CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. provisional patent application Serial Number 82/281 ,282, filed January 21 , 2016, the disclosure of which is incorporated herein by reference in its entirety,

Related patent applications by the inventor herein include:

US12/374908, PCTQ7/074978, WOG8/018978, "Vaccines and method for controlling adiposity"; US09/038546, PCT09/038548, WO09 120954, "Nicotine conjugates ^* ;

US 13/984230, PCT 12/000079, W012/108960, "Ghrelin mimetic polypeptide hapten

immunoconjugates having improved solubility and irnmunogenicity and methods of use thereof;

US13/261217, PCT 10/002489, W011/031327, "Nicotine haptens, immunoconjugates, and their uses";

US14/387511 , PGT11/001997, W013/095321, "Heroin haptens, immunoconjugates, and related uses ³¹ ;

US14/9Q1794, PCT14/045G19, WO15/002929, "Methods and compositions for treating obesity"; US14/901799, PCT14/045023, WO15/002391 , "Compositions and methods for treating obesity"; and, PCT15/0315S3, 015/179403, "Enantiopure haptens for nicotine vaccine development"; the disclosures of which are incorporated herein by reference in their entireties,

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under DA039834 awarded by the National Institute on Drug Abuse. The government has certain rights in the invention.

BACKGROUND

Fentanyi is an effective synthetic opioid that is used legally as a schedule II prescription pain reliever. However, fentanyl presents a significant abuse liability due to the euphoric feeling it induces via activation of mu-opioid receptors fMOR) in the brain; the same pharmacological target as the illegal schedule I opioid, heroinJ ¹ Excessive activation of OR results in respiratory depression which can be fatal. ^i2] Fentanyl exceeds the potency of heroin by >10-fold, and morphine by >80-foid posing a significant risk of overdose when it is consumed from unregulated sources, ^m Furthermore, the ease of fentanyl synthesis enables illegal production and the creation of designer drug analogues, ⁵ * The fact that the pharmacology of these analogues has yet to be properly characterized makes them particularly dangerous, especially when certain modifications, even methyl additions, can increase potency, notably at the 3- position, ^i¾

Last July, the National Institute on Drug Abuse (NIDA) reported an alarming surge in fentanyl overdose deaths:^ the latest update in a long stream of overdose cases starting with cs- mefhyrfanfanyl aka "China White" In the late I BSGs/ ³ A newer designer analogue,

acetyifentanyl, further exacerbates the opioid epidemic because of its deceptive sale as heroin or as a heroin mixture, ^[S] and it has been linked to a number of overdose deaths. ^m In addition to the US, fentanyl abuse is on the rise across Europe; while the most overdose deaths occurred in Estonia, the highest consumption of fentanyl per capita was reported in Germany and Austria. ^{E D3}

SUGARY

The invention provides, in various embodiments, a hapten for generating, when conjugated with a carrier, a vaccine for raising IgG antibodies in vivo, with specificity for fentanyl class drugs, comprising a compound of formula (!)

wherein R ~ CH ₂ CH ₂ C(=0)X and X ~ OH or an activated ester thereof; R ² - ^™ H or a (C ₁ ~C ₄ )alky! group, R ³ = H or a (CrC^alkyl group, and R ^4™ H or a (C ₁ -C ₄ )a kyl group; and a hapten conjugate for generating an in vivo immunoantagonist' to a fentanyl class drug, comprising a compound of formula (I) of claim 1 wherein X comprises a protein carrier or an amino- functionaiszed bead, the protein or the bead forming an amide bond with the hapten. For example, the protein carrier can be bovine serum albumin (BSA) or tetanus toxoid (ΤΓ), or the amino-functionalized bead can be a Dynabead Svl-270 Amine. The invention further provides a vaccine produced in a mammal by administration of an effective dose of the hapten conjugate described above. For example, the mamma! used for vaccine production can be a mouse, The vaccine can comprise IgG type antibodies.

In additional embodiments, the invention provides a method of effectively minimizing a concentration of a fentanyl class drug at a site of action in a patient, comprising administration of an effective dose of the vaccine to the patient. Additionally, administration of the effective dose of the vaccine imparts significant protection to the patient from an otherwise lethal dose of a fentanyi class drug. Further, administration of the effective dose of the vaccine to the patient reduces the addiction liability and overdose potential of the fentanyl ciass drug.

BRIEF DESCRIPTION OF THE FIGURES

Figyre 1. Structures of fentanyi immunoconjugate and analogues recognized by polyclonal antibodies with <100 nM affinity.

