Hapten conjugate vaccine against xylazine

Abstract

The application discloses compositions comprising a xylazine hapten having the Formula (I). The application further discloses xylazine hapten compositions and vaccines against xylazine. The application further discloses methods of preventing xylazine overdose. The application further discloses methods of preventing use disorder of xylazine. The application further discloses methods of generating antibodies against xylazine. The application further discloses methods of treating consumption of a substance laced with xylazine.

Description

HAPTEN CONJUGATE VACCINE AGAINST XYLAZINECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional patent application No. 63 / 569,247, which was filed on March 25, 2024, and which is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods for vaccines. More specifically, the present invention relates to vaccines against xylazine.BACKGROUND OF THE INVENTION

[0003] Xylazine is FDA-approved for veterinary use in animals as a sedative and pain reliever. Despite not being intended for human use, there is a growing popularity of individuals exposed to fentanyl, heroin, and other illicit drugs adulterated with xylazine. This phenomenon has prompted the Drug Enforcement Administration (DEA) to issue a public safety alert on March 20, 2023, reporting the widespread threat of fentanyl mixed with xylazine. Disturbingly, the DEA's findings reveal that approximately 23% of seized fentanyl powder and 7% of fentanyl pills contained xylazine. On average, xylazine was detected in 12.0% of fentanyl- involved deaths, with regional variations exhibiting up to 27.7%, with the highest prevalence observed in the Northeast. Moreover, the incidence of illicit manufactured fentanyl involved deaths with xylazine surged by 276% from January 2019 to June 2022. Simply stated fentanyl adulterated xylazine has greatly complicated the landscape of the opioid crisis.

[0004] Anecdotal reports from persons who have injected synthetic opioids suggested that xylazine helped lengthen the fentanyl “high” and delayed the onset of fentanyl withdrawal. Moreover, xylazine appears to compromise naloxone’s efficacy for overdose reversal due to naloxone’s inability to antagonize xylazine’ s non-opioid nature. Centrally acting, xylazine induces sedation, analgesia, and euphoria wherein it inhibits the outflow of norepinephrine and epinephrine, which can lead to a decrease peripheral heart rate and blood pressure. Documented human cases of xylazine overdose depict symptoms such as strong hypotension, bradycardia, hypothermia, and respiratory depression. Examining lethality, the fentanyl-xylazine drug combination produced a synergistic interaction represented by the decrease LD50 of each drugin the presence of the other. Rodent studies have put forward the notion that brain hypoxia as one of the mechanisms contributing to xylazine-positive opioid-overdose deaths. More perplexing, has been xylazine’s ability to cause conspicuous wounds that are distinct from typical fentanyl injected lesions. Although not fully clear the culprit behind these wounds, it has been surmised that lower tissue oxygenation leading to vasoconstriction and diminished perfusion to the skin is the cause. The outcome is abscesses, infection and skin ulceration / lesions and for this reason, xylazine has acquired the nickname “zombie drug.”

[0005] Xylazine is a weak base, that rapidly penetrates the blood-brain barrier and due to its lipophilic nature it also has a rapid onset of action undergoing distribution to different tissues. The pharmacology behind xylazine is that it primarily functions as an a2-adrenergic agonist. Yet, despite the existence of a2-adrenergic receptor antagonists such as yohimbine, studies have shown an inefficacy in preventing fentanyl-xylazine lethality. This ineffectiveness may arise from the mutual allosteric modulation between adrenergic and opioid molecules and receptors. Ultimately, the intricate interplay between opioids and the adrenergic system poses daunting challenges for a small molecule combatting the reversal of a xylazine related overdose.

[0006] In contrast, the concept of using monoclonal antibodies (mAbs) in drug sequestration has gained momentum recently with substance abuse monoclonal antibody therapeutics entering into clinical trials (NCT03336866, NCT06005402). Noteworthy is that antibodies offer advantages over small molecule antagonist treatments as they directly target the drug itself, bypassing complexities at the receptor level. Moreover, antibodies demonstrate higher safety profiles and selectivity against the targeted drug making them a viable emergency measure for opioid overdose, even in cases where the presence of xylazine is uncertain.

[0007] Opioid vaccines have been developed for many different compounds, such as fentanyl, that use a hapten of the opioid conjugated to a carrier. For example, U.S. Patent Application Publication No. 2022 / 0081400 to Janda, et al. discloses vaccines of fentanyl haptens and carfentanil haptens for use in treating opioid use disorder. However, no such treatment exists for xylazine.

[0008] There remains a need for methods of preventing xylazine addiction, misuse, and overdoses.SUMMARY OF THE INVENTION

[0009] The present invention provides for the present invention provides for a composition of a xylazine hapten.

[0010] The present invention also provides for a composition of a xylazine hapten conjugate including the xylazine hapten conjugated to a carrier.

[0011] The present invention provides for a vaccine including the xylazine hapten or the xylazine hapten conjugate and a pharmaceutically acceptable carrier.

[0012] The present invention provides for a method of treating a substance use disorder, by administering the xylazine hapten, xylazine hapten conjugate, or vaccine to an individual suffering from substance use disorder, and treating the individual.

[0013] The present invention provides for a method of preventing an overdose of xylazine or related compounds, by administering the xylazine hapten, xylazine hapten conjugate, or vaccine to an individual at risk of an overdose, and preventing an overdose in the individual.

[0014] The present invention also provides for a method of generating antibodies against xylazine in an individual, by administering the xylazine hapten, xylazine hapten conjugate, or vaccine to an individual, and generating antibodies in the individual.

[0015] The present invention provides for a method of treating an individual who has consumed a substance laced with xylazine, by administering the xylazine hapten, xylazine hapten conjugate, or vaccine to an individual, and treating the individual.DESCRIPTION OF THE DRAWINGS

[0016] Other advantages of the present invention are readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

[0017] FIGURE 1A shows the chemical structure of xylazine and FIGURE IB shows a conjugate of the xylazine hapten of Formula (I) for a vaccine study.

[0018] FIGURE 2 shows the synthetic route of a xylazine hapten of Formula (I).

[0019] FIGURE 3A is an NMR spectrum of the xylazine hapten of Formula (I), andFIGURE 3B is an NMR spectrum of the xylazine hapten of Formula (I).

[0020] FIGURE 4 shows a chemical structure of a side product in the synthesis of the xylazine hapten of Figure (I).

