Bivalent Conjugate Vaccine Induces Dual Immunogenic Response That Attenuates Heroin and Fentanyl Effects in Mice.

Opioid use disorders and fatal overdose due to consumption of fentanyl-laced heroin remain a major public health menace in the United States. Vaccination may serve as a promising potential remedy to combat accidental overdose and to mitigate the abuse potential of opioids. We previously reported the heroin and fentanyl monovalent vaccines carrying, respectively, a heroin hapten, 6-AmHap, and a fentanyl hapten, para-AmFenHap, conjugated to tetanus toxoid (TT). Herein, we describe the mixing of these antigens to formulate a bivalent vaccine adjuvanted with liposomes containing monophosphoryl lipid A (MPLA) adsorbed on aluminum hydroxide. Immunization of mice with the bivalent vaccine resulted in IgG titers of >105 against both haptens. The polyclonal sera bound heroin, 6-acetylmorphine, morphine, and fentanyl with dissociation constants (Kd) of 0.25 to 0.50 nM. Mice were protected from the anti-nociceptive effects of heroin, fentanyl, and heroin +9% (w/w) fentanyl. No cross-reactivity to methadone and buprenorphine was observed in vivo. Naloxone remained efficacious in immunized mice. These results highlighted the potential of combining TT-6-AmHap and TT-para-AmFenHap to yield an efficacious bivalent vaccine that could ablate heroin and fentanyl effects. This vaccine warrants further testing to establish its potential translatability to humans.

Experimental Colitis Enhances the Rate of Antinociceptive Tolerance to Morphine via Peripheral Opioid Receptors.

Opioids are highly effective analgesics, however, their therapeutic use is limited by adverse effects that include respiratory depression, dependence, and tolerance. Inflammation has been implicated as a significant driver for the development of tolerance to opioids. Recent studies show that chronic morphine in mice results in gut microbial dysbiosis and inflammation in the colon. In the present study, we examined whether colonic inflammation results in tolerance to the antinociceptive effects of morphine. Colonic inflammation was induced in mice by intrarectal administration of 2,4,6-trinitro-benzene sulfonic acid. The development of antinociceptive tolerance was determined by warm-water tail-immersion assay in mice implanted with 25-, 50-, or 75-mg morphine pellet. Colonic inflammation significantly enhanced the rate at which tolerance developed in each cohort of chronic morphine-treated mice. At the lowest dose of morphine pellet (25 mg), antinociceptive tolerance only developed in the presence of colonic inflammation, whereas in 50- and 75-mg pelleted mice, tolerance developed faster in the inflamed animals than in the noninflamed mice. The enhanced antinociceptive tolerance was attenuated with daily administration of peripheral opioid receptor antagonist, 6ß-N-heterocyclic-substituted naltrexamine derivative [17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6ß-[(4'pyridyl)acetamido]morphinan (NAP)], irrespective of colonic inflammation. Collectively, these findings show that the rate of tolerance to morphine antinociception is exaggerated in the presence of colonic inflammation, and tolerance is prevented by a peripheral µ-opioid receptor antagonist. These studies suggest a peripheral component to the development of antinociceptive tolerance to opioids. Furthermore, peripherally selective opioid antagonists may be useful adjuncts in opioid-based pain management. SIGNIFICANCE STATEMENT: This study supports the notion that inflammation influences the development of antinociceptive tolerance to chronic morphine exposure. We found that, in the presence of colonic inflammation, the rate of development of tolerance to the antinociceptive effects of morphine increased. We also found that treatment with a peripheral opioid receptor antagonist prevented morphine antinociceptive tolerance. Increasing opioid intake during an inflammatory state would result in decreased analgesia and enhanced analgesic tolerance, which puts patients with inflammatory bowel diseases, inflammatory joint diseases, and sickle cell anemia at risk for heavy opioid use.

Methylnaltrexone crosses the blood-brain barrier and attenuates centrally-mediated behavioral effects of morphine and oxycodone in mice.

