Patent Application
20230174468
Several classes of molecular container compounds are known, including cyclodextrins, calixarenes, cyclophanes, pillararenes, and cucurbiturils. These molecular container compounds bind to their target molecules in solution and thereby modulate the properties of the target including optical properties, solubility, odor, and even biological activity. Previous workers in the pillar[n]arene area have synthesized container molecules that feature a hydrophobic cavity and carboxylic acid solubilizing groups and showed that they bind with good affinity toward cationic targets in water. A challenge in the field is how to create new or modify existing molecular containers that maintain good solubility in water and simultaneously enhance their binding affinity toward their targets.
The present disclosure provides sulfated pillararenes. The present disclosure also provides methods of making sulfated pillararenes and uses thereof.
In this disclosure, it was shown that, for example, positioning anionic solubilizing groups (e.g., sulfate groups) at the rim of the pillararene cavity significantly enhances their binding affinity toward cationic targets in water and thereby enhances their abilities as sequestering agents for a variety of applications.
In an aspect, the present disclosure provides compounds. The compounds are sulfated pillararenes. A sulfated pillararene comprises a macrocycle core comprising a plurality of aryl groups, where adjacent aryl groups are covalently connected (e.g., linked) via alkyl linking groups (e.g., —CH2— groups). The alkyl linking groups are para on the aryl groups (e.g., 1,4-phenyl linkages). The linkages may be on different phenyl rings of an aryl group and correspond to a para linkage if the different phenyl rings were superimposed. In various examples, one or more or all of the adjacent aryl group(s) are not covalently connected by alkyl linking groups at meta positions on the aryl groups (e.g., 1,3-phenyl linkages (in the case where the linkages are on different phenyl rings or an aryl group the linkages do not correspond to a meta linkage if the different phenyl rings were superimposed)). Non-limiting examples of sulfated pillararenes are provided herein. Non-limiting examples of methods of making sulfated pillararenes are provided herein.
In an aspect, the present disclosure provides compositions comprising one or more sulfated pillararene(s). Non-limiting examples of compositions are described herein.
A composition may comprise one or more sulfated pillararene(s) and one or more pharmaceutical agent(s). In various examples, a pharmaceutical agent comprises one or more positively charged nitrogen atom(s) (e.g., ammonium ions, primary ammonium ions, secondary ammonium ions, tertiary ammonium ions, quaternary ammonium ions, or a combination thereof, where the non-hydrogen group(s) on the ammonium are chosen from aliphatic groups, alkyl groups, aryl groups, and combinations thereof).
In an aspect, the present disclosure provides uses of sulfated pillararenes. Non-limiting examples of uses of sulfated pillararenes are provided herein.
Sulfated pillararenes can be used to sequester various materials, which may be chemical compounds. In various non-limiting examples, one or more sulfated pillararene(s) is/are used to sequester one or more neuromuscular blocking agent(s) (such as, for example, rocuronium, tubocurarine, atracurium, (cis)atracurium besylate, mivacurium, gallamine, pancuronium, vecuronium, and rapacuronium, and the like); one or more anesthesia agent(s) (such as, for example, N-methyl D-aspartate (NMDA) receptor antagonists (e.g., ketamine and the like), short-acting anesthetic agents (e.g., etomidate and the like), and the like); one or more pharmaceutical agent(s) (such as, for example, a drug (e.g., anticoagulants, such as, for example, hexadimethrine and the like), drugs of abuse (e.g., methamphetamine, cocaine, fentanyl, carfentanil, and the like), and the like); one or more pesticide(s) (such as, for example, paraquat, diquat, organochlorines (e.g., DDT, aldrin, and the like), neonicotinoids (e.g., permethrin and the like), organophosphates (e.g., malathion, glyphosate, and the like), pyrethroids, triazines (e.g., atrazine and the like), and the like); one or more dyestuff(s) (such as, for example, methylene blue, nile red, crystal violet, thioflavin T, thiazole orange, proflavin, acridine orange, methylene violet, azure A, neutral red, cyanines, Direct orange 26, disperse dyes (e.g., disperse yellow 3, disperse blue 27, and the like), coumarins, congo red, and the like); one or more malodorous compound(s) (such as, for example, low molecular weight thiols (e.g., C1-C4 thiols), low molecular weight amines (e.g., triethylamine, putrescein, cadaverine, and the like), and the like); or one or more chemical warfare agent(s) (such as for example, nitrogen and sulfur mustards (e.g., bis(2-chloroethyl)ethylamine, bis(2-chloroethyl)methylamine, tris(2-chloroethyl)amine, bis(2-chloroethyl) sulfide, bis(2-chloroethylthioethyl) ether, and the like), nerve agents (such as, for example, those from the G, GV, and V series of nerve agents (e.g. tabun, sarin, soman, cyclosarin, 2-(dimethylamino)ethyl N,N-dimethylphosphoramidofluoridate (GV), novichok agents, VE, VG, VM, VX, and the like), and the like); or the like, or a combination thereof.
For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.
1H NMR spectra (600 MHz, D2O, 298K) recorded for the dilution of host P[6]AS (20.0-0.1 mM). Host P[6]AS is weakly self-associated in water, which is evidenced by the upfield chemical shift changes of the aromatic region at 7.34-7.38 ppm protons.
Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure.
Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value).
As used herein, unless otherwise stated, the term “group” refers to a chemical entity that is monovalent (i.e., has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalently bonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent, trivalent, and the like, radicals). Illustrative examples of groups include:
As used herein, unless otherwise indicated, the term “aryl group” refers to C5 to C18, including all integer numbers of carbons and ranges of numbers of carbons therebetween, aromatic or partially aromatic carbocyclic groups (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, and C18). An aryl group may also be referred to as an aromatic group. The aryl groups can comprise polyaryl groups such as, for example, fused ring or biaryl groups. The aryl group can be unsubstituted or substituted with one or more substituent(s). Examples of substituents include, but are not limited to, various substituents such as, for example, halogens (—F, —Cl, —Br, and —I), azide group, aliphatic groups (e.g., alkyl groups, alkene groups, alkyne groups, and the like), aryl groups, hydroxyl groups, alkoxide groups, carboxylate groups, carboxylic acid groups, ether groups, ester groups, amide groups, thioether groups, thioester groups, and the like, and combinations thereof. A substituent may be or further comprise a sulfonate group or a sulfate group. Examples of aryl groups include, but are not limited to, phenyl groups, biaryl groups (e.g., biphenyl groups and the like), and fused ring groups (e.g., naphthyl groups, anthracene groups, pyrenyl groups, and the like), which may be unsubstituted or substituted.
As used herein, unless otherwise indicated, the term “heteroaryl group” refers to a C1 to C18 monocyclic, polycyclic, or bicyclic ring groups (e.g., aryl groups) comprising one or two aromatic rings containing at least one heteroatom (e.g., nitrogen, oxygen, sulfur, and the like) in the aromatic ring(s), including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, and C18). The heteroaryl groups may be substituted or unsubstituted. Examples of heteroaryl groups include, but are not limited to, benzofuranyl groups, thienyl groups, furyl groups, pyridyl groups, pyrimidyl groups, oxazolyl groups, quinolyl groups, thiophenyl groups, isoquinolyl groups, indolyl groups, triazinyl groups, triazolyl groups, isothiazolyl groups, isoxazolyl groups, imidazolyl groups, benzothiazolyl groups, pyrazinyl groups, pyrimidinyl groups, thiazolyl groups, and thiadiazolyl groups, and the like. Examples of substituents include, but are not limited to, halogens (—F, —Cl, —Br, and —I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), aryl groups, alkoxide groups, amine groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and combinations thereof.
As used herein, unless otherwise indicated, the term “aliphatic” refers to branched or unbranched hydrocarbon groups that, optionally, contain one or more degree(s) of unsaturation. Degrees of unsaturation can arise from, but are not limited to, cyclic aliphatic groups. For example, the aliphatic groups/moieties are a C1 to C40 aliphatic group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, and C40). Aliphatic groups include, but are not limited to, alkyl groups, alkene groups, and alkyne groups. The aliphatic group can be unsubstituted or substituted with one or more substituent(s). Examples of substituents include, but are not limited to, various substituents such as, for example, halogens (—F, —Cl, —Br, and —I), azide group, aliphatic groups (e.g., alkyl groups, alkene groups, alkyne groups, and the like), aryl groups, hydroxyl groups, alkoxide groups, carboxylate groups, carboxylic acid groups, ether groups, ester groups, amide groups, thioether groups, thioester groups, and the like, and combinations thereof.
As used herein, unless otherwise indicated, the term “alkyl group” refers to branched or unbranched saturated hydrocarbon groups. Examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, n- and isopropyl groups, n-, iso-, sec-, and tert-butyl groups, and the like. For example, the alkyl group can be a C1 to C12, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, and C12). The alkyl group can be unsubstituted or substituted with one or more substituent(s). Examples of substituents include, but are not limited to, various substituents such as, for example, halogens (—F, —Cl, —Br, and —I), azide group, aliphatic groups (e.g., alkyl groups, alkene groups, alkyne groups, and the like), aryl groups, hydroxyl groups, alkoxide groups (—OR, where R is an alkyl group), carboxylate groups, carboxylic acid groups, ether groups, ester groups, amide groups, thioether groups, thioester groups, and the like, and combinations thereof.
The present disclosure provides sulfated pillararenes. The present disclosure also provides method making sulfated pillararenes and uses thereof.
In this disclosure, it was shown that, for example, positioning the anionic solubilizing groups at the rim of the pillararene cavity significantly enhances their binding affinity toward cationic targets in water and thereby enhances their abilities as sequestering agents for a variety of applications.
In an aspect, the present disclosure provides compounds. The compounds are sulfated pillararenes. A sulfated pillararene comprises a macrocycle core comprising a plurality of aryl groups, where adjacent aryl groups are covalently connected (e.g., linked) via alkyl linking groups (e.g., —CH2— groups). The alkyl linking groups are para on the aryl groups (e.g., 1,4-phenyl linkages). The linkages may be on different phenyl rings of an aryl group and correspond to a para linkage if the different phenyl rings were superimposed. In various examples, one or more or all of the adjacent aryl group(s) are not covalently connected by alkyl linking groups at meta positions on the aryl groups (e.g., 1,3-phenyl linkages (in the case where the linkages are on different phenyl rings or an aryl group the linkages do not correspond to a meta linkage if the different phenyl rings were superimposed)). Non-limiting examples of sulfated pillararenes are provided herein. Non-limiting examples of methods of making sulfated pillararenes are provided herein.
In various examples, a sulfated pillararene has the following structure:
where Ar is an aryl group attached (e.g., covalently bonded) in a para orientation to the adjacent methylene groups (e.g., 1,4-phenyl group linkage), which may be a part of a larger aryl group; each R is independently chosen from —OS(O)2O−M+ (where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS)), —OS(O)2OH, non-sulfate anionic groups (such as, for example, sulfonate (and corresponding acid) groups (e.g., —O(CH2)mS(O)2O−M+(where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS))/-O(CH2)mS(O)2OH, where m is 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8), —C6H5S(O)2OH, and the like and such groups where the terminal O is removed), carboxylate (and corresponding acid) groups (e.g., —O(CH2)mC(O)O−M+ (where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS))/-O(CH2)mC(O)OH, where n is 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8), and the like, such as for example, —OCH2CO2−M+/—OCH2CO2H groups and the like and such groups where the terminal O is removed), phosphonate (and corresponding acid) groups (e.g., —O(CH2)mP(O)(OH)2M+(where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS))/-O(CH2)mP(O)(OH)2, where m is 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8), and the like, such as for example, —O(CH2)2P(O)(OH)2 and the like and such groups where the terminal O is removed), phosphate groups —OP(O)(OH)2, and the like), substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, substituted or unsubstituted aliphatic groups, O-alkyl groups (comprising an alkyl group), azide groups, —H, substituted or unsubstituted alkyl groups, halogens (e.g., —Br, —F, —I, —Cl), amide groups, cyano groups, substituted or unsubstituted sulfur-containing aliphatic groups (e.g., —S-alkyl and poly thioethers, and the like), nitro groups, amino groups, substituted or unsubstituted nitrogen-containing aliphatic groups (e.g., polyamines, aliphatic groups comprising secondary and/or tertiary amines, and the like), substituted or unsubstituted polyethylene glycol groups, polyether groups, O-aryl groups (e.g., aryloxy groups), ester groups, carbamate groups, imine groups, aldehyde groups, —SO3H groups, —SO3Na groups, —OSO2F groups, —OSO2CF3 groups, —OSO2OR′″ groups (where R′″ are substituted or unsubstituted aryl groups or substituted or unsubstituted alkyl groups), and the like, and combinations thereof, x is 0, 1, 2, or 3; and y is independently at each occurrence 0, 1, 2, 3, or 4, with the proviso that at least one y is 1 and at least one R group is —OS(O)2O−M+(where M+ is Na+, K+) or —OS(O)2OH, or a salt, a partial salt, a hydrate, a polymorph, a stereoisomer, conformational isomer, or a mixture thereof. The R group(s) may be at any position(s) on an aryl group. In the case of an aryl group with multiple R groups, the individual R groups may be at any combination of positions of the aryl group. In various examples, all of the aryl groups comprise an R group that is independently —OS(O)2O−M+ (where M+ is Na+, K+, Ca2, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, and trishydroxymethyl aminomethane (TRIS)) or —OS(O)2OH. In various examples, at least one aryl group does not comprise an R group that is —OS(O)2O−M+(where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS)) or —OS(O)2OH. In various embodiments, the aryl groups may be further substituted with various substitutents, such as, for example, —H, alkyl groups, aliphatic groups, polyethylene glycol groups, or the like, or a combination thereof.
