Among vaccines aimed at treating substance use disorders, those targeting opioids present several unique medication development challenges. 1) Opioid overdose is a common complication of abuse, so it ...is desirable for an opioid vaccine to block the toxic as well as the addictive effects of opioids. 2) It is important that an opioid vaccine not interfere with the action of opioid antagonists used to reverse opioid overdose or treat addiction. 3) Some opioids are immunosuppressive and chronic ongoing opioid use could interfere with vaccine immunogenicity. 4) Although antibody-bound oxycodone is unable to enter the brain because of its size, it might still be able to activate peripheral opioid receptors. To assess vaccine impact on opioid toxicity, rats vaccinated with oxycodone conjugated to keyhole limpet hemocyanin subunit dimer (OXY-dKLH) adsorbed to alum or controls vaccinated with dKLH were compared with regard to oxycodone-induced hotplate analgesia and oxycodone-induced respiratory depression and bradycardia. Vaccination shifted the dose-response curves to the right, representing protection, for each of these endpoints. Naloxone was equally effective in both OXY-dKLH and control groups, providing complete and rapid reversal of respiratory depression. The administration of a long-acting naltrexone formulation during vaccination did not impair vaccine immunogenicity in mice. Similarly, serum anti-oxycodone antibody titers were not altered by continuous morphine infusion during vaccination compared to opioid-naïve controls. Competitive ELISA assay showed negligible or low affinity of immune antiserum for endogenous opioids or opioid antagonists. In vitro receptor binding assays showed that antibody-bound oxycodone does not activate mu opioid receptors. These data support further study of OXY-dKLH as a potential treatment for oxycodone abuse and suggest that vaccination might also reduce the severity of oxycodone overdose.
Therapeutic vaccines offer a viable strategy to treat opioid use disorders (OUD) complementary to current pharmacotherapies. The candidate Oxy(Gly)4-sKLH vaccine targeting oxycodone displayed ...pre-clinical proof of efficacy, selectivity and safety, and it is now undergoing clinical evaluation. To further support its implementation in the clinic, this study tested critical in vivo neuropsychopharmacological properties of the Oxy(Gly)4-sKLH vaccine in rats. While repeated immunizations with Oxy(Gly)4-sKLH were necessary to maintain the antibody response overtime, exposure to free oxycodone did not boost oxycodone-specific antibody levels in vaccinated rats, limiting concerns of immune-related side effects. Immunization with Oxy(Gly)4-sKLH achieved sustained antibody titers over a period of five months following initial vaccination, supporting its potential for providing long-lasting protection. In vivo studies of selectivity showed that vaccination prevented oxycodone-induced but not methadone-induced antinociception, while still preserving the opioid antagonist naloxone's pharmacological effects. Vaccination did not interfere with fentanyl-induced antinociception or fentanyl distribution to the brain. These in vivo data confirm the previously reported in vitro selectivity profile of Oxy(Gly)4-sKLH. Vaccination extended oxycodone's half-life up to 25 h compared to control. While vaccination reduced the reinforcing efficacy of oxycodone in an intravenous self-administration model, signs of toxicity were not observed. These rodent studies confirm that active immunization with Oxy(Gly)4-sKLH induces highly specific and long-lasting antibodies which are effective in decreasing the reinforcing effects of oxycodone while preserving the efficacy of medications used to treat OUD and overdose.
•Opioid use disorder is a worldwide problem and better treatments are needed.•This study characterizes an oxycodone vaccine currently undergoing clinical testing.•Exposure to oxycodone does not elicit antibody production in vaccinated animals.•Vaccine blunts oxycodone, but not methadone, fentanyl, or naloxone effects.•Vaccine alters oxycodone self-administration; no opioid-induced toxicity observed.
Summary
Cortical bone microstructure is an important parameter in the evaluation of bone strength. The aim of this study was to validate the characterization of human cortical bone microarchitecture ...using microcomputed tomography. In order to do this, microcomputed tomography structural measurements were compared with those obtained through histological examination (the gold standard). Moreover, to calculate structural parameters, microcomputed tomography images have to be binarized with the separation between bone and nonbone structures throughout a global thresholding. As the effect of the surrounding medium on the threshold value is not clear, an easy procedure to find the global uniform threshold for a given acquisition condition is applied. This work also compared the structural parameters of microcomputed tomography cortical sample scan in air or embedded in polymethylmethacrylate; histology was used as a reference. For each acquisition condition, a fixed threshold value was found and was applied on the corresponding microcomputed tomography image for the parameters assessment.
Twenty cortical bone samples were collected from human femur and tibia diaphyses. All samples were microcomputed tomography scanned in air, embedded in polymethylmethacrylate, rescanned by microcomputed tomography, examined by histology and finally compared.
A good correspondence between the microcomputed tomography images and the histological sections was found. Paired comparisons in cortical porosity, Haversian canal diameter and Haversian canal separation between histological sections and microcomputed tomography cross sections, first in air and then embedded in PolyMethylMethAcrylate, were made: no significant differences were found. None of the comparisons showed significant differences for cortical porosity, Haversian canal diameter and Haversian separation over a three‐dimensional volume of interest, between microcomputed tomography scans in air and with samples embedded in PolyMethylMethAcrylate.