[0021] FIGURE 5A is a graph of LC-MS of condensation with formic acid during activation, and FIGURE 5B is a graph of LC-MS of Lossen-rearrangement polymerization.

[0022] FIGURE 6 is a reaction scheme of side reactions in the synthesis of the xylazine hapten of Figure (I).

[0023] FIGURE 7 is an LC-MS graph showing side product Hapten-NHS-sulfo.

[0024] FIGURE 8A is a MALDI-TOF graph of BSA conjugate, and FIGURE 8B is a MALDI-TOF graph of CRM197 conjugate.

[0025] FIGURE 9 is a graph of hapten BSA-conjugate density measurements.

[0026] FIGURE 10 is an illustration of an immunization scheme using xylazine hapten vaccines.

[0027] FIGURE 11 is a graph of longitudinal locomotion results of xylazine at different drug concentrations.

[0028] FIGURE 12 is a graph of dose-response curves for xylazine-induced respiratory depression.

[0029] FIGURE 13 is a standard curve of blood-brain biodistribution.

[0030] FIGURE 14 is a graph showing titer response of the xylazine vaccine with three different carrier proteins.

[0031] FIGURE 15A is a graph of locomotion in vaccine groups, and FIGURE 15B is a graph of percentage of minute volume (MV) change post-drug challenge at 2 mg / kg.

[0032] FIGURE 16 is a graph showing blood-brain biodistribution of xylazine at 1 mg / kg, 15 minutes post-drug challenge.

[0033] FIGURE 17 is a reaction scheme for the synthesis of a xylazine hapten.DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention generally provides for compositions and methods for treating substance use disorder and overdose of xylazine in an individual with vaccines.

[0035] A "hapten" refers to a small molecule compound that is capable of generating or inducing the production of antibodies.

[0036] A “hapten conjugate” refers to a hapten conjugated to a larger carrier such as a protein that is capable of generating or inducing the production of antibodies.

[0037] The term "vaccine" refers to a composition that can provide active acquired immunity to and / or therapeutic effect (e.g. treatment) of a particular drug, disease, or pathogen. A vaccine typically contains one or more agents that can induce an immune response in an individual against a drug, pathogen, disease, i.e. a target drug, pathogen, or disease. The immunogenic agent stimulates the body's immune system to recognize the agent as a threat or indication of the presence of the target pathogen or disease, thereby inducing immunological memory so that the immune system can more easily recognize and destroy and / or eliminate any of the drug or pathogen on subsequent exposure. Vaccines can be prophylactic (e.g.preventing or ameliorating the effects of a future drug overdose) or therapeutic (e.g., treating substance use disorder in an individual). The administration of vaccines is referred to vaccination. A vaccine can include a hapten or a hapten-conjugate.

[0038] The terms "adjuvant" or "vaccine adjuvant" or "pharmaceutically acceptable adjuvant" refer to compounds used in a vaccine to enhance (e.g., increase, accelerate, prolong, and / or target) the specific immune response to the vaccine antigen / conjugate / hapten in order to enhance the individual's immune response to the vaccine.

[0039] More specifically, the present invention provides for a composition of a xylazine hapten, such as shown in FIGURE IB or in FIGURE 17. The xylazine hapten can include a xylazine epitope linked with a linker at the para-position of the dimethyl phenol amino ring with a three carbon spacer. It can be preferred to add a linker at a methyl group on an aromatic ring of xylazine. FIGURE 17 shows a method of synthesis of a xylazine hapten that produces a linker on one of the methyl groups of the benzene ring. The hapten can also include an analog or derivative of xylazine as the epitope, or a structurally similar compounds such as from the general class of phenothiazines. The xylazine hapten can also be deuterated at one or more hydrogen positions to improve an immune response.

[0040] The present invention also provides for a composition of a xylazine hapten conjugate including the xylazine hapten conjugated to a carrier. The carrier can be, but is not limited to, OVA (ovalbumin), BSA (bovine serum albumin), KLH (keyhole limpet hemocyanin), DT (diphtheria toxoid), CTB (cholera toxin subunit B), TT (tetanus toxoid), or CRM197. Conjugation of the hapten to the carrier can be performed by one skilled in the art.

[0041] The present invention provides for a vaccine including the xylazine hapten or the xylazine hapten conjugate and a pharmaceutically acceptable carrier. The vaccine can include at least one adjuvant, such as, but not limited to, aluminum salts (alum), MF59, AS01, AS03, AS04, dmLT, LTA1, squalene based oil-in-water, MPLA-SM, flagellin derivatives, calcium salts, iron salts, zinc salts, acylated tyrosine, acylated sugars, cationically or anionically derivatized saccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A derivatives, 3-0-deacylated MPL, quil A, saponin based adjuvants (TQL 1055, QS-21), tocol, Freund's Incomplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N. J.), AS-2 (Smith-Kline Beecham, Philadelphia, Pa.), toll like receptor agonists (e.g., CpG (cytosine phosphoguanine) ODNs), bioadhesives, mucoadhesives, microparticles, liposomes, polyoxyethylene ether formulations, polyoxyethylene ester formulations, muramyl peptides, imidazoquinolone compounds (e.g. imiquamod and its homologues), and the like. Human immunomodulators suitable for use asadjuvants include cytokines such as interleukins (e.g. IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc); macrophage colony stimulating factor; tumor necrosis factor, granulocyte; macrophage colony stimulating factor, and the like. The adjuvant can preferably be alum and CpG.

[0042] The compound of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.

[0043] In the method of the present invention, the compound of the present invention can be administered in various ways. It should be noted that it can be administered as the compound and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants and vehicles. The compounds can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, intratonsillar, and intranasal administration as well as intrathecal and infusion techniques. Implants of the compounds are also useful. The patient being treated is a warmblooded animal and, in particular, mammals including humans. The pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention.

[0044] The doses can be single doses or multiple doses over a period of several days, weeks, or months. The treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.

[0045] When administering the compound of the present invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

[0046] Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by theuse of surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for compound compositions. Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds.

[0047] Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.

[0048] A pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres. Examples of delivery systems useful in the present invention include: 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0049] The present invention provides for a method of treating a substance use disorder, by administering the xylazine hapten, xylazine hapten conjugate, or vaccine as described above to an individual suffering from substance use disorder, and treating the individual. Treating the individual can include generating antibodies against xylazine, analogs thereof, derivatives thereof, or structurally similar compounds. The method can include reducing any effect that the xylazine has on the individual’s body, reducing and / or preventing addiction of the xylazine, or reduce cravings for the xylazine.