In certain embodiments, M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS).
In certain embodiments, M+ is Na+, K+, and H4N+. In certain embodiments, M+ is Na+.
A sulfated pillararene can comprise various aryl groups. The aryl groups may all be the same or at least two of the aryl groups are different. Non-limiting examples of aryl groups are independently at each occurrence chosen from phenyl groups, fused-ring groups (e.g., naphthyl groups, anthracenyl groups, phenanthrenyl groups, tetracenyl groups, pentacenyl groups, and the like), biaryl groups (e.g., biphenyl groups and the like), terphenyl groups, and the like, and combinations thereof. For avoidance of doubt, a phenyl group, when it is not part of a larger aryl group, unless otherwise described, is a C6H4 group. A phenyl group may be referred to as a phenylene group.
Adjacent aryl groups can be linked by various linkages. The linkages are para-linked phenyl group linkages. In various examples, at least a portion or all of the linkages are 1,4-phenyl group linkage(s). Non-limiting examples of para-linked phenyl group linkages include:
and combinations thereof. These are illustrative examples. Other para-linked phenyl group linkages are within the scope of this disclosure. In various examples, the linkage is not a meta linkage.
An aryl group may comprise one or more phenyl group(s). In various non-limiting examples, at least two, at least three, or at least 4, or all of the one or more phenyl group(s) of one or more of the aryl group(s) comprising the cyclic core of the compound have at least 1 or at least 2 R groups independently chosen from —OS(O)2O−M+ (where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS)) and —OS(O)2OH. All of the aryl groups, one or more or all of which may be phenyl group(s), may comprise a sulfate group —OS(O)2O−M+ (where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS)) or —OS(O)2OH. In various examples, at least one aryl group, which may be a phenyl group, does not comprise a sulfate group (e.g., —OS(O)2O−M+ (where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS)) or —OS(O)2OH).
In various examples, a sulfated pillararene has the following structure:
In various examples, each R is —OS(O)2O−M+ (where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS)) and —OS(O)2OH.
In various examples, a sulfated pillararene has the following structure:
In various examples, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 of the R groups are independently —OS(O)2O−M+ groups (where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS)) or —OS(O)2OH groups.
In an aspect, the present disclosure provides compositions comprising one or more sulfated pillararene(s). Non-limiting examples of compositions are described herein.
A composition may comprise one or more sulfated pillararene(s) and one or more pharmaceutical agent(s). In various examples, a pharmaceutical agent comprises one or more positively charged nitrogen atom(s) (e.g., ammonium ions, primary ammonium ions, secondary ammonium ions, tertiary ammonium ions, quaternary ammonium ions, or a combination thereof, where the non-hydrogen group(s) on the ammonium are chosen from aliphatic groups, alkyl groups, aryl groups, and combinations thereof).
A composition may comprise one or more sulfated pillararene(s), one or more pharmaceutical carrier(s), and, optionally, one or more pharmaceutical agent(s). The compositions described herein can be with one or more pharmaceutically acceptable carrier(s). Suitable pharmaceutically acceptable carriers are known in the art. Some non-limiting examples of pharmaceutically acceptable carriers can be found in: Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, Pa. Lippincott Williams & Wilkins. In various examples, the pharmaceutical carrier is pure water or a buffer, such as PBS buffer or the like.
Compositions comprising one or more sulfated pillararene(s) combined with one or more pharmaceutical agent(s), which may form guest-host complexes, can be prepared at any point prior to use of the composition using any suitable technique. The compound-pharmaceutical agent complexes can be formed, for example, by mixing the compound and the pharmaceutical agent in a suitable solvent. It is desirable that the compound and pharmaceutical agent be soluble in the solvent such that the compound and agent form a non-covalent complex. Any suitable solvent can be used. In certain examples, the solvent is an aqueous solution, which includes, but is not necessarily limited to, water and various buffers (e.g., PBS buffer and the like). Non-aqueous solvents could also be used (e.g., MeOH, EtOH, and other organic solvents, and combinations thereof), and then removed and the compositions if desired can be re-dissolved in an aqueous solution for administration. In general, a solution of a compound(s) can be provided at a known concentration, examples of which include but are not limited to from 0.1 to 90 mM, inclusive and including all integers to the tenth decimal place there between, and a pharmaceutical agent for which enhanced solubility is desired is added to the solution. The agent(s) can be provided, for example, in a solid form. The combination can be shaken or stirred for a period of time and the amount of pharmaceutical agent that is dissolved is monitored. If all added agent goes into solution, more agent can be added until some detectable portion of it remains undissolved (e.g., a solid). The soluble compound-agent complex can then be isolated and analyzed by any suitable technique, such by recovering a centrifuged portion and analyzing it by NMR, to determine the concentration of pharmaceutical agent in solution. In various examples, a compound is provided in a composition comprising the drug at a ratio of at least 1 to 1 as pertains to the compound-agent stoichiometry (e.g., pillararene to drug ratio). In various examples, the pillararene (e.g., pillararene sulfate) to drug ratio is 100:1 to 1:5, including all ratio values and ranges therebetween (e.g., 100:1, 5:1, 1:2, 1:3, 1:4, or 1:5).
Compositions may be prepared at a patient's bedside or by a pharmaceutical manufacture. In the latter case, the compositions can be provided in any suitable container, such as, for example, a sealed sterile vial, ampoule, or the like, and may be further packaged (the combination of which may be referred to as a kit) to include instruction documents for use by a pharmacist, physician, other health care provider, or the like. The compositions can be provided as a liquid, or as a lyophilized or powder form that can be reconstituted if necessary when ready for use. In particular, the compositions can be provided in combination with any suitable delivery form or vehicle, examples of which include but are not limited to liquids, caplets, capsules, tablets, inhalants or aerosol, etc. The delivery devices may comprise components that facilitate release of the pharmaceutical agents over certain time periods and/or intervals, and can include compositions that enhance delivery of the pharmaceuticals, such as nanoparticle, microsphere or liposome formulations, a variety of which are known in the art and are commercially available. Further, each composition described herein can comprise one or more pharmaceutical agent(s).
Compositions of the present disclosure may comprise more than one pharmaceutical agent. Likewise, the compositions can comprise distinct host-guest complexes. For example, a first composition comprising one or more sulfated pillararene(s) and a first pharmaceutical agent can be separately prepared from a composition which comprises the same compound and a second pharmaceutical agent, and such preparations can be mixed to provide a two-pronged (or more) approach to achieving the desired prophylaxis or therapy in an individual. Further, compositions can be prepared using mixed preparations of any of the sulfated pillararene compounds disclosed herein.
A solid substrate may comprise one or more sulfated pillararene(s) disposed on (e.g., chemically bonded to) at least a portion of a surface of the substrate. At least a portion or all of the sulfated pillararenes may be chemically bonded to at least a portion of a surface by covalent bonds, non-covalent bonds, or a combination thereof. Methods of conjugating sulfated pillararenes to solid surfaces are known in the art. In various examples, sulfated pillararenes are conjugated to a surface by covalent bond- and/or non-covalent bond forming reactions including, but not limited to, amide bond formation, azide alkyne cycloaddition, gold thiol interactions, silicon alcohol condensations, and the like, and combinations thereof.
A solid substrate may comprise (or be) various materials. In various non-limiting examples, a solid substrate comprises or is silica (such as, for example, silica particles), polymer beads, polymer resins (such as, for example, polystyrene, poly NIPAM, polyacrylic acid, metal nanoparticles (e.g. gold nanoparticles, silver nanoparticles, magnetic nanoparticles), a metal (such as, for example, gold and the like), or the like, or a combination thereof.
In an aspect, the present disclosure provides uses of sulfated pillararenes. Non-limiting examples of uses of sulfated pillararenes are provided herein, for example, non-limiting examples of uses of sulfated pillararenes are described in the Statements and Examples.
Sulfated pillararenes can be used to sequester various materials, which may be chemical compounds. In various non-limiting examples, one or more sulfated pillararene(s) is/are used to sequester one or more neuromuscular blocking agent(s) (such as, for example, rocuronium, tubocurarine, atracurium, (cis)atracurium besylate, mivacurium, gallamine, pancuronium, vecuronium, and rapacuronium, and the like); one or more anesthesia agent(s) (such as, for example, N-methyl D-aspartate (NMDA) receptor antagonists (e.g., ketamine and the like), short-acting anesthetic agents (e.g., etomidate and the like), and the like); one or more pharmaceutical agent(s) (such as, for example, a drug (e.g., anticoagulants, such as, for example, hexadimethrine and the like), drugs of abuse (e.g., methamphetamine, cocaine, fentanyl, carfentanil, PCP, MDMA, heroin, and the like), and the like); one or more pesticide(s) (such as, for example, paraquat, diquat, organochlorines (e.g., DDT, aldrin, and the like), neonicotinoids (e.g., permethrin and the like), organophosphates (e.g., malathion, glyphosate, and the like), pyrethroids, triazines (e.g., atrazine and the like), and the like); one or more dyestuff(s) (such as, for example, methylene blue, nile red, crystal violet, thioflavin T, thiazole orange, proflavin, acridine orange, methylene violet, azure A, neutral red, cyanines, Direct orange 26, disperse dyes (e.g., disperse yellow 3, disperse blue 27, and the like), coumarins, congo red, and the like); one or more malodorous compound(s) (such as, for example, low molecular weight thiols (e.g., C1-C4 thiols), low molecular weight amines (e.g., triethylamine, putrescein, cadaverine, and the like), and the like); or one or more chemical warfare agent(s) (such as for example, nitrogen and sulfur mustards (e.g., bis(2-chloroethyl)ethylamine, bis(2-chloroethyl)methylamine, tris(2-chloroethyl)amine, bis(2-chloroethyl) sulfide, bis(2-chloroethylthioethyl) ether, and the like), nerve agents (such as, for example, those from the G, GV, and V series of nerve agents (e.g. tabun, sarin, soman, cyclosarin, 2-(dimethylamino)ethyl N,N-dimethylphosphoramidofluoridate (GV), novichok agents, VE, VG, VM, VX, and the like), and the like); one or more hallucinogen(s) (e.g., ergolines, lysergic acid diethylamide (LSD), psilocybin, tryptamines, dimethyltryptamine (DMT), phenethylamines, mescaline, ayahuasca, dextromethorphan, and the like); one or more toxin(s) (e.g., dioxins, perfluoralkylsulfonates (PFAS), perfluorooctanoic acid (PFOA), decabromobiphenyl ether (DECA), heavy metals (e.g. mercury), muscarine, tyramine, strychnine, tetrodotoxin, saxitoxin and the like, cholesterol, deoxycholic acid, N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, phenylalanine, tyrosine, arginine, histamine); one or more metabolite(s) (e.g., toxic metabolites, such as, for example, N-methyl-4-phenylpyridine, spermine, spermidine, N-nitroso compounds e.g. 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone); or the like, or a combination thereof.
A material, which may be a chemical compound, may comprise one or more cationic group. In various examples, a material, which may be a chemical compound, comprises one or more positively charged nitrogen atom(s) (e.g., ammonium ions, primary ammonium ions, secondary ammonium ions, tertiary ammonium ions, quaternary ammonium ions, or a combination thereof, where the non-hydrogen group(s) on the ammonium are chosen from aliphatic groups, alkyl groups, aryl groups, and combinations thereof).
