Materials and methods for preventing or reducing the toxicity of organophosphates and other toxic substances - Patents.com

Abstract

The present invention provides materials and methods for reducing organophosphate toxicity. More specifically, the present invention provides pharmaceutical compositions comprising the proteins and / or biologically active fragments and variants thereof, and methods of use thereof. TIFF2024545570000005.tif86134

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 276,091, filed November 5, 2021; and No. 63 / 335,336, filed April 27, 2022, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing The sequence listing of this application is referred to as "SeqList-as filed.xml", was created on November 7, 2022, and is 8 KB. This sequence listing is incorporated herein by reference in its entirety. [Background technology]

[0003] 2. Background of the Invention Organophosphates (OPs) are toxic compounds that pose a significant threat to human health worldwide, causing over 100,000 deaths annually resulting from hazardous accidental and occupational exposures (e.g., organophosphate pesticides), suicide attempts, or from weaponized OP agents used in terrorist attacks or for chemical warfare (CDC MMWR 1984, Lin et al., 2008; Meulenbelt and Nieuwenhiuzen, 2016; Mew et al., 2017; Bertolote et al., 2018; OPCW note 2020).

[0004] As potent acetylcholinesterase (AChE) inhibitors, organophosphates often rapidly and irreversibly block the enzyme, leading to the accumulation of acetylcholine in neuromuscular synapses and resulting in severe injury or death. Because of this rapid action of AChE inhibitors and the rapid onset of symptoms, victims of organophosphate poisoning have a very short time to be properly diagnosed and receive antidotes and medical treatment (Hulse et al., 2019).

[0005] The duration of exposure to OPs can be assessed using biomarkers to determine AChE levels. At low doses, OP agonists induce visual incapacitation. Local exposure of the eye to OPs leads to a reduction in cholinesterase activity in the iris and ciliary muscles, followed by an increase in acetylcholine in the anterior chamber of the eye. This cholinergic overstimulation effect on the pupillary sphincter leads to significant miosis with dimming of vision, reduced spatial visual ability with ciliary paralysis and reduced lens accommodation, and reduced adaptation to lighting changes due to desensitization of pupillary muscarinic receptors (Egoz 2017; Dabisch 2008; Genovese 2008; Lund-Karlsen and Fonneum 1976; Takayanagi 1993). Furthermore, cholinergic overstimulation induces spasms of the cholinergic ciliary muscles that can lead to blurred vision and myopia (Cannard, 2006; Nohara and Segawa, 1996; Rengstorff 1985; Smith and Smith 1980; Yanagisawa, 2006).

[0006] The United States Environmental Protection Agency (EPA) banned most non-agricultural uses of OPs in 2001 due to toxic properties, public health concerns, and environmental concerns associated with their use. Exposure to OP pesticides can occur through many routes, including food contamination, environmental and household contamination, proximity to agricultural land, and agricultural work. US farmworkers are one of the populations most susceptible to organophosphate exposure.

[0007] The use of OPs in chemical warfare was first documented by Gerhard Schrader in the 1930s. The first chemicals produced were called G-agents and included sarin, tabun, and soman. Subsequent research expanded on Gerhard Schrader's work to produce VX nerve agents, which are orders of magnitude more powerful than G-agents. Exposure to VX or G-agent poisons disrupts the body's signaling mechanisms between the nervous and muscular systems. This causes neuromuscular blockade, flaccid paralysis, and death by asphyxiation.

[0008] Currently, antidote options (e.g., oximes) and medical protocols for the treatment of organophosphate poisoning, such as intravenous atropine administration, are limited and not always available. Common antidotes used in exposure to OPs include atropine, which reduces the effects of poisoning by blocking muscarinic acetylcholine receptors and then preventing overstimulation of the receptors by excess acetylcholine accumulation. Pralidoxime (2-PAM), another possible treatment, works against organophosphate poisoning by re-cleaving phosphorylation. In severe cases, benzodiazepines can be prescribed. Occasionally, treatment with topical anticholinergics has been shown to induce mydriasis and worsen visual acuity and visual function, which is counterproductive to vision preservation, especially for soldiers. Direct identification of the toxic compound or nerve agent may not be readily available, which further complicates patient stabilization and the provision of appropriate medical care (John et al., 2018; Steindl et al., 2021).

[0009] There is a need for the development of easy-to-administer, non-toxic compounds that protect against organophosphate poisoning and preserve visual function. Ideally, such treatments would be suitable for immediate use by first responders or active military personnel when there is a risk of toxic exposure to organophosphates (e.g., industrial accidents, anti-terrorism actions, or activities in active combat zones where OP agents may be used or present).

[0010] Tetrodotoxin (TTX) is a potent neurotoxin. Its name comes from the Tetraodontiformes, an order that includes pufferfish, porcupine fish, sunfish, and triggerfish. Tetrodotoxin was discovered in these fish and has been found in several other animals, but it is actually produced by certain infectious or symbiotic bacteria, such as Pseudoalteromonas, Pseudomonas, and Vibrio.

[0011] Tetrodotoxin is a sodium channel blocker. It binds to voltage-gated sodium channels in nerve cell membranes and inhibits the firing of action potentials in neurons by blocking the movement of sodium ions (which are involved in the ascending phase of the action potential) into the neuron. This prevents the nervous system from transmitting messages, thereby preventing muscles from contracting in response to nerve stimulation.

[0012] Tetrodotoxin binds to what is known as site 1 of the fast voltage-gated sodium channel. Site 1 is located at the extracellular pore opening of the ion channel. Binding of any molecule to this site temporarily disables the function of the ion channel, thereby blocking the movement of sodium ions into the nerve cell.

[0013] TTX is highly toxic. Its oral LD50 for mice is 334 μg per kg. For comparison, the oral LD50 for mice of potassium cyanide is 334 μg per kg. 50 was 8.5 mg per kg, demonstrating that even orally, TTX is more toxic than cyanide.

[0014] The poison may enter the victim's body by ingestion, injection, or inhalation, or through abraded skin. Poisoning as a result of consuming the fish is very serious. Pufferfish organs (e.g., the liver) can contain levels of tetrodotoxin sufficient to cause paralysis of the diaphragm and subsequent death from respiratory failure.

[0015] Symptoms typically occur within 30 minutes of consumption, but may be delayed up to 4 hours; however, if the dose is lethal, symptoms usually appear within 17 minutes of consumption. Paresthesias of the lips and tongue are followed by paresthesias of the extremities, hypersalivation, sweating, headache, weakness, lethargy, incoordination, tremors, paralysis, cyanosis, aphonia, dysphagia, and seizures. Gastrointestinal symptoms are often severe and include nausea, vomiting, diarrhea, and abdominal pain; death is usually secondary to respiratory failure. Victims, even if completely paralyzed, may be conscious until shortly before death, which generally occurs within 4 to 6 hours, and in some cases may be completely sensible. If the patient survives 24 hours, recovery is usually expected over several days without any sequelae.

[0016] Therapy is supportive, symptom-based, with aggressive early airway management. If ingested, treatment may consist of emptying the stomach, giving the victim activated charcoal to bind the toxin, and administering standard life support measures to keep the victim alive until the effects of the toxin wear off. Alpha-adrenergic agonists are recommended in addition to intravenous fluids to address hypotension. Summary of the Invention

[0017] The present invention provides materials and methods for reducing toxicity caused by organophosphates and other toxic substances, including tetrodotoxin (TTX) toxicity. More specifically, in certain embodiments, the present invention provides pharmaceutical compositions comprising the protein and / or its biologically active fragments and variants, and methods of use thereof. In additional embodiments, the present invention provides pharmaceutical compositions comprising bacteria grown as biofilms and / or components of bacterial biofilms, including, for example, cells and extracellular matrix.

[0018] In a preferred embodiment, the present invention provides biologically active proteins capable of preventing or reducing organophosphate toxicity. In a specific embodiment, the present invention provides a protein "Qi611S" having the amino acid sequence set forth in SEQ ID NO. 1. In certain embodiments, the present invention also provides "Qi611S proteins" comprising Qi611S, as well as biologically active fragments and variants thereof.

[0019] In some embodiments, the Qi611S protein can be produced by a cell, preferably a bacterial cell. Thus, in a specific embodiment, the present invention provides a method for producing the Qi611S protein, comprising culturing a cell having a nucleotide sequence encoding all or part of SEQ ID NO. 1, or a variant or fragment thereof, under conditions favorable for expression of the protein. In a preferred embodiment, the nucleotide sequence is Qi611s (SEQ ID NO. 2). Optionally, the protein can be purified from the culture.

[0020] In one embodiment, the method utilizes a microorganism having the Qi611s nucleotide sequence (SEQ ID NO. 2), e.g., Lactobacillus fermentum Qi6. Qi611s encodes the amino acid sequence set forth in SEQ ID NO. 1 (Qi611S).

[0021] In another embodiment, the cell is a microorganism recombinantly altered to have the ability to express the Qi611S protein. In a specific embodiment, the microorganism has all or a portion of the Qi611s gene. Thus, in certain embodiments, the invention provides a recombinant cell capable of expressing a protein having all or a portion of the DNA sequence set forth in SEQ ID NO. 2 and / or having the amino acid sequence of SEQ ID NO. 1 or a fragment or variant of SEQ ID NO. 1. In an exemplary embodiment, the recombinant cell is E. coli BL21 or E. coli C43.

[0022] Such transformation of cells can be accomplished using techniques well known to those skilled in the art of microbiology. In one embodiment, the nucleotide sequence can be modified to optimize expression of the Qi611S protein.

