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Clinical trials have indicated that a vaccine must be immunogenic in genetically diverse human populations and that immunogenicity and protective efficacy in animal models are two key indices required for the approval of a new vaccine. Additionally, the immune response (immunogenicity) and immunoprotection are dependent on the mouse strain. Therefore, the objective of the present study was to determine the immune response (immunogenicity) and the protective efficacy (behavioral response) in three inbred mouse strains immunized with the M 6 TT vaccine. Female BALB/c, C57Bl/6, and DBA/2 inbred mice were immunized with the M 6 -TT vaccine. A solid-phase antibody-capture ELISA was used to monitor antibody titer responses after each booster dose in vaccinated animals. The study used tail-flick testing to evaluate the antinociceptive effects induced by heroin. Additionally, heroin-induced locomotor activity and place preference were evaluated. The M 6 -TT vaccine was able to generate a specific antibody titer in the three inbred mouse strains evaluated. The antibodies reduced the antinociceptive effect of different doses of heroin. In addition, they decreased the heroin-induced locomotor activity and place preference. These findings suggest that the M 6 -TT vaccine generates a powerful immunogenic response capable of reducing the antinociceptive and reinforcing effects of heroin in different inbred mouse strains, which supports its possible future use in clinical trials in genetically diverse human populations., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Opioids are effective analgesics; however, there are many negative consequences of chronic use. One important side effect of chronic opioid use is the continuous engagement of the immune response that can exacerbate chronic pain. The opioid, morphine, initiates a Toll-like receptor 4 (TLR4) signaling cascade that drives the activation of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome proteins, resulting in cytokine production and effectively creating a positive feedback loop for continuous TLR4 activation. In addition to driving cytokine production, morphine drives changes in proinflammatory lipid signaling. The alteration of both cytokine and lipid signaling systems by morphine suggests that its chronic use leads to a pathological immune response that would benefit from targeted therapy. Engaging the endogenous cannabinoid system has shown therapeutic benefit, particularly regarding its anti-inflammatory and immunosuppressive effects. Promising preclinical and clinical investigations suggest that cannabidiol (CBD) is an effective adjuvant for treatment of symptoms of opioid use disorders; however, the mechanism through which CBD drives this outcome is unclear. One potential source of insight into this mechanism is in how CBD regulates immune regulators such as cytokines and lipid signaling systems, including endocannabinoids and related immune-responsive lipids. In this review, we outline the immune response to chronic opioid use as well as CBD in the context of a lipopolysaccharide-induced immune response and speculate on the mechanism of CBD as a modulator of chronic opioid-induced immune system dysregulation.

Pain is a common symptom in patients with opioid use disorder (OUD), which increases synthetic and illicit synthetic opioid abuse and even fatalities due to opioid overdose. Many FDA-approved drugs are available for the treatment of OUD, however, the use of these medications is limited, mainly due to the development of various side effects. Active vaccination is a new therapeutic approach but the resulting antibodies may compromise the use and efficiency of opioid and non-opioid drugs. In this study, we evaluated whether the antibodies produced by the morphine/heroin vaccine (M-TT) would alter the antinociceptive effects of opioid and non-opioid drugs. Female Balb-c mice were immunized with the M-TT vaccine. A solid-phase antibody-capture ELISA was used for monitoring antibody titer responses after each booster dose in vaccinated animals, followed by tail-flick testing. This study found that the M-TT vaccine did not affect the antinociception induced by different doses of morphine or the ability of non-opioid and synthetic opioid drugs to decrease thermal pain. Moreover, the combination of vaccination and naloxone increased the time-course of morphine antagonism relative to either vaccination or naloxone alone. These results suggest that the antibody titers generated by the M-TT vaccine 1) are capable of reducing morphine-induced antinociception and 2) are selective enough not to alter antinociception induced by non-opioid or synthetic drugs. These characteristics support its potential as a treatment agent for patients with symptoms of pain comorbid to OUD., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Monoclonal antibodies (mAbs) against morphine are important in the development of immunotherapeutic and diagnostic methods for the treatment and prevention of drug addiction. By the surface plasmon resonance (SPR) and enzyme immunoassay techniques, we characterized two previously obtained mAbs 3K11 and 6G1 and showed their ability to recognize free morphine and morphine-containing antigens in different ways because of the epitope specificity thereof. Using the defined amino acid sequences, we obtained three-dimensional models of the variable regions of Fab fragments of these antibodies and compared them with the known sequence and spatial structure of the anti-morphine antibody 9B1. Docking simulations are performed to obtain models of the antibodies complexes with morphine. Differences in the models of 3K11 and 6G1 complexes with morphine correlate with their experimentally detected epitope specificity. The results, in particular, can be used for the structure-based design of the corresponding humanized antibodies. According to our modeling and docking results, the very different modes of morphine binding to mAbs 3K11 and 6G1 are qualitatively similar to those previously reported for cocaine and two anti-cocaine antibodies. Thus, the obtained structural information brings more insight into the hapten recognition diversity., (© 2020 John Wiley & Sons Ltd.)