In various examples, a method for sequestering one or more neuromuscular blocking agent(s), one or more anesthesia agent(s), one or more pharmaceutical agent(s), one or more pesticide(s), one or more dyestuff(s), one or more malodorous compound(s), one or more chemical warfare agent(s), one or more hallucinogen(s), one or more toxin(s), one or more metabolite(s) or the like, or a combination thereof comprises contacting the neuromuscular blocking agent(s), the anesthesia agent(s), the pharmaceutical agent(s), the pesticide(s), the dyestuff(s), the malodorous compound(s), the chemical warfare agent(s), the hallucinogen(s), the toxin(s), the metabolite(s), or a combination thereof with one or more sulfated pillararene(s) and/or one or more composition(s), where the neuromuscular blocking agent(s), the anesthesia agent(s), the pharmaceutical agent(s), the pesticide(s), the dyestuff(s), the malodorous compound(s), the chemical warfare agent(s), or a combination thereof are sequestered by the one or more sulfated pillararene(s) and/or one or more composition(s).
The neuromuscular blocking agent(s), the anesthesia agent(s), the pharmaceutical agent(s), the pesticide(s), the dyestuff(s), the malodorous compound(s), the chemical warfare agent(s), the hallucinogen(s), the toxin(s), the metabolite(s), or a combination thereof may be present in an aqueous sample, in a solid sample (such as, for example, a soil sample), in a gas sample, or the like. An aqueous sample may be derived (e.g., via extraction or other methods to isolate the neuromuscular blocking agent(s), the anesthesia agent(s), the pharmaceutical agent(s), the pesticide(s), the dyestuff(s), the malodorous compound(s), the chemical warfare agent(s), the hallucinogen(s), the toxin(s), the metabolite(s), or a combination thereof from the solid sample). The aqueous sample may be a wastewater sample (e.g., a municipal wastewater sample, industrial wastewater sample, and the like), an industrial water sample (e.g., water used to make a commercial product, such as, for example, a reagent, a solvent, or the like), a municipal water sample, or the like.
A composition may comprise one or more pharmaceutically active agent(s). In various non-limiting examples, at least a portion (or all) of the one or more compound(s) have a pharmaceutically active agent(s) disposed in the cavity of the one or more compound(s). Without intending to be bound by any particular theory, it is considered that a complex (which may be referred to as a guest-host complex) is formed from (e.g., one or more interaction(s) between (e.g., one or more non-covalent interactions, such as, for example, one or more non-covalent bond(s), is formed between) the compound(s), which may be referred to as hosts, and the neuromuscular blocking agent(s), the anesthesia agent(s), the pharmaceutical agent(s), which may be pharmaceutical agent(s) with undesirable (e.g., low) water solubility, the pesticide(s), the dyestuff(s), the malodorous compound(s), the chemical warfare agent(s), the hallucinogen(s), the toxin(s), the metabolite(s), or a combination thereof, which may be referred to a guest or guests. A guest-host complex can therefore be considered to be an organized chemical entity resulting from the association of the pharmaceutical agent(s) (guest(s)) and the host held together, for example, by non-covalent intermolecular forces.
A composition can comprise various pharmaceutically active agents. Non-limiting examples of pharmaceutical agents include drugs. The pharmaceutically active agent(s) may have various aqueous solubility. A pharmaceutically active agent may have hydrophobic, hydrophilic, or amphiphilic character.
The complexes may be removed from the aqueous sample, the solid sample, the gas sample, or the like. In various examples, the neuromuscular blocking agent(s), the anesthesia agent(s), the pharmaceutical agent(s), the pesticide(s), the dyestuff(s), the malodorous compound(s), the chemical warfare agent(s), the hallucinogen(s), the toxin(s), the metabolite(s), or a combination thereof are removed from the aqueous sample, the solid sample, the gas sample, or the like using a solid surface with one or more sulfated pillararene(s) disposed thereon.
Sulfated pillararenes can be used to sequester various materials in an individual. In various non-limiting examples, the neuromuscular blocking agent(s), the anesthesia agent(s), the pharmaceutical agent(s), the pesticide(s), the dyestuff(s), the malodorous compound(s), the chemical warfare agent(s), one or more hallucinogen(s), one or more toxin(s), one or more metabolite(s), or a combination thereof is present in an individual and the contacting comprises administration of the one or more compound(s) and/or one or more composition(s) to the individual.
Sulfated pillararenes can be used to reverse drug-induced neuromuscular block and/or anesthesia and/or the effects of one or more drug(s), which may be drugs of abuse in an individual.
In various non-limiting examples, a method for reversing drug-induced neuromuscular block and/or anesthesia and/or the effects of one or more pharmaceutical agent(s) (e.g., one or more drug(s) of abuse) in or on an individual comprising administering to an individual in need of reversal of neuromuscular block and/or reversal of anesthesia and/or reversal of the effects of the one or more pharmaceutical agent(s) (e.g., one or more drug(s) of abuse), one or more sulfated pillararenes, and/or one or more composition(s). The individual may be in need of reversal of drug-induced neuromuscular block. The individual may be in need of reversal of anesthesia. The individual may be in need of reversal of drug-induced neuromuscular block and anesthesia. The individual may be in need of reversal of the effects of one or more pharmaceutical agent(s), such as, for example, one or more drug(s), which may be drug(s) of abuse. The individual may have been exposed to the drug(s) of abuse (e.g., carfentanil and the like) in a terrorist attack.
The sulfated pillararene compounds may be used as containers to solubilize chemical compounds. Improvement of solubility for compounds in, for example, aqueous solutions, is desirable for studying drug compounds and for improvement of drug bioavailability for purposes such as, for example, therapeutic and/or prophylactic purposes. For example, the sulfated pillararenes are be used to enhance the stability (e.g., decrease degradation, increase shelf life, and the like) of drugs in water, the solid state, or both.
In certain examples, the sulfated pillararene compounds can be used to rescue promising drug candidates, which have undesirable solubility and bioavailability, and thus alleviate the attrition in the drug development process for anti-cancer agents and agents intended to treat other diseases. The containers may be used for targeted delivery of drugs to particular cell types, such as, for example, tumor cells and the like, to increase the effectiveness of existing drugs, reduce their toxic side effect(s), or both.
In various examples, a composition comprises one or more sulfated pillararene(s) and one or more pharmaceutical agent(s). Such compositions may be provided as pharmaceutical preparations as described herein.
It is important to emphasize that the pharmaceutical agent(s) that can be included in compositions comprising one or more sulfated pillararene(s) and one or more pharmaceutical agent(s) is not particularly limited. In certain examples, the pharmaceutical agent(s) combined with one or more sulfated pillararene(s) is/are a pharmaceutical agent or agents that is/are poorly water-soluble. In certain other examples, the pharmaceutical agent(s) combined with one or more sulfated pillararene(s) is/are a pharmaceutical agent or agents that is/are water soluble.
Solubility of any particular pharmaceutical agent can be determined, if desired, using any of a variety techniques that are well known to those skilled in the art. Solubility can be ascertained if desired at any pH, such as a physiological pH, and/or at any desired temperature. Suitable temperatures include, but are not necessarily limited to, from 4° C. to 70° C., inclusive, and including all integer ° C. values therebetween.
In connection with poorly soluble or low solubility pharmaceutical agents suitable for use in the present disclosure, in various examples, such agents are considered to be those which have a solubility of less than 100 μM in water or an aqueous buffer.
In various other examples, poorly soluble pharmaceutical agents are considered to include compounds, which are Biopharmaceutics Classification System (BCS) class 2 or class 4 drugs. The BCS is well known to those skilled in the art and is based on the aqueous solubility of drugs reported in readily available reference literature, and for drugs that are administered orally it includes a correlation of human intestinal membrane permeability. (See, for example, Takagi et al., (2006) Molecular Pharmaceutics, Vol. 3, No. 6, pp. 631-643.) The skilled artisan will therefore readily be able to recognize a drug as a member of BCS class 2 or class 4 from published literature, or can test a drug with an unknown BCS or other solubility value to determine whether it has properties consistent with either of those classifications, or for otherwise being suitable for use in the present disclosure. In an example, solubility is determined according to the parameters set forth in this matrix:
Thus, for the purposes of the present disclosure, a poorly soluble pharmaceutical agent that can be combined with one or more sulfated pillararene(s) can be any pharmaceutical agent that falls into the categories sparingly soluble, slightly soluble, very slightly soluble, and practically insoluble as set forth in the above matrix.
Again, it should be emphasized that other than being characterized as having low solubility in aqueous solution, the pharmaceutical agent with which one or more sulfated pillararene(s), which a compound can be combined is not limited. In this regard, at least one utility of the present disclosure is combination of one or more of a wide variety of distinct pharmaceutical agents with one or more sulfated pillararene(s), and as a consequence of combining these compounds with the pharmaceutical agent(s), solubility of the agent(s) is/are increased. In various examples, types of pharmaceutical agents suitable for solubilization include, but are not limited to, mitotic inhibitors (e.g., taxol, a mitotic inhibitor used in cancer chemotherapy, and the like); nitrogen mustard alkylating agents (e.g., Melphalan, trade name Alkeran used for chemotherapy, and the like); benzimidazoles (e.g., Albendazole, marketed as Albenza, Eskazole, Zentel and Andazol, for treatment of a variety of worm infestations, and the like); antagonists of the estrogen receptor in breast tissue which is used to treat breast cancers (e.g., Tamoxifen, which is an estrogen receptor antagonist when metabolized to its active form of hydroxytamoxifen, and the like); antihistamines (e.g., Cinnarizine, marketed as Stugeron and Stunarone for control of symptoms of motion sickness, and the like); thienopyridine class antiplatelet agents (e.g., Clopidogrel, marketed as Plavix for inhibiting blood clots in coronary artery disease and for other conditions, and the like); and antiarrhythmic agents (e.g., Amiodarone, used for treatment of tachyarrhythmias, and the like). Other pharmaceutical agents not expressly listed here are also included within the scope of the disclosure. Some examples of such agents include, but are not limited to, adjuvants for use in enhancing immunological responses, analgesic agents, detectably labeled agents used for diagnostic imaging, and the like. Combinations of any of these example pharmaceutical agents may be used. Sulfated pillararenes may be combined with and improve solubility of pharmaceutical agents that are members of vastly different classes of compounds which are characterized by disparate chemical structures and biological activities.
Compositions of the present disclosure can be administered to any human or non-human animal in need of therapy or prophylaxis for one or more condition(s) for which the pharmaceutical agent is intended to provide a prophylactic of therapeutic benefit. Thus, the individual can be diagnosed with, suspected of having, or be at risk for developing any of a variety of conditions for which a reduction in severity would be desirable. Non-limiting examples of such conditions include cancer, including solid tumors, blood cancers (e.g., leukemia, lymphoma, myeloma, and the like). Specific examples of cancers include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, pseudomyxoma peritonei, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, head and neck cancer, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oliodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, thymoma, Waldenstrom's macroglobulinemia, heavy chain disease, and the like.
In addition to various malignancies, compounds of the present disclosure are also suitable for providing a benefit for cardiovascular related disorders, examples of which include, but are not limited to, angina, arrhythmia, atherosclerosis, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, hypercholesterolemia/hyperlipidemia, mitral valve prolapse, peripheral artery disease, stroke, thrombosis, embolism, other forms of ischemic damage, and the like.
In addition, the compositions of the present disclosure can be used in connection with treating a variety of infectious diseases. It is expected that a variety of agents used to treat and/or inhibit infectious diseases caused by, for example, bacterial, protozoal, helminthic, fungal origins, viral origins, or the like can be aided by use of compositions of the present disclosure.
Various methods known to those skilled in the art can be used to introduce the compounds and/or compositions of the present disclosure to an individual. These methods include, but are not limited to, intravenous, intramuscular, intracranial, intrathecal, intradermal, subcutaneous, oral routes, and the like, and combinations thereof. The dose of the composition comprising a compound and a pharmaceutical agent will necessarily be dependent upon the needs of the individual to whom the composition is to be administered. These factors include, but are not necessarily limited to, the weight, age, sex, medical history, and nature and stage of the disease for which a therapeutic or prophylactic effect is desired. The compositions can be used in conjunction with any other conventional treatment modality designed to improve the disorder for which a desired therapeutic or prophylactic effect is intended, non-limiting examples of which include surgical interventions and radiation therapies. The compositions can be administered once, or over a series of administrations at various intervals determined using ordinary skill in the art, and given the benefit of the present disclosure.
Methods of the present disclosure may be used on various individuals. In various examples, an individual is a human or non-human mammal. Examples of non-human mammals include, but are not limited to, farm animals, such as, for example, cows, hogs, sheep, and the like, as well as pet or sport animals such as, for example, horses, dogs, cats, and the like. Additional non-limiting examples of individuals include, but are not limited to, rabbits, rats, mice, and the like.
