WO2024238940A1 - Vaccine compositions and methods of use thereof - Google Patents

Abstract

The present disclosure provides methamphetamine conjugate, and/or opioid conjugate(s) vaccine compositions and methods thereof. The present disclosure also provides multivalent vaccine compositions.

Description

VACCINE COMPOSITIONS AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to United States Provisional Application Number 63/467,228 filed on 17 May 2023, which is incorporated by reference herein.

GOVERNMENT FUNDING

This invention was made with government support under DA047711, 75N93020C00039, and DA048386 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Illicit drug mixtures containing opioids and stimulants have been responsible for the majority of fatal drug overdoses among individuals with opioid use disorder (OUD) and substance use disorders (SUD). Traditional FDA-approved pharmacotherapies have limitations in combating current opioid and/or substance use crisis. New compositions and methods are needed to provide additional tools for preventing or treating drug overdoses and disorders.

SUMMARY OF THE INVENTION

Certain embodiments of the invention provide a compound as described herein (e.g., (Compound 1).

Certain embodiments of the invention provide a conjugate as described herein.

Certain embodiments of the invention provide a conjugate comprising a methamphetamine and a carrier protein, wherein the methamphetamine and the carrier protein are linked via a linker.

Certain embodiments of the invention provide a methamphetamine conjugate having structure of Formula I:

(Formula I) wherein L is a linker, and C is a carrier protein, p is a hapten to carrier protein ratio number that is >=1 (e.g., 1-50, 1-30, or 1-10).

Certain embodiments of the invention provide a vaccine composition as described herein.

Certain embodiments of the invention provide a bivalent or multivalent vaccine composition comprising an opioid hapten-carrier protein conjugate, and a non-opioid haptencarrier protein conjugate (e.g., methamphetamine hapten-carrier protein conjugate).

Certain embodiments of the invention provide a vaccine composition comprising two or more hapten-carrier protein conjugates, wherein the two or more hapten-carrier protein conjugates comprise two or more different carrier proteins (e.g., KLH and CRM).

Certain embodiments of the invention provide a bivalent vaccine composition as described herein.

Certain embodiments of the invention provide a trivalent vaccine composition as described herein.

Certain embodiments of the invention provide a quadrivalent vaccine composition as described herein.

Certain embodiments of the invention provide a pentavalent vaccine composition as described herein.

Certain embodiments of the invention provide a vaccine composition comprising two or more opioid hapten-carrier protein conjugates, wherein the two or more opioid hapten-carrier protein conjugates comprise two or more different carrier proteins (e.g., KLH and CRM).

Certain embodiments of the invention provide a method of immunizing a subject (e.g., for preventing drug overdose, such as opioid overdose) comprising administering a vaccine composition as described herein to the subject.

Certain embodiments of the invention provide a method of treating opioid use disorder (OUD) and/or substance use disorders (SUD) of a subject comprising administering a vaccine composition as described herein to the subject.

Certain embodiments of the invention provide a conjugate or composition described herein for the prophylactic or therapeutic treatment of drug overdose, opioid use disorder (OUD) and/or substance use disorders (SUD).

Certain embodiments of the invention provide a conjugate or composition described herein for use in medical therapy.

Certain embodiments of the invention provide a method for preventing drug overdose or treating opioid use disorder (OUD) and/or substance use disorders (SUD) in a subject, comprising administering an effective amount of a conjugate or composition described herein to the subject. Certain embodiments of the invention provide the use of a conjugate or composition described herein to prepare a medicament for the treatment of opioid use disorder (OUD) and/or substance use disorders (SUD) or for the prevention of drug overdose in a subject.

Certain embodiments of the invention provide a kit comprising a conjugate or composition described herein, packaging material, and instructions for administering the conjugate or composition to a subject to treat opioid use disorder (OUD), substance use disorders (SUD), and/or to prevent drug overdose.

The invention also provides processes and intermediates disclosed herein (e.g., compounds 3, 5, 6 in Example 1) that are useful for preparing a compound, or conjugate described herein, as well as compositions described herein.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1D. The bivalent vaccine with different carrier proteins elicits haptenspecific IgG titers and attenuates drug distribution to the brain without interference. Sprague Dawley rats (n=5-6 per group) were vaccinated i.m. with CRM and sKLH as control, Fi- CRM, OXY-sKLH, or combination as bivalent by injecting two individual vaccines at different sites (co-administration). A week after the last vaccination, blood was collected to measure (Fig.lA) fentanyl-specific IgG titers and (Fig. IB) oxycodone-specific IgG titers. Immunized rats were challenged s.c. with a mixture of fentanyl and oxycodone, and analyzed for the drug distribution of fentanyl (Fig.lC) and oxycodone (Fig. ID). Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparisons post hoc test. Statistical symbols**** or #### p<0.0001, ^AAA p<0.001, ** or ## or ^AA p<0.01, and * or # or ^A p<0.05. Placement of * indicates significance compared to CRM, # indicates significance compared to F-CRM, and ^A indicates significance compared to OXY-sKLH.

Figures 2A-2I. The bivalent vaccine protects the subject animal from the combined pharmacological effects of fentanyl and oxycodone. Immunized rats were challenged s.c. with a mixture of fentanyl (0.05 mg/kg) and oxycodone (2.25 mg/kg). Oximetry and hotplate tests were used to measure opioid-induced antinociception, respiratory depression, and bradycardia at baseline and every 15 min up to 60 min post-drug administration. Statistical analysis was performed via two-way ANOVA paired with Dunnett’s (Figs.2A-C, E, F, H, I) or Bonferroni’s (Figs.2D, G) multiple comparisons post hoc test. Statistical symbols: ***/?<0.001, ** or ## or ^AA /?<0.01, * or # or ^A/?<0.05. Placement of * indicates significance between Fi-CRM and CRM, # indicates significance between F13-CRM and CRM, # (red) indicates significance between heterologous and CRM, and * (red) indicates significance between co-administration and CRM. Figures 3A-3H. Trivalent and quadrivalent vaccination induce each hapten-specific IgG titers and simultaneously block cognate drug distribution to the brain without interference in mice. Balb/c mice (n=5-6 per group) were vaccinated s.c. with Fi-CRM, F13- CRM, OXY-sKLH, or M-sKLH alone or as trivalent or quadrivalent formulations. A week after the last vaccination, blood was collected to measure (Fig.3 A) fentanyl-specific IgG titers, (Fig.3B) carfentanil-specific IgG titers, (Fig.3C) oxycodone-specific IgG titers, and (Fig.3D) heroinspecific IgG titers. 30 min post-drug challenge, blood and brain were collected to analyze drug distribution by calculating the braimserum ratio of (Fig.3E) fentanyl, (Fig.3F) carfentanil, (Fig.3G) oxycodone, and (Fig.3H) 6-MAM. Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparisons post hoc test, statistical symbols: **** p<0.0001, *** p<0.001, ** or ## p<0.01, and * or # p<0.05. Placement of * above columns indicate significance compared to control, # above columns indicate significance compared to the cognate monovalent vaccine.

Figures 4A-4H. Co-formulating a quadrivalent vaccine is more efficacious than coadministering a quadrivalent vaccine in inducing each hapten-specific IgG titers and simultaneously blocking cognate drug distribution to the brain without interference in rats. Sprague Dawley rats (n=6 per group) were vaccinated i.m. with Fi-CRM, F13-CRM, OXY-sKLH, M-sKLH, or combinations as quadrivalent by injecting four individual vaccines at different sites (co-administration) or injecting at a single site after co-formulating four conjugates in a single syringe (co-formulation). A week after the last vaccination, blood was collected to measure (Fig.4 A) fentanyl-specific IgG titers, (Fig.4B) carfentanil-specific IgG titers, (Fig.4C) oxycodonespecific IgG titers, and (Fig.4D) heroin-specific IgG titers. 30 min post-drug challenge, the brain and blood were collected to analyze the individual drug distribution by calculating the brain: serum ratio of (Fig.4E) fentanyl, (Fig.4F) carfentanil, (Fig.4G) oxycodone, and (Fig.4H) 6-MAM. Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparisons post hoc test. * above columns indicate significance compared to control, # above columns indicate significance compared to cognate monovalent vaccine, statistical symbols: **** p<0.0001, *** or ### p<0.001, **p<0.01, and * p<0.05.

Figures 5A-5E. Admixture of opioid and stimulant vaccines as pentavalent immunization by opioid and stimulant vaccines induces IgG titers specific to both opioid and stimulant haptens without interference in rats. Sprague Dawley rats (n=6 per group) were vaccinated i.m. with a combination of Fi-CRM, F13-CRM, OXY-sKLH, and M-sKLH as quadrivalent, or with an additional METH-sKLH as pentavalent. A week after the last vaccination, blood was collected to measure (Fig.5 A) fentanyl-specific IgG titers, (Fig.5B) carfentanil-specific IgG titers, (Fig.5C) oxycodone-specific IgG titers, (Fig.5D) heroin-specific IgG titers, and (Fig.5E) methamphetamine-specific IgG titers. Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparisons post hoc test. * above columns indicate significance as indicated by brackets, statistical symbols: * p<0.05.

Figures 6A-6C. Admixing opioid and stimulant vaccines as pentavalent simultaneously blocks both opioid and stimulant from entering the brain without interference. During the drug challenge, all groups of rats s.c. received a bolus of fentanyl (0.25 mg/kg), heroin (2.5 mg/kg), and methamphetamine (0.15 mg/kg). At 15- and 30-min post-drug administration, oximetry and hotplate tests were used to assess the opioid-induced (Fig.6A) antinociception, (Fig.6B) respiratory depression, and (Fig.6C) bradycardia. Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparisons post hoc test. * above columns indicate significance compared to control or as indicated by brackets, statistical symbols: **** p<o 0001, *** p<0.001, **p<0.01, and * p<0.05.

Figures 7A-7D. Representative opioid-carrier protein conjugates and METH-carrier protein conjugate (Fig.7A); exemplary oxy-KLH conjugate (Fig.7B); schematic graph of multivalent vaccine (Fig.7C); schematic graph of protection conferred by vaccine against certain target drug (Fig.7D).

Figures 8A-8C. Pentavalent vaccination elicits independent antibody responses in rats. During the drug challenge, all groups of rats subcutaneously received a bolus of fentanyl (0.25 mg/kg), heroin (2.5 mg/kg), and methamphetamine (0.15 mg/kg). At 15- and 30-min postdrug administration, oximetry and hotplate test were used to assess the opioid-induced (Fig.8A) antinociception, (Fig.8B) respiratory depression, and (Fig.8C) bradycardia. Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparisons post hoc test. * above columns indicate significance compared to control or as indicated by brackets, statistical symbols: **** p<o 0001, *** p<0.001, **p<0.01, and * p<0.05.

Figures 9A-9F. Pentavalent vaccine formulations are effective in attenuating the target drug distribution to the brain. 30 minutes after the multi -drug challenge in the same rats from Figure 2, blood and brain were collected for the LC-MS/MS analysis. The efficacy of pentavalent vaccination on blocking the drug distribution was analyzed by measuring the levels of (Fig.9A) serum fentanyl, (Fig.9B) serum 6-MAM, (Fig.9C) serum methamphetamine, (Fig.9D) brain fentanyl (Fig.9E) brain 6-MAM, and (Fig.9F) brain methamphetamine. Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparisons post hoc test. * above columns indicate significance compared to control or as indicated by brackets, Statistical symbols: **** p<o.QOOl, *** /?<0.001, **/?<0.01, and */?<0.05. -+ Figure 10. Quadrivalent vaccination prevent the antinociceptive effects of a mixture of fentanyl, carfentanil, oxycodone, and heroin in mice. Immunized mice were challenged s.c. with a mixture of fentanyl (0.25 mg/kg), carfentanil (0.005 mg/kg), oxycodone (1.125 mg/kg), and heroin (2.5 mg/kg). At 15 and 30 min post-drug administration, pulse oximetry and hotplate tests were used to assess the opioid-induced antinociception. Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparisons post hoc test. *above columns indicate significance compared to control or as indicated by brackets, statistical symbols: *** p<0.001.

