Enhancement of a Heroin Vaccine Through Hapten Deuteration

Abstract

The United States is in the midst of an unprecedented epidemic of opioid substance use disorder and while pharmacotherapies including opioid agonists and antagonists have shown success, they can be inadequate and frequently result in high recidivism. With these challenges facing opioid use disorder treatments immunopharmacotherapy is being explored as an alternative therapy option and is based upon antibody-opioid sequestering to block brain entry. Development of a heroin vaccine has become a major research focal point, however, producing an efficient vaccine against heroin has been particularly challenging because of the need to generate not only a potent immune response but one against heroin and its multiple psychoactive molecules. In this study, we explored the consequence of regioselective deuteration of a heroin-hapten and its impact upon the immune response against heroin and its psychoactive metabolites. Deuterium (H_dAc) and cognate protium heroin (H_Ac) haptens were compared head to head in an inclusive vaccine study. Strikingly the H_dAc vaccine granted greater efficacy in blunting heroin analgesia in murine behavioral models compared to the H_Ac vaccine. Binding studies confirmed that the H_dAc vaccine elicited both greater quantities as well as equivalent or higher affinity antibodies towards heroin and 6-AM. Blood-brain bio-distribution experiments corroborated these affinity tests. These findings suggest that regioselective hapten deuteration could be useful for the resurrection of previous drug of abuse vaccines that have met limited success in the past.

Graphical Abstract

Over the past two decades, mortality attributed to opioid dependence has increased and the opioid epidemic is now a priority for public health globally.^1,2 The epidemic has been fueled by a shift from prescription opioid use to heroin use, and more recently to illicitly manufactured fentanyl.³ Since changes in prescription of opioid guidelines, the use of opioid pain relievers have become less accessible. Consequently, the use of alternatives such as heroin, and/or synthetic opioids have skyrocketed.⁴ Traditional treatments for opioid use disorders have typically relied on either opioid agonists i.e., methadone/buprenorphine or opioid antagonists including naltrexone and naloxone. Although successful for many individuals, these options have drawbacks such as limited ability to attract and keep patients in treatment.⁵ In addition, these therapies may allow addiction to persist and even incur overdose; therefore, it is critical to trial alternatives.^{1, 6}

Immunopharmacotherapeutic interventions are antibody based strategies that target the drug molecule itself, aiming to reduce drug concentration at the site of action, minimizing any pharmacodynamic effect.⁷ Antidrug vaccines have been investigated for cocaine,⁸ nicotine,⁹ methamphetamine^{10, 11} as well as opioids and while created in many formats their overall success still depends upon three components: carrier protein (T-cell epitope), adjuvant and hapten.¹² With the opioid crisis in full view, opioid vaccines have taken center-stage^13–22 including heroin vaccines that have undergone extensive examination. Although success has been claimed with several heroin vaccines, our continued efforts to rationally design suitable heroin-hapten-drug candidates that are complementary to our pre-existing vaccine arsenal is equally important. Our over-arching goal is to deliver a repertoire of antibody centric therapeutics that could ultimately be evaluated in clinical trials.^{23, 24,25–35}

Heroin is sequentially metabolized into 6-monoacetylmorphine (6AM) and then to morphine (Figure 1) and it is often believed that morphine is responsible for the neural effects of heroin.³⁶ However, recent assessments have shown that 6-AM is primarily responsible for the rapid effects of this drug, although heroin does possess μ-opioid binding and receptor activation.^{17, 36, 37} Constructed upon these criteria an improved vaccine would be one that could elicit antibodies capable of blocking heroin and its psychoactive metabolities from entering the brain, thereby preventing heroin-induced reinforcement, pharmacokinetics and locomotor activity. Herein, we detail how hapten deuteration presents a viable tactic that meets these heroin-vaccine requirements as observed by titer, specificity, affinity, drug blood-brain distribution and antinociception evaluation.

Figure 1.

Metabolic pathway for heroin and key metabolites.