Figure 2, Timeline of experiments and anti-fentanyl antibody titers, Fent~TT (50 pg) was formulated with alum (750 ig) + CpG ODN 1828 (50 pg) and administered i.p. to each mouse (N~8). IgG titers were determined by EL!SA against fentanyi-BSA conjugate. Points denote means ± SE . Key: vaccine injection, a~antinociception assay, f-affinsty determination by SPR =blood/brain biodistribution study.

Figure 3. Fentanyi analogue dose-response curves and ED ₅₀ values in antinociception assays. Vaccinated and control mice (N~8 each) were cumulatively dosed with the specified drug and latency to nociception was measured by tail immersion (top) and hot plate (bottom) tests. Points denote means ± SEM. For all three drugs, p-vaiues were <0.001 in comparing control vs.

vaccine groups by an unpaired t test.

Figure 4. Biodistribution of fentanyl in blood and brain samples. Vaccinated and control mice (N ^™ 8 each) were dosed with 0.2 mg/kg fentanyl and tissue was harvested 15 min post-injection. Fentanyl quantification was performed by LCMS analysis. Bars denote means ÷ SEM. *** p < 0.001 , unpaired t test

Figure 5, Antiserum opioid binding curves and SPR sansorgrarrss. a) Diluted mouse serum from week 8 was incubated with serial dilutions of the listed opioids and injected into a Biacore 3000 containing a Fent-BSA loaded sensor chip, Signal produced by antibody binding to the SPR chip without drug present was used as a reference for 100% binding, Fentanyls used were racemic and 3-Me was ds. See Figure 1 for structures, b) Overlaid plots of sensorgrams obtained for the interaction between fentanyi (1000, 500, 250, 125, 82.5, 31.25, 15.63, 7.81 , 3.9, 1.95 and 0 nM) and immobilized anti-fentanyl antibodies at 2S X on a BiOptix 4Q4pi. Original experimental sensorgrarns are shown in black and fitted curves are traced in white.

DETAILED ESC IPTIO

To combat the harmful and addictive effects of fentanyl and its analogues, we pursued an immunopharrnacotherapeutic approach, similar to previous campaigns for addiction therapeutics against cocaine, ¹¹¹⁵ nicotine ¹²⁵ methamphetamine ^i1¾ and heroi .^ The basis of this strategy involves active vaccination of a protein-drug conjugate to generate an in wo 'irnmunoantagonis , which effectively minimizes concentrations of the target drug at the sites of action. As a result, the vaccine reduces the addiction liability and overdose potential of the specific drug. In this work, we report the first instance of an efficacious fentanyl conjugate vaccine. Upon immunization, this vaccine successfully stimulated endogenous generation of IgG antibodies with specificity for fentanyl class drugs. Moreover, mouse antiserum showed nanomolar affinity for a variety of fentanyl analogues by SPR-analytical methods. When mice were dosed with potentially lethal quantities of fentanyl analogues, the vaccine imparted significant protection. No other vaccines to date have demonstrated blockade of the acutely lethal effects of any drugs of abuse. Importantly, our research efforts have yielded significant progress for mitigating the pharmacodynamic effects of fentanyl class drugs.

in developing a fentanyl vaccine, hapten design presented the initial and possibly the most crucial challenge. As we have reported previously, small molecule haptens must faithfully preserve the natural structure of the target molecule to make a successful immunoconjugate, ^{! S3}

Confronted not only with fentanyl, but also designer analogues, our hapten incorporated the core A~(1~phenethylpiperidin-4-yl)-W-phenylacetamide scaffold In order to generate antibodies with broad immune specificity for virtually all fentanyl derivatives (Figure 1).

Furthermore, the propanoyi group in fentanyl was selected as the point of linker attachment because it would not sterically encumber the core structure {Figure 1). Hapten synthesis was achieved via replacement of the propanoyi group in fentanyl with a glutaric acid moiety, enabling standard bsoconjugate chemistry for amide coupling to an immunogenic carrier protein. Tetanus toxoid (TT) protein was chosen because of its use as a component of clinically-approved tetanus and glycoconjugate vaccines. Following conjugation of the fentanyl hapten to TT surface lysines, the resulting smmunoconjugate (termed Fent-TT, Figure 1) was administered to mice. Fent-TT was formulated with adjuvants alum and CpG ODN 1826 which have proven effective in boosting IgG antibody responses to a heroin conjugate vaccine. ⁵¹⁴⁸³ As shown in Figure 2, the Fent-TT vaccine induced very high anti-fentanyl antibody msd-point titers by ELISA (>100,000} even after one injection, providing ample In vivo neutralization capacity for fentanyis.