Figures 11A-11H. Trivalent and quadrivalent vaccination simultaneously reduce cognate drug distribution to the brain in mice. Balb/c mice (n=5-6 per group) were vaccinated s.c. with F-CRM, Carf-CRM, Oxy-sKLH, Her-sKLH, trivalent or quadrivalent formulations. At 30 min post-drug challenge, blood and brain were collected to analyze drug distribution by measuring the levels of serum: (Fig.11 A) fentanyl, (Fig.1 IB) carfentanil, (Fig. l lC) oxycodone, (Fig.1 ID) 6-MAM, and brain: (Fig. HE) fentanyl, (Fig.1 IF), carfentanil, (Fig.l lG) oxycodone, and (Fig.l lH) 6-MAM. Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparisons post hoc test. *above columns indicate significance compared to control, statistical symbols: **** p<0.0001, *** p<0.001, ** p<0.01, and * p<0.05.

Figures 12A-12B. While trending, the current formulations of quadrivalent vaccines did not significantly protect against the combined pharmacological effects of fentanyl, carfentanil, oxycodone, and heroin in rats. During the drug challenge, all groups of rats received a s.c. bolus of fentanyl (0.25 mg/kg), carfentanil (0.005 mg/kg), oxycodone (1.125 mg/kg), and heroin (2.5 mg/kg) to assess the opioid-induced (Fig. l2A) antinociception and (Fig. l2B) respiratory depression. Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparisons post hoc test.

Figures 13A-13B. Meth-sKLH is more effective than Meth-CRM in inducing methspecific IgG titers and inhibiting the distribution of methamphetamine to the brain in rats. Sprague Dawley rats (n=6 per group) were vaccinated i.m. with control, F-CRM, Meth-CRM, or Meth-sKLH. (Fig.13 A) A week after the last vaccination, blood was collected to measure methamphetamine-specific IgG titers. (Fig.l3B) At 30 min post-methamphetamine challenge, the brain and blood were collected to analyze the distribution of methamphetamine by calculating the brain: serum ratio of methamphetamine. Statistical analysis was conducted via one-way ANOVA with Tukey’s multiple comparisons post hoc test. *above columns indicate significance compared to control, # above columns indicate significance compared to F-CRM, ^A above columns indicate significance compared to Meth-CRM, statistical symbols: **** or #### p<0.0001, *** or ### p<0.001, ** or ## or ^AA p<0.01. Figure 14. Meth-sKLH does not provide adequate protection against methamphetamine-induced locomotor activity in mice. Female Balb/c mice (n=8-12/group) were vaccinated with either sKLH or Meth-sKLH on Days 0, 14, and 28. Blood was collected on day 35 and Meth-specific serum IgG titers were measured (inset). Meth-specific serum IgG titers were compared using an unpaired t test with Welch’s correction and data were expressed as Mean ± SD. On day 42, mice were baselined in locomotor activity chamber chambers (Med Associates, Fairfax, VT) for 60 min and then given a 2 mg/kg IP dose of methamphetamine. Mice were placed back in the locomotor activity chambers to measure distance traveled for an extra 60 min. Area- under-the-curve (AUC) was analyzed in Prism 10 using XY-analyses and “area under curve” throughout their total time in the chambers. The AUC was compared using unpaired t tests using Welch’s correction. Data were expressed as mean ± SEM. *p<0.05 different from control.

DETAILED DESCRIPTION

Conjugate

Certain embodiments of the invention provide a conjugate comprising a methamphetamine and a carrier protein, wherein the methamphetamine and the carrier protein are linked via a linker. The methamphetamine moiety can be bonded to the remainder of the conjugate as described herein by the removal of an atom such as a hydrogen atom from the methamphetamine. Removal of the atom (e.g., hydrogen) provides the open valency to be connected to the remainder of the conjugate.

Certain embodiments of the invention provide a methamphetamine conjugate having structure of Formula I:

(Formula I) wherein L is a linker, and C is a carrier protein, p is a hapten to carrier protein ratio number that is >=1.

In certain embodiments, p (i.e., hapten number per carrier protein, or haptenization ratio) is about 1-600, 1-500, 1-400, 1-300, 1-200, 1-100, 1-50, 1-40, 1-30, 1-20, or 1-10. In certain embodiments, p is about 10-50, 10-40, 10-30, or 10-20. In certain embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or higher number. In certain embodiments, p is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In certain embodiments, the linker L has a molecular weight of from about 20 daltons to about 20,000 daltons. In certain embodiments, the linker has a molecular weight of from about 20 daltons to about 10,000 daltons. In certain embodiments, the linker has a molecular weight of from about 20 daltons to about 5,000 daltons. In certain embodiments, the linker has a molecular weight of from about 20 daltons to about 3,000 daltons. In certain embodiments, the linker has a molecular weight of from about 20 daltons to about 2,000 daltons. In certain embodiments, the linker has a molecular weight of from about 20 daltons to about 1,000 daltons.

In certain embodiments, the linker L is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain having from 2 to 30 (e.g., 2-16 or 2-12) carbon atoms, wherein one or more of the carbon atoms is optionally replaced by a divalent heterocycle (e.g., pyrrolidinyl), (-O-), (-S-), or -N(Rb)-, wherein Rb is H or (Ci-Ce)alkyl, wherein the hydrocarbon chain is optionally substituted on carbon with one or more substituents selected from the group consisting of halo, hydroxy, and oxo(=O), wherein the divalent heterocycle is optionally substituted with one or more substituents selected from the group consisting of halo, hydroxy, and oxo(=O).

In certain embodiments, the linker L is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain having from 2 to 30 carbon atoms, wherein one or more of the carbon atoms is optionally replaced by (-O-), (-S-), -N(Rb)-, wherein Rb is H or (Ci-Ce)alkyl, wherein the hydrocarbon chain is optionally substituted on carbon with one or more substituents selected from the group consisting of halo, hydroxy, and oxo(=O).

In certain embodiments, the linker L is capable of forming an amide bond with the carrier protein.

In certain embodiments, the linker L is capable of forming an amide bond (-C=(O)-NH-) with an amine group (e.g., on the side chain of a lysine) of the carrier protein.

In certain embodiments, the linker L is capable of forming an amide bond (-NH-C=(O)-) with a carboxy group (e.g., on the side chain glutamic acid or aspartic acid) of the carrier protein.

Conjugation methods suitable for generating a conjugate described herein are known in the art and described herein. In certain embodiments, conjugation methods may utilize carbodiimide chemistry (e.g., see Example 1) to yield an amide bond (e.g., see Formula la) to achieve conjugation. In certain embodiments, conjugation methods may utilize maleimide-thiol chemistry to yield a thioester bond to achieve conjugation.

In certain embodiments, the linker comprises a segment

In certain embodiments, the linker comprises a segment

In certain embodiments, the linker L comprises, or consists of, a segment of

In certain embodiments, the conjugate is prepared by conjugating a carrier protein to a methamphetamine hapten compound, or salt thereof, having structure of

(Compound 1).

For example, the primary amine of the methamphetamine hapten Compound 1 may form an amide bond (-NH-C=(O)-) with a carboxy group (e.g., on the side chain glutamic acid or aspartic acid) of the carrier protein to provide a conjugate having structure of Formula la.

In certain embodiments, the conjugate has structure of Formula la:

(Formula la).

Certain embodiments of the invention provide a methamphetamine hapten compound, or salt thereof, having structure of

(Compound 1).

As used herein, “hapten” refers to a small molecule compound (MW no greater than lOOOg/mol) that, when conjugated to a carrier protein, could elicit a specific immune response or specific antibodies against the hapten. In certain embodiments, the hapten is a drug (e.g., an opioid compound, or a non-opioid compound such as methamphetamine) or derivative thereof. A drug derivative may have structural modification on the drug, for example, a drug derivative may comprise the drug moiety, and a functional group that may be a linker, or a portion of a linker.

As used herein, “carrier protein” refers to an immunogenic protein that is exogenous to a subject (e.g., a mammal) so that administration of the carrier protein to the subject may elicit an immune response against the carrier protein and/or a hapten conjugated to the carrier protein. Carrier proteins suitable for hapten conjugation are known in the art and described herein.

In certain embodiments, the carrier protein is Keyhole limpet hemocyanin (KLH), for example, native KLH (nKLH) or subunit KLH (sKLH).

In certain embodiments, the carrier protein is native KLH (nKLH). Native KLH may be in the form of didecamer (MW about 8MDa), and/or multidecamer (MW about 12-32 MDa).

In certain embodiments, the carrier protein is subunit KLH (sKLH). Subunit KLH (sKLH) may comprise KLH1 and/or KHL2 subuni t(s). Subunit KLH (sKLH) may have MW of about 350kDa to 400kDa.

In certain embodiments, the carrier protein is cross-reacting material (CRM, which is a non-toxic version of diphtheria toxin), tetanus toxoid (TT), meningococcal outer membrane protein complex (OMPC), diphtheria toxoid (DT), or H. influenzae protein D (HiD).

Vaccine composition (monovalent, bivalent, or multivalent vaccine)

Certain embodiments of the invention provide a vaccine composition comprising a methamphetamine conjugate described herein and a pharmaceutically acceptable carrier (e.g., aqueous medium).

In certain embodiments, the vaccine composition further comprises one or more haptencarrier protein conjugate. In certain embodiments, the vaccine composition further comprises one or more opioid hapten-carrier protein conjugate.

As used herein, “opioid” refers to a compound that is capable of activating mu-opioid receptor. An opioid compound may be a naturally occurring compound, or analog thereof. An opioid compound may be a synthetic compound. Exemplary opioid compounds, include but not limited to, morphine, heroin, oxycodone, fentanyl, or carfentanil, etc.

As used herein, “non-opioid” refers to a compound that is not capable of modulating mu- opioid receptor but may exert influence in the subject’s central nervous system. In certain embodiments, the non-opioid compound is a stimulant compound such as methamphetamine.

In certain embodiments, the one or more hapten is selected from the group consisting of methamphetamine, fentanyl, carfentanil, oxycodone, and morphine haptens.

In certain embodiments, the one or more opioid hapten is selected from the group consisting of fentanyl, carfentanil, oxycodone, and morphine haptens. In certain embodiments, the one or more opioid hapten-carrier protein conjugate is selected from the group consisting of fentanyl-carrier protein conjugate, carfentanil-carrier protein conjugate, oxycodone-carrier protein conjugate, and morphine-carrier protein conjugate.

In certain embodiments, the one or more opioid hapten-carrier protein conjugate is selected from the group consisting of fentanyl-CRM, carfentanil-CRM, oxycodone-sKLH, and morphine-sKLH.

Opioid hapten-carrier protein conjugates (e.g., fentanyl-CRM, carfentanil-CRM, oxycodone-KLH, and morphine-KLH) are known in the art (e.g., see Patent Application US2014/0093525, WO2021/092446, and WO2021/183913) and are also described herein (e.g., see Examples 1-2).

In certain embodiments, the vaccine composition is a bivalent vaccine having the methamphetamine conjugate described herein, and one opioid hapten-carrier protein conjugate.