The traditional mindset in drug-hapten design is to minimize structural differences between the target drug and the hapten, the idea being to hone the immune response to the drug versus non-psychoactive drug metabolites. With heroin this is complicated as the need is to generate antibodies not just to heroin but also the metabolite 6-AM.³⁶ Accordingly, we pondered whether introduction of deuterium regioselective into the heroin hapten could marshal in both improved antibody-affinity as well as broader specificity towards heroin and its psychoactive metabolites. We envisioned that while divergences between hydrogen and deuterium are subtle i.e. molar volume, lipophilicity and bond length.³⁸ Such variants could still perturb B cell recognition, which is thought to play a dominant role in the recognition of haptens.³⁹

To examine the effects of hapten deuteration on the immune response an unbiased approach was taken where we prepared two heroin-haptens that only differed by deuterium incorporation. The synthesis of protium-heroin hapten, 6, Scheme 1, represents a third-generation heroin-hapten that has undergone extensive examination both in its design and vaccine behavioral testing in rodents and non-human primates. Yet, as stated, vide supra, deficiencies have been observed, most noticeably in recent studies by Townsend and Schwienteck.^{24, 40} Indeed, using our most efficacious heroin-vaccine its was ascertained that the vaccine was less effective than the minimally effective continuous naltrexone dose. As such it was concluded that the vaccine would provide limited clinical effectiveness as an opioid use disorder therapy. Simply stated while our previous vaccine shows efficacy, it most likely will not meet critical end points needed for success in a clinical trial, hence our current efforts.

Scheme 1.

Synthesis and Conjugation of a heroin hapten and its deuterated counterpart.

The overall approach to the regiospecific deuterated heroin hapten 4 follows a similar methodology as used with the making of 6, Scheme 1. The, synthesis is initiated with morphine sulphate 1, wherein d₆-acetylation was installed by treatment with d₆-acetic an-hydride, affording d₆-ester 2. This was followed by demethylation using Olofson’s procedure granting norheroin 2a.⁴¹ Direct N-alkylation of crude 2a with bromobutanoate provided 3. Finally, deprotection under standard TFA conditions furnished the d₆-hapten 4. To generate the immunoconjugates (5 and 7) for vaccination, haptens 4 and 6 were coupled to the carrier protein keyhole limpet hemocyanin (KLH) based on previous protocols.²⁸ Importantly, no significant difference in hapten coupling efficiency was observed between either hapten as determined by MALDI-TOF MS. Finally, hapten bovine serum albumin (BSA) conjugates were prepared for binding assays.

The efficacy of vaccine 5, termed H_dAc and 7, termed H_Ac were assessed in BALB/C mice (n=6 per group). Considering sex as a biological variable, independent vaccine groups consisted of either all male (M) or all female (F) subjects. In total, the vaccine study was comprised of two KLH control groups (M/F) and four heroin hapten-KLH vaccine groups, H_dAc (M/F) and H_Ac (M/F). Each group was administered the appropriate vaccine formulation on day 0, 14, 28, and 51 and bled on day 21, 42, and 71 day (see SI). This vaccine schedule allowed us to examine titers to both haptens and potential cross-reactivity patterns between haptens and immune response to each. In general, the H_dAc-M/F vaccine groups generated higher antibody titers than the analogous H_Ac-(M/F) vaccine groups, Figure 2A. Moreover, the polyclonal antibody responses generated against the H_dAc-(M/F) haptens recognized the H_Ac-BSA conjugate better than the H_dAc-(M/F) antibodies. (SI).

Figure 2.

Effects of H_dAc and H_Ac vaccines on titer and affinity. A) Midpoint titer results from crossover ELISA experiments (week 11 sera). Each column represents the average antibody titer in the sera from 6 individual mice. Midpoint titers are reported as means ± standard error of the mean. Significance is denoted by an asterisk (for H_dAc-BSA and H_Ac-BSA as coating antigens) for midpoint titer from a one-way ANOVA and a Dunnetts post hoc test when comparing vaccinated groups to controls ***P ≤ 0.001; *P < 0.05 vs control. B) IC₅₀ values (nM) as determined by SPR for each vaccine group with pooled week 11 sera.

To complement the ELISA titer data, surface plasmon resonance (SPR) were conducted to examine antibody affinity and selectivity to heroin and 6-AM, (IC₅₀ values are shown in Figure 2B). While there appeared to be no real differences in affinity (both male and female mice) to 6-AM between the H_Ac and H_dAc vaccines; we saw large differences in affinity to heroin. The H_dAc vaccine generated tighter binding affinity to heroin whereas with the H_Ac vaccine poor affinity was seen to heroin (Figure 2B). We posit that the increase in effective binding generated by the H_dAc vaccine could arise from different T-cell dependant B-cell receptor activation (BCR) populations, to generate polyclonal antibodies that bind free drug in serum. Such differences in affinity have been observed with modified amino acids and suggests that while deuterium’s chemical differences are subtle, they appear to be readily differentiated by the immune system.^{42, 43}

