COMPOSITIONS, MATERIALS, AND METHODS FOR DEACTIVATING

TOXIC AGENTS

TECHNICAL FIELD

The present invention relates to compositions and materials incorporating a nucleophilic moiety for deactivating a toxic agent. In various aspects, the invention relates to compositions or articles incorporating the nucleophilic agent; and methods of making and using these compositions and articles.

BACKGROUND

A vast majority of agricultural and industrial workers are repeatedly exposed to pesticides in the field or during manufacture. While spraying a field, farm workers get exposed to pesticides through dermal route, by inhalation, or both. Organophosphate pesticides (methyl parathion, malathion, paraoxon and chlorpyrifos) are acetylcholinesterase (AChE) inhibitors. Acetylcholinesterase is found in both central and peripheral nervous system of humans and its inhibition can lead to the accumulation of acetylcholine (a neuro transmitter). Such accumulation of acetylcholine can result in neurological disorders, suffocation, paralysis, and in severe cases death. In addition to neurotoxic effects, AChE inhibition leads to cardiotoxicity, reduced immunity, infertility, and delayed sexual maturation. It is clear that systemic exposure to pesticides is a health hazard.

Although, personal protective equipment (PPE) such as suits, gloves, face masks, headgear, and boots are available; they are scarcely used, mainly due to high cost and discomfort. Moreover, the surface of these equipment gets exposed to pesticides and can by itself pose a safety hazard. These existing PPEs lack the ability to deactivate pesticides that come in contact with the PPE.

Accordingly, there is a need to minimize exposure of farm and factory workers to toxic agents such as pesticides.

The present invention in various aspects is directed to overcoming these and other deficiencies in the prior art.

SUMMARY OF THE INVENTION

In various aspects and embodiments, the present invention provides a composition or article suitable for deactivating a toxic agent, such as organophosphate-based or carbamate-based pesticides, insecticides, herbicides, fungicides and chemical warfare agents, among others. The composition or article incorporates a nucleophilic agent in an amount effective to deactivate the toxic agent. In various embodiments, the nucleophilic agent includes at least one nucleophilic moiety (Nu) that is conjugated or adsorbed to a surface or a fabric to deactivate the toxic agent as it comes into contact with the surface or fabric. Such embodiments include wearable articles. In other embodiments, the nucleophilic moiety is conjugated to a polymer or an aliphatic group or aromatic group providing for various topical compositions for protecting or washing the skin or surfaces before or after exposure to the toxic agent.

In various embodiments, the nucleophilic agent or its composition has activity to cleave/hydrolyze toxic agents before they can enter the subject and cause adverse effects. In various embodiments, the nucleophilic agent or its composition may serve as a prophylactic strategy to limit or reduce exposure of a subject to the toxic agent. The reactive nucleophilic agents disclosed in the present application are able to rapidly hydrolyze the toxic agents. Additionally, in some embodiments, the nucleophilic agents cleave the toxic agents catalytically instead of stoichiometrically. An exemplary nucleophilic moiety in accordance with the invention is pyridine-2- aldoxime, including in some embodiments “poly-Oxime” which involves functionalization of chitosan with the nucleophilic moiety.

In another aspect, the present invention provides a method of preventing a subject from bodily exposure to a toxic agent. This method includes a step of covering the subject or a body part of the subject with the nucleophilic deactivating agent. The subject or body part thereof may be covered using wearable articles that incorporate the nucleophilic agent as described herein. In some embodiments, these wearable articles are durable, and can withstand numerous detergent washes without substantial loss of activity. In other embodiments, the subject or body part is covered with a topical composition or cleanser that incorporates the nucleophilic agent and thereby deactivates the toxic agent on the skin, thereby preventing its penetration and subsequent biological impacts.

Another aspect of the present invention relates to a method of cleaning an object or a subject that has been exposed to a toxic agent. In various embodiments, the method comprises cleaning the object or the subject with a composition comprising the nucleophilic agent, to thereby reduce the accumulation of the toxic agent. In one embodiment, the exposure to toxic agent is prevented by applying the nucleophilic agent to a surface contaminated with the toxic agent such that the nucleophilic agent hydrolyzes the toxic agent.

Other aspects and embodiments of the invention will be apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-B show deactivation of pesticides by the conjugate of pyridine-2- aldoxime and chitosan. Figure 1A illustrates dermal penetration of methyl parathion (MPT) (an exemplary pesticide), and how the conjugate of pyridine-2-aldoxime and chitosan deactivates the MPT through hydrolysis. Dermal penetration of pesticide (MPT) leads to the inhibition of AChE, which plays a pivotal role in biological functions including neuronal signaling and neuromuscular coordination (NMC). MPT-mediated inhibition of AChE leads to severe toxicity including neuromuscular dysfunction, loss of endurance, and locomotor function. The presence of a nucleophile (an oxime) attached to a polymer (poly-Oxime) formulated as a topical gel could deactivate organophosphorus ester, MPT, through hydrolysis. This limits MPT penetration into the skin, which leads to the reduction of pesticide-induced toxicity. Figure 1B shows an exemplary method for preparing the conjugate of pyridine-2- aldoxime and chitosan where pyridine-2-aldoxime is connected to chitosan through acetamide linker. A topical gel was prepared with humectants like glycerin, propylene glycol, and carbopol 940. Two types of topical gels were prepared. Poly-Oxime is the active gel containing pyridine-2-aldoxime and un-functionalized chitosan is the control gel.

Figures 2A-I show deactivation of MPT to prevent MPT-mediated AChE inhibition, ex vivo. Figure 2A shows a schematic representation of Franz diffusion cell comprising dialysis membrane placed between donor and acceptor chamber. A thin layer of either poly-Oxime gel or sham gel was applied on a dialysis membrane, which was placed between donor and acceptor chambers. Figures 2B-E show concentration of MPT and pNP (hydrolytic degradation product of MPT) in donor and acceptor chambers. The presence of sham gel did not prevent the diffusion of MPT into the acceptor chamber and could not hydrolyze MPT to generate pNP, whereas poly- Oxime actively hydrolyzed to limit the penetration of toxic MPT into the acceptor chamber. Figures 2B and 2D illustrate the percentage of MPT and pNP in donor chamber, upon application of sham-gel and conjugate of pyridine-2-aldoxime and chitosan. Figures 2C and 2E illustrate the percentage of MPT and pNP in acceptor chamber, upon application of sham-gel and conjugate of pyridine-2-aldoxime and chitosan. Figures 2F and 2G show an ex-vivo assay to demonstrate the ability of poly- Oxime to limit MPT-induced assay AChE inhibition using rat blood. AChE containing rat blood was placed in the acceptor chamber, and MPT was added in the donor chamber in the presence of either poly-Oxime or sham gel. Active AChE was measured in the blood before and 3 hours after addition of MPT. In the absence of poly-Oxime gel, MPT diffused into an acceptor chamber and significantly inhibited AChE activity. However, poly-Oxime gel could hydrolyze MPT before diffusion, therefore limiting the MPT-induced inhibition of AChE. Data are means ± SD (n = 3, performed at least twice); P values were determined by one-way analysis of variance (ANOVA). P < 0.0001. ns, not significant. Figures 2H and 21 illustrate the effect of MPT on osteogenic differentiation of Human Mesenchymal Stem Cells (hMSCs) upon application of sham gel and conjugate of pyridine-2-aldoxime and chitosan, respectively.

Figures 3A-C show rheological analysis of: Carbopol gel (Figure 3A); sham gel (Figure 3B); and composition comprising the conjugate of pyridine-2-aldoxime and chitosan (poly-Oxime) (Figure 3C).

Figures 4A-F show AchE activity upon exposure to lethal dose of MPT, in vivo. It is shown that poly-Oxime gel limits AChE inhibition after exposure to the lethal dose of MPT. Figure 4A shows the effect of MPT at dose of 150 mg/kg applied directly on skin of rats, when MPT at dose of 220 mg/kg was applied in presence of sham gel and when MPT at dose of 220 mg/kg was applied in presence of conjugate of pyridine-2-aldoxime and chitosan, respectively. Figure 4B shows the quantity of active AChE in blood (AChE was quantified using Ellman’s assay). Direct exposure of MPT significantly reduced the active AChE in the blood. Sham gel could not limit MPT-induced AChE inhibition, while poly-Oxime gel deactivated MPT before entering into the skin, therefore reducing the MPT-induced AChE inhibition. Figures 4C-F illustrate quantification of AChE activity in brain, heart, liver and lungs, respectively. Dermal exposure of MPT either directly on the skin or in the presence of sham gel led to the decrease in the active AChE in all tissues such as brain, heart, liver, and lung, while poly-Oxime gel significantly reduced this MPT-mediated deactivation of AChE.

Figure 5 shows change in body weight in rats without exposure to MPT, with direct exposure to MPT and exposure to MPT upon application of conjugate of pyridine-2-aldoxime and chitosan.

Figures 6A-F illustrate prevention of mortality upon either daily or single application of conjugate of pyridine-2-aldoxime and chitosan, in vivo. Figure 6A illustrates quantification of AChE activity in blood and organ of rats upon (i) direct exposure of MPT and (ii) exposure of MPT after daily application of conjugate of pyridine-2-aldoxime and chitosan, respectively, using Ellman’s assay. Figure 6B illustrates Median survival time (MST) of rats. Figure 6C illustrates blood AChE activity in the rats. Figures 6D to 6F illustrate exposure of rats to MPT for 5 days directly and in presence of conjugate of pyridine-2-aldoxime and chitosan and percent survival of rats and activity of AChE in blood, upon mortality or after 21 days.

Figures 7A-G show that poly-Oxime gel prevented loss of endurance, NMC, nerve function impairment, and uncontrolled muscle activity in vivo. Figure 7A shows a rotarod experiment that was used to study endurance and NMC in MPT animals either directly or in the presence of poly-Oxime gel. Figure 7B shows the latency to fall that was measured by measuring the time the animal stayed on rotarod at a constant speed of 20 rpm, and it was normalized to the day of exposure. Directly exposed MPT animals showed significant reduction endurance that was prevented by poly-Oxime cream. Figure 7C shows that when animals were subjected to increasing speed from 2 to 60 rpm, the rpm reached before falling was taken as a measurement to assign NMC score, and data were normalized to the day of exposure. Poly-Oxime cream showed complete rescue of loss of NMC, which was observed with direct exposure MPT animals. Figures 7D and 7E show paw prints of animals that were exposed either directly or in the presence of poly-Oxime gel before and after exposure. Prints were used to calculate sciatic functional index (SFI). Direct exposure animals showed greatly reduced SFI that represents impairment of sciatic nerve function, which was again rescued by poly-Oxime cream. Figure 7F shows electromyograms (EMGs) recorded under 2.5% isoflurane anesthesia and Figure 7G shows EMGs when animals were awake. Poly-Oxime cream showed complete prevention of muscle spasm.

Figure 8 shows change in body temperature in rats without exposure to MPT, with direct exposure to MPT and exposure to MPT upon application of conjugate of pyridine-2-aldoxime and chitosan.

Figures 9A-B show brain AChE and body weight. Figure 9A shows the activity of brain AChE upon exposure to MPT directly and after daily (repeated) application of conjugate of pyridine-2-aldoxime and chitosan. Figure 9B shows the body weight upon exposure to MPT directly and after daily (repeated) application of conjugate of pyridine-2-aldoxime and chitosan.

Figures 10A-B show brain AChE and body weight. Figure 10A shows the activity of brain AChE upon exposure to MPT directly and after single application of conjugate of pyridine-2-aldoxime and chitosan. Figure 10B shows the body weight upon exposure to MPT directly and after single application of conjugate of pyridine - 2-aldoxime and chitosan.

Figures 11A-B show the non-toxicity profile of conjugate of pyridine-2- aldoxime and chitosan. Figure 11A shows the non-dermal penetrance capacity of conjugate of pyridine-2-aldoxime and chitosan. Figure 11B shows non-toxicity of conjugate of pyridine-2-aldoxime and chitosan to the keratinocytes.

Figure 12 shows prevention of AChE inhibition caused by carbaryl (carbamate), carbofuran (carbamate-based) and cypermethrin (ester-based) pesticides.

Figures 13A-D show ex- vivo Franz diffusion assay with commercial organophosphates. Franz diffusion assay was conducted with diluted rat blood in acceptor compartment separated from donor compartment by membrane (MWCO 3.5 kDa) without or with coating sham gel or poly-Oxime gel. 2 pmoles of each pesticide was added to donor compartment along with 500 pl phosphate buffer (pH 8.0). Figure 13A shows Metacil (MPT), Figure 13B shows Aalphos (Monocrotophos), Figure 13C shows Raise (Chlorpyrifos) and Figure 13D shows Profex Super (Profenophos). These pesticides were used to study efficiency of poly-Oxime to prevent AChE inhibition. The thickness of gels was 2 mm for all experiments except for profenophos (3 mm). Figures 14A-B show change in body weight and body temperature following acute exposure to MPT. As shown in Figure 14A, upon exposure to 150 mg/kg of MPT dermally, either directly or in the presence of sham gel, animals showed reduction of body weight by almost 20% in four days which represents severe toxicity. As shown in Figure 14B, when protected with poly-Oxime cream, this weight loss was completely prevented, and the animals showed increase in the body weight similar to naive animals. When exposed to 150 mg/kg of MPT, animals showed marked reduction in body temperature on day 3. Animals protected with poly-Oxime gel showed no decrease in body temperature as compared to unexposed animals.

Figure 15 shows that activated cloth prevented inhibition of AChE activity ex vivo when tested with rat blood using Franz diffusion cells. The efficiency of fabric to prevent AChE inhibition was intact even after 30 washes with strong SDS detergent. This suggests the robustness of the technology and strongly suggests its ability to be reused after repeated washing.

Figures 16A-B show catalyst activated mask showed protection against loss of AChE activity. Figure 16A: Blood AChE analysis after 96 hours of exposure revealed that exposure to MPT either directly or in the presence of normal (inactive) mask, significantly decreased the active AChE, whereas, in the presence of active mask, AChE activity did not decrease. Figure 16B: AChE activity in brain clearly suggest that direct MPT aerosol exposure leads to decreasing active AChE, while normal mask could not protect from MPT exposure. On the contrary, the presence of active mask prevented the reduction of active AChE. These results demonstrate that catalyst activated mask does not act like a typical physical barrier, but it hydrolytically deactivates organophosphate leading to prevention of AChE activity.