In certain embodiments, the vaccine composition is a trivalent vaccine having the methamphetamine conjugate described herein, and two opioid hapten-carrier protein conjugates.

In certain embodiments, the vaccine composition is a quadrivalent vaccine having the methamphetamine conjugate described herein, and three opioid hapten-carrier protein conjugates.

In certain embodiments, the vaccine composition is a pentavalent vaccine having the methamphetamine conjugate described herein, and four opioid hapten-carrier protein conjugates. In certain embodiments, the four opioid hapten-carrier protein conjugates are fentanyl-CRM, carfentanil-CRM, oxycodone-sKLH, and morphine-sKLH.

Certain embodiments of the invention provide a bivalent or multivalent vaccine composition comprising an opioid hapten-carrier protein conjugate, and a non-opioid haptencarrier protein conjugate.

Certain embodiments of the invention provide a bivalent or multivalent vaccine composition as described herein. In certain embodiments, the bivalent or multivalent vaccine composition comprises a methamphetamine conjugate described herein. In certain embodiments, the bivalent or multivalent vaccine composition does not comprise a methamphetamine conjugate described herein.

In certain embodiments, the vaccine composition is a trivalent vaccine composition (e.g., three opioid hapten-carrier protein conjugates). In certain embodiments, the vaccine composition is a quadrivalent vaccine composition (e.g., four opioid hapten-carrier protein conjugates). In certain embodiments, the vaccine composition is a pentavalent vaccine composition.

In certain embodiments, the bivalent or multivalent vaccine composition comprises two or more hapten-carrier protein conjugates, wherein the two or more hapten-carrier protein conjugates comprise two or more different carrier proteins. In certain embodiments, the bivalent or multivalent vaccine composition comprises two or more opioid hapten-carrier protein conjugates, wherein the two or more opioid hapten-carrier protein conjugates comprise two or more different carrier proteins.

In certain embodiments, the two or more different carrier proteins comprise KLH and CRM. For example, in certain embodiments, the two or more different carrier proteins comprise sKLH and CRM.

In certain embodiments, the bivalent or multivalent vaccine composition is a vaccine composition as described herein (e.g., see Examples 1-2).

In certain embodiments, the bivalent or multivalent vaccine composition comprises fentanyl-CRM and oxycodone-sKLH.

In certain embodiments, the two or more opioid haptens are selected from the group consisting of fentanyl, carfentanil, oxycodone, and morphine haptens.

In certain embodiments, the two or more opioid hapten-carrier protein conjugates are selected from the group consisting of fentanyl-cross-reacting material (CRM), carfentanil-CRM, oxycodone-sKLH, and morphine-sKLH.

In certain embodiments, the vaccine composition comprises four opioid hapten-carrier protein conjugates.

In certain embodiments, the vaccine composition further comprises a methamphetamine- carrier protein conjugate.

In certain embodiments, the vaccine composition described herein further comprises an adjuvant.

As used herein, the term “adjuvant” refers to substance or compound that could enhance immune response against an immunogen or antigen (e.g., including hapten-carrier protein conjugate). Exemplary adjuvants include, but not limited to, Complete Freund’s Adjuvant (CFA), Incomplete Freund's Adjuvant (IF A), squalene, AS04, aluminum salt (e.g., aluminum hydroxide, aluminum phosphate, alum (e.g., potassium aluminum sulfate), or mixed aluminum salts), and toll like receptor agonist (e.g., monophosphoryl lipid A (MPL-A), a lipopeptide, and a synthetic nucleic acid sequence such as CpG and poly(I:C)).

Methods

Certain embodiments of the invention provide a method of treating opioid use disorder (OUD) and/or substance use disorder (SUD) or immunizing a subject (e.g., for preventing drug overdose), comprising administering a vaccine composition described herein to the subject. For example, the vaccine composition is administered as a single bolus injection at a single site of injection, such as a single bolus intramuscular injection.

In certain embodiments, the method further comprises administering one or more booster single bolus injection(s).

In certain embodiments, the OUD is Heroin use disorder (HUD).

In certain embodiments, the method comprises administering a vaccine composition described herein to the subject, wherein morphine specific antibody, heroin specific antibody, and/or 6-AM specific antibody is induced in the subject.

In certain embodiments, the subject is a mammal (e.g., human, mouse, rat, hamster, monkey, mini-pig, rabbit, sheep, or horse).

In certain embodiments, the subject is immunized with 1 dose of the vaccine composition. In certain embodiments, the subject is immunized with one or more additional dose (e.g., 1, 2, 3, or more booster dose) of the vaccine composition. In certain embodiments, the subject is immunized with 1, 2, 3, 4 or more doses of the vaccine composition. A booster dose may be given 2, 3, 4, 5, 6, or more weeks after the original or a previous booster dose was administered. In certain embodiments, a sequence of booster doses (e.g., 2 or 3 booster doses) are administered wherein individual dose is administered every 2, 3, 4, 5 or 6 weeks.

In certain embodiments, the method or vaccine composition described herein does not interfere with mu-receptor antagonist (e.g., naloxone) based pharmacotherapy.

In certain embodiments, the method or vaccine composition described herein protects or is capable of protecting the subject against opioid-induced bradycardia.

In certain embodiments, the method or vaccine composition described herein protects or is capable of protecting the subject against opioid-induced respiratory depression.

In certain embodiments, the method or vaccine composition described herein reduces or is capable of reducing opioid-induced antinociception (e.g., as measured by antinociception latency to respond) in the subject.

In certain embodiments, the method or vaccine composition described herein induces or is capable of inducing in the subject opioid-specific antibody.

In certain embodiments, the method or vaccine composition described herein sequesters or is capable of sequestering opioid in the blood (e.g., as measured by serum opioid concentration after opioid exposure or challenge).

In certain embodiments, the method or vaccine composition described herein reduces or is capable of reducing opioid in the brain (e.g., as measured by brain opioid concentration after heroin exposure or challenge). In certain embodiments, the method or vaccine composition described herein reduces or is capable of reducing heroin or metabolite thereof (e.g., 6-MAM) in the brain (e.g., as measured by brain concentration, or as measured by brain to serum concentration ratio, after heroin exposure or challenge), for example, it is surprisingly found that the co-formulated multivalent vaccine was more effective at reducing 6-MAM distribution to the brain as compared to the co-administered multivalent vaccine (see Example 1).

In certain embodiments, the method or vaccine composition described herein induces or is capable of inducing in the subject methamphetamine-specific antibody.

In certain embodiments, the method or vaccine composition described herein reduces or is capable of reducing methamphetamine in the brain (e.g., as measured by brain methamphetamine concentration, or as measured by methamphetamine concentration brain to serum ratio, after methamphetamine exposure or challenge).

Administration

In certain embodiments, an effective amount of a conjugate, or composition described herein is administered to the subject. “Effective amount” or “therapeutically effective amount” or “immunologically effective amount” are used interchangeably herein, and refer to an amount of a conjugate, or composition, as described herein effective to achieve a particular biological result.

In certain embodiments, the vaccine composition is a liquid composition.

In certain embodiments, the composition is a solid composition (e.g., lyophilized) that may be reconstituted with suitable solvent (e.g., saline or Dextrose 5% in water) prior to administration.

In certain embodiments, a conjugate, or composition described herein is administered via intramuscular, intradermal, or subcutaneous delivery.

As used herein, “immunization” or “vaccination” are used interchangeably herein and are intended for prophylactic or therapeutic immunization or vaccination.

As used herein, “pharmaceutically acceptable” refers to those properties and/or substances which are acceptable to the subject or patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability. “Pharmaceutically acceptable carrier” refers to a medium (e.g., aqueous medium) that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered. In certain embodiments, a composition described herein comprises a pharmaceutically acceptable carrier (e.g., aqueous medium).

The conjugate, or composition of the invention may be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to the chosen route of administration, z.e., orally, intranasally, intradermally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

Thus, the present conjugate, or composition may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the conjugate, or composition may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of conjugate. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the conjugate in such therapeutically useful compositions is such that an effective dosage level will be obtained.

Lyophilized formulations may also contain carrier such as bulking agent (e.g., mannitol or glycine) and cryoprotectant/lyoprotectant (e.g., trehalose or sucrose). Lyophilized formulation can be reconstituted into a liquid dosage form using saline, 5% dextrose solution or sterile water before administration.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the conjugate, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the conjugate or vaccine composition may be incorporated into sustained-release preparations and devices.

The conjugate, or composition may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the conjugate can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the conjugate which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the conjugate in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the conjugate plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the present conjugate, or composition may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present conjugate can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Examples of useful dermatological compositions which can be used to deliver the conjugate, or composition to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

To immunize a subject, a conjugate, or composition described herein is administered parenterally, usually by intramuscular or subcutaneous injection in an appropriate vehicle. Other modes of administration, however, such as oral delivery or intranasal delivery, are also acceptable. Vaccine formulations will contain an effective amount of the active ingredient (conjugate) in a vehicle.

Formulations will contain an effective amount of the active ingredient in a vehicle. The active ingredient may typically range from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate. The amount for any particular application can vary depending on such factors as the severity of the condition. The quantity to be administered depends upon factors such as the age, weight and physical condition of the animal considered for vaccination and kind of concurrent treatment, if any. The quantity also depends upon the capacity of the animal's immune system to synthesize antibodies, and the degree of protection desired. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the composition, and animal models may be used to determine effective dosages for treatment of particular disorders. Various considerations are described, e.g., in Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Reminpton's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference. Additionally, effective dosages can be established by one of ordinary skill in the art through routine trials establishing dose response curves. The subject is immunized by administration of the composition thereof in one or more doses. Multiple doses may be administered as is required to maintain a state of immunity to the target. For example, the initial dose may be followed up with a booster dosage after a period of about four weeks to enhance the immunogenic response. Further booster dosages may also be administered. The composition may be administered multiple (e.g., 2, 3, 4 or 5) times at an interval of, e.g., about 14, or 21 days apart.

Intranasal formulations may include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, or may be presented dry in tablet form or a product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservative.

Thus, the present compositions may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the present compositions may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such preparations should contain at least 0.1% of the present composition. The percentage of the compositions may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of present composition in such therapeutically useful preparations is such that an effective dosage level will be obtained.

Useful dosages of the compositions of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. The amount of the compositions described herein required for use in treatment will vary with the route of administration and the age and condition of the subject and will be ultimately at the discretion of the attendant veterinarian or clinician.

Certain Definitions The term "amino acid," comprises the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Hyl, Hyp, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai) in D or L form, as well as unnatural amino acids (e.g. phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, a-methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also comprises natural and unnatural amino acids bearing a conventional amino protecting group (e.g. acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (Ci-Ce) alkyl, phenyl or benzyl ester or amide; or as an a-methylbenzyl amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T.W. Greene, Protecting Groups In Organic Synthesis,' Wiley: New York, 1981, and references cited therein). An amino acid can be linked to the remainder of a conjugate of formula I through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of a cysteine.

The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

The phrase "therapeutically effective amount" means an amount of a conjugate or vaccine of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

As used herein, a “subject” is an animal, e.g., a mammal, e.g., a human, monkey, dog, cat, horse, cow, pig, mini-pig, goat, rabbit, rat, hamster, or mouse.