The steps of the method described in the various examples disclosed herein are sufficient to carry out the methods of the present disclosure. Thus, in an example, the method consists essentially of a combination of the steps of the methods disclosed herein. In another example, the method consists of such steps.
In an aspect, the present disclosure provides articles comprising compounds of the present disclosure.
The articles may be articles of manufacture. Non-limiting examples of articles include wipes impregnated with one or more compounds of the present disclosure. For example, such a wipe is used to decontaminate a surface from any material capable of being sequestered by a compound (e.g., pillararene of the present disclosure). For example, the wipe is used to decontaminate a surface that has or was previously exposed to a toxin, abused drug, or the like, or a combination thereof.
The following Statements illustrate various embodiments of the present disclosure.
Statement 1. A compound having the following structure:
where Ar is an aryl group where adjacent aryl groups are linked by a para-linked phenyl group linkages (e.g., 1,4-phenyl group linkage(s)) (e.g., the aryl groups are attached in a para orientation to the adjacent methylene groups), which may be a part of a larger aryl group; each R is independently chosen from —OS(O)2O−M+ (where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS)), and —OS(O)2OH, non-sulfate anionic groups (such as, for example, sulfonate (and corresponding acid) groups (e.g., —O(CH2)mS(O)2O−M+(where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS))/-O(CH2)mS(O)2OH, where n is 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8), —C6H5S(O)2OH, and the like and such groups where the terminal O is removed), carboxylate (and corresponding acid) groups (e.g., —O(CH2)mC(O)O−M+ (where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS))/-O(CH2)mC(O)OH, where m is 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8), and the like, such as for example, —OCH2CO2−M+/-OCH2CO2H groups and the like and such groups where the terminal O is removed), phosphonate (and corresponding acid) groups (e.g., —O(CH2)mP(O)(OH)2M+(where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS))/-O(CH2)mP(O)(OH)2, where m is 1 to 8 (e.g., 1, 2 3, 4, 5, 6, 7, 8), and the like, such as for example, —O(CH2)2P(O)(OH)2 and the like and such groups where the terminal O is removed), phosphate groups —OP(O)(OH)2, and the like), substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, substituted or unsubstituted aliphatic groups, O-alkyl groups (comprising an alkyl group), polyether groups (e.g., polyethylene glycol (PEG) groups), azide groups, —H, substituted or unsubstituted alkyl groups, halogens (e.g., —Br, —F, —I, —Cl), amide groups, cyano groups, substituted or unsubstituted sulfur-containing aliphatic groups (e.g., —S-alkyl and poly thioethers, and the like), nitro groups, amino groups, substituted or unsubstituted nitrogen-containing aliphatic groups (e.g., polyamines, aliphatic groups comprising secondary and/or tertiary amines, and the like), substituted or unsubstituted polyethylene glycol groups, polyether groups, O-aryl groups (e.g., aryloxy groups), ester groups, carbamate groups, imine groups, aldehyde groups, —SO3H groups, —SO3Na groups, —OSO2F groups, —OSO2CF3 groups, —OSO2OR′″ groups (where R′″ are substituted or unsubstituted aryl groups or substituted or unsubstituted alkyl groups), and the like, and combinations thereof; x is 0, 1, 2, or 3; and y is independently at each occurrence 0, 1, 2, 3, or 4, with the proviso that at least one y is 1 and at least one R group is —OS(O)2O−M+ (where M+ is Na+, K+, Ca2+, Mg2+, Zn2+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+, or a cationic form of ethylenediamine, piperazine, or trishydroxymethyl aminomethane (TRIS)) or —OS(O)2OH, or a salt, a partial salt, a hydrate, a polymorph, a stereoisomer, conformational isomer, or a mixture thereof. The R group(s) may be at any position(s) on an aryl group. In the case of an aryl group with multiple R groups, the individual R groups may be at any combination of positions of the aryl group. In various embodiments, the aryl groups may be further substituted with various substituents.
Statement 2. A compound according to Statement 1, where the aryl groups are independently at each occurrence chosen from phenyl groups, fused-ring groups (e.g., naphthyl groups, anthracenyl groups, phenanthrenyl groups, tetracenyl groups, pentacenyl groups, and the like), biaryl groups (e.g., biphenyl groups and the like), terphenyl groups, and the like.
Statement 3. A compound according to Statements 1 or 2, where at least two, at least three, or at least 4, or all of the one or more phenyl group(s) of one or more of the aryl group(s) comprising the cyclic core of the compound have at least 1 or at least 2 R groups independently chosen from —OS(O)2O−M+ and —OS(O)2OH.
Statement 4. A compound according to Statement 3, where the compound has the following structure:
In various examples, each R is —OS(O)2O−M+ and —OS(O)2OH.
Statement 5. A compound according to any one of the preceding Statements, where all of the aryl groups comprise an R group that is independently —OS(O)2O−M+ or —OS(O)2OH.
Statement 6. A compound according to any one of Statements 1-3, where at least one aryl group does not comprise an R group that is —OS(O)2O−M+ or —OS(O)2OH.
Statement 7. A compound according to Statement 1, where the compound has the following structure:
Statement 8. A compound according to Statement 7, where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 of the R groups are independently —OS(O)2O−M+ groups or —OS(O)2OH groups.
Statement 9. A compound according to Statements 7 or 8, where each phenyl group comprising the cyclic core of the compound has at least 1 or at least 2 R groups independently chosen from —OS(O)2O−M+ and —OS(O)2OH.
Statement 10. A compound according to any one of Statements 7-9, where at least one phenyl group does not comprise an R group that is —OS(O)2O−M+ or —OS(O)2OH.
Statement 11. A composition comprising one of more compound(s) according to any one of the preceding Statements.
Statement 12. A composition according to Statement 11, further comprising a pharmaceutical carrier.
Statement 13. A composition according to Statement 11, where the one or more compound(s) are disposed (e.g., chemically bonded) to at least a portion of a solid substrate.
Statement 14. A composition according to Statement 13, where the solid substrate comprises (or is) silica (such as, for example, silica particles), polymer beads, polymer resins (such as, for example, polystyrene, poly NIPAM, polyacrylic acid), metal nanoparticles (e.g. gold nanoparticles, silver nanoparticles, magnetic nanoparticles), a metal (such as, for example, gold and the like), or the like, or a combination thereof.
Statement 15. A composition according to any one Statements 11-14, where at least a portion (or all) of the one or more compound(s) have a pharmaceutically active agent(s) disposed in the cavity of the one or more compound(s) (e.g., non-covalently complexed to the compound(s)).
Statement 16. A method for sequestering: one or more neuromuscular blocking agent(s) (such as, for example, rocuronium, tubocurarine, atracurium, (cis)atracurium besylate, mivacurium, gallamine, pancuronium, vecuronium, and rapacuronium, and the like); one or more anesthesia agent(s) (such as, for example, N-methyl D-aspartate (NMDA) receptor antagonists (e.g., ketamine and the like), short-acting anesthetic agents (e.g., etomidate and the like), and the like); one or more pharmaceutical agent(s) (such as, for example, a drug (e.g., anticoagulants, such as, for example, hexadimethrine and the like), drugs of abuse (e.g., methamphetamine, cocaine, fentanyl, carfentanil, PCP, MDMA, heroin, and the like), and the like); one or more pesticide(s) (such as, for example, paraquat, diquat, organochlorines (e.g., DDT, aldrin, and the like), neonicotinoids (e.g., permethrin and the like), organophosphates (e.g., malathion, glyphosate, and the like), pyrethroids, triazines (e.g., atrazine and the like), and the like); one or more dyestuff(s) (such as, for example, methylene blue, nile red, crystal violet, thioflavin T, thiazole orange, proflavin, acridine orange, methylene violet, azure A, neutral red, cyanines, Direct orange 26, disperse dyes (e.g., disperse yellow 3, disperse blue 27, and the like), coumarins, congo red, and the like); one or more malodorous compound(s) (such as, for example, low molecular weight thiols (e.g., C1-C4 thiols), low molecular weight amines (e.g., triethylamine, putrescein, cadaverine, and the like), and the like); or one or more chemical warfare agent(s) (such as for example, nitrogen and sulfur mustards (e.g., bis(2-chloroethyl)ethylamine, bis(2-chloroethyl)methylamine, tris(2-chloroethyl)amine, bis(2-chloroethyl) sulfide, bis(2-chloroethylthioethyl) ether, and the like), nerve agents (such as, for example, those from the G, GV, and V series of nerve agents (e.g. tabun, sarin, soman, cyclosarin, 2-(dimethylamino)ethyl N,N-dimethylphosphoramidofluoridate (GV), novichok agents, VE, VG, VM, VX, and the like), and the like); one or more hallucinogen(s) (e.g., ergolines, lysergic acid diethylamide (LSD), psilocybin, tryptamines, dimethyltryptamine (DMT), phenethylamines, mescaline, ayahuasca, dextromethorphan, and the like); one or more toxin(s) (e.g., dioxins, perfluoralkylsulfonates (PFAS), perfluorooctanoic acid (PFOA), decabromobiphenyl ether (DECA), heavy metals (e.g. mercury), muscarine, tyramine, strychnine, tetrodotoxin, saxitoxin and the like, cholesterol, deoxycholic acid, N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, phenylalanine, tyrosine, arginine, histamine); one or more metabolite(s) (e.g., toxic metabolites, such as, for example, N-methyl-4-phenylpyridine, spermine, spermidine, N-nitroso compounds e.g. 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone); or the like, or a combination thereof are sequestered by the one or more compound(s) according to any one of Statements 1-10 and/or one or more composition(s) according to any one of Statements 11-14.
Statement 17. A method according to Statement 16, where the neuromuscular blocking agent(s), the anesthesia agent(s), the pharmaceutical agent(s), the pesticide(s), the dyestuff(s), the malodorous compound(s), the chemical warfare agent(s), the hallucinogen(s), the toxin(s), the metabolite(s), or a combination thereof is present in an aqueous sample, in a solid sample (such as, for example, a soil sample), in a gas sample, on a solid surface, or the like.
Statement 18. A method according to Statement 17, where the aqueous sample is a wastewater sample (e.g., a municipal wastewater sample, industrial wastewater sample, and the like), an industrial water sample (e.g., water used to make a commercial product, such as, for example, a reagent, a solvent, or the like), a municipal water sample, or the like.
Statement 19. A method according to any one of Statements 16-18, where a complex is formed from (e.g., one or more interaction(s) between (e.g., one or more non-covalent bond(s) is formed between) the compound(s) and the neuromuscular blocking agent(s), the anesthesia agent(s), the pharmaceutical agent(s), the pesticide(s), the dyestuff(s), the malodorous compound(s), the chemical warfare agent(s), one or more hallucinogen(s), one or more toxin(s), one or more metabolite(s), or a combination thereof.
Statement 20. A method according to any one of Statements 16-19, where the complex is removed from the aqueous sample, the solid sample, the gas sample, or the like.
Statement 21. A method according to Statement 16, where the neuromuscular blocking agent(s), the anesthesia agent(s), the pharmaceutical agent(s), the pesticide(s), the dyestuff(s), the malodorous compound(s), the chemical warfare agent(s), one or more hallucinogen(s), one or more toxin(s), one or more metabolite(s), or a combination thereof is present in and/or on an individual and the contacting comprises administration of the one or more compound(s) and/or one or more composition(s) to the individual.
Statement 22. A method according to Statement 21, where the individual is a human or a non-human mammal.
Statement 23. A method for reversing drug-induced neuromuscular block and/or anesthesia and/or the effects of one or more pharmaceutical agent(s) (e.g., one or more drug(s) of abuse) in an individual comprising administering to an individual in need of reversal of neuromuscular block and/or reversal of anesthesia and/or reversal of the effects of one or more pharmaceutical agent(s) (e.g., one or more drug(s) of abuse) one or more compound(s) according to any one of Statements 1-10 and/or one or more composition(s) according to any one of Statements 11-14.
Statement 24. A method according to Statement 23, where the individual is in need of reversal of drug-induced neuromuscular block.
Statement 25. A method according to Statement 23, where the individual is in need of reversal of anesthesia.
Statement 26. A method according to Statement 23, where the individual is in need of reversal of drug-induced neuromuscular block and anesthesia.
Statement 27. A method according to Statement 23, where the individual is in need of reversal of the effects of one or more pharmaceutical agent(s) are chosen from one or more drug(s) of abuse, one or more pesticide(s), one or more chemical warfare agent(s), one or more nerve agent(s), one or more hallucinogen(s), one or more toxin(s), and/or one or more metabolite(s). In an example, the individual was exposed to the one or more drug(s) of abuse (e.g., carfentanil and the like), one or more pesticide(s), one or more chemical warfare agent(s), one or more nerve agent(s), one or more hallucinogen(s), one or more toxin(s), one or more metabolite(s) in a terrorist attack, and combinations thereof.