[0023] In a preferred embodiment, the present invention provides a composition comprising a cell comprising the Qi611S protein and / or all or part of the DNA sequence set forth in SEQ ID NO. 2, and, optionally, a pharma- ceutically acceptable carrier.

[0024] Additional preferred aspects of the present invention provide compositions comprising Lactobacillus fermentum bacterial strains grown in a biofilm phenotype and / or one or more bioactive extracts thereof, and methods of use thereof. The present invention also provides compositions comprising L. fermentum bacterial biofilms and / or bioactive extracts thereof in lyophilized, freeze-dried, and / or lysate form.

[0025] Pharmaceutically acceptable carriers can include materials used to administer the composition to a subject via a particular route, including, for example, oral administration, administration to the respiratory tract, administration to the eye, injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, and / or subcutaneous), and / or topical administration (e.g., via dermal absorption). In certain embodiments, the composition can be formulated as a lotion, cream, emulsion, ointment, oil, gel, aerosol, mists, inhalants, and / or combinations thereof.

[0026] In some embodiments, the protein can be extracted from the cell culture and, optionally, purified, prior to formulation into a pharmaceutical composition.

[0027] In some embodiments, the composition comprises cells capable of producing Qi611S protein. In one embodiment, the cells are in lyophilized, freeze-dried, and / or lysate form. In one embodiment, the composition comprises a cell culture in a biofilm state.

[0028] Thus, in certain embodiments, the compositions can be used to treat and / or prevent organophosphate toxicity or TTS toxicity in a subject.

[0029] The present invention further provides assays for assessing organophosphate-induced toxicity and the effectiveness of agents in preventing and / or reducing that toxicity. [Brief description of the drawings]

[0030] [Figure 1] 1 shows a map of the pET-15b vector utilized in accordance with an embodiment of the present invention. [Diagram 2] The pET-15b cloning / expression region of the coding strand transcribed by T7 RNA polymerase is shown. [Diagram 3] Figure 1 shows a silver stained SDS-PAGE gel that was used to confirm the presence of Qi611S synthesized by the recombinant E. coli strain. A sample of released protein from recombinant E. coli BL21 was loaded onto the SDS-PAGE gel, and the released protein is identified by the thick arrow on the right side of the gel. The E. coli strain was transformed with the Qi611s gene via the pET-15b expression vector. [Figure 4] 4 shows the hydrophilicity plot of Qi611S. [Diagram 5] 1 shows that saline, DMSO, or Qi611S alone do not significantly affect pupil size in ex vivo rabbit eyes. [Figure 6] 1 shows that saline solution of the Qi611S compound does not cause pupil constriction within 2 hours after treatment. [Figure 7]We show that paraoxon is able to reduce pupil size in an ex vivo rabbit model, reproducing the miosis typically seen as one of the hallmarks of organophosphate poisoning. A nylon ring containing the treatment was applied to the limbal area, and photographs were taken 10 minutes after exposure of the corneal surface to pOX. [Figure 8] FIG. 1 shows that compound Qi611S causes protection from paraoxon-induced miosis in an ex vivo young rabbit eye model. [Figure 9] 1 shows that prophylactic treatment with Qi611S protects AChE activity from paraoxon-induced inhibition. [Figure 10] Protection of AChE activity by Qi611S is shown to be rapid, as demonstrated by prophylactic treatment 10 and 30 min prior to paraoxon exposure. [Figure 11] Figure 1 shows that upregulation of PPAR-γ correlates with protection of AChE activity in ex vivo rabbit eyes treated with Qi611S. Paraoxon (P) concentrations of 1 mM and 10 mM were used in this experiment. [Figure 12] Figure 2 shows that prophylactic treatment with Qi611S prior to paraoxon exposure reduces tissue damage as demonstrated by LDH release assays. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] A brief description of the sequence SEQ ID NO:1 is the amino acid sequence of a protein designated as "Qi611S." SEQ ID NO:2 is the nucleotide sequence encoding Qi611s, i.e., the amino acid sequence of SEQ ID NO:1. SEQ ID NO: 3 is a forward primer useful according to the present invention, "Lacto F." SEQ ID NO: 4 is a reverse primer useful according to the present invention, "Lacto R." SEQ ID NO:5 is the nucleotide sequence of the cloning / expression region of the pET-15b vector. SEQ ID NO:6 is the amino acid sequence of the His-tagged recombinant protein encoded by the cloning / expression region of the pET-15b vector.

[0032] Detailed Description of the Invention The present invention provides materials and methods for reducing organophosphate toxicity. More specifically, the present invention provides pharmaceutical compositions comprising the proteins and / or biologically active fragments and variants thereof, and methods of use thereof.

[0033] The present invention provides a protein having the amino acid sequence set forth in SEQ ID NO: 1 ("Qi611S"), biologically active fragments and variants thereof, and therapeutic compositions and methods utilizing the novel protein and / or biologically active fragments and variants thereof to reduce organophosphate toxicity.

[0034] A further embodiment provides a cell capable of expressing the Qi611S protein. In certain embodiments, the cell is recombinantly altered to have a nucleotide sequence encoding all or a portion of SEQ ID NO: 1. In a specific embodiment, the nucleotide sequence has SEQ ID NO: 2.

[0035] In some embodiments, the bacterial strain is Lactobacillus fermentum Qi6, also referred to herein as Lf Qi6. In one embodiment, the present invention provides an isolated or biologically pure culture of Lf Qi6. In another embodiment, the present invention provides a culture of Lf Qi6 grown as a biofilm. Methods for inducing and identifying the biofilm phenotype are specifically taught herein. Additionally, methods for utilizing the biofilm phenotype, as well as extracts and lysates thereof, are provided herein. In a specific embodiment, the pharmaceutical composition comprises a bioactive extract of Lf Qi6 biofilm.

[0036] A culture of L. fermentum microorganism was deposited at the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 USA. The deposit was assigned the accession number ATCC number PTA-122195 by the collection and was deposited on June 10, 2015.

[0037] In certain embodiments, the composition comprises bacteria grown as a biofilm, preferably a strain of Lactobacillus fermentum bacteria. The present invention also provides compositions comprising biofilm extracts or extracellular matrix in lyophilized, freeze-dried, and / or lysate form.

[0038] The present invention further provides, in a preferred embodiment, an ex vivo eye model that uses simultaneous quantitative evaluation of two pathological endpoints in organophosphate (OP) intoxication: pupillary response (miosis) and acetylcholinesterase inhibition. Using this model and paraoxon, five characteristics of Qi611S in the setting of ocular OP challenge were demonstrated: 1) physiological protection by reduction of maximum miosis recorded after paraoxon challenge, 2) protection of AChE enzyme activity, 3) rapid onset of physiological and enzyme protection (10 min pretreatment of ex vivo eyes with Qi611S), 4) no toxicity of Qi611S to either acetylcholinesterase or attenuation of pupillary response, and 5) presence of Qi611S in intraocular fluids within 30 min of administration.

[0039] Other advantages of this model for OP toxicity range from the feasibility of using whole ex vivo eyes and the rapid onset of measurable outcomes, as paraoxon-induced miosis is visible within minutes of administration of the test OP compound. Furthermore, the amount of anterior chamber fluid collected per eye is sufficient to perform several different tests, and ocular tissue (e.g., corneal buttons following punch biopsy) is available for microscopic or immunohistochemical examination.

[0040] Because the Qi611S protein employs a multifunctional physiological host protection mechanism to mitigate organophosphate toxicity, these compounds can be used for broad-spectrum protection from multiple organophosphate agents and provide tissue barrier protection not only to the eye, but also to, for example, the skin and airways.

[0041] Selected Definitions "Qi611S" refers to a protein having the amino acid sequence of SEQ ID NO: 1. References to "Qi611S proteins" in the singular or plural form refer to Qi611S as well as biologically active fragments and variants of 611S.

[0042] As used herein, a "gene" refers to a segment of DNA or a nucleotide sequence capable of expressing a polypeptide and / or an amino acid chain. In certain embodiments, a gene includes regions such as a promoter region before and / or after the coding region.

[0043] As used herein, an "isolated" or "purified" compound is substantially free of other compounds with which it is naturally associated, e.g., cellular material. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) does not contain the sequences adjacent to it in its natural state. A purified or isolated polypeptide, for example, does not contain other cellular material with which it is thought to be naturally associated. "Isolated" in the context of a microbial strain means that the strain is removed from the environment in which it naturally occurs. Thus, an isolated strain can exist, for example, as a biologically pure culture or as a spore (or other form of the strain) bound to a carrier.

[0044] As used herein, a "biologically pure culture" is one that is isolated from other biologically active substances, including any substances that may be naturally associated with it. In a preferred embodiment, the culture is isolated from all other living cells. In a more preferred embodiment, the biologically pure culture has advantageous characteristics compared to the culture of the same microbial species that may exist in nature. The advantageous characteristics may be, for example, enhanced production of one or more desirable growth by-products.

[0045] In certain embodiments, the purified compound is at least 60% by weight of the compound of interest.Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the compound of interest.For example, the purified compound is preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% by weight (w / w) of the desired compound.Purity is measured by any suitable standard method, for example, by column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis.

[0046] "Metabolite" refers to any substance produced by metabolism (e.g., a growth by-product) or that is necessary to participate in a particular metabolic process. Examples of metabolites include, but are not limited to, biological surfactants, biopolymers, enzymes, acids, solvents, alcohols, proteins, vitamins, minerals, trace elements, and amino acids.

