RESUMO
Fentanyl and other synthetic opioids are the leading cause of drug-related deaths in the United States. mAbs that selectively target fentanyl and fentanyl analogues offer a promising strategy for treating both opioid-related overdoses and opioid use disorders. To increase the duration of efficacy of a candidate mAb against fentanyl, we selected three sets of mutations in the Fc region of an IgG1 anti-fentanyl mAb (HY6-F9DF215, HY6-F9DHS, HY6-F9YTE) to increase binding to the neonatal Fc receptor (FcRn). The mAb mutants were compared against unmodified (wild-type [WT], HY6-F9WT) anti-fentanyl mAb for fentanyl binding, thermal stability, and FcRn affinity in vitro, and for efficacy against fentanyl and mAb half-life in vivo in mice. Biolayer interferometry showed a >10-fold increase in the affinity for recombinant FcRn of the three mutant mAbs compared with HY6-F9WT. During an acute fentanyl challenge in mice, all FcRn-mutated mAbs provided equal protection against fentanyl-induced effects, and all mAbs reduced brain fentanyl levels compared with the saline group. Serum persistence of the mutant mAbs was tested in Tg276 transgenic mice expressing human FcRn. After administration of 40 mg/kg HY6-F9WT, HY6-F9DF215, HY6-F9DHS, and HY6-F9YTE, the mAbs showed half-lives of 6.3, 26.4, 14.7, and 6.9 d, respectively. These data suggest that modification of mAbs against fentanyl to bind to FcRn with higher affinity can increase their half-life relative to WT mAbs while maintaining efficacy against the toxic effects of fentanyl, further supporting their potential role as a therapeutic treatment option for opioid use disorder and overdose.
Assuntos
Anticorpos Monoclonais , Fentanila , Antígenos de Histocompatibilidade Classe I , Fragmentos Fc das Imunoglobulinas , Mutação , Receptores Fc , Fentanila/imunologia , Animais , Camundongos , Receptores Fc/genética , Receptores Fc/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Analgésicos Opioides , Meia-Vida , Engenharia de Proteínas , Imunoglobulina G/imunologiaRESUMO
Opioid use disorders (OUD) and overdoses are ever-evolving public health threats that continue to grow in incidence and prevalence in the United States and abroad. Current treatments consist of opioid receptor agonists and antagonists, which are safe and effective but still suffer from some limitations. Murine and humanized monoclonal antibodies (mAb) have emerged as an alternative and complementary strategy to reverse and prevent opioid-induced respiratory depression. To explore antibody applications beyond traditional heavy-light chain mAbs, we identified and biophysically characterized a novel single-domain antibody specific for fentanyl from a camelid variable-heavy-heavy (VHH) domain phage display library. Structural data suggested that VHH binding to fentanyl was facilitated by a unique domain-swapped dimerization mechanism, which accompanied a rearrangement of complementarity-determining region loops leading to the formation of a fentanyl-binding pocket. Structure-guided mutagenesis further identified an amino acid substitution that improved the affinity and relaxed the requirement for dimerization of the VHH in fentanyl binding. Our studies demonstrate VHH engagement of an opioid and inform on how to further engineer a VHH for enhanced stability and efficacy, laying the groundwork for exploring the in vivo applications of VHH-based biologics against OUD and overdose.
RESUMO
Opioid use disorders (OUDs) are a public health concern in the United States and worldwide. Current medications for OUDs may trigger side effects and are often heavily regulated. A novel treatment strategy to be used alone or in combination with existing medications is active immunization with antiopioid vaccines, which stimulate production of opioid-specific Abs that bind to the target drug and prevent its distribution to the brain. Although antiopioid vaccines have shown promising preclinical efficacy, prior clinical evaluations of vaccines targeting stimulants indicate that efficacy is limited to a subset of subjects who achieve optimal Ab responses. We have previously reported that depletion of IL-4 with a mAb increased opioid-specific IgG2a and total IgG, and it increased the number of germinal centers and germinal center T follicular helper cells in response to antiopioid vaccines via type I IL-4 signaling. The current study further investigates the mechanisms associated with IL-4-mediated increases in efficacy and whether IL-4 depletion affects specific processes involved in germinal center formation, including affinity maturation, class switching, and plasma cell differentiation in mice. Additionally, results demonstrate that preimmunization production of IL-4 after ex vivo whole blood stimulation predicted in vivo vaccine-induced Ab titers in outbred mice. Such mechanistic studies are critical for rational design of next-generation vaccine formulations, and they support the use of IL-4 as a predictive biomarker in ongoing OUD vaccine clinical studies.
