toxicity on days 4, 30, and 60 following sciatic injection of 15 mM Rop and 30 mM PPX. Data are means with standard deviations, n = 5.

[0054] FIG.33 is a collection of H&E-stained sections of muscles, nerve, and surrounding tissues from a microscopic tissue toxicity on days 4, 30, and 60 following sciatic injection of 15 mM Rop and 30 mM PPX. N = nerve, M = muscle, and inf = inflammation, mtox= myotoxicity.

[0055] FIG. 34 is a graph of block duration in minutes for liposomes including phospholipids encapsulating PPX.

[0056] FIG.35 are sciatic nerves that were harvested four days after injection of 30 mM PPX, embedded in Epon, and stained with toluidine blue.

[0057] FIG. 36 are posterior white matter of lumbar spinal cord sections harvested four days following intrathecal administration of 27.9 mM PPX and stained with toluidine blue.

[0058] FIG.37 is a graph of a time course of nerve block (%MPE) from 30 mM PPX + 30 mM Tween 20. The data from FIG.43 (PPX without Tween) are superimposed in grey. Data are means ± standard deviations, n = 4 except n = 6 for 15 mM ROP, n = 12 for 30 mM PPX and n = 8 for 45 mM and 60 mM PPX. *p < 0.05; ****p < 0.0001, ns = not statistically significant difference. comparison of sensory and motor blocks by paired t-test.

[0059] FIG. 38 is a collection of skeletal structure diagrams of conventional local anesthetics (CLAs) and 2’,6’-pipecoloxylidide (PPX), organized by length of carbon chain.

[0060] FIG.39 is an1H NMR of PPX hydrochloride (PPX HCl).1H NMR spectrum (300Attorney Docket No.701039-000139WOPT MHz) of PPX HCl was determined in deuterated water.

[0061] FIG. 40 is a time course of sensory and motor block (percent maximum possible effect, %MPE) after injection at the sciatic nerve with 15 mM ropivacaine (ROP).

[0062] FIG.41 shows an effect of ROP and PPX concentration on duration of nerve block.

[0063] FIG.42 shows an effect of ROP and PPX concentration on sensory to motor ratio (SMR) of nerve blocks. The “∞” values are due to the denominator being zero.

[0064] FIG.43 is a time course of nerve block (%MPE) from 30 mM PPX.

[0065] FIG.44 is a time course of sensory and motor block (percent maximum possible effect, %MPE) after injection at the sciatic nerve with 15 mM ROP.

[0066] FIG.45 shows duration of block with increasing concentrations of ROP and PPX.

[0067] FIG.46 is a time course of block (%MPE) from 30 mM PPX.

[0068] FIG. 47 is a cytotoxicity study comparing 24h exposure to increasing concentrations of ROP versus PPX in C2C12 cell lines. FIG.48 is a cytotoxicity study comparing 24h exposure to increasing concentrations of ROP versus PPX in PC12 cell lines. DETAILED DESCRIPTION

[0069] The present invention is described with reference to the attached figures. The figures are not drawn to scale, and they are provided merely to illustrate the instant invention. Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. In other instances, well- known structures or operations are not shown in detail to avoid obscuring the invention. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention. Definitions

[0070] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.Attorney Docket No.701039-000139WOPT

[0071] As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0072] The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0073] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0074] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.”

[0001] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0075] Where a range of values is provided, each numerical value between the upper and lower limits of the range is contemplated and disclosed herein.

[0076] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in molecular biology can be found in THE MERCK MANUAL OF DIAGNOSIS AND THERAPY, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); ROBERT S. PORTER ET AL. (EDS.), THE ENCYCLOPEDIA OF MOLECULAR CELL BIOLOGY AND MOLECULAR MEDICINE,Attorney Docket No.701039-000139WOPT published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and ROBERT A. MEYERS (ED.), MOLECULAR BIOLOGY AND BIOTECHNOLOGY: A COMPREHENSIVE DESK REFERENCE, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); IMMUNOLOGY BY WERNER LUTTMANN, published by Elsevier, 2006; LEWIN’S GENES XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); MICHAEL RICHARD GREEN AND JOSEPH SAMBROOK, MOLECULAR CLONING: A LABORATORY MANUAL, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); DAVIS ET AL., BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); LABORATORY METHODS IN ENZYMOLOGY: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), and CURRENT PROTOCOLS IN PROTEIN SCIENCE (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005 (ISBN 0471142735), the contents of which are all incorporated by reference herein in their entireties.

[0077] Compounds described herein can include one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, e.g., JACQUES ET AL., ENANTIOMERS, RACEMATES AND RESOLUTIONS (Wiley Interscience, New York, 1981); WILEN ET AL., TETRAHEDRON 33:2725 (1977); ELIEL, STEREOCHEMISTRY OF CARBON COMPOUNDS (McGraw— Hill, NY, 1962); and WILEN, TABLES OF RESOLVING AGENTS AND OPTICAL RESOLUTIONS 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0078] As used herein, the term “PPX” refers to 2’,6’-pipecoloxylidide and / or pharmaceutically acceptable salts thereof. A skeletal structure diagram of 2’,6’- pipecoloxylidide can be seen in FIG.1.

[0079] As used herein, the term “parent molecule” refers to a molecule from which another molecule, such as PPX, is derived. For example, ropivacaine and bupivacaine are parent molecules of PPX.Attorney Docket No.701039-000139WOPT

[0080] The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, BERGE ET AL., describe pharmaceutically acceptable salts in detail in J. PHARMACEUTICAL SCIENCES, 1977, 66, 1–19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4- salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0081] The terms “composition” and “formulation” are used interchangeably.

[0082] As used herein, the term “substantially free of” refers to a composition having less than about 1% by weight, e.g., less than about 0.5% by weight, less than about 0.1% by weight, less than about 0.05% by weight, less than about 0.01% by weight, less than about 0.009% by weight, less than about 0.008% by weight, less than about 0.007% by weight, less than about 0.006% by weight, less than about 0.005% by weight, less than about 0.004% by weight, lessAttorney Docket No.701039-000139WOPT than about 0.003% by weight, less than about 0.002% by weight, or less than about 0.001% by weight of the specified component., based on the total weight or volume of the composition.

[0083] A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and / or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and / or turkeys). In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal.

[0084] A “patient” refers to a human subject in need of treatment of a disease.

[0085] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” to a human patient shall be restricted to prescribing a controlled substance that will be administer to the patent by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” includes both methods practiced on the human body and also the foregoing activities.

[0086] The terms “administer,” “administering,” or “administration,” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing an inventive compound, or a pharmaceutical composition thereof. Stated another way, the terms “administer,” “administering,” or “administration,” refer to the placement of the PPX or a composition comprising the same into a subject by a method or route which results in at least partial localization of the PPX or composition at a desired site such that desired effect is produced.

[0087] PPX or a composition comprising same can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal,Attorney Docket No.701039-000139WOPT intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion.

[0088] It is noted that administration can be local or systemic. In some embodiments, administration will generally be local rather than systemic. In some embodiments, administering is intravenous (IV) or intraperitoneal (IP) administration. In some embodiments, administering is oral administration.

[0089] In some embodiments, the PPX, or pharmaceutical composition is administered parenterally, i.e., parenteral administration. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection, infusion and other injection or infusion techniques, without limitation.

[0090] An “effective amount” of an agent described herein refers to an amount sufficient to elicit the desired biological response. In certain embodiments, an effective amount is an amount of PPX sufficient to elicit the desired biological response. An effective amount of PPX described herein may vary depending on such factors as the desired biological endpoint, pharmacokinetics, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of an agent described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of an agent described herein in multiple doses. When an effective amount of a composition is referred herein, it means the amount is prophylactically and / or therapeutically effective, depending on the subject and / or the disease to be treated. Determining the effective amount or dosage is within the abilities of one skilled in the art.

[0091] Depending on the route of administration, effective doses can be calculated according to the body weight, body surface area, or organ size of the subject to be treated. Optimization of the appropriate dosages can readily be made by one skilled in the art in light of pharmacokinetic data observed in canine laboratory testing and clinical trials. Alternatively, or additionally, the dosage to be administered can be determined from studies using animal models for the particular type of condition to be treated, and / or from animal or human data obtained from agents which are known to exhibit similar pharmacological activities. The finalAttorney Docket No.701039-000139WOPT dosage regimen will be determined by the attending surgeon or physician, considering various factors which modify the action of active agent, e.g., the agent’s specific activity, the agent’s specific half-life in vivo, the severity of the condition and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any present infection, time of administration, the use (or not) of other concomitant therapies, and other clinical factors.

[0092] Determination of an effective amount is well within the capability of those skilled in the art. Generally, the actual effective amount can vary with the specific compound, the use or application technique, the desired effect, the duration of the effect and side effects, the subject’s history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents. Accordingly, an effective dose of compound described herein is an amount sufficient to produce at least some desired therapeutic effect in a subject.

[0093] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in dogs. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of use or administration utilized.

[0094] The effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the therapeutic which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The effective plasma concentration for the PPX or a metabolite thereof can be about 0.01 µM to about 10 µM, about 0.2 µM to about 5 µM, or about 0.8 to about 3 µM in a subject, such as mammal, e.g., human, a non-human primate, a rodent, or a dog.

[0095] Generally, the compositions are administered so that PPX is used or given at a dose from 50 µg / kg to 1000 mg / kg; 1 µg / kg to 500 mg / kg; 1 µg / kg to 150 mg / kg, 1 µg / kg to 100 mg / kg, 1 µg / kg to 50 mg / kg, 1 µg / kg to 20 mg / kg, 1 µg / kg to 10 mg / kg, 1µg / kg to 1mg / kg, 100 µg / kg to 100 mg / kg, 100 µg / kg to 50 mg / kg, 100 µg / kg to 20 mg / kg, 100 µg / kg to 10 mg / kg, 100µg / kg to 1mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 10 mg / kg, 10 mg / kg to 100 mg / kg, 10 mg / kg to 50 mg / kg, or 10 mg / kg to 20 mg / kg. It is to be understood that ranges given here include all intermediate ranges, for example, theAttorney Docket No.701039-000139WOPT range 1 mg / kg to 10 mg / kg includes 1mg / kg to 2 mg / kg, 1mg / kg to 3 mg / kg, 1mg / kg to 4 mg / kg, 1mg / kg to 5 mg / kg, 1mg / kg to 6 mg / kg, 1mg / kg to 7 mg / kg, 1mg / kg to 8 mg / kg, 1mg / kg to 9 mg / kg, 2mg / kg to 10mg / kg, 3mg / kg to 10mg / kg, 4mg / kg to 10mg / kg, 5mg / kg to 10mg / kg, 6mg / kg to 10mg / kg, 7mg / kg to 10mg / kg, 8mg / kg to 10mg / kg, 9mg / kg to 10mg / kg, and the like. Further contemplated is a dose (either as a bolus or continuous infusion) of about 0.1 mg / kg to about 10 mg / kg, about 0.3 mg / kg to about 5 mg / kg, or 0.5 mg / kg to about 3 mg / kg. It is to be further understood that the ranges intermediate to those given above are also within the scope of this disclosure, for example, in the range 1 mg / kg to 10 mg / kg, for example use or dose ranges such as 2mg / kg to 8 mg / kg, 3mg / kg to 7 mg / kg, 4mg / kg to 6mg / kg, and the like.

[0096] Typically, a dose of 500 mgs, 1 gram, 2.5 grams or more is used for a subject of about 75 kg. For example, about 200 mgs to about 3 grams, or more of the PPX can be administered to the subject. A dose of 1 gram corresponds to about 13.3 mgs / kg body weight, and the dosing can be scaled appropriately depending on the size of the subject. Thus, in some embodiments, a dose of about 6.7 mg / kg, 13.3 mg / kg, or 33.3 mg / kg or more can be used.

[0097] The PPX can be administered at once, or the dose can be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment will be a function of the location of where the PPX or a composition comprising same is administered, the carrier and other variables that can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values can also vary with the age of the individual treated. It is to be further understood that for any particular subject, specific dosage regimens can need to be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations. Hence, the concentration ranges set forth herein are intended to be exemplary and are not intended to limit the scope or practice of the claimed formulations.

[0098] The PPX or a composition comprising same can be administered as a single bolus or multiple boluses, as a continuous infusion, or a combination thereof. For example, the PPX or a composition comprising same can be administered as a single bolus initially, and then administered as a continuous infusion following the bolus. The rate of the infusion can be any rate sufficient to maintain effective concentration, for example, to maintain effective plasma concentration. Some contemplated infusion rates include from 1 µg / kg / min to 100 mg / kg / min, or from 1 µg / kg / hr to 1000 mg / kg / hr. Rates of infusion can include 0.2 to 1.5 mg / kg / min, or more specifically 0.25 to 1 mg / kg / min, or even more specifically 0.25 to 0.5 mg / kg / min. It will be appreciated that the rate of infusion can be determined based upon the dose necessaryAttorney Docket No.701039-000139WOPT to maintain effective plasma concentration and the rate of elimination of the compound, such that the compound is administered via infusion at a rate sufficient to safely maintain a sufficient effective plasma concentration of compound in the bloodstream.

[0099] As used herein, “sensory block” refers to a decrease in sensitivity to sensory stimuli in a subject. Without wishing to be bound by a theory, “sensory block” may include blocking of transmission of impulses to the center by nerve endings in a sensory site; thereby, resulting in loss of perception. The term “sensory block” includes loss of pain perception. For example, sensory block includes a decrease in pain in a subject.

[0100] As used herein, “sensory block duration” refers to an amount of time during which sensory block occurs in a subject. For example, sensory block duration may refer to an amount of time in which the subject experiences a decrease in sensation or pain in a range from about 1% to 99%, from about 5% to about 95%, from about 10% to about 90%, from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 60%, and / or about 50%.

[0101] As used herein, “motor block” refers to a decrease in motor function of a subject (i.e. a decrease in the ability or power of a subject to move). Without wishing to be bound by a theory, “motor block” may include blocking of transmission of nerve impulses to the surroundings; thereby, impairing motor function and limiting activity, e.g., movement is blocked. For example, motor block includes inhibiting a subject from being able to move their legs.

[0102] As used herein, “motor block duration” refers to an amount of time during which motor block occurs in a subject. For example, sensory block duration may refer to an amount of time in which the subject experiences a decrease in motor function in a range from about 1% to 99%, from about 5% to about 95%, from about 10% to about 90%, from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 60%, and / or about 50%.

[0103] As used herein, “anesthesia” refers to a loss of feeling or inability to feel pain. Anesthesia can include mitigation of pain, alteration of consciousness, and / or sedation. Generally, anesthesia is a temporary state with one or more of the following characteristics: analgesia (relief from or prevention of pain), immobility, paralysis (extreme muscle relaxation), amnesia (loss of memory), unconsciousness, and sedation. As used herein, the term “sedation” refers to calming through effects on the central nervous system (CNS), and it includes tranquillization, immobilization, muscle relaxation and analgesia.

[0104] As used herein, “local anesthesia” refers to reversible absence of pain sensation in a target area of the body. The term “local anesthesia” includes all anesthesia of afferent and efferent nerves and includes nerve blocks, infiltration anesthesia, dermal anesthesia, and topicalAttorney Docket No.701039-000139WOPT anesthesia. The term “nerve block” as used herein refers to the total or partial blockade of nerve transmission. The term “infiltration anesthesia” refers to anesthesia of thin nerve fibers and nerve endings by infiltrating the tissues containing such nerves or nerve endings with PPX or a composition comprising PPX. The term “dermal anesthesia” refers to treatment of nociceptive or neuropathic pain in the skin. The term “topical anesthesia” means local anesthesia of mucosal membranes, such as for examples those of the eye, the ear, the mouth, the nose, the rectal area and the urogenital tract.

[0105] “Hydrogel” described herein means a hydrophilic or amphiphilic polymeric network composed of homopolymers or copolymers, which is insoluble due to the presence of covalent chemical crosslinks. The crosslinks provide the network structure and physical integrity. Hydrogels exhibit a thermodynamic compatibility with water which allows them to swell in aqueous media. Generally, a hydrogel is a three-dimensional polymeric structure that is insoluble in water or some other liquid but which is capable of absorbing and retaining large quantities of water or some other liquid to form a stable, often soft and pliable, structure. In some embodiments, water or some other liquid can penetrate in between the polymer chains of the polymer network, subsequently causing swelling and the formation of a hydrogel. Methods of Inducing Anesthesia

[0106] In some embodiments, the method of inducing anesthesia, e.g., local anesthesia or systemic anesthesia in a subject includes administering to the subject in need thereof 2’,6’- pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia, e.g., local anesthesia or systemic anesthesia in the subject. An active molecule that is metabolized to PPX in the subject is not administered to the subject with the PPX.

[0107] In some embodiments, the PPX is in a pharmaceutical composition. The pharmaceutical composition includes the PPX and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition does not include a parent molecule of the PPX.

[0108] In some embodiments, the pharmaceutical composition comprises PPX and a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition is substantially free of a parent molecule of the PPX.

[0109] In some embodiments, the pharmaceutical composition comprises PPX and a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition is substantially free of a molecule that can be metabolized to PPX after administering to a subject.Attorney Docket No.701039-000139WOPT

[0110] In some embodiments, the pharmaceutical composition comprises PPX and a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition is substantially free of a molecule that can be metabolized to 3’-hodroxy-2’,6’-pipecoloxylidide after administering to a subject.

[0111] In some embodiments, the pharmaceutical composition comprises PPX and a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition is substantially free of ropivacaine, bupivacaine, levobupivacaine, mepivacaine, or a pharmaceutically acceptable salt, or an analog or derivative thereof.

[0112] In some embodiments, a method of inducing anesthesia, e.g., local anesthesia or systemic anesthesia in a subject includes administering to the subject in need thereof an effective amount of a pharmaceutical composition. The pharmaceutical composition includes: (i) 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof; and (ii) an active molecule capable of inducing anesthesia in the subject and being metabolized to PPX in the subject. The pharmaceutical composition includes the PPX in an amount that is at least about 5% or more of the amount of the active molecule in the composition.

[0113] In some embodiments, the pharmaceutical composition includes the PPX in an amount from about 0.001% to about 99%, from about 0.01% to about 10%, from about 0.1% to about 2%, from about 0.2% to about 0.7%, and / or about 0.5% by weight of the pharmaceutical composition. In some embodiments, the pharmaceutical composition includes the PPX in an amount from about 0.001% to about 99%, from about 0.01% to about 10%, from about 0.1% to about 2%, from about 0.2% to about 0.7%, and / or about 0.5% weight by volume of the pharmaceutical composition. In some embodiments, the pharmaceutical composition also includes epinephrine and / or dexamethasone.

[0114] In some embodiments, the pharmaceutical composition includes saline and / or any other suitable solution. For example, and in some embodiments, the solution is chosen based on baricity.

[0115] In some embodiments, the PPX in the pharmaceutical composition is in a concentration from about 5 mmol to about 100 mmol, from about 10 mmol to about 75 mmol, from about 20 mmol to about 50 mmol, or from about 22.5 mmol to about 30 mmol.