The term “heterocycle” or “heterocycloalkyl” refers to a single saturated or partially unsaturated ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; the term also includes multiple condensed ring systems that have at least one such saturated or partially unsaturated ring, which multiple condensed ring systems are further described below. Thus, the term includes single saturated or partially unsaturated rings (e.g., 3, 4, 5, 6 or 7-membered rings) from about 1 to 6 carbon atoms and from about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. The sulfur and nitrogen atoms may also be present in their oxidized forms. Exemplary heterocycles include but are not limited to azetidinyl, tetrahydrofuranyl and piperidinyl. The term “heterocycle” or “heterocycloalkyl” also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a single heterocycle ring (as defined above) can be condensed with one or more groups selected from cycloalkyl, aryl, and heterocycle to form the multiple condensed ring system. The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the individual rings of the multiple condensed ring system may be connected in any order relative to one another. It is also to be understood that the point of attachment of a multiple condensed ring system (as defined above for a heterocycle) can be at any position of the multiple condensed ring system including a heterocycle, aryl and carbocycle portion of the ring. In one embodiment the term heterocycle includes a 3-15 membered heterocycle. In one embodiment the term heterocycle includes a 3-10 membered heterocycle. In one embodiment the term heterocycle includes a 3-8 membered heterocycle. In one embodiment the term heterocycle includes a 3-7 membered heterocycle. In one embodiment the term heterocycle includes a 3-6 membered heterocycle. In one embodiment the term heterocycle includes a 4-6 membered heterocycle. In one embodiment the term heterocycle includes a 3-10 membered monocyclic or bicyclic heterocycle comprising 1 to 4 heteroatoms. In one embodiment the term heterocycle includes a 3-8 membered monocyclic or bicyclic heterocycle heterocycle comprising 1 to 3 heteroatoms. In one embodiment the term heterocycle includes a 3-6 membered monocyclic heterocycle comprising 1 to 2 heteroatoms. In one embodiment the term heterocycle includes a 4-6 membered monocyclic heterocycle comprising 1 to 2 heteroatoms. Exemplary heterocycles include, but are not limited to aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2, 3, 4- tetrahydroquinolyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3 -dihydrobenzofuranyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro[cyclopropane-l,l'-isoindolinyl]-3'-one, isoindolinyl-l-one, 2-oxa-6- azaspiro[3.3]heptanyl, imidazolidin-2-one imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, di oxolane, phthalimide, and 1,4-di oxane.

It will be appreciated by those skilled in the art that certain compounds described herein have a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase). When a bond in a compound formula herein is drawn in a non-stereochemical manner (e.g. flat), the atom to which the bond is attached includes all stereochemical possibilities.

The invention will now be illustrated by the following non-limiting Examples.

EXAMPLE 1. Multivalent vaccination strategies against opioids and psychoactive drug mixtures in mice and rats

Illicit drug mixtures containing opioids and stimulants have been responsible for the majority of fatal drug overdoses among individuals with opioid use disorder (OUD) and substance use disorders (SUE)). Amid the opioid epidemics, active immunization with conjugate vaccines has been proposed as a complementary approach to traditional FDA-approved pharmacotherapies to treat OUD. Vaccination against drugs of abuse could help address the limitations of current medications in patient access, compliance, abuse liability, and safety and provide an additional tool to prevent drug overdoses. However, more research is needed to fully understand the potential benefits and limitations of vaccines for addiction and overdose and to inform how to deploy this strategy in the field. In previous reports, we have shown the possibility of combining certain vaccines into bivalent vaccine formulations to target multiple drugs at once. Here, multiple individual candidate monovalent vaccines were combined in multivalent vaccine formulations to simultaneously target fentanyl, carfentanil, oxycodone, heroin, and methamphetamine, respectively. Tri- and quadrivalent vaccinations induced independent serum IgG titers against their respective opioid targets without interference and selectively attenuated the distribution of target drugs to the brain in mice and rats. In respect to multivalent formulation, a single injection of an admixed multivalent vaccine yielded higher immune responses than co-injecting multiple monovalent vaccines at multiple sites. Finally, adding a methamphetamine conjugate vaccine to the opioid quadrivalent vaccine as pentavalent did not interfere with the independent production of anti-opioid IgG titers and attenuation of target opioids distribution to the brain. Amid opioid epidemics, multivalent vaccination will provide a broader spectrum of protection against multiple drugs with a single vaccination.

INTRODUCTION

The number of annual fatal drug overdoses reached >100,000 between April 2020 and present, of which the majority involved synthetic opioids such as fentanyl presented alone or in poly drug mixtures containing other opioids or psychostimulants such as methamphetamine¹. Respiratory arrest and cardiovascular deficits are the major symptoms of fatal opioid overdose^{2 5}. Full opioid agonists like heroin, fentanyl, and more potent fentanyl analogs such as carfentanil cross the blood-brain barrier (BBB) and activate the brain p-opioid receptor (MOR) to induce respiratory depression and bradycardia². Emerging literature indicates that activation of opioid receptors in the periphery⁶ as well as the unique properties of the phenylpiperidines (fentanyl- like) chemical family to activate receptor systems beyond the MOR signaling pathways^9,10 are contributing to the complexity of opioids’ effects on the pulmonary and cardiac systems.

The high incidence of overdose from polydrug use is driven by the widespread availability of street mixtures of multiple opioids, or opioids and stimulants (e.g., heroin/fentanyl; methamphetamine/fentanyl)¹. Although FDA-approved MOR agonists, partial agonists, and antagonists provide a safe and effective medication-assisted therapy (MAT) for OUD and overdose significant limitations are still associated with each of them¹¹. For instance, the MOR antagonist naloxone is not as efficient in counteracting effects of fentanyl(s), possibly due to their non-MOR effects on airways^9,10. In addition, no FDA-approved medications are available for overdose involving stimulants, such as methamphetamine. Because of their different mechanisms of action, opioids and stimulants, as well as their combination, offer a challenge in developing a mechanism-agnostic pharmacotherapeutic intervention suitable to counteract both opioids and stimulants.

As an alternative or complementary strategy to pharmacotherapies, vaccines may provide broad, yet selective, protection against a wide variety of drugs by stimulating the generation of highly specific polyclonal antibodies that prevent drugs from crossing the BBB, and limit drug- induced adverse effects^{12 l 5}. Vaccines could be designed to target either individual drugs (monovalent) or multiple drugs at once (multivalent)^{16 l9}. Our laboratory has demonstrated preclinical proof of efficacy, selectivity, and safety for a series of conjugate vaccines targeting heroin, oxycodone, fentanyl, and their analogs or metabolites^{13, 20 23}. One such vaccine against oxycodone (OXY-sKLH) is currently in Phase I clinical trials (NCT04458545). Individual vaccines comprise haptens, small molecules that mimic the structure of the targeted drug, conjugated to carrier proteins, such as detoxified diphtheria toxin (CRM) or keyhole limpet hemocyanin (KLH). Pre-clinical studies identified lead haptens targeting fentanyl (Fi, or F), carfentanil (F13, or Carf), oxycodone (OXY), heroin/morphine (M), and methamphetamine (METH). Preclinical studies showed the efficacy of each individual vaccine (Fi-CRM, F13-CRM, OXY-sKLH, and M-sKLH) in blocking their respective target drug distribution to the brain and their behavioral effects in mice and rats¹³'^{20 23}. Throughout this Example, the M hapten and its conjugated form, M-sKLH, may also be referred to as “Her” and “Her-sKLH”, as they target heroin, 6-acetylmorphine (6-MAM), and morphine²³. Individual vaccines can be combined in bivalent vaccines of OXY-sKLH plus M-sKLH, or Fi-CRM plus F13-CRM¹⁶’²². Similarly, individual nicotine vaccines could be combined in trivalent formulations²⁴. Other groups have successfully explored the possibility of combining vaccines in either co-admixed multivalent or multidisplay formulations against fentanyl/heroin^{25 27} and morphine/heroin^{28 30}. Here, we further expand upon this concept by demonstrating that individual vaccines could be combined in bivalent, trivalent, quadrivalent, and pentavalent formulations to target multiple drugs simultaneously. While it is commonly accepted that multivalent vaccine formulations are a safe and effective strategy against infectious diseases^{17 l9}, multivalent vaccination strategies against polydrug use have not yet been fully explored at the pharmacokinetic and pharmacodynamic (PK/PD) interface. Here, multiple conjugate vaccines have been administered as co-administration or admix on mice and rats to test the individual efficacy of the monovalent vaccine in the multivalent formulations.

MATERIALS AND METHODS

Synthesis of fentanyl-, carfentanil-, oxycodone-, morphine-based haptens, and methamphetamine-based haptens. Fentanyl- (Fi), carfentanil- (F13), oxycodone- (OXY), and heroin- (M) based haptens were synthesized as previously described^13,21’²²’ ^28,31.

As used herein, “heroin-based hapten” or “morphine-based hapten” are used interchangeably (e.g., see this Example, and Figure 7A), which refers to a hapten comprising morphine or derivative thereof. Likewise, her-sKLH or M-sKLH are used interchangeably (e.g., see this Example, and Figure 7A), which refers to a hapten comprising morphine or derivative thereof that is linked to a KLH carrier protein (e.g., sKLH).

The synthesis of methamphetamine-based hapten is as followed: Scheme 1

te/7-Butyl (3-acrylamidopropyl)carbamate (3): To a 0 °C solution of acrylic acid 2 (0.500 g, 6.94 mmol, 1.0 equiv) in 40 mL of anhydrous CH2CI2 was added A-Boc diaminopropane (1.80 g, 10.4 mmol, 1.5 equiv) followed by EDC (3.32 g, 17.3 mmol, 2.5 equiv) and DMAP (85 mg, 0.694 mmol, 10 mol%). The resulting clear solution was maintained at ambient temperature for 20 h. TLC analysis (5% MeOH/CFLCL, KMnCU stain) showed the consumption of the starting acid. The reaction mixture was diluted with 100 mL of CH2CI2 and transferred to a separatory funnel. The organics were washed with saturated aqueous NaHCCL (100 mL), H2O (100 mL), and brine (100 mL). Dried over MgSCU, filtered, and concentrated. Purification by flash chromatography on SiCL (110 g, 5% MeOEECEECh) afforded 1.05 g of a pale yellow oil (66%) that became a white solid upon storage at -20 °C. 'H NMR (500 MHz, DMSO-< 5 8.09-8.00 (m, 1H), 6.83- 6.75 (m, 1H), 6.19 (dd, J = 17.1, 10.2 Hz, 1H), 6.06 (dd, J = 17.1, 2.3 Hz, 1H), 5.56 (dd, J = 10.1, 2.3 Hz, 1H), 3.14-3.07 (m, 2H), 2.95-2.89 (m, 2H), 1.57-1.48 (m, 2H), 1.37 (s, 9H).

Chemical Formula: C₃H₄O₂ Chemical Formula: C₈H₁₈N₂O₂ Exact Mass: 72.02 Chemical Formula: CII H Exact Mass: 174.14 ₂ON₂0₃ Molecular Weight: 72.06 Exact Mass: 228.15

Molecular Weight: 174.24 Molecular Weight: 228.29

/‘ /V-Butyl (E)-(3-(3-(4-(2-oxopropyl)phenyl)acrylamido)propyl)carbamate (5): To a solution of ketone 4 (0.400 g, 1.88 mmol, 1.0 equiv) in 13 mL of anhydrous DMF under N2 was added Pd(OAc)2 (8 mg, 0.0375 mmol, 2 mol%), EtsN (0.785 mL, 5.63 mmol, 3.0 equiv), P(o-tolyl)3 (52 mg, 0.169 mmol, 9 mol%) and acrylamide 3 (0.514 g, 2.25 mmol, 1.2 equiv). The resulting yellow solution was heated at 100 °C for 90 min. LC-MS analysis showed the reaction was complete. After cooling to ambient temperature, the reaction was diluted with 30 mL of H2O and transferred to a separatory funnel. Extracted with EtOAc (3 x 40 mL) then washed with H2O (2 x 40 mL) and brine. The combined organics were dried over MgSCh, filtered, and concentrated. Purification by flash chromatography on SiO? (100 g, 70% EtOAc/hexanes) afforded 495 mg (73%) of a white solid. 'H NMR (500 MHz, DMSO-t/e): 8 8.07 (t, J = 5.6 Hz, 1H), 7.49 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 15.9 Hz, 1H), 7.22 (d, J = 8.2 Hz, 2H), 6.80 (t, J = 5.4 Hz, 1H), 6.58 (d, J = 15.8 Hz, 1H), 3.79 (s, 2H), 3.19-3.12 (m, 2H), 2.98-2.92 (m, 2H), 2.14 (s, 3H), 1.61-1.52 (m, 2H), 1.38 (s, 9H).