Assuntos
Interleucina-4 , Vacinas , Camundongos , Animais , Analgésicos Opioides , Centro Germinativo , Células Produtoras de Anticorpos , Proliferação de CélulasRESUMO
Currently approved pharmacotherapies for opioid use disorders (OUDs) and overdose reversal agents are insufficient to slow the spread of OUDs due to the proliferation of fentanyl. This is evident in the 31% rise in drug overdose deaths from 2019 to 2022, with rates increasing from 21.6 to 28.3 overdoses per 100,000 deaths. Vaccines are a potential alternative or adjunct therapy for the treatment of several substance use disorders (nicotine, cocaine) but have shown limited clinical success due to suboptimal antibody titers. In this study, we demonstrate that coconjugation of a Toll-like receptor 7/8 (TLR7/8) agonist (UM-3006) alongside a fentanyl-based hapten (F1) on the surface of the carrier protein cross-reactive material 197 (CRM) significantly increased generation of high-affinity fentanyl-specific antibodies. This demonstrated enhanced protection against fentanyl challenges relative to an unconjugated (admix) adjuvant control in mice. Inclusion of aluminum hydroxide (alum) adjuvant further increased titers and enhanced protection, as determined by analysis of fentanyl concentration in serum and brain tissue. Collectively, our findings present a promising approach to enhance the efficacy of antiopioid vaccines, underscoring the need for extensive exploration of TLR7/8 agonist conjugates as a compelling strategy to combat opioid use disorders.
Assuntos
Transtornos Relacionados ao Uso de Opioides , Vacinas , Animais , Camundongos , Receptor 7 Toll-Like/agonistas , Fentanila/uso terapêutico , Adjuvantes Imunológicos/uso terapêutico , Antígenos/uso terapêutico , Haptenos , Transtornos Relacionados ao Uso de Opioides/prevenção & controle , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Analgésicos Opioides/uso terapêuticoRESUMO
Vaccines and mAbs offer promising strategies to treat substance use disorders (SUDs) and prevent overdose. Despite vaccines and mAbs against SUDs demonstrating proof of efficacy, selectivity, and safety in animal models, it is unknown whether the mechanism of action of these immunotherapeutics relies exclusively on the formation of Ab/drug complexes, or also involves Ab-mediated effector functions. Hence, this study tested whether the efficacy of active and passive immunization against drugs of abuse requires phagocytosis, the intact Fc portion of the anti-drug Ab, FcγRs, or the neonatal FcR (FcRn). The efficacy of a lead vaccine against oxycodone was not diminished in mice after depletion of macrophages or granulocytes. Anti-oxycodone F(ab')2 fragments resulted in lower serum levels of F(ab')2 compared with intact mAbs, and F(ab')2s were not as effective as the parent mAbs in reducing distribution of oxycodone to the brain. The efficacy of vaccines and mAbs against oxycodone was preserved in either FcγIII or FcγI-IV ablated mice, suggesting that FcγRs are not required for Ab efficacy. Finally, both active and passive immunization against oxycodone in FcRn-/- mice yielded reduced efficacy compared with wild-type control mice. These data identified a role for FcRn, but not for phagocytosis or Fc-dependent effector functions, in mediating the efficacy of vaccines and mAbs against SUD. This study supports rational design of vaccines and mAbs engineered for maximal neutralization activity and optimal FcRn binding.