Statement 28. A method according to any one of Statements 23-27, wherein the individual in need is a human.
Statement 29. A method according to any one of Statements 23-27, where the individual in need is a non-human mammal.
Statement 30. A method according to any one of Statements 27-29, where the drug of abuse is fentanyl.
Statement 31. A method according to Statement 30, wherein the one or more compound(s) are administered at least five minutes after administration of the fentanyl.
Statement 32. A method for prophylaxis and/or therapy of a condition in an individual comprising administering to an individual in need of the prophylaxis and/or the therapy one or more compound(s) according to any of Statements 1-10 and one or more pharmaceutical agent(s), where the compound(s) and the pharmaceutical agent(s) are present as complex (or a composition, which may be a pharmaceutical composition, comprising the complex(es)), where subsequent to the administration the therapy and/or the prophylaxis of the condition in the individual occurs.
Statement 33. A method according to Statement 32, where one or more of the pharmaceutical agent(s) has/have a solubility of less than 100 μM in an aqueous solvent.
Statement 34. A compound according to any one of Statements 1-10, a composition according to any one of Statements 11-15, or a method according to any one of Statements 16-33, where M+ is Na+, K+, H4N+, Et3NH+, Me4N+, (HOCH2CH2)3NH+.
Statement 35. A compound according to any one of Statements 1-10, a composition according to any one of Statements 11-15, or a method according to any one of Statements 16-34, where M+ is Na+.
The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any manner.
This example provides a description of compounds of the present disclosure, methods of making the compounds, characterization of the compounds, and uses of the compositions.
General experimental details. Starting materials were purchased from commercial suppliers and were used without further purification or were prepared by literature procedures. Melting points were measured on a Meltemp apparatus in open capillary tubes and are uncorrected. IR spectra were recorded on a JASCO FT/IR 4100 spectrometer and are reported in cm−1. 1H NMR spectra were measured on Bruker instruments operating at 400 or 600 MHz for 1H and 100 MHz for 13C. Mass spectrometry was performed using a JEOL AccuTOF electrospray instrument (ESI). ITC data was collected on a Malvern Microcal PEAQ-ITC instrument.
Synthetic procedures and characterization data.
Host P[5]AS.
The first two compounds (2 and 3) were synthesized by using methods adapted from methods known in the art. The procedure for the last step was: to a mixture of compound 3 (0.200 g, 0.328 mmol) and pyridine sulfur trioxide complex (1.050 g, 6.56 mmol) was added dry pyridine (10 mL). The resulting mixture was stirred at 90° C. under N2 for 24 hours. The reaction mixture was cooled to RT. The product precipitated out of the solution and was collected by filtration. The solid was slurried in water (5 mL), and the pH was adjusted to 8.4 by slow addition of saturated aqueous NaHCO3. After addition of EtOH (35 mL), the crude product was collected by centrifugation 7000 rpm×7 min. The precipitate was suspended in ethanol (20 mL×2), sonicated for 30 minutes, and solid collected by centrifugation. The crude solid was redissolved in minimum amount of water (2 ml) and purified by size exclusion chromatography using Sephadex® G25 resin (30 mm×200 mm) and eluted by water. Pure product was collected as the front fractions. After drying under high vacuum, the compound P[5]AS was obtained as a white solid (0.374 g, 0.229 mmol, 70% yield). M.p.>310° C. (decomposed). IR (ATR, cm−1): 3490w, 1630m, 1497m, 1399m, 1234s, 1116s, 1042s, 995m, 941m, 858m, 806m. 1H NMR (600 MHz, D2O): 7.31 (s, 10H), 4.00 (s, 10H). 13C NMR (150 MHz, D2O, EtOH as internal reference): 147.4, 134.1, 125.6, 30.8. MS (ESI): m/z 791.78179 ([M-2Na]2−), calculated 791.79597.
Host P[5]ACS.
A solution of P[5]A (0.200 g, 0.28 mmol) in NaOH (10 wt %, 2 mL) was treated dropwise with a solution of propane sultone (0.687 g, 5.63 mmol) in acetone (4 mL). This solution was stirred at RT for 5 days (d) and then EtOH (25 mL) was added to the mixture to yield the crude product as a precipitate. The precipitate was obtained by filtration, the solid was dissolved in H2O (0.5 mL), and then re-precipitated by the addition of EtOH (5 mL) to yield P[5]ACS as a light yellow solid (45 mg, 0.022 mmol, 8%). M.p.>300° C. (decomposed). IR (ATR, cm−1): 3452w, 2936w, 1725m, 1625m, 1479m, 1471m, 1406m, 1181s, 1035s, 951w, 798m, 756m. 1H NMR (400 MHz, D2O): 6.76 (s, 10H), 3.90 (m, 10H), 3.86 (s, 10H), 3.68 (m, 10H), 3.05 (m, 20H), 2.08 (m, 20H). 13C NMR (150 MHz, D2O, EtOH as internal reference): δ 150.9, 129.8, 116.7, 68.5, 49.1, 31.1, 25.5. HR-MS (ESI): m/z 1002.03215 ([M-2Na]2−), calculated 1002.03072.
Host P[6]AS.
The first two compounds (7 and 8) were synthesized by using methods adapted those known in the art. The procedure for the last step was: to a mixture of compound 8 (0.200 g, 0.27 mmol) and pyridine sulfur trioxide complex (1.090 g, 6.83 mmol) was added dry pyridine (10 mL). The resulting mixture was stirred at 70° C. under N2 for 24 hours. The reaction mixture was cooled to RT. The product precipitated out of the solution and was collected by filtration. The solid was slurried in water (5 mL), and the pH was adjusted to 8.4 by slow addition of saturated aqueous NaHCO3. After addition of EtOH (35 mL), the crude product was collected by centrifugation 7000 rpm×7 min. The precipitate was suspended in ethanol (20 mL×2), sonicated for 30 minutes, and solid collected by centrifugation. The crude solid was redissolved in minimum amount of water (2 ml) and purified by size exclusion chromatography using Sephadex® G25 resin (30 mm×200 mm) and eluted by water. Pure product was collected as the front fractions. After drying under high vacuum, the compound P[6]AS was obtained as a white solid (0.352 g, 0.18 mmol, 66% yield). M.p.>290° C. (decomposed). IR (ATR, cm−1): 3509w, 1712m, 1630m, 1498m, 1364m, 1237m, 1113s, 1045s, 995m, 942m, 861m, 813m. 1H NMR (600 MHz, D2O): 7.35 (s, 12H), 4.11 (s, 12H). 13C NMR (150 MHz, D2O and CD3OD 10:1): δ 148.1, 133.6, 125.6, 31.5. MS (ESI): m/z 954.7593 ([M-2Na]2−), calculated 954.7537.
Host P[6]A8S.
The first three compounds were synthesized by using methods adapted from those known in the art. The procedure for the last step was: to a mixture of compound octahydroxy pillar[6]arene (0.100 g, 0.15 mmol) and pyridine sulfur trioxide complex (0.479 g, 3 mmol) was added dry pyridine (5 mL). The resulting mixture was stirred at 70° C. under N2 for 24 hours. The reaction mixture was cooled to RT. The product precipitated out of the solution and was collected by filtration. The solid was slurried in water (4 mL), and the pH was adjusted to 8.4 by slow addition of saturated aqueous Na2CO3. After addition of EtOH (10 mL), the crude product was collected by centrifugation 7000 rpm×7 min. The precipitate was suspended in ethanol (10 mL×2), sonicated for 30 minutes, and solid collected by centrifugation. The crude solid was redissolved in minimum amount of water (0.5 ml) and purified by size exclusion chromatography using Sephadex® G25 resin (30 mm×200 mm) and eluted by water. Pure product was collected as the front fractions. After drying under high vacuum, the compound P[6]A8S (10) was obtained as a white solid (0.075 g, 0.051 mmol, 33% yield). M.p.>285° C. (decomposed). IR (ATR, cm−1): 3491w, 1630m, 1440s, 1234s, 1078m, 1043s, 941m, 878m, 800m, 667m. 1H NMR (600 MHz, D2O): 7.39 (s, 4H), 7.25 (s, 4H), 7.04 (s, 8H), 4.07 (s, 4H), 3.99 (s, 8H). 13C NMR (150 MHz, D2O, EtOH as internal reference): δ 147.7, 147.6, 138.7, 134.2, 133.2, 129.2, 125.3, 125.1, 35.7, 31.1. MS (ESI): m/z 718.88334 ([M-2Na]2−), calculated 718.88632.
Host P[7]AS.
The first two compounds were synthesized by using methods adapted from those known in the art. The procedure for the last step was: to a mixture of compound (HO)14 pillar[7]arene (0.020 g, 0.023 mmol) and pyridine sulfur trioxide complex (0.375 g, 2.34 mmol) was added dry pyridine (3 mL). The resulting mixture was stirred at 70° C. under N2 for 24 hours. The reaction mixture was cooled to RT. The product precipitated out of the solution and was collected by filtration. The solid was slurried in water (1 mL), and the pH was adjusted to 8.4 by slow addition of saturated aqueous Na2CO3. After addition of EtOH (10 mL), the crude product was collected by centrifugation 7000 rpm×7 min. The precipitate was suspended in ethanol (10 mL×2), sonicated for 30 minutes, and solid collected by centrifugation. The crude solid was redissolved in minimum amount of water (0.5 ml) and purified by size exclusion chromatography using Sephadex® G25 resin (30 mm×200 mm) and eluted by water. Pure product was collected as the front fractions. After drying under high vacuum, the compound P[7]AS was obtained as a white solid (0.025 g, 0.011 mmol, 46% yield). M.p.>290° C. (decomposed). IR (ATR, cm−1): 3494w, 1624m, 1444s, 1244m, 1102s, 1049s, 995m, 875m, 807m, 614s. 1H NMR (600 MHz, D2O): 7.29 (s, 14H), 4.14 (s, 14H). 13C NMR (150 MHz, D2O, Dioxane as external reference): δ 147.6, 132.8, 124.7, 30.8. MS (ESI): m/z 547.36023 ([M-4Na]4−), calculated 547.36080.
Rim-P[5]AS.
The starting material 2-(Benzyloxy)-5-methoxybenzyl alcohol was synthesized based on methods known in the art. The penta-hydroxy pillar[5]arene compound was synthesized by using the methods known in the art. To a mixture of penta-hydroxy pillar[5]arene (0.200 g, 0.328 mmol) and pyridine sulfur trioxide complex (1.050 g, 6.56 mmol) was added dry pyridine (10 mL). The resulting mixture was stirred at 70° C. under N2 for 24 hours. The reaction mixture was cooled to RT. The product precipitated out of the solution and was collected by filtration. The solid was slurried in water (5 mL), and the pH was adjusted to 9 by slow addition of saturated aqueous NaHCO3. Addition of EtOH (EtOH/H2O v/v=2:1) gave a precipitate which was removed by centrifugation (7000 rpm×10 min). The filtrate was collected as the crude product and was redissolved in a minimum amount of water (2 mL) and purified by size exclusion chromatography using Sephadex® G25 resin (5 cm×50 cm) with water as eluent. The front fractions eluting from the column contain pure product. After drying under high vacuum, Rim-P[5]AS was obtained as a white solid (0.374 g, 0.229 mmol, 67% yield, content: ˜92%, determined by using sodium 2-bromoethanesulfonate as 1H NMR internal standard). M.p.>300° C. (decomposed). 1H NMR (400 MHz, D2O): 7.21 (s, 5H), 6.56 (s, 5H), 3.90 (s, 10H), 3.24 (s, 15H). 13C NMR (150 MHz, D2O, EtOH as internal reference): 155.3, 143.2, 134.5, 129.6, 124.7, 114.9, 56.7, 30.6.
Solubility Determination.
Determination of the solubility of P[5]AS in water. Compound P[5]AS was added in excess to 0.5 mL deuterium oxide. This suspension was magnetically stirred at room temperature overnight and then centrifuged (4500 rpm) twice for 10 min each time. Supernatant (50 μL) and sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TMSP) (10 mM, 50 L in D2O) were added into 0.4 mL deuterium solvent. The concentration of P[5]AS was measured with 1H NMR and calculated using sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TMSP) as internal reference.
Determination of the solubility of P[6]AS in water. Compound P[6]AS was added in excess to 0.5 mL deuterium oxide. This suspension was magnetically stirred at room temperature overnight and then centrifuged (4500 rpm) twice for 10 min each time. Supernatant (50 μL) and sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TMSP) (10 mM, 50 L in D2O) were added into 0.4 mL deuterium solvent. The concentration of P[6]AS was measured with 1H NMR and calculated using sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TMSP) as internal reference.