To assess vaccine performance, we employed antinociception assays that are a standard method for measuring the analgesic potential of opioid drugs in rodent modeis; ^{E1 a: 1t>i} opioids such as fentanyl increase pain thresholds in a dose-responsive manner, and these thresholds oan be quantified by measuring animal latency to nociception induced by a hot surface, A drug vaccine will blunt the pharmacological action of the target drug via serum antibody-mediated immunoarttago ism of an administered dose; therefore, a successful vaccine should shift the drug dose-response curve in antinociception assays to the 'right'. Comparison of drug EDso doses in vaccinated and non-vaccinated mice serves as a useful metric for drug vaccine efficacy. Previously, we have reported vaccine-induced shifts of about 5-10 fold which caused heroin-dependent rats to extinguish drug self-administration j ^{1 a} ' ^i4bj in the current study, we observed large fentanyl ED _se shifts of over 30-fold. Remarkably, during the initial week 8 testing session, fentanyl dosing was incapable of overriding the protective capacity of the vaccine. One month later (week 10), anti-drug titers in vaccinated mice had decreased to a point where smaller fentanyl doses could be used to generate full dose-response curves for EDs ₀ determination; large vaccine-mediated shifts were observed (33-fold in the tail immersion test, Figure 3). Strikingly, at the two largest doses that were safely administered to vaccinated mice (2.2 and 4.4 mg/kg), untreated mice experienced a 18 and 55% fatality rate respectively, thus demonstrating the ability of the vaccine to block lethal fentanyl doses.

As a testament to the vaccine's ability to neutralize other fentanyl analogues, Fent-TT immunized mice showed protection from two of the most common illegal fentanyl derivatives, 3- methylf ntanyl and -methyifentanyl aka "China White" (see Figure 1 for structures). The a-Me analogue was equipotent with the parent compound, and the vaccine was able to shift the a- e ED ₅₀ by about 8-fold (Figure 3). On the other hand, the 3-Me analogue was extraordinarily potent (about 10-fold greater than fentanyl), yet the vaccine still produced a 4-fold ED _S0 shift (Figure 3). Overall, our behavioral results indicate that the Fent-TT vaccine provided ample attenuation of large fentanyl doses in vivo while demonstrating a therapeutically useful level of cross-reactivity to fentanyl analogues. Clinically, these results implicate Fent-TT as an effective addiction therapy for curbing fentanyl abuse and overdose-induced lethality.

From a pharmacokinetic standpoint, we investigated the effect of the Fent-TT vaccine on the biodistribution of a fentanyl dose. Following administration of a fentanyl dose, we sacrificed both control and vaccinated mice at roughly the t _max (time at peak drug plasma concentrations) and measured fentanyl concentrations in both brain and blood samples by LCMS. Our results clearly show how serum antibodies in vaccinated mice act as a depot to bind 45-tlmes the amount of fentanyl relative to serum proteins in control mice. This translated to a significant reduction in fentanyl brain concentrations of vaccinated mice, lending to a pharmacological explanation of how the vaccine attenuates fentanyl psychoactivity.

Behavioral and pharmacokinetic results were corroborated with thorough biochemical analysis of antiserum derived from Fent~TT vaccinated mice. To achieve this, we employed surface plasmon resonance (SPR) spectroscopy, a highly sensitive technique for investigating protein-protein or protein-small molecule binding interactions. ⁵¹⁷⁵ in a new application of SPR, we measured binding affinities of polyclonal antibodies in vaccinated mouse serum for various fentanyl analogues. Diluted mouse serum was preincubated with a series of concentrations of selected fentanyl derivatives and then injected into a Biacore 3000 containing a Fent-BSA coated chip. Essentially, this method is a more sophisticated version of competitive ELISA in which serially-diluted free drug competes with an immobilized drug hapten for antibody binding. Our results from the SPR competition experiment (Figure 5a) indicated that antibodies from Fent-TT immunized mice have high affinity for fentanyl derivatives, generating binding curves with low nanomolar values and limits of detection in the pM range (see Figure 5a and raw sensorgrams, Figure S2). Relative affinities between analogues with different R-i a!kyf groups were very similar, likely due to the fact that the position was used as a linker attachment point. As expected, methylation at other positions resulted in lower affinity but still IC ₅ Q values were <100 nM. Furthermore, the SPR iC¾ ₀ s mirrored the results in behavioral assays, and in both cases followed a trend of Fent>a-lv1e> s-3-fv1a. Since the Fent-TT vaccine gave broad specificity to fentanyl class drugs, two clinically used opioids methadone (IvleD) and oxycodone (Qx ) were tested to ensure minimal cross reactivity, Indeed, affinities for these opioids were >7,500 times lower compared to fentanyl (Figure 5a), demonstrating that they could still be used in Fent-TT vaccinated subjects.