Assuntos
Transtornos Relacionados ao Uso de Opioides , Vacinas , Animais , Anticorpos Monoclonais , Camundongos , OxicodonaRESUMO
The incidence of fatal drug overdoses in the United States is an alarming public health threat that has been exacerbated by the COVID-19 pandemic, resulting in over 100,000 deaths between April 2020 and April 2021. A significant portion of this is attributable to widespread access to fentanyl and other synthetic opioids, alone or in combination with heroin or psychostimulants, such as cocaine or methamphetamine. Monoclonal antibodies (mAb) offer prophylactic and therapeutic interventions against opioid overdose by binding opioids in serum, reducing distribution of drug to the brain and other organs. Here, we investigated the efficacy of a leading antifentanyl mAb, clone HY6-F9, in reversal and prevention of fentanyl-induced toxicity compared with the opioid receptor antagonist naloxone (NLX) in rats. In postexposure models, rats were challenged with fentanyl, followed by HY6-F9, NLX, or both. HY6-F9 reversed fentanyl-induced antinociception, respiratory depression, and bradycardia, and rats retained protection against additional challenges for at least 1 week. Although intravenous NLX reversed fentanyl-induced respiratory depression more rapidly than mAb alone, kinetics of reversal by intravenous mAb were similar to subcutaneous NLX. Coadministration of mAb and NLX provided greater protection than individual treatments against high doses of fentanyl. Prophylactic administration of mAb reduced the ED50 of NLX approximately twofold against 2.25 mg/kg of fentanyl. Finally, mAb sequestered fentanyl and its metabolite norfentanyl in serum and reduced brain concentrations of fentanyl. These results support the translation of mAb as medical interventions alone or in combination with NLX to prevent and reverse fentanyl-related overdose. SIGNIFICANCE STATEMENT: Fentanyl-related overdoses have increased dramatically in the US and worldwide. Currently, approved pharmacotherapies for treatment of opioid use disorder and reversal of overdose are not sufficient to curb the incidence of opioid-related deaths. Additionally, fentanyl and its potent analogs present a potential risk from use in deliberate poisoning or chemical attacks. This study demonstrates the use of monoclonal antibodies as a countermeasure to fentanyl-induced toxicity in pre- and postexposure scenarios, supporting their use in combination with the opioid antagonist naloxone.
Assuntos
COVID-19 , Overdose de Drogas , Insuficiência Respiratória , Analgésicos Opioides/uso terapêutico , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Overdose de Drogas/tratamento farmacológico , Fentanila , Humanos , Naloxona/farmacologia , Naloxona/uso terapêutico , Antagonistas de Entorpecentes/farmacologia , Pandemias , Ratos , Insuficiência Respiratória/tratamento farmacológicoRESUMO
Increased opioid use and misuse have imposed large analytical demands across clinical and forensic sectors. Due to the absence of affordable, accurate, and simple on-site tests (e.g., point of interdiction and bedside), analysis is primarily conducted in centralized laboratories via time-consuming, labor-intensive methods. Many healthcare facilities do not have such analytical capabilities and must send samples to commercial laboratories, increasing turnaround time and care costs, as well as delaying public health warnings regarding the emergence of specific substances. Enzyme-linked immunosorbent assays (ELISAs) are used ubiquitously, despite lengthy workflows that require substantial manual intervention. Faster, reliable analytics are desperately needed to mitigate the mortality and morbidity associated with the current substance use epidemic. We describe one such alternativeâa portable centrifugal microfluidic ELISA system that supplants repetitive pipetting with rotationally controlled fluidics. Embedded cellulosic membranes act as microvalves, permitting flow only when centrifugally generated hydraulic pressure exceeds their liquid entry pressure. These features enable stepwise reagent introduction, incubation, and removal simply by tuning rotational frequency. We demonstrate the success of this platform through sensitive, specific colorimetric detection of opiates, a subclass of opioids naturally derived from the opium poppy. Objective image analysis eliminated subjectivity in human color perception and permitted reliable detection of opiates in buffer and artificial urine at the ng/µL range. Opiates were clearly differentiated from other drug classes without interference from common adulterants known to cause false positive results in current colorimetric field tests. Eight samples were simultaneously analyzed in under 1 h, a marked reduction from the traditional multiday timeline. This approach could permit rapid, automatable ELISA-based drug detection outside of traditional laboratories by nontechnical personnel.