Figures 17A-B show catalyst activated fabric protected against weight loss and reduction in blood AChE activity in rats exposed to 100 mg/kg/day of MPT every day for 4 days. Figure 17A shows that animals exposed either directly or upon normal clothing showed nearly 15% reduction in body weight in just 4 days while animals protected with active fabric showed no loss of weigh by the end of day 4. Figure 17B shows blood AChE analysis on day 4 of exposure. It shows that exposure to MPT either directly or in the presence of normal (inactive) clothing significantly decreased the active AChE, whereas, in the presence of active mask, AChE activity did not decrease.

Figure 18 shows that the activated fabric limited loss of sciatic function due to pesticide exposure. Animals exposed to pesticides either directly or through normal cloth showed significant drop in SFI suggesting severe impairment in sciatic nerve function. The animals who were exposed through activated cloth did not show any drop in SFI confirming the protection offered by catalytic nucleophiles.

Figures 19A-B show that o-amphi oxime (Figure 19A) and p-amphi oxime (Figure 19B) showed maximum activity at catalyst:CTABr ratio of 1:10 and 1:0.75 respectively.

Figures 20A-B show that the log k _0bs vs. pH profiles for the hydrolysis of substrate [MPT] =2.5* 10 ^-5 M by catalyst 1 [o-amphi-oxime] = 2.5* 10 ^-4 M; and catalyst 2 [p-amphi oxime] =2.5* 10 ^-4 M in the micellar medium.

Figures 21A-B show time dependent formation of p-nitro phenol upon hydrolysis of MPT by catalyst 1 [o-amphi oxime] and catalyst 2 [o-amphi oxime]/CTABr in the presence of excess substrate over catalyst.

Figures 22A-B show ceramic surface decontamination using micellar o- amphi-oxime; a 10 cmx10 cm ceramic surface was contaminated with MPT followed by decontamination with micellar o-amphi-oxime. SDS/HEPES buffer and only CTAM served as controls (Figure 22A). The amount of MPT at the end of 2 hrs post treatment (Figure 22B). The amount of pNP is 2 hrs post treatment.

Figures 23A-B show the rate of formation of Para nitro phenol by Catalyst 1 [o-amphi oxime] with respect to time on ceramic surface. Figure 23 A shows that Catalystl [o-amphi oxime] can cleave MPT in 15 mins. Conditions: 25+0. l°C, catalyst [1 & 2] = 10 mM, [CTABr] = 100 mM. 50 mM Hepes buffer, pH=8.2. The substrate concentration [MPT] = 1 mM. o-amphi oxime showed complete conversion of MPT within 15 mins (Figure 23 A) which was also reflected in corresponding increase of pNP (Figure 23B). This data substantiates the efficiency of catalyst to rapidly decontaminate the surfaces.

Figures 24A-B show the rate of formation of Para nitro phenol by Catalyst 2[p-amphi oxime] with respect to time on ceramic surface shows that it can cleave MPT sprayed on the surface in 1.5 hours (Figure 24A). Conditions: 25+0. l°C, catalyst [1 & 2] = 10 mM, [CTABr] = 100 mM. 50 mM Hepes buffer, pH=8.2. The substrate concentration [MPT] = 1 mM. o-amphi oxime showed complete conversion of MPT within 15 mins (Figure 24 A) which was also reflected in corresponding increase of pNP (Figure 24B). This data substantiates the efficiency of catalyst to rapidly decontaminate the surfaces.

Figure 25 shows that a spray of catalyst 1 [o-amphi oximes] decontaminates the cage surface exposed with lethal dose of MPT in vivo. A dose of MPT (1 mM) was sprayed on the surface, one day before introducing animals into the cages with and without the present of catalyst 1 [o-amphi oxime]. Active AChE in the blood was quantified using Ellman’s assay. Direct exposure of MPT significantly reduced the active AChE in the blood. Amphi Oxime spray deactivated MPT on the surface before it enters into the rats, therefore reducing the MPT-induced AChE inhibition.

Figures 26A-D show AchE concentration in different tissues of rats upon surface exposure to MPT. On day 15, rats were sacrificed, tissue was collected, and the amount of active AChE was quantified. Surface exposure of MPT led to the decrease in the active AChE in all tissues such as brain (Figure 26A), heart (Figure 26D), liver (Figure 26C), and lung (Figure 26B), while the spray of amphi oxime significantly reduced this MPT-mediated deactivation of AChE.

Figure 27: Catalyst 1 [o-amphi oxime] spray prevented loss of endurance in vivo. Rota rod was used to study endurance and NMC in MPT animals either directly or in the presence of amphi oxime spray. Latency to fall was measured increasing speed from 2 to 60 rpm, the rpm reached before falling was taken as a measurement to assign NMC score, and data were normalized to the day of exposure. The animals kept in contaminated cages significantly lost their neuro-muscular coordination and showed reduced latency to fall scores. The animals introduced in cages decontaminated with micellar amphi-oxime catalyst showed no alternation in latency to fall.

Figure 28 is a schematic showing no systemic entry of the pesticide into the subject upon employing the article disclosed herein (e.g., a face mask) comprising the conjugate. Figure 29 shows a method of making of the conjugate of organosilicon compound and nucleophile. The organosilicon compound is triethoxy silane and the nucleophile is pyridine-2-aldoxime.

Figure 30 shows a method for preparing the articles where a nucleophile is attached to a fabric.

Figures 31A-B show Franz diffusion cell assay to demonstrate the ability of the article in prevention of AChE inactivation. Figure 31A shows the Franz diffusion cell apparatus and Figure 31B shows the percentage of AChE activity retained after application if MPT in the presence of no cloth, an inactive cloth or an active cloth.

Figure 32 shows various linkers for functionalizing articles such as fabrics with the nucleophilic agents. As shown in the figure, the linker can be conjugated to a free hydroxyl via an ether linking group, an ester linking group, a carbonate linking group, a carbamate linking group, a silane ether linking group, a thioester linking group, or polyethylene linking group.

Figure 33 shows various linker schemes for functionalizing articles and polymers having a free amine for functionalization. As shown, the nucleophilic agent reacts with the polymer to form a polymer-linker-nucleophile structure.

Figure 34 shows various linkers for functionalizing polymers with the nucleophilic agents. In this figure, the polymer has free hydroxyl groups for functionalization. As shown, the linker can be conjugated to free hydroxyls via a silane ether linking group, an ether linking group, an ester linking group, a carbonate linking group, and a carbamate linking group.

Figure 35 shows various linkers for functionalizing a polymer comprising carboxylic acid functional groups.

DETAILED DESCRIPTION

In various aspects and embodiments, the present invention provides a composition or article suitable for deactivating a toxic agent, including but not limited to organophosphate-based and carbamate-based pesticides, insecticides, herbicides, fungicides and chemical warfare agents. The composition or article incorporates a nucleophilic agent in an amount effective to deactivate the toxic agent. In various embodiments, the nucleophilic agent includes at least one nucleophilic moiety that is conjugated or adsorbed to a surface or a fabric to deactivate the toxic agent as it comes into contact with the surface or fabric. Such embodiments include wearable articles. In other embodiments, the nucleophilic moiety is conjugated to a polymer or an aliphatic group or aromatic group providing for various topical compositions for protecting or washing the skin or surfaces before or after exposure to the toxic agent. In various embodiments, the nucleophilic agent or its composition has activity to cleave/hydrolyze toxic agents, and are regenerated, allowing the nucleophilic agent to cleave the toxic agents catalytically instead of stoichiometrically. An exemplary nucleophilic agent as described herein comprises a pyridine-2-aldoxime nucleophilic group, which can functionalize various surfaces, polymers, and/or carriers as described in detail below. In some embodiments, pyridine-2-aldoxime functionalizes a non-crosslinked polymer such as chitosan, forming a poly-Oxime compound that is suitable for topical application, and which can provide effective protection from the toxic agent with application of only a thin film.

In some embodiments, the nucleophilic agent is conjugated to a surface or a fabric. The nucleophilic agent may be bonded directly to the surface or the fabric or may be indirectly bonded through a linker (Z) group. Alternatively, the nucleophilic agent can be non-covalently adsorbed to the surface or to the fabric. These embodiments provide for functionalized materials, including wearable articles, that deactivate the toxic agent to limit the exposure of individuals at risk. Types of wearable materials, including durable materials in which the functionalization can withstand numerous detergent washes, are described herein.

In some embodiments, the nucleophilic agent is conjugated to a polymer (including but not limited to chitosan), an aliphatic group, or an aromatic group directly or indirectly through a linker (Z). In these embodiments, the invention provides compositions suitable for topical application, as well as soaps and detergents, for both reducing exposure as well as deactivating toxic agents to prevent their accumulation on surfaces and materials.

In one embodiment, the linker (Z) is covalently bonded to the nucleophilic moiety (Nu) at either a neutral atom or a positively charged atom. In some embodiments, the nucleophilic agent has the structure according to formula A-Z-Nu; wherein (A) is a surface, a fabric, or polymer; (Z) is a linker; and (Nu) is a nucleophilic moiety.

The association between the nucleophilic agent (or the nucleophilic moiety) and the fabric or the polymer can be non-covalent (e.g., an ionic interaction, such as an ionic bond or a hydrogen bond(s)), or is via covalent bonds. In some embodiments, the interaction between the nucleophilic moiety and the fabric or the polymer is by adsorption. Adsorption is the adhesion of atoms, ions or molecules from the nucleophile to the surface, the fabric or the polymer. In some embodiments, the nucleophile is bonded or conjugated to the fabric or the polymer covalently via a linker (Z). In some embodiments, the nucleophile is a N-hydroxy alpha nucleophile.

In some embodiments, the deactivating agent has one of the following structures:

where Ri is an aromatic or aliphatic group. In some embodiments, Ri is a hydrocarbon aromatic group or a hetero-aromatic group.

In some embodiments, the deactivating agent has a structure selected from one or more of:

In some embodiments, the deactivating agent (such as those above) is covalently bonded to a surface, a polymer, a fabric, or an aliphatic group through any available atom such that the nucleophilic functionality is preserved. In one embodiment, the nucleophilic moiety is pyridine-2-aldoxime.

In some embodiments, the nucleophilic deactivating agent is a compound of Formula (I):

wherein:

Ri, R ₂ , R3, and R ₄ are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, hydroxyl, hydroxylalkyl, halogen, -CN, -O-alkyl, -C(O)-alkyl, -C(O)0-alkyl, -C(O)0H, -C(O)NH- alkyl, -NH ₂ , -N0 ₂ , -CF ₃ , -NH-alkyl, -N(alkyl) ₂ , -NHC(O) -alkyl and aryl, wherein alkly, alkenyl, alkynyl and aryl are each optionally substituted;

R ⁵ and R ⁷ are each independently hydrogen or an optionally substituted alkyl group having 1 to 6 carbon atoms;

L is selected from the group consisting of a bond, or a divalent group such as -0-, -NH-, -(CH ₂ CH ₂ O) _0- (where o is an integer of 1 to 100), and - (CFh)pNHC(O)- (where p is an integer of 1 to 20); Y is a functional group, such as -OH, -NH ₂ , -N0 ₂ , -SH, -C(O)OH, - C(O)0-alkyl, and halogen;

n is an integer of 0 to 100;

X- is an organic or inorganic counter anion.

In some embodiments, the nucleophilic deactivating agent is a compound of

Formula 1(a):

wherein L is a bond or a divalent group, such as -O-, -NH-, -(CH ₂ CH ₂ O) _o- (where o is an integer of 1 to 100), and -(CH ₂ )pNHC(O)- (where p is an integer of 1 to 20);

Y is a functional group, such as a functional group, and/or may be selected from -OH, -NH ₂ , -N0 ₂ , -SH, -C(O)OH, -C(O)0-alkyl, and halogen;

n is an integer of 0 to 100; and

X- is an organic or inorganic counter anion.

In some embodiments, the nucleophilic deactivating agent is a compound of

Formula 1(b):

wherein L is a bond, or a divalent group, such as a divalent group selected from -0-, -NH-, -(CH ₂ CH ₂ O) _0- (where o is an integer of 1 to 100), and - (CH ₂ )pNHC(O)- (where p is an integer of 1 to 20);

n is an integer of 0 to 100; and X is an organic or inorganic counter anion.

In some embodiments, the nucleophilic agent is compound of formula 1(b) wherein L is -O- or -NH-.

In some embodiments, the nucleophilic agent is a compound of Formula 1(c):

wherein Y is selected from -OH, -NH ₂ , -NO2, -SH, -C(O)0H, -C(O)0-alkyl, and halogen;

n is an integer of 0 to 100; and

X- is an organic or inorganic counter anion.

In some embodiments, the nucleophilic agent is compound of formula 1(c) wherein Y is -OH, -NH ₂ , -SH, or -Br.

In some embodiments, the nucleophilic deactivating agent is a compound of Formula 1(d):

wherein Y is selected from -OH, -NH ₂ , -N0 ₂ , -SH, -C(O)OH, -C(O)0-alkyl, and halogen;

o is an integer of 1 to 100; and

X- is an organic or inorganic counter anion.

In one embodiment, the nucleophilic deactivating agent is compound of formula 1(d) wherein Y is -OH, and o is an integer of 2. In some embodiments, the nucleophilic deactivating agent is a compound of Formula 1(e):

wherein Y is selected from -OH, -NH ₂ , -NO2, -SH, C(O)0H, C(O)0-alkyl, alkyl, alkenyl, and halogen; p is an integer of 1 to 20; and

X- is an organic or inorganic counter anion.

In some embodiments, the nucleophilic deactivating agent is compound of formula 1(e) wherein Y is halo, such as -Br, and p is an integer of 3.

In some embodiments, the nucleophilic deactivating agent of formula 1(e), where Y is a hydrocarbon moiety, such as an alkyl or alkenyl group. For example, the nucleophilic deactivating agent may be a compound having one of the following chemical structures:

In some embodiments, the nucleophilic moiety (Nu) includes a linker wherein the linker is attached to any neutral atom or positively charged atom in the nucleophilic moiety, which in some embodiments is a nitrogen. In some embodiments, the linker is attached to the nucleophilic moiety, and in such embodiments the nucleophilic functionality is preserved.