Basic behavioral tests including antinociception hot plate and tail-flick assays were carried out to compare the efficacy of the two vaccines, Figure 3A. The H_dAc vaccine outperformed the H_Ac vaccine in both hot plate and tail flick assays (see SI for tail-flick data). The improved antinociception results for the H_dAc vaccine are readily explained by the improved titers as well as a potent affinity to 6-AM and the additional binding seen to heroin. We contrast this to a previous morphine vaccine that had a poor affinity for 6-AM/heroin and was not effective³⁵. Blood-brain distribution analysis (Figure 3B) revealed that the H_dAc vaccine sequestered more active metabolite 6-AM and contained a lower blood to brain ratio compared to the original heroin vaccine. This also held for both sexes of animals tested. In trying to rationalize the genesis of this phenomenon it is recognized that deuteration is a well-established means to slow down drug metabolism, especially cytochrome P450 mediated transformations.^{38, 44} However, the case at hand the deuterated esters contained within the H_dAc hapten would not be susceptible to P450 relegation. We have also investigated simple aqueous hydrolysis of heroin and the heroin-d6 ester and their first order rate constants appear indistinguishable (SI). So, stability to aqueous hydrolysis appears not responsible for the results seen.

Figure 3.

Comparison of H_dAc and H_Ac vaccines on heroin vaccine efficacy in antinociception and blood brain bio-distribution of 6AM. A) Left panel hot plate data after week 11 for both male and female BALB/C mice. Bars represent ± SEM, where each vaccine group is comprised of 6 mice and control groups are comprised of 4 mice. B) Right panel blood brain distribution study of pooled sera from each vaccine group.

From an immunological perspective we would suggest that our findings cannot be readily evoked with a singular unifying principle. Rather, we envision two scenarios that could account for the data presented. We speculate that the deuteration of the heroin hapten could modify or improve the binding of hapten to BCRs improving activation of B-cell populations. Equally plausible is that hapten-deuteration of the carrier protein could modify or improve T-cell receptor recognition. Alternatively heroin-haptenic sequence fragments displayed upon the major histocompatibility complex II (MHCII) / T-cell epitope(s) may also be altered, and presented contrarily to the (MHC) receptor sites.^{42, 43} Either outcome could play a role towards an improvement in hapten immunogenicity. Moreover, there is strong evidence that rationally modified antigens can boost BCR binding and overcome the poor antigenicity of native antigens.^{45, 46} Minimal isosteric replacement by changing the hydrogen atom to a fluorine atom has improved vaccine efficacy against cocaine and provoked additional investigations into electronic properties of various halogenated 4-substituted benzoyl groups.⁴⁷ Hapten fluorination data suggest that a combination of electrostatics and hydrophobicity are the main determinants for how fluorine can modulate BCR binding affinity.⁴⁷ Similar principles could be educed for the H_dAc vaccine.

A second course that could also account in part for the results seen comes from the dendritic cell processing of the hapten-carrier-complex, which involves denaturation, reduction and/or proteolysis.^{48, 49} It is this later processing where deuteration could compensate for the hydrogen esters embedded within heroin. Clopidogrel-d₃ methyl ester has been shown to be about 4.5 times more stable than its protium counterpart in rodent studies.⁵⁰ Major proteases are involved in antigen processing within the dendric cell. Based on this enabled logic, we hypothesize that the d₆-heroin esters could be more stable against enzymatic hydrolysis and therefore improve heroin APC presentation by dendritic cells and macrophages. This would also help support the SPR data we have presented where the H_dAc vaccine provides antibodies with excellent affinity to heroin while the H_Ac vaccine generates poor affinity antibodies to heroin.

By numerous measures, rates of opioid misuse and overdose have reached new heights in recent years. This has been fueled by an abundance of prescription opioids and recent spikes in heroin and synthetic opioid use. We have presented data showing how regioselective hapten deuteration can improve a heroin vaccines efficacy as viewed from titer, affinity, specificity and behavioral models including antinociception and drug-bio-distribution. Regioselective hapten deuteration has never been examined at any level of immune response and/or its recognition. At, the onset of our research we expected that hapten deuteration could alter antibody affinity/specificity as antibody affinity/specificity is largely driven by the antigen. However, we were surprised to see antibody titers affected by hapten deuteration as titers are largely influenced by the adjuvant.⁵¹ A further understanding of how regioselective hapten-deuteration modulates the immune response could prove valuable for reviving other drug of abuse vaccines that have failed in clinical trials.^{52, 53} Finally, not lost upon us is that persons with an opioid use disorder are especially vulnerable to COVID-19 and thus colliding epidemics.^{2, 54} An opioid treatment that could counteract one of these risks would reduce the diversion of resources from those requiring acute care from COVID-19.