Further validation of the SPR method was pursued to confirm that the generated ICs ₀ values were representative of actual _D ; hapten affinity does not always reflect free drug affinity. To address this problem, we affinity purified anti-fentanyl antibodies and loaded them onto an SPR chip for direct measurement of free fentanyl binding kinetics. As shown in Figure 5b sensorgrams, the fentanyl _D of purified antibodies (2 nfvT), is in close agreement with the IC ₅₀ value determined by the competition method (5 nM). Thus, we have demonstrated the SPR competition method as an accurate way to measure drug affinities of polyclonal serum antibodies, A crucial aspect of immunopharmaeotherapy is proper characterization of anti-drug antibodies, and the SPR method could help to facilitate this facet of drug vaccine development.

8 Additionally, the method could be used to screen biological samples e.g. blood or urine for the presence of a wide variety of fentanyl derivatives, especially since the limit of detection for many fentanyl analogues is <1 nM (Figure 5a).

The current study has yielded a potential therapeutic that could assist in combatting the rise of opioid abuse. An effective fentanyl conjugate vaccine was developed that easiiy ablates small doses needed to achieve a normal drug-induced 'high ¹ but also attenuates large, potentially iethal doses of fentanyl class drugs. Furthermore, the success of this vaccine helps to advance immunopharmacotherap from an academic novelty towards a practical therapy, and influences the creation of vaccines against other destructive designer drugs such as "bath salts' J ⁴ ' ^Sj

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Nuclear magnetic resonance ( ¹ H NMR (400 MHz), ¹³ C NMR (100 MHz)} spectra were determined on a Bruker 400 instrument unless otherwise noted. Chemical shifts for NMR are reported in parts per million (ppm) relative to chloroform (7.26 pprn) and coupling constants are in hertz (Hz). The following abbreviations are used for spin multiplicity: s - singlet, d ~ doublet, t = triplet, q = quartet, m ~ mu tlplet, br ~ broad. Chemical shifts for ¹³ C HMR were reported in ppm relative to the center line of a triplet at 77.0 ppm for chloroform. Electrospray Ionization (ESI) mass were obtained on a ThermoFinnigan LTQ Ion Trap, Matrix-assisted Laser

Desorption/ionizafion (MALDI) mass spectra were obtained on an Applied Biosystems Voyager DE. Analytical thin layer chromatography (TLC) was performed on Merck precoated analytical plates, 0,25 mm thick, silica gel 80 F _2$ *- Preparative TLC (PTLC) separations were performed on Merck analytical plates (0.50 mm thick) precoated with silica gel 60 F25 . Flash

chromatography separations were performed on Aldrich silica gel (catalog # 717185, 60 A pore size, 40-63 pm particle size, 230-400 mesh) unless otherwise noted.

S 8-8 week old male Swiss Webster mice (n ~ 8/group) were obtained from laconic Farms (Germantown, NY). Mice were group-housed in an AAALAC-accradited vivarium containing temperature- and humidity-controlled rooms, with mice kept on a reverse light cycle (lights on: SPM-QAIVI). All experiments were performed during the dark phase, generally between 1 PM- 4PM. General health was monitored by both the scientists and veterinary staff of Scripps Research Institute, and all studies were performed in compliance with the Scripps Institutional Animal Care and Use Committee, and were in concordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Biood serum samples for titer quantification were performed using tail-tip amputation (<1 cm) in order to collect between 100- 150 ΐ whole blood, and samples then centrifuged at 7500 rpm for 8 min to separate serum. Fenian f Hapten Sy th s s

1 a

To a solution of (200 mg, 714 pmol) In CH ₂ CI ₂ (7.0 mL) were added pyridine (115 μ5_, 1.42 mmol) and glutaric acid monomethyl ester chloride (110 pL, 780 pmol) at 0 °C. After stirring at 0 °C for 5 min, the reaction mixture was allowed to warm to rt After stirring at rt for 1 ,5 h, the reaction mixture was quenched with saturated aqueous aHC0 ₃ . and extracted with EtOAc. The aqueous layer was extracted with EtGAc three times. The combined organic extracts were dried over a ₂ S0 ₄ , filtered, and evaporated in vacuo. The residual oil was purified by flush column chromatography (Si0 ₂ ; r?-hexane:EtOAc ~ 1 :1 to 1 :2 to 0:1) to afford 2 (288 mg, 98.7%) as a pale yellow solid.