[0047] As used herein, the term "extract" refers to a composition obtained by processing a cultured microorganism. Processing can involve, for example, physical and / or chemical treatment. Physical and / or chemical treatment can include, for example, filtration, centrifugation, sonication, pressure treatment, irradiation treatment, lysis, treatment with solvents or other chemicals, and combinations of these treatments. The extract can be in the form of a supernatant, such as that produced by centrifugation. The extract can also include cell mass obtained by centrifugation. The cells can be intact or non-intact, viable or non-viable. The extract can include cell membrane components and / or intracellular components. In certain embodiments, the extract is at least 80, 85, 90, or 95% cell mass by weight. In certain embodiments, at least 95% of the intact cells are non-viable. In certain embodiments, less than 10% of the cell mass in the extract is intact cells.

[0048] "Planktonic" refers to a phenotype typical of microorganisms (bacteria, fungi, and / or protozoa with associated bacteriophages and other viruses) that float freely in liquid media. On the other hand, "biofilm" is an accumulation of microorganisms embedded in an extracellular polymeric matrix (EPS) and attached to a solid biological or abiotic surface. In addition to having a typical planktonic phenotype, Lf Qi6 is capable of forming biofilms.

[0049] Methods for growing biofilms are known in the art and are described, for example, in WO 2012 / 118535, which is incorporated by reference in its entirety, including the publications cited therein, such as those cited on pages 26-31.

[0050] As used herein, "modulate" means to cause a change (eg, an increase or decrease).

[0051] As used herein, "pharmaceutical," "health-promoting compound," or "health-promoting substance" refers to a compound useful as a medicinal and / or therapeutic agent.

[0052] As used herein, a "polypeptide," "peptide," or "protein" refers to a polymer of amino acid residues.

[0053] As used herein, the term "subject" refers to an animal. An animal may be, for example, a human, a pig, a horse, a goat, a cat, a mouse, a rat, a dog, a monkey, a fish, a chimpanzee, an orangutan, a guinea pig, a hamster, a cow, a sheep, a bird (including a chicken), and any other vertebrate or invertebrate animal.

[0054] In the present invention, the preferred subject is a human of any age and / or sex. In some embodiments, the subject suffers from a health condition, disease, or disorder caused by organophosphate toxicity, while in some embodiments, the subject is in good health (e.g., free of injury or disease) but desires protection from organophosphate toxicity.

[0055] As used herein, the terms "treat" and "treatment" refer to eradicating, reducing, improving, or reversing to any extent the signs or symptoms of a health condition, disease, or disorder, including, but not requiring, a complete cure of the condition, disease, or disorder. Treatment can be curing, improving, or partially improving the disorder. Treatment can also include improving or enhancing a condition or characteristic, for example, bringing the function of a particular system in the body to an enhanced state of health or homeostasis.

[0056] As used herein, "preventing" a health condition, disease, or disorder refers to avoiding, delaying, forestalling, or minimizing the onset of a particular sign or symptom of a condition, disease, or disorder.Prevention can be absolute or complete, but is not required, meaning that the sign or symptom may still occur later.Prevention can include reducing the severity of the onset of such a condition, disease, or disorder, and / or inhibiting the progression of a condition, disease, or disorder to a more severe condition, disease, or disorder.

[0057] Ranges provided herein are understood to be shorthand notations for all values ​​within the range. For example, a range of 1-20 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, as well as all intervening decimal values ​​between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. For subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1-50 can include 1-10, 1-20, 1-30, and 1-40 in one direction, or 50-40, 50-30, 50-20, and 50-10 in the other direction.

[0058] As used herein, a "recombinant" cell has been modified by the introduction of a heterologous nucleic acid molecule or by alteration of a native nucleic acid molecule. Thus, for example, a recombinant cell can express genes that are not found in the native (non-recombinant) form of the cell, or can express native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all.

[0059] As used herein, the term "reduce" refers to a negative change and the term "increase" refers to a positive change, respectively, of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0060] As used herein, "reference" refers to a standard or control condition.

[0061] The transitional term "comprising," which is synonymous with "including" or "containing," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not explicitly stated in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the materials or steps explicitly stated and those that do not substantially affect the "basic and novel characteristics" of the claimed invention, e.g., the ability to prevent bacterial growth. Use of the term "comprising" contemplates embodiments that "consist" and "consist essentially" of the recited ingredients.

[0062] As used herein, unless otherwise stated or clear from context, the term "or" is understood to be inclusive. As used herein, unless otherwise stated or clear from context, the terms "a," "an," and "the" are understood to be singular or plural.

[0063] Unless otherwise stated or clear from the context, the term "about" as used herein is understood to mean within the normal tolerance in the art, for example, within 2 standard deviations of the mean. The term "about" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[0064] The recitation of a list of chemical groups in any definition of a variable herein includes a definition of that variable as any single group or combination of listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0065] Any composition or method provided herein can be combined with any of the other compositions and methods provided herein.

[0066] Other features and advantages of the present invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. All references cited herein are incorporated by reference in their entirety.

[0067] Organophosphates Organophosphates are a class of organophosphorus compounds with the general structure O=P(OR)3, a central phosphate molecule with alkyl or aromatic substituents.

[0068] Organophosphate pesticides, like some nerve agents, inhibit acetylcholinesterase, which is widely essential for the normal functioning of insects as well as humans and many other animals. Organophosphates affect this enzyme in a variety of ways, primarily through irreversible covalent inhibition, which can lead to various degrees of toxicity. The brain sends neurotransmitters to nerve endings in the body; organophosphates prevent this process from happening. Acetylcholinesterase breaks down the acetylcholine neurotransmitter, which sends signals to other nerve endings in the body. Commonly used organophosphates include: Parathion Malathion Methyl parathion Chlorpyrifos Diazinon Dichlorvos ·Hosmet Fenitrothion Tetrachlorvinphos Azamethiphos Azinphos methyl Terbukhos Includes:

[0069] In specific embodiments, the organophosphate has the following structural characteristics: · A terminal oxygen attached to phosphorus by a double bond, i.e. a phosphoryl group; · two lipophilic groups attached to phosphorus; and A leaving group attached to phosphorus, often a halide has.

[0070] Many organophosphates are potent nerve agents that function by inhibiting the action of acetylcholinesterase (AChE) in nerve cells. The first such nerve agents produced were called G agents, which include sarin, tabun, and soman. Subsequent research produced VX nerve agents, which are orders of magnitude more potent than the G agents. Exposure to VX or G agent toxins disrupts the body's signaling mechanisms between the nervous and muscular systems. This causes neuromuscular blockade, flaccid paralysis, and death by asphyxiation. Novichok is another organophosphate nerve agent. Organophosphate nerve agents are described in more detail by Mukherjee et al., which is incorporated herein by reference in its entirety.

[0071] Polynucleotide sequences encoding Qi611S and 611S proteins In a preferred embodiment, the present invention provides proteins, and fragments and variants thereof, that are useful for ameliorating the toxicity of organophosphates. The present invention further provides nucleotide sequences encoding the proteins, and fragments and variants thereof.

[0072] In certain specific embodiments, the protein of the present invention, referred to as "Qi611S", has a molecular weight of about 8.0 kDA. "Qi611S protein", including Qi611S and its biologically active fragments and variants, can be characterized according to several parameters, including biological activity, such as: reducing organophosphate toxicity; antibacterial activity; inhibiting the growth and attachment of pathogenic biofilms; promoting the detachment of pathogenic biofilms; promoting the growth of commensal biofilms; modulating metabolism; enhancing skin barrier function and innate immune function; inhibiting cancer cell growth; and / or inducing the expression and / or activity of various receptors and / or kinases. In specific embodiments, Qi611S protein has anticancer, antibacterial (e.g., antibacterial, antifungal, and / or antiviral), anti-inflammatory, metabolic modulation, and / or skin immunomodulatory activity.

[0073] In certain embodiments, the Qi611S protein can directly or indirectly induce the expression of and / or act as an agonist for one or more molecules selected from, for example, peroxisome proliferator-activated receptors (PPARs) (e.g., PPARα, PPARβ / δ, and / or PPARγ); extracellular signal-regulated kinase (ERK 1 / 2); and glucocorticoid receptor (GR). Whether the Qi611S protein acts as an inducer or an agonist can depend, for example, on the biological or metabolic system and / or type of cell involved (e.g., bacterial cells, cancer cells, or immune cells).

[0074] The Qi611S protein can be further defined by its amino acid sequence. In a specific embodiment (Qi611S), the protein has a sequence of 74 amino acids, as shown as SEQ ID NO:1.

[0075] In certain aspects, the proteins provided herein can be identified based on immunoreactivity with certain antibodies, as well as other methods described below.

[0076] In certain embodiments, the Qi611S protein can be produced by the Lactobacillus fermentum Qi6 bacterial strain when laboratory growth conditions are used to force the growth into a biofilm phenotype. In a preferred embodiment, the bacterial strain has a Qi611S DNA sequence (SEQ ID NO: 2) that is capable of expressing a protein having SEQ ID NO: 1 under conditions of the biofilm phenotype.

[0077] In a further embodiment, a polynucleotide encoding the Qi611S protein can be defined, for example, by its ability to hybridize to or be amplified by certain exemplified probes and primers (e.g., SEQ ID NOs.: 3-4).

[0078] Lactobacillus fermentum is a gram-positive bacillus. Lactobacillus fermentum Qi6 (Lf Qi6) can grow in MRS medium at 37°C.

[0079] A culture of L. fermentum Qi6 microorganism was deposited at the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 USA. The deposit was assigned the accession number ATCC number PTA-122195 by the collection and was deposited on June 10, 2015.