[0116] In some embodiments, the PPX is administered in an amount from about 0.1 mg / kg to about 100 mg / kg, from about 0.5 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 7 mg / kg, from about 2 mg / kg to about 5 mg / kg, or from about 3 mg / kg to about 4 mg / kg of the subject.Attorney Docket No.701039-000139WOPT

[0117] In some embodiments, the pharmaceutical composition is formulated for local administration. In some other embodiments, the pharmaceutical composition is formulated for systemic administration.

[0118] In some embodiments, the pharmaceutical composition is formulated for administration via injection and / or sustained release. In some embodiments, the pharmaceutical composition includes liposomes, nanoparticles, and / or hydrogels.

[0119] In some embodiments, the pharmaceutical composition is formulated for oral delivery.

[0120] In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0121] In some embodiments, the pharmaceutical composition is in form of a capsule, pill, tablet, aqueous or non-aqueous solution or suspension, gavage, lozenges, dragees, boluses, powder, granule, or a paste.

[0122] In some embodiments, the administering occurs repeatedly at fixed and / or varied time intervals. In some embodiments, the administering occurs continuously over a period of time, for example, to maintain and / or increase an anesthetic effect as the PPX wears off.

[0123] In some embodiments, a ratio of sensory block duration to motor block duration (S:M) caused by the administration of PPX is at least about 1.25, at least about 5, at least about 10, at least about 100, at least about 1,000, at least about 10,000, at least about 50,000, at least about 100,000, at least about 1,000,000, or at least about 10,000,000.

[0124] In some embodiments, the PPX selectively induces sensory block and does not induce motor block. For example, and in some embodiments, the PPX is administered in an amount that is less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the amount of PPX capable of inducing motor block.

[0125] In some embodiments, the PPX, the pharmaceutically acceptable salt, and / or the pharmaceutical composition is administered in an amount that is less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of the amount of a toxic amount of PPX, pharmaceutically acceptable salt, and / or pharmaceutical composition.Attorney Docket No.701039-000139WOPT

[0126] Administration can be done in a variety of ways. For example, and in some embodiments, administering is done intravenously, intrathecally, intraperitoneally, epidurally, orally, via suppository, via inhalation, via absorption, enterally, intramuscularly, subcutaneously, and / or any other suitable method of administration. In some embodiments, administration is via any suitable drug delivery vehicle. For example, and administration is via particulates, liposomes, formulations composed of synthetic hydrophobic polymers, lipid- protein-sugar particles, lipospheres, hydrogels, cyclodextrins, injectable liquid polymers, injectable lipid matrix, hybrid formulations, polymeric particles, polymeric micro and / or nanoparticles, polymeric microparticle hydrogel, liposome-hydrogel, macroscopic systems, covalent modification, triggered systems, pharmacological approaches, and / or calcium phosphate apatite matrix, as described in McAlvin, J. Brian, and Daniel S. Kohane. "Prolonged duration local anesthesia." Focal Controlled Drug Delivery (2013): 653-677.; C.M. Santamaria, et al., Mater. Today (2016), dx.doi.org / 10.1016 / j.mattod.2016.11.019; and Y. Li, G. E. Owens, D. S. Kohane, ChemMedChem 2023, 18, e202300009, which are herein incorporated by reference.

[0127] In some embodiments, the subject is a human. However, this is not necessary, and in some embodiments, the subject is an animal such as a pig, cow, horse, rabbit, dog, or cat. In some embodiments, the subject is in labor and / or giving birth. Pharmaceutical Compositions

[0128] For administration to a subject, the PPX can be formulated into pharmaceutically acceptable compositions / formulations. These pharmaceutically acceptable compositions comprise PPX, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. As described in detail below, the pharmaceutical compositions described herein can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), gavages, lozenges, dragees, capsules, pills, tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, for example, as a pessary, cream or foam; (4) sublingually; (5) transdermally; (6) transmucosally; or (7) nasally. Additionally, compounds can be implanted into a patient or injected using a drug delivery system. See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-Attorney Docket No.701039-000139WOPT 236 (1984); Lewis, ed. “Controlled Release of Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No.3,773,919; and U.S. Pat. No.353,270,960, content of all of which is herein incorporated by reference.

[0129] As used here, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0130] As used here, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.Attorney Docket No.701039-000139WOPT

[0131] Examples of solid carriers include starch, sugar, bentonite, silica, and other commonly used carriers. Further non-limiting examples of carriers and diluents which can be used in the formulations comprising PPX include saline, syrup, dextrose, and water.

[0132] Pharmaceutically acceptable antioxidants include, but are not limited to, (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lectithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acids, and the like.

[0133] The PPX can be formulated in a gelatin capsule, in tablet form, dragee, syrup, suspension, topical cream, suppository, injectable solution, or kits for the preparation of syrups, suspension, topical cream, suppository or injectable solution just prior to use. Also, PPX can be included in composites, which facilitate its slow release into the blood stream, e.g., silicon disc, polymer beads.

[0134] The formulations can conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques, excipients and formulations generally are found in, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1985, 17th edition, Nema et al., PDA J. Pharm. Sci. Tech. 1997 51:166-171. Methods to make invention formulations include the step of bringing into association or contacting PPX with one or more excipients or carriers. In general, the formulations are prepared by uniformly and intimately bringing into association PPX with liquid excipients or finely divided solid excipients or both, and then, if appropriate, shaping the product.

[0135] The preparative procedure may include the sterilization of the pharmaceutical preparations. The PPX may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, salts for influencing osmotic pressure, etc., which do not react deleteriously with the compounds.

[0136] Examples of injectable form include solutions, suspensions and emulsions. Injectable forms also include sterile powders for extemporaneous preparation of injectable solutions, suspensions or emulsions. The PPX can be injected in association with a pharmaceutical carrier such as normal saline, physiological saline, bacteriostatic water, CremophorTMEL (BASF, Parsippany, N.J.), phosphate buffered saline (PBS), Ringer's solution, dextrose solution, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof, and other aqueous carriersAttorney Docket No.701039-000139WOPT known in the art. Appropriate non-aqueous carriers may also be used and examples include fixed oils and ethyl oleate. In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. A suitable carrier is 5% dextrose in saline. Frequently, it is desirable to include additives in the carrier such as buffers and preservatives or other substances to enhance isotonicity and chemical stability.

[0137] In some embodiments, PPX can be administrated encapsulated within liposomes. The manufacture of such liposomes and insertion of molecules into such liposomes being well known in the art, for example, as described in US Pat. No.4,522,811. Liposomal suspensions (including liposomes targeted to particular cells, e.g., a pituitary cell) can also be used as pharmaceutically acceptable carriers.

[0138] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the composition can be administered in a sustained release formulation.Attorney Docket No.701039-000139WOPT

[0139] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0140] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient, e.g., PPX) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0141] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185; content of each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS®(Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.

[0142] In some embodiments, the PPX is prepared with carriers that will protect the PPX against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,Attorney Docket No.701039-000139WOPT polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.

[0143] In the case of oral ingestion, excipients useful for solid preparations for oral administration are those generally used in the art, and the useful examples are excipients such as lactose, sucrose, sodium chloride, starches, calcium carbonate, kaolin, crystalline cellulose, methyl cellulose, glycerin, sodium alginate, gum arabic and the like, binders such as polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, ethyl cellulose, gum arabic, shellac, sucrose, water, ethanol, propanol, carboxymethyl cellulose, potassium phosphate and the like, lubricants such as magnesium stearate, talc and the like, and further include additives such as usual known coloring agents, disintegrators such as alginic acid and PrimogelTM, and the like. The PPX can be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the PPX may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% of compound. The percentage of the agent in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit. The amount of the PPX in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions according to the present invention are prepared so that an oral dosage unit contains between about 100 and 2000 mg of the PPX. Examples of bases useful for formulation of suppositories are oleaginous bases such as cacao butter, polyethylene glycol, lanolin, fatty acid triglycerides, witepsol (trademark, Dynamite Nobel Co. Ltd.) and the like. Liquid preparations may be in the form of aqueous or oleaginous suspension, solution, syrup, elixir and the like, which can be prepared by a conventional way using additives. The compositions can be given as a bolus dose, to maximize the circulating levels for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.

[0144] The PPX can also be administered parenterally. Solutions or suspensions of the PPX can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, areAttorney Docket No.701039-000139WOPT preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0145] It may be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, “dosage unit” refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the PPX calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0146] Administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0147] For oral or enteral formulations as disclosed herein for use with the present invention, tablets can be formulated in accordance with conventional procedures employing solid carriers well-known in the art. Capsules employed for oral formulations to be used with the methods of the present invention can be made from any pharmaceutically acceptable material, such as gelatin or cellulose derivatives. Sustained release oral delivery systems and / or enteric coatings for orally administered dosage forms are also contemplated, such as those described in U.S. Pat. No. 4,704,295, “Enteric Film-Coating Compositions,” issued Nov. 3, 1987; U.S. Pat. No. 4, 556,552, “Enteric Film- Coating Compositions,” issued Dec. 3, 1985; U.S. Pat. No. 4,309,404, “Sustained Release Pharmaceutical Compositions,” issued Jan. 5, 1982; and U.S. Pat. No.4,309,406, “Sustained Release Pharmaceutical Compositions,” issued Jan.5, 1982.

[0148] As regards formulations for administering PPX, one particularly useful embodiment is a tablet formulation comprising a compound of PPX with an enteric polymer casing. An example of such a preparation can be found in WO2005 / 021002. The active material in the core can be present in a micronised or solubilised form. In addition to active materials the core can contain additives conventional to the art of compressed tablets. Appropriate additives in such a tablet can comprise diluents such as anhydrous lactose, lactose monohydrate, calcium carbonate, magnesium carbonate, dicalcium phosphate or mixtures thereof; binders such as microcrystalline cellulose, hydroxypropylmethylcellulose, hydroxypropyl-cellulose,Attorney Docket No.701039-000139WOPT polyvinylpyrrolidone, pre-gelatinised starch or gum acacia or mixtures thereof; disintegrants such as microcrystalline cellulose (fulfilling both binder and disintegrant functions) cross- linked polyvinylpyrrolidone, sodium starch glycollate, croscarmellose sodium or mixtures thereof; lubricants, such as magnesium stearate or stearic acid, glidants or flow aids, such as colloidal silica, talc or starch, and stabilisers such as desiccating amorphous silica, colouring agents, flavours etc. Preferably the tablet comprises lactose as diluent. When a binder is present, it is preferably hydroxypropylmethyl cellulose. Preferably, the tablet comprises magnesium stearate as lubricant. Preferably the tablet comprises croscarmellose sodium as disintegrant. Preferably, the tablet comprises microcrystalline cellulose.

[0149] The diluent can be present in a range of 10 – 80% by weight of the core. The lubricant can be present in a range of 0.25 – 2% by weight of the core. The disintegrant can be present in a range of 1 – 10% by weight of the core. Microcrystalline cellulose, if present, can be present in a range of 10 – 80% by weight of the core.

[0150] The active ingredient, e.g., PPX preferably comprises between 10 and 50% of the weight of the core, more preferably between 15 and 35% of the weight of the core (calculated as free base equivalent). The core can contain any therapeutically suitable dosage level of the active ingredient, but preferably contains up to 150mg of the active ingredient. Particularly preferably, the core contains 20, 30, 40, 50, 60, 80 or 100mg of the active ingredient. The active ingredient can be present as is or as any pharmaceutically acceptable salt. If the active ingredient is present as a salt, the weight is adjusted such that the tablet contains the desired amount of active ingredient, calculated as free base or free acid of the salt.

[0151] The core can be made from a compacted mixture of its components. The components can be directly compressed, or can be granulated before compression. Such granules can be formed by a conventional granulating process as known in the art. In an alternative embodiment, the granules can be individually coated with an enteric casing, and then enclosed in a standard capsule casing.

[0152] The core is surrounded by a casing which comprises an enteric polymer. Examples of enteric polymers are cellulose acetate phthalate, cellulose acetate succinate, methylcellulose phthalate, ethylhydroxycellulose phthalate, polyvinylacetate pthalate, polyvinylbutyrate acetate, vinyl acetate-maleic anhydride copolymer, styrene-maleic mono-ester copolymer, methyl acrylate-methacrylic acid copolymer or methacrylate-methacrylic acid-octyl acrylate copolymer. These can be used either alone or in combination, or together with other polymers than those mentioned above. The casing can also include insoluble substances which are neither decomposed nor solubilised in living bodies, such as alkyl cellulose derivatives such asAttorney Docket No.701039-000139WOPT ethyl cellulose, crosslinked polymers such as styrene-divinylbenzene copolymer, polysaccharides having hydroxyl groups such as dextran, cellulose derivatives which are treated with bifunctional crosslinking agents such as epichlorohydrin, dichlorohydrin or 1, 2-, 3, 4-diepoxybutane. The casing can also include starch and / or dextrin.

[0153] In some embodiments, an enteric coating materials are the commercially available Eudragit^ enteric polymers such as Eudragit^ L, Eudragit^ S and Eudragit^ NE used alone or with a plasticiser. Such coatings are normally applied using a liquid medium, and the nature of the plasticiser depends upon whether the medium is aqueous or non-aqueous. Plasticisers for use with aqueous medium include propylene glycol, triethyl citrate, acetyl triethyl citrate or Citroflex^ or Citroflex^ A2. Non-aqueous plasticisers include these, and also diethyl and dibutyl phthalate and dibutyl sebacate. A preferred plasticiser is Triethyl citrate. The quantity of plasticiser included will be apparent to those skilled in the art.

[0154] The casing can also include an anti-tack agent such as talc, silica or glyceryl monostearate. Preferably the anti-tack agent is glyceryl monostearate. Typically, the casing can include around 5 – 25 wt% Plasticizers and up to around 50 wt % of anti-tack agent, preferably 1-10 wt % of anti-tack agent.

[0155] If desired, a surfactant can be included to aid with forming an aqueous suspension of the polymer. Many examples of possible surfactants are known to the person skilled in the art. Preferred examples of surfactants are polysorbate 80, polysorbate 20, or sodium lauryl sulphate. If present, a surfactant can form 0.1 – 10% of the casing, preferably 0.2 – 5% and particularly preferably 0.5 – 2%.

[0156] A seal coat can also be included between the core and the enteric coating. A seal coat is a coating material which can be used to protect the enteric casing from possible chemical attack by any alkaline ingredients in the core. The seal coat can also provide a smoother surface, thereby allowing easier attachment of the enteric casing. A person skilled in the art would be aware of suitable coatings. Preferably the seal coat is made of an Opadry coating, and particularly preferably it is Opadry White OY-S-28876. Other enteric-coated preparations of this sort can be prepared by one skilled in the art, using these materials or their equivalents. Aqueous formulations

[0157] In some embodiments, PPX is typically administered by injection or infusion. Accordingly, in some embodiments, the PPX is formulated in a liquid or aqueousAttorney Docket No.701039-000139WOPT pharmaceutical formulation e.g., for intramuscular (IM), intraperitoneal (IP), or subcutaneous (SQ or Sub-Q) administration.

[0158] Generally, the aqueous formulation comprises the PPX and a buffer or a buffering agent. Some exemplary buffers include, but are not limited to, sodium phosphate buffer, potassium phosphate buffer, acetate buffer, TRIS buffer, HEPES buffer, 2-(N- morpholino)ethanesulfonic acid (MES) buffer, cacodylate buffer, hydrochloride buffer, arginine buffer, glycine buffer, citrate buffer, histidine buffer, sodium succinate buffer, and TES buffer. It is noted that a buffering agent in the buffer can be present in any amount suitable to maintain the pH of the aqueous formulation at a predetermined level. In some embodiments, the pH buffering concentration is about 0.1 mM to about 1000 mM, e.g., about 0.5 mM to about 500 mM. For example, the amount of the buffering agent in the aqueous formulation is at least about 0.1 mM, about 0.5 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.2 mM, about 1.5 mM, about 1.7 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, about 200 mM, about 205 mM, about 210 mM, about 215 mM, about 220 mM, about 225 mM, about 230 mM, about 235 mM, about 240 mM, about 245 mM, about 250 mM, about 255 mM, about 260 mM, about 265 mM, about 270 mM, about 275 mM, about 280 mM, about 285 mM, about 290 mM, about 295 mM, about 300 mM, about 305 mM, about 310 mM, about 315 mM, about 320 mM, about 325 mM, about 330 mM, about 335 mM, about 340 mM, about 345 mM, about 3150 mM, about 355 mM, about 360 mM, about 365 mM, about 370 mM, about 375 mM, about 380 mM, about 385 mM, about 390 mM, about 395 mM, about 400 mM, about 405 mM, about 410 mM, about 415 mM, about 420 mM, about 425 mM, about 430 mM, about 435 mM, about 440 mM, about 445 mM, about 4150 mM, about 455 mM, about 460 mM, about 465 mM, about 470 mM, about 475 mM, about 480 mM, about 485 mM, about 490 mM, about 495 mM, or about 500 mM.

[0159] Typically, the pharmaceutical formulation has a pH of about 5 to about 8. For example, the aqueous pharmaceutical formulation has a pH of about 5.5 to about 8, about 6 toAttorney Docket No.701039-000139WOPT about 8, about 6.5 to about 8, about 6.5 to about 7.5, about 7 to about 8. In some embodiments, the aqueous formulation has a pH of about 7.2 to about 7.6, or about 7.4. In some embodiments, the aqueous formulation has a pH of about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8 or about 7.9. In some embodiments, the aqueous formulation has a pH of about 7.2 to about 7.6, or about 7.4. In some embodiments, the aqueous formulation has a pH of about 5.5 to about 6.5. For example, the aqueous formulation has a pH of about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5.

[0160] In some embodiments, the aqueous formulation has a pH of about 6.5 to about 7.5. For example, the aqueous formulation has a pH of about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5.

[0161] A stabilizer can be added to the aqueous formulation to prevent or reduce storage- induced aggregation and chemical degradation. As used herein, the term “stabilizer” means an excipient capable of preventing aggregation or physical degradation, including chemical degradation (for example, autolysis, deamidation, oxidation, etc.) in an aqueous state. Some exemplary stabilizers include, but are not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCL, poly-hydroxy compounds, including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrins, N-methyl pyrollidene, cellulose and hyaluronic acid, sodium chloride. See, e.g., Carpenter et al., Develop. Biol. Standard 74:225, (1991). The aqueous formulation can comprise a stabilizer in an amount from about 0.1 mM to about 500 mM, e.g., about 0.5 mM to about 250 mM. For example, the amount of the stabilizer in the aqueous formulation can be at least about 0.1 mM, about 0.5 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.2 mM, about 1.5 mM, about 1.7 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, about 200 mM, about 205 mM, about 210 mM, about 215 mM, about 220 mM, about 225 mM, about 230 mM,Attorney Docket No.701039-000139WOPT about 235 mM, about 240 mM, about 245 mM, about 250 mM, about 255 mM, about 260 mM, about 265 mM, about 270 mM, about 275 mM, about 280 mM, about 285 mM, about 290 mM, about 295 mM, about 300 mM, about 305 mM, about 310 mM, about 315 mM, about 320 mM, about 325 mM, about 330 mM, about 335 mM, about 340 mM, about 345 mM, about 3150 mM, about 355 mM, about 360 mM, about 365 mM, about 370 mM, about 375 mM, about 380 mM, about 385 mM, about 390 mM, about 395 mM, about 400 mM, about 405 mM, about 410 mM, about 415 mM, about 420 mM, about 425 mM, about 430 mM, about 435 mM, about 440 mM, about 445 mM, about 4150 mM, about 455 mM, about 460 mM, about 465 mM, about 470 mM, about 475 mM, about 480 mM, about 485 mM, about 490 mM, about 495 mM, or about 500 mM.