H-NMR (400 MHz, CDCi ₃ ) δ 7.41-7.35 (m, 3H), 7.27-7.23 (m, 2H), 7.19-7.13 (m, 3H), 7.08-7.05 (m, 2H), 4.67 (tt, =12.G, 4.0 Hz, 1 H), 3.S9 (s, 3H), 2.99 (br d, J=12.8 Hz, 2H), 272 (dd, J=8A, 8.0 Hz, 1 H), 2.72 (d, J=8,4 Hz, 1 H), 2.53 (dd _: J-8.4, 7.2 Hz, 1 H), 2.28 (t, =7.2 Hz, 2H), 2,15 (td, -12.4, 2.4 Hz, 2H), 1.98-1.93 (m, 2H), 1.87 (qd, J=7.8, 1 ,6 Hz, 3H), 1.85-1.77 (m, 2H), 1.42 (qd, 12.4, 3.8 Hz, 2H); ³ C ^» NMR (100 MHz, CDCi ₃ ) δ 173.5, 171 ,7, 139.7, 138.4, 130.2 (2C), 129.3 (2C), 128.5 (2C), 128.3, 128.3 (2C), 128.0, 59,9, 52.8 (2C), 51.9, 51 ,3, 33.9, 33.2, 33.1 , 29.9 (2C), 20,5; HRMS (ESI+) 409.2487 {calcd for C ₂₅ H _ss N ₂ 0 ₃ 409,2491).

To a solution of 2 (49.9 mg, 122 pmoi) in MeOH (1.0 mL) was added 1 ,0 aqueous LiOH (250 pL) at rt. After stirring at rt for 4 h, the reaction mixture was washed with hexane. The aqueous layer was acidified with 2.0 M aqueous HCI, and extracted with EtOAc three times. The combined organic extracts were dried over Na ₂ S0 _i filtered, and evaporated in vacuo. The residual white solid 3 ^ί¾ (52.0 mg, quant) was used for the next step without further purification. The spectroscopic data for 3 were collected after purification by PTLC (Ss0 ₂ ; CH ₂ Ci ₂ :MeOH™ 9:1).

¹ H~NMR (400 MHz, CDCI ₃ ) δ 7,41-7,35 (m, 3H), 7.29-7,24 (m, 2H), 7.22-7.18 (m, 1 H), 7.17- 7.15 (m, 2H), 7.06-7.04 (m, 2H), 4.83 (br s, 1 H), 4.69 (tt, J-12.Q, 4.0 Hz, 1 H), 3.35 (br d, J-1 1 ,8 Hz, 2H) _S 2.98-2.85 (m, 4H), 2.56 (br t, J-10,8 Hz, 2H), 2.16 (t, J=7.2 Hz, 2H) _S 1 ,98 (t, J^7.2 Hz, 2H) _S 1.87 (br d, J=12.4 Hz, 2H), 183-1.74 (m, 2H), 180 (t, J-6.8 Hz, 2H); ¹ ¾ NMR (100 MHz, CDCk) 5 176,9, 172,3, 139.0, 138.4, 130.0 (2C), 129.5 (2C), 128.6 (2C), 128.8, 128,5 (2C), 126.3, 59.2, 52.3 (2C), 517, 34.3, 34.1 , 32.3, 29.2 {2C}, 20.9; HRMS (ESI+) 395,2327 (caicd for

FentssnvS Hapten Conjugation to Bovine Ssrum bumin fBSA) a d Tetanus Toxoid (TT)

To a solution of 3 (2.7 mg, 6.8 μϊηοΙ} in DMF (144 pL) and H ₂ 0 (16 pi) were added NHS (rV-hydroxysuccinimide} (4.8 mg, 40,4 pmol) and EDC (W-(3-dimethylaminopropy!)-A ~ ethy!carbodiimide) (7,8 mg, 40.8 μηιοΙ) at rt. After stirring at rt for 1 ,5 h, additional EDC (4.2 mg, 21.9 ΓηοΙ) was added. After additional stirring at rt for 2 h, the reaction mixture was divided info two portions. One portion (80 μί_) was added into a solution of BSA (Thermo Scientific) in PBS buffer (pH 7.4) (660 μΙ_, 1.50 mg/mL) at rt, and the other portion (80 uL) was added into a solution of IT (Statens Serum !nstiiut) in P8S buffer (pH 7.4) (850 μΐ, 1.53 mg/mL) at rt. After stirring at rt for 15 h, each of the reaction mixture was diaiyzed against PSS buffer (pH 7.4) at rt using a SHde-A-Lyzer 10 MWGO dialysis device. The buffer was exchanged every 2 h for 6 h. and then dialysis was continued for 12 h at 4 °C. The conjugate concentrations were quantified by 8CA assay and stored at 4 °C.