[0080] The cultures have been deposited under conditions assuring that access to the cultures will be available during the pendency of this application for patent to those determined to be entitled thereto by the Director of the Patent and Trademark Office under 37 CFR 1.14 and 35 USC 122. The deposit will be available as required by foreign patent laws in countries in which counterpart or successor applications to this application may be filed. It will be understood, however, that the availability of the deposit does not constitute a license to practice the invention in derogation of patent rights granted by governmental action.

[0081] Furthermore, the culture deposit will be preserved and made available to the public in accordance with the provisions of the Budapest Treaty on the Deposit of Microorganisms, i.e., preserved under all controls necessary to keep it in a viable and uncontaminated state for a period of at least five years from the most recent request to provide a sample of the deposit, and in any event for a period of at least thirty years from the date of deposit, or for the enforceable term of any patent which may result in disclosure of the culture. The depositor acknowledges that it will be obligated to replace the deposit in the unlikely event that the depository institution is unable to provide a sample when requested, because of the condition of the deposit. All restrictions on the public availability of the culture deposit will be irrevocably removed upon the grant of a patent disclosing it.

[0082] In some embodiments, Lf Qi6 can grow in biofilm phenotype.As used herein, "biofilm" is a complex aggregate of microorganisms, such as bacteria, in which cells adhere to each other using a matrix that is usually composed of, but not limited to, polysaccharide material.Compared with the planktonic cells of the same organism, which are single cells that can float or move around in liquid or gas medium, or can exist on or in solid or semi-solid surfaces, the cells in biofilm have physiologically different characteristics.

[0083] In certain embodiments, the present invention provides isolated polynucleotide sequences or genes encoding therapeutically and / or cosmetically useful Qi611S proteins. Additionally, in some embodiments, the present invention provides methods for using the polynucleotide sequences to generate recombinant hosts expressing Qi611S proteins.

[0084] In certain embodiments, the polynucleotide sequence is Qi611S, which is 222 base pairs and encodes Qi611S; however, in certain embodiments, different DNA sequences can encode the amino acid sequences disclosed herein, for example, due to redundancy in the genetic code. It is well within the skill of the artisan to generate these alternative DNA sequences that encode the Qi611S protein.

[0085] As used herein, a "mutant" of a protein refers to a sequence that has one or more amino acid substitutions, deletions, additions, or insertions. In a preferred embodiment, these substitutions, deletions, additions, or insertions do not substantially adversely affect the therapeutic activity of Qi611S. Mutants that retain one or more biological activities of Qi611S are within the scope of the present invention. Preferably, the one or more biological activities are selected from: reducing organophosphate toxicity; antibacterial activity; inhibiting the growth and attachment of pathogenic biofilms; promoting the detachment of pathogenic biofilms; promoting the growth of commensal biofilms; modulating metabolism; enhancing skin barrier function and innate immune function; inhibiting cancer cell proliferation; and / or inducing the expression and / or activity of various receptors and / or kinases.

[0086] "Fragments" of Qi611S and its variants are also within the scope of the Qi611S protein, so long as the fragment retains one or more biological properties of Qi611S. Preferably, the one or more biological activities are selected from: reducing organophosphate toxicity; antibacterial activity; inhibiting pathogenic biofilm growth and attachment; promoting detachment of pathogenic biofilm; promoting commensal biofilm growth; modulating metabolism; enhancing skin barrier function and innate immune function; inhibiting cancer cell growth; and / or inducing the expression and / or activity of various receptors and / or kinases. Preferably, the fragment is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the full-length Qi611S. The fragment can, for example, comprise one or more hydrophilic domains of Qi611S or variants. These domains may be directly connected with the removal of intervening amino acids. Hydrophilic domains can be easily identified using standard procedures known in the art and as illustrated in FIG. 4.

[0087] The present invention further contemplates fusion constructs in which the Qi611S protein is linked directly or indirectly (e.g., via a linker) to another moiety which may be, for example, a targeting moiety (e.g., a ligand, antibody, or aptamer), a toxin, a carrier, a label, or an activity promoter.

[0088] The present invention further contemplates antibodies (e.g., polyclonal, monoclonal, chimeric, and humanized) against the Qi611S protein. These antibodies can be easily prepared by those skilled in the art given the teachings provided herein. These antibodies can be used, for example, for therapy, diagnosis, and protein purification.

[0089] In certain embodiments, the polynucleotide encoding the Qi611S protein can be isolated, amplified, and ligated into a vector. A "vector," "plasmid," or "plasmid vector" is a DNA molecule used to transfer DNA into a cell, often from one cell to another (host cell). A vector can replicate in a host cell; or a vector can be a means to introduce DNA into a cell (or remove DNA from a cell). A variety of means can be used to introduce a vector into a host cell. Some cells can take up a vector without any action by the skilled artisan other than placing the vector in cell culture. Other cells require chemical modification. Regardless of the means by which a cell can take up a vector, once a host cell has the ability to do so, it is a "competent" cell.

[0090] One example of a commercially available vector is pET-15b, and using restriction enzyme digestion, one of skill in the art can create a vector carrying Qi611S or other polynucleotides encoding the Qi611S protein. The pET-15b vector carries an N-terminal His·Tag® sequence followed by a thrombin site and three cloning sites (SEQ ID NO. 6). The unique sites are shown in the circular map depicted in FIG. 1. The cloning / expression region of the coding strand transcribed by T7 RNA polymerase is shown in FIG. 2 (SEQ ID NO. 5).

[0091] In certain embodiments, the present invention relates to genetic transformation of host cells to confer the ability of these cells to produce Qi611S protein. For example, host cells (e.g., microbial cells, plant cells, eucaryotic cells and / or animal cells) may be transformed with a vector carrying Qi611S (or other polynucleotides encoding Qi611S protein), thereby allowing the recombinant cells to be used for the production of Qi611S protein.

[0092] In a preferred embodiment, the host cell is a strain of E. coli, such as E. coli BL21 or E. coli C43. Alternatively, the ability to convert cells other than E. coli into competent cells is well understood in the art, and includes cells selected based on, for example, their transformation ability, ability and efficiency for expression of heterologous proteins, stability of the protein in the host, presence of auxiliary genetic capabilities, lack of mammalian toxicity, ease of killing and fixing without damage to the protein, ease of culture and / or formulation, ease of handling, economics, storage stability, etc.

[0093] Bacterial hosts of particular interest include, for example, Escherichia, Lactobacillus, and Bacillus.

[0094] Eukaryotic hosts include, for example, Phycomycetes and yeasts, such as Ascomycetes, including Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeasts, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.

[0095] pET-15b vector, for example, codes for a polyhistidine tag and a thrombin cleavage site.Synthesis can be assessed through methods such as a polyhistidine tag that is coded alongside the protein to allow affinity chromatography, also referred to as affinity purification.The tag can remain attached to the Qi611S protein that is synthesized by the host, or preferably, can be cleaved from the protein by the thrombin cleavage site before recombinant protein is used.

[0096] Also within the scope of the present invention is a vector or expression cassette that comprises the polynucleotide that encodes the gene construct described herein or the polypeptide described above, which is functionally linked to a regulatory element.Vector and expression cassette can further comprise a selection marker.Vector and expression cassette can also comprise additional transcriptional control sequences, such as a strong promoter, to enhance the expression of cloned genes.

[0097] An expression cassette typically comprises, in the 5'-3' direction of transcription, a transcription and translation initiation region, a coding sequence of the present invention, and a transcription and translation termination region. The transcription initiation region, promoter may be native or analogous to the host cell, or may be foreign or heterologous. By "foreign" it is intended that the transcription initiation region is not found in the organism into which it is introduced.

[0098] The present invention also provides detection probes (e.g., fragments of the disclosed polynucleotide sequences) for hybridization with a target sequence or an amplification product generated from a target sequence. Such detection probes include a contiguous / contiguous span of at least 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides of SEQ ID NO:2.

[0099] The present invention also provides a method for producing a Qi611S protein by culturing a host cell transformed with a polynucleotide of the present invention (e.g., SEQ ID NO: 2) under conditions allowing expression of the polypeptide, and optionally recovering the expressed polypeptide.

[0100] In certain embodiments, host cells are transformed to express polypeptides in enhanced amounts.For example, DNA vectors can contain strong transcription promoter sequences immediately before the gene to be cloned.In certain embodiments, the strong promoters are trp operon, lac operon, T7 promoter, and / or pL promoter.

[0101] Figure 3 shows a silver stained SDS-PAGE gel used to confirm the presence of Qi611S synthesized by the recombinant E. coli strain. Samples of released proteins from recombinant E. coli BL21 were loaded onto the SDS-PAGE gel. The E. coli strain was transformed with the Qi611S gene via the pET-15b expression vector.

[0102] In certain embodiments, tags such as polyhistidine tag and glutathione-S-transferase can be used to purify Qi611S protein from the culture in which it is synthesized. Other means of protein purification can be used in conjunction with or without affinity chromatography. Some alternative methods include centrifugation, filtration, sonication, and fractionation. Proteins can be precipitated from the culture by adding ammonium sulfate. Also, various other chromatographic methods can be used, such as ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, or immobilized metal affinity chromatography. These protein purification methods can be used alone or in conjunction with each other. Each method or combination of methods has various advantages and disadvantages that will be understood by those skilled in the art.

[0103] It will be recognized by those skilled in the art that the DNA sequences of the present invention may vary due to the degeneracy of the genetic code and codon usage. All DNA sequences encoding Qi611S protein are contemplated. Thus, all polynucleotide sequences encoding Qi611S protein are included in the present invention, including DNA encoding SEQ ID NO: 1 (optionally including ATG immediately preceding the coding region). The present invention also includes polynucleotides with codons that are optimized for expression in host cells, including any of the specific types of cells mentioned herein. Various techniques for generating optimized sequences are known in the art.