[0050] The present invention provides for a method of preventing an overdose of xylazine or related compounds, by administering the xylazine hapten, xylazine hapten conjugate, or vaccine as described above to an individual at risk of an overdose, and preventing an overdosein the individual. Preventing an overdose can include generating antibodies against xylazine, analogs thereof, derivatives thereof, or structurally similar compounds. The compositions of the present invention can provide an antibody effect strong enough such that the individual does not die after ingesting xylazine, and so that additional medical attention can be sought if needed.

[0051] The present invention also provides generally for a method of generating antibodies against xylazine in an individual, by administering the xylazine hapten, xylazine hapten conjugate, or vaccine as described above to an individual, and generating antibodies in the individual. Antibody generation can be confirmed by taking a sample (such as a blood sample) from the individual and performing an assay (such as an ELISA).

[0052] The hapten, hapten conjugate, or vaccine can be administered in combination with an opioid hapten, opioid hapten conjugate, or opioid vaccine, or a vaccine for any other abused substance. This can be useful in treating an individual who has consumed an opioid such as fentanyl, carfentanyl, morphine, heroin, codeine, oxycodone, or hydrocodone laced with xylazine. Therefore, the present invention provides for a method of treating an individual who has consumed a substance laced with xylazine, by administering the xylazine hapten, xylazine hapten conjugate, or vaccine as described above to an individual, and treating the individual. Treating the individual can include generating antibodies against xylazine, analogs thereof, derivatives thereof, or structurally similar compounds as well as against the opioid or other abused substance.

[0053] The present invention provides several advantages. Antibodies offer advantages over small molecule antagonist treatments as they directly target the drug itself, bypassing complexities at the receptor level. Moreover, antibodies demonstrate higher safety profiles and selectivity against the targeted drug, making them a viable emergency measure for drug overdose, even in cases where the presence of drug mixture is uncertain. The mechanism of action underlying antibody therapy allows for its potential application across different drugs, thereby streamlining the development of therapeutics against emerging substances and those with multifaceted receptors like xylazine.EMBODIMENTS

[0054] Embodiment 1. A composition comprising a xylazine hapten and optionally comprising an adjuvant.

[0055] Embodiment 2. The composition comprising a xylazine hapten of Embodiment 1having the Formula (I)(I) .

[0056] Embodiment 3. The composition of either Embodiment 1 or Embodiment 2, wherein the xylazine hapten is conjugated to a pharmaceutically acceptable protein or peptide carrier.

[0057] Embodiment 4. The composition of Embodiment 3, wherein the protein carrier is KLH.

[0058] Embodiment 5. The composition of Embodiment 3, wherein the protein carrier is TT.

[0059] Embodiment 6. The composition of Embodiment 3, wherein the protein carrier is CRM197.

[0060] Embodiment 7. A vaccine comprising the xylazine hapten composition of Embodiment 1.

[0061] Embodiment 8. A vaccine comprising the xylazine hapten composition of Embodiment 2.

[0062] Embodiment 9. A vaccine comprising the xylazine hapten composition of Embodiment 3.

[0063] Embodiment 10. The vaccine of Embodiment 9, wherein the carrier protein is KLH.

[0064] Embodiment 11. The vaccine of Embodiment 9, wherein the carrier protein is TT.

[0065] Embodiment 12. The vaccine of Embodiment 9, wherein the carrier protein isCRM197.

[0066] Embodiment 13. A method of preventing an overdose of xylene comprising administering the xylazine hapten composition of Embodiment 1 to a subject in need thereof.

[0067] Embodiment 14. A method of preventing an overdose of xylene comprising administering the xylazine hapten composition of Embodiment 2 to a subject in need thereof.

[0068] Embodiment 15. A method of preventing an overdose of xylene comprising administering the xylazine hapten composition of any one of Embodiments 3-6 to a subject in need thereof.

[0069] Embodiment 16. A method of preventing an overdose of xylene comprising administering the xylazine hapten vaccine of any one of Embodiments 7-12 to a subject in need thereof.

[0070] Embodiment 17. A method of treating a substance use disorder comprising administering the xylazine hapten composition of Embodiment 1 to a subject in need thereof.

[0071] Embodiment 18. A method of treating a substance use disorder comprising administering the xylazine hapten composition of Embodiment 2 to a subject in need thereof.

[0072] Embodiment 19. A method of treating a substance use disorder comprising administering the xylazine hapten composition of any one of Embodiments 3-6 to a subject in need thereof.

[0073] Embodiment 20. A method of treating a substance use disorder comprising administering the xylazine hapten vaccine of any one of Embodiments 7-12 to a subject in need thereof.

[0074] Embodiment 21. A method of generating antibodies against xylazine comprising administering the xylazine hapten composition of Embodiment 1 to a subject in need thereof.

[0075] Embodiment 22. A method of generating antibodies against xylazine comprising administering the xylazine hapten composition of Embodiment 2 to a subject in need thereof.

[0076] Embodiment 23. A method of generating antibodies against xylazine comprising administering the xylazine hapten composition of any one of Embodiments 3-6 to a subject in need thereof.

[0077] Embodiment 24. A method of generating antibodies against xylazine comprising administering the xylazine hapten vaccine of any one of Embodiments 7-12 to a subject in need thereof.

[0078] Embodiment 25. A method of treating consumption of a substance laced with xylazine comprising administering the xylazine hapten composition of Embodiment 1 to a subject in need thereof.

[0079] Embodiment 26. A method of treating consumption of a substance laced with xylazine comprising administering the xylazine hapten composition of Embodiment 2 to a subject in need thereof.

[0080] Embodiment 27. A method of treating consumption of a substance laced with xylazine comprising administering the xylazine hapten composition of any one of Embodiments 3-6 to a subject in need thereof.

[0081] Embodiment 28. A method of treating consumption of a substance laced with xylazine comprising administering the xylazine hapten composition of any one of Embodiments 7-12 to a subject in need thereof.

