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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• • A—HUMAN NECESSITIES
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  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
  • A61K47/6817—Toxins
  • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• immunoconjugates having improved solubility and irnmunogenicity and methods of use thereof;
• Fentanyi is an effective synthetic opioid that is used legally as a schedule II prescription pain reliever.
• 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.
• 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 (!
• the protein carrier can be bovine serum albumin (BSA) or tetanus toxoid ( ⁇ ), or the amino-functionalized bead can be a Dynabead Svl-270 Amine.
• BSA bovine serum albumin
• ⁇ tetanus toxoid
• the invention further provides a vaccine produced in a mammal by administration of an effective dose of the hapten conjugate described above.
• the mamma! used for vaccine production can be a mouse
• the vaccine can comprise IgG type antibodies.
• 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.
• Figyre Structures of fentanyi immunoconjugate and analogues recognized by polyclonal antibodies with ⁇ 100 nM affinity.
• FIG. 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.
• FIG. 4 Biodistribution of fentanyl in blood and brain samples. Vaccinated and control mice (N TM 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
• this 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.
• 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
• 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.
• the resulting smmunoconjugate 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.
• 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'.
• the SPR iC3 ⁇ 4 0 s mirrored the results in behavioral assays, and in both cases followed a trend of Fent>a-lv1e> s-3-fv1a.
• 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.
• 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).
• mice 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 ⁇ whole blood, and samples then centrifuged at 7500 rpm for 8 min to separate serum.
• tail-tip amputation ⁇ 1 cm
• 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.
• 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
• 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
• 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.
• Tissue Tearor Biospec; Bartlesville, OK
• sample 400 pL, blood or homogenised brain
• fentanyl-d 5 20 pL, 50 ng/mL
• MeOH MeOH
• H 2 0 400 pL
• Basification was obtained by addition of 0.1 M aqueous K 2 C0 3 solution (400 ⁇ ) followed by agitation using a vortex mixer.
• Extraction was conducted with mixture of n ⁇ hexane and EtOAc (7:3) (2,8 mL).
• 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 !
• 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.
• 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 ⁇ 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.
• 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).
• 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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