[0104] Furthermore, it will be recognized by those skilled in the art that allelic variations may occur in the DNA sequences that would not significantly alter the activity of the amino acid sequence of the peptide that it encodes, and all such mutant DNA sequences are included within the scope of the present invention.

[0105] Those skilled in the art will understand that the exemplified sequences can be used to identify, produce, and use additional nucleotide sequences encoding Qi611S proteins. Mutant DNA sequences having at least 90% or at least 95% identity to the listed DNA sequences and encoding Qi611S proteins are included in the present invention. Other numerical ranges for mutant polynucleotide and amino acid sequences are provided below (e.g., 50-99%). Following the teachings of this specification and using knowledge and techniques well known in the art, it is believed that those skilled in the art will be able to generate many effective embodiments having mutant DNA sequences without undue experimentation. Homologs from other strains or species are specifically contemplated.

[0106] The fragments and mutational, insertional and deletional variants of the polynucleotides and amino acid sequences of the present invention can be used in the same manner as the exemplified sequences, so long as the fragments and variants have substantial sequence similarity with the original sequence. As used herein, substantial sequence similarity refers to the degree of similarity of the nucleotide or amino acid sequence that is sufficient to allow the variant or fragment sequence to function in the same capacity as the original sequence. Preferably, the similarity is greater than 50%; more preferably, the similarity is greater than 75%; most preferably, the similarity is greater than 90%. The degree of similarity required for the variant to function in its intended capacity will depend on the intended use of the sequence. It will be within the skill of one of ordinary skill in the art to create mutational, insertional and deletional mutations that are designed to improve the function of the sequence or provide methodological advantages. Identity and / or similarity can also be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% compared to the sequences exemplified herein.

[0107] Amino acid identity / similarity and / or homology is typically highest in the key regions of proteins that are responsible for and / or determine the three-dimensional configuration that ultimately contributes to biological activity. In this context, certain amino acid substitutions may be tolerated and predicted if they are in regions that are not important for activity or are conservative amino acid substitutions that do not affect the three-dimensional configuration of the molecule. For example, amino acids can be divided into the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions in which an amino acid of a class is replaced with another amino acid of the same type are within the scope of the present invention as long as the substitution does not substantially change the biological activity of the compound. Below (Table 1) is a list of examples of amino acids that belong to each class.

[0108] Table 1. Classification of amino acids based on physical properties TIFF2024545570000002.tif41154

[0109] In some cases, non-conservative substitutions can also be made. The important factor is that these substitutions should not significantly reduce the biological activity of the protein.

[0110] Therapeutic Uses of Qi611S Proteins, Cell Cultures, Cell Components, and Extracts The Qi611S proteins, cell cultures, cell components, and extracts of the present invention may be useful in therapeutic and / or prophylactic applications.

[0111] As used herein, "therapeutic" means useful for treating and / or preventing a disease, condition, or disorder.

[0112] In preferred embodiments, the present invention provides pharmaceutical compositions comprising Qi611S protein (e.g., SEQ ID NO: 1) and / or cells capable of producing Qi611S protein (as well as components of such cells), and methods of use thereof. In certain embodiments, the compositions can optionally include a pharma- ceutically acceptable carrier.

[0113] In a further aspect, the invention provides a method of providing a therapeutic benefit to a subject in need thereof, wherein a composition according to the invention is administered to the subject. In some aspects, a therapeutically effective dose of the composition may be administered to the subject, for example, one, two, three or more times daily, for as long as required or until the desired benefit is achieved.

[0114] As used herein, a "therapeutically effective" amount or dose is an amount or dose of a compound or composition that, when administered to a subject, is capable of treating or ameliorating a condition, disease, or disorder in the subject, or of conferring enhanced health or function to an organ, tissue, or body system. Preferably, the condition is organophosphate toxicity.

[0115] The actual amount will vary depending on several factors, including, but not limited to, the particular condition, disease, or disorder being treated or ameliorated; the severity of the condition; the particular organ, tissue, or body system for which enhanced health or function is desired; the weight, height, age, and health of the patient; and the route of administration. Prescription of treatment, such as determining dosage, is within the purview of general practitioners and other physicians, and typically takes into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to experts.

[0116] In certain embodiments, the therapeutic benefit is the treatment and / or prevention of organophosphate toxicity.

[0117] In some embodiments, the compositions and methods utilize a biologically pure culture of a cell capable of producing the Qi611S protein. The cell may be a microorganism having SEQ ID NO: 2, such as L. fermentum Qi6; and / or the cell may be a recombinant cell engineered to have a polynucleotide sequence encoding the Qi611S protein, such as SEQ ID NO: 2. In exemplary embodiments, the recombinant cell is E. coli BL21 or E. coli C43.

[0118] In certain embodiments, the cells are bacterial strains capable of growing in both planktonic and biofilm phenotypes. In one embodiment, the cells are in lyophilized, freeze-dried, and / or lysed form.

[0119] In one embodiment, proteins can be extracted from cell cultures and optionally purified before use in the compositions and methods. As used herein, the term "extract" refers to processing cell cultures to obtain one or more desired compounds. Processing can include, for example, physical and / or chemical treatments, such as filtration, centrifugation, sonication, pressure treatment, irradiation treatment, lysis, treatment with solvents or other chemicals, and combinations of these treatments. The resulting extract can be in the form of a supernatant, such as that produced by centrifugation. The extract can also include cell mass obtained by centrifugation. The cells can be intact or not intact, viable or not viable. The extract can include cell membrane components and / or intracellular components. In certain embodiments, the extract is at least 80, 85, 90, or 95% cell mass by weight. In certain other embodiments, the extract is at least 80, 85, 90, or 95% protein by weight.

[0120] The compositions provided herein can include a single (unit) dose of cells or lysates or proteins extracted therefrom. In some embodiments, the compositions according to the present invention can include a dose of at least about 0.01% to about 100% by weight of Qi611S protein, or at least about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about The composition may contain about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, about 50.0%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% Qi611S protein by weight.

[0121] In some embodiments, the composition can comprise a dosage of at least about 0.01% to about 30% by weight, about 0.01% to about 20% by weight, about 0.01% to about 5% by weight, about 0.1% to about 30% by weight, about 0.1% to about 20% by weight, about 0.1% to about 15% by weight, about 0.1% to about 10% by weight, about 0.1% to about 5% by weight, about 0.2% to about 5% by weight, about 0.3% to about 5% by weight, about 0.4% to about 5% by weight, about 0.5% to about 5% by weight, or about 1% to about 5% by weight of Qi611S protein.

[0122] The appropriate dose of bacteria (intact, lysed, or extracted) is 10 4 ~10 12 CFU, e.g., 10 4 ~10 10 , 10 5 ~10 8 , 10 6~10 12 , 10 6 ~10 10 , or 10 6 ~10 8 CFU. In the present invention, the abbreviation CFU is intended to designate "colony forming units" and is defined as the number of bacterial cells revealed by the microbial count on an agar plate.

[0123] Anti-inflammatory activity and immune modulation In some embodiments, the Qi611S protein and / or cells comprising a nucleotide sequence encoding the Qi611S protein can modulate the expression of skin innate immune peptides and / or cytokines involved in inflammation (e.g., PPARγ, interleukin-10 (IL-10), tumor necrosis factor alpha (TNFα), PERK, Toll-like receptors (TLRs), and / or filaggrin). Thus, in certain embodiments, the compositions and methods can be used to enhance skin and other innate immune functions. Thus, in one embodiment, the compositions and methods can enhance skin barrier function by upregulating the expression of skin barrier proteins.

[0124] Advantageously, the compositions of the invention can be administered systemically or locally and can be used to reduce the dose of glucocorticoids required, thereby mitigating side effects, including immunosuppression.

[0125] dermatological use Human skin comprises two compartments, namely the deep compartment (dermis) and the superficial compartment (epidermis). The skin constitutes a barrier against external aggressions, in particular chemical, mechanical or infectious aggressions, and some defense reactions against environmental factors, such as climate, UV radiation and tobacco, and / or xenobiotic factors, such as microorganisms. This property is called the skin barrier function and is mainly provided by the outermost layer of the epidermis, namely the stratum corneum. Adverse changes in the barrier can be reflected, for example, by skin discomfort, sensory phenomena, and / or skin dryness.

[0126] In some embodiments, repair or regeneration of the barrier comprises repair or regeneration of mucosa, including those of the mouth (including those of the cheeks, soft palate, tongue, including the underside of the tongue and floor of the mouth), nose, throat (including those of the pharynx, larynx, trachea, and esophagus), bronchi, lungs, eyes, ears, digestive tract, vagina, penis, urethra, bladder, and anus.

[0127] The active agent, eg, Qi611S protein, may be present at 0.01% to 2%, or any range therebetween, including, for example, 0.1 to 0.5%.

[0128] ophthalmological use There is a need in the field of ophthalmology for non-GC GR activators that do not damage ocular tissues, increase intraocular pressure, and do not predispose to glaucoma or cataract formation. These are all known complications of the use of GCs. Non-GC GR activators combined with PPAR activation will be particularly used in the topical and intraocular treatment of ocular inflammation and ocular diseases. PPAR signaling is an activation pathway important for the development and maintenance of several ocular tissue types, including meibomian glands, conjunctival goblet cells, limbal stem cells, trabecular meshwork, uveal tissue, and choroid. In addition, angiotensin receptors can be activated by PPAR gamma ligands, which this unique peptide can act on.