Heroin, marijuana and cocaine are widely abused drugs. Their use can be readily detected by analyzing urine for the metabolites morphine (MOR), tetrahydrocannabinol (THC) or benzoylecgonine (BZC). A multiplex immunosensor is described here for detection of MOR, THC and BZC using screen printed carbon array electrodes modified with gold nanoparticles. Antibodies against MOR, THC and BZC were immobilized on eight electrodes in a sensor array simultaneously, and a competitive assay was used for the detection. The free analytes in the sample compete with bovine serum albumin-conjugated analytes for the immobilized antibodies on the sensor surface. The array is capable of detecting the three drugs simultaneously within 20-40 min. The method has a high sensitivity, with detection limits as low as 1.2, 7.0, and 8.0 pg.mL -1 for MOR, THC and BZC, respectively. Cross reactivity testing was preformed to monitor any nonspecific binding. The results revealed good selectivity. Urine samples were spiked with the 3 drugs and tested with the multiplexed immunosensor. Recovery percentages ranged between 88 to 115%. Graphical abstract Schematic presentation of the multiplexed immunosensor for drugs of abuse,viz. tetrahydrocannabinol (THC), morphine (MOR), and benzoylecgonine (BZC)) by using an array of modified electrodes.

We describe for the first time a method that utilizes microscale thermophoresis (MST) technology to determine polyclonal antibody affinities to small molecules. Using a novel type of heterologous MST, we have accurately measured a solution-based binding affinity of serum antibodies to heroin which was previously impossible with other currently available methods. Moreover, this mismatch approach (i.e., using a cross-reactive hapten tracer) has never been reported in the literature. When compared with equilibrium dialysis combined with ultra-performance liquid chromatography/tandem mass spectrometry (ED-UPLC/MS/MS), this novel MST method yields similar binding affinity values for polyclonal antibodies to the major heroin metabolites 6-AM and morphine. Additionally, we herein report the method of synthesis of this novel cross-reactive hapten, MorHap-acetamide-a useful analog for the study of heroin hapten-antibody interactions. Using heterologous MST, we were able to determine the affinities, down to nanomolar accuracies, of polyclonal antibodies to various abused opioids. While optimizing this method, we further discovered that heroin is protected from serum esterase degradation by the presence of these antibodies in a concentration-dependent manner. Lastly, using affinity data for a number of structurally different opioids, we were able to dissect the moieties that are crucial to antibody binding. The novel MST method that is presented herein can be extended to the analysis of any ligand that is prone to degradation and can be applied not only to the development of vaccines to substances of abuse but also to the analysis of small molecule/protein interactions in the presence of serum. Graphical abstract Strategy for the determination of hapten-induced antibody affinities using Microscale thermophoresis.