Preparation of FerstanyJ Hapten Coated Dynabeads ^® Amine

To a solution of 3 (3.0 mg, 7.6 Mmol) in DMF (225 μ ) and H ₂ 0 (25 pL) were added NHS (9.0 mg, 75.8 Mmoi) and EDC (14.8 rng, 76.1 μητίο!) at rt. After stirring at rt for 12 h, a 50 iL aliquot of the reaction mixture was added into 1.0 mL of Dynabeads ^® M-270 Amine (washed 4 times with PBS buffer (pH 7.4) prior to use) in PBS buffer at rt. After stirring at rt for 1.5 h, the beads were washed with PBS buffer (pH 7,4) for two times and stored at 4°C.

In order to quantify copy number (hapten density) for each fentanyl hapten-BSA and TT conjugates prepared in this study, samples were submitted for MALDI-TGF analysis and compared MW of feniany! hapten-BSA and TT conjugates with M)N of unmodified BSA and TT, respectively, as per the formula:

CO number / (M f _erliafl yi hapien ~ W _w¾ief )

MWferitanyi hapten™ 3^5 Da, MW _¾¾i ef = 18 Da

MW _{fentan ! hapten-B} sA = 80,939 Da, M _BSA = 66,500 Da

hapjsfrTT ~ 168,757 Da, MWTT = 153,500 Da

CO numbetfentanyi hapters-BSA ~ 38

COpy numbe fsntanyi hapten-TT ^™ 40

ELiSA Pipetting and washing steps were performed on a Biomek 3000 liquid handling robot. PBS was used throughout the assay at pH 7.4 and was prepared from a 10X powder packet from Fisher Scientific. First, half-area high-binding 98-welS microliter plates (Costar 3890) were coated with 25 pg of Fent-BSA per well overnight at 37 X, aiiowing the liquid to evaporate. Foiiowing blocking with skim milk for 1 h at ii, vaccinated mouse serum was serially diluted 1 :1 in 2% BSA solution across the 1 columns starting at 1 :5000. After a 2 h incubation at rt, the p!ates were washed 5X and donkey anti-mouse IgG HRP secondary (Jackson

I munoResearch) at a 1 :10,000 dilution in 2% BSA was added and incubated for 1 h at rt, SX washing was performed and TMB substrate (Thermo Pierce) was added, followed by 2M h S(¾ 5 min after 1MB addition. Plates were allowed to incubate 20 min before their absorbances were read at 450 nm. In GraphPad PRISM, absorbance values were normalized to the highest absorbance value per sample, and a curve was fit using the log(inhibifor) vs. normalized response - variable slop© equation to determine the 50% titer and standard errors. on~ vaccinate mice did not contain any detectable anti-fenfanyi titers.

Animal Procedure for Blood/Srasrs Biodlstr&ytson Study

Mice (n-8 vaccinated and n-8 control) were injected with subcutaneously with 0.2 mg/kg feniany!, an established fully analgesic dose in naive mice, in a 10 mL/kg volume of

physioiogicai sterile saline, 15 min following injection, mice were fully anesthetized using nose cones constructed from 50 mL Falcon ^® conical centrifuge tubes (Corning, NY) containing gauze pads soaked in isoflurane, Mica were then opened along the midline Just below the sternum and the diaphragm peeled back to expose the heart. Cardiac puncture yielded roughly 1.5 mL of whole blood. Immediately following cardiac puncture, mice were rapidly decapitated using large surgical scissors and brain extracted with rongeurs. The brain was then weighed (typically between 4.0-8.0 g) and lightly washed in a 1.0 mL solution of ice-cold standard PBS to remove excess external blood; however, mice were not perfused to fully remove all blood that may have been contained within ventricfes and internally within the brain matter. Brains were then added to 1.0 mL fresh ice-cold PBS in 5,0 mL conical sample tubes and homogenized using a Tissue Tearor (Biospec; Bartlesville, OK), Samples of both brain and serum were then frozen until sample prep for LCMS analysis.