[0162] In some embodiments, the aqueous formulation can comprise a viscosity modifier. Some exemplary viscosity modifiers include, but are not limited to, arginine, lysine, histidine, polyvinyl alcohol, polyalkyl cellulose, hydroxyalkyl cellulose (e.g., hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropyl methyl cellulose), carbopol, glycerin, polyethylene glycol, glucose, dextrose, and sucrose. In some embodiments, the viscosity modifier is arginine, lysine or histidine. In some embodiments, the viscosity modifier is arginine. In some embodiments, the viscosity modifier comprises a salt form, for example a salt of arginine, lysine or histidine. In some embodiments, the viscosity modifier is an amino acid, e.g., an L- form amino acid such as L-arginine, L-lysine, or L-histidine. In some embodiments, the viscosity modifier is in a concentration of about 50 mM to about 400 mM, or about 100 mM to about 250 mM. In some embodiments, the viscosity modifier is in a concentration of about 190 mM. In some embodiments, the viscosity modifier is arginine in a concentration of about 100 mM to about 250 mM. In some embodiments, the viscosity modifier is in a concentration of about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, about 200 mM, about 205 mM, about 210 mM, about 215 mM, about 220 mM, about 225 mM, about 230 mM, about 235 mM, about 240 mM, about 245 mM, about 250 mM, about 255 mM, about 260 mM, about 265 mM, about 270 mM, about 275 mM, about 280 mM, about 285 mM, about 290 mM, about 295 mM, about 300 mM, about 305 mM, about 310 mM, about 315 mM, about 320 mM, about 325 mM, about 330 mM, about 335 mM, about 340 mM, about 345 mM,Attorney Docket No.701039-000139WOPT about 3150 mM, about 355 mM, about 360 mM, about 365 mM, about 370 mM, about 375 mM, about 380 mM, about 385 mM, about 390 mM, about 395 mM, or about 400 mM.

[0163] Generally, the viscosity modifier is present in the aqueous formulation in an amount to obtain a viscosity of less than about 40 cP, less than about 30 cP, less than about 25 cP, or less than about 20 cP at ambient temperature, such as at a temperature between 20°C and 30°C, e.g., at temperature of about 21oC, about 22oC, about 23oC, about 24oC, about 25oC, about 26oC, about 27oC, about 28oC, or about 28oC, preferably at a temperature of about 22oC, about 23oC, about 24oC, about 25oC, about 26oC, or about 27oC, and more preferably at a temperature of about 22oC, about 23oC, or about 24oC. In some embodiments, viscosity modifier is added in an amount to obtain a viscosity of about 1 cP to about 40 cP, or about 2 cP to about 30 cP, about 5 cP to about 25 cP, or about 10 cP to about 20 cP, or about 1 cP to about 20 cP, or about 1 cP to about 15 cP, or about 1 cP to about 10 cP, or about 1 cP to about 8 cP, or about 1 cP to about 6 cP at ambient temperature, such as at a temperature between 20°C and 30°C, e.g., at temperature of about 21oC, about 22oC, about 23oC, about 24oC, about 25oC, about 26oC, about 27oC, about 28oC, or about 28oC, preferably at a temperature of about 22oC, about 23oC, about 24oC, about 25oC, about 26oC, or about 27oC, and more preferably at a temperature of about 22oC, about 23oC, or about 24oC. Viscosity measurements can be done by a cone and plate technique with a Peltier element set at a defined temperature, such ambient temperature, such as at a temperature between 20°C and 30°C, e.g., at temperature of about 21oC, about 22oC, about 23oC, about 24oC, about 25oC, about 26oC, about 27oC, about 28oC, or about 28oC, preferably at a temperature of about 22oC, about 23oC, about 24oC, about 25oC, about 26oC, or about 27oC, and more preferably at a temperature of about 22oC, about 23oC, or about 24oC

[0164] The aqueous formulation can also include a surfactant. Various surfactants are known to those in the art. In some embodiments, the surfactant is a non-ionic surfactant. In some embodiments, the surfactant is a non-ionic surfactant. Exemplary non-ionic surfactants include, but are not limited to, polyoxyethylene fatty alcohol ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkylglucosides, alkyl phenol ethoxylates, preferably polysorbates, polyoxyethylene alkyl phenyl ethers, and any combinations thereof. In some embodiments, the non-ionic surfactant can be selected from the group of Brij 010, BRIJ 020, Brij Cl 0, Brij S20, BrijSl0, ECOSURF EH-14, ECOSURF EH- 9, ECOSURF SA-15, IGEPAL CA-720, IGEPAL CO-630, IGEPAL CO-720, MERPOL HCS surfactant, MERPOL OJ surfactant, MERPOL SH surfactant, nonoxynol-9, polyoxamer, Pluronic 10R5, Pluronic 10R5, Pluronic F-68, Poloxamer P 188, Poloxamer P 338, Poloxamer P188, Poloxamer P407, Polyoxyethylene (12) tridecyl ether, polysorbates (e.g., polysorbate-Attorney Docket No.701039-000139WOPT 20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, or polysorbate-85), sorbitan monostearate, stearyl alcohol, TERGITOL 15-S-12, TERGITOL 15-S-7, TERGITOL 15-S-9, TERGITOL L-64, TERGITOL NP-10, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-9.5, TERGITOL15-S-9, TERGITOLNP-13, TERGITOLNP-7, Triton (e.g., Triton X-100), Tween 80, TWEEN 60, sodium dodecyl sulfate (SDS); sodium laurel sulfate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl- sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine, cocamidopropyl- betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmidopropyl-betaine, isostearamidopropyl-betaine (e.g. lauroamidopropyl), myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine, sodium methyl cocoyl-, or disodium methyl ofeyl- taurate, and the Monaquat™ series (Mona Industries, Inc., Paterson, N.J.). Generally, the amount of the surfactant in the aqueous formulation is such that it reduces aggregation of the reconstituted PPX to an acceptable level as assayed using, e.g., SEC-HPLC of HMW species or LMW species, and minimizes the formation of particulates after reconstitution of a lyophilate of an the PPX formulation. For example, the surfactant can be present in the aqueous formulation in an amount from about 0.001% to about 1% (v / v or w / v), e.g., from about 0.005% to 0.05%, from about 0.005% to about 0.2%, from about 0.01% to 0.2%, from about 0.002% to about 0.4%, from 0.005% to about 0.15%, from about 0.002% to about 0.2%, from about 0.01% to about 0.1%, or from about 0.02% to about 0.08% (v / v or w / v). For example, the surfactant can be present in an amount of about 0.04% (v / v or w / v).

[0165] The aqueous formulation can also include a cryoprotectant. A “cryoprotectant” is a molecule that, when combined with a protein of interest, significantly prevents or reduces the chemical and / or physical instability of a protein upon lyophilization and subsequent storage. Exemplary cryoprotectants include, but are not limited to, sugars such as sucrose, sorbitol, or trehalose; amino acids such as monosodium glutamate or histidine; methylamine such as betaine; lyophilic salts such as magnesium sulfate; polyols such as trihydric or higher sugar alcohols such as glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; Pluronic; and combinations thereof. Typically, the cryoprotectant is a non-reducing sugar such as trehalose or sucrose. The cryoprotectant is added to the aqueous formulation in a “cryoprotecting amount” which means that, following lyophilization of the PPX in the presence of the cryoprotecting amount of the cryoprotectant, the PPX essentially retains its physical and chemical stability and integrity upon lyophilizationAttorney Docket No.701039-000139WOPT and storage. The aqueous formulation can comprise a cryoprotectant in an amount from about 0.1 mM to about 500 mM, e.g., about 0.5 mM to about 250 mM. For example, the amount of the cryoprotectant in the aqueous formulation can be at least about 0.1 mM, about 0.5 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.2 mM, about 1.5 mM, about 1.7 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, about 200 mM, about 205 mM, about 210 mM, about 215 mM, about 220 mM, about 225 mM, about 230 mM, about 235 mM, about 240 mM, about 245 mM, about 250 mM, about 255 mM, about 260 mM, about 265 mM, about 270 mM, about 275 mM, about 280 mM, about 285 mM, about 290 mM, about 295 mM, about 300 mM, about 305 mM, about 310 mM, about 315 mM, about 320 mM, about 325 mM, about 330 mM, about 335 mM, about 340 mM, about 345 mM, about 3150 mM, about 355 mM, about 360 mM, about 365 mM, about 370 mM, about 375 mM, about 380 mM, about 385 mM, about 390 mM, about 395 mM, about 400 mM, about 405 mM, about 410 mM, about 415 mM, about 420 mM, about 425 mM, about 430 mM, about 435 mM, about 440 mM, about 445 mM, about 4150 mM, about 455 mM, about 460 mM, about 465 mM, about 470 mM, about 475 mM, about 480 mM, about 485 mM, about 490 mM, about 495 mM, or about 500 mM.

[0166] The aqueous formulation can also include a preservative. Exemplary preservatives include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antimicrobial agents include, but are not limited to, parabens, isothiazolinone, phenolics, acidic preservatives, halogenated compounds, quarternia, and alcohol. Exemplary parabens can include, but are not limited to, parabens and paraben salts. Exemplary isothiazolinones can include, but are not limited to, methylchloroisothiazolinone, methylisothiazolinone, benzisothiazolinone ProClin 150, ProClin 200, ProClin 300, and ProClin 950. Exemplary phenolic types can include, but are not limited to, phenoxyethanol, benzyl alcohol, and phenethyl alcohol. Exemplary acidicAttorney Docket No.701039-000139WOPT preservatives can include, but are not limited to, dehydroacetic acid, benzoic acid, sorbic acid, salicylic acid, formic acid, propionic acid. Exemplary halogenated compounds can include, but are not limited to, 2-bromo-2-nitropropane-1, 3-diol, chloroacetamide, chlorobutanol, chloroxylenol, chlorphenesin, dichlorobenzyl alcohol, iodopropynyl butylcarbamate, methyldibromo glutaronitrile. Exemplary quaternia can include, but are not limited to, benzalkonium chloride, benzethonium chloride, chlorhexidine, hexamidine diisethionate, and polyaminopropyl biguanide. Exemplary alcohols can include, but are not limited to, ethyl alcohol and isopropyl alcohol. Examples thereof include, but are not limited to, triazine antimicrobial agents, thiazole bactericidal agents (for example, benzisothiazolone etc.), pyrithione, pyridine bactericidal agents (for example, 1-hydroxy pyridine-2-thiosodium etc.), 2-phenoxyethanol, and the like. Exemplary phenolic preservatives include phenoxyethanol, benzyl alcohol and phenethyl alcohol. In some embodiments, the preservative is selected from the group consisting of octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which alkyl groups are long chain compounds), benzethonium chloride, aromatic alcohols (such as phenol, butyl and benzyl alcohol), alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0167] Various additional excipients, such as a salt, e.g., a NaCl, or KCl salt can be present in the aqueous formulation. The salt can be present in the aqueous formulation in a concentration from about 50 mM to about 750 mM. For example, the salt can be present at a concentration from about 75 mM to about 700 mM, from about 100 mM to about 650 mM, from about 120 mM to about 600 mM, from about 140 mM to about 550 mM, or from about 150mM to about 400mM. For example, the salt can be present at a concentration of about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, or about 475 mM.

[0168] The ionic strength of the aqueous formulation can be at least about 100 mM. For example, the ionic strength of the aqueous formulation can be from about 125 mM to about 750 mM, or from about 150 mM to about 500 mM, or from about 175 mM to about 700 mM, from about 200mM to about 600 mM, or from about 225 mM to about 550 mM, or from about 250 mM to about 500 mM, or from about 275 mM to about 450 mM, or from about 300 mM to about 400 mM, or from about 100 mM to about 200. In another example, the ionic strength of the aqueous formulation is at least about 125 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, at least about 225 mM, at least about 250 mM, at least about 275Attorney Docket No.701039-000139WOPT mM, at least about 300 mM, at least about 325 mM, at least about 350 mM, at least about 375 mM, at least about 400 mM, at least about 425 mM, at least about 450 mM, at least about 475 mM or at least about 500 mM. In yet some other non-limiting examples, the ionic strength of the aqueous formulation is less than 100mM, for example about 95mM, about 90mM, about 85mM, about 80mM, about 75mM, about 70mM, about 65mM, about 60mM, about 55mM, about 50mM, or even less.

[0169] Generally, the osmolarity of the aqueous formulation is maintained at near isotonic levels. For example, the osmolarity of the aqueous formulation can be from about 100 mOsm to about 600 mOsm, such as from about 125 mOsm to about 500 mOsm, or, from about 130 mOsm to about 350 mOsm, or, from about 140 mOsm to about 400 mOsm, or, from about 140 mOsm to about 350 mOsm, or from about 200 mOsm to about 400 mOsm, or from about 500 mOsm to about 600 mOsm, or from about 200 mOsm to about 600 mOsm, or from about 300 mOsm to about 600 mOsm, or from about 200 mOsm to about 500 mOsm, or from about 300 mOsm to about 400 mOsm, or from about 150 mOsm to about 350 mOsm, or from about 175 mOsm to about 300 mOsm. In some non-limiting examples, the osmolarity of the aqueous formulation can be from about 300 mOsm to about 375 mOsm, or from about 200 mOsm to about 350 mOsm, or from about 225 mOsm to about 325 mOs, or from about 525 mOsm to about 590 mOsm. In some other non-limiting examples, the osmolarity of the aqueous formulation is about 125 mOsm, about 126 mOsm, about 127 mOsm, about 128 mOsm, about 129 mOsm, about 130 mOsm, about 131 mOsm, about 132 mOsm, about 133 mOsm, about 134 mOsm, about 135 mOsm, about 136 mOsm, about 137 mOsm, about 138 mOsm, about 139 mOsm, about 140 mOsm, about 141 mOsm, about 142 mOsm, about 143 mOsm, about 144 mOsm, about 145 mOsm, about 146 mOsm, about 147 mOsm, about 148 mOsm, about 149 mOsm, about 150 mOsm, about 151 mOsm, about 152 mOsm, about 153 mOsm, about 154 mOsm, about 155 mOsm, about 160 mOsm, about 165 mOsm, about 170 mOsm, about 175 mOsm, about 180 mOsm, about 185 mOsm, about 190 mOsm, about 191mOsm, about 192mOsm, about 193mOsm, about 194mOsm, about 195 mOsm, about 200 mOsm, about 205 mOsm, about 210, mOsm, about 215 mOsm, about 220 mOsm, about 225 mOsm, about 250 mOsm, about 275 mOsm, about 280 mOsm, about 285 mOsm, about 290 mOsm, about 295 mOsm, about 300 mOsm, about 301 mOsm, about 302 mOsm, about 303 mOsm, about 304 mOsm, about 305 mOsm, about 306 mOsm, about 307 mOsm, about 308 mOsm, about 309 mOsm, about 310 mOsm, about 311 mOsm, about 312 mOsm, about 313 mOsm, about 314 mOsm, about 315 mOsm, about 316 mOsm, about 317 mOsm, about 318 mOsm, about 319 mOsm, about 320 mOsm, about 321 mOsm, about 322 mOsm, about 323 mOsm, about 324Attorney Docket No.701039-000139WOPT mOsm, about 325 mOsm, about 326 mOsm, about 327 mOsm, about 328 mOsm, about 329 mOsm, about 330 mOsm, about 331 mOsm, about 332 mOsm, about 333 mOsm, about 334 mOsm, about 335 mOsm, about 336 mOsm, about 337 mOsm, about 338 mOsm, about 339 mOsm, about 340 mOsm, about 341 mOsm, about 342 mOsm, about 343 mOsm, about 344 mOsm, about 345 mOsm, , about 346 mOsm, about 347 mOsm, about 348 mOsm, about 349 mOsm, about 350 mOsm, about 355 mOsm, about 360 mOsm, about 365 mOsm, about 370 mOsm, about 375 mOsm, about 400 mOsm, about 425 mOsm, about 450 mOsm, about 475 mOsm, about 500 mOsm, about 525 mOsm, about 530 mOsm, about 540 mOsm, about 550 mOsm, about 560 mOsm, about 570 mOsm, about 580 mOsm, or about 590 mOsm.

[0170] The aqueous formulation described herein may be suitable for storage for extended periods of time. For example, the PPX formulation described herein is stable upon storage at about 40°C for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 1 year, or at least about 18 months. In another example, the aqueous formulation is stable upon storage at about 40°C for about 2 weeks to about 1 year, about 1 month to about 1 year, about 2 months to about 1 year, or about 3 months to about 1 year. For example, the aqueous formulation described herein is stable upon storage at about 40°C for about 2 weeks to about 6 months, about 1 month to about 6 months, about 2 months to about 6 months, or about 3 months to about 6 months. Delivery device

[0171] The PPX or a composition / formulation comprising same can be present in an administration or delivery device. As used herein, the term "administration device" and “delivery device” mean a device which is designed to deliver a controlled definable or fixed amount of a substance, e.g., PPX or a composition / formulation comprising the PPX to an internal tissue or a mucosal tissue of the mammal. It is noted that the delivery device can have a single dose or multiple doses of the PPX.

[0172] In some embodiments, the delivery device is a pre-filled syringe, an autoinjector, or a large volume infusion device. For example, MyDose™ product from Roche, a single use infusion device that enables the subcutaneous administration of large quantities of liquid medication, may be used as the administration device. Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of PPX. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf,Attorney Docket No.701039-000139WOPT Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of PPX include, but are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), YPSOMATE™, YPSOMATE 2.25™, VAIROJECT™ (Ypsomed AG, Burgdorf, Switzerland) to name only a few. Examples of large-volume delivery devices (e.g., large-volume injectors) include, but are not limited to, bolus injectors such as, e.g., BD Libertas West SmartDose, Enable Injections, SteadyMed PatchPump, Sensile SenseTrial, YPsomed YpsoDose, Bespak Lapas, and the like Additional information relating to example delivery devices that could be used with PPX are described, for example, in WO2009 / 040602, WO2016 / 169748, WO2016 / 179713, contents of all which are incorporated herein by reference in their entireties.

[0173] In some embodiments, the pharmaceutical composition capable of inducing anesthesia in a subject includes 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce local anesthesia in a subject; and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition does not include an active molecule that is metabolized to PPX in the subject.

[0174] In some embodiments, the pharmaceutical composition capable of inducing anesthesia in a subject, includes 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce local anesthesia in a subject; an active molecule capable of inducing anesthesia in the subject and being metabolized to PPX in the subject; and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition includes PPX in an amount that is at least about 5% or more of the amount of the active molecule in the composition.

[0175] In some embodiments, the pharmaceutical composition includes the PPX in an amount from about 0.001% to about 99%, from about 0.01% to about 10%, from about 0.1% to about 2%, from about 0.2% to about 0.7%, and / or about 0.5% weight by weight of the pharmaceutical composition. In some embodiments, the pharmaceutical composition includes the PPX in an amount from about 0.001% to about 99%, from about 0.01% to about 10%, fromAttorney Docket No.701039-000139WOPT about 0.1% to about 2%, from about 0.2% to about 0.7%, and / or about 0.5% weight by volume of the pharmaceutical composition. In some embodiments, the pharmaceutical composition also includes epinephrine and / or dexamethasone.