Pd(OAc)₂, P(o-tol l)

/‘c/V-Butyl (E)-(3-(3-(4-(2-(methylamino)propyl)phenyl)acrylmido)propyl)carbamate (6): To a mixture of methyl ketone 5 (0.120 g, 0.333 mmol, 1.0 equiv) in 1.7 mL of anhydrous DCE was added a 2.0 M solution of Mebflh in MeOH via syringe at ambient temperature. The mixture was then treated with NaBH(OAc)s (0.106 g, 0.500 mmol, 1.5 equiv) and AcOH (20 uL, 0.361 mmol, 1.0 equiv). The reaction was maintained at ambient temperature for 20 h. LC-MS showed the reaction was not complete. Added another 0.250 mL MebflL and 0.106 g of NaBH(OAc)s and maintained for 3 h. LC-MS still showed some unreacted starting material. Added another 0.170 mL MeNH2 and 110 mg of NaBH(OAc)3. After 2 h, the reaction looked complete by LC-MS. Saturated aqueous NaHCCf (20 mL) was slowly added to quench the reaction mixture. The crude mixture was extracted with 20 mL EtOAc then further extracted with EtOAc (2 x 15 mL). The organics were washed with brine. The combined EtOAc layers were then washed with 1 M HC1 (2 x 15 mL then 1 x 10 mL). Basified acid layer to pH 10 with 5 N NaOH. Extracted with EtOAc (2 x 30 mL). Washed with brine. Dried over Na2SO4, filtered, and concentrated. Obtained 101 mg (81%) of a viscous pale yellow oil that became a white solid upon storage at -20 °C. 'H NMR (500 MHz, DMSO-t/e): 6 8.05 (t, J = 5.6 Hz, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.37 (d, J = 15.7 Hz, 1H), 7.21 (d. J = 8.1 Hz, 2H), 6.80 (t, J = 5.4 Hz, 1H), 6.56 (d, J = 15.8 Hz, 1H), 3.19-3.12 (m, 2H), 2.99-2.91 (m, 2H), 2.78-2.65 (m, 2H), 2.44 (dd, J = 12.8, 7.0 Hz, 1H), 2.28 (s. 3H), 1.60- 1.51 (m, 2H), 1.38 (s, 9H), 0.89 (d, J = 6.1 Hz, 3H).

g . (E)-A-(3-aminopropyl)-3-(4-(2-(methylamino)propyl)phenyl)acrylamide dihydrochloride (Meth): A solution of Boc amine 6 (81 mg, 0.216 mmol, 1.0 equiv) in 0.5 mL of anhydrous dioxane was cooled to 0 °C and treated with 4.3 mL of 4 M HC1 in dioxane. The resulting reaction mixture was maintained at ambient temperature for 45 min. LC-MS showed the reaction was complete. After 1 h of total reaction time, the reaction was concentrated under reduced pressure (20 mbar) then triturated with dry Et2O to obtain a white solid. Decanted off Et2O and washed with additional Et2O (3 x 5 mL). Dried under reduced pressure. The solid was dissolved in 10 mL of H2O and lyophilized. Obtained 64 mg (85%) of an off-white foam. 'H NMR (500 MHz, DMSO- d₆) 5 9.15-8.97 (m, 2H), 8.42 (t, J = 5.8 Hz, 1H), 7.98 (br, 3H), 7.53 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 15.8 Hz, 1H), 7.31 (d, J = 8.2 Hz, 2H), 6.65 (d, J = 15.8 Hz, 1H), 3.43-3.32 (m, 1H), 3.28-3.16 (m, 3H), 2.86-2.76 (m, 2H), 2.68 (dd, J = 13.1, 10.0 Hz, 1H), 2.56 (t, J = 5.5 Hz, 3H), 1.81-1.72 (m, 2H), 1.10 (d, J = 6.4 Hz, 3H).

Chemical Formula: C21H33N3O3 Chemical Formula: C16H25N3O Exact Mass: 375.25 Exact Mass: 275.20 Molecular Weight: 375.51 Molecular Weight: 275.40 MW salt: 348.31

Conjugation of vaccines Fi, F13, OXY, M, and METH haptens were conjugated to either subunit keyhole limpet hemocyanin (sKLH) or cross-reactive material (CRM) as previously described^13,32 to generate F-CRM, Carf-CRM, Oxy-sKLH, Her-sKLH, and METH-sKLH. For antibody analysis, haptens were conjugated to bovine serum albumin (BSA) as previously described³². Briefly, the haptens were dissolved in a 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer with a pH of 4.5 (M-sKLH) or 5.0 (Fi-CRM, F13-CRM, OXY-sKLH, and METH-sKLH) with 10% DMSO (Fi-CRM) at a concentration of 5.2 mM. Carbodiimide cross-linking chemistry was prepared by activating the haptens with 208 mM N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC). After 15 minutes of stirring at room temperature, CRM, sKLH, or BSA was added at a final concentration of 2.8 mg/mL and stirred for 3 hours at room temperature. To terminate the reaction, the conjugate mixture was washed with PBS buffer with a pH of 7.2 using a filter unit with a 30 kDa (CRM and BSA conjugates) or 100 kDa (sKLH conjugates) molecular weight cutoff, and then resuspended with PBS buffer with a pH of 7.2 to a final concentration of 2.5 mg/mL before being stored at 4°C. Additionally, CRM conjugates received 250 mM sucrose as a stabilizing agent in the MES and PBS buffers for the conjugation reaction and storage. The haptenation ratios of the CRM and BSA conjugates were measured by MALDI-TOF (ABSCIEX 5800, Foster City, CA) and estimated using a previously described formula: [(MW_COnju_gate - MW_carrierprotein)/MWhapten]³². For sKLH conjugates, it was not feasible to employ MALDI-TOF to determine the haptenation ratio due to its substantial size (approximately 5 to 8 million Da). Instead, dynamic light scattering (DLS) on a Zetasizer (Malvern Panalytical Inc., United Kingdom) was used to characterize size and aggregation status of the sKLH conjugates as previously described³³. The unconjugated protein and conjugate vaccines were mixed with aluminum hydroxide adjuvant before conducting in vivo immunization studies as outlined in the formulation section.

Drugs. Fentanyl citrate was obtained from the University of Minnesota Boynton Pharmacy, and carfentanil hydrochloride was obtained from the NIDA Supply at RTI International (Research Triangle Park, NC). Oxycodone, heroin HC1, and methamphetamine were obtained from Sigma Aldrich (St. Louis, MO).

Animal subjects. Male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) and Sprague Dawley rats (Envigo, Indianapolis, IN) were eight weeks old upon arrival at A AAL AC -approved facilities at the University of Minnesota. The mice and rats had free access to water and food and were habituated to a 14-hour light/10-hour dark cycle prior to the experiments. The animal experiments were conducted with the approval of the University of Minnesota Animal Care and Use Committee.

Formulation and vaccinations. Vaccines comprise conjugates adsorbed onto aluminum hydroxide adjuvant (alum, Alhydrogel 2%, InvivoGen, San Diego, CA) and diluted in sterile PBS. Vaccines were formulated at 30 pg conjugate to 150 pg alum (ratio of 1 :5) for mice and 30 pg conjugate to 45 pg alum (ratio of 1 : 1.5) for rats. Rodents were immunized 3 times either 2 weeks (mice) or 3 weeks (rats) apart. In mice, Fi-CRM, OXY-sKLH, F13-CRM and M-sKLH were subcutaneously (s.c) injected as monovalent immunization, or co-injected at separate sites for trivalent and quadrivalent vaccination. Trivalent immunization has Fi-CRM, OXY-sKLH, and M- sKLH (90 pg of conjugate and 450 pg of alum, total), while quadrivalent immunization has all 4 conjugates (120 pg of conjugate and 600 pg of alum, total). Mice were immunized intramuscularly (i.m.) with 60 pg of Meth-sKLH adsorbed on 30 pg of alum in the methamphetamine-induced locomotor activity study. In rats, four individual monovalent vaccines, Fi-CRM, F13-CRM, OXY- sKLH, and M-sKLH, were intramuscularly (i.m.) injected alone (monovalent), co-injected at four separate sites on hind legs (co-admini strati on) or were individually absorbed in aluminum salts, then co-formulated in a single syringe for a single injection (co-formulation). Furthermore, METH-sKLH was co-formulated with the opioid quadrivalent vaccine as pentavalent (150 pg of conjugate and 225 pg of alum total) and injected i.m. at a single site.

Antibody analysis. Blood obtained from the vaccinated animals by facial bleeding on day 34 (mouse) or tail bleeding on day 49 (rat) was analyzed for hapten-specific serum IgG antibody titers by ELISA³². Briefly, 96-well plates were coated with 5 ng/well of F1-, F13-, OXY-, M-, or METH-BSA conjugates to bind fentanyl-, carfentanil-, oxycodone-, heroin/morphine-, and methamphetamine-specific antibodies. Unconjugated BSA was used as a negative control. After blocking plates, serum from immunized animals was serially diluted starting at 1 :200. Plates were incubated overnight with goat-anti-mouse (1 :30,000) or goat anti-rat (1 :35,000) secondary antibodies conjugated to HRP, and were developed using SIGMAFAST™ OPD substrate (Sigma Aldrich, St. Louis, MO).

In vivo efficacy: antinociception, respiratory depression, and bradycardia. Oximetry and hot plate test were used to measure opioid-induced respiratory depression, bradycardia, and antinociception as previously described³². Briefly, to measure opioid-induced respiratory depression and bradycardia, oxygen saturation (%SaO2) and heart rate (bpm) of the animals were monitored by oximetry collar (MouseOX, Starr Life Sciences Corp, PA) at the baseline and 15 min of intervals up to 30 min post-drug challenge. Immediately after each oximetry measurement, the animals were placed on a hot plate (Columbus Instruments, OH) at 54°C to measure latency to respond (as thermal pain indicated by a lift or flick of the hind paw) with a maximum cutoff of 30 sec for rats and 60 sec for mice to prevent thermal damage.

Pharmacokinetic analysis. After the last measurement in the drug challenge, the mice and rats were euthanized for post-mortem analysis of the concentration of the drugs in their brain and serum. The drug concentration was analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the drug distribution from the blood to the brain as previously described³².

Statistical analysis. Mean oxygen saturation, heart rate, and latency to respond between groups at baseline and each time point post-drug challenge were analyzed by two-way ANOVA with Geisser-Greenhouse correction. Hapten-specific IgG titers and drug concentration in serum and brain between groups were compared by one-way ANOVA paired with Tukey’s multiple comparisons post-hoc test. Statistical analyses were performed using Graphpad Prism v.9 (GraphPad, La Jolla, CA).

