Enhancing Efficacy and Stability of an Anti-Heroin Vaccine: Examination of Antinociception, Opioid Binding Profile, and Lethality Candy S Hwang, Paul T
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Article Enhancing Efficacy and Stability of an Anti-Heroin Vaccine: Examination of Antinociception, Opioid Binding Profile, and Lethality Candy S Hwang, Paul T. Bremer, Cody J Wenthur, Sam On Ho, SuMing Chiang, Beverly Ellis, Bin Zhou, Gary Fujii, and Kim D Janda Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00933 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. 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ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts. Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Page 1 of 13 Molecular Pharmaceutics 1 2 3 4 5 6 7 Enhancing Efficacy and Stability of an Anti-Heroin Vaccine: 8 Examination of Antinociception, Opioid Binding Profile, and 9 10 Lethality 11 12 Candy S. Hwang1, Paul T. Bremer1, Cody J. Wenthur1, Sam On Ho2, SuMing Chiang2, Beverly Ellis1, Bin Zhou1, 13 Gary Fujii2, Kim D. Janda1,* 14 15 1Departments of Chemistry, Immunology and Microbial Science, Skaggs Institute for Chemical Biology; The Scripps Research Insti- 16 tute, La Jolla, CA, USA; 2 Molecular Express, Inc., Rancho Dominguez, CA, USA 17 18 19 KEYWORDS: heroin, opioids, vaccine, immunopharmacotherapy, adjuvants 20 Supporting Information Placeholder 21 22 strongest risk factor for initiating heroin abuse, and OPR users 23 ABSTRACT: In recent years, drug conjugate vaccines have are 40 times more likely to abuse heroin.3, 4 This phenomenon is 24 shown promise as therapeutics for substance use disorder. As driven by the relatively low cost of heroin and its wide availabil- 25 a means to improve the efficacy of a heroin conjugate vac- ity.3, 4 Current treatments for heroin addiction involve opioid 26 cine, we systematically explored twenty vaccine formulations replacement therapy e.g., methadone administration, to pro- 27 with varying combinations of carrier proteins and adjuvants. mote heroin detoxification.5 Unfortunately, the addictive nature 28 In regard to adjuvants, we explored a Toll-like receptor 9 of heroin and other opioids, combined with the adverse effects 29 (TLR9) agonist and a TLR3 agonist in the presence of alum. of withdrawal and high cost of treatment, lead to a high inci- 30 The TLR9 agonist was cytosine-guanine oligodeoxynucleo- dence of drug relapse.5 3, 4 In the face of increasing opioid abuse 31 tide 1826 (CpG ODN 1826), while the TLR3 agonist was and overdose, the development of improved therapies that can 32 virus-derived genomic doubled-stranded RNA (dsRNA). attenuate the effects of opioids is crucial. 33 The vaccine formulations containing TLR3 or TLR9 agonist Vaccination is a promising strategy to promote cessation of 34 alone elicited strong anti-heroin antibody titers and blockade heroin abuse and prevent relapse. Implementation of this strat- 35 of heroin-induced antinociception when formulated with 36 egy involves active immunization using a small molecule- alum; however, a combination of TLR3 and 9 adjuvants did protein conjugate, which elicits high-affinity, drug-specific anti- 37 not result in improved efficacy. Investigation of month-long 38 bodies. These polyclonal IgG antibodies sequester free drug in stability of the two lead formulations revealed that the TLR9 39 the blood and prevent access to the brain, subsequently reduc- but not the TLR3 formulation was stable when stored as a 40 ing the drug compound’s psychoactive effects. This approach lyophilized solid or as a liquid over 30 days. Furthermore, 6, 7 41 has been pre-clinically validated for vaccines against nicotine, 8, 9 10, 11 42 mice immunized with the TLR9 + alum heroin vaccine cocaine, and methamphetamine. For heroin specifically, 43 gained significant protection from lethal heroin doses, sug- vaccination efficacy has been repeatedly demonstrated in mice, 12-18 44 gesting that this vaccine formulation is suitable for mitigating rats, and non-human primates. 