[0082] The invention is further described in detail by reference to the following experimental examples. These examples are provided for the purpose of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.EXAMPLES

[0083] To establish if the immune repertoire could block the pharmacological effects from xylazine, a drug-vaccine study was initiated using a xylazine-conjugate and the consequences of its formulation was examined with a series of protein-carriers and adjuvants. To gauge these formulations, vaccination was undertaken and their efficacy in titer and affinity were evaluated; furthermore, these vaccines were appraised in locomotor, respiratory depression and biodistribution rodent models.Chemicals and Instruments

[0084] 4-Hydroxy xylazine was purchased from Expert Synthesis Solutions (ESS0030), xylazine hydrochloride from TCI Chemicals and xylazine-de from Cayman Chemical Company. All other chemicals and reagents were purchased from Aldrich and Combi-Block and used without further purification, unless otherwise stated. All solvents were American Chemical Society (ACS) grade or better and used without further purification. Analytical thin layer chromatography (TLC) was performed with glass backed silica gel (0.25 mM thick, 60 A) plates purchased from Sigma and visualized under UV irradiation at 254 nm and / or by staining with potassium permanganate solution followed by heating. Flash automated column chromatography was performed using a CombiFlash Rf + Luman (Teledyne Isco) purification system with flash silica RediSep Rf columns for normal phase (NP) or RediSep Rf Gold Cis HP columns for reverse phase (RP). Analytical LCMS was performed on an Agilent ESLToF (LC / MSD ToF) with an Agilent Zorbax 300SB-C8 (4.6 x 50 mm), 5 pm column at a flow rate of 0.5 mL / min (Solvent A: 0.1% formic acid in H2O, Solvent B: 0.1% formic acid in acetonitrile) for ten minutes (0-6 min: 5-95% Solvent B, 6-10 min: 95% Solvent B). High resolution mass spectra (HRMS) were obtained in the Scripps Centre of Mass Spectrometry.HPLC spectra were recorded on an Agilent Systems 1260 using a Poroshell 120 EC-C8 column.Hapten Synthesis

[0085] The synthetic route is shown in FIGURE 2. A solution was prepared by combining 50 mg of 4-hydroxy xylazine and 100 mg of CsCCh (3 equivalents) in 4 mL of DMF. To this mixture, methyl 4-bromobutanoate (27 pL, 1.2 equivalents) was added. The reaction proceeded at room temperature for 3 hours. Following standard extraction and wash procedures, flash chromatography purification was carried out using RediSep® Rf Normal -phase Silica Flash Columns (60 A pore size, 35-70 microns particle size, 230 to 400 mesh) with a gradient of hexane and EtOAc as the mobile phase.

[0086] The collected product was dissolved in 5 mL of methanol, and 5 mL of 2M KOH solution was added. The reaction was allowed to continue at 50 degrees overnight. The solvent was then evaporated using a Rotavap, and the residue was loaded onto a Reversed-phase chromatography column (RediSep Rf Gold® Reversed-phase Cl 8) using a gradient of H2O (0.1% formic acid) and CH3CN (0.1% formic acid) as the mobile phase. A white solid was obtained with a total yield of 41%.

[0087] NMR data:XH NMR (600 MHz, CDCh) 5 8.41 (s, 1H), 6.57 (s, 2H), 3.92 (t, J= 6.3 Hz, 2H), 3.55 (t, J= 5.6 Hz, 2H), 3.01 - 2.96 (m, 2H), 2.42 (t, J = 7.2 Hz, 2H), 2.17 (s, 6H), 2.13 - 2.09 (m, 2H), 2.01 (p, J= 6.8 Hz, 2H).13C NMR (151 MHz, CDCh) 5 177.35, 168.34, 167.36, 158.95, 138.33, 125.39, 114.23, 67.03, 40.76, 31.20, 26.43, 24.80, 21.03, 18.34. HRMS (ESI): calcd for C16H22N2O3S ([M+H]+), 323.1429, found 323.1428. FIGURES 3A and 3B show NMR data.

[0088] In the hapten design, it was considered that the thiourea moiety and the phenyl ring entrenched within xylazine to be the key “chemical epitopes” that would be responsible for a stout antibody-hapten union (FIGURE 1 A-1B). Based on these considerations, the linker was installed within the para-position of the dimethyl phenol amino ring with a three carbon spacer. It was hypothesized that the phenyl ether linkage would not only simplify the hapten’s synthesis but also facilitate a stronger antibody-drug pi-pi interaction owing to its electron donating property. While the overall synthetic route to the xylazine hapten was straightforward, (see FIGURE 2), some minor complications arose wherein the thiourea moiety exhibited a propensity to form a formic acid adduct during purification with standard reverse phase chromatography (see13C NMR data in FIGURES 3 A and 3B). Moreover, during hapten activation, depending on the solvent used, amide condensation was observed as was a Loosen rearrangement that led to polymerization. Thus, recourse was taken wherein activation wasaccomplished under aqueous conditions (pH = 6) for a short period, which proved to be optimal.Bioconjugation

[0089] Due to the presence of a nucleophilic amine structure in the molecule, the hapten could not be preactivated in DMF for extended periods. Instead, activation was achieved by mixing the hapten with EDC / NHS-sulfo in H2O for 15 minutes before adding the carrier protein solution. The reaction then proceeded at room temperature for 2 hours. Mass studies revealed the occurrence of a side reaction between the hapten and NHS in the presence of EDC as a result of an elongated activation, resulting in the formation of the Lessen rearrangement side product, shown in FIGURE 4. The resulting amine could further react with another NHS molecule, leading to the formation of a series of polymers at higher EDC and NHS concentrations and longer incubation times. This phenomenon is due to the activation effect of formic acid on the thiourea. If purified without formic acid, this phenomenon was not observed.

[0090] To address this, the amount of EDC was minimized to 2 equivalents of the hapten, ensuring that the side reaction was controlled to less than 10% of the total conversion. The final reaction procedure for 1 mg of hapten involved mixing 1 mg of EDC and 1 mg of NHS-sulfo with the hapten and allowing the reaction to proceed in 200 pL of H2O for 15 minutes. Subsequently, 1 mg of protein, diluted in PBS buffer, pH = 7.4, was added to the mixture. After two hours of conjugation at room temperature, LC-MS analysis was employed to assess the percentage of side reaction (shown in FIGURES 5 A and 5B). The solution was then subjected to extensive dialysis against PBS buffer, pH = 7.4. FIGURE 6 shows a proposed reaction scheme.