Sample Preparation and Extraction for LC-MS Analysis of Fentanyl in Blood and Br ^ ³

To an aliquot of sample (400 pL, blood or homogenised brain) was added fentanyl-d ₅ (20 pL, 50 ng/mL) in MeOH as an infernal standard, and then the mixture was vortex mixed and allowed to equilibrate. After 30 min for equiiibration, H ₂ 0 (400 pL) was added to the mixture. Basification was obtained by addition of 0.1 M aqueous K ₂ C0 ₃ solution (400 μί) followed by agitation using a vortex mixer. Extraction was conducted with mixture of n~hexane and EtOAc (7:3) (2,8 mL). After vortex mixing for 2 min and centrifugation at 3000 rpm for 5 min, the organic layer was evaporated using GENEVAC® An 8 pL aliquot was injected into LC-fU S system equipped with an Agilent Porosheli 120 S8-C8 column using 5 mfvl H OAc pH

4/acetonstrile mobile phase, A blank was injected before every sampie. Deuterated and non- deuterated masses were extracted in MassHunter and resulting peaks were integrated, Using the ratio of non-deuterated to deuterated integration values, fentanyf concentrations were determined via a six point standard curve (see bebw). Standards for the calibration curve were prepared in the same manner as tissue samples except with sampies of known non-deuterated fentanyl concentrations,

Vaccine Formulation ¾nd Administration

On a per mouse basis, 50 pg Fent-TT + 50 pg CpG OD 1828 (Eurofins) in 75 pL pH 7.4 PBS was combined with 75 pL (0.75 mg) Aih drogei (fnvivogen) and mixed for 30 min. The suspension (150 pL per mouse) was injected intraperitoneal^ to 8 mice at weeks 0, 2, 4, Mice were bled at weeks 2, 4, 8, 10 and 12.

Opioid Antinociceptive Poterscy Testing

At least 2 days following a bleed, mice were tested for cumulative fentanyl response in primarily supraspinal (hot plate) and spinal (tail immersion} behavioral tests as previously described, ^{l ]} The hot plate test was measured by placing the mouse in an acrylic cylinder (14 cm diameter * 22 cm) on a 54 ^e C surface and timing latency to perform one of the following nociceptive responses: licking of hindpaw _! shaking/withdrawal of hindpaw, or jumping, Typical baseline latency was between 8-15 s and a 35 s cutoff was imposed to prevent tissue damage; after response mice were removed from the plate, The tail immersion test was administered by lightly restraining mice in a small pouch constructed from absorbent laboratory underpads and dipping 1 cm of the tip of the tail into a heated water bath, with the time to withdrawal timed.

Typical baseline response was 1-2 s and a cutoff of 10 s was used to prevent tissue damage. Since tail immersion is a more reflexive behavior, testing order was always hot plate first followed by tail immersion. Immediately following completion of both antinociceptive assays, fentanyl (5.0 mlikg in normal saline) was immediately injected subcutaneously. Testing was repeated roughly 10 min following each injection, and this cycle of testing and injections was repeated with increasing cumulative fentanyl dosing until full antinociception (i.e. cutoff times surpassed) was observed in both assays, Upon completion of all testing, mice were administered a cocktail of 1.0 mg/kg naloxone and naltrexone in saline to prevent subsequent consequences of potential overdose. Male Swiss Webster mice {N=11) were injected intraperitoneal with 2.2 mg/kg fentanyl HCI In pH 7.4 PBS after which 2/11 died of overdose. A second 2.2 ng/kg fentany! HCI dose was administered 15 min later to the remaining 9 mice after which four more (8/11) died of overdose. The same experiment { =12) was performed subcutaneously with cumulative dosing of OA 0.8, 1.8, 2.4 and 4.4 mg/kg over 1 h inducing overdose deaths in 2/12 of the mice.

Statists©®

Statistical analysis was performed in GraphPad Prism 6 (La Jolla, CA). Ali values are reported as means ± SEM, Antinociceptive data was transformed from time to % maximum possible effect <%IV3PE), which is calculated as: %MPE ~ (test - baseline) / (cutoff - baseline) * 100. This data was then fit using a log{agonist) vs. normalized response non-linear regression. ED _S0 values and 95% confidence intervals of the ED _S o were calculated for each pain test and individual treatment group to determine potency ratios. An unpaired f test was used for verification of statistically significant differences (a < 0.001) between control and vaccinated groups for blood/brain fentanyl concentrations and fentanyl ED _S0 shifts.