BACKGROUND: Antagonism of peripheral opioid receptors by methylnaltrexone (MNTX) was recently proposed as a potential mechanism to attenuate the development of opioid analgesic tolerance based on experiments conducted in mice. However, reports indicate that MNTX is demethylated to naltrexone (NTX) in mice, and NTX may subsequently cross the blood-brain barrier to antagonize centrally-mediated opioid effects. The goal of this study was to determine whether MNTX alters centrally-mediated behaviors elicited by the opioid analgesics, morphine and oxycodone, and to quantify concentrations of MNTX and NTX in blood and brain following their administration in mice. METHODS: Combinations of MNTX and morphine were tested under acute and chronic conditions in thermal nociceptive assays. Effects of MNTX and NTX pretreatment were assessed in an oxycodone discrimination operant procedure. Blood and brain concentrations of these antagonists were quantified after their administration using liquid chromatography-mass spectrometry. RESULTS: MNTX dose-dependently attenuated acute and chronic morphine antinociception. MNTX and NTX dose-dependently antagonized the discriminative stimulus effects of oxycodone. MNTX and NTX were detected in both blood and brain after administration of MNTX, confirming its demethylation and demonstrating that MNTX itself can cross the blood-brain barrier. CONCLUSIONS: These results provide converging behavioral and analytical evidence that MNTX administration in mice attenuates centrally-mediated effects produced by opioid analgesics and results in functional concentrations of MNTX and NTX in blood and brain. Collectively, these findings indicate that MNTX cannot be administered systemically in mice for making inferences that its effects are peripherally restricted.

Morphine Exacerbates Experimental Colitis-Induced Depression of Nesting in Mice.

Opioids and non-steroidal anti-inflammatory drugs (NSAIDs) are excellent analgesics, but recent clinical evidence suggests that these drugs might worsen disease severity in Crohn's disease patients, limiting their clinical utility for treating Inflammatory Bowel Disease (IBD). One indicator of change in well-being from conditions such as IBD is behavioral depression and disruption to activities of daily living. Preclinical measures of behavioral depression can provide an indicator of changes in quality of life and subsequent modification by candidate analgesics. In mice, nesting is an adaptive unconditioned behavior that is susceptible to disruption by noxious stimuli, and some types of pain related nesting depression are responsive to opioid and NSAID analgesics. Here we show that a 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) model of experimental colitis depresses nesting behavior in mice, and we evaluated effects of morphine, an opioid, and ketoprofen, a NSAID, on TNBS-induced nesting depression. In Swiss Webster mice, TNBS significantly reduced nesting that peaked on Day 3 and recovered in a time-dependent manner with complete recovery by Day 7. In the absence of colonic inflammation, daily treatment with morphine (1-10 mg/kg) did not decrease nesting except at 10mg/kg/day. However, in TNBS-treated mice 3.2 mg/kg/day morphine significantly exacerbated TNBS-induced nesting depression and delayed recovery. While 3.2 mg/kg/day morphine alone did not alter locomotor activity and TNBS-induced depression of locomotion recovered, the combination of TNBS and 3.2 mg/kg/day morphine significantly attenuated locomotion and prevented recovery. Daily treatment with 3.2 or 10 mg/kg ketoprofen in TNBS-treated mice did not prevent depression of nesting. These data suggest that opioid analgesics but not NSAIDS worsen colonic inflammation-induced behavioral depression. Furthermore, these findings highlight the importance of evaluating analgesic effects in models of colonic inflammation induced depression of behavior.

Novel chimeric monoclonal antibodies that block fentanyl effects and alter fentanyl biodistribution in mice.

The prevalence and societal impact of opioid use disorder (OUD) is an acknowledged public health crisis that is further aggravated by the current pandemic. One of the devastating consequences of OUD is opioid overdose deaths. While multiple medications are now available to treat OUD, given the prevalence and societal burden, additional well-tolerated and effective therapies are still needed. To this point, we have developed chimeric monoclonal antibodies (mAb) that will specifically complex with fentanyl and its analogs in the periphery, thereby preventing them from reaching the central nervous system. Additionally, mAb-based passive immunotherapy offers a high degree of specificity to drugs of abuse and does not interfere with an individual's ability to use any of the medications used to treat OUD. We hypothesized that sequestering fentanyl and its analogs in the periphery will mitigate their negative effects on the brain and peripheral organs. This study is the first report of chimeric mAb against fentanyl and its analogs. We have discovered, engineered the chimeric versions, and identified the selectivity of these antibodies, through in vitro characterization and in vivo animal challenge studies. Two mAb candidates with very high (0.1-1.3 nM) binding affinities to fentanyl and its analogs were found to be effective in engaging fentanyl in the periphery and blocking its effects in challenged animals. Results presented in this work constitute a major contribution in the field of novel therapeutics targeting OUD.