Determination of Ka between various hosts and cationic guests or drugs of abuse or neuromuscular blocking agents using Isothermal Titration Calorimetry (ITC). All ITC experiments were conducted in the 200 μL working volume of the sample cell of the PEAQ ITC instrument. We used an injection syringe of 40 μL capacity. In each case, the host and guest solutions were prepared in a 20 mM NaH2PO4 buffer (pH 7.4). The sample cell was filled to capacity (200 μL) with the host solution and the guest solution was titrated in (first injection=0.4 μL, subsequent 18 injections=2 μL). The binding data was fitted using the 1:1 binding model in MicroCal PEAQ-ITC analysis software. In cases where Ka was too large to determine by direct titration, competition ITC titrations were performed where a competitive guest of known Ka and ΔH was included in the ITC cell along with the host which was then titrated with the guest whose Ka was to be determined.
WP5, WP6, and the following were compounds used in comparative examples:
1H NMR spectra of selected drugs with hosts.
1H NMR spectra of competition binding.
The crystal structures P[6]AS was also determined.
11 + Y − X, 1 − X, +Z;
21 − Y, +X − Y, +Z;
31 − Y + X, 1 − Y, 1/2 − Z;
4+X, +X − Y, 1/2 + Z;
52 − X, 1 − Y, 1 − Z;
61 − X, 1 − Y, 1 − Z;
72 − X, 1 − X + Y, 1/2 − Z;
81 + Y − X, +Y, 1/2 + Z;
91 − Y, 1 + X − Y, +Z;
10+Y, +X, 1/2 − Z;
11−X, −Y, 1 − Z;
12−Y + X, +X, 1 − Z;
13+Y, −X + Y, 1 − Z
11 + Y − X, 1 − X, +Z;
21 − Y, +X − Y, +Z;
31 − Y + X, 1 − Y, 1/2 − Z;
4+X, +X − Y, 1/2 + Z;
52 − X, 1 − Y, 1 − Z;
61 − X, 1 − Y, 1 − Z;
72 − X, 1 − X + Y, 1/2 − Z;
81 + Y − X, +Y, 1/2 + Z;
91 − Y, 1 + X − Y, +Z;
10+Y, +X, 1/2 − Z;
11+X, +X − Y, −1/2 + Z;
121 + Y − X, +Y, −1/2 + Z;
13−Y + X, 1 − Y, 1/2 − Z;
14−X, −Y, 1 − Z;
15−Y + X , +X, 1 − Z;
16+Y, −X + Y, 1 − Z;
17+Y − X, 1 − X, +Z
11 − Y, +X − Y, +Z;
22 − X, 1 − Y, 1 − Z;
32 − X, 1 − X + Y, 1/2 − Z;
4+Y, +X, 1/2 − Z;
5+X, +X − Y, −1/2 + Z;
61 + Y − X, +Y, −1/2 + Z;
7−Y + X, 1 − Y, 1/2 − Z;
81 − X, 1 − Y, 1 − Z;
91 + Y − X, 1 − X, +Z;
101 + Y − X, +Y, 1/2 + Z;
111 − Y, 1 + X − Y, +Z;
121 + X, 1 + Y, +Z;
13−Y + X , +X, 1 − Z;
14−X, −Y, 1 − Z;
15+Y, −X + Y, 1 − Z;
16+Y − X, 1 − X, +Z;
17+Y − X, −X, +Z;
18−Y, +X − Y, +Z
Experimental. A suitable single crystal of P[6]AS was selected and measured on a Bruker Smart Apex2 diffractometer. The crystal corresponded to C45.44H24Na12O65.65S12. The crystal was kept at 150(2) K during data collection. The integral intensity were correct for absorption using SADABS software using multi-scan method. Resulting minimum and maximum transmission are 0.634 and 0.959 respectively. The structure was solved with the ShelXT-2014 (Sheldrick, 2015a) program and refined with the ShelXL-2015 (Sheldrick, 2015c) program and least-square minimisation using ShelX software package. Number of restraints used=387.
Crystal structure determination. Crystal data for C45.44H24Na12O65.65S12 (M=2280.86 g/mol): trigonal, space group P-3cl (no. 165), a=15.112(2) Å, c=20.365(3) Å, V=4027.8(13) Å3, Z=2, T=150(2) K, μ(MoKα)=0.519 mm−1, Dcalc=1.881 g/cm3, 23483 reflections measured (4°≤2Θ≤53.178°), 2810 unique (Rint=0.0602, Rsig=0.0410) which were used in all calculations. The final R1 was 0.0448 (I>2σ(I)) and wR2 was 0.1097 (all data).
Refinement details. H atoms (except those in disordered solvent) were located from difference Fourier map and freely refined including Uiso. Water and ethanol solvent is heavily disordered and was modelled with partially occupied O and C atoms.
This Example provides synthesis, x-ray crystal structure, and molecular recognition properties of pillar[n]arene derivative P[6]AS which is referred to herein from time to time as Pillar[6]MaxQ, along with analogues P[5]AS and P[7]AS toward guests 11-28. This Example demonstrates ultratight binding affinity of P[5]AS and P[6]AS toward quaternary (di)ammonium ions, which supports their use for in vitro and in vivo non-covalent bioconjugation for imaging and delivery applications and as in vivo sequestration agents.
In more detail, it will be recognized by those skilled in the art that progress in the construction of supramolecular systems for biological (e.g., imaging and drug delivery) and chemical applications (e.g., sensing, catalysis, separations) depends critically on the availability of a library of building blocks that can be easily integrated into more complex and functional systems. Molecular containers-whether prepared by covalent bond forming reactions or by self-assembly processes-occupy a central space within the field. Some of the most popular molecular containers include cyclodextrins, calix[n]arenes, crown ethers, cyclophanes, coordination cages, molecular clips and tweezers, cucurbit[n]urils (CB[n]), and H-bonded capsules. Within this group, the CB[n] family (
Contemplating the creation of new ultratight binding hosts based on pillar[n]arenes lead us to ponder the relevant structural features of CB[n] (
The inherent aqueous solubility of the two most potent hosts (P[5]AS: 100 mM; P[6]AS: 20 mM; vide infra) were measured by integrating the 1H NMR resonances for a solution of host against the methyl resonance for sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 as internal standard of known concentration. Before proceeding to investigate the host.guest properties of the new hosts, we performed dilution experiments monitored by 1H NMR spectroscopy to quantify their intermolecular self-association. The spectra were recorded for P[5]AS and P[6]AS as a function of concentration (P[5]AS: 20-0.1 mM; P[6]AS: 20-0.1 mM) used to calculate Ks values (P[5]AS: 19.7 M−1; P[6]AS: 16.2 M−1) by using a standard 2-fold self-association model. These Ks values ensure that the hosts remain monomeric at the mM concentrations used in the NMR and ITC experiments described below in this Example. Crystals of both P[6]AS and P[5]ACS were obtained and their structures as solved by x-ray diffraction measurements (
Initially, the molecular recognition properties of the new hosts toward guests 11-28 (
The extensive dataset presented in
Related comparisons can be made between hosts WP[6] and P[6]AS which exhibit 1:1 host: guest complexation toward all the guests used in this study. For example, P[6]AS is the superior host toward 20 out of the 23 guests studied with exceptions including 1° ammonium ions 24 and 27. Similar to P[5]AS, P[6]AS is highly selective based on guest length (e.g. 14 vs 28 vs 17; 15 vs 16 vs 20) and on the degree of methylation of the diammonium ion (e.g. 17 vs 20; Ka=1.43×109 vs 1.20×1011 M−1). Interestingly, the binding affinity of P[6]AS toward Me4N+(26, Ka=2.32×106 M−1) is 75-fold stronger than P[5]AS which suggests P[6]AS should be regarded as a powerful host for quaternary ammonium ions. In fact the Ka values of P[6]AS toward the guest panel lie in the single digit μM to 1 μM range which places P[6]AS squarely alongside CB[n] as one of the highest affinity synthetic host.guest systems in water although the balance between complexation driving forces (e.g. electrostatic versus hydrophobic effect) obviously differs. Finally,
Given the demonstrated preference of P[5]AS and P[6]AS toward quaternary diammonium ions the guest panel extended to include the clinically important neuromuscular blocking agents roc, vec, pan, and cis as well as acetyl choline (ACh). Macrocyclic receptors (e.g., γ-cyclodextrin derivative Sugammadex marketed by Merck as Bridion™, acyclic CB[n]-type receptor M2, and WP[6]) have previously been used as in vivo sequestration agents for NMBAs. Accordingly, the binding affinities of P[5]AS-P[7]AS, WP[5], and WP[6] were measured toward the NMBAs (
In summary, this Example describes the synthesis of P[5]AS-P[7]AS, the x-ray crystal structures of P[5]ACS and P[6]AS, and their molecular recognition properties toward (di)ammonium ions in aqueous solution. P[n]AS packs 2n negative charges into a small volume near the portals of the receptors which augments the electrostatic contributions to binding free energy. It was found that P[5]AS and P[6]AS display significantly higher binding affinity than WP[5] and WP[6] toward (bis)quaternary (di)ammonium ions. Accordingly, the suggested family name is Pillar[n]MaxQ. The picomolar affinity of P[6]AS toward roc and vec greatly exceeds that of acyclic CB[n]-type receptor M2 and Sugammadex which is used in clinical practice under the trade name BRIDION™. The ultratight binding (e.g. picomolar Ka) displayed by P[5]AS and P[6]AS places them alongside CB[n] as some of the most potent synthetic receptors in water. The ultratight binding of P[5]AS and P[6]AS suggests that sulfated pillararenes and their functionalized derivatives may be used as non-covalent connectors for bioconjugation, in (bio)chemical separations, for theranostics, as well as for sequestration and remediation in chemical and biological systems.
Determination of Ka between various hosts and cationic guests using Isothermal Titration Calorimetry (ITC). All ITC experiments were conducted in the 200 μL working volume of the sample cell of the PEAQ ITC instrument. An injection syringe of 40 L capacity was used. In each case, the host and guest solutions were prepared in a 20 mM NaH2PO4 buffer (pH 7.4). The sample cell was filled to capacity (200 μL) with the host solution and the guest solution was titrated in (first injection=0.4 μL, subsequent 18 injections=2 μL). The binding data was fitted using the 1:1 binding model or the competitive binding models in MicroCal PEAQ-ITC analysis software.
This Example provides in vivo effects of P[6]AS on reversal of methamphetamine induced hyperlocomotion in a pertinent mouse model. This Example also provides results from an in vivo toxicology study of P[5]AS and P[6]AS.
Cell Cytotoxicity Data for P[5]AS and P[6]AS. To test the Cytotoxicity and Cell Viability of the above compounds we used two different assays: an MTS (CellTiter 96 AQueous Kit®) assay that measures cellular metabolism, and the AK (Toxilight®BioAssay Kit) assay that measures cell death through release of the cytosolic enzyme adenylate kinase into the supernatant. Both assays were performed with two different cell lines. HEK293 and Hep G2cells, are frequently used in drug toxicity studies. HEK293, a human kidney cell line, is used to evaluate the effect of the drug on the renal system and Hep G2, a human hepatocyte cell line, is used to assess the response of liver cells where drugs are metabolized. The MTS and AK assays for both cell lines were conducted after 24 h of incubation with the compounds at concentrations of 0.01 mM, 0.03 mM, 0.1 mM, 0.3 mM, and 1 mM. Eight technical replicates were designated for untreated cells and four technical replicates were designated for the cells treated with each compound and staurosporine (apoptosis inducer).
The collected absorbance and relative luminescence data were normalized to percent cell viability (MTS) and percent cell death (AK) using equations 1 and 2:
% cell viability=(Abs sample/Average Abs UT)×100 1)
% cell death=(RLU samples/Average RLU Distilled water)×100 2)
Toxicity studies using the MTS and AK assays for the liver cell line, HepG2 suggests that P[5]AS demonstrates low cytotoxicity up to a concentration of 1 mM and high cell tolerance up to a concentration of 0.3 mM (
Similarly toxicity studies performed on human kidney (HEK293) cells suggest that P[5]AS demonstrates low cytotoxicity up to a concentration of 1 mM and high cell tolerance up to a concentration of 0.1 mM (
In Vivo Maximum Tolerated Dose Study (MTD). Animals studies were performed at the University of Maryland, Microbiology Building under the supervision of Dr. Volker Briken (IACUC #R-JAN-17-25). A total of 20 female Swiss Webster were used for this study. Three different concentrations of P[6]AS (11.31 mM, 7.54 mM, 3.77 mM) were used. A PBS control group was also included. Each concentration and control group contained 5 mice. The mice received the compound in 0.150 ml of PBS via tail vein injection, with 48 hours between injections. The weight and health status of the mice were monitored for 2 weeks following the last injection. Behavior summary: 11.31 mM dose group showed dose-dependent adverse effects in the form of freeze ups and some labored breathing. The 11.31 mM dose group returned to baseline behavior (that observed with the PBS control) ≈2-3 hours after injection. The lowest dose group 3.77 mM overall exhibited no adverse effects and behavior on par with the PBS control group.