Background: Investigation of immediate hypersensitivity reactions in the perioperative setting involves skin testing and measurement of specific IgE (sIgE) as standard practice. In the case of the neuromuscular blocking agents (NMBAs), the main allergenic epitopes have been shown to be substituted ammonium groups. Commercial assays are available for detection of sIgE to these epitopes using morphine and pholcodine substrates but questions have been raised about the effectiveness of these assays in the diagnosis of benzylisoquinoline anaphylaxis. This study was therefore undertaken to assess the effectiveness of these assays in the diagnosis of hypersensitivity reactions to this group of NMBAs., Methods: Analysis was carried out on all available results for patients assessed at the Royal North Shore Hospital Anaesthetic Allergy Clinic during the period June 2009 to June 2016. Standardised intradermal skin tests were performed with a panel of NMBAs. Measurement of sIgE to morphine and pholcodine was performed via the Phadia ImmunoCAP ® system., Results: For all patients with positive skin test results to NMBAs which included a benzylisoquinoline NMBA (n = 24), 75% exhibited negative sIgE to both morphine and pholcodine. Where patients were reactive to benzylisoquinoline NMBAs alone (n = 12), 100% exhibited negative sIgE results, indicating 0% sensitivity of the assays relative to skin testing, in this subgroup., Conclusion: Use of sIgE testing to morphine and pholcodine in the assessment of NMBA immediate hypersensitivity is a valuable tool particularly in the case of reactions to the aminosteroid NMBAs. However, these assays are unreliable in detecting sensitisation to benzylisoquinoline NMBAs., (© 2018 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Quantum dots (QDs)-labeled antibody fluorescence immunoassays (FLISA) for the detection of morphine were developed. Quantum dots (CdSe/ZnS), which contained carboxyl, were used to label antimorphine antibody by 1-ethyl-3-(3-dimethylaminoprophyl) carbodiimide hydrochloride/N-hydroxysulfosuccinimide, which were used as coupling reagents. The CdSe/ZnS QDs labeled antimorphine antibody (QDs labeled Ab) was characterized by fluorescence spectrum and gel electrophoresis. Plate-based FLISA and nitrocellulose membrane-based flow-through FLISA were developed and applied to quantitative and qualitative detection of morphine. Under the optimal conditions for plate-based FLISA, the linear range spanned from 3.2 × 10 -4 to 1 mg/L (R 2 = 0.9905), and the detection limit was 2.7 × 10 -4 mg/L. The visual detection limit for morphine by membrane-based flow-through FLISA was 0.01 mg/L. These results demonstrated that the developed fluorescence immunoassays could be applied as highly sensitive and convenient tools for rapid detection of morphine, which make it ideally suited for on-site screening of poppy shell added illegally in hot pot soup base.

Background: Nerve growth factor (NGF) is known to induce inflammation and pain; however its role in opioid-induced tolerance has not been studied. This study investigated the effects of an anti-NGF neutralizing antibody on the development of tolerance following chronic morphine treatment in naïve rats., Methods: Four groups of rats were used in this study; one treated with saline alone, one with 10 mg/kg of morphine, one with 10 μg of anti-NGF and the other with 10 mg/kg of morphine + 10 μg of anti-NGF, twice per day for 5 days. The route of treatment was subcutaneous (S.C.) for morphine and saline, and intraperitoneal (i.p.) for anti-NGF. Response to a noxious thermal stimulus during the course of drug treatment was assessed (Hargreaves' test). Further, the change in the NGF levels in the lumbar spinal cord was measured by ELISA., Results: Our results showed that repeated administration of morphine produced an apparent tolerance which was significantly attenuated by co-administration of anti-NGF (P < 0.001). Additionally, the area under the curve (AUC) of the analgesic effect produced by the combination of morphine and anti-NGF was significantly (P < 0.001) greater than for saline controls and chronic morphine treated rats. Moreover, the level of NGF in the spinal cord of chronic morphine treated rats was significantly higher (P < 0.05) than in both the saline control group and the group receiving simultaneous administration of anti-NGF with morphine. These results indicate that anti-NGF has the potential to attenuate morphine-induced tolerance behavior by attenuating the effects of NGF at the spinal level., Conclusion: Taken together, our study strongly suggests that the NGF signaling system is a potential novel target for treating opioid-induced tolerance.

The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (K d) of antibodies to 6-AM and morphine is described. The method can readily detect antibodies with K d in the low nanomolar range. Since heroin is rapidly degraded in sera, esterase inhibitors were included in the assay, greatly reducing heroin hydrolysis. MS/MS detection directly measured the heroin in the assay after overnight ED, thereby allowing the quantitation of % bound heroin in lieu of K d as an alternative measurement to assess heroin binding to polyclonal antibody sera. This is the first report that utilizes a solution-based assay to quantify heroin-antibody binding without being confounded by the presence of 6-AM and morphine and to measure K d of polyclonal antibody to 6-AM. Hapten surrogates 6-AcMorHap, 6-PrOxyHap, MorHap, DiAmHap, and DiPrOxyHap coupled to tetanus toxoid (TT) were used to generate high affinity antibodies to heroin, 6-AM, and morphine. In comparison to competition ED-UPLC/MS/MS which gave K d values in the nanomolar range, the commonly used competition enzyme-linked immunosorbent assay (ELISA) measured the 50% inhibition concentration (IC50) values in the micromolar range. Despite the differences in K d and IC50 values, similar trends in affinities of hapten antibodies to heroin, 6-AM, and morphine were observed by both methods. Competition ED-UPLC/MS/MS revealed that among the five TT-hapten bioconjugates, TT-6-AcMorHap and TT-6-PrOxyHap induced antibodies that bound heroin, 6-AM, and morphine. In contrast, TT-MorHap induced antibodies that poorly bound heroin, while TT-DiAmHap and TT-DiPrOxyHap induced antibodies either did not bind or poorly bound to heroin, 6-AM, and morphine. This simple and nonradioactive method can be extended to other platforms, such as oxycodone, cocaine, nicotine, and methamphetamine for the selection of the lead hapten design during substance abuse vaccine development.