[0129] Administration to subjects The present invention provides a method of providing a therapeutic benefit to a subject in need thereof, wherein a therapeutically effective amount of a composition comprising Qi611S protein and / or cells capable of producing Qi611S protein is administered to the subject in need thereof. In some embodiments, the composition can comprise a pharma- ceutically acceptable carrier and / or can be administered simultaneously with a pharma- ceutically acceptable carrier.

[0130] In one embodiment, subject has already been exposed to organophosphate.Such exposure can be known, for example, by observing exposure, detecting exposure (for example, by chemical testing), and / or by the presence of symptoms of organophosphate exposure.The most important ocular signs of OP poisoning are pupil constriction (miosis), impaired visual function, and lacrimation; all of these physiological outcomes are the direct result of the increase in acetylcholine concentration in the synaptic cleft of neuromuscular junction in ocular tissue.

[0131] The duration of exposure to OPs can be assessed using biomarkers to determine AChE levels. At low doses, OP agonists induce visual loss. Local exposure of the eye to OPs leads to a reduction in cholinesterase activity in the iris and ciliary muscles, followed by an increase in acetylcholine in the anterior chamber of the eye. This cholinergic overstimulation effect on the pupillary sphincter leads to significant miosis with impaired visual acuity, reduced spatial visual ability with ciliary paralysis and reduced lens accommodation, and reduced adaptation to lighting changes due to desensitization of pupillary muscarinic receptors (Egoz 2017; Dabisch 2008; Genovese 2008; Lund-Karlsen and Fonneum 1976; Takayanagi 1993). Furthermore, cholinergic overstimulation induces spasms of the cholinergic ciliary muscles that can lead to blurred vision and myopia (Cannard, 2006; Nohara and Segawa, 1996; Rengstorff 1985; Smith and Smith 1980; Yanagisawa, 2006).

[0132] In other embodiments, the subject is at risk for organophosphate exposure. Evidence of such risk can include detection of the presence of organophosphate, use of organophosphate, imminent exposure to organophosphate, presence of symptoms of organophosphate exposure, and others are close proximity to the subject. Close proximity can be, for example, less than 1 mile, 1,500 feet, 500 feet, 100 feet, 50 feet, 25 feet, 10 feet, or 3 feet.

[0133] The composition can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated.The composition provided herein can be dissolved in, suspended in, or mixed with one or more other active or inactive ingredients.In certain embodiments, the composition can be formulated as food, capsule, pill, drinking liquid, lotion, cream, emulsion, ointment, oil, gel, serum, mist, inhalant, and / or combination thereof.The composition can also be provided in liposome or other microparticles.

[0134] The compositions provided herein can also include other pharma- ceutically acceptable components known to those of skill in the art, including, but not limited to, carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, and others described below. The formulations can further include other active agents, including, for example, other therapeutic or prophylactic agents.

[0135] As provided herein, "pharmaceutical acceptable" refers to approved or approvable by a regulatory agency of the U.S. Federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, including humans.

[0136] As used herein, "dermatologically acceptable" and "topically acceptable" are used interchangeably and are intended to mean that a particular ingredient is safe and non-toxic for application to the integument (e.g., skin) at the levels used. In one embodiment, the ingredients of the composition are recognized as "Generally Regarded as Safe" (GRAS).

[0137] A "pharmaceutical acceptable" carrier or adjuvant is one that can be administered to a subject together with an active ingredient, does not destroy its pharmacological or cosmetic activity, respectively, and is non-toxic when administered in a dose sufficient to deliver a therapeutic or cosmetic amount of the composition provided herein. As used herein, "carrier" includes excipients.

[0138] Carriers and / or adjuvants can include substances used to administer a composition according to a particular route, including, for example, oral administration, injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, and / or subcutaneous), and / or topical administration (e.g., via dermal absorption).

[0139] Carriers according to the present invention include any and all solvents, diluents, buffers (e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), oil-in-water or water-in-oil emulsions, aqueous compositions with or without organic co-solvents, e.g., suitable for IV use, solubilizers (e.g., polysorbate 65, polysorbate 80), colloids, dispersion media, vehicles, fillers, chelating agents (e.g., EDTA or glutathione, ), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavorings, fragrances, thickeners (e.g., carbomer, gelatin, or sodium alginate), coating agents, preservatives (e.g., thimerosal, benzyl alcohol, polyquaterium), antioxidants (e.g., ascorbic acid, sodium metabisulfite), osmotic regulators, absorption retarders, adjuvants, bulking agents (e.g., lactose, mannitol), and the like. The use of carriers and / or excipients in the pharmaceutical, cosmetic, and dietary supplement fields is well known.

[0140] In one embodiment, the method is used to treat dermatological disorders and / or to enhance the health of skin exposed to or at risk of being exposed to organophosphates.

[0141] In a preferred embodiment, the composition is formulated for local administration, in particular for use or application to or on the skin.As used herein, "local" means suitable for external local application to the skin or for dermal application.In other words, the topical composition is not intended to be applied to a subject via oral, intravenous, intramuscular, intrathecal, subcutaneous, sublingual, buccal, rectal, vaginal, inhalation, ocular or aural routes.

[0142] Formulations suitable for topical, dermal, and / or transdermal administration include, but are not limited to, gels, pastes, ointments, creams, lotions, oils, patches, bandages, bandages, dressings, depots, cements, adhesives, reservoirs, rinses, sprays, drops, foams, powders, sponges, tapes, inhalants, tinctures, and transdermal patches.

[0143] An ointment is typically prepared from a cosmetic composition provided herein and a paraffinic or water-miscible ointment base.

[0144] Creams are typically prepared from the cosmetic composition provided herein and an oil-in-water cream base.If desired, the aqueous phase of cream base can contain, for example, at least about 30% w / w of polyhydric alcohol, i.e., alcohol with two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol, polyethylene glycol, and mixtures thereof.As will be easily recognized by those skilled in the art, the formulation according to the present invention can also contain other alcohols, such as isopropyl alcohol or ethanol, and can also cover other alcohol-based formulations, such as alcohol-based hand sanitizers.

[0145] Topical formulations can include compounds that enhance absorption or penetration of the active compound through the skin or other affected areas. Examples of such skin penetration enhancers include dimethylsulfoxide and related analogues.

[0146] Suitable emulgents and emulsion stabilizers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate.Since the solubility of active compounds in most oils that may be used in pharmaceutical emulsion formulations may be very low, the selection of suitable oils or fats for formulation is based on achieving the desired cosmetic properties.Therefore, creams should preferably be non-greasy, non-staining, and washable products with suitable consistency to avoid leakage from tubes or other containers. Linear or branched mono- or dibasic alkyl esters can be used, such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a blend of branched esters known as Crodamol CAP, the last three being the preferred esters. These can be used alone or in combination, depending on the properties required. Alternatively, high melting point lipids can be used, such as white soft paraffin and / or liquid paraffin, or other mineral oils.

[0147] Additional topical ingredients can include, for example, emollients such as carnauba wax, cetyl alcohol, cetyl ester wax, emulsifying wax, hydrous lanolin, lanolin, lanolin alcohol, microcrystalline wax, paraffin, petrolatum, polyethylene glycol, stearic acid, stearyl alcohol, white beeswax, or yellow beeswax. Additionally, the composition can include humectants such as glycerin, propylene glycol, polyethylene glycol, sorbitol solution, and 1,2,6 hexanetriol, or permeation enhancers such as ethanol, isopropyl alcohol, or oleic acid.

[0148] In some embodiments, the compositions are formulated for administration via other non-topical routes, such as, for example, via oral, intravenous, intramuscular, intrathecal, subcutaneous, sublingual, buccal, rectal, vaginal, inhalation, ocular and / or otic routes. Administration may be systemic and / or local.

[0149] In one embodiment, the composition is formulated as an orally consumable product, such as a food product, a capsule, a pill, or a drinking liquid. An orally deliverable health-promoting compound is any bioactive substance that is delivered via initial absorption in the digestive tract or through the mucous membranes of the mouth.

[0150] The compositions may also be formulated to be administered via injection, including, for example, intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously. The compositions may also be administered sublingually, bucally, rectally, or vaginally. Additionally, the compositions may be sprayed intranasally for absorption through the nasal membranes, may be atomized or inhaled through the mouth or nose, or may be administered to the eye or ear.

[0151] Formulations can include, for example, orally consumable products, emulsions, tablets, capsules, powders, foams, granules, solutions, swabs, drops, suspensions, suppositories, injections, inhalants, and aerosols.

[0152] An orally consumable product according to the present invention is any preparation or composition suitable for consumption, nutrition, oral hygiene or pleasure, which is intended to be introduced into the oral cavity of a human or animal, to remain there for a certain period of time and then either swallowed (e.g., a ready-to-consume food or a pill) or removed again from the oral cavity (e.g., a chewing gum or an oral hygiene product or a medicinal mouthwash).

[0153] Orally consumable products include any substance or product that is intended to be ingested by humans or animals in a processed, semi-processed, or raw state. It also includes substances that are added to orally consumable products (especially food and pharmaceutical products) during their production, treatment, or processing, and that are intended to be introduced into the oral cavity of humans or animals.

[0154] Orally consumable products can also include materials that are intended to be swallowed by a human or animal and then digested in an unchanged, prepared, or processed state. Orally consumable products according to the present invention also include casings, coatings, or other encapsulation that are intended or expected to be swallowed along with the product.

[0155] In one embodiment, the orally consumable product is a capsule, pill, syrup, emulsion, or liquid suspension containing the desired orally deliverable substance (e.g., Qi611S protein). In one embodiment, the orally consumable product can include a powdered orally deliverable substance that can be mixed with water or another liquid to produce a drinkable orally consumable product.