MTD study performed for P[6]AS. Female Swiss Webster mice (n=5 per group) were dosed via tail vein on days 0 and 2 (denoted by *) with different concentrations of P[6]AS or phosphate buffered saline (PBS). The normalized average weight change per study group is indicated. Error bars represent SEM.
In Vivo Reversal of Methamphetamine Induced Hyperlocomotion by P[6]AS
Animals. Eight male Swiss Webster (CFW) mice were obtained from Charles River Laboratories that weighed ˜30 g upon arrival. Mice were individually housed in a temperature- and humidity-controlled room on a 12 h light/dark schedule with lights on at 6:00 am EST. For the duration of both experiments mice had ad libitum access to food and water. All behavioral testing occurred between 6:30 am and 2:00 pm EST, and all experimental procedures were approved by the University of Maryland Animal Care and Use Committee and conformed to the guidelines set forth by the National Research Council
Surgical Procedures. Mice were anesthetized with an intraperitoneal (IP) injection of ketamine (100 mg/kg)/xylazine (10 mg/kg) (n=8) and were implanted with jugular catheters with head-mounted ports. All surgical procedures were conducted using aseptic technique, with body temperature monitored and maintained throughout surgery. Catheters were placed in the right jugular vein with the port passed subcutaneously out towards the top of skull. Ports (5 MM Up Pedestal; P1 Technologies) were fixed to the skull with a combination of super glue (Loctite) and dental cement. Following surgery, mice received an immediate injection of Rimadyl (5 mg/kg) and 0.4 mL of warm sterile saline. Mice were treated post-operatively for two days with Rimadyl (5 mg/kg) and given a minimum of 5 days to recover before resuming training. Catheters were flushed daily with 0.1 mL sterile saline solution containing gentamycin (0.33 mg/mL) and 0.1 mL sterile saline solution containing heparin (20 IU/mL) in order to reduce clotting and maintain catheter patency. Catheter patency was assessed daily from the first day following surgery until the end of testing. Any mouse whose catheter exhibited significant flowback on a majority of days was excluded from analysis.
Behavioral Testing. Mice were trained on a standard autoshaping task described previously. All behavioral procedures were conducted in a Med Associates test chamber equipped with a food cup, a retractable lever, and 4 floor IR photobeams. Time stamps were generated from head entries into the food cup, downward deflections of the lever, or disruption of floor beams and recorded by the behavioral computer.
Mice were given one day of magazine training that consisted of the delivery of thirty 20 mg sucrose pellets (Bioserv) randomly delivered on a variable interval 30±15 schedule, in order to habituate mice to the box and pellet delivery. In order to minimize the impact of novelty-induced suppression of feeding, mice were given five to six 20 mg sucrose pellets each in their home cage for 2-3 days prior to the beginning of training.
Following magazine training, mice began Pavlovian training sessions, which consisted of the presentation of the lever (CS) for 8 s, which was immediately followed by the delivery of a sucrose pellet and the retraction of the lever. The CS was presented on a random interval of 90±30 s schedule. Each Pavlovian session consisted of 30 trials. Pavlovian training continued for 4 days prior to surgery. Following surgery and recovery, mice underwent Pavlovian training for an additional 8 days while being exposed to various treatments.
Experimental Design. P[6]AS's efficacy was assessed using a semi-counterbalanced design where all mice received each possible experimental treatment. The purpose of the experiments was to: (1) verify that binding of methamphetamine by P[6]AS would not be compromised in vivo, (2) verify that P[6]AS would not alter locomotor behavior, and (3) to demonstrate that P[6]AS can sequester methamphetamine in vivo. On the first day, regardless of experiment, mice underwent a refresher session free of treatment. On the following six sessions mice were treated with one of six possible treatments: 0.01M PBS (0.2 mL infused), P[6]AS only (4 mM; 0.178 mL infused), methamphetamine only (0.5 mg/kg; 0.022 mL infused), a premixed solution of P[6]AS and methamphetamine (Premix; ˜7:1 P[6]AS:Meth; 0.178 mL P[6]AS+0.022 mL Meth infused), P[6]AS followed by methamphetamine administered 30 s later (0.178 mL P[6]AS, 0.022 mL Meth infused), and methamphetamine followed by P[6]AS administered 30 s later (0.022 mL Meth, 0.178 mL P[6]AS infused). Mice only received only one infusion per day. The dose of methamphetamine was chosen based on previously published values that observed reliable hyperlocomotion in mice. It was sought to choose smallest dosage that reliably induced hyperlocomotion.
Following completion of the first six sessions, mice completed another two days of behavioral testing. On day 7, half of the mice (n=4) received P[6]AS followed by methamphetamine administered 5 minutes later (0.178 mL P[6]AS, 0.022 mL Meth infused), followed by infusion of methamphetamine followed by P[6]AS administered 5 minutes later (0.022 mL Meth, 0.178 mL P[6]AS infused) administered on the eighth day of testing. The other half of the mice (n=4) received the same exact treatment but in reverse order across days 7 and 8.
For each experiment, total locomotion counts (i.e., the total number of beam breaks) were obtained for each mouse across the entirety of each training session. For each experiment, locomotion counts were then analyzed across treatments using one-way repeated measures ANOVAs with tukey-corrected pairwise post-hoc t-tests in Graphpad Prism (Version 9.0.0).
In vivo reversal of methamphetamine-induced hyperlocomotion effects observed after 5 minute delay between treatment with methamphetamine and P[6]AS administration. On day 7 and 8 mice (n=8) received methamphetamine followed by an infusion of 0.01M PBS administered 5 minutes later (REV-C; 0.022 mL Meth, 0.2 mL PBS infused) or methamphetamine followed by P[6]AS administered 5 minutes later (REV-5; 0.022 mL Meth, 0.178 mL P[6]AS infused) in counterbalanced manner. Administration of P[6]AS 5 minutes after exposure to methamphetamine reduced hyperlocomotion (paired t-test, t(7)=2.757, p=0.0282). Bars represent average locomotion counts. Error bars represent the standard error of the mean (SEM). Dots represent counts for each mouse (n=8).
It will be recognized from the foregoing that this Example provides an analysis of the efficacy of P[6]AS in the sequestration of methamphetamine in vivo. Eight male Swiss Webster (CFW) mice were trained on an Pavlovian autoshaping task described previously and locomotion values were obtained and analyzed accordingly. To establish methamphetamine induced hyperlocomotion and examine the efficacy of P[6]AS mice were first treated single infusions of PBS (0.01M), P[6]AS only, methamphetamine only, a premixed solution of P[6]AS and methamphetamine, P[6]AS followed by methamphetamine administration 30s later, or methamphetamine followed by P[6]AS administered 30s later in counterbalanced manner.
Although the results of this first analysis are suggestive of the potential efficacy of P[6]AS in the sequestration of methamphetamine and inducing behavioral change, it is possible that the 30 second (s) interval between methamphetamine administration and P[6]AS administration in the reversal condition is too short to be ethologically relevant. To address this, a follow up experiment was conducted where on days 7 and 8 of testing, mice (n=8) were administered either methamphetamine followed by administration of 0.01 M PBS 5 minutes later (REV-C) or methamphetamine followed by P[6]AS 5 minutes later (REV-5) in a counterbalanced manner before completing the autoshaping task.
The following example shows ITC data for the binding of various drugs with hosts of the present disclosure.
a Measured by the ITC competition titration of Host (0.1 mM) and 1,3-propanediammonium chloride (0.15 mM) in the cell with Guest (1 mM) in the syringe.
b Measured directly by the ITC titration of Host (10 μM) in the cell with Guest (100 μM) in the syringe.
c Measured directly by the ITC titration of Host (0.1 mM) in the cell with Guest (1 mM) in the syringe.
The following example shows use of pillararene sulfates as in vivo reversal.
Human ether-a-go-go (hERG) Ion Channel Inhibition Assay. The hERG ion channel is a voltage-gated potassium channel in cardiac cells that is essential for cardiac repolarization. With the inhibition of this channel, the electrical depolarization and repolarization of the heart ventricles can be extended, leading to potentially fatal cardiac malfunction. The ability of P6AS at six concentrations (0.008 μM to 25 μM) to inhibit the hERG ion channel function was evaluated via the patch-clamp technique (QPatch HTX). The patch clamp hERG assay was conducted using mammalian cells (HEK-293) expressing the hERG channel.
Ames Test. In order to assess the potential mutagenicity of P6AS, the Ames fluctuation test and the associated bacterial cytotoxicity assays were performed. The Ames fluctuation test is a reverse mutation assay that utilizes four different S. typhimurium strains (TA98, TA100, TA1535, TA1537) which possess unique mutations within the histidine operon. Compounds that induce reverse mutations allow these strains to grow in the absence of histidine which is measured spectroscopically. The S. typhimurium strain TA1535 contains a T to C missense mutation in the hisG gene (his G46) leading to a leucine to proline amino acid substitution. With the reversal of this mutation, TA1535 can detect compounds that cause base pair mutations. The TA1537 strain detects compounds that induce a +1 frameshift mutation on the his C gene (his C3076). This allows frameshift mutagens to be detected. The TA98 strain detects +1 frameshift mutation on the his D gene (his D3052) and also contains the pkM101 plasmid, which increases the sensitivity of the strain to mutagenic compounds. Finally, TA100 contains the same mutation as TA1535 plus the pkM101 plasmid. The Ames fluctuation test also employs rat liver enzyme fractions (S9) to assess the potential mutagenicity of metabolites produced by the action of the liver enzymes on the test compound.
Initially, bacterial cytotoxicity assays were performed to determine whether P6AS was cytotoxic toward the histidine revertant tester strains (TA98R, TA100R, TA1535R, TA1537R) which would cause false negatives in the Ames fluctuation test. For this purpose, the four tester strains were cultured overnight at 37° C. in media containing Davis Mingoli salts, D-glucose, D-biotin, and low level histidine at pH 7.0 yielding OD650 from 0.60 to 1.10. The cultures were then incubated with eight different concentrations of P6AS (0.6, 1.2, 2.5, 5, 10, 25, 50, 100 μM; n=3) for 96 hours followed by measurement of OD650. Compounds that exhibit OD650 values less than 60% of control (not treated with compound) are deemed cytotoxic and do not proceed to the Ames fluctuation test. The known cytotoxic compound mitomycin C (IC50≤100 nM toward the tester strains) is used as a positive control. P6AS did not exhibit bacterial cytotoxicity toward any of the four tester strains at concentrations up to 100 μM (
Given the absence of bacterial cytotoxicity for P6AS, the Ames fluctuation test was subsequently performed. For this purpose, the four tester strains of bacteria were cultured overnight in media containing Davis Mingoli salts, D-glucose, D-biotin, and low level histidine at pH 7.0 yielding OD650 from 0.60 to 1.10. The cultures were then incubated in the absence of P6AS or in the presence of P6AS (5, 10, 50, 100 μM; n=48) both with and without Arochlor-induced rat liver S9 fraction (0.2 mg mL−1) for 96 hours. Bromocresol purple is included as a colorimetric pH indicator that responds to the pH drop resulting from bacterial growth upon reverse mutation. After 96 hours, the OD430 and OD570 values are measured and the number of positive wells with OD430/OD570≥1 is determined as surrogate for reverse mutation. The significance of the number of positive wells in the treatment groups (P6AS present) versus the control group (P6AS absent) is calculated using the one-tailed Fisher's exact test and classified as follows: p<0.001 (very strong positive, +++); 0.001<p <0.01 (strong positive, ++); 0.01<p<0.05 (weak positive, +); p >0.05 (negative, −). Control compounds known to induce reverse mutation (2-aminoanthracene (2-AA), 9-aminoacridine (9-AA), Quercetin, Streptozotocin) were tested as positive controls. Table 11 presents the results of the Ames fluctuation test. As can be seen, compared to background, none of the P6AS treatments result in a statistically significant increase in the number of positive wells. This indicates that P6AS does not significantly increase the rate of reverse mutation and is not genotoxic. Conversely, the genotoxic control compounds Streptozotocin, 2-AA, Quercetin, and 9-AA all display the expected increase in genotoxicity in one or more bacterial strains.