The deactivating agent may form an amphiphilic molecule in an aqueous environment, e.g., forming micelles. In these embodiments, the deactivating agent can be employed as/with detergent compositions, as described further below. As demonstrated herein, the nucleophilic agent deactivates a toxic agent, such as but not limited to an organophosphate-based or carbamate -based toxic agent, by nucleophilic hydrolysis. Further, after such hydrolysis, the nucleophile functionality is regenerated, such that the deactivating property is not merely a stoichiometric reaction. In this manner, the deactivating property of the compositions and articles disclosed herein is not quickly exhausted, but in fact persists.

In some embodiments, the nucleophilic agent is conjugated to a surface, a fabric, a polymer, or an aliphatic or aromatic group indirectly through a linker (Z). For example, such a composition or article may have a structure according to formula A— Z— Nu; wherein (A) is a fabric or polymer, (Z) is a linker, and (Nu) is the nucleophilic moiety. In one embodiment, the nucleophilic agent is bonded to an aromatic or aliphatic group, optionally, through a linker (Z).

In one embodiment, the nucleophilic agent is a conjugate of pyridine-2- aldoxime and chitosan, which may be linked by an acetamide moiety. In some embodiments, the chitosan is functionalized with the nucleophilic moiety at from about 10% to about 50% of available amines, or in some embodiments, from about 10 to about 40% of available amines, or from about 10% to about 30% of available amines. In another embodiment, the nucleophilic agent is a conjugate of pyridine-2- aldoxime and triethoxy silane, which is optionally linked by an acetamide moiety. In these embodiments, a fabric can be functionalized with the nucleophilic moiety, providing for a durable, functional fabric that can withstand numerous detergent washes. Alternative linking groups and conjugation schemes are described herein.

In another embodiment, the linker is a divalent group that is covalently attached to the nucleophilic moiety and is also covalently attached to, e.g., a polymer, a fabric, or a surface in order to make the following structures (as illustrated in Figures 32-35):

1. ) Polymer— Linker— N ucleophilic moiety ;

2.) Fabric— Linker— Nucleophilic moiety; or

3.) Surface— Linker— Nucleophilic moiety.

The linker is attached to the nucleophilic moiety such that the nucleophilic functionality is preserved. The linker is further conjugated to an available reactive group on the surface, the polymer or the fabric. For example, the linker may be attached covalently to a functional group selected from hydroxyl groups, amine groups, or carboxylic groups on the surface, the polymer or the fabric.

In some embodiments, the nucleophilic moiety is linked to a surface, fabric, or polymer through a linking moiety selected from: an acetamide; a carbamate, such as a thiocarbamate; a silane ether such as a triethoxysilane moiety; an ether; and a hydrocarbon linker such as an alkyl, alkenyl, alkynyl, or ethylene oxide moiety (e.g., in a polyethylene glycol linker). In one embodiment, the linker is a triethoxy silane. In another embodiment, the linker is an acetamide moiety.

In some embodiments, the linker (Z) is a linker moiety having one of the following structures:

; where n is an integer of 1 to 100, and R denotes the

position where the linker (Z) is attached to the nucleophilic moiety (Nu).

In some embodiments, the deactivating agent is incorporated into a composition along with one or more carrier compounds or suitable excipients. In various embodiments, the conjugate in the composition does not penetrate the skin and the composition is biocompatible. In various embodiments, the excipient may be, for example, one or more of carbopol 940, glycerin, propylene glycol, disodium EDTA, water, methyl parahydroxy benzoate, stearic acid, liquid paraffin, lanolin, cetostearyl alcohol, glycerol monostearate, isopropyl myristate, propyl parahydroxy benzoate, or potassium hydroxide. In an embodiment, the excipients include one or more of Carbopol 940, glycerin and propylene glycol.

In some embodiments, the composition is in the form of liquid, solid, powder, gel, ointment, lotion, roll-on, paste, spray, aerosol, emulsion, micellar solution, a cream, foam, shower gel, hand wash, shampoo, hair gel, conditioner, soap, detergent, or fabric conditioner. In one embodiment, the composition is a gel. In another embodiment, the composition is in the form a micellar solution.

In some embodiments, the composition also includes carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, gelling agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, antibacterial agents, antifungal agents, lubricating agents and/or dispensing agents, depending on the nature of the mode of application.

Examples of suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances. Various antibacterial and antifungal agents can be included, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Examples of suitable carriers, diluents, solvents, or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and organic esters such as ethyl oleate. Examples of excipients include sodium citrate, calcium carbonate, and dicalcium phosphate. Examples of disintegrating agents include starch, alginic acids, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols.

In an embodiment, the composition comprises the conjugate of pyridine-2- aldoxime and a polymer, such as chitosan, alginate, or cellulose (or derivatives thereof) and an excipient selected from one or more of Carbopol 940, glycerin and propylene glycol.

In some embodiments, the composition is a topical composition that contains from about 0.5% to about 50% (w/w) of the conjugate of pyridine-2-aldoxime and chitosan. In some embodiments, the topical composition contains from about 1% to about 30% (w/w) of the conjugate of pyridine-2-aldoxime and chitosan. In some embodiments, the topical composition contains from about 1% to about 20% (w/w) of the conjugate of pyridine-2-aldoxime and chitosan. In some embodiments, the topical composition contains from about 1% to about 10% (w/w) of the conjugate of pyridine-2-aldoxime and chitosan (e.g., about 2-5%). In an embodiment, the composition comprises about 0.5% to 50% (by weight) of the conjugate of pyridine-2-aldoxime and chitosan, about 1% to 4.5% (by weight) carbopol 940, about 2.5% to 15% (by weight) glycerin, and about 3% to 14% (by weight) propylene glycol.

In an embodiment, the composition comprises about 0.5 to 50% (by weight) of the conjugate of formula 1, about 0.05 to 0.5% (by weight) of disodium EDTA, about 0.05 to 1% (by weight) of methyl parahydroxy benzoate, about 2 to 10% (by weight) of glycerin, about 1 to 8% (by weight) of stearic acid, about 5 to 9% (by weight) of light liquid paraffin, about 0.5 to 2% (by weight) of lanolin, about 1 to 4% (by weight) of cetostearyl alcohol, about 1 to 5% (by weight) of glycerol monostearate, about 0.5 to 4.5% (by weight) of isopropyl myristate, about 0.05 to 1% (by weight) of propyl parahydroxy benzoate, about 0.02 to 1.2% (by weight) of potassium hydroxide, and 50 to 80% (by weight) water.

In some embodiments, the composition comprises a conjugate of pyridine-2- aldoxime and triethoxy silane, and one or more excipients selected from Carbopol 940, glycerin and propylene glycol.

The present disclosure further relates to a process of preparing the composition, by mixing the ingredients described above. For example, in an embodiment, the process of preparing the composition comprises mixing the nucleophilic agent disclosed herein with the excipients, such as Carbopol 940, glycerin or propylene glycol, or any combination thereof. In an embodiment, the process of preparing the composition comprises mixing about 1% to 4.5% (by weight) of Carbopol, about 2.5% to 15% (by weight) of glycerin, about 3% to 14% (by weight) of propylene glycol, and about 0.5% to 50% (by weight) of the nucleophilic agent, in about 50 to about 80% water to obtain the composition.

In one embodiment, the pH of the composition is about 6 to about 9, such as about 8. The pH of the composition may be adjusted using various acids or alkali known in the art. In one embodiment, the pH may be adjusted using triethylanolamine (TEA).

In various embodiments, the composition is capable of deactivating the toxic agents, such as pesticides, insecticides, herbicides and warfare agents at various temperatures and climates, i.e., it is effective in tropical as well as in cold conditions. In various embodiments, the deactivating agent (as incorporated into a composition or article), provides a rate of reaction with one or more organophosphate toxins (i.e., kobs) at a temperature of about 18 °C of at least about 3.37x10 ^-4 s ^-1 (±10%). In various embodiments, the deactivating agent (as incorporated into a composition or article), provides a rate of reaction with one or more organophosphate toxins (i.e.. k ₀ bs) at temperature of about 40 °C of at least about 3.64x10 ^-4 s ^-1 (±10%).

In some embodiments, the nucleophilic deactivating agent is bonded (directly or indirectly) or adsorbed to a polymer. The term polymer as used herein includes a polymer composition, a polymeric surface, or an object made of polymer. In various embodiments, the polymer as provided in a topical composition prevents the toxic agent from entering the skin of a subject. In some embodiments, the polymer enhances the rate of hydrolysis of the toxic agent. The polymer could enhance the rate of hydrolysis of the toxic agent by e.g., about two fold, about three fold, about four fold or more, as compared to the unconjugated nucleophilic moiety. In some embodiments, the composition prevents or protects a subject from toxic agent induced mortality, toxic agent induced neuromuscular dysfunction, toxic agent induced loss of endurance, and toxic agent induced loss of locomotor coordination.

In some embodiments, the polymer may be a natural polymer or a synthetic polymer. Suitable natural polymers include, for example, dextrans, cellulose, chitosan, chitin, gelatins, collagens, lignins, cyclodextrins, carrageenans, gums and mucilage isolated from plant (guar gum, xantham gum, arabic gum, acacia gum, cashew gum, almond gum, pectin), alginic acid, heparin, starch, glucan, inulin, agar, hyaluronic acid, pullulan, or combinations thereof. In some embodiments, the polymer is a polysaccharide.

Synthetic polymers include, for example, Polyethyleneimine (Branched and linear), Poly(vinyl amine), Poly(4-amino styrene), Poly(N-methyl vinyl amine), Poly (vinyl alcohol), Poly (maleic anhydride), Poly (maleic anhydride alt- 1 - octadecene), poly(isobutylene-alt-maleic anhydride), poly (ethylene-alt-maleic anhydride), poly (methyl vinyl ether- alt-maleic anhydride), Poly(acrylonitrile), Poly(2-dimethylamino-ethylmethacrylate) (PDMAEMA), Polyamidoamine (PAMAM) dendrimers, Polyallyl amine, Polypyrrole, Poly acrylic acid, Poly acryloyl chloride, Poly(n-acryl amino acids), Polymethacrylic acid, Poly (methyl methacrylic acid), Poly(hydroxyl ethyl methyl methacrylate), Polystyrene-block-poly(ethylene- ran-butylene)-block-polystyrene-graft-maleic anhydride, Poly (acrylonitrile-co- butadiene), Polyamino acids and their copolymers, sequential copolymers (e.g., poly lysine, poly-ornithine, poly-aspartic acid, poly-glutamic acid).

In some embodiments, the polymer comprises functional groups for attachment of the deactivating agent, and the functional groups may be hydroxyl group, amine group, and/or carboxylic groups. In some embodiments, about 5% to about 80% (or about 10% to about 50%) of the polymer functional groups are functionalized with the deactivating agent.

In some embodiments, the nucleophilic deactivating agent is bonded or adsorbed on to a fabric. In some embodiments, the fabric enhances the rate of hydrolysis of the toxic agent. The fabric may enhance the rate of hydrolysis by e.g., about two fold, about three fold, about four fold or more. In some embodiments, the fabric prevents or protects a subject from toxic agent induced mortality, toxic agent induced neuromuscular dysfunction, toxic agent induced loss of endurance, and toxic agent induced loss of locomotor coordination. In some embodiments, the fabric could be a natural fabric or a synthetic fabric. Exemplary fabrics include cotton, linen, silk, rubber, semi-silk, polyester, acrylic, nylon, lycra, wool, jute, coir or a combination thereof.

In some embodiments, the fabric comprises reactive functional group for conjugation of the deactivating agent, such as a functional group selected from hydroxyl groups, amine groups, and/or carboxylic groups. In some embodiments, about 5% to about 80% (or about 10% to about 50%) of the fabric functional groups are functionalized with the deactivating agent.

In some embodiments, the article of the present invention includes functionalized equipment, clothing or instruments used in agriculture. In one embodiment, the article is a protective mask covering the mouth and/or nose. In another embodiment, the article is piece of protective equipment or gear worn by a farm worker, a factory worker, or other a subject who is at risk of being exposed to a toxic agent. In other embodiments, the article of the present invention includes any object that may be contaminated or has a risk or potential of being contaminated with a toxic agent. The articles include, for example, an air filter, water filter, suit, overalls, innerwear, mask, socks, gloves, clothing, head caps, shirts, pants, coats, jackets, ponchos, shoes, shoe-covers, towels, handkerchief, unstitched wearable clothes, apparel, bedding, canvas, carpets, rugs, coated fabrics, fleece fabrics, hosiery, mattress ticking, medical textiles, mops, non-wovens, pillows with fiberfill, shower curtains, sportswear, underwear, uniforms, towels, trousers, jackets, knits, jersey, shirts, T-shirts, shapeware, seat covers, handbags, and saree. In other embodiments, the article includes a wipe, a filter and surface cleaning cloth. Other examples of the article include glass, a wearable, a windshield, a household item, a sprayer, a vehicle, a living area, a protective box, or an observation box. In one embodiment, the article is a face mask (Figure 28). Figure 28 shows one embodiment of a method of protecting a subject from exposure to a toxic agent. In this Figure 28, the mask prevents the toxic agent from entering the subject’s lungs, oral exposure, or exposure to internal soft tissue.

In one embodiment, the article of the present invention does not lose their ability to deactivate a toxic agent despite multiple uses or multiple washing. In such embodiments, the article may be used repeatedly, e.g., for the purpose of protecting a subject from exposure to a toxic agent or for the purpose of removing a toxic agent. In one example, the article may be washed or rinsed with water (and optionally detergent) and used repeatedly, e.g., for the purpose of protecting a subject from exposure to a toxic agent or for the purpose of cleaning or removing a toxic agent (i.e., decontaminating a surface).

In one embodiment, the article includes a substrate and a conjugate of pyridine-2-aldoxime. In some embodiments, the article includes a substrate and a conjugate of pyridine-2-aldoxime, optionally through a triethoxy silane linkage. In some embodiments, the substrate is a fabric that is functionalized with the nucleophilic moiety, providing for a durable, functional fabric that can withstand numerous detergent washes.