A monoclonal antibody (MAb) was produced by immunization of a BALB/c mouse with a conjugated morphine C6-hemisuccinated derivative (MHS) to cationized bovine serum albumin (cBSA). The hybridoma clones were screened by indirect ELISA using MHS-BSA. The best hybridoma clone was subcloned thrice by limiting dilution. This hybridoma was found to be of IgG2b class and subclass and contained lambda light chain. The affinity of the MAb to morphine was obtained 2.8×10(9) M(-1). The titer of the cell culture supernatant was at least 1:800. The MAb was cross-reacted with codeine (100%) and apomorphine (16.5%), but not with heroin, naloxone, naltrexone, or papaverine. Morphine was conjugated to HRP using a mixed anhydride method and a direct competitive ELISA was designed using anti-morphine MAb. The assay was sensitive over the 50 ng/mL to 5 μg/mL concentration range. In conclusion, this MAb is useful for the development of immunoassays to measure morphine in urine.

Background: Correct diagnosis of genuine IgE-mediated opiate allergy poses a significant challenge, mainly because of uncertainties associated with opiate skin testing and the unavailability of drug-specific IgE (sIgE) assays. Recently, it has been suggested that sIgE to poppy seed extract and morphine would be reliable in the diagnosis of opiate allergy. However, given the high prevalence of sIgE antibodies to these compounds in an allergic population, the predictive value of these tests leaves significant doubts., Objective: This study aims at verifying the predictive value of positive poppy seed and morphine sIgE assays results., Methods: A total of 22 individuals with a positive sIgE to poppy seed or morphine were selected. All had controlled drug challenges with increasing doses of morphine and/or codeine. Of these, 18 patients had an additional basophil activation test (BAT) with morphine and codeine., Results: None of the 22 patients demonstrated objective or subjective symptoms on provocation with morphine and/or codeine. Regarding BAT with morphine and codeine, expression of CD63 on basophils from 14 opiate tolerant individuals remained comparable to spontaneous expression by resting cells. The remaining 4 patients were classified as nonresponders., Conclusion: Positive sIgE results to poppy seed and morphine are not per se predictive for genuine opiate allergy and should not be used in isolation to diagnose morphine or codeine allergy. To avoid overdiagnosis, for the time being, we propose to supplement serological diagnosis with an oral provocation test. Whether BAT might help to discriminate between clinical reactivity and sensitization remains to be confirmed in larger collaborative studies., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Currently approved medications for opioid addiction have shown clinical efficacy, but undesired side effects, dependence induced by the medications themselves, and low treatment compliance necessitate the need for novel therapies., Methods: A novel morphine-keyhole limpet hemocyanin conjugate vaccine was synthesized with 6-glutarylmorphine as the hapten and a lengthened linker of 6 carbon atoms. The titer and specificity of the triggered antibody were assessed by enzyme-linked immunosorbent assay. The effects of the vaccine on the morphine-induced elevation of dopamine levels in the nucleus accumbens were determined by high-performance liquid chromatography. The effects of the vaccine on morphine-induced locomotor sensitization and heroin-primed reinstatement of heroin self-administration were also assessed., Results: After subcutaneous administration in rats, the vaccine triggered a high antibody titer, with comparable specificity for morphine, 6-acetylmorphine, and heroin, but no interaction with dissimilar therapeutic opioid compounds, including buprenorphine, naloxone, and nalorphine, was observed. The vaccine significantly prevented the elevation of dopamine levels in the nucleus accumbens induced by a single morphine challenge. Moreover, the vaccine prevented the expression of morphine-induced locomotor sensitization and heroin-primed reinstatement of heroin seeking, suggesting its potential for preventing relapse., Conclusion: These results demonstrate that active immunization with the present vaccine induces a robust morphine/heroin-specific antibody response in rats and attenuates the behavioral effects of morphine and heroin., (© The Author 2015. Published by Oxford University Press on behalf of CINP.)