In Vivo Reversal of Fentanyl Induced Hyperlocomotion by P6AS—Experiment #1.
Animals. Nine male Swiss Webster (CFW) mice were obtained from Charles River Laboratories that weighed ˜35 g upon arrival. Mice were individually housed in a temperature- and humidity-controlled room on a 12 h (hour) light/dark schedule with lights on at 6:00 am EST. For the duration of both experiments mice had ad libitum access to food and water. All behavioral testing occurred between 6:30 am and 2:00 pm EST, and all experimental procedures were approved by the University of Maryland Animal Care and Use Committee and conformed to the guidelines set forth by the National Research Council.
Surgical Procedures. Mice were anesthetized with an intraperitoneal (IP) injection of ketamine (100 mg/kg)/xylazine (10 mg/kg) (n=9) and were implanted with jugular catheters with head-mounted ports. All surgical procedures were conducted using aseptic technique, with body temperature monitored and maintained throughout surgery. Catheters were placed in the right jugular vein with the port passed subcutaneously out towards the top of skull. Ports (5 MM Up Pedestal; P1 Technologies) were fixed to the skull with a combination of super glue (Loctite) and dental cement. Following surgery, mice received an immediate injection of Rimadyl (5 mg/kg) and 0.4 mL of warm sterile saline. Mice were treated post-operatively for two days with Rimadyl (5 mg/kg) and given a minimum of 5 days to recover before testing. Catheters were flushed daily with 0.1 mL sterile saline solution containing gentamycin (0.33 mg/mL) and 0.1 mL sterile saline solution containing heparin (20 IU/mL) in order to reduce clotting and maintain catheter patency. Catheter patency was assessed daily from the first day following surgery till the end of testing. Any mouse whose catheter exhibited significant flowback on a majority of days was excluded from analysis.
Behavioral Testing. Mice were trained on a standard autoshaping task. All behavioral procedures were conducted in a Med Associates test chamber equipped with a food cup, a retractable lever, and 4 floor IR photobeams. Time stamps were generated from head entries into the food cup, downward deflections of the lever, or disruption of floor beams and recorded by the behavioral computer.
In order to minimize the impact of novelty-induced suppression of feeding, mice were given five to six 20 mg sucrose pellets (Bioserv) each in their home cage for 2-3 days prior to the beginning of training. Mice were weighted and handled daily upon arrival until the completion of testing.
Following surgery, mice were habituated to the behavioral box and underwent one session of autoshaping to establish baseline locomotion levels before treatment began. Pavlovian training sessions which consisted of the presentation of the lever (CS) for 8 s, which was immediately followed by the delivery of a sucrose pellet and the retraction of the lever. The CS was presented on a random interval of 90±30 s schedule. Each Pavlovian session consisted of 30 trials. In total baseline plus testing lasted 11 days.
Experimental Design. P6AS efficacy was assessed using a semi-counterbalanced design where all mice received each possible experimental treatment. The purpose of the experiments was to: (1) verify that binding of fentanyl by P6AS would not be compromised in vivo, (2) verify that P6AS would not alter locomotor behavior, and (3) to demonstrate that P6AS can sequester fentanyl in vivo. On the first day of testing, regardless of experiment, mice underwent a baseline session free of treatment. On the following six sessions mice were treated with one of six possible treatments: PBS (0.2 mL infused), P6AS only (4 mM; 0.178 mL infused), fentanyl only (0.17 mg/mL; 0.022 mL infused), a premixed solution of P6AS and fentanyl (Premix; ˜68.34:1 P6AS:Fentanyl; 0.178 mL P6AS+0.022 mL fentanyl infused), P6AS followed by fentanyl administered 30 s later (0.178 mL P6AS, 0.022 mL fentanyl infused), and fentanyl followed by P6AS administered 30 s later (0.022 mL fentanyl, 0.178 mL P6AS infused). Mice received only one treatment per day. The dose of fentanyl was chosen based on previously published values that observed reliable hyperlocomotion in mice. The smallest dosage that reliably induced hyperlocomotion was selected.
Following completion of the first six sessions, mice completed another two days of behavioral testing. On day 8, half of the mice (n=5) received fentanyl followed by P6AS administered 5 minutes later (0.022 mL fentanyl infused, 0.178 mL P6AS), followed by infusion of and fentanyl followed by PBS administered 5 minutes later (0.022 mL fentanyl, 0.178 mL PBS infused) administered on the ninth day of testing. The other half of the mice (n=4) received the same exact treatment but in reverse order across days 8 and 9. On days 10 and 11, mice received the same treatment order but the time between fentanyl and PBS or P6AS was extended to 15 minutes.
For each experiment, total locomotion counts (i.e., the total number of beam breaks) were obtained for each mouse across the entirety of each training session. For each experiment, locomotion counts were then analyzed across treatments using one-way repeated measures ANOVAs with tukey-corrected pairwise post-hoc t-tests in Graphpad Prism (Version 9.0.0). For 5 minute and 15-minute reversal experiments data were analyzed with paired t-tests.
Results.
Applicable to
Discussion.
The efficacy of P6AS in the sequestration of fentanyl was examined in vivo. Nine male Swiss Webster (CFW) mice were trained on an Pavlovian autoshaping task described previously and locomotion values were obtained and analyzed accordingly. To establish fentanyl induced hyperlocomotion and examine the efficacy of P6AS, mice were first treated single infusions of PBS (0.01 M), P6AS only, fentanyl only, a premixed solution of P6AS and fentanyl, P6AS followed by fentanyl administration 30s later, or fentanyl followed by P6AS administered 30s later in counterbalanced manner.
Although the results of this first analysis are suggestive of the potential efficacy of P6AS in the sequestration of fentanyl and inducing behavioral change, it is possible that the 30s interval between fentanyl administration and P6AS administration in the reversal condition is too short to be ethologically relevant. To address this, two follow up experiment were conducted, where on days 8/9 of testing, and days 10/11 of testing, mice (n=9) were administered either fentanyl followed by administration of 0.01M PBS or fentanyl followed by P6AS either 5 or 15 minutes later in a counterbalanced manner before completing the autoshaping task.
Although not directly comparable from an experimental design perspective, locomotion levels in the 5-minute reversal and 15-minute reversal using P6AS condition closely approximated those observed in control conditions on Day 1-7, while locomotion counts in the PBS condition approximate those observed with the fentanyl only treatment. Collectively these findings suggest that P6AS is capable of sequestering fentanyl in vivo and reversing fentanyl-induced hyperlocomotion, with little to no effect on the locomotor behavior of the animal itself.
Given the desirable results obtained in these experiments, it was tested whether lower doses of P6AS would be similarly effective and also included active comparators in the form of Naloxone and Motor1.
Comparison of Lower Doses of P6AS, Naloxone, and TetM1 as Countermeasures to Fentanyl-Induced Hyperlocomotion in Mice—Experiment #2.
Animals. Eleven male Swiss Webster (CFW) mice were obtained from Charles River Laboratories weighting 32.36±1.2 g upon arrival. Mice were individually housed in a temperature- and humidity-controlled room on a 12 h light/dark schedule with lights on at 6:00 am EST. For the duration of the experiment mice were given ad libitum access to food and water. All behavioral testing occurred between 6:00 am and 2:00 pm EST, and all experimental procedures were approved by the University of Maryland Animal Care and Use Committee and conformed to the guidelines set forth by the National Research Council.
Surgical Procedures. Mice were anesthetized with an intraperitoneal (IP) injection of ketamine (100 mg/kg)/xylazine (10 mg/kg) (n=11) and were implanted with jugular catheters with head-mounted ports. All surgical procedures were conducted using aseptic technique, with body temperature monitored and maintained throughout surgery. Catheters were placed in the right jugular vein with the port passed subcutaneously out towards the top of skull. Ports (5 MM Up Pedestal; P1 Technologies) were fixed to the skull with a combination of super glue (Loctite) and dental cement. Following surgery, mice received an immediate injection of Rimadyl (5 mg/kg) and 0.4 mL of warm 0.9% sterile saline. Mice were treated post-operatively for two days with Rimadyl (5 mg/kg) and given a minimum of 7 days to recover before testing. Catheters were flushed daily with 0.1 mL 0.9% sterile saline solution containing gentamycin (0.33 mg/mL) and 0.1 mL sterile saline solution containing heparin (20 IU/mL) in order to reduce clotting and maintain catheter patency. Catheter patency was assessed daily from the first day following surgery until the end of testing. Any mouse whose catheter exhibited significant flowback on a majority of days was excluded from analysis.
Behavioral Testing. Mice were trained on a standard autoshaping task described previously. All behavioral procedures were conducted in a Med Associates test chamber equipped with a food cup, a retractable lever, and 4 floor IR photobeams. Time stamps were generated from head entries into the food cup, downward deflections of the lever, or disruption of floor beams and recorded by the behavioral computer.
In order to minimize the impact of novelty-induced suppression of feeding, mice were given five to six 20 mg sucrose pellets (Bioserv) each in their home cage for 2-3 days prior to the beginning of training. Mice were weighted and handled daily upon arrival until the completion of testing.
Following surgery, mice were habituated to the behavioral box and underwent one session of autoshaping to establish baseline locomotion levels before treatment began. Pavlovian training sessions which consisted of the presentation of the lever (CS) for 8 s, which was immediately followed by the delivery of a sucrose pellet and the retraction of the lever. The CS was presented on a random interval of 90±30 s schedule. Each Pavlovian session consisted of 30 trials. In total baseline plus testing lasted 11 days.
Experimental Design. P6AS efficacy was assessed using a semi-counterbalanced design where all mice received each possible experimental treatment. The purpose of the experiments was to: (1) verify that binding of fentanyl by P6AS would not be compromised in vivo, (2) test lower doses of P6AS (0.5 mM and 1.5 mM) than previously used (4.0 mM) to modulate locomotor behavior, and (3) to compare the efficacy of P6AS sequester fentanyl to alternative countermeasures, naloxone (4.37 mM) and TetM1 (1.507 mM), in vivo. On the first day of testing, regardless of experiment, mice underwent a baseline session free of treatment. During the following six sessions mice underwent 15 minute reversals where either a single 0.022 mL infusion of PBS or 0.1591 mg/mL fentanyl followed by a 0.178 mL infusion of a candidate countermeasure. The possible six treatments included PBS followed by PBS, fentanyl followed by 1.5 mM P6AS, fentanyl followed by 0.5 mM P6AS, fentanyl followed by 4.37 mM naloxone, fentanyl followed by 1.507 mM TetM1, or fentanyl followed by PBS. Mice received only one treatment per day, and treatments were counterbalanced across animals. The dose of fentanyl used was chosen based on previously published values that observed reliable hyperlocomotion in mice.
TetM1 is Also Known as Motor 1 or Calabadion 1.
Total locomotion counts (i.e., the total number of beam breaks) were obtained for each mouse across the entirety of each behavioral session. Locomotion counts were analyzed across treatments using one-way repeated measures ANOVAs with tukey-corrected pairwise post-hoc t-tests in Graphpad Prism (Version 9.0.0).
Discussion The efficacy of P6AS in the sequestration of fentanyl was investigated, as well as comparison its efficacy to other known countermeasures in vivo. Eleven male Swiss Webster (CFW) mice were trained on an Pavlovian autoshaping task and locomotion values were obtained and analyzed accordingly. Mice underwent 15 minutes reversals in which they were first treated with either PBS or 0.1591 mg/mL of fentanyl before receiving one of six possible countermeasures 15 minutes later.
The results establish that P6AS is a desirable in vivo sequestrant for fentanyl that is capable of reversing the hyperlocomotion observed for animals treated with fentanyl. Reversal using P6AS, TetM1, and Naloxone displayed no statistically significant differences. The fact that low dose P6AS (5 mg/kg) is capable of effecting reversal is a finding with translational potential.
Although the present disclosure has been described with respect to one or more particular example(s), it will be understood that other examples of the present disclosure may be made without departing from the scope of the present disclosure.
This application claims priority to U.S. Provisional Application No. 63/344,559, filed on May 21, 2022, and is a continuation-in-part of International Application No. PCT/US2021/020333, filed on Mar. 1, 2021, which claims priority to U.S. Provisional Application No. 62/982,460, filed on Feb. 27, 2020, and to U.S. Provisional Application No. 63/013,336, filed on Apr. 21, 2020, the disclosures of each of which are incorporated by reference herein.
This invention was made with government support under CHE1404911 awarded by National Science Foundation. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63344559 | May 2022 | US | |
| 61013336 | Dec 2007 | US | |
| 62982460 | Feb 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2021/020333 | Mar 2021 | US |
| Child | 17822863 | US |