In one aspect, the present invention provides a method of making the article disclosed herein. In an embodiment, the process of preparing the article comprises the step of contacting the nucleophilic agent with a substrate to obtain the article. In one embodiment, the nucleophilic agent is dissolved in a solvent, e.g., methanol or other alcohol solvent, to obtain a nucleophilic agent concentration of about 20 mg/ml. In one embodiment, the solution containing the nucleophilic agent is added to the substrate such that the concentration of the nucleophilic agent on the substrate is about 10 mg/cm ² . Thereafter, in one embodiment, the substrate is dried at a temperature ranging from about 25 °C to 45 °C. This drying step may be followed by incubating the substrate in hot air oven at a temperature of about 70 °C to 120 °C. In some embodiments, any excess solution comprising the composition or the nucleophilic agent is removed by washing the substrate in excess solvent. Figure 30 illustrates one embodiment of a method for preparing the article.

In some embodiments, the nucleophilic agent, as incorporated into compositions or articles for example, deactivate various types of toxic agent, including organophosphates. This deactivation is by a nucleophilic hydrolysis of the toxic agent. In various embodiments, the toxic agent is a carbamate -based pesticide, an organophosphate -based pesticide, an ester-based pesticide, a chemical warfare agent. In some embodiments, the toxic agent comprises an herbicide, a fungicide, and/or an insecticide.

In an embodiment, the composition deactivates organophosphate or organophosphate-based pesticides, insecticides, herbicides, fungicides and chemical warfare agents selected from acephate, acethion, acetophos, amidithion, amiton, aspon, athidathion, azamethiophos, azinphos-ethyl, azinphos-methyl, azothoate, bensulide, bomyl, bromfenvinfos, bromophos, bromophos-ethyl, butathiofos, cadusafos, calvinphos, carbophenothion, cadusafos, chlorethoxyfos, chlorfenvinphos, chlormephos, Chlorphoxim, chlorprazophos, Chlorpyrifos, Chlorpyrifos-methyl, Chlorthiophos, colophonate, Coumaphos, coumithoate, Crotoxyphos, Crufomate, Cyanofenphos, cyanophos, cyanthoate, cythioate, dematon, dematon-S-methyl, dematon-O-methyl, dematon-S-methylsulphon, demephion, demephion-O, demephion-S, dialifor, dialifos, diazinon, dicapthon, Dichlofenthion, dichlorvos, dicrotophos, dicresyl-butylphosphate, dicresyl phenyl phosphate, dimefos, Dimethoate, dimethylvinphos, dioxabenzofos, dioxathion, dioxabenzofos, Disulfoton, dithicrofos, edifenphos, endothion, etaphos, Ethion, ethoate-methyl, Ethoprop, ethoprophos, ethyl-parathion, parathion, Etrimfos, etrimfos, famphur, fenamiphos, fenchlorphos, Fenitrothion, fenophosphon, fensulfothion, fenthion, fenthion-ethyl, Fonofos, formothion, fosmethilan, Fospirate, fosthietan, Heptenophos, heterophos, iodofenphos, isazofos, isofenfos, isothioate, isoxathion, jodfenphos, leptophos, lirimfos, lythidathion, malahion, mecarbam, menazon, mephosfolan, merphos, mesulfenfos, methacrifos, Methamidophos, methidathion, methocrotophos, methyl- parathion, methylacetophos, Mevinphos, mipafox, Monocrotophos, morphothion, naftalofos, Naftalophos, naled, omethoate, oxydemeton-methyl, oxydeprofos, oxydisulfoton, phenkapton, phenthoate, Phorate, phosalone, phosfolan, phosmet, phosnichlor, Phosphamidon, phospholan, phoxim, phoxim-methyl, pirimioxyphos, pirimiphos ethyl, pirimiphos methyl, primidophos, profenofos, Propaphos, propetamphos, propyl thiopyrophosphate, prothidathion, prothiofos, prothoate, pyraclofos, pyrazophos, pyrazothion, pyridaphenthion, pyrimitate, Quinalphos, Quinalphos-methyl, quinothion, Ronnel, schradan, sophamide, sulfotep, Sulprofos, tebupirimfos, temephos, Terbufos, tetrachlorvinphos, thicrofos, thiometon, thionazin, triazophos, tribufos, Trichlorfon, trichlormetaphos-3, trifenofos, vamidothion, xiaochongliulin, zolaprofos , Ampropylfos, Ditalimfos, Edifenphos, Fosetyl, Hexylthiofos, iprobenfos, pyrazophos, tolclofos-methyl, triamiphos, inezin, izopamfos, kejunlin, phosdiphen, amiprofos -methyl, amiprofos, anilofos, bilanafos, butamifos, clacyfos, fosamine, huangcaoling and piperophos, or any combination thereof.

In some embodiments, the deactivating agent (as incorporated into compositions and articles, for example) deactivates carbamate or carbamate based pesticides, insecticides, herbicides, fungicides and chemical warfare agents selected from alanycarb, aldicarb, aldoxycarb, allyxycarb, aminocarb, bendiocarb, benfuracarb, benomyl, bufencarb, butacarb, butocarboxim, butoxycarboxim, carbanolate, carbaryl, carbofuran, carbosulfan, cloethocarb, decarbofuran, dimetan, dimethacarb, dimetilan, dioxacarb, ethiofencarb, fenethacarb, fenobucarb, furathiocarb, hyquincarb, isolan, isoprocarb, methiocarb, methomyl, metolcarb, mexacarbate, nitrilacarb, oxamyl, pirimicarb, promacyl, promecarb, propoxur, pyramat, pyrolan, tazimcarb, thiocarboxime, thiodicarb, thiofanox, trimethacarb, xylylcarb, benthiavalicarb, furophanate, iodocarb, iprovalicarb, picarbutrazox, propamocarb, pyribencarb, thiophanate, thiophanate-methyl, tolprocarb, albendazole, carbendazim, cypendazole, debacarb, mecarbinzid, diethofencarb, pyraclostrobin, pyrametostrobin, triclopyricarb, asulam, carboxazole, chlorprocarb, dichlormate, fenasulam, karbutilate, terucarb, barban, carbasulam, carbetamide, chlorbufam, chlorpropham, desmedipham, methyl 3,4-dichlorcarbanilate, phenisopham, phenmedipham, phenmedipham-ethyl, and propham, or any combination thereof.

In some embodiments, the deactivating agent (as incorporated into compositions or articles, for example) deactivates ester or ester-based pesticides, insecticides, herbicides, fungicides and chemical warfare agents selected from a group comprising allethrin, acrinathrin, barthrin, bifenthrin, bioethanomethrin, brofenvalerate, brofluthrinate, bromethrin, butethrin, chlorempenthrin, cyclethrin, cycloprothrin, cyfluthrin, cyhalothrin, cypermethrin, cyphenothrin, deltamethrin, dimefluthrin, dimethrin, empenthrin, d-fanshiluquebingjuzhi, chloroprallethrin, fenfluthrin, fenpirithrin, fenpropathrin, fenvalerate, flucythrinate, fluvalinate, furamethrin, furethrin, heptafluthrin, imiprothrin, japothrins, kadethrin, methothrin, metofluthrin, momfluorothrin, pentmethrin, permethrin, phenothrin, prallethrin, profluthrin, proparthrin, pyresmethrin, renofluthrin, resmethrin, tefluthrin, terallethrin, tetramethrin, tetramethylfluthrin, tralocythrin, tralomethrin, and transfluthrin, or any combination thereof.

In some embodiments, the deactivating agent (as incorporated into compositions and articles, for example) deactivates chemical warfare agents selected from sarin, tabun, cyclosarin, VE (S-(Diethylamino)ethyl O-ethyl ethylphosphonothioate), VG, (O,O-diethyl S-[2-(diethylamino)ethyl] phosphorothioate), VM, S-[2-(Diethylamino)ethyl] O-ethyl methylphosphonothioate, VR, (N,N-diethyl-2-(methyl-(2-methylpropoxy)phosphoryl)sulfanyle thanamine),

VX, (Ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl)(methyl)phosphinat e), and soman, or any combination thereof.

In some embodiments, the compositions or nucleophilic agents could be used to make, e.g., gel, ointment, creams, detergent, fabric conditioner, fabric activator, fabric soak, fabric damp, lotions, spray-on, roll-on, application tissues, wet tissues, dry power, dust, aerosol, spray, paints, tiles, ceramic tiles, glass, metal surface, mist, fog, splash, vapors, surface decontaminant, air filters, water filters, eye gel/drops, nasal gel/drops, oral gel/drops, ear gel/drops for use by a subject or in a condition where there is a risk of exposure to a toxic agent or where there is known exposure to a toxic agent. In some embodiments, the compositions or nucleophilic agents could be used, e.g., in treatment of waste contaminated with toxic agents or at risk of contamination with toxic agents, in cleaning water bodies or soil contaminated with toxic agents or at risk of contamination with toxic agents, in cleaning food items, including, fruits, grains, tubers, vegetables and flowers contaminated with toxic agents or at risk of contamination with toxic agents (before or after harvest).

In some embodiments, the compositions or nucleophilic agents could be used, e.g., in decontaminating glass as wearable, windshields, housing, sprayer vehicle, cubicle, protective box, observation box. In some embodiments, the compositions or nucleophilic agents as disclosed herein could be used for protection of animals (e.g., domestic pets, farm animals, or insects). In one embodiment, the compositions or nucleophilic agents could be used for making insect feed or pollinator protector. The compositions or nucleophilic agents could be used as a prophylactic or therapeutic for a subject.

In some embodiments, the compositions or nucleophilic agents could be used, e.g., in detection of exposure to a toxic agent or to evaluate the level of contamination by the toxic agent. In some embodiments, the compositions or nucleophilic agents could be used, e.g., as a food additive, in a surface coating, in an animal wash or dip, in a hand sanitizer, in a floor cleaner, or in an equipment cleaner.

As used herein, the term“about” means + or - 10% of the associated numerical value.

The present invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1: Methods

In-vitro: Hydrolytic cleavage of pesticides using a nucleophilic topical gel was studied for preventing inhibition of AChE using Franz diffusion cells.

In-vivo: A series of experiments were designed to recapitulate pesticide-induced systemic AChE inhibition, loss of endurance, loss of NMC, sciatic nerve damage, and lethality in rats upon dermal exposure of pesticide, MPT. The hypothesis that presence of nucleophilic poly-Oxime gel can deactivate pesticides on the skin and prevent pesticide-induced toxicity and mortality was tested.

Rats: For all experiments, 10- to l4-week-old Sprague-Dawley albino rats were used. Animals were provided by the animal house at the National Centre for Biological Sciences, Bengaluru. Animals were caged (maximum, four per cage) before the experiment and individually after starting the experiment. Food and water were offered ad libitum. All rat studies were performed according to institutional and national guidelines for humane animal use. Experimental protocols were approved by the Institute Animal Ethical Committee at the Institute for Stem Cell Biology and Regenerative Medicine (INS -IAE-2016/09).

Materials: Pyridine-2-aldoxime, bromoacetic acid, chitosan (medium molecular weight), dimethyl formamide, potassium carbonate, O(benzotriazol-l-yl)- N N N N’-tctramcthyluronium hexafluorophosphate (HBTU), parathion methyl (PESTANAL), methyl paraoxon (PESTANAL), chlorpyrifos (PESTANAL), carbopol 940, glycerin, propylene glycol, triethanolamine (TEA; S.D. Fine-Chem Limited, Bangalore), snake skin dialysis membrane (MWCO, 3.5 kDa; Thermo Fisher Scientific), trifluoroacetic acid, pNP (Alfa Aesar), isoflurane (Isotroy), Triton X100, EDTA (Thermo Fisher Scientific), 5,5'-dithiobis(2-nitrobenzoicacid) (DTNB), acetylthiocholine iodide (ASChl), and deionized water were used. Unless mentioned otherwise, all chemicals were procured from Sigma- Aldrich.

Catalytic efficiency of poly-Oxime: A solution of poly-Oxime (2 mg/ml) in 1% acetic acid was used to study its catalytic ability to cleave pesticides. The reactions were studied spectrophotometrically by monitoring the hydrolysis of pesticides (MPT, methyl paraoxon, or chlorpyrifos) at 25 ± 0.1 °C and pH 8.2 by measuring the absorbance at 400 nm as a function of time. Pseudo-first-order rate constants were obtained by using an excess of nucleophile (2.5 x 10 ^-4 M) over the substrate (2.5 x 10- ⁵ M). For all the kinetic runs, the absorbance/time result fits very well to the first-order rate equation. To study temperature stability, the same reactions were carried out at 20°, 30°, and 40 °C.

Preparation of poly-Oxime gel: The dermal gel was formulated by using carbopol 940 (1.8%), glycerin (6.81%), propylene glycol (3.74%), and poly-Oxime polymer (2%) mixed in water (85.6%). The pH of the gel was maintained at 8 by TEA. For sham gel, the un-functionalized chitosan polymer, instead of the poly-Oxime polymer, was used, serving as a control gel with all ingredients but no catalytic activity.

In vitro and ex vivo efficacy of poly-Oxime dermal gel to reduce permeation of pesticide: The dialysis membrane (MWCO, 3.5 kDa) was hydrated overnight in the deionized water at room temperature. It was then placed between the donor and acceptor compartments of the Franz diffusion cells (DBK Diffusion apparatus) and clamped to avoid any leakage. The acceptor compartment was filled with PBS (pH

7.4), making uniform contact with a dialysis membrane. The experiment was performed in three groups (only membrane, sham gel, and poly-Oxime gel) comprising three diffusion cells in each set. Poly-Oxime or sham gel (220 mg) was applied uniformly on 10 cm ² area on the donor compartment side of the dialysis membrane. On the gel, 1 mg of MPT was added in 500 ml of PBS, and in the direct exposure, MPT was added directly on the dialysis membrane. The temperature of the acceptor chamber was maintained at 37 ± 0.5 °C using a thermostatic water bath under constant stirring. Samples were withdrawn from acceptor (1 ml) and donor chambers (20 ml) at a regular interval of 1 hour, and an equal amount of phosphate buffer (pH

7.4) was replaced. The amount of MPT that permeated through the membrane and the amount of pNP that formed at each time interval were analyzed using UFLC (photodiode array: SPD-M20A, C18 reverse-phase column: LC-20AD Prominence Chromatograph, Shimadzu). MPT was detected using 60% acetonitrile in double- distilled water (DDW) as mobile phase at 1 ml/min, with a retention time of 5.5 min at 280 nm while maintaining the column at 40 °C. For detection of pNP, 22% acetonitrile, 0.5% triethylamine, and 1% trifluoroacetic acid in DDW was used as mobile phase at a flow rate of 0.5 ml/min through a column maintained at 40 °C with a retention time of 3.6 min. Similarly, in another set of experiment, the solution in the acceptor chamber was replaced with rat blood 1000X diluted in phosphate buffer (pH 8.0). The entire diffusion apparatus was maintained at 5 °C to maintain the blood AChE activity. Samples were collected at regular intervals and analyzed using Ellman’s method (as mentioned elsewhere) for AChE activity as a proxy for the MPT exposure.