[0176] In some embodiments, the pharmaceutical composition includes saline and / or any other suitable solution. For example, and in some embodiments, the solution is chosen based on baricity.

[0177] Baricity is the ratio of a substance's density to the density of cerebrospinal fluid (CSF). It is known in the art that CSF comprises a baricity, or density of solution, of approximately 1 g / mL at 37°C. Accordingly, in some embodiments, the pharmaceutical composition is an isobaric and wherein the baricity of the composition at 37°C is about 1 g / mL. In some embodiments, the pharmaceutical composition is hypobaric and wherein the baricity of the composition at 37°C is less than 1 g / mL. In some embodiments, the pharmaceutical composition is hyperbaric composition and wherein the baricity of the composition at 37°C is greater than 1 g / mL.

[0178] In some embodiments, the PPX in the pharmaceutical composition is in a concentration from about 5 mmol to about 100 mmol, from about 10 mmol to about 75 mmol, from about 20 mmol to about 50 mmol, or from about 22.5 mmol to about 30 mmol.

[0179] In some embodiments, the PPX in the pharmaceutical composition is in an amount from about 0.1 mg / kg to about 100 mg / kg, from about 0.5 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 7 mg / kg, from about 2 mg / kg to about 5 mg / kg, or from about 3 mg / kg to about 4 mg / kg of the subject.

[0180] In some embodiments, the pharmaceutical composition is formulated for administration via injection and / or sustained release. In some embodiments, the pharmaceutical composition includes nanoparticles (e.g., lipid nanoparticles, such as liposomes), and / or hydrogels.

[0181] In some embodiments, a ratio of sensory block duration to motor block duration (S:M) caused by administration of the pharmaceutical composition to the subject is at least about 1.25, at least about 5, at least about 10, at least about 100, at least about 1,000, at least about 10,000, at least about 50,000, at least about 100,000, at least about 1,000,000, or at least about 10,000,000.

[0182] In some embodiments, the pharmaceutical composition selectively induces sensory block and does not induce motor block. For example, and in some embodiments, the pharmaceutical composition includes PPX in an amount that is less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than aboutAttorney Docket No.701039-000139WOPT 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the amount of PPX capable of inducing motor block.

[0183] In some embodiments, an amount of the PPX, or the pharmaceutically acceptable salt thereof, in the pharmaceutical composition is less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of a toxic amount of PPX, or pharmaceutically acceptable salt thereof.

[0184] In some embodiments, the pharmaceutical composition is formulated for intravenous, intrathecal, intraperitoneal, epidural, oral, suppository, inhalation, absorption, enteral, intramuscular, subcutaneous, and / or any other suitable form of administration. The pharmaceutical composition may be administered via any suitable drug delivery vehicle. For example, and in some embodiments, the pharmaceutical composition is administered via particulates, liposomes, formulations composed of synthetic hydrophobic polymers, lipid- protein-sugar particles, lipospheres, hydrogels, cyclodextrins, injectable liquid polymers, injectable lipid matrix, hybrid formulations, polymeric particles, polymeric micro and / or nanoparticles, polymeric microparticle hydrogel, liposome-hydrogel, macroscopic systems, covalent modification, triggered systems, pharmacological approaches, and / or calcium phosphate apatite matrix, as described in McAlvin, J. Brian, and Daniel S. Kohane. "Prolonged duration local anesthesia." Focal Controlled Drug Delivery (2013): 653-677.; C.M. Santamaria, et al., Mater. Today (2016), dx.doi.org / 10.1016 / j.mattod.2016.11.019; and Y. Li, G. E. Owens, D. S. Kohane, ChemMedChem 2023, 18, e202300009, which are herein incorporated by reference. Lipid nanoparticles

[0185] The PPX can be comprised in or formulated into lipid nanoparticles. As used herein, the term “lipid nanoparticle” or “LNP” refers to particles that contain one or more types of lipid molecules. LNPs can have a diameter in the range from about 1 nm to about 500 nm, e.g., from about 5 nm to about 475 nm, from about 10 nm to about 450 nm, from about 15 nm to about 425 nm, from about 20 nm to about 400 nm, or from about 30 nm to about 300 nm. LNPs include, but are not limited to, liposomes, micelles and lipid aggregates.

[0186] As used herein, the term “nanoparticle” refers to particles that are on the order of 10-9 or one billionth of a meter and below 10-6 or 1 millionth of a meter in size. The term “nanoparticle” includes nanospheres; nanorods; nanoshells; and nanoprisms; and theseAttorney Docket No.701039-000139WOPT nanoparticles may be part of a nanonetwork. The nanoparticle can be a regular or irregular shape. For example, the nanoparticle can be a spheroid, hollow spheroid, cube, polyhedron, prism, cylinder, rod, disc, lenticular, or other geometric or irregular shape. The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle. The particles may be, e.g., monodisperse or polydisperse and the variation in diameter of the particles of a given dispersion may vary, e.g., particle diameter of between about 0.1 to 100 nm.

[0187] As used herein, the term “liposome” encompasses any compartment enclosed by a lipid bilayer. The term liposome includes unilamellar vesicles which are comprised of a single lipid bilayer and generally have a diameter in the range of about 20 to about 400 nm. Liposomes can also be multilamellar, which generally have a diameter in the range of 1 to 10 pm. In some embodiments, liposomes can include multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and small unilamellar vesicles (SUV). Generally, the liposomes comprise an aqueous compartment enclosed by at least one lipid bilayer. When lipids that include a hydrophilic headgroup are dispersed in water they can spontaneously form bilayer membranes referred to as lamellae. The lamellae are composed of two monolayer sheets of lipid molecules with their non-polar (hydrophobic) surfaces facing each other and their polar (hydrophilic) surfaces facing the aqueous medium. The term liposome includes unilamellar vesicles which are comprised of a single lipid bilayer and generally have a diameter in the range of about 20 to about 400 nm, about 50 to about 300 nm, or about 100 to 200 nm. Liposomes can also be multilamellar, which generally have a diameter in the range of 1 to 10 μm with anywhere from two to hundreds of concentric lipid bilayers alternating with layers of an aqueous phase. In some embodiments, liposomes can include multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and small unilamellar vesicles (SUV). The lipids of the liposome can be cationic, zwitterionic, neutral or anionic, or any mixture thereof.

[0188] As used herein, the term “micelle” refers to an aggregate of amphiphilic molecules such as lipids, assembled so as to form a particle with a hydrophobic interior and a hydrophilic exterior. Micelles are generally spherical assemblies with diameters below 100 nm, although a range of micelle diameters and varying micelle shapes, such as discoid micelles, are known in the art.

[0189] As used herein, the term “lipid” refers to lipid molecules that can include fats, waxes, steroids, cholesterol, fat-soluble vitamins, monoglycerides, diglycerides, phospholipids, sphingolipids, glycolipids, cationic or anionic lipids, derivatized lipids, and the like, as described in detail below.Attorney Docket No.701039-000139WOPT

[0190] The lipid nanoparticles, e.g., liposomes and micelles can contain any suitable lipid, including cationic lipids, zwitterionic lipids, neutral lipids, or anionic lipids as described above. Suitable lipids can include fats, waxes, steroids, cholesterol, fat-soluble vitamins, monoglycerides, diglycerides, phospholipids, sphingolipids, glycolipids, cationic or anionic lipids, derivatized lipids, and the like.

[0191] In some embodiments, the lipid nanoparticles, e.g., liposomes and micelles comprise a phospholipid. Some exemplary phospholipids include but are not limited to phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidylinositol (P1), dimyristoyl phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl serine (DSPS), dioleoyl phosphatidyl serine (DOPS), dipalmitoyl phosphatidyl serine (DPPS), dioleoyl phosphatidyl ethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl- phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), 1,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE), and cardiolipin. Lipid extracts, such as egg PC, heart extract, brain extract, liver extract, and soy PC (e.g., Hydro Soy PC (HSPC)), can also be used. In certain embodiments, the lipids can include derivatized lipids, such as PEGylated lipids. Derivatized lipids can include, for example, DSPE-PEG2000, cholesterol-PEG2000, DSPE- polyglycerol, or other derivatives generally known in the art.

[0192] In some embodiments, the phospholipids derivatized with an aromatic group can be used. For example, the lipid nanoparticle, e.g., liposome or micelle comprises phenoxy- conjugated dipalmitoylphosphatidylcholine (Ph-DPPC) or coumarin-conjugated dipalmitoylphosphatidylcholine (CM-DPPC). Exemplary phospholipids modified with aromatic groups, e.g., phenoxy-conjugated and coumarin-conjugated phospholipids, such as dipalmitoylphosphatidylcholine are described are described in Li, Y. et al. Aromatized liposomes for sustained drug delivery. Nat Commun 14, 6659 (2023), contents of which are incorporated herein by reference in its entirety.Attorney Docket No.701039-000139WOPT

[0193] In some embodiments, the lipid nanoparticles, e.g., liposomes and micelles can contain steroids. Examples of steroids include, but are not limited to, cholesterol, cholic acid, progesterone, cortisone, aldosterone, estradiol, testosterone, dehydroepiandrosterone. Synthetic steroids and derivatives thereof are also can also be used.

[0194] In some embodiments, the lipid nanoparticles, e.g., liposomes and micelle can comprise a cationic lipid. Cationic lipids contain positively charged functional groups under physiological conditions. Cationic lipids include, but are not limited to, N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDA13), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N- (1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3- ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1- (2,3,dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide (DORIE), 3B— [N—(N′,N′-dimethylaminoethane) carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB) and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA).

[0195] The lipid nanoparticles, e.g., liposomes and micelles described herein can contain surfactants including non-ionic surfactants. Without wishing to be bound by a theory, surfactants can act as triggering agents to facilitate release of the LNP's cargo, e.g., PPX. Examples of non-ionic surfactants include, but are not limited to, ethoxylated alkylphenols, ethoxylated fatty esters, sorbitan derivatives, and tocopherol derivatives. Some exemplary surfactants include, but are not limited to, D-α-tocopherol polyethylene glycol succinate (TPGS), which is available having different polyethylene glycol sizes. In some embodiments, the molecular weight range for polyethylene glycol in TPGS is 400-5000. In still other embodiments, the molecular weight range for polyethylene glycol in TPGS is 800-2000. In yet other embodiments, the molecular weight range for polyethylene glycol in TPGS is 800-1500. One particularly useful TPGS is TPGS(1000), in which to total molecular weight of the D-α- tocopherol polyethylene glycol succinate is about 1543. Other useful non-ionic surfactants include, but are not limited to, polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, polyoxyethylene (2) isooctylphenyl ether, polyoxyethylene (150) dinonylphenyl ether, dodecanoic acid 2,3-dihydroxypropyl ester, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (20) sorbitan monooleate, and the like.

[0196] Any suitable combination of lipids can be used in the lipid nanoparticles, e.g., liposomes and micelles described herein. The lipid compositions can be tailored to affectAttorney Docket No.701039-000139WOPT characteristics such as leakage rates, stability, particle size, zeta potential, protein binding, in vivo circulation, and / or accumulation in tissues or organs. For example, DSPC and / or cholesterol can be used to decrease leakage from lipid nanoparticles, e.g., liposomes and micelles. Negatively or positively lipids, such as DSPG and / or DOTAP, can be included to affect the surface charge of lipid nanoparticles, e.g., liposomes and micelles. In some embodiments, the lipid nanoparticles, e.g., liposomes and micelles can include about ten or fewer types of lipids, or about five or fewer types of lipids, or about three or fewer types of lipids. In some embodiments, the molar percentage (mol %) of a specific type of lipid present typically comprises from about 0% to about 10%, from about 10% to about 30%, from about 30% to about 50%, from about 50% to about 70%, from about 70% to about 90%, from about 90% to 100% of the total lipid present in the lipid nanoparticle, e.g., liposome or micelles. One of skill in art will recognize that the lipid compositions can be adjusted to modulate the release properties or other characteristics of the LNPs as required by a given application.

[0197] Without limitation, nanoparticles amenable to the invention can be composed of any material. In some embodiments of this and other aspects of the invention, the nanoparticle comprises a polymer, e.g. a biocompatible polymer. The average molecular weight of the polymer, as determined by gel permeation chromatography, can range from 20,000 to about 500,000.

[0198] As used herein, the term “biocompatible” means exhibition of essentially no cytotoxicity or immunogenicity while in contact with body fluids or tissues. As used herein, the term “polymer” refers to oligomers, co-oligomers, polymers and co-polymers, e.g., random block, multiblock, star, grafted, gradient copolymers and combination thereof.

[0199] The term “biocompatible polymer” refers to polymers which are non-toxic, chemically inert, and substantially non-immunogenic when used internally in a subject and which are substantially insoluble in blood. The biocompatible polymer can be either non- biodegradable or preferably biodegradable. Preferably, the biocompatible polymer is also noninflammatory when employed in situ.

[0200] Biodegradable polymers are disclosed in the art. Examples of suitable biodegradable polymers include, but are not limited to, linear-chain polymers such as polypeptides, polynucleotides, polysaccharides, polylactides, polyglycolides, polycaprolactones, copolymers of polylactic acid and polyglycolic acid, polyanhydrides, polyepsilon caprolactone, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polydihydropyrans, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates,Attorney Docket No.701039-000139WOPT polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, polymethyl methacrylate, chitin, chitosan, copolymers of polylactic acid and polyglycolic acid, poly(glycerol sebacate) (PGS), fumaric acid, sebacic acid, and copolymers, terpolymers including one or more of the foregoing. Other biodegradable polymers include, for example, gelatin, collagen, silk, chitosan, alginate, cellulose, poly-nucleic acids, etc.

[0201] Suitable non-biodegradable biocompatible polymers include, by way of example, cellulose acetates (including cellulose diacetate), polyethylene, polypropylene, polybutylene, polyethylene terphthalate (PET), polyvinyl chloride, polystyrene, polyamides, nylon, polycarbonates, polysulfides, polysulfones, hydrogels (e.g., acrylics), polyacrylonitrile, polyvinylacetate, cellulose acetate butyrate, nitrocellulose, copolymers of urethane / carbonate, copolymers of styrene / maleic acid, poly(ethylenimine), Poloxamers (e.g. Pluronic such as Poloxamers 407 and 188), Hyaluron, heparin, agarose, Pullulan, and copolymers including one or more of the foregoing, such as ethylene / vinyl alcohol copolymers (EVOH).

[0202] In some embodiments, the biocompatible polymer is a copolymer of polylactic acid and polyglycolic acid, poly(glycerol sebacate) (PGS), poly(ethylenimine), Pluronic (Poloxamers 407, 188), Hyaluron, heparin, agarose, or Pullulan.

[0203] Various aspects of the disclosure can be embodied in any of the embodiments described herein, including, but not limited to, the following numbered embodiments:

[0204] Embodiment 1: A method of inducing anesthesia in a subject, comprising: administering to the subject in need thereof 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia in the subject, and wherein an active molecule that is metabolized to PPX in the subject is not administered to the subject with the PPX.

[0205] Embodiment 2: The method of Embodiment 1, wherein the PPX is comprised in a pharmaceutical composition comprising the PPX and a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition does not comprise a molecule that is metabolized to PPX.

[0206] Embodiment 3: A method of inducing anesthesia in a subject, comprising: administering to the subject in need thereof an effective amount of a pharmaceutical composition comprising: (i) 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof; and (ii) an active molecule capable of inducing anesthesia in the subject and being metabolized to PPX in the subject, and wherein the pharmaceutical composition comprises theAttorney Docket No.701039-000139WOPT PPX in an amount that is at least about 5% or more of the amount of the active molecule in the composition.

[0207] Embodiment 4: A method of inducing anesthesia in a subject, comprising: administering to the subject in need thereof 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia in the subject, and wherein an active molecule that is metabolized to 3’-hydroxy-2’,6’-pipecoloxylidide in the subject is not administered to the subject with the PPX.

[0208] Embodiment 5: The method of Embodiment 4, wherein the PPX is comprised in a pharmaceutical composition comprising the PPX and a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition does not comprise a molecule that is metabolized to 3’-hydroxy-2’,6’-pipecoloxylidide.

[0209] Embodiment 6: A method of inducing anesthesia in a subject, comprising: administering to the subject in need thereof 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia in the subject, and wherein ropivacaine, bupivacaine, levobupivacaine, mepivacaine, or a pharmaceutically acceptable salt, or an analog or derivative thereof is not administered to the subject with the PPX.

[0210] Embodiment 7: The method of Embodiment 6, wherein the PPX is comprised in a pharmaceutical composition comprising the PPX and a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition does not comprise ropivacaine, bupivacaine, levobupivacaine, mepivacaine, or a pharmaceutically acceptable salt, or an analog or derivative thereof.

[0211] Embodiment 8: A method of inducing anesthesia in a subject, comprising: administering to the subject in need thereof an effective amount of a pharmaceutical composition comprising: (i) 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof; and (ii) an active molecule capable of inducing anesthesia in the subject and being metabolized to PPX in the subject, and wherein the pharmaceutical composition comprises the PPX in an amount that is at least about 5% or more of the amount of the active molecule in the composition, and wherein the active molecule is ropivacaine, bupivacaine, levobupivacaine, mepivacaine, or a pharmaceutically acceptable salt, or an analog or derivative thereof.

[0212] Embodiment 9: The method of any one of Embodiments 2-8, wherein the pharmaceutical composition comprises the PPX at a concentration of 15 mM or higher, e.g., at least about 17.5 mM, at least about 20 mM, at least about 22.5 mM, at least about 25 mM, at least about 27.5 mM, at least 30 mM or higher.Attorney Docket No.701039-000139WOPT

[0213] Embodiment 10: The method of Embodiment 9, wherein pharmaceutical composition comprises the PPX at a concentration of from higher than 15 mM to about 250 mM, e.g., from about 17.5 mM to about 200 mM, from about 20 mM to about 150 mM, from about 25 mm to about 100 mM, or from about 22.5 mM to about 42.5 mM.

[0214] Embodiment 11: The method of any one of Embodiments 2-8, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.001% to about 99% by weight / weight or weight / volume of the pharmaceutical composition.

[0215] Embodiment 12: The method of Embodiment 11, wherein the pharmaceutical composition comprises PPX in an amount from about 60% to about 80% by weight / weight or weight / volume of the pharmaceutical composition.

[0216] Embodiment 13: The method of Embodiment 11, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.01% to about 10% by weight / weight or weight / volume of the pharmaceutical composition.

[0217] Embodiment 14: The method of Embodiment 11, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.3% to about 4% by weight / weight or weight / volume of the pharmaceutical composition.

[0218] Embodiment 15: The method of Embodiment 13, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.1% to about 2% by weight / weight or weight / volume of the pharmaceutical composition.

[0219] Embodiment 16: The method of any one of Embodiment 2-15, wherein the pharmaceutical composition further comprises epinephrine.

[0220] Embodiment 17: The method of any one of Embodiments 2-15, wherein the pharmaceutical composition further comprises dexamethasone.

[0221] Embodiment 18: The method of any one of Embodiments 1-17, wherein PPX is administered locally, and wherein the therapeutic effect is local or systemic.