[0091] To optimize conditions to minimize the side reaction and conjugate impurity, a solution of Img EDC, Img NHS-sulfo and Img hapten (with formic acid adduct) was preactivated for 15 minutes in H2O, followed by addition of Img BSA and 2 hours at room temperature. FIGURE 7 is a graph showing side product < 1: 10.

[0092] Haptens are covalently linked to protein or peptide carriers, enabling their recognition by T-cell receptors wherein they are processed by antigen-presenting cells, loaded on to MHCs ultimately eliciting a specific immune response against the hapten. With this said typically within the sphere of drug-vaccine design several protein carriers are commonly examined, including OVA, BSA, KLH, DT (diphtheria toxoid), CTB (cholera toxin subunit B), TT (tetanus toxoid), and CRM197, a nontoxic mutant of DT. These carriers vary in protein size, cost, and immunogenicity. The two least effective carriers, OVA and BSA have modest immunogenicity and are typically used in ELISA coatings for determining hapten-specific titerresponses. KLH has been extensively studied in preclinical practice, along with other evaluated options such as recombinant CTB. However, KLH has yet to be approved due to the complexity in its multi-subunit structure. DT, TT, and CRM197 on the other hand have already gained approval for clinical use in polysaccharide conjugate vaccines. Previous comparison between these three carriers uncovered a trend in immunogenicity in the following orders: KLH < CRM197 < TT.23-25 Thus to find the most suitable carrier protein for preparing a vaccine these three proteins were investigated.

[0093] Coupling of the hapten to the carrier protein is dictated by the functional groups present on the hapten / carrier as well as solubility / aggregation compatibility. While BSA, CRM197 and TT presented no problems during hapten-protein coupling, some issues were observed in the case of KLH. Thus, at a commonly used concentration (1 mg / mL hapten and protein) significant precipitation of the conjugate occurred that is believed to be unrelated to a hapten-hydrophobicity effect that has been noted previously. Moreover, this phenomenon was exclusive to the addition of activated hapten to the protein solution rather than the hapten itself. This could be attributed to protein aggregation as KLH is a large molecule prone to form aggregates and / or precipitates. To circumvent this difficulty, the KLH was filtered through a 0.22 pm filter before adding the activated hapten material. As anticipated this process transformed the KLH to a lower aggregation state as indicated by the opalescent blue color, which persisted throughout the conjugation and purification process.

[0094] For hapten-carrier protein conjugation'SEpjanalytics MALDLTOF was utilized fofsEPthe characterization of BSA (copyisEpjnumber = 17) (FIGURE 8A) and CRM197 (copyisEpjn umber = 12) (FIGURE 8B). Given that th e / h / detoxifi cation process of tetanus toxoidisEpj(TT) involves formaldehyde treatment, resulting in the loss of mass signal, an indirect ELISA assay was used to estimate the surface hapten density of TT by referencing the well- characterized BSA and CRM197 conjugates. In sum, CRM197 exhibited superior hapten loading, while TT showed comparable density to the BSA standard at the same coating concentration (FIGURE 9).Vaccine Preparation

[0095] Solutions of BSA / KLH / CRM197 conjugates were concentrated using AMICON® Ultra Centrifugal Filters 10K MWCO (Sigma-Aldrich) at 5,000 rpm until protein concentrations reached >0.5 mg / mL. Conjugate protein concentrations were verified by bicinchoninic acid (BCA) assays using the appropriate protein standard BSA, KLH, TT, or CRM197 and the concentrated conjugate solutions were stored at 4 °C until formulated. For each mouse, 50 pg of hapten conjugates were formulated with 500 mg of alum (alhydrogel)and 50 pg of CpG 1826 and then gently rotating at room temperature for 1 hour prior to immunizations to ensure sufficient absorption of the conjugate onto the alum surface.

[0096] To augment the immune response an adjuvant was needed and for all three carrier proteins the well-established adjuvant formulation of Alum and CpG was relied upon, which was conducted intraperitoneally using BALB / cByJ mice.Animals

[0097] Seven week old male BALB / cByJ mice (n = 6 / vaccine group) were obtained from Jackson Laboratories and allowed to acclimate for approximately 1 week before vaccination. Mice were group housed in an AAALAC-accredited vivarium containing temperature- and humidity-controlled rooms, with mice kept on a reverse light cycle (lights on: 9PM to 9AM). All experiments were performed during the dark phase. General health was monitored by both the scientists and veterinary staff of The Scripps Research Institute. All animal studies were performed in compliance with The Scripps Institutional Animal Care and Use Committee (Protocol#08-0127) and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals .Immunization was carried out according to FIGURE 10.ELISA

[0098] To measure the antibody response in mouse serum, 96-well plates (Corning 3690) were coated with 25 ng of hapten-BSA conjugate in PBS pH 7.4 (1 pg / mL, 25 pL per well) and incubated overnight at 4 °C. The plates were then washed with dH2O and blocked with 5% skim milk in PBS, pH 7.4 at room temperature for 45 minutes. Mouse serum samples were added to the first column of the plate, and a 1 : 1 serial dilution in 1% BSA-PBS, pH 7.4 was performed across each row starting with a dilution of 1 :200. After incubation at 37 °C for 2 hours, the plates were washed 10 times with dH2O, then incubated 1 hour at 37 °C with horseradish peroxidase-conjugated secondary antibody (donkey anti-mouse IgG, Jackson ImmunoResearch 715-035-151) diluted 1 : 10,000 in 1% BSA-PBS, pH 7.4. Plates were again washed 10 times with dH2O before being developed with 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Thermo Scientific). The TMB substrate (40 pL) was incubated for 8-10 minutes at room temperature, and the reaction was quenched with 2 M aq. H2SO4 (40 pL). Absorbance at 450 nm was recorded, and the absorbance values were normalized to the highest absorbance value per assay using GraphPad PRISM 8. Data was fit using the log(inhibitor) vs normalized response-variable slope equation to determine midpoint titer values.