Protection from acute exposure to MPT: Rats were randomized to one of four experimental groups (n =6 in each group): (i) no exposure (no MPT was given), (ii) direct exposure (MPT, 150 mg/kg), (iii) sham gel (220 mg of gel; MPT, 150 mg/kg), and (iv) poly-Oxime gel (220 mg of gel; MPT, 150 mg/kg). Polymer concentration in the gel was 2% (w/w). The dorsal coat was clipped using a hair clipper under mild anesthesia (2.5% isoflurane) 24 hours before the dermal pesticide application, taking care not to damage the integrity of the skin. Unless specified otherwise, the total area of 10 cm ² was marked and used for dermal exposure experiments. In gel treatment groups, 220 mg of gel was uniformly applied 1 hour before the pesticide exposure. Before exposure, 200 ml of blood was collected using retro-orbital puncture and evaluated for various parameters as an internal control. After 96 hours of post exposure, blood was collected using cardiac puncture under anesthesia. After sacrificing the animals, organs such as brain, heart, lungs, and liver were harvested. Organ tissue was homogenized (Polytron PT-MR 2100, 15,000 rpm) in nine volumes of solution D [1 M NaCl, 1% Triton X-100, 0.01 M tris-HCl, 0.01 M EDTA (pH 7.4)] and incubated on ice for 1 hour, followed by centrifugation at 13,300 rpm for 45 min. The supernatant was used to quantitate active AChE. Whole blood diluted 1: 1000 in phosphate buffer was used for AChE quantification. The activity of AChE in blood and organs was quantified by modified Ellman’s assay. In this assay, DTNB and ASChl was used, which is specific for AChE. For the colorimetric assay, according to Ellman’s method, reaction mixtures were made up in 0.1 mM phosphate buffer (pH 7.4) containing 0.5 mM DTNB and ASChl at a final concentration of 20 mM. The reaction was performed at 25 °C and monitored at 405 nm.

Survival and AChE inhibition study in rats exposed to MPT multiple times: Rats were randomized to three groups (n = 6 rats per group): (i) direct exposure (no gel, MPT given dermally, 100 mg/kg per day for 4 days), (ii) sham gel (220 mg of sham gel applied daily dermally 30 min before the MPT exposure), and (iii) poly-Oxime gel [220 mg of gel applied daily dermally before applying MPT (100 mg/kg per day) daily for 4 days] (Figure 6A). In all animals before exposure, 200 ml of blood was collected using retro-orbital puncture and evaluated for AChE activity as an internal control. Immediately after mortality or on day 30, animals were sacrificed, and their organs were collected to quantify the AChE level as described above. Similarly, another set of a single application of gel experiments was performed (n = 6 rats per group). In this experiment, 220 mg of sham gel or poly-Oxime gel was applied only once, i.e., on day 0, and on the same gels, these animals received MPT (100 mg/kg per day) for 4 days (Figure 6D). Rotarod test: A total of 12 male Sprague-Dawley rats (10 weeks) were used to study the loss of endurance in MPT-exposed animals and the ability of poly-Oxime gel to prevent the same. Animals were placed on a Rotarod treadmill (Rotamex-5 1.4, Columbus Instruments; lane width, 9.3 cm; diameter of rod, 7 cm; fall distance, 48.3 cm), subjected to a uniform increase in acceleration between 0 and 20 rpm, and allowed to run at 20 rpm until the animal got tired and fell off the rod, and the time of fall was recorded. Rats were trained for 3 days before the exposure, where animals were randomly grouped into three groups: (i) direct exposure, (ii) poly-Oxime gel- treated, and (iii) control. On day 4, animals of the direct exposure group were exposed to a dosage of 150 mg/kg, and the animals of treatment group were given poly-Oxime gel and then exposed to the same dosage of MPT. Latency to fall was calculated as the time taken to fall on any day with respect to day 4 (before exposure) and was converted to percentage. Similarly, to study NMC, 15 animals were trained on an increasing rotarod acceleration between 2 and 60 rpm, with an acceleration step of 4 rpm every 8 s. On day 5 of training, animals were divided into three groups: (i) direct exposure, (ii) poly-Oxime gel-treated, and (iii) naive control. On day 5, animals of the direct exposure group were exposed to a dosage of 150 mg/kg, and the animals of treatment group were given poly-Oxime gel and then exposed to the same dosage of MPT. Animals were given a score of +1 for each step up in the acceleration (increase by 4 rpm) before the fall, and the percentage of NMC was calculated with respect to day 5 (before exposure).

Gait analysis and EMG: Gait analysis was performed on 15 male rats (10 weeks old) to evaluate their walking pattern. To obtain footprints, four paws were colored with different nontoxic water colors and animal was trained to walk through an ally (width, 8 cm; length, 120 cm; height, 10 cm), leading to its home cage. After training, animals were divided randomly into three groups: (i) direct exposure, (ii) poly-Oxime gel- treated, and (iii) naive control. Footprints were analyzed manually, and SFI was calculated using following formula:

where N indicates normal/before exposure, E indicates after exposure, TOF is the distance to the opposite foot, PL is the distance from the heel to the third toe (the print length), TS is the distance from the first toe to the fifth toe (the toe spread), and GG is the distance from the second toe to the fourth toe (the intermediate toe spread). EMG of animals from these three groups was recorded using Muscle SpikerBox (Backyard Brains). Skin surface electrodes were used with adhesive pads and conductive gel to facilitate the recordings. To track the muscle spasms under anesthesia (2.5% isoflurane in carbogen), EMG from biceps femoris of left hind limb was recorded. To avoid animal from disturbing the electrodes, EMG was recorded between spino trapezius and gluteus maximus when the animal was awake.

Statistical Analysis: The two-tailed Student’s t test was used to compare differences between two experimental groups. In experiments with multiple groups, one-way ANOVA with Tukey post hoc test was used. In survival experiments, Mantel-Cox test was used. P < 0.05 was considered as a statistically significant difference. Statistical analysis and graphing were performed with Prism 6 (GraphPad Software).

Osteogenic Differentiation of MSCs: Osteogenic differentiation was induced in 90% confluent human bone marrow derived mesenchymal stem cells (hBM-MSC) for up to 21 days in alpha-MEM media (Himedia) supplemented with, 1% heat-inactivated FBS (Himedia), IX antibiotic and antimycotic solution (Himedia), 5 mM b- glycerophosphate (Alfa-aesar), 50 mg/ml L-ascorbic acid (Sigma- Aldrich), and 10 nM dexamethasone (Himedia). Osteogenic differentiation was detected by alkaline phosphatase levels in media followed by Von Kossa staining to detect mineralization post 21 days. For Von-Kossa staining, cells were fixed in 1% glutaraldehyde solution (Sigma) for 1 hour at room temperature. After two washes with PBS and one wash with deionized water cells were incubated in 1% silver nitrate (Sigma -Aldrich) and exposed to UV for 60 minutes. The cells were then incubated for 5 minutes with 2.5 wt.% Sodium thiosulphate (Fisher Scientific), washed thrice with deionized water and images were taken. Quantitative determination of alkaline phosphatase activity was performed by measuring the absorbance of the reaction product from the conversion of para-nitrophenol phosphate (Sigma-Aldrich) by alkaline phosphatase. Cells were fixed in 95% ethanol for 5 min and incubated in a buffer containing 20 mM NaHC0 ₃ , 3 mM MgCl ₂ , and 1 mg/ml para -nitrophenol, pH 9.5 for 10-30 min. Absorbance of buffer was measured using 96-well plate at 405 nm using Multi-Mode Reader SPECTRAMAX.

Synthesis of poly-Qxime and poly-methoxyOxime:

Scheme for the synthesis of poly-Oxime:

Synthesis of l-(carboxymethyl)-2-((hydroxyimino)methyl)pyridin- l-ium bromide (3): To a stirred solution of 2-picolinaldehyde oxime 1 (5 g, 40 mmol) in 175 ml of acetone, 2-bromoacetic acid 2 (5.68 g, 40 mmol) was added at room temperature. The reaction mixture was refluxed for 48 hours. The product formation of reaction was monitored by TLC. Upon cooling the reaction mixture to room temperature, light brown color solid was formed. The solid was filtered under vacuum and washed with acetone (4X with 50 ml) and dried under vacuum which gave 2 g of l-(carboxymethyl)-2- ((hydroxyimino)methyl)pyridin- l-ium bromide 3 as a light brown solid (19% yield).

Characterization:

1H-NMR (DMSO-d6 600 MHz) d = 13.22 (1H, s), 9.05-9.04 (1H, m), 8.67 (1H, s), 8.66-8.65 (1H, m), 8.46-8.45 (1H, m), 8.15-8.15 (1H, m), 5.75 (2H, s).

¹³ C-NMR (DMSO-d6 150 MHz) d= 167.68, 148.12, 147.74, 146.77, 142.77, 127.68, 126.66, 59.64.

MS (M+l) = 181.2

Synthesis of poly-Oxime (5):

To a stirred solution of l-(carboxymethyl)-2-((hydroxyimino)methyl)pyridin- l-ium bromide 3 (2 g, 7.6 mmol) in 40 ml of DMF, Potassium Carbonate (4.2 g, 30.7 mmol), HBTU (3.4 g, 9.23 mmols) was added. Stirring was continued at room temperature for 15 mins. To this activated carboxylic acid, Chitosan (1.66 g, 9.23 mmols, which was dissolved in 65 ml of 1% acetic acid) was added dropwise using a dropping funnel over a period of 15 mins, and stirred at room temperature for 48 hours. Initially the reaction mixture was completely dissolved and slowly it was precipitated. The reaction mixture was transferred to a dialysis bag (MWCO 3.5 kDa) to remove unreacted reactant. After dialyzing in 1M NaCl for 24 hours, dialysis was continued in deionized water for 48 hrs. After completion of dialysis the reaction mixture was lyophilized to get 2.4 g of poly- Oxime (5).

1H-NMR (TFA 600 MHz): 8.2-7.8 (2.5H, br, Aromatic Protons), 5.57 (0.9H, br, - CH ₂ -), 5.34 (1H, br, Chitosan protons), 4.0-3.2 (6H, Chitosan Protons). Based on the NMR data, the calculated conjugation of the nucleophile to chitosan polymer is -40%.

Scheme for the synthesis of poly -methoxy Oxime :

Synthesis of (E)-l-(2-( tert-butoxy)-2-oxoethyl )-2-( ( hydroxyimino jmethyl )pyridin-l - ium bromide (7): To a stirred solution of 2-picolinaldehyde oxime 1 (5g, 40 mmol) in 150 ml of acetonitrile, tert-butyl 2-bromoacetate 6 (7.9 g, 40 mmol) was added at room temperature. The reaction mixture was refluxed at 90 °C for 48 hours. The product formation of reaction was monitored by TLC. Upon cooling the reaction mixture to room temperature, solid was precipitated. The solid were filtered under vacuum and washed with acetonitrile (4 times with 50 ml) and dried under vacuum to get 5.5 g of (E)-l-(2-(tert-butoxy)-2-oxoethyl)-2-((hydroxyimino)methyl)p yridin-l- ium bromide 7 (43% Yield).

Characterization: 1H-NMR (DMSO-d6 600 MHz) d= 13.16 (1H, s), 9.05-9.04 (1H, m), 8.69 (1H, s), 8.46-8.44 (1H, m), 8.33-8.32 (1H, m), 8.19-8.17 (1H, m), 5.77 (2H, s), 1.43 (9H, s).

¹³ C-NMR (DMSO-d6 150 MHz) d= 165.21, 147.96, 147.03, 142.69, 127.77, 127.19, 84.37, 79.69, 60.03, 27.95.

MS (M+l)=237.2

Synthesis of (E)-l-(2-(tert-butoxy)-2-oxoethyl)-2-((methoxyimino)methyl)p yridin-l- ium bromide (8): To a suspension of (E)-l-(2-(tert-butoxy)-2-oxoethyl)-2- ((hydroxyimino)methyl)pyridin-l-ium bromide 7 (2g, 6.3 mmols) in acetonitrile, Potassium Carbonate (0.88g, 6.3 mmols) and methyl iodide (1.07 g, 7.59 mmols) were added, and stirred at 100 °C for 12 hrs in a pressure tube. The product formation was confirmed by mass spectroscopy. Upon cooling the reaction mixture to room temperature, solid was formed. Subsequently, filtered the reaction mixture to separate solid potassium carbonate, washed the residue with acetonitrile (4 times in 50 ml) and evaporated the filtrate with reduced pressure to get 1.8 g of (E)-l-(2-(tert-butoxy)-2- oxoethyl)-2-((methoxyimino)methyl)pyridin-l-ium bromide, 8 (86% Yield).

Characterization: 1H-NMR (DMSO-d6 600 MHz) d= 8.97-9.95 (1H, m), 8.69 (1H, s), 8.65-8.62 (1H, m), 8.35-8.34 (1H, m), 8.15-8.12 (1H, m), 5.65 (2H, s), 4.01 (3H, s), 1.34 (9H, s).

¹³ C-NMR (DMSO-d6 150 MHz) d= 165.12, 148.53, 147.44, 146.57, 143.33, 128.36, 128.04, 79.66, 60.52, 49.02, 28.00.

MS (M+l)=25l.2.