[0156] In one embodiment, the composition can be made into an aerosol formulation, for example, so that the composition can be sprayed or inhaled. Pharmaceutical formulations suitable for administration in the form of aerosol or spray are, for example, solutions, suspensions, or emulsions. Formulations for oral or nasal aerosol or inhalation administration can also be formulated with exemplary carriers, including, for example, saline, polyethylene glycol or glycol, DPPC, methylcellulose, or in a mixture with powdered dispersants or fluorocarbons. Aerosol formulations can be placed in pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Illustratively, delivery can be by using a disposable delivery device, a mist nebulizer, a breath-actuated powder inhaler, an aerosol metered dose inhaler (MDI), or any other of the many nebulizer delivery devices available in the art. In addition, mist tents, or direct administration through an endotracheal tube can also be used.

[0157] In one embodiment, the composition can be formulated for administration via injection, for example, as a solution or suspension.The solution or suspension can include suitable non-toxic parenterally acceptable diluent or solvent, for example, mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting agent and suspending agent, for example, sterile non-irritating fixed oil, including synthetic mono- or diglyceride, and fatty acid, including oleic acid.One example of carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600, and the rest is USP water for injection (WFI). Other exemplary carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyldiphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral vegetable oil-in-water emulsions. Water or saline and aqueous dextrose and glycerol solutions may be preferably used as carriers, particularly for injectable solutions. Illustrative carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solutions, 5% dextrose in WFI and 0.01-0.1% triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a 1:2 or 1:4 mixture of 10% USP ethanol, 40% propylene glycol, with the remainder being an acceptable isotonic solution such as 5% dextrose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyldiphosphatidylcholine and 1-10% squalene in USP WFI or parenteral vegetable oil-in-water emulsions.

[0158] Additional formulations contemplated for administration to a subject include ear drops and eye drops, e.g., to treat ear and / or eye infections and / or dry eye.

[0159] For ophthalmic use, exemplary formulations that may be combined with the Qi611S protein are as follows: Isotonic sodium chloride solution: Sodium chloride 0.9% Benzalkonium chloride 1:100,000 sterile water buffer solution Boric acid 1.9% Benzalkonium chloride 1:100,000 Sodium sulfite, anhydrous 0.1% Phenylmercuric nitrate 1:50,000 Artificial tear solution: Polyvinyl alcohol 1.5% Povidone 0.5% Chlorobutanol 0.5% Ethylenediaminetetraacetic acid 0.01% Disodium edetate 0.05% White Vaseline 55% Mineral oil 41% Lanolin 2% Thimerosal 0.002% Ammonium acetate 0.0077% Human Albumin 0.1% Vehicle: An average drop is 25-50 microliters.

[0160] Additional components determined by those skilled in the art can be added to the compositions described herein, such as buffers, carriers, viscosity adjusters, preservatives, flavorings, dyes, and other components specific to intended use.Those skilled in the art will recognize that the above description is illustrative rather than exhaustive.In fact, many additional formulation techniques and pharmacologic acceptable excipients suitable for specific administration modes are well known to those skilled in the art.

[0161] All patents, patent applications, provisional applications, and publications referenced or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent not inconsistent with the explicit teachings of this specification. EXAMPLES

[0162] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that in light thereof, various modifications or changes will be suggested to those skilled in the art, which are included within the spirit and scope of the present application.

[0163] Example 1 - Isolation of the Qi611S gene and transformation of E. coli BL21 The gene encoding Qi611S, Qi611S, can be isolated from Lactobacillus fermentum Qi6 and transformed into E. coli BL21. As shown in Figure 3, E. coli BL21 can synthesize Qi611S protein.

[0164] The primers used to amplify Qi611S from LF Qi6 can be used to confirm the presence of Qi611S in E. coli cells. The primers used for cloning Qi611S into the pET-15b vector are listed in Table 2 (see also Figures 1-2). These specially designed primers were obtained from Integrated DNA Technologies (IDT) (Coralville, Iowa).

[0165] Table 2 Primers used for cloning Qi611S for transformation into E. coli BL21 TIFF2024545570000003.tif37128

[0166] DNA was isolated and amplified from the Lf Qi6 chromosome encoding Qi611S using the primers listed in Table 2. The primers allowed the use of two restriction enzymes, NdeI and BamHI, for restriction enzyme digestion of the amplified Qi611S gene, as well as the multiple cloning site of the pET-15b vector.

[0167] Once the amplified DNA and vector are digested using restriction enzymes, the Qi611S gene is ligated into the vector, which can then be transformed into E. coli BL21.

[0168] The transformed E. coli culture was incubated for 37 o The bacteria are grown overnight in C and individual colonies are tested for the presence of Qi611S using PCR. Once a positive clone is identified, the bacteria can then be grown to confirm that Qi611S successfully encodes the synthesis of Qi611S.

[0169] Qi611S protein can be detected by silver staining on SDS-PAGE gels. Protein samples released from E. coli BL21 expressing Qi611S are loaded onto SDS-PAGE gels. The pET-15b vector encodes a polyhistidine tag, which allows identification of the synthesized protein without the use of an antibody specific for Qi611S. In addition, pET-15b encodes a thrombin protease cleavage site (Leu-Val-Pro-Arg-Gly-Ser) that allows removal of the polyhistidine tag.

[0170] Example 2 - Assay to test the effects of ocular organophosphate toxicity induced by exposure to paraoxon Paraoxon, an organophosphate compound that acts as an irreversible AChE inhibitor and can be used as a surrogate for military-grade nerve agents such as sarin, VX, or novichok, was used to study organophosphate toxicity in an ex vivo rabbit eye model. The study involved the use of the compound Qi611 to evaluate its efficacy in protecting ocular AChE from paraoxon-induced inhibition and in activating the ocular peroxisome proliferator-activated receptor PPAR-γ.

[0171] material and method 1. AChE Inhibitor Loading of Rabbit Eyes Ex vivo whole eyes including conjunctivae from young (8-12 weeks old) New Zealand White rabbits used in this study were purchased from Pel-Freez, LLC (Rogers, AR, USA). Custom organ collection allowed one eye from the same animal to serve as a control (e.g., saline or DMSO) and the other eye to be subjected to 611 prophylactic treatment followed by paraoxon challenge. This setup facilitated limiting possible confounding factors regarding the animals used in the study, e.g., discrepancies in animal age, sex bias, or genetic differences, that may collectively affect tissue susceptibility to OP challenge. At least six eyes were assigned to each treatment group.

[0172] Eyes were transported on wet ice in drug- and serum-free DMEM cell culture medium and kept at 4 °C until ready for processing. Paraoxon-ethyl (pOX) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and aliquots were freshly prepared before each experiment due to the limited stability of the OP compound. Dimethyl sulfoxide (DMSO) and sterile saline were used to prepare serial dilutions of paraoxon-ethyl for use in the experiments, and all experiments were performed under a laminar flow hood to reduce the risk of contamination. Compound 611S was resuspended in sterile saline.

[0173] Briefly, rabbit eyes were removed from transport medium, transferred to 3 mL of fresh drug-free DMEM-F12 Ham's 1:1 culture medium, and placed in 6-well tissue culture plates, one eye per well. Eyes were allowed to warm to 20°C (usually within approximately 15 min) before any experimental procedures were performed, and temperature measurements were taken with a non-contact infrared thermometer (Braun BNT100CN, Kaz USA, Inc.). Using surgical forceps, the eyelids were pulled back to expose the orbit, followed by gently rinsing the cornea with 1 mL of drug- and serum-free DMEM-F12 culture medium. A sterile nylon ring (φ15 mm) was gently placed around the limbal region to contain saline, DMSO, or 611 treatment (volume of 200 μL per eye). The treatment solution was left on the corneal surface for 10 min, followed by removing the ring and draining the treatment solution. After drainage, a fresh sterile nylon ring was placed around the corneal area and paraoxon was applied as a 20 μL drop directly onto the corneal surface, with a total of 3 drops applied at 5 min intervals to allow tissue penetration of the paraoxon solution into the ocular tissue.

[0174] Pupil size was monitored for 40 min after application of the first dose of paraoxon, with photographs taken every 5 min. Digital photographs of the pupils were used for subsequent ImageJ (NIH, USA) analysis of changes in pupil area size over time, in addition to physical measurements of pupil diameter with an electronic caliper (Digital Caliper 15-719-8, Swiss Precision Instruments was used in this study).

[0175] After paraoxon challenge, anterior chamber fluid was withdrawn from the rabbit eyes using a hubless U-100 insulin syringe and a 31G needle, and the fluid samples were aliquoted into Eppendorf tubes and immediately placed at -80°C until required for further analysis. The volume of anterior chamber fluid ranged from 100 to 250 μl depending on the size of the eye.

[0176] 2. Colorimetric test for AChE enzyme activity Acetylcholinesterase assay colorimetric kit ab138871 (Abcam, USA) was used to measure AChE activity in anterior chamber fluid samples. The assay followed the manufacturer's recommendations included in the kit. Acetylcholinesterase standards (1 mU / ml to 1,000 mU / ml) replicates, experimental samples (5 μL of the indicated concentrations were added to 45 μL reaction buffer), and blank controls (assay buffer only) are added in a total volume of 50 μL to the indicated microplate wells. Paraoxon (5 μL added to 45 μL reaction buffer) control and calibration curve were applied to validate the model. Samples are incubated for 10 min, followed by adding 50 μL of acetylthiocholine reaction mixture to each well and incubating for 30 min at room temperature protected from light. The increase in absorbance at 410 nm is analyzed with a microplate reader. Corrected readings are obtained by subtracting background absorbance (blank wells) and by a trend line equation created from the calibration curve.