Novel synthetic compounds similar to heroin and its major active metabolites, 6-acetylmorphine and morphine, were examined as potential surrogate haptens for the ability to interface with the immune system for a heroin vaccine. Recent studies have suggested that heroin-like haptens must degrade hydrolytically to induce independent immune responses both to heroin and to the metabolites, resulting in antisera containing mixtures of antibodies (type 2 cross-reactivity). To test this concept, two unique hydrolytically stable haptens were created based on presumed structural facial similarities to heroin or to its active metabolites. After conjugation of a heroin-like hapten (DiAmHap) to tetanus toxoid and mixing with liposomes containing monophosphoryl lipid A, high titers of antibodies after two injections in mice had complementary binding sites that exhibited strong type 1 ("true") specific cross-reactivity with heroin and with both of its physiologically active metabolites. Mice immunized with each surrogate hapten exhibited reduced antinociceptive effects caused by injection of heroin. This approach obviates the need to create hydrolytically unstable synthetic heroin-like compounds to induce independent immune responses to heroin and its active metabolites for vaccine development. Facial recognition of hydrolytically stable surrogate haptens by antibodies together with type 1 cross-reactivities with heroin and its metabolites can help to guide synthetic chemical strategies for efficient development of a heroin vaccine., (Copyright © 2014. Published by Elsevier Ltd.)

Unlabelled: Rengalin liquid formulation on the basis of antibodies to bradikinin histamine and morphine was specially designed for the treatment of cough in children. The three-component combination in therapeutically active against both dry and wet cough due to effect on diverse pathogenetic aspects of the cough reflex. The aim of the multicenter, comparative, randomized clinical trial was to estimate the efficacy and safety of rengalin in the treatment of cough in patients with acute respiratory infection (ARI) of the upper respiratory tract., Methods: One hundred forty six patients at the age of 3 to 17 years (the average age of 8.2 ± 3.6 years) from 14 medical centres of Russia were observed. The patients suffered from dry/nonproductive, frequent, sore cough preventing from day-time activity and/or night sleep (≥ 4 by the Cough Severity Scale). The cough duration ranged from 12 hours to 3 days. For 3 days the patients of group 1 (n = 71) and group 2 (n = 75) were treated with rengalin and sinekod (butamirate) respectively. For the following 4 days the patients (in case of viscid expectoration were treated with ambroxole in the age doses. The results of the Per Protokol Analysis (n = 67 rengalin group and n = 73 sinekod group) with an account of the Non-Infectiority Design are presented., Results: In 3 days the number of the group 1 patients with significant improvement/recovery by the day and night estimates amounted to 90% and 88% respectively (vs. 81% and 88% in the group 2 patients, no night opisodes of cough after 3-days rengalin use being recorded in 52% of the patients vs. 34% in the sinekod group patients (p = 0.0003). On the 7th day of the treatment with rengalin the number of the children with significant improvement of or recovery from day-time cought amounted to 99%and that of the patients with significant improvement of or recovery from night-time cough amounted to 93%, in 90% of them no night-time cough being recorded (p = 0.0008). As for the patients of the reference group, the respective values were 93% and 90%, no night-time cough being recorded in 81% of the patients. The time required for development of productive/moist cough during the 3-day treatment course in the patients of both the group was the same (2.9 ± 0.3 days in the patients of group 1 and 2.9 ± 0.4 days in the group 2 patients. Moreover, in 34% of the rengalin dry cough became residual (as rare episode of tussiculation with scantly exudation). After 3-day course of the rengalin therapy, 66% of the patients was treated with ambroxole (versus 95% in sinecod group (p < 0.0001) based on comparative analysis and χ2 = 17.7, p > 0.0001 by the results of the frequency analysis). The total duration of cough in the patients of groups 1 and 2 was 6.5 ± 0.8 and 6.7 ± 0.7 days respectively (the comparability truth, p = 0.0001). The severity of the day-time cough by the area under the curve estimates for 7 days of the treatment in the rengalin group patients was equel to 14.3 ± 5.6 numbers--days and that of the patients of the sinekod? group was equal to 15.9?6.1 numbers - days. The severity of the night-time cough was equal to 4.2 ± 2.7 number--days respectively. In 2 patients (3%) treated with sinekod signs of ARI generalization was observed after the 3-day treatment (p > 0.0001). The research physicians-investigators (CGI-EL Scale) the combination of the anti- and protussive activities in one drug to be efficient and absolutely safe for the chilgren. The therapeutic efficacy in the patients of the rengalin group was higher in 3 days (2.1 ± 0.5 numbers) and even in 7 days (2.7 ± 0.5 numbers). The results value in the patients of the sinekod group being 1.8 ± 0.4 and 2.5 ± 0.6 numbers (one-wayANOVA for repeated estimates ANOVA: Visit - F(1/138) = 146, p < 0.0001, TREATMENT--F(1/138) = 9.0, p = 0.003). The factor of the side effects in the patients of the rengalin group was zero (no side effects due to the treatment were recorded in the patients), whereas in the patients treated with sinekod for 3 days the respective value was 0.1 ± 0.3 (true superiority of rengalin by the ANOVA data. TREATMENT--F(1/138) = 4.7, p = 0.03). The efficacy factor of the rengalin was also in its favour (ANOVA: Visit--F(1/138) = 182, p < 0.0001, TREATMENT--F(1y138) = 7.3, p = 0.008). In the patients treated with rengalin there were defected no deviations in the biochemical and general clinical analyses of blood and urine, no adverse reactions characteristic of antitussive drugs of the action. 100-percent adherence to the therapy was stated., Conclusion: He antitussive effect of rengalin in the treatment of frequent dry day-time and night-time cough was observed earlier and proved to be comparable with that of butamirate (sinekod). Rengalin prevented significant exudation and viscid expectoration in many patients, promoted rapid residual in the patients with dry cough and the patients recovery. The use of rengalin for 3 days significantly lowered the percentage of the patients requiring treatment with mucolytics at the subsequent stages of ARI.