[0099] For hapten density measurements, varying concentrations of BSA-conjugates were used to coat 96-well plates (4pg / mL, 2pg / mL, Ipg / mL, 0.5pg / mL, 0.25pg / mL, 0.125pg / mL)and the same protocol was followed vide supra. Absorbance measurements in the plateau region were plotted against the antigen coating concentrations to determine hapten density (FIGURE 9).Surface plasmon resonance[000100] A competitive binding assay was conducted using SPR on a Biacore 3000 instrument (GE Healthcare Life Sciences) equipped with a research-grade CM5 sensor chip. Ligands were immobilized on the chip surface using standard NHSZEDC coupling chemistry with BSA in the reference flow cell (FC1) and hapten-BSA conjugate in FC2. Mouse serum was diluted in running buffer (HBS-EP+ buffer: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v / v Surfactant P20) and titrated on the hapten-BSA immobilized flow cell minus the reference (FC2-1) to generate a response of -100 RU following the dissociation phase. For competitive binding studies, serum diluted at the predetermined concentration was incubated with analyte (xylazine) at room temperature for at least 30 minutes on a 600 rpm shaker. Following pre-incubation, samples were injected for 5 minutes followed by a 2.5 minutes dissociation phase. The chip surface was then regenerated by injection of 10 mM Glycine-HCl (pH 1.5) for 30 seconds, followed by a stabilization period before the next assay. Due to the carryover of the analyte, the assay was performed at a sequence of increasing inhibitor concentration, and a blank injection of 5% DMSO in HBS-EP+ buffer was performed after each serum sample. Inhibitor concentrations ranged from 100 pM to 0 nM with two-fold dilution for a total of 12 points. This is summarized in TABLE 1 below.TABLE 1TT 1 TT 2 CRM 1 CRM 2 KLH 1 Ik LU 2XYLAZINE (l:20K) (l:20K) (l:10K) (l:10K) (1 / 25K) (1 / 25K)0 210.74 148.62 114.62 114.42 104.4 124.519.8 185.49 119.59 107.14 100.02 82.22 88.6519.5 175.48 113.14 105.73 96.30 74.51 77.7239 161.45 101.17 99.08 88.59 66.37 67.3578 144.08 87.39 91.82 79.40 56.51 56.07156 122.05 71.67 80.41 65.53 47.72 45.28312 103.94 57.36 69.84 58.34 37.96 36.61625 81.71 44.43 58.24 48.69 31.86 27.971250 62.94 32.47 47.31 39.03 24.3 22.012500 45.03 23.38 37.50 29.74 18.58 16.395000 32.61 16.35 29.12 22.12 14.87 12.6310000 23.19 11.65 21.85 17.62 11.59 9.71IC50(nM) 276.30 133.40 486.20 277.40 92.14 46.69Locomotor activity[000101] Mice were acclimated to the testing room with a white light source in their home cages for at least 10 minutes. Subsequently, mice were placed in individual plastic cages (267 x 483 x 203 mm), enclosed with clear ventilated acrylic top, for a habituation period of 1 hour. Movement was tracked and recorded with overhead cameras using ANY-Maze video tracking software (Stoelting Co., Wood Dale, IL). Following the habituation period, mice were injected IP with vehicle or drug (1, 3, 6 mg / kg) and returned to the cage for a 1 hour testing period. The distance traveled was binned into 3-minute intervals, totaling 40 segments over 120 minutes (n = 4) and plotted against time in FIGURE 11.Plethysmography[000102] Respiration was measured in freely moving mice using whole-body mouse plethysmography chambers (EMKA Technologies, France) under a 5% CO2 atmosphere. One day before the experiment, mice were habituated to the chambers for 30 minutes while breathing air. On the day of the experiment, baseline respiration was recorded for each mouse over a 20-minute period before drug challenge IP. Using the IOX software by EMKA, data was recorded and binned in 5-minute intervals. Changes in minute volume were used to assess respiratory effects after acute drug administration. For each mouse, the percent change in minute volume was calculated as the percentage or pre-drug baseline. Statistics were analyzed using Two-Way ANOVA (repeated measure, each row represents a different time points), together with imbedded multiple comparison, within each row comparing columns with Bonferroni’s post-test. Data were graphed in FIGURE 12 and analyzed using GraphPad Prism 8, setting p <0.05 as the critical value. All data are displayed as mean ± SEM. *P < 0.05,**P < 0 oi,***p < 0.001. Significance between xylazine groups and the no drug control group is denoted by asterisks (n = 8).Blood-brain biodistribution[000103] In this study, blood and brain samples were collected from mice to investigate drug biodistribution. Specifically, 15 minutes after IP drug injection, mice were fully anesthetized with isoflurane and rapidly decapitated using a sharp guillotine. Trunk blood was collected,and the brain was surgically separated, weighed (typically 0.4-0.5 g), and added to a tube containing zirconium oxide beads (Next Advance ZrOB05). PBS, pH 7.4 was added to the brain sample at a 1 : 1 weight ratio (i.e., 1 mL PBS for 1 g brain tissue), and the sample was homogenized using a Bullet Blender homogenizer (Next Advance). The samples were then stored at -80 °C until analysis.[000104] To generate a standard curve, 100 pL of blank serum or brain homogenate samples were spiked with 50 pL of 50 ng / mL deuterated xylazine as an internal standard and 50 pL of xylazine at concentrations of 63, 125, 250, 500, and 1000 ng / mL in ACN and samples were mixed thoroughly through intensive vortexing. The sample pH was adjusted by adding 100 pL of 50 mM sodium carbonate (Na2COs), and extraction was performed by vortexing extensively with 700 pL of EtOAc. After centrifugation at 3,000 rpm for 5 minutes, 500 pL of the top solvent layer was removed, transferred to a new tube, and evaporated to dryness. The resulting residue was reconstituted in 100 pL of MeOH before analysis by LC-MS. The standard curve was produced by plotting drug concentrations divided by the factor of two (ratio of sample volume over spiked drug volume), which is 31, 63, 125, 250, and 500 ng / mL, against the corresponding ratio of drug / internal standard signal, shown in FIGURE 13. Linear regression fit, r2= 0.99.[000105] For experimental samples, frozen samples were thawed at room temperature, and 100 pL of serum orbrain homogenate samples were spiked with 50 pL of 50 ng / mL deuterated internal standard and 50 pL of pure PBS. Following the same sample processing methods as previously stated, the drug concentration in the samples was calculated by referencing the standard curve using the ratio of drug / internal standard signal. The values in the brain samples should be multiplied by two due to the 1 : 1 PBS dilution.Results[000106] The collected serum after the 1st and 2nd boosts were evaluated using ELISA (n = 6 / group) and SPR providing antibody titer and affinity to xylazine, (FIGURE 14, TABLE 2). Results of the two bleeds are shown as mean ± SEM. As seen in FIGURE 14, the second boost with all three vaccine preparations had a minimal impact on the overall titer but notably enhanced binding affinity as antibody maturation progressed. Contrary to previous reported trends, KLH afforded better affinity and titer compared to CRM197. This finding aligns with Applicants’ previous data, presenting evidence that CRM’s immune response appears more hapten concentric. In contrast TT elicited titers twice that of KLH but at the expense of lower affinity. Considering that the effect of the carrier protein mainly manifests through antibodytiter, TT still maintained merit as a superior carrier protein providing a robust titer response.TABLE 2 - IC50 values of anti-xylazine serum as defined by SPR[000107] Drug-receptor affinity typically serves as a guidepost as to what is required for a vaccine to neutralize a drugs pharmacology. The reported affinity of xylazine on the alpha2- adrenergic receptor exceeds 100 nM, which categorizes it as a weak agonist. Therefore, an immune response with affinities approximating or better than this value should readily antagonize drug-receptor binding. However, in making this statement hand-in-glove with affinity is the quantity of circulating antibody (titer) to the drug.