[0222] Embodiment 19: The method of any one of Embodiments 1-8, wherein the PPX administered systemically, and wherein the therapeutic effect is local or systemic.

[0223] Embodiment 20: The method of Embodiment 18 or 19, wherein the therapeutic effect is mitigation of pain, alteration of consciousness, sedation, or anesthesia.

[0224] Embodiment 21: The method of any one of Embodiments 1-20, wherein the PPX is administered in an amount from about 0.1 mg / kg to about 100 mg / kg of the subject.

[0225] Embodiment 22: The method of Embodiment 21, wherein the PPX is administered in an amount from about 0.5 mg / kg to about 10 mg / kg of the subject.Attorney Docket No.701039-000139WOPT

[0226] Embodiment 23: The method of Embodiment 22, wherein the PPX is administered in an amount from about 1 mg / kg to about 7 mg / kg of the subject.

[0227] Embodiment 24: The method of Embodiment 23, wherein the PPX is administered in an amount from about 2 mg / kg to about 5 mg / kg of the subject.

[0228] Embodiment 25: The method of Embodiment 24, wherein the PPX is administered in an amount from about 3 mg / kg to about 4 mg / kg of the subject.

[0229] Embodiment 26: The method of any one of Embodiments 2-25, wherein the pharmaceutical composition is formulated for local administration.

[0230] Embodiment 27: The method of any one of Embodiments 2-26, wherein the pharmaceutical composition is formulated for systemic administration.

[0231] Embodiment 28: The method of any one of Embodiments 2-27, wherein the composition is formulated for oral or intravenous administration.

[0232] Embodiment 29: The method of any one of Embodiments 2-28, wherein the composition is in form of a capsule, pill, tablet, aqueous or non-aqueous solution or suspension, gavage, lozenges, dragees, boluses, powder, granule, or a paste.

[0233] Embodiment 30: The method of any one of Embodiments 2–29, wherein the pharmaceutical composition is formulated for administration via injection, sustained release, or a combination thereof.

[0234] Embodiment 31: The method of any one of Embodiments 2–30, wherein the pharmaceutical composition comprises liposomes, nanoparticles, hydrogels, or a combination thereof.

[0235] Embodiment 32: The method of any of Embodiments 1–31, wherein a ratio of sensory block duration to motor block duration (S:M) caused by the administration of PPX is at least about 1.25.

[0236] Embodiment 33: The method of Embodiment 32, wherein a ratio of sensory block duration to motor block duration caused by the administration of PPX is at least about 5.

[0237] Embodiment 34: The method of Embodiment 33, wherein a ratio of sensory block duration to motor block duration caused by the administration of PPX is at least about 10.

[0238] Embodiment 35: The method of Embodiment 34, wherein a ratio of sensory block duration to motor block duration caused by the administration of PPX is at least about 100.

[0239] Embodiment 36: The method of Embodiment 35, wherein a ratio of sensory block duration to motor block duration caused by the administration of PPX is at least about 1,000.

[0240] Embodiment 37: The method of any one of Embodiments 1–36, wherein the PPX selectively induces sensory block and does not induce motor block.Attorney Docket No.701039-000139WOPT

[0241] Embodiment 38: The method of Embodiment 37, wherein the PPX is administered in an amount that is less than about 90% of the amount of PPX capable of inducing motor block.

[0242] Embodiment 39: The method of Embodiment 38, wherein the PPX is administered in an amount that is less than about 85% of the amount of PPX capable of inducing motor block.

[0243] Embodiment 40: The method of Embodiment 39, wherein the PPX is administered in an amount that is less than about 80% of the amount of PPX capable of inducing motor block.

[0244] Embodiment 41: The method of Embodiment 40, wherein the PPX is administered in an amount that is less than about 75% of the amount of PPX capable of inducing motor block.

[0245] Embodiment 42: The method of Embodiment 41, wherein the PPX is administered in an amount that is less than about 70% of the amount of PPX capable of inducing motor block.

[0246] Embodiment 43: The method of Embodiment 42, wherein the PPX is administered in an amount that is less than about 50% of the amount of PPX capable of inducing motor block.

[0247] Embodiment 44: The method of any of Embodiments 1–43, wherein the administering comprises administering intravenously.

[0248] Embodiment 45: The method of any of Embodiments 1–43, wherein the administering comprises administering intrathecally.

[0249] Embodiment 46: The method of any of Embodiments 1–43, wherein the administering comprises administering intraperitoneally.

[0250] Embodiment 47: The method of any of Embodiments 1–43, wherein the administering comprises administering epidurally.

[0251] Embodiment 48: The method of any one of Embodiments 1-43, wherein the administering comprises administering orally or intravenously.

[0252] Embodiment 49: The method of any of Embodiments 1–48, wherein the subject is a human.

[0253] Embodiment 50: The method of any of Embodiments 1–49, wherein the subject is in labor.

[0254] Embodiment 51: A pharmaceutical composition capable of inducing anesthesia in a subject, comprising: (i) 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable saltAttorney Docket No.701039-000139WOPT thereof in an amount effective to induce anesthesia in a subject; and (ii) a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition does not comprise an active molecule that is metabolized to PPX in the subject.

[0255] Embodiment 52: A pharmaceutical composition capable of inducing anesthesia in a subject, comprising: (i) 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia in a subject; and (ii) a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition does not comprise an active molecule that is metabolized to 3’-hydroxy-2’,6’-pipecoloxylidide in the subject.

[0256] Embodiment 53: A pharmaceutical composition capable of inducing anesthesia in a subject, comprising: (i) 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia in a subject; (ii) an active molecule capable of inducing anesthesia in the subject and being metabolized to PPX in the subject; and (iii) a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition comprises PPX in an amount that is at least about 5% or more of the amount of the active molecule in the composition.

[0257] Embodiment 54: The pharmaceutical composition of any one of Embodiments 51- 53, wherein the pharmaceutical composition comprises the PPX at a concentration of 15 mM or higher, e.g., at least about 17.5 mM, at least about 20 mM, at least about 22.5 mM, at least about 25 mM, at least about 27.5 mM, at least 30 mM or higher.

[0258] Embodiment 55: The pharmaceutical composition of Embodiment 54, wherein pharmaceutical composition comprises the PPX at a concentration of from higher than 15 mM to about 250 mM, e.g., from about 17.5 mM to about 200 mM, from about 20 mM to about 150 mM, from about 25 mm to about 100 mM, or from about 22.5 mM to about 42.5 mM.

[0259] Embodiment 56: The pharmaceutical composition of any one of Embodiments 51- 53, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.001% to about 99% by weight / weight or weight / volume of the pharmaceutical composition.

[0260] Embodiment 57: The pharmaceutical composition of Embodiment 56, wherein the pharmaceutical composition comprises PPX in an amount from about 60% to about 80% by weight / weight or weight / volume of the pharmaceutical composition.

[0261] Embodiment 58: The pharmaceutical composition of any one of Embodiments 51- 56, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.01% to about 10% by weight / weight or weight / volume of the pharmaceutical composition.Attorney Docket No.701039-000139WOPT

[0262] Embodiment 59: The pharmaceutical composition of Embodiment 58, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.3% to about 4% by weight / weight or weight / volume of the pharmaceutical composition.

[0263] Embodiment 60: The pharmaceutical composition of Embodiment 58, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.1% to about 2% by weight / weight or weight / volume of the pharmaceutical composition.

[0264] Embodiment 61: The pharmaceutical composition of any one of Embodiment 51- 60, wherein the pharmaceutical composition further comprises epinephrine.

[0265] Embodiment 62: The pharmaceutical composition of any one of Embodiments 51- 61, wherein the pharmaceutical composition further comprises dexamethasone.

[0266] Embodiment 63: The pharmaceutical composition of any one of Embodiments 51- 62, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.1 mg / kg to about 100 mg / kg of the subject.

[0267] Embodiment 64: The pharmaceutical composition of any one of Embodiments 51- 63, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.5 mg / kg to about 10 mg / kg of the subject.

[0268] Embodiment 65: The pharmaceutical composition of any one of Embodiments 51- 64, wherein the pharmaceutical composition comprises the PPX in an amount from about 1 mg / kg to about 7 mg / kg of the subject.

[0269] Embodiment 66: The pharmaceutical composition of any one of Embodiments 51- 65, wherein the pharmaceutical composition comprises the PPX in an amount from about 2 mg / kg to about 5 mg / kg of the subject.

[0270] Embodiment 67: The pharmaceutical composition of any one of Embodiments 51- 66, wherein the pharmaceutical composition comprises the PPX in an amount from about 3 mg / kg to about 4 mg / kg of the subject.

[0271] Embodiment 68: The pharmaceutical composition of any one of Embodiments 51- 67, wherein the pharmaceutical composition is formulated for administration via injection, sustained release, or a combination thereof.

[0272] Embodiment 69: The pharmaceutical composition of any one of Embodiments 51- 68, wherein the pharmaceutical composition further comprises liposomes, nanoparticles, hydrogels, or a combination thereof.

[0273] Embodiment 70: The pharmaceutical composition of any one of Embodiments 51- 69, wherein the composition is in form of a capsule, pill, tablet, aqueous or non-aqueous solution or suspension, gavage, lozenges, dragees, boluses, powder, granule, or a paste.Attorney Docket No.701039-000139WOPT

[0274] Embodiment 71: The pharmaceutical composition of any one of Embodiments 51- 70, wherein administration of the pharmaceutical composition to the subject is capable of causing a ratio of sensory block duration to motor block duration (S:M) of at least about 1.25.

[0275] Embodiment 72: The pharmaceutical composition of Embodiment 71, wherein administration of the pharmaceutical composition to the subject is capable of causing a ratio of sensory block duration to motor block duration of at least about 5.

[0276] Embodiment 73: The pharmaceutical composition of Embodiment 72, wherein administration of the pharmaceutical composition to the subject is capable of causing a ratio of sensory block duration to motor block duration of at least about 10.

[0277] Embodiment 74: The pharmaceutical composition of Embodiment 73, wherein administration of the pharmaceutical composition to the subject is capable of causing a ratio of sensory block duration to motor block duration of at least about 100.

[0278] Embodiment 75: The pharmaceutical composition of Embodiment 74, wherein administration of the pharmaceutical composition to the subject is capable of causing a ratio of sensory block duration to motor block duration of at least about 1,000.

[0279] Embodiment 76: The pharmaceutical composition of any one of Embodiments 51- 75, wherein the pharmaceutical composition selectively induces sensory block and does not induce motor block when administered to the subject.

[0280] Embodiment 77: The pharmaceutical composition of Embodiment 76, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 90% of the amount of PPX capable of inducing motor block.

[0281] Embodiment 78: The pharmaceutical composition of Embodiment 77, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 85% of the amount of PPX capable of inducing motor block.

[0282] Embodiment 79: The pharmaceutical composition of Embodiment 78, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 80% of the amount of PPX capable of inducing motor block.

[0283] Embodiment 80: The pharmaceutical composition of Embodiment 79, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 75% of the amount of PPX capable of inducing motor block.

[0284] Embodiment 81: The pharmaceutical composition of Embodiment 80, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 70% of the amount of PPX capable of inducing motor block.Attorney Docket No.701039-000139WOPT

[0285] Embodiment 82: The pharmaceutical composition of Embodiment 81, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 50% of the amount of PPX capable of inducing motor block.

[0286] Embodiment 83: The pharmaceutical composition of any one of Embodiments 51- 82, wherein the pharmaceutical composition is formulated for local administration.

[0287] Embodiment 84: The pharmaceutical composition of any one of Embodiments 51- 83, wherein the pharmaceutical composition is formulated for systemic administration.

[0288] Embodiment 85: The pharmaceutical composition of any one of Embodiments 51- 84, wherein the pharmaceutical composition is formulated for intravenous administration.

[0289] Embodiment 86: The pharmaceutical composition of any one of Embodiments 51- 84, wherein the pharmaceutical composition is formulated for intrathecal administration.

[0290] Embodiment 87: The pharmaceutical composition of any one of Embodiments 51- 84, wherein the pharmaceutical composition is formulated for intraperitoneal administration.

[0291] Embodiment 88: The pharmaceutical composition of any one of Embodiments 51- 84, wherein the pharmaceutical composition is formulated for epidural administration.

[0292] Embodiment 89: The pharmaceutical composition of any one of Embodiments 51- 84, wherein the composition is formulated for oral or intravenous administration.

[0293] Embodiment 90: The method of any of Embodiments 1-50, wherein an effect of the effective amount of PPX is altered by a second agent co-administered to the subject.

[0294] Embodiment 91: The Method of Embodiment 90, wherein the second agent is selected from the group consisting of vasoconstrictors, glucocorticoid receptor agonists, and chemical permeation enhancers. Examples

[0295] In order for the present disclosure to be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the pharmaceutical compositions and methods provided herein and are not to be construed in any way as limiting their scope.

[0296] Materials Used. All chemical products commercially available were purchased from Sigma-Aldrich (Burlington, MA, USA), TCI America (Portland, OR, USA), and Thermo Scientific Chemicals (Waltham, MA, USA) and used without further purification. Dichloromethane (DCM) was purchased in its anhydrous form and used without further purification.Attorney Docket No.701039-000139WOPT

[0297] Instruments and characterizations. Reactions were monitored by thin-layer chromatography carried out on silica 250 µm (Silicycle) using UV light (254 nm) as visualizing agent. Flash column chromatography was performed on Sorbtech silica gel (40-63 µm particle size) using ACS grade solvents purchased from Fisher Scientific (Waltham, MA, USA).1H NMR experiments were performed on a Varian 400 MHz NMR spectrometer (Palo Alto, CA, USA), recorded at 300MHz, and calibrated from solvent (D2O: δ 4.79 ppm) resonance.13C NMR experiments were performed on a Varian 400 MHz NMR spectrometer (Palo Alto, CA, USA), and spectra were recorded at 75 MHz. Chemical shifts (δ) are reported in parts per million (ppm) relative to the residual solvent resonance and coupling constants (J) are reported in hertz (Hz). Splitting patterns are designated as singlet (s), broad singlet (br. s), doublet (d), triplet (t), quartet (q), quintet (quint), heptuplet (hept), multiplet (m). Splitting patterns that could not be interpreted or easily visualized are designated as multiplet (m). The Agilent InfinityLab LC / MSD XT single quadrupole mass spectrometer (Santa Clara, CA, USA) was used for molecular weight determinations. Synthesis of 2’,6’-pipecoloxylidide (PPX)

[0298] A skeletal diagram of the synthesis of PPX as described below is shown in FIG.9.

[0299] (I) Synthesis of tert-butyl (S)-2-((2,6-dimethylphenyl)carbamoyl)piperidine-1- carboxylate. (S)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid (10 g, 43.61 mmol), triethylamine (TEA) (9.1 ml, 65.42 mmol), and anhydrous dichloromethane (DCM) (90 mL) were added to a 250 mL round bottom flask equipped with a stirrer. The white solid is dissolved at room temperature and the mixture was cooled to 0 ºC with an ice-bath. Isobutyl chlorocarbonate (6.22 mL, 47.97 mmol) was added dropwise. The solution gradually appears a white precipitate. After adding isobutyl chlorocarbonate, the mixture was reacted at 0°C for 1 hour. Then, 2,6-dimethylaniline (5.9 mL, 47.97 mmol) was added drop by drop and the mixture allowed to return to room temperature. The mixture was stirred at the room temperature for 24 hours. The mixture was washed with saturated aqueous NaCl and Na2CO3solution in sequence, dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and the resulting residue is used directly in the next reaction step without purification.

[0300] (II) Synthesis of (S)-N-(2,6-dimethylphenyl)piperidine-2-carboxamide. The residue obtained from the previous step was dissolved in anhydrous dichloromethane (100 ml). Trifluoroacetic acid (16.2 mL, 218.05 mmol) was added dropwise, and the mixture was stirred at the room temperature for 24 hours. The mixture was concentrated under vacuum, the crude mixture was diluted with water and the pH is adjusted to pH = 2 with 1 M NaOH solution. The aqueous layer was extracted with DCM (3 x 100 mL), the combined organic layers wereAttorney Docket No.701039-000139WOPT washed with saturated NaHCO3(2 x 100 mL) and dried over Na2SO4. The organic phase was evaporated under vacuum and the resulting oil was purified by column chromatography (n- Hex / EA, 1:1), obtaining 4.86g (48%) of a white solid.

[0301] (III) (PPX) Synthesis of (S)-2-((2,6-dimethylphenyl)carbamoyl)piperidin-1-ium chloride. (S)-N-(2,6-dimethylphenyl)piperidine-2-carboxamide II (1 g, 4.30 mmol) was suspended in ether hydrochloride solution (21.5 mL). The mixture was stirred for 2 hours and filtered. The solid was washed with acetonitrile (3 x 5 mL) and dried under high vacuum affording 1.13 g (98%) of a white powder.1H NMR (300 MHz, D2O, δ ppm): 7.16 (m, 3H), 4.14 (dd, 1H), 3.46 (d, 1H), 3.06 (td, 1H), 2.38 (d, 1H), 2.12 (s, 6H), 1.92 (m, 3H), 1.66 (m, 2H).13C NMR (75 MHz, D2O, δ ppm): 169.45 (1CO), 135.87 (1C), 132.04 (2C), 128.29 (2CH), 128.12 (1CH), 57.69 (1CH), 43.86 (2CH2), 27.57 (2CH2), 21.59 (2CH2), 21.52 (CH2), 17.06 (CH3). HR-MS (ESI+, m / z): [M+H]+= C14H21N2O, calculated.: 233.1648; found 233.16423. Methods Used

[0302] Ex vivo multi-channel single-fiber recordings from mouse sciatic nerve. Detailed methods were reported previously. See References 27, 28. Briefly, sciatic nerves were harvested from C57BL / 6 mice of both sexes aged 10–16 weeks, 20–35 g (Taconic, Germantown, NJ, USA). Mice were anesthetized by isoflurane inhalation, euthanized by exsanguination via perforating the right atrium, and transcardially perfused with oxygenated (95 % O2, 5% CO2) Krebs solution (in mM: 117.9 NaCl, 4.7 KCl, 25 NaHCO3, 1.3 NaH2PO4, 1.2 MgSO4, 2.5 CaCl2, and 11.1 D-glucose at room temperature). In a dissection chamber circulated with oxygenated ice-cold Krebs solution, the whole length of sciatic nerves (~30 mm) was harvested bilaterally from their proximal projection to the L3 spinal cord to their distal branches innervating gastrocnemius muscles. Sciatic nerves were then transferred to a custom-built tissue perfusing and recording platform, consisting of a tissue chamber perfused (5-8 mL / min) with oxygenated Krebs solution at 29 - 31⁰C and an adjacent recording chamber filled with paraffin oil (Fisher Scientifics). The proximal sciatic nerve was placed in the tissue compartment, while the distal segment was pulled into the recording compartment through a mouse hole under a plastic gate. On a hydrophilic glass surface at the bottom of the recording compartment, the ~5 mm distal nerve segment was gently de-sheathed by using two pairs of fine forceps (11252-00, Fine Science Tools, Inc., CA, USA). The nerve was then manually split into fine filaments of 10 to 20 µm thick, which were individually mounted onto a four- channel wire electrode array for multi-channel single-fiber recordings. Action potentials were evoked by electrically stimulating the proximal end of the sciatic nerve in the tissueAttorney Docket No.701039-000139WOPT compartment by a suction electrode fabricated with quartz glass capillary (cathodic, 0.4-3 mA, 0.2 ms duration, 0.5Hz) and a stimulus isolator (Model 4100, A-M Systems). In the recording compartment, single-unit action potentials from all five electrodes were recorded simultaneously, pre-amplified (x192), digitized at 20 kHz (16-bit ADC, 0.195 µV recording resolution), and stored on a PC by a 32-channel neural recording system (RHD C3324 / C3100, Intan Technologies LLC, CA, USA).