Assuntos
Preparações Farmacêuticas , Detecção do Abuso de Substâncias , Colorimetria , Ensaio de Imunoadsorção Enzimática , Humanos , MicrofluídicaRESUMO
Opioid use disorders (OUDs) and opioid-related fatal overdoses are a significant public health concern in the United States and worldwide. To offer more effective medical interventions to treat or prevent OUD, antiopioid vaccines are in development that reduce the distribution of the targeted opioids to brain and subsequently reduce the associated behavioral and toxic effects. It is of critical importance that antiopioid vaccines do not interfere with medications that treat OUD. Hence, this study tested the preclinical proof of concept of combining a candidate oxycodone vaccine [oxycodone-keyhole limpet hemocyanin (OXY-KLH)] with an FDA-approved extended-release naltrexone (XR-NTX) depot formulation in rats. The effects of XR-NTX on oxycodone-induced motor activity and antinociception were first assessed in nonvaccinated naïve rats to establish a baseline for subsequent studies. Next, OXY-KLH and XR-NTX were coadministered to determine whether the combination would affect the efficacy of each individual treatment, and it was found that the combination of OXY-KLH and XR-NTX offered greater efficacy in reducing oxycodone-induced motor activity, thigmotaxis, antinociception, and respiratory depression over a range of repeated or escalating oxycodone doses in rats. These data support the feasibility of combining antibody-based therapies with opioid receptor antagonists to provide greater or prolonged protection against opioid-related toxicity or overdose. Combining antiopioid vaccines with XR-NTX may provide prophylactic measures to subjects at risk of relapse and accidental or deliberate exposure. Combination therapy may extend to other biologics (e.g., monoclonal antibodies) and medications against substance use disorders. SIGNIFICANCE STATEMENT: Opioid use disorders (OUDs) remain a major problem worldwide, and new therapies are needed. This study reports on the combination of an oxycodone vaccine [oxycodone-keyhole limpet hemocyanin (OXY-KLH)] with a currently approved OUD therapy, extended-release naltrexone (XR-NTX). Results demonstrated that XR-NTX did not interfere with OXY-KLH efficacy, and combination of low doses of XR-NTX with vaccine was more effective than each individual treatment alone to reduce behavioral and toxic effects of oxycodone, suggesting that combining OXY-KLH with XR-NTX may improve OUD outcomes.
Assuntos
Preparações de Ação Retardada/farmacologia , Naltrexona/farmacologia , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Oxicodona/farmacologia , Vacinas/administração & dosagem , Analgésicos Opioides/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Hemocianinas/farmacologia , Masculino , Antagonistas de Entorpecentes/farmacologia , RatosRESUMO
Monoclonal antibodies (mAbs) and vaccines have been proposed as medical countermeasures to treat opioid use disorder (OUD) and prevent opioid overdose. In contrast to current pharmacotherapies (e.g., methadone, buprenorphine, naltrexone, and naloxone) for OUD and overdose, which target brain opioid receptors, mAbs and vaccine-generated polyclonal antibodies sequester the target opioid in the serum and reduce drug distribution to the brain. Furthermore, mAbs offer several potential clinical benefits over approved medications, such as longer serum half-life, higher selectivity, reduced side effects, and no abuse liability. Using magnetic enrichment to isolate opioid-specific B cell lymphocytes prior to fusion with myeloma partners, this study identified a series of murine hybridoma cell lines expressing mAbs with high affinity for opioids of clinical interest, including oxycodone, heroin and its active metabolites, and fentanyl. In mice, passive immunization with lead mAbs against oxycodone, heroin, and fentanyl reduced drug-induced antinociception and the distribution of the target opioid to the brain. In mice and rats, mAb pretreatment reduced fentanyl-induced respiratory depression and bradycardia, two risk factors for opioid-related overdose fatality. Overall, these results support use of mAbs to counteract toxic effects of opioids and other chemical threats. SIGNIFICANCE STATEMENT: The incidence of fatal overdoses due to the widespread access to heroin, prescription opioids, and fentanyl suggests that current Food and Drug Administration-approved countermeasures are not sufficient to mitigate the opioid epidemic. Monoclonal antibodies (mAbs) may provide acute protection from overdose by binding to circulating opioids in serum. Use of mAbs prophylactically, or after exposure in combination with naloxone, may reduce hospitalization and increase survival.
Assuntos
Analgésicos Opioides/imunologia , Analgésicos Opioides/toxicidade , Anticorpos Monoclonais/imunologia , Comportamento Animal , Animais , Feminino , Imunização , Masculino , Camundongos , RatosRESUMO
The opioid epidemic has become a severe public health problem, with approximately 130 opioid-induced deaths occurring each day in the United States. Prescription opioids are responsible for approximately 40% of these deaths. Oxycodone is one of the most commonly abused prescription opioids, but despite its prevalent misuse, the number of preclinical studies investigating oxycodone-seeking behaviors is relatively limited. Furthermore, preclinical oxycodone studies that include female subjects are even more scarce, and it is critical that future work includes both sexes. Additionally, the oral route of administration is one of the most common routes for recreational users, especially in the early stages of drug experimentation. However, currently, only two studies have been published investigating operant oral oxycodone self-administration in rodents. Therefore, the primary goal of the present study was to establish an oral oxycodone operant self-administration model in adult male and female rats, as well as to examine a potential mechanism of stress-primed reinstatement. We found that females consumed significantly more oral oxycodone than males in operant self-administration sessions. We also found that active oxycodone self-administration was reduced by mu opioid receptor antagonism and by substitution of water for oxycodone solution. Lastly, we induced stress-primed reinstatement and found that this behavior was significantly attenuated by antagonism of the neurokinin-1 receptor, consistent with our prior work examining stress-induced reinstatement of alcohol- and cocaine-seeking.