[000108] To evaluate antibody-xylazine titer-affinity metrics from the three carrier proteins examined, behavioral experiments were conducted with mice involving xylazine induced hypolocomotion and respiratory depression. As benchmarks confirmed in the locomotor model, a minimum of 3 mg / kg of xylazine was required to observe a two-fold decrease distance traveled, whereas with plethysmography, 1-2 mg / kg was sufficient to detect significance, indicating that plethysmography provides a superior sensitivity index. In both models, the drug's behavioral effects were most profound around 10 minutes after injection. Challenging the vaccinated mice with 3 mg / kg in the locomotor model, elevated activity was observed in both TT and KLH groups, but not in the CRM197 group (FIGURE 15 A). FIGURE 15 A shows the sum of distance traveled in the 7-37 minutes window after drug challenge at 3 mg / kg. Significance is denoted by asterisks determined by unpaired t test, P = 0.0189. However, significance was only observed with the TT vaccinated group as the KLH vaccine displayed a large group variation. In the plethysmography model, both TT and KLH groups demonstrated overlapping performance and significant recovery from the 2 mg / kg dose (FIGURE 15B). Statistical comparison between groups was made by two-way ANOVA [Ftreatment (3, 20) = 21.74; P < 0.0001] with Bonferroni’s comparison; **P < 0.01. Data are presented as the mean ± SEM. Conclusions from these data indicate that TT and KLH produced similar outcomes,and because mAb therapy would not be limited by titer (antibody amounts), mAb affinity will be the crucial component for mAb development against xylazine.[000109] To further quantify the binding capacity for each vaccine group, a blood-brain biodistribution study was carried out, shown in FIGURE 16. Based on the results from the behavior studies, a dose of 1 mg / kg was applied to prevent antibody saturation and thus ensuring a more meaningful comparison across vaccine groups. As stated, vide supra, xylazine has a strong distribution bias towards the brain region and this is confirmed by a sizable brain- to-blood drug gradient in the control group indicating a high occurrence of binding events in the brain region associated with various subtypes of adrenergic receptors. Consistent with the behavioral results, both the TT and KLH groups displayed nearly identical binding capacities, (FIGURE 4). All bars are shown as mean ± SEM (n=6). Significance is denoted by asterisks determined by one-way ANOVA analyzing blood and brain separately (***P<0.001, **P<0.01, *P<0.05). Despite CRM197 also demonstrating a substantial increase in blood drug concentration, the impact on brain concentration was not significant, which aligned with its insufficient titer and affinity values to counteract brain binding events.[000110] In summary, the prophylactic effect brought about by vaccine xylazine-targeting has been successfully demonstrated. Lessons learned from this process underscore that while xylazine does not exhibit high affinity against the alpha2-adrenergic receptors as is seen with the synthetic opioids, it is compensated by a multiple receptor targeting regime. There is currently no antidote for xylazine poisoning other than supportive care, underscoring the need for effective measures to treat acute toxicity caused by xylazine. The xylazine vaccine data presented provides a therapeutic strategy for combating xylazine toxicity.REFERENCES:1. J. Johnson, L. Pizzicato, C. Johnson and K. Viner, Inj Prev, 2021, 27, 395-398.2. K. Ruiz-Colon, C. Chavez-Arias, J. E. Diaz-Alcala and M. A. Martinez, Forensic Sci Ini, 2014, 240, 1-8.3. M. Kariisa, J. O'Donnell, S. Kumar, C. L. Mattson and B. A. Goldberger, Mrnwr- MorbidMortal W, 2023, 72, 721-727.4. J. Friedman, F. Montero, P. Bourgois, R. Wahbi, D. Dye, D. Goodman-Meza and C. Shover, Drug Alcohol Depen, 2022, 233, 109380.5. R. Gupta, D. R. Holtgrave and M. A. Ashburn, New Engl J Med, 2023, 388, 2209-2212.6. A. J. Capraro, J. F. Wiley and J. R. Tucker, Pediatr Emerg Care, 2001, 17, 447-448.7. M. A. Smith, S. L. Biancorosso, J. D. Camp, S. H. Hailu, A. N. Johansen, M. H. Morris and H. N. Carlson, Front Pharmacol, 2023, 14, 1280289.8. P. Acosta-Mares, V. Viol ante- Sori a, T. Browne, Jr. and S. L. Cruz, Drug Alcohol Depend, 2023, 253, 110993.9. S. Choi, M. R. Irwin and E. A. Kiyatkin, Psychopharmacology, 2023, 240, 1561-1571.10. J. C. Reyes, J. L. Negron, H. M. Colon, A. M. Padilla, M. Y. Millan, T. D. Matos and R. R. Robles, J Urban Health, 2012, 89, 519-526.11. S. V. Malayala, B. N. Papudesi, R. Bobb and A. Wimbush, Cureus J Med Science,2022, 14, e28160.12. K. A. Moore, M. G. Ripple, S. Sakinedzad, B. Levine and D. R. Fowler, J Anal Toxicol, 2003, 27, 110-112.13. D. D. Schwartz and T. P. Clark, J Vet Pharmacol Ther, 1998, 21, 107-111.14. R. Root-Bernstein, in The Neurobiology, Physiology, and Psychology of Pain, eds. R. Rajendram, V. B. Patel, V. R. Preedy and C. R. Martin, Academic Press, 2022, DOI: https: / / doi .org / 10.1016 / B978-0-12-820589-l .000Q8-7, pp. 79-89.15. R. Liu, J. Grothusen, T. T. Joseph and A. McKinstry -Wu, Transl Perioper Pain Med,2023, 10, 522-525.16. R. Root-Bernstein, B. Churchill, M. Turke, U. K. T. Subhramanyam and J. Labahn, Int JMolSci, 2019, 20, 4137.17. ClinicalTrials.gov. National Library of Medicine (U.S.)., Identifier: NCT03336866,18. ClinicalTrials.gov. National Library of Medicine (U.S.)., Identifier: NCT06005402,19. T. T. Mai, Y. Y. Zhang and S. Q. Zhao, Toxics, 2023, 11.20. L. L. Chen, X. F. Hu, Y. N. Sun, Y. R. Xing and G. P. Zhang, Food Chem, 2022, 383, 132293.21. M. E. Pichichero, Hum Vacc Immunother, 2013, 9, 2505-2523.22. M. Pravetoni, J. S. Vervacke, M. D. Distefano, A. M. Tucker, M. Laudenbach and P.R. Pentel, Pios One, 2014, 9, e96547.23. P. T. Bremer, J. E. Schlosburg, M. L. Banks, F. F. Steele, B. Zhou, J. L. Poklis and K. D. Janda, J Am Chem Soc, 2017, 139, 8601-8611.24. A. Y. Moreno, M. R. Azar, N. A. Warren, T. J. Dickerson, G. F. Koob and K. D. Janda, Mol Pharmaceut, 2010, 7, 431-441.25. F. Baruffaldi, A. H. Kelcher, M. Laudenbach, V. Gradinati, A. Limkar, M. Roslawski, A. Birnbaum, A. Lees, C. Hassler, S. Runyon and M. Pravetoni, Mol Pharmaceut, 2018, 15, 4947-4962.26. M. L. Lin, J. C. Lee, S. Blake, B. Ellis, L. M. Eubanks and K. D. Janda, JACS Au, 2021,I, 31-40.27. P. T. Bremer, J. E. Schlosburg, J. M. Lively and K. D. Janda, Mol Pharmaceut, 2014,II, 1075-1080.28. M. L. Lin, A. Marin, B. Ellis, L. M. Eubanks, A. K. Andrianov and K. D. Janda, Mol Pharmaceut, 2022, 19, 3358-3366.29. R. Virtanen, J. M. Savoia, V. Saano and L. Nyman, Eur J Pharmacol, 1988, 150, 9-14.30. K. A. Totaro, X. L. Liao, K. Bhattacharya, J. I. Finneman, J. B. Sperry, M. A. Massa, J. Thorn, S. V. Ho and B. L. Pentelute, Bioconjugate Chem, 2016, 27, 994-1004.31. P T. Bremer, A. Kimishima, J. E. Schlosburg, B. Zhou, K. C. Collins and K. D. Janda, Angew Chem Int Edit, 2016, 55, 3772-3775.[000111] Throughout this application, various publications, including United States patents, are referenced by author and year and patents by number. Full citations for the publications are listed herein. The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.[000112] The invention has been described in an illustrative manner and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.[000113] Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention can be practiced otherwise than as specifically described.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a xylazine hapten and optionally comprising an adjuvant.