RESULTS

Vaccination with different carrier proteins does not interfere with opioid vaccine efficacy. Prior to developing a multivalent vaccine that employs various vaccines with distinct types of carrier proteins, it was essential to verify whether blending vaccines with diverse carrier proteins would not drive opposing immune responses in the field of opioid vaccine that could be detrimental to antibody production and vaccine efficacy. For example, previous studies have reported various carrier proteins in multivalent vaccines for nicotine²⁴. To assess whether immunization with vaccines having two different carrier proteins (CRM and sKLH) interferes with vaccine efficacy, rats were immunized i.m. with CRM and sKLH as a negative control, Fi- CRM, OXY-sKLH, or a combination of two vaccines (Fi-CRM and OXY-sKLH) adsorbed to alum. Vaccination with these different formulations successfully induced IgG titers specific to the cognate haptens and the rats that received both Fi-CRM and OXY-sKLH vaccines induced separate IgG titers specific to fentanyl and oxycodone (Figure 1A, IB). In fact, bivalent immunization led to a significant increase in oxycodone-specific IgG titers when compared to the OXY-sKLH monovalent immunization, indicating that vaccine formulations having conjugates with different carrier proteins may further stimulate the immune response and lead to additive increases in antibody titers (Figure IB). After immunization, rats were challenged s.c with either fentanyl (0.1 mg/kg), oxycodone (4.5 mg/kg), or a mixture of two drugs (fentanyl (0.05 mg/kg) and oxycodone (2.25 mg/kg)), and monitored for opioid-induced respiratory depression and antinociception. All vaccines provided selective attenuation from the cognate opioid-induced effects, but only the rats that were co-immunized with Fi-CRM and OXY-sKLH were protected against the mixture of fentanyl and oxycodone (Figure 2). In particular, during the challenge with oxycodone and fentanyl, rats in the monovalent vaccine groups were not protected in any metrics at any time point (Figure 2A-2I), while bivalent immunized animals were protected against bradycardia (p=0.0267 at t= 15 min; p=0.0214 at t=30 min) and antinociception (p=0.0456 at t=30 min) (Figure 2G, 21). While not statistically significant, there was also a trend (p=0.1275 at t=30 min; p=0.0929 at t=45 min) in protection against respiratory depression in the bivalent group compared to controls (Figure 2H). However, fentanyl and oxycodone distribution to brain was reduced in the respective monovalent vaccine groups, as well as in the bivalent group, compared to controls. Specifically, LC-MS/MS analysis showed that each vaccination selectively sequestered the cognate drug in the bloodstream and blocked its distribution to the brain while only the co-vaccination with Fi-CRM and OXY-sKLH blocked both fentanyl and oxycodone from entering the brain without interference (Figure 1C, ID).

Trivalent and quadrivalent vaccinations elicit IgG titers without interference and protect against opioid mixtures in mice. After testing co-immunization with different carrier proteins did not interfere with the vaccine efficacy, the study aimed to evaluate the effectiveness of trivalent and quadrivalent conjugate vaccines in mice as a next step towards developing a multivalent vaccine. Mice were immunized with vaccines targeting fentanyl (Fl -CRM), carfentanil (Fl 3 -CRM), oxycodone (OXY-sKLH), heroin (M-sKLH), or combinations (trivalent and quadrivalent) thereof. Vaccinations with these different formulations successfully induced serum IgG titers specific to the cognate haptens without interference (Figure 3A, 3B, 3C, 3D). Compared to immunization with monovalent vaccines, trivalent and quadrivalent vaccinations elicited significantly greater heroin-specific serum IgG titers, suggesting the possible additive effects of combining multiple monovalent vaccines (Figure 3D).

After immunization, mice were challenged s.c with a mixture of fentanyl (0.025 mg/kg), carfentanil (0.005 mg/kg), oxycodone (1.125 mg/kg), and heroin (0.25 mg/kg) to assess the opioid-induced effects and drug distribution across the groups. Against the mixture of four opioids, only the mice that were immunized with the quadrivalent vaccine showed a significant reduction in antinociceptive response (p=0.0003) compared to the control group at 30 min postdrug challenge (Figure 10). The drug distribution analysis by LC-MS/MS showed that each vaccination selectively retained the cognate target opioid in the bloodstream and blocked its distribution to the brain, while trivalent and quadrivalent vaccinations simultaneously prevented the entry of three and four opioids into the brain, respectively (Figure 3E, F, G, H). Additionally, the brain distribution of each targeted opioid may have further attenuated in the mice that were immunized with the multivalent vaccines compared to monovalent immunization, consistent with the observed higher antibody titers. Compared to the monovalent vaccination groups, the mean brain concentrations of fentanyl (Figure HE), oxycodone (Figure 11G), and heroin’s primary metabolite 6-MAM (Figure 11H) were lower in trivalent [mean difference=0.3733±1.9467 (fentanyl); mean difference=23.33±209.07 (oxycodone); mean difference=1.594±13.496 (6- MAM)] and quadrivalent [mean difference=0.4224±2.0416 (fentanyl); mean difference=138.7±219.2 (oxycodone); mean difference=7.544±14.096 (6-MAM)] vaccine groups. The mean fentanyl concentrations in the brain of the trivalent and quadrivalent vaccine groups were 10.6% and 12.0% lower than the Fi-CRM vaccine group. Similarly, in the trivalent and quadrivalent groups, the mean brain concentrations of oxycodone were lowered by 5.8% and 34.7%, and the mean brain concentration of 6-MAM was lowered by 25.9% and 27.0% than their cognate monovalent vaccination. The LC-MS/MS analysis demonstrated that the efficacy of individual monovalent vaccines was not only preserved but also enhanced for several monovalent vaccines in the multivalent vaccination. As efficacy of individual monovalent vaccine was preserved in the quadrivalent vaccination, trivalent vaccine was not tested in the follow-up studies.

Co-formulation of monovalent vaccines is more effective than the co-administration of individual monovalent vaccines in rats. Optimizing the delivery regimen between multiple vaccines is crucial for increasing vaccine efficacy and patient convenience when developing a clinically available multivalent vaccine^34,35. In the previous experiment conducted on mice, monovalent vaccines were co-administered s.c at different sites using individual syringes. However, administering the vaccine formulation in a single syringe would be clinically advantageous to minimize injections and increase patient compliance. To assess whether multivalent vaccination could be equally and more efficacious when co-formulated in a single syringe, rats were vaccinated i.m. with Fl-CRM, F13-CRM, OXY-sKLH, M-sKLH alone, or all four conjugates (quadrivalent) by injecting four individual vaccines at different sites (coadministration) or injecting at a single site after co-formulating four conjugates in a single syringe (co-formulation). Both the co-administrated and co-formulated quadrivalent vaccines elicited IgG titers comparable to cognate monovalent vaccines (Figure 4A, B, C, D). However, IgG titers specific to fentanyl and oxycodone (Figure 4A, C) showed a trend towards higher serum IgG titers with the co-formulated quadrivalent vaccine compared to the co-administered vaccine.

To evaluate opioid-induced effects and drug distribution, the immunized rats were challenged s.c with a mixture of fentanyl (0.025 mg/kg), carfentanil (0.005 mg/kg), oxycodone (1.125 mg/kg), and heroin (0.25 mg/kg). In contrast to the outcomes of the previous mouse study, the co-administration and co-formulation of quadrivalent vaccines did not lead to a significant inhibition of opioid-induced antinociception 30 min post-drug challenge. Although not statistically significant, the rats that received the co-formulated quadrivalent vaccine showed a trend toward protection from opioid-induced antinociception and respiratory depression at 15 min and 30 min post-drug challenge (Figure 11A, 1 IB) [at t=15, mean difference=31.88±18,99; at t=30, mean difference^! 9.76± l 4.79 compared to control group (%MPE); at t=30, mean difference=-12.52±8.102 compared to control group (SaCh)]. This result implies that if the efficacy of each individual vaccine was improved, it could potentially provide simultaneous protection against multiple opioids. The rats that received the co-administered quadrivalent vaccine or other monovalent vaccines did not exhibit any trend of protection against opioid- induced pharmacological effects.

Similar to the previous mouse experiment, LC-MS/MS analysis demonstrated a marked reduction in the brain: serum ratio of fentanyl, carfentanil, oxycodone, or 6-MAM in all groups vaccinated with Fl-CRM, F13-CRM, OXY-sKLH, or M-sKLH, respectively (Figure 4E, F, G, H). However, unlike the previous mouse experiment, co-administered quadrivalent vaccination showed insufficient reductions in the brain: serum ratio of carfentanil and heroin compared to the cognate monovalent vaccination in rats (Figure 4F, H). In contrast, rats that received the coformulated quadrivalent vaccine showed a significant reduction in the brain: serum ratio of all four opioids comparable to the cognate monovalent vaccination. Surprisingly, the co-formulated quadrivalent vaccine was more effective at reducing 6-MAM distribution to the brain compared to the co-administered quadrivalent vaccine in rats (p=0.0285) (Figure 4H). The effectiveness of delivering a multivalent vaccine through co-formulation was higher than that of co-administration, considering the outcomes of both titer and drug distribution analyses. These results highlighted that optimizing the vaccination regimen could enhance efficacy even if the composition is identical between multivalent formulations. These results suggest that delivering a multivalent vaccine through co-formulation may be a more effective immunization strategy than the coadministration of individual vaccines at different sites.

Admixing an opioid quadrivalent conjugate vaccine with a stimulant conjugate vaccine does not interfere with the vaccine efficacy, but simultaneously blocks both opioid and stimulant. The incidence of fatal opioid overdoses involving stimulants in street mixtures has been rapidly increasing. Between January and June 2019, about 85% of overdose deaths were associated with the use of illicitly produced fentanyl, heroin, cocaine, or methamphetamine, either alone or in combinations³⁶. Therefore, a clinically available multivalent vaccine for substance use disorder (SUD) should comprise opioid and stimulant vaccines to offer protection from both classes of drugs. Here, we first developed a novel methamphetamine conjugate vaccine (Meth-sKLH) and tested its efficacy in reducing methamphetamine distribution to brain in rats (Figures 13A and 13B) and methamphetamine-induced locomotor activity in mice (Figure 14). Meth-sKLH reduced brain distribution of methamphetamine compared to controls in rats, but did not reduce methamphetamine-induced motor stimulating effects compared to controls in mice, possibly due to differences in vaccine formulations between these two experiments. Therefore, Meth-sKLH was admixed, based on the formulation from the drug distribution study in rats, with the opioid quadrivalent vaccine to create a pentavalent vaccine.

To determine whether an opioid multivalent vaccine can be co-formulated with a stimulant vaccine without compromising efficacy, the METH-sKLH methamphetamine vaccine was admixed with the previously tested opioid quadrivalent vaccine to create a pentavalent vaccine. Using the same protocols as previously described, rats were immunized with a control group comprising CRM and sKLH, a quadrivalent vaccine containing four opioid vaccines, and a pentavalent vaccine containing four opioid vaccines and METH-sKLH.