Assuntos
Analgésicos Opioides/administração & dosagem , Oxicodona/administração & dosagem , Uso Indevido de Medicamentos sob Prescrição , Receptores da Neurocinina-1/fisiologia , Autoadministração , Analgésicos Opioides/sangue , Animais , Comportamento de Procura de Droga/efeitos dos fármacos , Ciclo Estral/efeitos dos fármacos , Extinção Psicológica/efeitos dos fármacos , Feminino , Masculino , Naloxona/administração & dosagem , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Oxicodona/sangue , Ratos , Ratos Wistar , Receptores Opioides mu/antagonistas & inibidoresRESUMO
Fentanyl is an extremely potent synthetic opioid that has been increasingly used to adulterate heroin, cocaine, and counterfeit prescription pills, leading to an increase in opioid-induced fatal overdoses in the United States, Canada, and Europe. A vaccine targeting fentanyl could offer protection against the toxic effects of fentanyl in both recreational drug users and others in professions at risk of accidental exposure. This study focuses on the development of a vaccine consisting of a fentanyl-based hapten (F) conjugated to keyhole limpet hemocyanin (KLH) carrier protein or to GMP-grade subunit KLH (sKLH). Immunization with F-KLH in mice and rats reduced fentanyl-induced hotplate antinociception, and in rats reduced fentanyl distribution to the brain compared with controls. F-KLH did not reduce the antinociceptive effects of equianalgesic doses of heroin or oxycodone in rats. To assess the vaccine effect on fentanyl toxicity, rats immunized with F-sKLH or unconjugated sKLH were exposed to increasing subcutaneous doses of fentanyl. Vaccination with F-sKLH shifted the dose-response curves to the right for both fentanyl-induced antinociception and respiratory depression. Naloxone reversed fentanyl effects in both groups, showing that its ability to reverse respiratory depression was preserved. These data demonstrate preclinical selectivity and efficacy of a fentanyl vaccine and suggest that vaccines may offer a therapeutic option in reducing fentanyl-induced side effects.
Assuntos
Analgésicos Opioides/antagonistas & inibidores , Analgésicos Opioides/metabolismo , Fentanila/antagonistas & inibidores , Fentanila/metabolismo , Vacinas/farmacologia , Analgésicos Opioides/toxicidade , Animais , Bradicardia/sangue , Bradicardia/induzido quimicamente , Bradicardia/prevenção & controle , Fentanila/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Sprague-Dawley , Insuficiência Respiratória/sangue , Insuficiência Respiratória/induzido quimicamente , Insuficiência Respiratória/prevenção & controle , Vacinas/uso terapêuticoRESUMO
Heroin and oxycodone abuse occurs over a wide range of drug doses and by various routes of administration characterized by differing rates of drug absorption. The current study addressed the efficacy of a heroin vaccine [morphine hapten conjugated to keyhole limpet hemocyanin (M-KLH)] or oxycodone vaccine [oxycodone hapten conjugated to keyhole limpet hemocyanin (OXY-KLH)] for reducing drug distribution to brain after intravenous heroin or oxycodone, or subcutaneous oxycodone. Rats immunized with M-KLH or keyhole limpet hemocyanin (KLH) control received an intravenous bolus dose of 0.26 or 2.6 mg/kg heroin. Vaccination with M-KLH increased retention of heroin and its active metabolites 6-acetylmorphine (6-AM) and morphine in plasma compared with KLH controls, and reduced total opioid (heroin + 6-AM + morphine) distribution to brain but only at the lower heroin dose. Immunization also protected against respiratory depression at the lower heroin dose. Rats immunized with OXY-KLH or KLH control received 0.22 or 2.2 mg/kg oxycodone intravenously, the molar equivalent of the heroin doses. Immunization with OXY-KLH significantly reduced oxycodone distribution to brain after either oxycodone dose, although the magnitude of effect of immunization at the higher oxycodone dose was small (12%). By contrast, vaccination with OXY-KLH was more effective when oxycodone was administered subcutaneously rather than intravenously, reducing oxycodone distribution to brain by 44% after an oxycodone dose of 2.3 mg/kg. Vaccination also reduced oxycodone-induced antinociception. These data suggest that the efficacy of OXY-KLH and M-KLH opioid vaccines is highly dependent upon opioid dose and route of administration.