Synthesis of (E)-l -(carboxymethyl)-2-((methoxyimino)methyl)pyridin-l -ium bromide (9): To a stirred solution of (E)-l-(2-(tert-butoxy)-2-oxoethyl)-2-((methoxyimino) methyl)pyridin- 1 -ium bromide 8 (l.6g, 4.84 mmols) in 10 ml of Dichloromethane, 10 ml of TFA was added at 0 °C. The reaction mixture was stirred at room temperature for overnight. Completion of the reaction was monitored by mass spectroscopy. After completion of the reaction, the solvent was evaporated under reduced pressure to get a gummy residue. Upon addition of «-hexane solid was precipitated. The solid was filtered and dried with vacuum to get 1.2 g of (E)-l-(carboxymethyl)-2- ((methoxyimino)methyl)pyridin-l-ium bromide, 9 (90% yield).

Characterization:

1H-NMR (DMSO-d6 600 MHz) d= 9.06-9.05 (1H, m), 8.79 (1H, s), 8.69-8.67 (1H, m), 8.45-8.43 (1H, m), 8.21-8.19 (1H, m), 5.73 (2H, s), 4.08 (3H, s).

¹³ C-NMR (DMSO-d6 150 MHz) d= 167.74, 149.87, 146.18, 144.89, 142.90, 128.29, 127.42, 60.02, 49.10

MS (M+l)=l95.2

Synthesis of poly-methoxyOxime (10): To a stirred solution of l-(carboxymethyl)-2- ((hydroxyimino)methyl)pyridin-l-ium bromide 3 (1 g, 3.64 mmol) in 20 ml of DMF, Potassium Carbonate (2 g, 14.5 mmol) and HBTU (1.65 g, 4.37 mmol) were added. The reaction mixture was stirred at room temperature for 15 mins. To this reaction mixture, Chitosan (0.788 g 4.37 mmol which was dissolved in 32 ml of 1% acetic acid) was added dropwise using a dropping funnel over a period of 10 mins, and stirred at room temperature for 48 hours. After completion of the reaction, the reaction mixture was transferred to a dialysis bag (MWCO 3.5 kDa) to remove the unreacted reactant. After dialyzing in 1M NaCl for 24 hours, dialysis was continued in deionized water for 48 hrs. After completion of dialysis the reaction mixture was lyophilized to get 1.2 g of poly-methoxyOxime (10).

Characterization:

1H-NMR (TFA 600 MHz): 8.2-7.9 (5H, br, Aromatic Protons), 5.61 (1.88 H, br, - CH2-), 5.38 (1H, br, Chitosan protons), 4.06 (2.8H, s, OCH3), 4.0-3.3 (9H, Chitosan Protons). Based on the NMR, the calculated conjugation of the methoxy-oxime to chitosan polymer is between -50% to 55%.

Example 2: Synthesis of poly-Qxime Gel and Deactivation of Pesticides In-vitro and Ex-vivo

Oxime (A-hydroxy compound) is a potent a-effect (9-nucleophile. Pyridine-2- aldoxime is a powerful a-nucleophile which can be functionalized on a biopolymer, Chitosan (Figure 1B). Conjugation of pyridine-2-aldoxime to chitosan through an acetamide linker in solution phase generated poly- Oxime (Figure 1B). The degree of functionalization was 20%. The kinetics of MPT hydrolysis was obtained by monitoring the appearance of p-nitrophenoxide spectrophotometrically at 400 nm (Figure 1A). The observed pseudo-first-order (k ₀ bs) values for the cleavage of MPT, paraoxon, and chlorpyrifos were 4.59, 2.28 and 5.55 x10 ^-4 s ^-1 , respectively. Data shows that poly- Oxime is an effective catalyst for the hydrolytically deactivation of multiple organophosphates, in that they afford more than two orders of magnitude rate enhancement over the background reaction. Under similar reaction conditions, native chitosan without oxime groups (sham polymer) did not deactivate pesticides suggesting that poly- Oxime a-nucleophile is essential for the reactivity. The poly- Oxime also showed activity at different temperatures, (at l8°C, k _{0 bs} 3.37 x10 ^-4 s ^-1 ; at 40°C, k _obs 3.64 x10 ^-4 s ^-1 ). To evaluate poly- Oxime gel’s ability to prevent pesticide penetration into the skin, the poly- Oxime (2% w/w) was formulated into a topical gel using additives, glycerin (6.8% w/w), propylene glycol (3.74% w/w) and carbopol 940 (1.8% w/w). Sham-gel, as a control, was prepared using the same composition with unmodified chitosan replacing the poly- Oxime. Both gels have shown standard gel-like behavior in rheology studies (Figure 3), and could be applied on the skin. Franz diffusion apparatus was used to mimic the transdermal penetration (Figure 2A). Uncoated dialysis membrane (MWCO: 3.5kD) or coated with a thin layer of sham-gel or poly- Oxime gel was placed between donor and acceptor chambers. Subsequently, MPT was added into the donor chamber, and acceptor chamber was filled with phosphate buffered saline (PBS). Chamber’s temperature was maintained at 37 °C. At different time points (0, 1, 2 and 3 h) concentration of MPT and its hydrolytically degraded product -nitrophcnol (pNP) was quantified in both donor and acceptor chambers using ultra-fast liquid chromatography (UFLC) (Figures 2B to E). In the presence of uncoated membrane or sham-gel coated membrane, MPT was quantitatively penetrated into the acceptor chamber within 3 h, whereas poly- Oxime gel prevented the MPT penetration (Figures 2B, 2C). It suggests that sham- gel does not act as a physical barrier, and MPT can easily pass through the gel. Data in Figures 2D and 2E shows that poly- Oxime gel was able to degrade MPT into pNP which was quantified in both donor and acceptor chambers. Interestingly, pNP was not generated in any of those chambers in the absence of poly- Oxime gel. These results suggest that poly- Oxime gel prevents penetration of MPT not as a physical barrier, but through chemically hydrolyzing MPT.

An ex vivo assay was carried out using rat blood in Franz diffusion cell to evaluate the poly- Oxime gel’s ability to prevent MPT-induced AChE inhibition (Figure 2F). Akin to the previous experiment, three groups were taken (uncoated, sham-gel and poly- Oxime gel coated membranes between both chambers) along with no MPT group, and 50x diluted rat blood was taken in acceptor chamber, and % of the AChE activity was quantified using modified colorimetric Ellman’s assay (see Methods). In the absence of MPT, no change was observed in the activity of AChE after 3 h (Figure 2G). The addition of MPT (1 mM) in donor chamber led to a significant inhibition of AChE activity in blood. The presence of native membrane or sham-gel coated membrane did not prevent MPT mediated AChE inhibition (Figure 2G). On the contrary, poly- Oxime gel deactivated MPT before entering into the acceptor chamber, thereby completely preventing MPT-induced inhibition of blood AChE activity. These results clearly suggest that nucleophilic poly- Oxime gel could protect from pesticide-induced AChE inhibition by chemically deactivating organophosphates.

AChE is known to be expressed on human mesenchymal stem cells (hMSC). Pesticide exposure to the cultured hMSCs is known to reduce their ability to differentiate into osteogenic lineage. In a trans-well based in vitro assay, the ability of the poly- Oxime gel to prevent MPT-induced inhibition of hMSC’s osteogenic differentiation was investigated (Figure 2H). Bone marrow-derived hMSCs were cultured in osteogenic media (see Methods). During 21 days differentiation protocol, cells were constantly exposed to MPT (100 mM) through trans-well either directly or through an uniform layer of poly- Oxime gel applied on the trans-well membrane. Osteogenic differentiation was assessed by staining mineral deposits using Von Kossa stain (Figure 2H). Reduced mineral deposition was observed in the directly MPT exposed cells, while MPT exposure through the poly- Oxime gel layer did not reduce the mineral deposition. Additionally, quantitative analysis of alkaline phosphatase (ALP) activity was done. ALP activity was restored to 84% by the poly- Oxime gel which was reduced to only 30% in MPT exposed group. (Figure 21). These results suggest that the poly- Oxime gel prevents the MPT induced inhibition of hMSC’s osteogenic differentiation.

Example 3: Poly-Oxime Gel Prevents Pesticide-induced AChE Inhibition in Blood and Tissue, In-vivo

To evaluate the ability of poly- Oxime gel to prevent MPT-induced AChE inhibition, 18 Sprague-Dawley (SD) rats (10-13 weeks) were randomly divided into 3 groups (n=6 per group. Dorsal coat was clipped and 10 cm ² area was exposed to MPT directly, or in presence of sham-gel or poly- Oxime gel layer (see Methods). According to the WHO, pesticides which have LD50 of 10-100 mg/kg of body weights in rats (dermal exposure) are considered as‘highly hazardous, class lb. To test the robustness of poly- Oxime gel, all groups were exposed dermally to 150 mg/kg of MPT (Figure 4A). Quantification of active AChE in the blood at pre-exposure (0 h) and post-exposure (90 h) revealed that dermal exposure of MPT alone or in the presence of sham-gel, significantly decreased the active AChE, whereas, in the presence of poly- Oxime gel no such decrease was seen (Figure 4B). Animals those were exposed to MPT alone or in the presence of sham-gel lost -20% their initial body weight by day 4, while animals exposed to MPT in the presence of poly- Oxime gel did not lose their weight and showed normal weight gain (Figure 5). Additionally, on day 4, animals were sacrificed, and tissues like brain, heart, liver and lung were isolated and quantified for active AChE in comparison with the naive rat tissues. Data in Figures 4C-F clearly suggest that MPT exposure leads to decrease in active AChE levels, while sham-gel could not protect from MPT exposure. On the contrary, the presence of poly- Oxime gel prevented the reduction of active AChE. These results demonstrate that poly- Oxime gel does not act like a typical barrier cream, but it hydrolytically deactivates organophosphate leading to prevention of AChE inhibition.

Example 4: Poly-Oxime Gel Prevents Pesticide-induced Mortality, In-vivo

As multiple exposures of MPT could cause mortality, the ability of the poly- Oxime gel to prevent MPT-induced mortality was evaluated in rats. Twelve SD rats were randomized into two groups (n=6), both groups received MPT (100 mg/kg/day) once a day for consecutive 4 days. Group 1 animals received MPT without any gel, while poly- Oxime gel was applied on group 2 animals, 30 min prior to the MPT exposure (Figure 6A). Animals in Group 1 which received MPT directly, showed the characteristics of organophosphate pesticide poisoning in the rats; symptoms include salivation, muscular fibrillation, diarrhea, lacrimation, respiratory distress, gasping, decreased body temperature and tremors (Figure 8). On the contrary, Group 2 animals which received MPT after application of prophylactic poly- Oxime gel did not show any such signs of toxicity. All animals directly exposed MPT group died in 5 days with median survival time (MST) of 4 days, while all animals which received MPT post poly- Oxime gel application survived (Figure 6B). A 100% survival was observed in the presence of poly- Oxime gel (n=6, P = 0.0005, Mantel-Cox test). The blood was collected before exposing to MPT (day-0), and at terminal stage (on day 3 and 30, from group- 1 and 2, respectively). Although animals in group-2 did not die, the study was terminated at day 30. Quantification of active AChE in the blood revealed that direct exposure of MPT decreased the active AChE while poly- Oxime gel prevented the inhibition of AChE activity (Figure 6C). Additionally, brain tissue was collected from Group 1 animals immediately after they died, and since not even single animal died in Group 2, they were sacrificed on day 30 and brain tissue was harvested. The active AChE in tissue was quantified. Direct exposure of MPT significantly decreased the active AChE, while poly- Oxime gel prevented reduction of active AChE in the brain (Figure 9A). Additionally, while animals which received MPT lost their body weight, Group 2 animals that were exposed to MPT in the presence of poly- Oxime gel continue to gain weight normally till the end of the study (Figure 9B).

An oxime could hydrolyze multiple organophosphate molecules in a truly catalytic manner. After cleaving one molecule of organophosphate, an oxime nucleophile could regenerate and cleave more organophosphate molecules (Figure 1A). To test the catalytic nature of poly- Oxime gel, in vivo ten SD rats were randomized into two groups (n=5), both groups received MPT (50 mg/kg/day) once a day for six consecutive days. Group 1 animals received MPT without any gel, while poly- Oxime gel was applied only single time on Group 2 animals on day 0 (Figure 6D). In this experiment, compared to oxime group, two-fold excess molar equivalent of MPT was applied. All the animals in direct MPT exposure group died within 10 days with MST of 6 days, whereas, a 100% survival observed in poly- Oxime gel treated group (Figure 6E, n=5, P = 0.0018, Mantel-Cox test). Analysis of active AChE in the blood revealed that direct dermal exposure significantly reduced the active AChE in the blood and brain tissue, compared to the presence of prophylactic poly- Oxime gel (Figure 6F and Figure 10A). Additionally, animals in poly- Oxime gel group did not lose their body weight compared to the direct exposure of MPT (Figure 10B). When poly- Oxime gel was applied prior to MPT exposure, in addition to providing 100% survival there were no visible signs such as shivering and stress induced porphyrin discharge which are hallmarks of pesticide-induced toxicity and stress. Cumulatively, these results suggest that poly- Oxime gel can deactivate organophosphate pesticides in a catalytic manner to prevent pesticide-induce toxicity and mortality. This property of poly- Oxime gel will be protective for farmers or workers getting exposed to high doses of pesticides. The catalytic poly- Oxime gel will serve as an efficient prophylactic topical material.

Example 5: Polv-Oxime Gel Prevents Pesticide-induced Foss of Motor Coordination and Altered Neuromuscular Signaling The endurance and motor coordination of rats exposed to MPT with and without poly- Oxime gel were tested using rotarod experiment. Three groups of male SD rats (n=4 in each group), were trained on a rotarod for four days (Figure 7A), and while training, animals in all the groups showed similar latency to fall (time to fall from rotating rod with 2-20 rpm). Latency to fall on the day 4 was considered as 100%. On day 4, Group 1 was left unexposed, Group 2 animals were directly exposed to MPT, and Group 3 animals received MPT in the presence of poly- Oxime gel. Post- MPT exposure, Group 2 animals showed a significant drop in the latency to fall, thus indicating a MPT-induced reduction in endurance (Figure 7B). On the contrary, Group 3 animals which received MPT in the presence of prophylactic poly- Oxime gel did not show a reduction in latency to fall, and continued improvement in performance like unexposed Group 1, which suggests that poly- Oxime gel prevents pesticide- induced reduction in endurance. Acceleration in rotation speed from 2 rpm to 60 rpm, with a step size of 4 rpm was used to study the neuromuscular coordination (NMC) of rats under constant need to readjust the muscle recruitment. After training for six days, animals were similarly divided in three groups (n=5 per group) and tested for NMC. Animals in group 2 showed significant drop in NMC, while poly- Oxime gel could prevent this drop of NMC and showed performance similar to unexposed animals (Figure 7C).