[0177] 3. PPAR-γ levels in ocular tissues Measurement of peroxisome proliferator-activated receptor-gamma upregulation in rabbit eyes in response to paraoxon challenge was performed using immunoblotting with anti-PPAR-γ mAb (Abcam, USA). Briefly, anterior chamber fluid samples were separated by SDS-PAGE on NuPAGE 4–12% Bis-Tris midi-protein gels (Invitrogen, USA), followed by protein transfer to PVDF Immobilon-P membranes (Millipore, USA). Membranes were blocked overnight at 4°C in 20 mL of PBS-T buffer containing 5% w / v nonfat dry milk (Publix, USA). After blocking, membranes were incubated with mouse monoclonal anti-PON1 or anti-PPARy antibodies (1:5,000 titer) for 2 h with gentle rocking, followed by rinsing the membranes three times for 5 min with PBS-T. The membrane was then placed in 20 mL of fresh PBS-T buffer containing 5% w / v nonfat dry milk and incubated with secondary anti-mouse HRP-conjugated antibody (1:10,000) for 1 h with gentle rocking. A SuperSignal Femto kit was used for immunoblot signal development and visualization (Thermo Scientific, USA).

[0178] 4. LDH Assay Anterior chamber fluid samples were used for quantification of lactate dehydrogenase (LDH) activity using a colorimetric LDH assay kit ab102526 (Abcam, USA) according to the manufacturer's recommended protocol, and 2 μl of sample was used in the assay.

[0179] 5.Statistical analysis For evaluation of the effect of paraoxon treatment on pupil size (maximum miosis observed) of ex vivo rabbit eyes and on the activity levels of AChE, PPAR-γ, or LDH detected in the anterior chamber fluid, one-way ANOVA and linear regression models with Dunnett's post hoc test were used. Statistical significance was assumed at a P value <0.05.

[0180] result 1. Assessment of Paraoxon-Induced Miosis in Ex Vivo Rabbit Eyes Custom collections of eyes were used in the experiments to reduce variability associated with phenotypic and genotypic differences. Constituting treatment groups based on per animal rather than per number of eyes, using standardized eye temperatures in the experiments, validating digitally measured pupil size by physical measurements with digital calipers, and using small batches of eyes to limit the risks associated with tissue decay over time and working with designated people for each step of the experiment to limit operator error (e.g., collection of anterior chamber fluid) contributed to reproducible study results. Comparison of pupil sizes recorded 40 min (t40) after the addition of the first drop of paraoxon (t0, basal value) showed no significant differences between saline-treated eyes, DMSO-treated eyes, or 611-treated eyes (Figure 5).

[0181] Figure 5. Saline, DMSO, or 611 alone do not significantly affect pupil size in ex vivo rabbit eyes.

[0182] Furthermore, saline preparations of Qi611S did not induce significant changes in pupil size, even within 2 h after a 10 min pretreatment with Qi611S, demonstrating that Qi611S alone does not act as a miotic or mydriatic agent (Figure 6).

[0183] Eyes subjected to paraoxon treatment at concentrations of 5 mM or 10 mM showed significant miosis with a significant reduction in pupil size when compared to eyes treated with saline or 611 alone (p<0.005). Furthermore, ex vivo rabbit eyes exposed to a 250 μg dose of 611 for 10 min and later challenged with 5 mM or 10 mM paraoxon showed significantly less miosis when compared to paraoxon-challenged eyes pretreated with saline for 10 min (FIGS. 7, 8). This attenuation of OP-induced miosis correlates with the preservation of AChE activity and the upregulation of PPAR-γ.

[0184] 2.AChE enzyme activity - colorimetric test data Anterior chamber fluid collected from eyes treated with Qi611S and exposed to paraoxon exhibits significantly higher AChE activity, whereas saline-treated eyes exhibit an approximately 80% reduction in enzyme activity, consistent with the inhibition of AChE typically seen following addition of OP compounds (Figure 9). Depending on the paraoxon concentration, 100 µg of 611 can preserve 50-80% of the acetylcholinesterase enzyme activity.

[0185] Figure 10. Protection of AChE activity by Qi611S is rapid, as demonstrated by prophylactic treatment 10 and 30 min prior to paraoxon exposure.

[0186] 3. PPARy-γ Activity – Western Blotting Eyes treated with Qi611S prior to paraoxon exposure showed a 3-4 fold higher upregulation of PPAR-γ as demonstrated by immunoblot (Figure 11). These results were consistent when using eyes from different cohorts of rabbits, indicating a role for Qi611S as a conditional PPAR-γ agonist. Upregulation of PPAR-γ correlates with protection of AChE activity in ex vivo rabbit eyes treated with Qi611S. Concentrations of 1 mM and 10 mM paraoxon (P) were used in this experiment.

[0187] FIG. 12 shows that prophylactic treatment with Qi611S prior to paraoxon exposure reduces tissue damage as demonstrated by LDH release assay.

[0188] Example 3 - Biofilm Production Cultivation of LfQi6 biofilms After isolation and identification, LfQi6 was cultured on MRS agar plates. The culture was then incubated in 5 ml of MRS broth at 37°C for 24 hours. 1 ml of the culture was transferred into a T-150 tissue culture plate containing 25 ml of MRS broth. The biofilm of LfQi6 was grown as a lawn on the bottom surface of the culture plate with 25 ml of MRS medium replaced every 48 hours. The culture was then grown for 7 days to generate a thick biofilm layer. The grown biofilm was subsequently detached and suspended in fresh medium. Freezer stacks were made with glycerol and stored at -80°C.

[0189] Frozen stocks of LfQi6 with biofilm phenotype were cultured in 10 ml of fresh MRS medium at 37°C for 24 h. 10 ml of culture was inoculated into 25 L of MRS medium containing 500 g of sterile glass wool. Biofilms were then cultured under static conditions at 37°C for 72 h. Cultures were mixed every 24 h by gentle shaking, after which the medium and glass wool were collected.

[0190] The biofil cells were then detached from the glass wool by sonication. The cells were further centrifuged to concentrate the Lf Qi6 biofilm, which was then suspended in sterile water. This scale-up resulted in a biofilm culture with a concentration of 50g / 25L. The growth of Lf Qi6 biofilm is further illustrated in Figure 4, where the biofilm was grown on a substrate in the scale-up culture described herein.

[0191] Downstream processing of LfQi6 biofilms 50 g of biofilm phenotype Lf Qi6 was suspended in 1 L of sterile water. The suspension was gently mixed at room temperature for 24 hours to allow passive release of multiple bioactive substances. The mixture was then sonicated (50 KHz, 200 watts) for 30 minutes using an OmniSonic Ruptor 400 to produce a homogenous lysate. The sonicated lysate was then frozen and lyophilized to a fine powder.

[0192] References TIFF2024545570000004.tif199154

Claims

1. 1. A method for reducing toxicity caused by a toxic agent, comprising: i) SEQ ID NO: 1; ii) a variant of SEQ ID NO: 1; and iii) A fragment of i) or ii) to a subject in need of such reduction in toxicity, wherein the variant or fragment reduces toxicity.

2. 2. The method of claim 1, wherein the protein comprises SEQ ID NO:1 or is a variant of SEQ ID NO:1 having at least 75% sequence identity to SEQ ID NO:1, or is a fragment of SEQ ID NO:1 or said variant, wherein the fragment has a length of at least 20% of the length of SEQ ID NO:

1.

3. The method of claim 1, wherein the protein comprises SEQ ID NO:

1.

4. The method of claim 1, wherein the protein has SEQ ID NO:

1.

5. The method of claim 1, wherein the protein comprises at least a 70% fragment of SEQ ID NO:

1.

6. 2. The method of claim 1, wherein the toxic agent is an organophosphate.

7. 7. The method of claim 6, wherein the organophosphate is a pesticide.

8. 7. The method of claim 6, wherein the organophosphate is a neuroactive agent.

9. 2. The method of claim 1, wherein the toxin is tetrodotoxin (TTX).

10. A method for assessing toxicity caused by an organophosphate, the assay comprising administering the organophosphate to an ex vivo rabbit eye and measuring miosis and AChE enzyme activity in the rabbit eye, wherein the presence of miosis and reduced AChE enzyme activity indicates organophosphate toxicity.

11. used to evaluate the ability of candidate compositions to reduce organophosphate toxicity; the use of two eyes of an ex vivo rabbit, where one of the eyes serves as a control and receives the organophosphate but not the candidate composition, and the other eye serves as a test and receives both the candidate composition and the organophosphate, the degree of miosis and AChE activity in each eye being measured and compared to each other, with less miosis and more AChE activity in the test eye indicating that the candidate compound reduces organophosphate toxicity.

11. The method of claim 10.

12. A method for reducing toxicity caused by a toxic substance, comprising administering to a subject in need of such reduction in toxicity a composition comprising Lactobacillus fermentum bacteria grown in a biofilm phenotype and / or an extract from said L. fermentum and / or said biofilm.

13. 13. The method of claim 12, wherein the toxicant is an organophosphate.

14. 14. The method of claim 13, wherein the organophosphate is a pesticide.

15. 14. The method of claim 13, wherein the organophosphate is a neuroactive agent.

16. 13. The method of claim 12, wherein the toxin is tetrodotoxin (TTX).

17. The method of claim 12, wherein the L. fermentum is LFQi6 (ATCC number PTA-122195).

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