[0303] Application of chemicals to the mouse sciatic nerve trunk (ex vivo). The effect of chemicals on nerve conduction was assessed by local application to the nerve trunk in the tissue chamber. Similar to a previous pharmacological study (see Reference 29), a brass tubing (4x4 square x 16 mm high tubing) with bottom edges lined with petrolatum was placed over a short segment of the sciatic nerve in the tissue chamber (~3 mm); two semi-circular openings at the bottom edges allowed the sciatic nerve to go through the tubing without being mechanically compressed. The Krebs solution within the tubing was removed by gentle suction and replaced by 150 µL of solution containing the assessed chemical for 10 minutes. Then, the chemical solution was removed, and the tubing lifted to re-expose the sciatic nerve segment to Krebs solution. Two chemicals were tested, i.e., ropivacaine (Rop, 0.5 mg / mL) and PPX (4 mg / mL), which were all dissolved in phosphate-buffered saline (PBS, pH 7.4). The vehicle effect (PBS) was assessed in some single-fiber recordings.

[0304] In Vitro Effects on hERG, Nav1.5, Nav1.7, Nav1.8, and Nav1.9 currents. Currents were measured using the whole-cell variant of the patch clamp method. Glass pipettes were pulled from borosilicate glass by a horizontal puller (Sutter Instruments, USA). Pipette tip resistance was approximately 1 to 2 MΩ when filled with internal solutions. Series resistance was compensated electronically by approximately 60-80%. Bath temperature was measured by a thermistor placed near the cell under study. An Axopatch 1-B amplifier (Axon Instruments, Foster City, CA, USA) was used for whole-cell voltage clamping. Creation of voltage clamp pulses and data acquisition was controlled by a computer running pClamp software (ver 9.2 Axon Instruments). After rupture of the cell membrane (entering whole-cell mode), current kinetics and amplitudes were allowed to stabilize as the cell was dialyzed with internal solution and paced every 10 seconds (typically 2 to 3 minutes). Peak INawas elicited using a pulse pattern with fixed amplitudes (holding potential = -95 mV, test step to –15 mV for Nav1.5, holding potential = -130 mV, test step to –80 mV for Nav1.7; holding potential = -40 mV, test step to –100 mV for Nav1.8; (holding potential = 100 mV, test step to –60 mV for Nav1.9). hERG current was elicited by a pulse to +40mV from a holding potential of -Attorney Docket No.701039-000139WOPT 90mV. Stably transfected HEK cells were perfused at each testing concentration until a steady- state was reached.

[0305] Cell culture for cytotoxicity studies. Cell culture of C2C12 mouse myoblasts (American Type Culture Collection (ATCC), Manassas, VA, USA) and PC12 rat adrenal gland pheochromocytoma cells (ATCC, Manassas, VA, USA) were performed as previously reported. In brief, C2C12 cells were seeded into a 96-well plate at 1,000 cells per well in DMEM with 20% FBS and 1% Penicillin Streptomycin and incubated for 10–14 days in DMEM with 2% horse serum and 1% Penicillin Streptomycin for differentiation into myotubules. PC12 cells were seeded into 96-well plate at 4,000 cells per well in DMEM with 2.5% FBS, 12.5% horse serum, and 1% Penicillin Streptomycin and incubated for 7 days in DMEM with 1% horse serum, 50 ng / mL nerve growth factor, and 1% Penicillin Streptomycin.

[0306] Cytotoxicity Studies. The cytotoxicity of PPX was evaluated with the 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) colorimetric assay. The C2C12 and PC12 cells were treated with varying doses of PPX with bupivacaine used as a comparison. After 24 hours of incubation, 40μL of the MTS-based CellTiter 96®AQueousOne Solution Reagent was added to each well. The cells were incubated for another 4 hours, and the absorbances (490nm) were measured on a BioTek®Synergy™ Mx microplate reader (BioTek Inc., VT, USA). Survival is expressed as percentages of results in untreated cells.

[0307] Preparation of Test Solutions for in vivo experiments. For sciatic nerve injection, ropivacaine hydrochloride (United States Pharmacopeia, North Bethesda, MD, USA) and synthesized PPX were prepared in normal saline solution (0.9% NaCl) at the desired concentration. For intrathecal administration, ropivacaine hydrochloride and synthesized PPX were prepared in distilled water. All preparations were made within 15 minutes preceding administration. Animal Care

[0308] For sciatic nerve injection. Adult male Sprague-Dawley rats (350-450 g) were obtained from Charles River Laboratories (CLR; Wilmington, MA, USA) or Taconic Biosciences (Germantown, NY, USA). They were housed in groups of two per cage on a 7 am to 7 pm light / dark cycle. Animals were fed with standard laboratory chow and water ad libitum. All animals were cared for in accordance with protocols approved by the Boston Children’s Hospital Animal Care and Use Committee and the Guide for the Care and Use of Laboratory Animals of the US National Research Council.Attorney Docket No.701039-000139WOPT

[0309] For intrathecal injection. Adult male Sprague-Dawley rats (350-400 g) were obtained from CRL (Wilmington, MA, USA) with an intrathecal catheter in place (Instech PinPort™). Briefly, the intrathecal catheter had been placed using a caudal advancement of catheter placement technique. The animal was placed in ventral recumbency with an incision made over midline to expose the T13 spinous process. The catheter is inserted between T13 and L1, advanced caudally to reach L3-L5, and secured. The external portion of the catheter was tunneled subcutaneously, exteriorized, and secured just caudal to the scapular region using wound clips. Animals were housed at CRL for at least 2 days following surgery. Surgery and postoperative animal care at CRL was performed in accordance with protocols approved by the CRL Institutional Animal Care and Use Committee. Upon arrival to the institution, animals were singly housed and fed with standard laboratory chow and water ad libitum. Catheter maintenance was performed daily for increased patency. All animals were cared for in accordance with protocols approved by the Boston Children’s Hospital Animal Care and Use Committee and the Guide for the Care and Use of Laboratory Animals of the US National Research Council.

[0310] Sciatic Nerve Injection Technique. As previously described, after anesthetizing rats using isoflurane in oxygen, a 23-gauge needle was advanced posteromedial to the greater trochanter, pointing in an anteromedial direction (see References 30, 31). Following contact with the bone, 0.3 mL of the test solution was injected, depositing the injectate over the sciatic nerve. All injections were performed on the left leg with the right leg serving as an untreated, negative control. For histopathologic analysis only, the injectate volume was increased to 1.5 mL with the goals of (1) reducing the risk of sampling error and (2) reducing the risk of sampling tissue traumatized by the needle (see Reference 32). All injections were performed by the same experimenter who demonstrated >99% successful sciatic nerve blocks with 0.1 mL of 0.5% bupivacaine prior to beginning the experiments reported in this study, suggesting that differences in successful nerve blockade reflect local anesthetic efficacy, not operator error (i.e., “missing”).

[0311] Intrathecal Injection Technique. All solutions were created at the specified concentration within 15 minutes prior to administration. Rats were anesthetized using isoflurane in oxygen. The Instech PinPort™ button was wiped with 70% alcohol wipes. Using a sterile Nanofil syringe, animals were bolused with 30µL of the solution over a period of 15- 30 s. This was followed by injection of artificial cerebrospinal fluid (Tocris Bioscience, Bristol, United Kingdom) corresponding to the dead volume of the catheter. Repeat dosing wasAttorney Docket No.701039-000139WOPT performed in a similar fashion with at least 30 min between doses. Catheter maintenance was performed daily.

[0312] Given that intrathecal catheters had not been placed or confirmed by the experimenters themselves and that animals traveled to the experimenter’s institution following surgery, the following measure was taken to avoid false negative nerve blockade occurring secondary to catheter misplacement, occlusion, or dislodgement. Any animals with a 0 minute sensory nerve blockade following administration of the compound being investigated in this study (i.e., PPX) were allowed to recover for at least 30 min and were then administered 0.75% ropivacaine which has been previously described as an effective dose in adult rats (see References 33–35). Data from animals without nerve blockade following intrathecal ropivacaine administration were excluded from analysis.

[0313] Sciatic Nerve Blockade Assessment. A modified hot plate test was used to determine the presence and degree of sciatic sensory nerve blockade (i.e., thermal nociception), as previously described (see References 36–37). The experimenter, blinded to the treatment the animal had received, held the animal above the hot plate and placed the animals’ hind paws in triplicate on a 56°C hot plate (model 39D Hot Plate Analgesia meter; IITC Inc., Woodland Hills, CA, USA). The time (thermal latency) to the animal’s withdrawal of its hind paw was measured (the experimenter removed the hind paw from the hot plate if the animal did not withdraw after 12 seconds to avoid injury of development of hyperalgesia). Following sciatic nerve injection, assessment was conducted at 15 minutes, 30 minutes, 60 minutes, and then hourly after injection until resolution of nerve block. Following intrathecal injection, assessment was conducted every 5 minutes until resolution of nerve block. Duration of thermal nociceptive block (i.e., sensory block) was defined as the time required for thermal latency to return to an average value of less than 7 seconds. This corresponds to the midpoint between baseline thermal latency (2 seconds in adult rats) and maximal latency (12 seconds).

[0314] The extensor postural thrust (EPT) test was used to assess motor blockade. The experimenter suspended the animal’s hind paws over a digital balance in sequence (left then right). The maximum weight that the animal could bear was measured in triplicate. The duration of motor block was defined as the time required for weight-bearing to return halfway to normal from the maximum block.

[0315] Tissue Harvesting and Histology. Animals were euthanized by carbon dioxide inhalation four, thirty, and sixty days following injection for evaluation of local anesthetic inflammation and toxicity (see Reference 38). For animals that had been injected at the sciatic nerve, the sciatic nerve and adjacent connective tissues were harvested by the nerve blockAttorney Docket No.701039-000139WOPT injection site. For animals that had been injected intrathecally, the spinal cord was harvested by hydraulic extrusion (see Reference 39).

[0316] Muscle samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard techniques. Four independent sections were assessed and graded in a blinded fashion based on the area of most severe damage. Samples were scored for degree of inflammation (0-4) and myotoxicity (0-6), as previously described (see References 6, 40, 41). Inflammation scoring reflects a subjective assessment of the severity of inflammation: 0=none, 1=minimal, 2=mild, 3=moderate, and 4=severe inflammation. Myotoxicity scoring considers two markers of local anesthetic myotoxicity, specifically nuclear internalization (characterized by normal myocyte size and cytoplasm chromicity but with nuclei located away from their normal peripheral location) and myocyte regeneration (characterized by a shrunken appearance with basophilic cytoplasm): 0=normal, 1=perifascicular internalization, 2=deep internalization (>5 cell layers), 3=perifascicular regeneration, 4=deep regeneration, 5=hemifascicular regeneration, and 6=holofascicular regeneration.

[0317] Sciatic nerves and lumbar spinal cord samples were fixed in Karnovsky’s KII solution, processed and epon embedded for toluidine blue staining. They were assessed by light microscopy in a blinded fashion. Statistical Analysis

[0318] For in vitro multichannel single-unit recordings of action potentials from mouse sciatic nerve. Data was processed off-line using customized MATLAB programs (Mathworks R2021). The single-fiber recordings were digitally filtered at 300 – 5000 Hz for extracting the single-unit spike waveforms. Root-mean-square (RMS) value of 5 ms pre-stimulation noise was calculated and 3 times of that value was set as the detection threshold for action potential spikes. The time of first exceeding the threshold was deemed as on the onset of the action potential. To avoid confusion, only easily discriminable single-unit spikes temporally separated from adjacent spikes in the record are analyzed. Conduction delays were measured as time between the onset of stimulus artifact and onset of recorded action potentials. Conduction velocity was computed from the conduction delay and distance between stimulating and recording electrodes. Data are presented as means ± SE. One-way ANOVA with repeated measures was performed as appropriate using SigmaPlot v9.0 (Systat Software, San Jose, CA, USA). Post-doc multiple comparisons were conducted using the Bonferroni t-tests. Differences were considered significant when p < 0.05.Attorney Docket No.701039-000139WOPT

[0319] For in vitro effects on hERG, Nav1.5, Nav1.7, Nav1.8, and Nav1.9 currents. Data are presented as means with standard deviations with percent reduction of current amplitude. This was measured as current reduction after a steady-state effect had been reached in the presence of drug relative to current amplitude before drug was introduced (control). Each cell served as its own control. A nonlinear curve-fitting routine was utilized to fit a three-parameter Hill equation to the results using GraphPad Prism Version 10.0.2. Statistical analysis was performed by the independent t-test. Statistical significance was defined as p < 0.05.

[0320] For neurobehavioral data and histopathological scoring. Neurobehavioral data and tissue injury scores are presented as means with standard deviations. Statistical analysis was performed by the independent t-test (paired t-test when comparing motor and sensory block duration of the same animal; unpaired when comparing different compounds) with statistical significance defined as p < 0.05. Example 1 – Synthesis and characterization of 2’,6’-pipecoloxylidide (PPX)

[0321] Although the chemical synthesis of 2’,6’-pipecoloxylidide (PPX) has been previously described, optimization was performed to improve synthetic yield and product purity. Synthesis is shown in FIG.9 with synthetic procedures detailed in the methods section herein. In brief, (S)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid and 2,6- dimethylaniline were reacted to synthesize (I) tert-butyl (S)-2-((2,6- dimethylphenyl)carbamoyl)piperidine-1-carboxylate. This product (I) then modified to produce (II) (S)-N-(2,6-dimethylphenyl)piperidine-2-carboxamide. The resulting product (II) was then suspended in ether hydrochloride solution, followed by filtering, washing, and drying to yield a solid product of 2’,6’-pipecoloxylidide (PPX).1H NMR (FIGS.10 and 39) and13C NMR (FIG. 11) demonstrated the successful synthesis of PPX. This was also confirmed by HR-MS (FIG.12). Example 2 – Efficacy of PPX as a local anesthetic for peripheral nerve blockade

[0322] PPX was evaluated for its local anesthetic properties using a well-established rodent sciatic nerve block model.

[0323] Sensory nerve block was assessed by modified hotplate testing, where maximal nerve block (100% maximal peak effect, MPE) was a latency of 12 s and baseline (0% MPE) was 2 s. Motor block was assessed by a weight bearing test, where 100% MPE was weight bearing of ≤ 4 g, and 0% MPE was the highest weight the animal could bear when not blocked. The duration of block was calculated as the time to return to 50% MPE. Blocks that did not reach 50% MPE were considered unsuccessful and were counted as having durations of zero.Attorney Docket No.701039-000139WOPT Please see Methods for further details on neurobehavioral testing. Conversion of anesthetic concentrations in mM to the %w / v used in clinical practice is provided in Table 1. Table 1:

[0324] PPX did not yield a sensory or motor block at a concentration of 15mM, a clinically used concentration of Ropivacaine (Rop) that produces a sensory and motor block of 149.7±12.9 minutes and 151.4±11.1 minutes in this model, respectively (FIG. 40). The duration of block from ROP increased with increasing concentration (FIG.41). There was no statistically significant difference between the durations of sensory and motor block at any concentration (p > 0.05), as reflected in a ratio of ̴ 1 of the durations of sensory and motor block (referred to as the sensory-motor ratio, or SMR) across the range of concentrations, with a very narrow distribution (FIG.42).

[0325] Increasing concentrations of PPX were therefore tested (FIG. 13). At concentrations of 22.5 and 30mM, PPX produced 100% sensory MPE (FIG.43, showing 30 mM PPX) and yielded sensory nerve duration of 34.5±14.9 minutes and 67.4 ± 17.4 minutes, respectively (FIG. 41). However, no motor block was produced at either of these concentrations (p = 0.02 and p < 0.0001 for comparison of sensory and motor block with 22.5 and 30mM PPX, respectively). This is in contrast to Rop, which produces equal sensory and motor block durations at 15 mM (p = 0.21) and 30mM (p = 0.45). Injection of 45mM and 60mM PPX produced both sensory and motor nerve block. However, the motor block was of significantly shorter duration than the sensory block (p = 0.04 for 45mM, p = 0.03 for 60mM). At 120mM PPX, the sensory and motor block durations were similar (p = 0.12).Attorney Docket No.701039-000139WOPT

[0326] With ROP, only minor deficits in weight bearing were noted following administration of PPX (e.g., 14.7 ± 9.0 % MPE at 30 min with 30 mM PPX). With 30 mM PPX, only 2 out of 12 animals developed motor block as defined here (i.e., ≥50% MPE), and for short durations (10 min and 13 min). Injection of increasing concentrations of PPX led to increasing degrees of motor block. At 45 mM PPX, the SMR was 1.4 (p = 0.04), and 1.3 at 60 mM PPX, (p = 0.03; FIGS.41 and 42). At 120 mM PPX, the sensory and motor block durations were not statistically significantly different (p = 0.12). Note that at 45 mM and 60 mM PPX, there were still animals with SMRs >> 1, in contrast to the findings with ROP. At all concentrations, the durations of block from PPX were shorter than those from ROP.

[0327] The sensory to motor block duration ratios (S:M ratio) were compared between Rop and PPX (FIG.14). Rop, one of the clinically used CLAs, has S:M ratio between 0.99 and 1.01 at all tested concentrations (15mM to 90mM). In contrast, PPX has ∞ S:M ratio at concentrations of 22.5mM and 30mM and S:MR of 1.23 to 1.24 at higher concentrations (45mM to 120mM). No neurobehavioral deficits were detected in the contralateral (uninjected) limb in any animal injected with Rop or PPX. Deficits in the contralateral extremity are a sign of systemic drug distribution. All nerve blocks resolved completely as indicated by return to a latency of 2 seconds. Example 3 – Effect of PPX on single-unit recordings of action potentials from mouse sciatic nerve

[0328] A custom-built tissue perfusing and recording platform was created as shown in FIG.15. Action potentials were evoked by electrical stimulation from the proximal end of the sciatic nerve and recorded from split nerve filaments at the distal end. A representative single- fiber recording from one filament, which includes a fast-conducting A-fiber (blue arrow) and a slow-conducting C-fiber (red arrow), as displayed in FIG. 16. Recordings were conducted before (baseline), immediately after chemical application to the nerve trunk (drug), and 30 - 60 minutes after removal of the chemical (wash). The vehicle effect on action potential transmission was conducted on 3 A-fibers and 3 C-fibers by applying PBS in the tubing for 10 minutes. The normalized conduction delay recorded before and after the PBS applications showed no statistical differences (100% vs.101.9 ±1%, t-test, p = 0.2).