2. The composition comprising a xylazine hapten of Claim 1 having the Formula(I) (I) •3. The composition of either Claim 1 or Claim 2, wherein the xylazine hapten is conjugated to a pharmaceutically acceptable protein or peptide carrier.

4. The composition of Claim 3, wherein the protein carrier is KLH.

5. The composition of Claim 3, wherein the protein carrier is TT.

6. The composition of Claim 3, wherein the protein carrier is CRM197.

7. A vaccine comprising the xylazine hapten composition of Claim 1.

8. A vaccine comprising the xylazine hapten composition of Claim 2.

9. A vaccine comprising the xylazine hapten composition of Claim 3.

10. The vaccine of Claim 9, wherein the carrier protein is KLH.

11. The vaccine of Claim 9, wherein the carrier protein is TT.

12. The vaccine of Claim 9, wherein the carrier protein is CRM197.

13. A method of preventing an overdose of xylene comprising administering the xylazine hapten composition of Claim 1 to a subject in need thereof.

14. A method of preventing an overdose of xylene comprising administering the xylazine hapten composition of Claim 2 to a subject in need thereof.

15. A method of preventing an overdose of xylene comprising administering the xylazine hapten composition of any one of Claims 3-6 to a subject in need thereof.

16. A method of preventing an overdose of xylene comprising administering the xylazine hapten vaccine of any one of Claims 7-12 to a subject in need thereof.

17. A method of treating a substance use disorder comprising administering the xylazine hapten composition of Claim 1 to a subject in need thereof.

18. A method of treating a substance use disorder comprising administering the xylazine hapten composition of Claim 2 to a subject in need thereof.

19. A method of treating a substance use disorder comprising administering the xylazine hapten composition of any one of Claims 3-6 to a subject in need thereof.

20. A method of treating a substance use disorder comprising administering the xylazine hapten vaccine of any one of Claims 7-12 to a subject in need thereof.

21. A method of generating antibodies against xylazine comprising administering the xylazine hapten composition of Claim 1 to a subject in need thereof.

22. A method of generating antibodies against xylazine comprising administering the xylazine hapten composition of Claim 2 to a subject in need thereof.

23. A method of generating antibodies against xylazine comprising administering the xylazine hapten composition of any one of Claims 3-6 to a subject in need thereof.

24. A method of generating antibodies against xylazine comprising administering the xylazine hapten vaccine of any one of Claims 7-12 to a subject in need thereof.

25. A method of treating consumption of a substance laced with xylazine comprising administering the xylazine hapten composition of Claim 1 to a subject in need thereof.

26. A method of treating consumption of a substance laced with xylazine comprising administering the xylazine hapten composition of Claim 2 to a subject in need thereof.

27. A method of treating consumption of a substance laced with xylazine comprising administering the xylazine hapten composition of any one of Claims 3-6 to a subject in need thereof.

28. A method of treating consumption of a substance laced with xylazine comprising administering the xylazine hapten composition of any one of Claims 7-12 to a subject in need thereof.

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