The very good correlation between bone structural measures obtained from microcomputed tomography datasets and from two‐dimensional histological sections confirms that microcomputed tomography may be an efficient tool for the characterization of cortical bone microstructure. Moreover, when the corresponding threshold value for each condition is used, structural parameters determined by microcomputed tomography are not affected by the surrounding medium (PolyMethylMethAcrylate).
•This study was aimed at mimicking in vitro a psoriatic microenvironment.•We used a three-dimensional model of organotypic human skin cultures.•Tumor necrosis factor-alpha and interleukin-17 were ...added to the culture medium.•A decrease of epidermal proliferation was induced by both proinflammatory cytokines.•No synergistic effects on cell proliferation were found with the co-administration of both cytokines.
Among all cytokines involved in the pathogenesis and in the progression of psoriasis, Tumor Necrosis Factor (TNF)-alpha and interleukin (IL)-17 play a pivotal role.
The aim of the present study was to mimic a psoriatic microenvironment and to investigate the early effects of TNF-alpha and IL-17 in a three-dimensional model of organotypic normal human skin.
Human skin explants were obtained from plastic aesthetic surgery of healthy young women 20–40years old (n=7). The study was approved by the Institutional Review Board and written informed consent was obtained from all subjects. Bioptic fragments were cultured at the air–liquid interface overnight in a Transwell system and further divided before adding either 50ng/ml IL-17 or 100ng/ml TNF-alpha or a combination of both cytokines. For each subject, a control sample was cultured without any cytokine. Samples were harvested 24 or 48h after cytokine incubation. At both time points and for all cytokine treatments a bioptic fragment obtained from each patient was processed. Epidermal proliferation, expressions of terminal differentiation (keratin 10, K10, and 14, K14) and of intercellular adhesion (occludin for tight junctions and E-cadherin for adherens junctions) biomarkers were investigated by indirect immunofluorescence.
IL-17 and TNF-alpha induced an early and statistically significant inhibition of keratinocyte proliferation (more than 80% compared with their respective controls). At 24h, the combination of both cytokines did not further reduce cell proliferation. Starting from 24h of incubation, a non-continuous occludin expression in the granular layer was observed after both IL-17 and TNF-alpha exposure. Immunolabelling for E-cadherin in adherens junctions, for K10 in the suprabasal layers, and for K14 in the basal layer was similar in all experimental groups and unaffected after cytokine treatment.
These results suggest that in this experimental model IL-17 and TNF-alpha induced an early alteration of the homeostasis of the inner proliferative layer and of the upper granular layer, as shown by cell proliferation inhibition and occludin expression.
Abstract Human cancellous bone is a heterogeneous material. Despite this, most of the published studies report correlations between mechanical properties and morphometric parameters averaged on the ...whole specimen. This work investigated whether local variations in morphometric parameters were linked to the localized failure regions of cancellous bone. Additionally, it was examined whether local values of morphometric parameters can predict the ultimate stress better than the average bone volume fraction (BV/TV). Cylindrical cancellous bone specimens extracted along the primary compressive group of human femoral heads were studied. These were microCT-imaged to assess the morphometric parameters, compressed to determine the ultimate stress, and rescanned by microCT to visualize the failure region. Failure involved slightly less than half of the free height of the specimens. Significant differences were found in the morphometric parameters calculated in the failure and in the non-failure regions. The cross-sections containing minimum BV/TV values were those most often located inside the failure region (83%, p <0.001). Regression analysis confirmed that variations in BV/TV best describe variations in ultimate stress ( R2 =0.84) out of the averaged morphometric parameters. The prediction of ultimate stress increased when minimum or maximum values of the morphometric parameters were taken, with the highest prediction found by considering the minimum BV/TV ( R2 =0.95). In conclusion, due to the heterogeneity of cancellous bone, there may exist regions characterized by a different microarchitecture, where the bone is weaker and consequently is more likely to fail. These regions mostly contain minimum values in BV/TV, which were found to predict ultimate stress better than average BV/TV.
This study focused on formulating conjugate vaccines targeting oxycodone and heroin for technology transfer, good manufacturing practice (GMP), and clinical evaluation. Lead vaccines used the highly ...immunogenic carrier protein keyhole limpet hemocyanin (KLH), which poses formulation problems because of its size. To address this barrier to translation, an oxycodone-based hapten conjugated to GMP-grade subunit KLH (OXY-sKLH) and adsorbed on alum adjuvant was studied with regard to carbodiimide coupling reaction time, buffer composition, purification methods for conjugates, conjugate size, state of aggregation, and protein/alum ratio. Vaccine formulations were screened for post-immunization antibody levels and efficacy in reducing oxycodone distribution to the brain in rats. While larger conjugates were more immunogenic, their size prevented characterization of the haptenation ratio by standard analytical methods and sterilization by filtration. To address this issue, conjugation chemistry and vaccine formulation were optimized for maximal efficacy, and conjugate size was measured by dynamic light scattering prior to adsorption to alum. An analogous heroin vaccine (M-sKLH) was also optimized for conjugation chemistry, formulated in alum, and characterized for potency against heroin in rats. Finally, this study found that the efficacy of OXY-sKLH was preserved when co-administered with M-sKLH, supporting the proof of concept for a bivalent vaccine formulation targeting both heroin and oxycodone. This study suggests methods for addressing the unique formulation and characterization challenges posed by conjugating small molecules to sKLH while preserving vaccine efficacy.
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