Assuntos
Heroína/administração & dosagem , Transtornos Relacionados ao Uso de Opioides/prevenção & controle , Oxicodona/administração & dosagem , Vacinas/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Heroína/metabolismo , Heroína/farmacologia , Masculino , Oxicodona/metabolismo , Oxicodona/farmacologia , RatosRESUMO
Vaccines may offer a new treatment strategy for opioid use disorders and opioid-related overdoses. To speed translation, this study evaluates opioid conjugate vaccines containing components suitable for pharmaceutical manufacturing and compares analytical assays for conjugate characterization. Three oxycodone-based haptens (OXY) containing either PEGylated or tetraglycine [(Gly)4] linkers were conjugated to a keyhole limpet hemocyanin (KLH) carrier protein via carbodiimide (EDAC) or maleimide chemistry. The EDAC-conjugated OXY(Gly)4-KLH was most effective in reducing oxycodone distribution to the brain in mice. Vaccine efficacy was T cell-dependent. The lead OXY hapten was conjugated to the KLH, tetanus toxoid, diphtheria cross-reactive material (CRM), as well as the E. coli-expressed CRM (EcoCRM) and nontoxic tetanus toxin heavy chain fragment C (rTTHc) carrier proteins. All vaccines induced early hapten-specific B cell expansion and showed equivalent efficacy against oxycodone in mice. However, some hapten-protein conjugates were easier to characterize for molecular weight and size. Finally, heroin vaccines formulated with either EcoCRM or KLH were equally effective in reducing heroin-induced antinociception and distribution to the brain of heroin and its metabolites in mice. This study identifies vaccine candidates and vaccine components for further development.
Assuntos
Portadores de Fármacos/química , Overdose de Drogas/terapia , Heroína/administração & dosagem , Transtornos Relacionados ao Uso de Opioides/terapia , Oxicodona/administração & dosagem , Adjuvantes Imunológicos/química , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteínas de Transporte/química , Modelos Animais de Doenças , Composição de Medicamentos/métodos , Haptenos/química , Hemocianinas/química , Heroína/química , Heroína/imunologia , Heroína/farmacocinética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nociceptividade/efeitos dos fármacos , Transtornos Relacionados ao Uso de Opioides/imunologia , Oxicodona/química , Oxicodona/imunologia , Oxicodona/farmacocinética , Distribuição Tecidual , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologiaRESUMO
Translation of therapeutic vaccines for addiction, cancer, or other chronic noncommunicable diseases has been slow because only a small subset of immunized subjects achieved effective Ab levels. We hypothesize that individual variability in the number of naive and early-activated hapten-specific B cells determines postvaccination serum Ab levels and vaccine efficacy. Using a model vaccine against the highly abused prescription opioid oxycodone, the polyclonal B cell population specific for an oxycodone-based hapten (6OXY) was analyzed by flow cytometry paired with Ag-based magnetic enrichment. A higher frequency of 6OXY-specific B cells in either spleen biopsies or blood, before and after immunization, correlated to subsequent greater oxycodone-specific serum Ab titers and their efficacy in blocking oxycodone distribution to the brain and oxycodone-induced behavior in mice. The magnitude of 6OXY-specific B cell activation and vaccine efficacy was tightly correlated to the size of the CD4(+) T cell population. The frequency of enriched 6OXY-specific B cells was consistent across various mouse tissues. These data provide novel evidence that variations in the frequency of naive or early-activated vaccine-specific B and T cells can account for individual responses to vaccines and may predict the clinical efficacy of a therapeutic vaccine.