[0329] The effects of PPX compared to Rop on the conduction velocities (CV) of both A- fibers and C-fibers in the sciatic nerve are shown in FIG. 17; blocked fibers show zero CV. Rop (0.5 mg / mL) reversibly blocked A-fibers (F2,8 = 44.45, p<0.001; post-hoc comparison, p<0.05 for Rop vs. baseline and Rop vs. wash). Similarly, Rop (0.5 mg / mL) reversibly blockedAttorney Docket No.701039-000139WOPT C-fibers (F2, 20 = 32.97, p<0.001; post-hoc comparison, p<0.05 for Rop vs. baseline and Rop vs. wash). PPX at 4 mg / mL had no significant effects on A-fibers (F2,10 = 0.51, p=0.62) but reversibly blocked C-fibers (F2, 10 = 25.69, p<0.001; post-hoc comparison, p<0.05 for PPX vs. baseline and PPX vs. wash). Example 4 – Effect of PPX on Nav1.7, Nav1.8, and Nav1.9

[0330] Sensory selective anesthesia can be achieved by several different pharmacological approaches. To characterize how the sensory selective effect of PPX is attained, specific voltage gated sodium channels were investigated. Specifically, Nav1.7, Nav1.8, and Nav1.9 were chosen given that they are located selectively or preferentially in sensory neurons (see References 42–45). The effects of PPX on Nav1.7, Nav1.8, and Nav1.9 currents in stably transfected HEK cells were compared to the effects of Rop.

[0331] Rop blocked all three channels, confirming that it acts as a non-selective Nav channel blocker. As shown in FIG. 19, PPX similarly inhibited the Nav1.8 current at both a low concentration (9.3±2.0% blockade by PPX vs. 9.7±4.0% blockade by Rop at 1µM; p = 0.87) and high concentration (86.4±2.9% blockade by PPX vs.90.4±2.6% blockade by Rop at 100µM; p = 0.15).

[0332] In contrast, PPX led to minimal blockade of Nav 1.7 across the tested concentration range (1-100µM; FIG.18): PPX inhibited 6.1±2.8% Nav1.7 at 10µM (compared to 45.5±4.6% blockade by 10µM Rop, p = 0.0002) and 10.8±4.1% Nav1.7 at 100µM (compared to 84.9±2.9% blockade by 100µM Rop, p < 0.0001). Similarly, PPX minimally affected Nav1.9 currents across the tested concentration range (1-100µM; FIG.20): PPX inhibited 2.6±4.6% Nav1.9 at 10µM (compared to 21.6±6.5% blockade by 10µM Rop, p = 0.01) and 7.6±5.4% Nav1.9 at 100µM (compared to 49.5±7.5% blockade by 100µM Rop, p < 0.0001).

[0333] The mean ratios of current inhibition by Rop compared to PPX are shown in FIG. 21. Particularly at higher concentrations (10-100µM), Rop blocks Nav1.7 and Nav1.9 currents 7.7-fold and 7.4-fold more potently than PPX, respectively. By comparison, Rop and PPX cause similar inhibition of Nav1.8 currents (ratios 1.1-1.8). Overall, while Rop non-selectively blocked Nav1.7, Nav1.8, and Nav1.9, PPX most potently blocked Nav1.8 with minimal effect on Nav1.7 and Nav1.9 even at high concentrations. Example 5 – Efficacy of PPX for intrathecal anesthesia

[0334] Given the efficacy of PPX as a sensory selective local anesthetic for peripheral anesthesia, we proceeded to examine the effect and properties of PPX for neuraxial anesthesia. Neuraxial anesthesia with ROP and PPX was assessed using animals with surgically placedAttorney Docket No.701039-000139WOPT intrathecal catheters. Animals were treated with 30 µL ROP or PPX and then underwent neurobehavioral testing of the hindpaws as performed after sciatic nerve blockade.

[0335] Intrathecal administration of 15 mM ROP produced 100% sensory MPE with a sensory block duration of 18.8 ± 7.8 min (FIG.44).100% motor MPE was also achieved, with a duration of 15.2 ± 5.4 min. Intrathecal ROP had an average SMR of 1.23 at 15 mM ROP (p = 0.24 for comparison of durations of sensory and motor block).

[0336] Intrathecal administration of 30 mM PPX produced maximal sensory latency, yielding a sensory block lasting 24.8 ± 8.7min (FIG.45), similar to (1.3-fold) the duration of sensory block from 15 mM ROP (p = 0.08). However, unlike intrathecal block with ROP, there were only very minor motor deficits (e.g., 12.0 ± 7.8 % MPE at 10 min). None of the animals developed motor block as defined here (> 50% MPE).

[0337] Increasing concentrations of intrathecal PPX produced increasing durations of sensory block (FIG. 46): sensory nerve block from 80 mM PPX lasted 43.4 ± 6.5min (p < 0.0001 compared to the duration of sensory block from 30 mM PPX). Above 60 mM PPX, animals also had brief motor block, but significantly shorter than their respective sensory blocks (2.84-fold shorter for 60 mM and 2.48-fold shorter for 80 mM PPX, p < 0.0001 for both). All nerve blocks resolved completely as indicated by return to 0% MPE.

[0338] To mimic the effect of a continuous infusion, animals were given 3 repeated doses of 30 mM PPX (FIG.24) at 35 min intervals, which all resulted in sensory block (durations of 21.9 ± 4.2 min, 25.5 ± 4.1 min, 27.7 ± 3.8 min for each repeated dose) without any motor block. To provide a comparison between PPX and ROP in the same animal, the three PPX doses were followed (after nerve block from PPX recovered completely) by a single dose of 15 mM ROP. That dose was selected because it provided a similar duration of sensory block to 30 mM PPX (FIGS. 44 and 46). The ROP dose was not sensory-selective, highlighting the difference between the two compounds.

[0339] The effects of clinically used Rop as an intrathecally administered anesthetic are well-described in the rodent model (see References 33–35). In our model, we confirmed similar findings for 0.75% intrathecal Rop, producing both a sensory block (48.2±5.4min) and a motor block (23.8±3.1min) (FIG. 22). Intrathecal administration of an equal concentration of PPX (0.75%) notably produced a sensory block with an average duration of 24.8±8.7 minutes, demonstrating that PPX exerts anesthetic effects both for peripheral and neuraxial anesthesia. The duration of sensory blockade was significantly shorter than that of Rop (p < 0.0001). Most notably, mirroring results following sciatic nerve injection, intrathecal administration of 0.75%Attorney Docket No.701039-000139WOPT PPX did not produce a motor block. Following recovery from inhalational anesthesia, animals did not demonstrate any difficulties with ambulation, in stark contrast to Rop where animals dragged their bilateral hind limbs.

[0340] Administration of increasing concentrations of intrathecal PPX produced increasing durations of sensory blockade (FIG. 23): 1.5% PPX resulted in a mean sensory block of 31.4±7.5 minutes and 2.0% PPX resulted in a mean sensory block of 43.4±6.5 minutes (p = 0.11 and p < 0.0001, respectively, compared to sensory block duration with 0.75% PPX). At higher concentrations of PPX (1.5-2.0%), animals demonstrated a short duration of motor block, lasting 10.0±2.3 minutes for 1.5% PPX and 17.2±2.2 minutes for 2.0% PPX, significantly shorter than their respective sensory block durations (p < 0.0001 for both 1.5% and 2.0% PPX). All nerve blocks resolved completely as indicated by return to a latency of 2 seconds.

[0341] The effect of repeated administrations of 0.75% PPX (i.e., a sensory selective concentration of PPX) was additionally examined: animals were given 3 repeated doses of 0.75% PPX at 35 minute intervals, followed by a single dose of 0.75% Rop administered 50 minutes following the third dose of PPX (to allow complete recovery of sensory function) (FIG. 24). Following administration of each of the three PPX doses, complete sensory blockade was observed without an accompanying motor block in any hind limb. However, upon administration of 0.75% Rop, all tested hind limbs demonstrated both sensory and motor blockade. Example 6 – Systemic Toxicity of PPX

[0342] Systemic CLA (conventional local anesthetics) toxicity is well-described, typically occurring due to inadvertent intravascular injection or infiltration of an excessive dose. Early symptoms can include perioral paresthesia and audio-visual disturbances, progressing to seizures, dysrhythmias, and potentially lethal cardiovascular collapse (see Reference 5). Cardiac toxicity is mediated by Nav1.5, a TTX-resistant voltage gated sodium channel predominantly located on cardiac muscle, and hERG, a cardiac potassium channel (see References 14, 46).

[0343] To assess the potential for cardiotoxicity with PPX, inhibition of Nav1.5 and hERG currents were measured in vitro with exposure to Rop or PPX (FIGS.25–26). PPX produced minimal inhibition of the hERG current despite exposure to a range of concentrations (1- 100µM). At 100µM, Rop inhibited 82.3±2.6% of hERG currents (similar to published values) (see Reference 47), compared to only 19.4±2.8% hERG inhibition with PPX (p < 0.0001).Attorney Docket No.701039-000139WOPT Further, PPX showed significantly less Nav1.5 current inhibition than Rop: at 100µM, Rop inhibited 44.7±5.6% of Nav1.5 currents (similar to published values) (see Reference 48) compared to only 23.4±3.7% inhibition with PPX (p < 0.0001). Mean ratios of current inhibition by Rop compared to PPX are shown in FIG.27. Rop leads to 4.2 to 5.9-fold higher inhibition of hERG currents compared to PPX. At highest concentrations, Rop exerts 1.9-fold increased inhibition of Nav1.5 compared to PPX. This suggests that PPX could have much less systemic toxicity in vivo than clinically used CLAs.

[0344] Prior studies aimed to investigate the safety of PPX as a metabolite of CLAs have demonstrated that PPX causes significantly less cardiotoxicity, neurotoxicity, and subsequently decreased mortality compared to the CLA bupivacaine (Bup) when administered via the intravenous or intraperitoneal route (see References 19, 20). Therefore, we sought to examine the systemic toxicity of intramuscular (i.e., sciatic) administration of PPX compared to the CLA Rop. CLAs lead to a predictable progression of systemic toxicity, from respiratory distress to contralateral latency, followed by seizures and ultimately terminal apnea with increasing doses (see Reference 49). The LD50of Rop in our model has been demonstrated to be 56±6 mg / kg in adult rats (see Reference 49) and was not repeated in this study for ethical reasons. We instead elected to compare a lower concentration of Rop (~40mg / kg) that is likely to demonstrate some signs of acute systemic toxicity to a very high dose of PPX (75.3±3.2mg / kg) that would be universally fatal for Rop and Bup (see Reference 49). As shown in FIG.28, intramuscular administration of 39.0±1.8 mg / kg Rop to four animals led to 100% respiratory distress (n = 4), 50% contralateral latency (n = 2), and 25% seizures (n =1). In contrast, administration of 75.3±3.2 mg / kg PPX to four animals did not cause respiratory distress, contralateral latency, seizures, or mortality in any of the animals (0%). Example 7 – Cytotoxicity of PPX

[0345] The in vitro cytotoxicity of PPX was evaluated in differentiated C2C12 myotubes and in the pheochromocytoma PC12 cell line, assessing for myotoxicity and neurotoxicity, respectively, by MTS colorimetric assay. Cytotoxicity of PPX was compared to ROP, a clinically used CLA with defined cytotoxicity. C2C12 cell viability following 24h exposure to ROP or PPX was concentration-dependent (FIG. 47). Cell viability was significantly lower with ROP exposure than PPX exposure at all tested concentrations in C2C12 cells (1-3 mM; p < 0.01 for all concentrations). PC12 cell viability was not significantly different following 24 h exposure to ROP or PPX at all tested concentrations (1-3 mM; p > 0.05 for all concentrations) (FIG.48).Attorney Docket No.701039-000139WOPT

[0346] Cell viability in response to PPX was concentration-dependent in both cell lines (FIG. 29). Cytotoxicity of PPX was also compared to bupivacaine (Bup), a clinically used CLA with high cytotoxicity compared to both ropivacaine and lidocaine (see References 50– 52). The IC50(i.e., the concentration required to inhibit growth of half of the cells) was determined after incubation of each cell line with PPX and Bup for 24 hours. The IC50was significantly higher for PPX than Bup in both C2C12 and PC12 cell lines (p < 0.0001), as can be seen in FIG.30. Example 8 – Biocompatibility of PPX

[0347] Tissue reaction to an effective, fully sensory selective concentration (i.e., 30mM) PPX was assessed at 4, 30, and 60 days following sciatic injection. Inflammation and myotoxicity is typically expected to be highest at 4 days following injection (see Reference 53); therefore, tissue reaction at this timepoint was compared to the reaction observed with a clinically effective dose of Rop (15mM). On necropsy, none of the animals demonstrated gross abnormalities in their tissues.

[0348] On day 4, inflammation in rats injected with PPX was minimal (score 0.8±0.4 out of maximum score of 4), similar to the inflammation observed with Rop (score 1.3±0.5; p = 0.2) (FIGS.31 and 33). Inflammation was localized to perifascicular area adjacent to the nerve, likely representing the site of injection. No residual inflammation was observed on days 30 and 60 following PPX injection.

[0349] On day 4, all animals showed some evidence of muscle injury with injection of PPX, ranging from edematous muscle cells with centralized nuclei to shrunken myofibers with basophilic cytoplasm, i.e., regenerating myocytes (FIGS. 32–33). However, regenerating myocytes were limited only to the perifascicular areas with no evidence of myotoxicity deeper in the muscle tissue (score 1.8±1.1 out of maximum of 6). This was similar to myotoxicity observed with Rop injection (3.0±0, p = 0.07). Muscle tissues demonstrated improvement by 30 and 60 days following injection, with only sparse residual cells affected by perifascicular internalization (score 0.6±0.5 at both timepoints). Example 9 – PPX Liposomes

[0350] Liposomes including phospholipids modified with a phenoxy aromatic group phospholipids (Lipo-Ph) were synthesized as described in Reference 62 (Li, Y. et al. Aromatized liposomes for sustained drug delivery. Nat Commun 14, 6659 (2023)). Lipo-Ph encapsulating PPX (PPX@lipo-Ph) were created using the thin-film technique. A drug loading efficiency of 61.6 ± 2.1 % was achieved. Injection of PPX@lipo-Ph at the sciatic nerveAttorney Docket No.701039-000139WOPT produced a sensory nerve blockade of 300.6 ± 51.8 minutes (FIG.34) with no detectable motor nerve blockade. Example 10 – PPX Neurotoxicity

[0351] Because H&E staining has relatively low sensitivity for nerve injury, sciatic nerves were harvested four days after injection of 30 mM PPX, embedded in Epon, and stained with toluidine blue. No nerve injury was observed. Toluidine blue-stained section of nerves can be seen in FIG.35.

[0352] Lumbar spinal cord sections were harvested four days following intrathecal administration of 27.9 mM PPX. Histologic damage in spinal cords following intrathecal administration of CLAs has been demonstrated and is characterized by edema and axonal degeneration at the posterior white matter and roots. No injury was observed in any animal following PPX administration throughout the spinal cord, including in the posterior white matter. Toluidine blue-stained section of posterior white matter of spinal cords on day four can be seen in FIG.36. Example 11 – Effect of chemical permeation enhancers on PPX

[0353] Differences in the sensory selectivity of conventional local anesthetics and similar compounds are generally attributed to differences in hydrophilic-hydrophobic balance, especially those with structures similar to CLAs (such as the amino-amide agents in FIG.38). In this view, the hydrophobicity of the drug affects its ability to penetrate myelin (Schwann cells), which surrounds motor Aα fibers. The relative sensory selectivity of ropivacaine over bupivacaine is ascribed to its hydrophilicity impeding access to myelinated Aα fibers, while less- or un-myelinated Aδ or C fibers are not protected (Drugs with the right balance would be sensory selective as they would be able to penetrate to the relatively unmyelinated Aδ or C fibers (sensory) nerve fibers, but not to the myelinated Aα (motor) fibers.). SSLA from specific concentrations of QX-314 co-administered with a narrow range CPE concentrations, and the modest SSLA from Tonicaine (N-beta-phenylethyl lidocaine) were also attributed to HHB. Chemical permeation enhancers (CPEs) are compounds that can enhance the penetration of some local anesthetics to nerves, in some cases altering their sensory-selectivity. It was hypothesized that the addition of CPEs, by increasing flux to nerve, would impart more motor block to PPX. Previous attempts to achieve clinically relevant local anesthetic formulations with sensory selectivity have been limited by local and systemic toxicity.

[0354] Rats were injected at the sciatic nerve with 30 mM PPX co-dissolved with 30 mM of the CPE Tween 20, producing 100% sensory MPE with a block duration of 69 ± 14 minAttorney Docket No.701039-000139WOPT (FIG.37).100% motor MPE was also achieved, with a duration of 60 ± 23 min. (p > 0.05). The average SMR was 1.14, i.e. co-injection of a CPE with PPX abolished sensory-selectivity.

[0355] It is noted that a variety of mechanisms have been developed for sensory-selective nerve block. In particular, differences in hydrophilic-hydrophobic balance (HHB) have been invoked to explain differences in the degree to which compounds such as amino-ester and amino-amide local anesthetics affect sensory and motor nerve function. PPX is structurally similar to other amino-amide local anesthetics (Figure 38). Without wishing to be bound by a theory, the HHB would affect penetration of compounds to the various axon types, resulting in differential block. Thus, the addition of chemical permeation enhancers (CPEs) such as Tween 20 can negate the effect of PPX’s HHB by permeabilizing the barriers its penetration, such as myelin sheaths on motor neurons. See, for example Sagie I, & Kohane DS, “Prolonged sensory-selective nerve blockade.” Proc Natl Acad Sci USA (2010), vol. 107, pp.3740-3745 and Simons et al. “Effect of chemical permeation enhancers on nerve blockade” Mol Pharmaceutics (2009), vol. 6, pp. 265-273. It has been previously shown that low concentrations of CPEs can impart sensory selectivity to the quaternary lidocaine derivative QX-314, and higher concentrations can take that selectivity away (Sagie I, & Kohane DS, “Prolonged sensory-selective nerve blockade.” Proc Natl Acad Sci USA (2010), vol.107, pp. 3740-3745). Here, 30 mM Tween 20 abolished the sensory-selectivity of PPX. Without wishing to be bound by a theory, this suggests a role for HHB in the sensory-specificity of PPX. However, it is possible that other mechanisms can be involved in the sensory-specificity of PPX. Examples Overview and Summary

[0356] Animals peripherally injected with PPX, a compound best known for being a metabolite of CLA breakdown, exhibited sensory selective nerve blockade at low concentration windows. This was mirrored by ex vivo application of PPX to murine sciatic nerve A-fibers and C-fibers. Perhaps more importantly, similar sensory selectivity was demonstrated in animals treated with PPX intrathecally. In contrast to previously reported formulations achieving sensory selective nerve blockade (see Reference 12), PPX was found to have good biocompatibility with similar minor degrees of inflammation and myotoxicity to CLAs in clinical use, such as ropivacaine. Further, of critical value is the finding that PPX has decreased neurologic and cardiac toxicity compared to CLAs, likely mediated at least in part by significantly lower inhibition of Nav1.5 and hERG channels. We had elected to examine theAttorney Docket No.701039-000139WOPT local anesthetic effects of PPX as part of a larger study assessing CLA derivates and thus admittedly had not anticipated the sensory selectivity that was observed. We therefore examined PPX inhibition of various voltage gated sodium channels as a potential mechanism for sensory selectivity and propose that PPX may achieve its sensory selectivity for both regional and neuraxial anesthesia through selective inhibition Nav1.8 channels.