ELISA analysis of serum titers following vaccination indicated that both the quadrivalent and pentavalent vaccines elicited similar levels of serum IgG titers specific to each of the four opioid haptens (Figure 5A, B, C, D, E). This result suggests that the addition of a stimulant conjugate vaccine did not interfere the production of anti -opioid IgG antibodies. To assess the efficacy of pentavalent immunization against a common form of street mixtures of opioids and stimulants, the rats were challenged with a mixture of fentanyl (0.025 mg/kg), heroin (0.25 mg/kg), and methamphetamine (0.15 mg/kg). Results showed that pentavalent vaccination was as effective as the previous opioid quadrivalent vaccine in blocking opioid-induced antinociception (p=0.0128 at t=15 min), respiratory depression (p=0.0004 at t=15 min), and bradycardia (p=0.0157 at t=15 min; p=0.0127 at t=30 min) (Figure 8A, 8B, 8C). These findings confirm that adding a stimulant conjugate vaccine in the multivalent formulation does not affect individual efficacy of the opioid vaccines. Furthermore, drug distribution analysis by LC-MS/MS showed that pentavalent vaccination effectively blocked the entry of fentanyl, heroin metabolite 6-MAM, and reduced entry of methamphetamine into the brain without any interference (Figure 6A, 6B, 6C). However, the serum IgG (Figure 5D) induced by METH-sKLH exhibited some cross-reactivity with heroin, and, to a lesser extent, fentanyl (Figure 6A, 6B), indicating that further optimization of the hapten design may be required.

DISCUSSION

Carrier-induced epitopic suppression (CIES) is a phenomenon in which the immune response to a vaccine is suppressed when multiple vaccines that use the same carrier protein are administered^37,38. Therefore, the previous studies investigated the phenomenon of CIES in vaccine response by multiple conjugate vaccines. Two separate bivalent vaccines were designed to simultaneously block two different opioids without decreasing the efficacy of the individual monovalent vaccines. The first bivalent vaccine contained the OXY and M haptens, both conjugated to sKLH to target oxycodone and heroin, respectively¹⁶. The second bivalent vaccine contained the Fl and Fl 3 haptens, both conjugated to CRM to block fentanyl and carfentanil²². Each bivalent vaccine was effective against its cognate opioids, indicating that CIES was not observed in these formulations.

Prior to developing a multivalent vaccine that employs various vaccines with distinct types of carrier proteins, it was essential to verify whether blending vaccines with diverse carrier proteins would not compromise the vaccine efficacy. Because previous studies have reported that incorporating distinct carrier proteins may affect the efficacy of multivalent nicotine vaccine²⁴, the current study investigated the effect of combining two different carrier proteins, CRM and sKLH, in a bivalent vaccine and whether a bivalent vaccine against fentanyl and oxycodone using two different carrier proteins, CRM and sKLH would reduce their efficacy. The study found that co-immunization with Fl -CRM and OXY-sKLH as a bivalent vaccine was effective in producing independent serum IgG titers specific for fentanyl and oxycodone (Figure 1). The co- immunization effectively attenuated drug distribution to the brain and protected rats from opioid- induced antinociception, respiratory depression, and bradycardia when challenged with a mixture of fentanyl and oxycodone (Figure 2). This suggests that combining different carrier proteins in a bivalent vaccine does not interfere with individual vaccine efficacy.

The study aimed to evaluate the effectiveness of trivalent and quadrivalent conjugate vaccines in mice as a next step towards developing a multivalent vaccine. The co-injection of Fl- CRM, F13-CRM, OXY-sKLH, and M-sKLH as a quadrivalent vaccination successfully elicited independent hapten-specific serum IgG titers without interference. Only the mice immunized with a quadrivalent vaccine showed a significant reduction in antinociception and efficient mitigation of drug distribution to the brain (Figure 3), providing selectivity of the vaccine-induced antibodies for the drug target. When monovalent vaccines were co-administered as trivalent and quadrivalent, we observed synergistic efficacy of individual vaccines in eliciting IgG titers and mitigating drug distribution in mice. These data are consistent with a previous study reporting increased efficacy in a dose-matched multivalent nicotine vaccine²⁴. The overall results suggest that simultaneous immunization is necessary for broader protection against opioid combinations, and the efficacy of individual monovalent vaccines is preserved in the multivalent vaccination. The observed overall increase in efficacy of the quadrivalent vaccine in mice could potentially be attributed to multiple factors, including the engagement of non-overlapping cross reactive antigen-specific B and CD4⁺ T cell populations, resulting in cross-reactivity of antibodies elicited by vaccines targeting structurally similar compounds (e.g., between fentanyl and its analog carfentanil, as well as between the morphinans, oxycodone and heroin). Additionally, the administration of a larger total vaccine dose might have contributed to a non-specific overstimulation of the immune system. These results suggest that simultaneous immunization using a multivalent formulation may be beneficial for broader protection against opioid combinations, and the efficacy of individual monovalent vaccines is preserved in multivalent vaccines.

When developing a clinically available multivalent vaccine, it is crucial to optimize the delivery regimen between multiple vaccines to increase vaccine efficacy and patient compliance^34,35. In a previous experiment with mice, monovalent vaccines were administered s.c at different sites using individual syringes. However, administering the vaccine formulation in a single syringe would be clinically advantageous as it would minimize injections and increase patient compliance. In addition, previous studies showed that i.m. injection of a hepatitis B conjugate vaccine resulted in a slower decay of antibodies and higher seroconversion rate compared to s.c injection and therefore higher efficacy, thus, vaccination regimen plays a key role in determining the vaccine efficacy³⁴. To improve the vaccination regimen, the previous four monovalent vaccines were co-formulated in a single syringe as a quadrivalent vaccine and injected i.m. into rats. The results showed that co-formulated quadrivalent vaccination induced greater drug-specific serum IgG titers and reduced drug distribution to the brain to a greater extent than co-administering individual vaccines at different injection sites (Figure 4). This suggests that a single injection of an admixed multivalent vaccine is not only more convenient but also more efficacious than co-injecting multiple individual vaccines at different sites.

Although the quadrivalent vaccine showed potential in reducing the distribution of target opioids to the brain (Figure 4), the current quadrivalent formulation did not demonstrate significant benefits of the vaccine in reducing pharmacological effects on opioid-induced antinociception and respiratory depression in rats. These findings are inconsistent with a previous study conducted on mice, where a clear reduction in opioid-induced antinociception was observed 30 minutes after the drug challenge. Additionally, the synergistic efficacy of the multivalent vaccine on eliciting IgG titers observed in mice (Figure 3) was not evident in rats (Figure 4), suggesting species-specific differences in vaccine efficacy, species-specific drug-drug interactions in the context of poly drug use, or variations in immune responses due to distinct routes of immunization (s.c. injection in mice vs. i.m. injection in rats) may have contributed to the differences observed. While the experimental design incorporated optimized parameters for vaccine and drug dosage, as well as a 30-minute terminal time point for brain and blood sample collection, which were intended for drug distribution analysis by LC-MS/MS, further optimization is necessary to investigate the efficacy of the quadrivalent vaccine on the opioid-induced behavioral effects by the mixture of four different opioids. Although no statistical significance was observed in reducing the opioid-induced pharmacological effects, the data from the brain and serum clearly indicate that the drug is being sequestered away from the brain and accumulating in serum, suggesting that the absence of significance may be attributed to higher levels of individual variability in physiological responses. Highlighting how individual variability could impact pharmacological and behavioral responses to these drugs, both quadrivalent and pentavalent vaccines in the subsequent study exhibited significant pharmacological protection against the combination of opioids and methamphetamine. Given that the quadrivalent formulation has been shown to not hinder the ability of the individual monovalent vaccine to produce titers and reduce drug distribution to the brain in rats (Figure 4), we imply that increasing the efficacy of the individual vaccine could enable concurrent protection against opioid-induced antinociception and respiratory depression. Adding TLR agonists, for example, has been previously shown to increase the efficacy of conjugate vaccines^{39 42}.

Lastly, adding a stimulant conjugate vaccine (METH-sKLH) as a pentavalent formulation did not decrease the efficacy of the quadrivalent vaccine in eliciting opioid-specific titers (Figure 5) and mitigating the opioid distribution to the brain (Figure 6). The combined results show that different conjugate vaccines can be co-formulated as multivalent vaccines to provide protection against polydrug use by different classes of drugs and suggest a potential therapeutic to reduce overdose related to polydrug use.

Abbreviations. Opioid use disorder (OUD), mu-opioid receptor (MOR), cross-reactive material (CRM), keyhole limpet hemocyanin (sKLH), oxygen saturation (SaO?), beats per minute (bpm), subcutaneous (s.c), intramuscular (i.m.), substance use disorder (SUD)

The entire content of D. Song, et al., ACS Pharmacol Transl Sci. 2024, 7, 2, 363-374. doi: 10.1021/acsptsci.3c00228 (titled “Multivalent Vaccination Strategies Protect against Exposure to Polydrug Opioid and Stimulant Mixtures in Mice and Rats”) is incorporated by reference herein.

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21. Raleigh, M. D. et al. A Fentanyl Vaccine Alters Fentanyl Distribution and Protects against Fentanyl-Induced Effects in Mice and Rats. J. Pharmacol. Exp. Ther. 368, 282-291 (2019). 22. Crouse, B. et al. Efficacy and Selectivity of Monovalent and Bivalent Vaccination Strategies to Protect against Exposure to Carfentanil, Fentanyl, and Their Mixtures in Rats. ACS Pharmacol. Transl. Sci. 5, 331-343 (2022).

23. Raleigh, M. D., Pravetoni, M., Harris, A. C., Birnbaum, A. K. & Pentel, P. R. Selective effects of a morphine conjugate vaccine on heroin and metabolite distribution and heroin- induced behaviors in rats. J. Pharmacol. Exp. Ther. 344, 397-406 (2013).

24. de Villiers, S. H. L., Cornish, K. E., Troska, A. J., Pravetoni, M. & Pentel, P. R.

Increased efficacy of a trivalent nicotine vaccine compared to a dose-matched monovalent vaccine when formulated with alum. Vaccine 31, 6185-6193 (2013).

25. Hwang, C. S. et al. Efficacious Vaccine against Heroin Contaminated with Fentanyl. ACS Chem. Neurosci. 9, 1269-1275 (2018).

26. Barrientos, R. C. et al. Bivalent Conjugate Vaccine Induces Dual Immunogenic Response That Attenuates Heroin and Fentanyl Effects in Mice. Bioconjug. Chem. 32, 2295- 2306 (2021).

27. Natori, Y. et al. A chemically contiguous hapten approach for a heroin-fentanyl vaccine. BeilsteinJ. Org. Chem. 15, 1020-1031 (2019).

28. Anton, B. & Leff, P. A novel bivalent morphine/heroin vaccine that prevents relapse to heroin addiction in rodents. Vaccine 24, 3232-3240 (2006).

29. Stowe, G. N. et al. Developing a vaccine against multiple psychoactive targets: a case study of heroin. CNS Neurol. Disord. Drug Targets 10, 865-875 (2011).

30. Li, Q.-Q. et al. A morphine/heroin vaccine with new hapten design attenuates behavioral effects in rats. J. Neurochem. 119, 1271-1281 (2011).

31. Butcher, K. J. & Hurst, J. Aromatic amines as nucleophiles in the Bargellini reaction. Tetrahedron Lett. 2497-2500 (2009) doi: 10.1016/j.tetlet.2009.03.044.

32. Robinson, C. et al. Therapeutic and Prophylactic Vaccines to Counteract Fentanyl Use Disorders and Toxicity. J. Med. Chem. 63, 14647-14667 (2020).

33. Baruffaldi, F. et al. Preclinical Efficacy and Characterization of Candidate Vaccines for Treatment of Opioid Use Disorders Using Clinically Viable Carrier Proteins. Mol. Pharm. 15, 4947-4962 (2018).

34. Shaw, F. E. et al. Effect of anatomic injection site, age and smoking on the immune response to hepatitis B vaccination. Vaccine 7, 425-430 (1989).