Pefermmation of B ding !C _ss s for H$©us® antf-Ferstanyl lmm r¾ogto u ms |IGs|

The binding !C ₅₀ for mouse IGs and free fentanyl was determined by competitive binding assay via surface plasmon resonance using a Biacore 3000 instrument (GE Healthcare) equipped with a research-grade CM3 sensor chip. The iigand, fentanyl-BSA conjugate, was immobilized using NHS, EDC coupling reaction. The surface of all two flow cells (flow cells 1 and 2) were activated for 7 min with a 1 :1 mixture of 0,1 M NHS and 0.1 Svl EDC at a flow rate of 5 pL/min. The Iigand resuspended in 10 mivl sodium acetate (pH 4.0) was immobilized at a density of 2,000 RU on flow cell 2; whereas flow cell 2 was immobilized with BSA at the same density to serve as a reference surface. All the surfaces were blocked with a 7 min injection of 1.0 M ethanolamine-HCI (pH 8.5), The mouse IGs were diluted in running buffer {HBS~EP+ buffer) and titrated on both coated flow cells, so as to give a response of ~ 60 RU with 3 min of injection and 2.5 min dissociation at a flow rate of 30 pL/min. The mouse IGs prepared in HBS- EP+ buffer at determined concentration was incubated with a series concentration of compounds for 1 h at room temperature before conducting the competitive binding assay. The compounds and their concentration series are as follows: a) fentanyl, ranging from 10 μΜ to 169 pM with a three-fold dilution series; b) acetylfentanyl, butyryifentanyf, and p-tol ifentanyl, ranging from 50 mM to 850 pfvt with three-fold dilution; c) c/s-3-meth ifentan l, and a- methy!f ntan l {China White), ranging from 100 mivl to 95 pM, four-fold dilution series; d) methadone and oxycodone, ranging from 100 mM to 10 nM, ten-fold dilution series. Note, all fentany!s were racemic. To collect binding data, the analyta, the mouse IGs and compound mixture, was injected over the two flow ceils at a flow rate of 30 μί/πιΐη at 25 ^e C for 3 ruin and was dissociated in buffer for 2.5 min before regeneration, The chip surface was regenerated by injection of 10 mM G! -HCI (pH1.5) for 30 seconds before the next round of assay. The response at the end of dissociation phase for each cycle of binding analysis was used to calculate the fC _S0 value for each compound by GraphPad Prism 6 software. The binding curves are illustrated in Figure 5a.

Determf ation of Binding Kinetics for Purified Ksyse aotl-Fenfarsyl Immunoglobu ins; |IGs|

The binding kinetics between mouse IGs (purified directly from week 8 bleed using magnetic fentanyl-coupled Dynabeads) and free fentanyi were determined by surface plasmon resonance using a BiOptix 404pi instrument {BiOptix Diagnostics, Inc., Boulder, Co,} equipped with a CMD200m sensor chip. The Iigand, mouse anti-fentanyl IgGs (-150 kDa), were immobilized using NHS, EDC coupling reaction. The surface of all two flow cells (flow cells 2 and 3, assay set at 2 * 2 injection mode) were activated for 7 min with a 1 :1 mixture of 0.1 M NHS and 0.1 M EDC at a flow rate of 5 pL min. The Iigand resuspended in 10 mM sodium acetate (pH 4.5) was immobilized at a density of 12,000 RU on flow call 3; whereas flow cell 2 was immobilized with a non~reiated antibody at the same density to serve as a reference surface. All the surfaces were blocked with a 7 min injection of 1.0 M afhanolamina-HCI (pH 8.5). To collect kinetic data, the analyte, fentanyi (338.48 Da) prepared in HBS-EP+ buffer (10 mM HEPES, 150 mM aCl, 0.05% P20 (pH 7.4)), was injected over the two flow cells at concentration range from 1 ,000 - 1.95 nM (two-fold dilution series) at a flow rate of 50 pL min at a temperature of 25 X. The complex was allowed to associate and dissociate for 300 and 900 s, respectively. Duplicate injections (in random order) of each analyte sample and blank buffer injections were fiowed over the two surfaces. Data were collected, double referenced, and were fit to a 1 :1 interaction model using the global data analysis by Scrubber 2. The kinetic data are shown in Figure 5b.

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While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements will be apparent to those skilled in the art without departing from the spirit and scope of the claims.

All patents and publications referred to herein are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended ciaims.