Assuntos
Subpopulações de Linfócitos B/imunologia , Haptenos/imunologia , Transtornos Relacionados ao Uso de Opioides/prevenção & controle , Vacinas/imunologia , Animais , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Modelos Animais de Doenças , Centro Germinativo/imunologia , Humanos , Imunização , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Masculino , Camundongos , Oxicodona/química , Oxicodona/imunologia , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Vacinas/administração & dosagemRESUMO
G-protein-gated inwardly rectifying K(+) (GIRK/Kir3) channel activation underlies key physiological effects of opioids, including analgesia and dependence. GIRK channel activation has also been implicated in the opioid-induced inhibition of midbrain GABA neurons and consequent disinhibition of dopamine (DA) neurons in the ventral tegmental area (VTA). Drug-induced disinhibition of VTA DA neurons has been linked to reward-related behaviors and underlies opioid-induced motor activation. Here, we demonstrate that mouse VTA GABA neurons express a GIRK channel formed by GIRK1 and GIRK2 subunits. Nevertheless, neither constitutive genetic ablation of Girk1 or Girk2, nor the selective ablation of GIRK channels in GABA neurons, diminished morphine-induced motor activity in mice. Moreover, direct activation of GIRK channels in midbrain GABA neurons did not enhance motor activity. In contrast, genetic manipulations that selectively enhanced or suppressed GIRK channel function in midbrain DA neurons correlated with decreased and increased sensitivity, respectively, to the motor-stimulatory effect of systemic morphine. Collectively, these data support the contention that the unique GIRK channel subtype in VTA DA neurons, the GIRK2/GIRK3 heteromer, regulates the sensitivity of the mouse mesolimbic DA system to drugs with addictive potential.
Assuntos
Analgésicos Opioides/farmacologia , Neurônios Dopaminérgicos/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/fisiologia , Neurônios GABAérgicos/fisiologia , Atividade Motora/fisiologia , Animais , Neurônios Dopaminérgicos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Neurônios GABAérgicos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Subunidades Proteicas/fisiologia , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/fisiologiaRESUMO
The current opioid epidemic is one of the most profound public health crises facing the United States. Despite that it has been under the spotlight for years, available treatments for opioid use disorder (OUD) and overdose are limited to opioid receptor ligands such as the agonist methadone and the overdose reversing drugs such as naloxone. Vaccines are emerging as an alternative strategy to combat OUD and prevent relapse and overdose. Most vaccine candidates consist of a conjugate structure containing the target opioid attached to an immunogenic carrier protein. However, conjugate vaccines have demonstrated some intrinsic shortfalls, such as fast degradation and poor recognition by immune cells. To overcome these challenges, we proposed a lipid-PLGA hybrid nanoparticle (hNP)-based vaccine against oxycodone (OXY), which is one of the most frequently misused opioid analgesics. The hNP-based OXY vaccine exhibited superior immunogenicity and pharmacokinetic efficacy in comparison to its conjugate vaccine counterpart. Specifically, the hNP-based OXY vaccine formulated with subunit keyhole limpet hemocyanin (sKLH) as the carrier protein and aluminum hydroxide (Alum) as the adjuvant (OXY-sKLH-hNP(Alum)) elicited the most potent OXY-specific antibody response in mice. The induced antibodies efficiently bound with OXY molecules in blood and suppressed their entry into the brain. In a following dose-response study, OXY-sKLH-hNP(Alum) equivalent to 60 µg of sKLH was determined to be the most promising OXY vaccine candidate moving forward. This study provides evidence that hybrid nanoparticle-based vaccines may be superior vaccine candidates than conjugate vaccines and will be beneficial in treating those suffering from OUD.
RESUMO
Illicit drug mixtures containing opioids and stimulants have been responsible for the majority of fatal drug overdoses among occasional users, and those with either opioid use disorder (OUD) or substance use disorder (SUD). As a complementary strategy to current pharmacotherapies, active immunization with conjugate vaccines has been proposed as a viable intervention to treat OUD as well as other SUD for which there are either limited or no treatment options. Vaccination against opioids and stimulants could help address the limitations of current medications (e.g., patient access, compliance, misuse liability, and safety) by providing an additional tool to prevent drug misuse and/or overdoses. However, more research is needed to fully understand the potential benefits and limitations of using vaccines to treat SUD and overdose and to inform us on how to deploy this strategy in the field. Previous reports have shown promise by combining two vaccines into bivalent vaccine formulations to concurrently target multiple drugs. Here, multiple individual candidate monovalent vaccines were incrementally combined in multivalent vaccine formulations to simultaneously target fentanyl, carfentanil, oxycodone, heroin, methamphetamine, and their analogs or metabolites. Bi-, tri-, and quadrivalent vaccine formulations induced the formation of independent serum antibody responses against their respective opioid targets and selectively attenuated the distribution of each individual drug to the brain in mice and rats. Results indicate that a single injection of an admixed multivalent vaccine formulation may be more effective than coinjecting multiple monovalent vaccines at multiple sites. Finally, adding a methamphetamine conjugate vaccine to an quadrivalent opioid vaccine in a pentavalent formulation did not interfere with the production of effective antiopioid IgG antibodies. Multivalent vaccines could provide multifaceted, yet selective, protection against polydrug use and exposure.