35. Groswasser, J. et al. Needle length and injection technique for efficient intramuscular vaccine delivery in infants and children evaluated through an ultrasonographic determination of subcutaneous and muscle layer thickness. Pediatrics 100, 400-403 (1997). 36. O’Donnell, J., Gladden, R. M., Mattson, C. L., Hunter, C. T. & Davis, N. L. Vital Signs: Characteristics of Drug Overdose Deaths Involving Opioids and Stimulants — 24 States and the District of Columbia, January-June 2019. MMWR Mor b. Mortal. Wkly. Rep. 69, 1189-1197 (2020).

37. Herzenberg, L. A. & Tokuhisa, T. Epitope-specific regulation. I. Carrier-specific induction of suppression for IgG anti-hapten antibody responses. J. Exp. Med. 155, 1730-1740 (1982).

38. Herzenberg, L. A., Tokuhisa, T., Parks, D. R. & Herzenberg, L. A. Epitope-specific regulation. II. A bistable, Igh-restricted regulatory mechanism central to immunologic memory. J. Exp. Med. 155, 1741-1753 (1982).

39. Vasilakos, J. P. & Tomai, M. A. The use of Toll-like receptor 7/8 agonists as vaccine adjuvants. Expert Rev. Vaccines 12, 809-819 (2013).

40. Bremer, P. T., Schlosburg, J. E., Lively, J. M. & Janda, K. D. Injection Route and TLR9 Agonist Addition Significantly Impact Heroin Vaccine Efficacy. Mol. Pharm. 11, 1075-1080 (2014).

41. Pravetoni, M. et al. Effect of Currently Approved Carriers and Adjuvants on the Pre- Clinical Efficacy of a Conjugate Vaccine against Oxycodone in Mice and Rats. PLoS ONE 9, e96547 (2014).

42. Hwang, C. S. et al. Enhancing Efficacy and Stability of an Antiheroin Vaccine: Examination of Antinociception, Opioid Binding Profile, and Lethality. Mol. Pharm. 15, 1062- 1072 (2018).

Example 2. Multivalent vaccination strategies against polydrug use in Opioid Use Disorder

More than 107,000 cases of fatal drug overdose were reported in 2021, with around 70% of cases involving opioids. Individuals with opioid use disorder often administer polydrug street mixtures that contain multiple opioids (fentanyl and oxycodone) and non-opioids (including stimulants such as methamphetamine), leading to detrimental health outcomes. Active vaccination is being pursued as a treatment against substance use disorders (including fentanyl, carfentanil, oxycodone, heroin, and methamphetamine) and has shown efficacy in preclinical models, although the efficacy against polydrug use is not well established. To develop these vaccines, conjugates having haptens crosslinked to immunogenic carrier proteins induce the T- cell-dependent generation of hapten-specific antibodies. These antibodies bind to target drugs in the circulatory system and block the drugs from crossing the blood-brain barrier, attenuating drug effects in the central nervous system. Previously, four monovalent vaccines against four different opioids have been co-administered as a quadrivalent vaccine without decreasing their individual efficacy in mice. In the current study, these four monovalent vaccines were coformulated in a single syringe and injected into Sprague Dawley rats as a new vaccination regimen. Results showed that co-formulated quadrivalent vaccination induced greater drugspecific serum IgG titers and reduced drug distribution to the brain to a greater extent than coadministering individual vaccines at different injection sites. In addition, adding a stimulant conjugate vaccine as a pentavalent formulation did not decrease the efficacy of the quadrivalent vaccine against opioids. The combined results show that different conjugate vaccines can be coformulated as multivalent vaccines to provide protection against polydrug use by different classes of drugs and suggest a potential therapeutic to reduce overdose related to polydrug use.

A series of conjugate vaccines against fentanyl¹, carfentanil², oxycodone^3,4,5’⁶ and heroin^5,6,7 have shown preclinical proof of efficacy, selectivity, and safety. These hapten-based conjugate vaccines can be combined in multivalent formulation to target multiple opioids without interference simultaneously.

Pentavalent vaccination elicits independent production of IgG titers against five target drugs of abuse (Fig.5). Pentavalent vaccination blocks opioid-induced pharmacological effects: antinociception, respiratory depression, and bradycardia (Fig.8). Pentavalent vaccination selectively sequesters target drugs in the bloodstream and blocks drug distribution to the brain (Fig.9).

In conclusion, five different conjugate vaccines were combined as a pentavalent vaccine in this Example. Pentavalent vaccination produced independent serum IgG titers against five haptens without interference in rats. Pentavalent vaccination blocked opioid-induced pharmacological effects when the rats were challenged by the mixture of opioids and a stimulant. Pentavalent vaccination concurrently attenuated multiple target drug distributions to the brain.

References cited in Example 2:

1. Robinson C, Gradinati V, Hamid F, Baehr C, Crouse B, Averick S, Kovaliov M, Harris D, Runyon S, Baruffaldi F, LeSage M, Comer S, Pravetoni M. Therapeutic and Prophylactic Vaccines to Counteract Fentanyl Use Disorders and Toxicity. J Med Chem. 2020 Dec 10;63(23): 14647-14667. doi: 10.1021/acs.jmedchem.0c01042. Epub 2020 Nov 20. PMID: 33215913; PMCID: PMC8193686

2. Crouse B, Wu MM, Gradinati V, Kassic AJ, Song D, Averick S, Runyon S, Comer SD, Pravetoni M. Monovalent and bivalent vaccination strategies to protect against deliberate and accidental exposure to carfentanil and mixtures of carfentanil and fentanyl in rats. ASC Pharmacol and Transl Sci.

3. Raleigh MD, King SJ, Baruffaldi F, Saykao A, Hamid FA, Winston S, LeSage MG, Pentel PR, Pravetoni M. Pharmacological mechanisms underlying the efficacy of antibodies generated by a vaccine to treat oxycodone use disorder. Neuropharmacology. 2021 Sep 1; 195: 108653. doi: 10.1016/j.neuropharm.2021.108653. Epub 2021 Jun 11. PMID: 34126123; PMCID: PMC8410661.

4. Raleigh MD, Peterson SJ, Laudenbach M, Baruffaldi F, Carroll FI, Comer SD, Navarro HA, Langston TL, Runyon SP, Winston S, Pravetoni M, Pentel PR. Safety and efficacy of an oxycodone vaccine: Addressing some of the unique considerations posed by opioid abuse. PLoS One. 2017 Dec l;12(12):e0184876. doi: 10.1371/joumal.pone.0184876. PMID: 29194445; PMCID: PMC5711015

5. Baruffaldi F, Raleigh MD, King SJ, Roslawski MJ, Birnbaum AK, Hassler C, Carroll FI, Runyon SP, Winston S, Pentel PR, Pravetoni M. Formulation and Characterization of Conjugate Vaccines to Reduce Opioid Use Disorders Suitable for Pharmaceutical Manufacturing and Clinical Evaluation. Mol Pharm. 2019 Jun 3;16(6):2364-2375. doi: 10.1021/acs.molpharmaceut.8b01296. Epub 2019 May 14. PMID: 31018096; PMCID: PMC6598681.

6. Raleigh MD, Laudenbach M, Baruffaldi F, Peterson SJ, Roslawski MJ, Birnbaum AK, Carroll FI, Runyon SP, Winston S, Pentel PR, Pravetoni M. Opioid Dose- and Route- Dependent Efficacy of Oxycodone and Heroin Vaccines in Rats. J Pharmacol Exp Ther. 2018 May;365(2):346-353. doi: 10.1124/jpet.l 17.247049. Epub 2018 Mar 13. PMID: 29535156; PMCID: PMC5884377.

7. Raleigh MD, Pravetoni M, Harris AC, Birnbaum AK, Pentel PR. Selective effects of a morphine conjugate vaccine on heroin and metabolite distribution and heroin-induced behaviors in rats. J Pharmacol Exp Ther. 2013 Feb; 344(2): 397-406. doi:

10.1124/jpet. l 12.201194. Epub 2012 Dec 7. PMID: 23220743; PMCID: PMC3558830.

All publications, patents and patent applications cited herein are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Unless defined otherwise, the meanings of all technical and scientific terms used herein are those commonly understood by one of skill in the art to which this invention belongs. One of skill in the art will also appreciate that any methods and materials similar or equivalent to those described herein can also be used to practice or test the invention. Further, all publications mentioned herein are incorporated by reference in their entireties.

Claims

CLAIMS What is claimed is:

1. A methamphetamine conjugate having structure of Formula I:

(Formula I) wherein L is a linker, and C is a carrier protein, p is a hapten to carrier protein ratio number that is >=1 (e.g., 1-50, 1-30, or 1-10).

2. The methamphetamine conjugate of claim 1, wherein L comprises a segment of

3. The methamphetamine conjugate of claim 1 or 2, having structure of Formula la:

(Formula la).

4. The methamphetamine conjugate of claim 1, wherein the carrier protein is Keyhole limpet hemocyanin (KLH) (e.g., native KLH, or subunit KLH).

5. A vaccine composition comprising the methamphetamine conjugate according to any one of claims 1-4, and a pharmaceutically acceptable carrier (e.g., aqueous medium).

6. The vaccine composition of claim 5, further comprising one or more opioid haptencarrier protein conjugate.

7. The vaccine composition of claim 6, wherein the one or more opioid hapten is selected from the group consisting of fentanyl, carfentanil, oxycodone, and morphine haptens.

8. The vaccine composition of claim 6 or 7, wherein the one or more opioid hapten-carrier protein conjugate is selected from the group consisting of fentanyl-CRM, carfentanil- CRM, oxycodone-sKLH, and morphine-sKLH.

9. The vaccine composition of any one of claims 6-8, wherein the vaccine composition is a pentavalent vaccine having the methamphetamine conjugate, and four opioid haptencarrier protein conjugates.

10. The vaccine composition of claim 6, comprising two or more opioid hapten-carrier protein conjugates, wherein the two or more opioid hapten-carrier protein conjugates comprise two or more different carrier proteins.

11. The vaccine composition of claim 10, wherein the two or more different carrier proteins comprises KLH and CRM.

12. A bivalent or multivalent vaccine composition comprising an opioid hapten-carrier protein conjugate, and a non-opioid hapten-carrier protein conjugate.

13. A vaccine composition comprising two or more hapten-carrier protein conjugates, wherein the two or more hapten-carrier protein conjugates comprise two or more different carrier proteins (e.g., KLH and CRM).

14. The vaccine composition of claim 12 or 13, wherein the hapten(s) are selected from the group consisting of methamphetamine, fentanyl, carfentanil, oxycodone, and morphine haptens.

15. The vaccine composition of any one of claims 13-14, wherein the hapten-carrier protein conjugates are selected from the group consisting of fentanyl-CRM, carfentanil -CRM, oxycodone-sKLH, and morphine-sKLH.

16. The vaccine composition of any one of claims 12-15, comprising four opioid haptencarrier protein conjugates.

17. The vaccine composition of any one of claims 12-16, comprising four opioid haptencarrier protein conjugates and one non-opioid hapten-carrier protein conjugates.

18. The vaccine composition of claim 17, wherein the non-opioid hapten-carrier protein conjugate is a methamphetamine-carrier protein conjugate.

19. A method of treating OUD or SUD or immunizing a subject (e.g., for preventing drug overdose), comprising administering a vaccine composition according to any of claims 5-18 to the subject.

20. The method of claim 19, further comprising administering one or more booster dose of the vaccine composition.

21. A methamphetamine hapten compound, or salt thereof, having structure of

(Compound 1).