Effect of Preexisting Immunity to Tetanus Toxoid on the Efficacy of Tetanus Toxoid-Conjugated Heroin Vaccine in Mice.

Opioid use disorder (OUD) is a serious health problem that has dramatically increased over the last decade. Although current therapies for the management of OUD can be effective, they have limitations. The complementary strategy to combat the opioid crisis is the development of a conjugate vaccine to generate high affinity antibodies in order to neutralize opioids in circulation before reaching the brain. The components of an opioid vaccine include an opioid hapten (6-AmHap) that is conjugated to a carrier protein (tetanus toxoid) with the addition of adjuvants (Army Liposome Formulation adsorbed to aluminum hydroxide-ALFA). There is no consensus in the literature as to whether preexisting immunity to the carrier protein may impact the immunogenicity of the conjugate vaccine by inducing an enhanced or suppressed immune response to the hapten. Here, we investigated whether pre-exposure to tetanus toxoid would affect the immunogenicity and efficacy of the heroin vaccine, TT-6-AmHap. Mice were primed with diphtheria, tetanus, and acellular pertussis (DTaP) vaccine at weeks -4 and -2, then immunized with TT-6-AmHap vaccine at weeks 0, 3, and 6. Using ELISA and behavioral assays, we found that preexisting immunity to tetanus toxoid had no influence on the immunogenicity and efficacy of the TT-6-AmHap vaccine.

Methylation Products of 6ß- N-Heterocyclic Substituted Naltrexamine Derivatives as Potential Peripheral Opioid Receptor Modulators.

Two 6ß- N-heterocyclic naltrexamine derivatives, NAP and NMP, have been identified as peripherally selective mu opioid receptor (MOR) antagonists. To further enhance the peripheral selectivity of both compounds, the 17-amino group and the nitrogen atom of the pyridine ring in both NAP and NMP were methylated to obtain dMNAP and dMNMP, respectively. Compared with NAP and NMP, the binding affinities of dMNAP and dMNMP shifted to MOR and KOR (kappa opioid receptor) dual selective and they acted as moderate efficacy partial agonists. The results from radioligand binding studies were further confirmed by molecular docking studies. In vivo studies demonstrated that dMNAP and dMNMP did not produce antinociception nor did they antagonize morphine's antinociceptive activity, indicating that these compounds did not act on the central nervous system. Meanwhile, both dMNAP and dMNMP significantly slowed down fecal excretion, which indicated that they were peripherally acting opioid receptor agonists. All together, these results suggested that dMNAP and dMNMP acted as peripheral mu/kappa opioid receptor modulators and may be applicable in the treatment of diarrhea in patients with bowel dysfunction.

The effect of gut microbiome on tolerance to morphine mediated antinociception in mice.

There is growing appreciation for the importance of gastrointestinal microbiota in many physiological and pathophysiological processes. While morphine and other narcotics are the most widely prescribed therapy for moderate to severe pain clinically, they have been noted to alter microbial composition and promote bacterial translocation to other tissues. Here we examined the pharmacodynamic properties of chronic morphine in mice following bacterial depletion with oral gavage of an antibiotic cocktail (ABX). ABX significantly reduced gut bacteria and prevented chronic morphine induced increases in gut permeability, colonic mucosal destruction, and colonic IL-1ß expression. In addition, ABX prevented the development of antinociceptive tolerance to chronic morphine in both the tail-immersion and acetic acid stretch assays. Morphine tolerance was also reduced by oral vancomycin that has 0% bioavailability. These findings were recapitulated in primary afferent neurons isolated from dorsal root ganglia (DRG) innervating the lower gastrointestinal tract, wherein in-vivo administration of ABX prevented tolerance to morphine-induced hypoexcitability. Finally, though ABX repeatedly demonstrated an ability to prevent tolerance, we show that it did not alter susceptibility to precipitation of withdrawal by naloxone. Collectively, these finding indicate that the gastrointestinal microbiome is an important modulator of physiological responses induced by chronic morphine administration.