Gait analysis is a conventionally used noninvasive method to study sciatic nerve injury and recovery. The pattern of walking is decided by the posture of the foot, angle form the ground, and the force exerted. These things combined will establish a particular print length, toe spread, and inter toe spread of an animal and it gets affected if there is any impairment of nerve function (Figure 7D). When analyzed for Sciatic Functional Index (SFI), which is an empirically derived formula to evaluate nerve function (see Methods), the direct exposure animals showed values decreasing to -30 to -60 which indicates partial impairment of Sciatic nerve function. However, the poly- Oxime gel protected animals showed no significant reduction in SFI, suggesting complete prevention of nerve function impairment (Figure 7E).

Visible muscular spasms were observed in the animals directly exposed to MPT. Electromyogram (EMG) was recorded for bicep femoris of left hind limb under 2.5% isofluorane anesthesia and between spinotrapezius and gluteus maximus when the animal was awake. In either cases EMG showed frequent muscle activity. Such involuntary muscle activity was completely absent in the animals protected with poly- Oxime gel before application of MPT (Figures 7F and 7G).

Additionally, the biocompatibility of poly- Oxime was investigated. Results confirmed that poly- Oxime is an inert, non-immunogenic polymer and did not induce secretion of pro-inflammatory cytokines such as tumor necrosis factor-a, interleukin- 6 and interleukin- 1b (Figure 11A-B). Additionally, by using YO-PRO-l iodide dye conjugated poly- Oxime, it was shown that poly- Oxime is confined to the outermost layer stratum corneum and does not penetrate into the skin (Figure 11 A).

In conclusion, the poly- Oxime topical gel is able to prevent pesticide-induced AChE inhibition, loss of motor coordination, altered neuromuscular function and mortality. Thus, it shows a significant promise for the application in farmers and workers using pesticides. The ability of poly- Oxime gel to deactivate organophosphate in a catalytic manner also has a potential to be used for protection against organophosphate-based chemical warfare agents.

Discussion of Examples 1-5

These examples 1-5 show embodiments where a polymeric super- nucleophile (a- nucleophile) mediates hydrolysis of pesticides, e.g., on the skin. Poly-Oxime could be formulated into the dermal gel using excipients. The data suggest that a thin layer of poly-Oxime gel can hydrolyze organophosphate s on, e.g., the skin; therefore, it can prevent AChE inhibition quantitatively in blood and in all internal organs such as brain, lung, liver, and heart. Previously, there were no examples that demonstrated prevention of AChE inhibition in vivo. The examples demonstrate that poly-Oxime gel does not act as a physical barrier. Instead, it hydrolyzes organophosphate ester, MPT, into non-harmful hydrolytic products such as dimethyl phosphate and pNP, which cannot inhibit AChE. Poly-Oxime gel can hydrolytically cleave a wide range of pesticides, including commercial organophosphate formulations such as chlorpyriphos, prophenophos, and monocrotophos, and therefore can prevent pesticide-induced AChE inhibition. The activity of poly-Oxime gel against a broader range of pesticides shows its robustness.

As farmers spray pesticides in all seasons including cold winters and hot summers, the prophylactic gel should be active at a wide range of temperature. The fact that the poly-Oxime showed an efficient catalytic activity at temperatures ranging from 20° to 40 °C and retained its activity even after prolonged exposure to ultraviolet (UV) light shows stability and ability to function in varying weather conditions. In rat models, it was shown that pesticide exposure can lead to the loss of endurance and NMC. These examples demonstrate that the presence of poly-Oxime gel completely prevented pesticide induced loss of endurance and NMC. In addition, pesticide exposure can severely damage nerves such as the sciatic nerve. Using Gait analysis, it was demonstrated that direct exposure to pesticide significantly damaged the sciatic nerve, while the presence of poly-Oxime gel prevented this damage. To demonstrate the efficiency of poly-Oxime gel to prevent pesticide induced mortality, two sets of experiments were done. In the first set, rats were repeatedly exposed to a pesticide, while every day a thin layer of poly-Oxime gel was applied before pesticide exposure. While rats died in the absence of poly-Oxime gel (MST, 4 days), the presence of poly- Oxime gel prevented mortality and a 100% survival was observed. In the second set, poly-Oxime gel was applied only once on day 0 and repeatedly exposed to pesticide for 4 days. Even a single application of poly-Oxime gel completely prevented mortality with a 100% survival. A daily or single application of sham gel led to 100% mortality with an MST of 7 and 6 days, respectively. This demonstrates the stability, robustness, and true catalytic nature of poly-Oxime gel.

The prophylactic topical gel (poly-Oxime) can limit pesticide-induced systemic AChE inhibition and therefore can prevent loss of motor coordination, loss of endurance, altered neuromuscular function, and mortality. Thus, this technology offers a way for farmers and workers who use pesticides to protect themselves from exposure to toxic agents such as pesticides. The ability of poly-Oxime gel to deactivate organophosphate in a catalytic manner also has a potential to be used for protection against organophosphate based Chemical Warfare Agents.

Example 6: Catalytic Efficiency of Conjugate of Pyridine-2-aldoxime and Chitosan (poly-Oxime)

To assess the catalytic activity of conjugate of Pyridine-2-aldoxime and Chitosan, the conjugate was reacted with the pesticides, such as methyl parathion (MPT) and chlorpyrifos, respectively. The reactions were studied spectrophotometrically by monitoring the hydrolysis of methyl parathion and chlorpyrifos, respectively at temperature ranging from about 24 °C to 26 °C and at a pH of about 8.2, by measuring the absorbance at about 400 nm for the release of p- nitrophenoxide (pNP) ion as a function of time. The Pseudo-first-order-rate constants were obtained by reacting the conjugate of about 2.5 x 10 ^-4 M with about 2.5x10 ^-5 M pesticide, such as methyl parathion and chlorpyrifos, respectively. Table 1 illustrates the pseudo first order rate constants for hydrolysis of the said pesticides by the conjugate of Pyridine-2-aldoxime and Chitosan. To study temperature stability, same reactions were carried out at 18 °C, 20 °C, 30 °C, 40 °C, and 45 °C.

Table 1:

Data in Table 1 suggest that poly-Oxime conjugate can deactivate a wide spectrum of pesticides, e.g., methyl parathion, methyl paraoxon, and chlorpyrifos.

Example 7: Preparation of Composition Comprising the Conjugate of Pyridine-2- aldoxime and Chitosan

The dermal gel (composition) was formulated by mixing about 1.8% of Carbopol 940, about 6.81% of glycerin, about 3.74% of propylene glycol and about 2.02% of conjugate of Pyridine-2-aldoxime and Chitosan was mixed in about 85.6% of water. The pH of the gel was maintained at about 8 by triethanolamine (TEA). For sham gel, the un-functionalized chitosan polymer, instead of the poly-Oxime polymer, was used, serving as a control gel with all ingredients but no catalytic activity.

Example 8: Preparation of Conjugate of Pyridine-2-aldoxime and Triethoxy Silane. About one equivalence of tri-ethoxy silane and about one equivalence of bromo-acetyl bromide was reacted in dichloromethane at temperature of about 25 °C to 45 °C under nitrogen environment for about 72 hours. To the reaction mixture about one equivalence of pyridine-2-aldoxime was added dropwise by dissolving acetone, while maintaining nitrogen environment. After about 24 hours of the reaction, the reaction mixture was filtered through Whatman filter paper under inert environment to collect precipitated conjugate of pyridine-2-aldoxime and triethoxy silane. Excess solvent was evaporated under vacuum and the conjugate was stored in a temperature of about -20 °C.

Example 9: Preparation of the Article Comprising the Conjugate of Pyridine-2- aldoxime and Triethoxy silane

The conjugate of pyridine-2-aldoxime and triethoxy silane was dissolved in methanol at a concentration of about 20 mg/ml. The solution was added on substrate, such as fabric at a rate of about 10 mg/cm ² , ensuring uniform spreading and allowed the fabric to dry at a temperature of about 25 °C to 45 °C. Thereafter, the fabric was incubated at a temperature of about 70 °C, in hot air oven, overnight. The excess unreacted conjugate was removed by washing the fabric in excess methanol.

Example 10: Experiment to Demonstrate Deactivation of Carbamate and Ester Based Pesticides by Pyridine-2-aldoxime and Chitosan (polyoximc).

Pyridine-2-aldoxime and Chitosan was evaluated to deactivate pesticides such as carbamates and esters. Pyridine-2-aldoxime and Chitosan (polyoxime) was added to the samples comprising carbaryl (carbamate), carbofuran (carbamate-based) and cypermethrin (ester-based), respectively. Figure 12 illustrates the deactivation carbaryl (carbamate), carbofuran (carbamate -based) and cypermethrin (ester-based) induced AChE inhibition.

Example 11: The poly-Oxime Gel Could Hydrolytically Cleave a Wide Range of Commercial Qrganophosphate Formulations

The poly-Oxime gel could hydrolytically cleave a wide range of commercial organophosphate formulations (Figure 13A-D). Under similar reaction conditions, sham gel which was made with native chitosan without oxime groups did not deactivate pesticides suggesting that poly-Oxime alpha-nucleophile is essential for the reactivity (Figure 13A-D). These results clearly suggest that nucleophilic poly-Oxime gel could limit pesticide-induced AChE inhibition by chemically deactivating organophosphates.

Example 12: poly-Qxime Gel Limited Weight Loss and Drop of Body Temperature Upon Lethal Exposure

Different groups of animals were exposed to lethal 150 mg/kg dermal dose of methyl parathion in the presence or absence of sham or poly-oxime gel. Animals those were exposed to MPT alone or in the presence of sham-gel lost -20% their initial body weight by day 4, while animals exposed to MPT in the presence of poly-Oxime gel did not lose their weight and showed normal weight gain (Ligure 14A-B). Animals exposed directly or in the presence of sham gel also showed significant decrease in body temperature which was not observed in animals protected with poly-Oxime gel.

Example 13: Catalytic Nucleophile Activated Labric for Pesticide Decontamination

Following study is aimed towards developing face masks and clothing which can decontaminate organophosphates and thereby eliminate chances of increased exposure.

Synthesis of Silane based activator

Schematic: - Synthesis of quaternary salt with Silane

To a solution of 2-pyridine aldoxime (4g, 32.7 mmol) in 60 mL of dry acetone, 3- chloropropyltriethoxysilane (7.88g, 32.7 mmol) was added under N2. To this solution potassium iodide (2.7 lg, 16.3 mmol) was added and was kept under stirring for 48 hrs. at 80°C under N2. The reaction was confirmed complete using TLC (hexane: ethyl acetate - 1: 1; Rf - 0.1). The precipitate that was formed was filtered and washed several times with acetone and dried.

Yield - 8% (900 mg) 1H NMR (DMSO-d6, 600 MHz):d 8.5-8.49 (d, 1H), 8.21 (s, 1H), 7.88-7.85 (t, 21H), 7.72-7.71 (d, 1H), 7.44-7.42 (t, 1H), 3.86-3.82 (m, 6H), 3.57-3.55 (t, 2H), 1.84-1.80 (m, 2H), 1.19-1.17 (t, 9H), 0.86-0.83 (m, 2H).

¹³ C NMR (DMSO- d6, 150 MHz):d 152, 148, 139, 125, 122, 59, 25, 17, 6.6

Schematic: Fabric activation with Silane based activator

The fabric was washed several times with detergent and dried. A solution of functionalized silane was made to a final concentration of 5 mg/ml in methanol. This solution was loaded on 110 mg of fabric/ml of the solution by repeated soaking and drying. The loaded fabric was baked at 70 °C for 4 hours and washed repeatedly with methanol to remove any un-conjugated silane. Finally, the functionalized fabric was dried at 70 °C to constant weight and used for further experiments.

Example 14: Catalytic Fabric Prevented Blood AChE Inhibition ex-vivo Up to 30 Washes

Efficiency of activated cloth to decontaminate pesticides was evaluated using Franz diffusion apparatus. The transdermal penetration of pesticide was evaluated in the presence or absence of cloth (active or normal) using Franz diffusion cells. Pesticides coming in contact with catalytic oxime groups on fabric in donor chamber will be cleaved and therefore will not be able to enter acceptor chamber. For ex-vivo evaluation, the donor compartment was filled with diluted rat blood and change in blood AChE activity with time was used as a proxy for pesticide exposure. As seen in Figure 15, the activated cloth showed significant protection against inhibition of AChE activity ex vivo as compared to normal cloth. This suggests that, the normal cloth does not act as a potential physical barrier against pesticide exposure. In order to test the reusability of activated fabric, it was washed 15, 30 times with 1% SDS detergent followed by milliQ wash and its ability to prevent pesticide was studied as mentioned above. As evident from the Figure 15 the cloth retained its activity to prevent pesticide exposure even after multiple detergent washes, suggesting strong covalent activation of the fabric.

Example 15: Catalyst Activated Mask Limited Toxicity in Rat Exposed Repeatedly to Methyl Parathion Aerosol

To evaluate the ability of catalyst activated mask to prevent MPT-induced AChE inhibition, 18 Sprague Dawley (SD) rats (10-13 weeks) were randomly divided into 3 groups (n=4 per group). Animals were exposed to 300 pg/cm ³ of methyl parathion aerosol in acetonitrile and water mixture (8:2) for 1.5 hrs under general anaesthesia induced with Ketamine (91 mg/kg) + Xylazine (9.1 mg/kg) IP. Group 1 received the aerosol directly while group 2 and 3 inhaled aerosol through single layer of either normal (inactive) or catalyst activated (active) mask respectively. Quantification of active AChE in the blood at pre-exposure (0 hr) was considered to be 100%. Additionally, on day 4, animals were sacrificed, and brain was isolated and quantified for active AChE, and compared with the naive rat tissue without exposing to MPT (Figures 16A-B).