[0357] PPX is commercially available from vendors such as TCI America (D4348; Portland, OR, USA) and Sigma Aldrich (M0370020 and 1078529; Burlington, MA, USA). However, PPX garnered attention in the scientific and clinical community as a metabolite of CLAs and therefore scientific inquiry has focused on pharmacokinetics and systemic toxicity of this compound (see References 19–25). Whether PPX inhibits voltage gated sodium channels and thereby exerts local anesthetic properties had not previously been considered. Yet, prior investigations are highly relevant and add promise in considering the clinical relevance of our findings. Although sensory selective anesthetics are highly sought after, the potential of systemic toxicity must be considered. CLAs can produce severe cardiac and neurologic toxicities and lead to death in severe cases (see Reference 5). PPX has been found to have decreased systemic toxicity compared to CLAs when administered intravenously and intraperitoneally (see References 19, 20). We were additionally able to confirm these findings when PPX is administered via an intramuscular route. This parallels findings of decreased hERG and Nav1.5 current inhibition by PPX compared to ropivacaine, providing a physiologic explanation of the decreased cardiotoxicity observed.

[0358] Sensory selective local anesthesia requires blockade of nociceptive nerve fibers, including small, thinly myelinated Aδ fibers or unmyelinated C-fibers, but sparing of large, well-myelinated Aα motor nerve fibers (see Reference 54). Various approaches to achieve this have been considered, including alteration of the hydrophilic and hydrophobic balance which can mediate access to myelinated versus unmyelinated fibers, as well as targeting of specific channels, such as TRPV1 and voltage-gated sodium channels (see References 7, 11). In this study, we worked to elucidate the pathophysiological mechanism driving the unexpected sensory selectivity observed with PPX and found that PPX exerts selective Nav1.8 inhibition at tested concentrations, contrasting with CLAs that non-selectively inhibit Nav1.7, Nav1.8, and Nav1.9 at various concentrations. Notably, Nav1.8 channels are highly expressed in small- diameter sensory neurons (see Reference 55). Specifically, strong Nav1.8 expression has been identified in C-, Aδ- and Aα- / β nociceptive units but not in muscle spindle afferent units (see Reference 56). Genetic knockout studies (see Reference 57), antisense knockdown studies (seeAttorney Docket No.701039-000139WOPT Reference 58), and recent discovery of a human gain-of-function mutation in Nav1.8 channels (see Reference 59) provider further support that Nav1.8 contributes to neuropathic, inflammatory, and visceral pain (see References 16, 17). Therefore, it seems plausible that PPX is able to achieve sensory selectivity through its selective inhibition of Nav1.8 at lower concentrations, although higher concentrations may lead to inhibition of additional pathways thereby producing motor blockade in addition to sensory blockade.

[0359] A similar hypothesis is proposed for the sensory selectivity observed following intrathecal administration of PPX. Intrathecal administration of A-803467 or ambroxol (selective Nav1.8 inhibitors) have previously been found to prevent induction of persistent pain in a rodent model, specifically mechanical allodynia and thermal hyperalgesia (see Reference 60). We hypothesize that intrathecal PPX blocks presynaptic Nav1.8 currents via central axons of primary afferent neurons in the spinal cord. Due to the predilection of Nav1.8 channels in nociceptive neurons, this results in a sensory selective nerve blockade at low concentrations, but similar to loss of full sensory selectivity at higher concentrations in peripheral nerve blockade, additional currents may be inhibited following intrathecal administration to account for the motor blockade observed at higher concentrations.

[0360] The sensory selective nerve blockade produced by PPX by peripheral nerve block and intrathecal administration is shorter in duration than the sensory blockade produced by administration of equivalent ropivacaine doses. Higher concentrations of PPX were required for clinical effect, suggesting that PPX is less potent than ropivacaine. However, although tissue toxicity is frequently concentration dependent, clinically effective doses of PPX (30mM) and ropivacaine (15mM) at the sciatic nerve demonstrated similar levels of tissue injury. Ultimately, for clinical use, prolonged sensory nerve blockade in the peripheral and neuraxial setting would be optimal. It is possible that this could be achieved by delayed release formulations (i.e., liposomes, nanoparticles) or continuous catheter-based release systems and will be a major consideration in future studies.

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[0362] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfiedAttorney Docket No.701039-000139WOPT if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0363] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the disclosure, or aspects described herein, is / are referred to as comprising particular elements and / or features, certain embodiments described herein or aspects described herein consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.

[0364] It is also noted that the terms “comprising” and “containing” are intended to be open and permit the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments described herein, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0365] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein.

[0366] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to theAttorney Docket No.701039-000139WOPT above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

Attorney Docket No.701039-000139WOPT CLAIMS WHAT IS CLAIMED IS:

1. A method of inducing anesthesia in a subject, comprising: administering to the subject in need thereof 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia in the subject, and wherein an active molecule that is metabolized to PPX in the subject is not administered to the subject with the PPX.

2. The method of claim 1, wherein the PPX is comprised in a pharmaceutical composition comprising the PPX and a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition does not comprise a molecule that is metabolized to PPX.

3. A method of inducing anesthesia in a subject, comprising: administering to the subject in need thereof an effective amount of a pharmaceutical composition comprising: (i) 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof; and (ii) an active molecule capable of inducing anesthesia in the subject and being metabolized to PPX in the subject, and wherein the pharmaceutical composition comprises the PPX in an amount that is at least about 5% or more of the amount of the active molecule in the composition.

4. A method of inducing anesthesia in a subject, comprising: administering to the subject in need thereof 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia in the subject, and wherein an active molecule that is metabolized to 3’- hydroxy-2’,6’-pipecoloxylidide in the subject is not administered to the subject with the PPX.

5. The method of claim 4, wherein the PPX is comprised in a pharmaceutical composition comprising the PPX and a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition does not comprise a molecule that is metabolized to 3’-hydroxy-2’,6’-pipecoloxylidide.

6. A method of inducing anesthesia in a subject, comprising: administering to the subject in need thereof 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia in the subject, and wherein ropivacaine, bupivacaine, levobupivacaine,Attorney Docket No.701039-000139WOPT mepivacaine, or a pharmaceutically acceptable salt, or an analog or derivative thereof is not administered to the subject with the PPX.

7. The method of claim 6, wherein the PPX is comprised in a pharmaceutical composition comprising the PPX and a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition does not comprise ropivacaine, bupivacaine, levobupivacaine, mepivacaine, or a pharmaceutically acceptable salt, or an analog or derivative thereof.

8. A method of inducing anesthesia in a subject, comprising: administering to the subject in need thereof an effective amount of a pharmaceutical composition comprising: (i) 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof; and (ii) an active molecule capable of inducing anesthesia in the subject and being metabolized to PPX in the subject, and wherein the pharmaceutical composition comprises the PPX in an amount that is at least about 5% or more of the amount of the active molecule in the composition, and wherein the active molecule is ropivacaine, bupivacaine, levobupivacaine, mepivacaine, or a pharmaceutically acceptable salt, or an analog or derivative thereof.

9. The method of any one of claims 2-8, wherein the pharmaceutical composition comprises the PPX at a concentration of 15 mM or higher, e.g., at least about 17.5 mM, at least about 20 mM, at least about 22.5 mM, at least about 25 mM, at least about 27.5 mM, at least 30 mM or higher.

10. The method of claim 9, wherein pharmaceutical composition comprises the PPX at a concentration of from higher than 15 mM to about 250 mM, e.g., from about 17.5 mM to about 200 mM, from about 20 mM to about 150 mM, from about 25 mm to about 100 mM, or from about 22.5 mM to about 42.5 mM.

11. The method of any one of claims 2-8, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.001% to about 99% by weight / weight or weight / volume of the pharmaceutical composition.

12. The method of claim 11, wherein the pharmaceutical composition comprises PPX in an amount from about 60% to about 80% by weight / weight or weight / volume of the pharmaceutical composition.

13. The method of claim 11, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.01% to about 10% by weight / weight or weight / volume of the pharmaceutical composition.Attorney Docket No.701039-000139WOPT 14. The method of claim 11, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.3% to about 4% by weight / weight or weight / volume of the pharmaceutical composition.

15. The method of claim 13, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.1% to about 2% by weight / weight or weight / volume of the pharmaceutical composition.

16. The method of any one of claim 2-15, wherein the pharmaceutical composition further comprises epinephrine.

17. The method of any one of claims 2-15, wherein the pharmaceutical composition further comprises dexamethasone.

18. The method of any one of claims 1-17, wherein PPX is administered locally, and wherein the therapeutic effect is local or systemic.

19. The method of any one of claims 1-8, wherein the PPX administered systemically, and wherein the therapeutic effect is local or systemic.

20. The method of claim 18 or 19, wherein the therapeutic effect is mitigation of pain, alteration of consciousness, sedation, or anesthesia.

21. The method of any one of claims 1-20, wherein the PPX is administered in an amount from about 0.1 mg / kg to about 100 mg / kg of the subject.

22. The method of claim 21, wherein the PPX is administered in an amount from about 0.5 mg / kg to about 10 mg / kg of the subject.

23. The method of claim 22, wherein the PPX is administered in an amount from about 1 mg / kg to about 7 mg / kg of the subject.

24. The method of claim 23, wherein the PPX is administered in an amount from about 2 mg / kg to about 5 mg / kg of the subject.

25. The method of claim 24, wherein the PPX is administered in an amount from about 3 mg / kg to about 4 mg / kg of the subject.

26. The method of any one of claims 2-25, wherein the pharmaceutical composition is formulated for local administration.

27. The method of any one of claims 2-26, wherein the pharmaceutical composition is formulated for systemic administration.

28. The method of any one of claims 2-27, wherein the composition is formulated for oral or intravenous administration.Attorney Docket No.701039-000139WOPT 29. The method of any one of claims 2-28, wherein the composition is in form of a capsule, pill, tablet, aqueous or non-aqueous solution or suspension, gavage, lozenges, dragees, boluses, powder, granule, or a paste.

30. The method of any one of claims 2–29, wherein the pharmaceutical composition is formulated for administration via injection, sustained release, or a combination thereof.

31. The method of any one of claims 2–30, wherein the pharmaceutical composition comprises liposomes, nanoparticles, hydrogels, or a combination thereof.

32. The method of any of claims 1–31, wherein a ratio of sensory block duration to motor block duration (S:M) caused by the administration of PPX is at least about 1.

25.

33. The method of claim 32, wherein a ratio of sensory block duration to motor block duration caused by the administration of PPX is at least about 5.

34. The method of claim 33, wherein a ratio of sensory block duration to motor block duration caused by the administration of PPX is at least about 10.

35. The method of claim 34, wherein a ratio of sensory block duration to motor block duration caused by the administration of PPX is at least about 100.

36. The method of claim 35, wherein a ratio of sensory block duration to motor block duration caused by the administration of PPX is at least about 1,000.

37. The method of any one of claims 1–36, wherein the PPX selectively induces sensory block and does not induce motor block.

38. The method of claim 37, wherein the PPX is administered in an amount that is less than about 90% of the amount of PPX capable of inducing motor block.

39. The method of claim 38, wherein the PPX is administered in an amount that is less than about 85% of the amount of PPX capable of inducing motor block.

40. The method of claim 39, wherein the PPX is administered in an amount that is less than about 80% of the amount of PPX capable of inducing motor block.

41. The method of claim 40, wherein the PPX is administered in an amount that is less than about 75% of the amount of PPX capable of inducing motor block.

42. The method of claim 41, wherein the PPX is administered in an amount that is less than about 70% of the amount of PPX capable of inducing motor block.

43. The method of claim 42, wherein the PPX is administered in an amount that is less than about 50% of the amount of PPX capable of inducing motor block.

44. The method of any of claims 1–43, wherein the administering comprises administering intravenously.Attorney Docket No.701039-000139WOPT 45. The method of any of claims 1–43, wherein the administering comprises administering intrathecally.

46. The method of any of claims 1–43, wherein the administering comprises administering intraperitoneally.

47. The method of any of claims 1–43, wherein the administering comprises administering epidurally.

48. The method of any one of claims 1-43, wherein the administering comprises administering orally or intravenously.

49. The method of any of claims 1–48, wherein the subject is a human.

50. The method of any of claims 1–49, wherein the subject is in labor.

51. A pharmaceutical composition capable of inducing anesthesia in a subject, comprising: (i) 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia in a subject; and (ii) a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition does not comprise an active molecule that is metabolized to PPX in the subject.

52. A pharmaceutical composition capable of inducing anesthesia in a subject, comprising: (iii)2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia in a subject; and (iv) a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition does not comprise an active molecule that is metabolized to 3’-hydroxy-2’,6’-pipecoloxylidide in the subject.

53. A pharmaceutical composition capable of inducing anesthesia in a subject, comprising: (i) 2’,6’-pipecoloxylidide (PPX) or a pharmaceutically acceptable salt thereof in an amount effective to induce anesthesia in a subject; (ii) an active molecule capable of inducing anesthesia in the subject and being metabolized to PPX in the subject; and (iii)a pharmaceutically acceptable carrier or excipient, and wherein the pharmaceutical composition comprises PPX in an amount that is at least about 5% or more of the amount of the active molecule in the composition.Attorney Docket No.701039-000139WOPT 54. The pharmaceutical composition of any one of claims 51-53, wherein the pharmaceutical composition comprises the PPX at a concentration of 15 mM or higher, e.g., at least about 17.5 mM, at least about 20 mM, at least about 22.5 mM, at least about 25 mM, at least about 27.5 mM, at least 30 mM or higher.

55. The pharmaceutical composition of claim 54, wherein pharmaceutical composition comprises the PPX at a concentration of from higher than 15 mM to about 250 mM, e.g., from about 17.5 mM to about 200 mM, from about 20 mM to about 150 mM, from about 25 mm to about 100 mM, or from about 22.5 mM to about 42.5 mM.

56. The pharmaceutical composition of any one of claims 51-53, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.001% to about 99% by weight / weight or weight / volume of the pharmaceutical composition.

57. The pharmaceutical composition of claim 56, wherein the pharmaceutical composition comprises PPX in an amount from about 60% to about 80% by weight / weight or weight / volume of the pharmaceutical composition.

58. The pharmaceutical composition of any one of claims 51-56, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.01% to about 10% by weight / weight or weight / volume of the pharmaceutical composition.

59. The pharmaceutical composition of claim 58, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.3% to about 4% by weight / weight or weight / volume of the pharmaceutical composition.

60. The pharmaceutical composition of claim 58, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.1% to about 2% by weight / weight or weight / volume of the pharmaceutical composition.

61. The pharmaceutical composition of any one of claim 51-60, wherein the pharmaceutical composition further comprises epinephrine.

62. The pharmaceutical composition of any one of claims 51-61, wherein the pharmaceutical composition further comprises dexamethasone.

63. The pharmaceutical composition of any one of claims 51-62, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.1 mg / kg to about 100 mg / kg of the subject.

64. The pharmaceutical composition of any one of claims 51-63, wherein the pharmaceutical composition comprises the PPX in an amount from about 0.5 mg / kg to about 10 mg / kg of the subject.Attorney Docket No.701039-000139WOPT 65. The pharmaceutical composition of any one of claims 51-64, wherein the pharmaceutical composition comprises the PPX in an amount from about 1 mg / kg to about 7 mg / kg of the subject.

66. The pharmaceutical composition of any one of claims 51-65, wherein the pharmaceutical composition comprises the PPX in an amount from about 2 mg / kg to about 5 mg / kg of the subject.

67. The pharmaceutical composition of any one of claims 51-66, wherein the pharmaceutical composition comprises the PPX in an amount from about 3 mg / kg to about 4 mg / kg of the subject.

68. The pharmaceutical composition of any one of claims 51-67, wherein the pharmaceutical composition is formulated for administration via injection, sustained release, or a combination thereof.

69. The pharmaceutical composition of any one of claims 51-68, wherein the pharmaceutical composition further comprises liposomes, nanoparticles, hydrogels, or a combination thereof.

70. The pharmaceutical composition of any one of claims 51-69, wherein the composition is in form of a capsule, pill, tablet, aqueous or non-aqueous solution or suspension, gavage, lozenges, dragees, boluses, powder, granule, or a paste.

71. The pharmaceutical composition of any one of claims 51-70, wherein administration of the pharmaceutical composition to the subject is capable of causing a ratio of sensory block duration to motor block duration (S:M) of at least about 1.

25.

72. The pharmaceutical composition of claim 71, wherein administration of the pharmaceutical composition to the subject is capable of causing a ratio of sensory block duration to motor block duration of at least about 5.

73. The pharmaceutical composition of claim 72, wherein administration of the pharmaceutical composition to the subject is capable of causing a ratio of sensory block duration to motor block duration of at least about 10.

74. The pharmaceutical composition of claim 73, wherein administration of the pharmaceutical composition to the subject is capable of causing a ratio of sensory block duration to motor block duration of at least about 100.

75. The pharmaceutical composition of claim 74, wherein administration of the pharmaceutical composition to the subject is capable of causing a ratio of sensory block duration to motor block duration of at least about 1,000.Attorney Docket No.701039-000139WOPT 76. The pharmaceutical composition of any one of claims 51-75, wherein the pharmaceutical composition selectively induces sensory block and does not induce motor block when administered to the subject.

77. The pharmaceutical composition of claim 76, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 90% of the amount of PPX capable of inducing motor block.

78. The pharmaceutical composition of claim 77, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 85% of the amount of PPX capable of inducing motor block.

79. The pharmaceutical composition of claim 78, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 80% of the amount of PPX capable of inducing motor block.

80. The pharmaceutical composition of claim 79, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 75% of the amount of PPX capable of inducing motor block.

81. The pharmaceutical composition of claim 80, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 70% of the amount of PPX capable of inducing motor block.

82. The pharmaceutical composition of claim 81, wherein the pharmaceutical composition comprises PPX in an amount that is less than about 50% of the amount of PPX capable of inducing motor block.

83. The pharmaceutical composition of any one of claims 51-82, wherein the pharmaceutical composition is formulated for local administration.

84. The pharmaceutical composition of any one of claims 51-83, wherein the pharmaceutical composition is formulated for systemic administration.

85. The pharmaceutical composition of any one of claims 51-84, wherein the pharmaceutical composition is formulated for intravenous administration.

86. The pharmaceutical composition of any one of claims 51-84, wherein the pharmaceutical composition is formulated for intrathecal administration.

87. The pharmaceutical composition of any one of claims 51-84, wherein the pharmaceutical composition is formulated for intraperitoneal administration.

88. The pharmaceutical composition of any one of claims 51-84, wherein the pharmaceutical composition is formulated for epidural administration.Attorney Docket No.701039-000139WOPT 89. The pharmaceutical composition of any one of claims 51-84, wherein the composition is formulated for oral or intravenous administration.

90. The method of any of claims 1-50, wherein an effect of the effective amount of PPX is altered by a second agent co-administered to the subject.

91. The Method of claim 90, wherein the second agent is selected from the group consisting of vasoconstrictors, glucocorticoid receptor agonists, and chemical permeation enhancers.