RESUMO
Introduction: Fentanyl and fentanyl analogs (F/FA) have become increasingly common adulterants in counterfeit prescription pills and illicit street drug mixtures due to their ease of synthesis and exceedingly high potency. The ongoing epidemic of fatal overdoses fueled by F/FA continues to highlight the need for longer-acting therapies than naloxone (NLX), the current gold-standard for reversing opioid overdoses, which shows limited efficacy to prevent renarcotization associated with F/FA toxicity. A novel opioid reversal agent based on covalent naloxone nanoparticles (cNLX-NP) has been shown to blunt fentanyl-induced respiratory depression out to 48 hr, demonstrating its potential therapeutic utility. The purpose of this study was to characterize how rapidly cNLX-NP reverses fentanyl-induced respiratory effects as well as the duration of its protective effects. Methods: Sprague Dawley male rats (n=6/group) were tested on an oximeter for baseline percent arterial oxygen saturation (%SaO2) challenged with 0.1 mg/kg SC fentanyl and 15 min later given 10 mg/kg IM doses of NLX, nalmefene (NLMF), or cNLX-NP and continuously monitored via oximetry for 10 minutes. One week later the experiment was repeated using a 1:1 mixture of NLX:cNLX-NP as the reversal agent in the rats that previously received NLX alone. Results: While both NLX and NLMF rapidly reversed %SaO2 to baseline within 1 min, rats that received cNLX-NP did not return to >90% SaO2 levels until 9 min after administration. Similarly, heart and breath rates returned to baseline within 1 min of treatment with NLX and NLMF but did not return to baseline until 10 minutes after cNLX-NP administration. In contrast, NLX:cNLX-NP reversed all fentanyl-induced respiratory depressive effects within one minute. Discussion: While cNLX-NP alone may not sufficiently reverse F/FA overdose in a timely manner, mixing free NLX with cNLX-NP can provide a mechanism to both rapidly reverse fentanyl-related effects and maintain extended protection against synthetic opioid toxicity. These data support further development of cNLX-NP as a fast-acting and long-lasting antidote to treat F/FA-induced respiratory depression and overdose, and potentially prevent renarcotization in humans.
RESUMO
SARS-CoV-2 virus has continued to evolve over time necessitating the adaptation of vaccines to maintain efficacy. Monoclonal antibodies (mAbs) against SARS-CoV-2 were a key line of defense for unvaccinated or immunocompromised individuals. However, these mAbs are now ineffective against current SARS-CoV-2 variants. Here, we tested three aspects of αSARS-CoV-2 therapeutics. First, we tested whether Fc engagement is necessary for in vivo clearance of SARS-CoV-2. Secondly, we tested bi-specific killer engagers (BiKEs) that simultaneously engage SARS-CoV-2 and a specific Fc receptor. Benefits of these engagers include the ease of manufacturing, stability, more cell-specific targeting, and high affinity binding to Fc receptors. Using both mAbs and BiKEs, we found that both neutralization and Fc receptor engagement were necessary for effective SARS-CoV-2 clearance. Thirdly, due to ACE2 being necessary for viral entry, ACE2 will maintain binding to SARS-CoV-2 despite viral evolution. Therefore, we used an ACE2 decoy Fc-fusion or BiKE, instead of an anti-SARS-CoV-2 antibody sequence, as a potential therapeutic that would withstand viral evolution. We found that the ACE2 decoy approach also required Fc receptor engagement and, unlike traditional neutralizing antibodies against specific variants, enabled the clearance of two distinct SARS-CoV-2 variants. These data show the importance of Fc engagement for mAbs, the utility of BiKEs as therapies for infectious disease, and the in vivo effectiveness of the ACE2 decoy approach. With further studies, we predict combining neutralization, the cellular response, and this ACE2 decoy approach will benefit individuals with ineffective antibody levels. Abbreviations: ACE2, scFv, mAb, BiKE, COVID-19, Fc, CD16, CD32b, CD64, d.p.i. Key points: With equal dosing, both neutralization and Fc engagement are necessary for the optimal efficacy of in vivo antibodies and bi-specific killer engagers (BiKEs) against SARS-CoV-2. BiKEs can clear SARS-CoV-2 virus and protect against severe infection in the hACE2-K18 mouse model. ACE2 decoys as part of Fc-fusions or BiKEs provide in vivo clearance of two disparate SARS-CoV-2 variants.