Example 16: Catalyst Activated Clothing Limited Toxicity in Rat Repeatedly Exposed to Methyl Parathion via Dermal Route

To evaluate the ability of catalyst activated fabric clothing to prevent MPT- induced AChE inhibition, 18 Sprague Dawley (SD) rats (10-13 weeks) were randomly divided into 3 groups (n=4 per group). Animals were exposed to 100 mg/kg/day of MPT until 50% mortality in direct exposure group was observed (4 days). Methyl parathion was applied dermally either in the presence or absence of clothing as 10 mg/ml solution in acetonitrile and water mixture (8:2) for 1.5 hrs under general anaesthesia induced with isofluorane 2.5%. Group 1 received the MPT directly on the skin while group 2 and 3 were exposed through single layer of either normal (inactive) or catalyst activated clothing respectively. Quantification of active AChE in the blood at pre-exposure (0 hr) was considered to be 100%. Animal weights were recorded every day and percent change in weight was calculated with respect to day 0. These results suggest that active fabric does not just act as a physical barrier but can actively cleave organophosphate molecules and prevent weight loss and loss of AChE activity in blood (Figures 17A-B).

Example 17: Active Fabric Prevented Loss of Sciatic Nerve Function

Exposure to organophosphate is known to cause accumulation of acetylcholine which leads to impairment of nerve function. Therefore, the ability of activated cloth to prevent impairment of nerve function in SD rats was studied. As per the protocols mentioned previously, animal feet were pained with non-toxic water colors and their footprints were obtained. Further the footprints were analysed to calculate Sciatic Functional Index (SFI) (Figure 18).

Example 18: Amphiphilic Micellar Nucleophiles to Decontaminate Surfaces with Pesticides

Experiments concerning decontamination of pesticide contaminated surfaces with the use of amphiphilic micellar nucleophiles are presented in this example.

Synthesis of amphi-oxime

a.) Tetra-decyl amine (300 mg, 1.4 mmol) was dissolved in dichloromethane (10 mL). Potassium carbonate (290 mg, 2.1 mmol) was dissolved in water (Quantity required), and the aqueous solution was added to the organic solution. The resulting two-phase solution was cooled to 5 °C. A solution of (b.) bromoacetyl bromide (423 mg, 2.1 mmol) in dichloromethane (5 ml) was added drop wise to the cooled solution for about 30 min while maintaining the temperature at 5 °C. Then the reaction mixture was stirred at room temperature for about 2 h. The aqueous solution was separated and washed with dichloromethane (2 x 5 ml). All the organic solutions were mixed together and washed with water (2 x 5 ml) and passed over anhydrous Na ₂ S0 ₄ and concentrated to yield a white product (c.) quantitatively.

To a stirred solution of 27.8 mg (0.227 mmol) of 2-pyridinealdoxime (d.) in 1 ml of tetrahydrofuran (THF), two molar equivalents (16.4 mmol) of corresponding alkyl bromides were added drop wise at RT. The reaction was monitored for 12 h by TLC using Merck Kiesel gel F254 silica-gel plate (0.25 mm thickness) as the stationary phase, ethyl acetate as the mobile phase, and ultraviolet absorption at 254 nm as the detecting method. The Rf values of 2-pyridinealdoxime was 0.6. The mixture was refluxed until the spot of 2- pyridine aldoxime on TLC disappeared. The product (e) was given hexane and ethyl acetate wash twice. Similar protocol was used for the synthesis of product (g). The synthesized compounds were characterized by NMR spectroscopy.

Example 19: Amphi-Qximes Showed Several Fold Higher Decontamination Efficiency Compared to Hydroxide Ions

To evaluate the potency of catalysts, hydrophobic reactive phosphotriester MPT was taken as a substrate that is widely used as a pesticide in many developing countries like India, Sri Lanka. The amphiphilic nucleophiles were synthesized by conjugating ortho and para positioned pyridine aldoxime with aliphatic chain that adds on hydrophobic character to molecules while retaining its hydrophilic properties as well. The activities of ortho and para-amphi oxime were investigated at the fixed concentration of catalyst and varying concentration of commercial available CTABr from the ratio of 1:0.25 to 1:30 (Figures 19A-B). From Figure 19A it can be inferred that o-amphi oxime showed the maximum activity at 1: 10 ratio whereas highest activity was seen at 1:0.75 ratio. In both the cases, highest activities have been seen at lower ratio but there is a significant drop in their activity at higher ratios. The reason behind this is the dilution of catalyst concentration and increase in the concentration of surfactant in each micellar environment available for decontamination reaction.

Through kinetics studies, the cleaving ability of catalyst 2-4 (see Table 2) were compared against MPT in aqueous and micellar medium. The release of p- nitrophenoxide ion as a product of MPT cleavage was examined at 400 nm. At pH 8.2, the hydrolysis of MPT (2.5x10 ^-5 M) by o-amphi oxime (2.5x10 ^-5 M) has been accelerated by the factor of 3958 relative to hydroxide ion whereas p- amphi oxime showed 280 folds higher activity. In other case, the para and ortho monopyridium oxime displayed 267 and 116 fold greater reactivity over OH- against MPT in micellar condition. To see whether CTABr is playing a major role in forming the micellar environment or not, the CMC of synthesized amphiphiles was checked. Figure 20 gave answer to all queries where CMC of amphi oxime are determined by using pyrene molecule as a fluorescent probe. From the graph, it can see that 13/11 values of p-amphi oxime are constant till 200 mM then its start increasing. This shows that there is an increase in 13/11 value only when pyrene molecule mobilizes from aqueous phase to pseudo phase. The minimum concentration at which there is change in 13/11 value is the CMC of the compound. Thus, the CMC of p-amphi oxime is around 200 mM that sheds light on all the queries that this example tried to address through kinetics experiments. In o-amphi oxime, the CMC value is around 1.1 mM that gives complete justification to its higher activity at 1: 10 where concentration of CTABr is more than its CMC. With this CMC graph it can be concluded that p- Amphi oxime even in lower concentration can get tightly packed in micellar environment without CTABr due to its terminal positioned oxime without steric hindrance. On the other hand, p-amphi oxime forms CMC at higher concentration because of its structural dynamics.

From Figure 20, it was evident that the NO- forms of catalyst acts as a nucleophilic species in the hydrolysis of MPT. To determine the pk _a value of these amphiphillic catalysts in micellar conditions, the pseudo first order constants of MPT cleavage at 25 °C were determined at several pH values from 6.2 to 8.5. In Figure 20, the plots of log k ₀ bs vs pH showed discontinuities at definite pH values, which were taken as systemic pKa values for amphiphiles. For this experiment, the concentration of MPT and catalyst (4-5) was taken 2.5 x10 ^-5 M and 2.5 x10 ^-4 M in micellar CTABr (1* 10 ^-2 M) solution at 25 °C. The plots of log K ₀ bs vs pH showed the breaks at 6.85 for o-amphi oxime and 7.5 for o-amphi oxime. A comparison of these values to the reported pK _a values of ortho and para monopyridium oxime which are 7.91+ 0.02 and 8.36 + 0.01 respectively, shows that amphiphillic oximes are highly catalytic at the physiological pH of 6.5-7.5. This can be due to catalyst’s amphiphillic property where functional group in the molecules presumably remains projected towards MPT and approximately align their aliphatic chain with CTABr surfactant tails embedded in the micelles. As the aforementioned kinetics studies were performed at pH 8.2, it explains the higher activity of amphi- oxime than mono-oximes. This was due to the lower pKa value of amphi-oxime as they were all assumed to be in deprotonated form at pH 8.2 compared to mono oximes. Figure 20 also reveals that o-amphi oxime has lower pk _a value than the -amphi oxime due to the high pH of stem layer where NO- species are projected which makes it the most reactive catalyst in the micellar medium.

In next experiment, to investigate the real catalytic activity of ortho and para positioned amphi-oxime in presence of excess substrate were performed with MPT in pseudo phase system. In the reaction condition of pH 8.2 (50 mM) at 25 °C, catalysts (1* 10 ^-5 M) in CTABr (1* 10 ^-3 M) with 10-fold excess substrate (MPT) w.r.t catalyst were taken. In Figure 21, it can be observed that there was a nearly quantitative, non exponential release of p-nitrophenoxide ion with no evidence of“burst” kinetics. It shows that the turnover of catalyst was very fast as it was quickly regenerating in the aqueous micellar medium. To determine the rate limiting step, the kinetic studies of amphi oxime with ten times higher concentration of MPT was performed. Either the formation of N-0 phosphorylation species or its decay in order to regenerate the catalyst can be the rate limiting step. Since there was no accumulation of intermediate species in the reaction media, it can be assumed that the attacking of hydroxide ion on the phosphorylated species must be quick. Thus, it is evident through Figure 21 that catalyst cleaves intermediate species fast enough and continuously regenerates the catalyst which was further free to react with new available substrate molecules. Thus, decay of N-0 phosphorylated species and regeneration of the catalyst was a slow process which was the rate determining step. The time dependent formation of p-nitro phenol for the cleavage of excess substrate revealed perfect pseudo first order kinetics behavior.

Example 20: Surface Decontamination with Amphi-Oximes

This test was done to understand the efficiency of decontamination on different surfaces as a proxy to the reality. A 10 cmx10 cm surface (metal/ceramic/glass) was contaminated with known amount of methyl parathion (100 uM) and allowed to dry. After 2 hrs of contamination, the surface was sprayed with micellar catalyst solution and the decontamination was allowed to happen for 2 hours. To terminate the experiment, the surface was wiped with 80% acetonitrile in water and MPT and pNP was extracted in 20 ml of 80% ACN. The extract was then used for HPLC quantification using conditions mentioned elsewhere. As observed in Figures 22A-B, the micellar o-amphi-oxime and p-amphi-oxime could completely convert MPT to pNP as opposed to SDS, HEPES and CTABr controls. This confirms that it can be used as a very promising way to decontaminate pesticides from surfaces.

Example 21: Decontamination Kinetics with Micellar Poly-Oximes

This test was done to study the kinetics of pesticide deactivation with micellar amphi-oximes. Akin to previous experiment, the micellar solution was used to decontaminate ceramic surfaces contaminated with known quantities of MPT. Reactions were terminated at different time points by wiping with 80% CAN water mixture and extent of reaction was evaluated using HPLC (Figures 23A-B and 24A- B). In order to evaluate efficiency of surface decontamination in an in vivo setup mimicking the real life residential exposure scenario, contaminated surface of a rat cage with known quantities of MPT was tested. MPT was dissolved in 80% CAN to water mixture and sprayed uniformly cover the surface of cage bottom. The solvent was allowed to evaporate for 12 hrs. Next day, either the cages were kept undisturbed or were sprayed with solution of micellar amphi-oxime catalyst. Decontamination was allowed to happen for 24 hours and animals were introduced in these cages without bedding material. Bedding material was introduced after further 24 hours (Figure 25). Animals were expected to get exposed via oral and dermal route. Similar exposure scenario was repeated on day 4. Animals were observed for blood AChE and rotarod with accelerated speed for evaluating extent of toxicity. On day 15, animals were sacrificed and organs were collected to perform organ AchE (Figures 26A-D). Rota rod was used to study endurance and NMC in MPT animals either directly or in the presence of amphi oxime spray (Figure 27). Latency to fall was measured increasing speed from 2 to 60 rpm, the rpm reached before falling was taken as a measurement to assign NMC score, and data were normalized to the day of exposure. The animals kept in contaminated cages significantly lost their neuro-muscular coordination and showed reduced latency to fall scores. The animals introduced in cages decontaminated with micellar amphi-oxime catalyst showed no alternation in latency to fall. Example 22: Evaluating Different Classes of Nucleophiles for Pesticide Decontamination

In the process of identifying better catalysts for building platform technologies, nucleophiles from various classes of pesticides were designed and synthesized. Their ability to hydrolyze methyl parathion was studied in vitro by following formation of para nitro phenol in pseudo-first order reaction kinetics. The Table 3 shows the molecular structure of the nucleophiles, their chemical name, the active group, the reaction rate for hydrolysis of methyl parathion, and reaction conditions.

Table 3:

Example 23: Development of Nucleophilic Catalytic Mask and Clothing to Prevent Inhalation and Dermal Exposure to Pesticides.

This example is aimed at developing face masks and clothing (Figure 12) which can deactivate or decontaminate organophosphates and thereby eliminate chances of increased exposure. Single pot synthesis was carried out to synthesize nucleophilic silane fabric activator. One equivalence of each, tri-ethoxy silane (1) and bromo-acetyl bromide were reacted in dichloromethane at room temperature under N ₂ environment for 72 hrs. To the reaction mixture (2), one equivalence of pyridine -2- aldoxime was added drop wise by dissolving acetone whilst maintaining nitrogen environment. After 24 hrs reaction mixture was filtered through Whatman filter paper under inert environment to collect precipitated compound (3). Excess solvent was evaporated under vacuum and stored in -20 °C until further use (Figure 29).

The nucleophilic silane fabric activator was dissolved in methanol at concentration of 20 mg/ml. The solution was added on fabric or mask slowly (10 mg/cm ² of fabric), ensuring uniform spreading and allowed to dry at room temperature. Thereafter, fabric was incubated at 70 °C, in hot air oven, overnight. To remove unconjugated activator, fabric was washed thrice in excess methanol (Figure 30).

Franz diffusion (FD) assay was carried out to study efficiency of activated fabric to prevent from organophosphate exposure. Typically, the donor and acceptor chamber of FD cells were separated by dialysis membrane (MWCO: 3.5 kDa), and acceptor compartment was filled with rat blood diluted 1000 times in phosphate buffer pH 8 (Figures 31A-B). The dialysis membrane was either covered with activated or inactive cloth while only dialysis membrane served as control. In the donor chamber, 1 pmole of methyl parathion (MPT) was added (in 1 ml HEPES buffer, pH 8.0). Samples from acceptor chamber were collected and AChE activity was quantified using modified colorimetric Ellman’s assay. Activated fabric showed more than 80% retention of AChE activity. Inactive fabric and no fabric similar reduction in AChE activity, suggesting that only fabric does not act as a barrier to MPT exposure but requires functionalization with nucleophilic silane activator to prevent MPT exposure.

EQUIVALENTS

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.