45 the harmful effects of heroin, even following month-long In general, formulation of a vaccine with an adjuvant is an at- 46 storage at room temperature. tractive approach to enhance the magnitude and length of vac- 47 cine immunity against the target antigen by stimulating antigen 48 presenting cells, T-cells or B-cells. Historically, Alhydrogel (al- 49 um) has been the most commonly used adjuvant, but numerous Introduction 50 alternatives have been pursued in recent years.19 Adjuvants can 51 Heroin is a schedule I, highly addictive opioid drug and a sig- act as pathogen-associated molecular patterns (PAMPs), which 52 nificant public health concern. In the US, drug overdose deaths activate Toll-like receptors (TLRs) resulting in upregulation of 1 53 have nearly tripled between 1999 and 2014. In 2015, 52,404 an immune response. Specific PAMPs include lipopolysaccha- 1 54 overdose deaths were reported, 63% of which involved opioids. rides (LPS), double-stranded RNA (dsRNA) and unmethylated 55 Recently, there has been a marked increase in prescriptions of cytosine-guanine (CpG) motifs.19 However, at this time only a 56 synthetic opioid pain relievers (OPRs) for management of limited number of adjuvants are approved for use in humans. By 2 57 chronic pain. Evidence suggests that misuse of OPRs is the exploring new adjuvants or combinations of adjuvants, we can 58 1 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 2 of 13 rationally design vaccines with enhanced immunogenicity di- rected toward production of heroin-neutralizing antibodies. 1 2 3 4 5 6 7 8 9 Figure 1. Structures of the heroin haptens, corresponding immunoconjugates and the general vaccine approach. The structure of 10 heroin is highlighted in red. 11 CpG oligodeoxynucleotide (ODN) 1826 is a B-class ODN an immune epitope with high structural congruence to heroin. 12 that stimulates B-cell responses though TLR920, 21 and was (7, Figure 1 and Scheme S1).22 We also prepared a heroin 13 recently shown to elicit robust titers in anti-heroin vaccine hapten with a truncated linker at this same position to probe 14 studies.13, 22 Natural or synthetic dsRNA, e.g., polyinosi- the effect of linker length on vaccine efficacy (11, Figures 1 15 ic:polycytidylicacid (poly I:C), is a molecular pattern associ- and S1). The haptens were activated and conjugated to carrier 16 ated with viral replication, which elicits an immune response protein tetanus toxoid (TT) or a mutant diphtheria toxoid 17 via TLR3 and has been used as an effective adjuvant in several (CRM), using an EDC-mediated coupling reaction (Figure 18 vaccine studies.23-25 Given the potent immunostimulatory 1), followed by dialysis against pH 7.4 phosphate buffered 19 capacity of viral or bacterial PAMPs, we were interested in saline (PBS). The degree of haptenation was determined by 20 evaluating the efficacy of a yeast-derived viral dsRNA genome MALDI-ToF mass spectrometry, using a heroin-bovine serum 21 relative to CpG ODN, using a well-studied dsRNA virus of albumin (Her-BSA) immunoconjugate as a surrogate for de- 22 Saccharomyces cerevisiae, L-A.26 To date, only the L-BC viral termining hapten density (Figures S9-14). Immunoconjugates 23 dsRNA genome generated from infected S. cerevisiae has been were stored at -80 ˚C until the day of formulation and vaccina- 24 used as an adjuvant, where it increased immunogenicity of a tion. More information on the synthesis and characterization 25 prophylactic viral vaccine in mice.27 data are described in the supplemental information. 26 Here, we investigate L-A-derived dsRNA in combination Vaccine Formulation 27 with alum and/or CpG ODN in the context of our drug of After conjugation of the proteins, immunoconjugates were 28 abuse vaccine. Although alum is not necessary for TLR activa- formulated with different adjuvants as described in Tables S1- 29 tion, it is one of the few adjuvants used in FDA-approved vac- 5. The adjuvants were CpG ODN 1826, dsRNA, Alhydrogel 30 cines and has shown promising activity in anti-drug vaccines. (alum), and VesiVax® CALV.28 CpG ODN 1826 is a phos- 31 In comparison to alum, we selected