Vaccines for opioid dependence may provide a treatment that would reduce or slow the distribution of the drug to brain, thus reducing the drug's reinforcing effects. We tested whether a conjugate vaccine against morphine (keyhole limpet hemocyanin-6-succinylmorphine; KLH-6-SM) administered to rats would produce antibodies and show specificity for morphine or other heroin metabolites. The functional effects of the vaccine were tested with antinociceptive and conditioned place preference (CPP) tests. Rats were either vaccinated with KLH-6-SM and received two boosts 3 and 16 weeks later or served as controls and received KLH alone. Anti-morphine antibodies were produced in vaccinated rats; levels increased and were sustained at moderate levels through 24 weeks. Antibody binding was inhibited by free morphine and other heroin metabolites as demonstrated by competitive inhibition ELISA. Vaccinated rats showed reduced morphine CPP, tested during weeks 4 to 6, and decreased antinociceptive responses to morphine, tested at week 7. Brain morphine levels, assessed using gas-chromatography coupled to mass spectrometry (GC-MS) on samples obtained at 26 weeks, were significantly lower in vaccinated rats. This suggests that morphine entry into the brain was reduced or slowed. These results provide support for KLH-6-SM as a candidate vaccine for opioid dependence., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Heroin addiction, a chronic relapsing disorder characterized by excessive drug taking and seeking, requires constant psychotherapeutic and pharmacotherapeutic interventions to minimize the potential for further abuse. Vaccine strategies against many drugs of abuse are being developed that generate antibodies that bind drug in the bloodstream, preventing entry into the brain and nullifying psychoactivity. However, this strategy is complicated by heroin's rapid metabolism to 6-acetylmorphine and morphine. We recently developed a "dynamic" vaccine that creates antibodies against heroin and its psychoactive metabolites by presenting multihaptenic structures to the immune system that match heroin's metabolism. The current study presents evidence of effective and continuous sequestration of brain-permeable constituents of heroin in the bloodstream following vaccination. The result is efficient blockade of heroin activity in treated rats, preventing various features of drugs of abuse: heroin reward, drug-induced reinstatement of drug seeking, and reescalation of compulsive heroin self-administration following abstinence in dependent rats. The dynamic vaccine shows the capability to significantly devalue the reinforcing and motivating properties of heroin, even in subjects with a history of dependence. In addition, targeting a less brain-permeable downstream metabolite, morphine, is insufficient to prevent heroin-induced activity in these models, suggesting that heroin and 6-acetylmorphine are critical players in heroin's psychoactivity. Because the heroin vaccine does not target opioid receptors or common opioid pharmacotherapeutics, it can be used in conjunction with available treatment options. Thus, our vaccine represents a promising adjunct therapy for heroin addiction, providing continuous heroin antagonism, requiring minimal medical monitoring and patient compliance.

Background: Immunoglubulin E antibody-mediated allergic reactions to opioids are rare and difficult to document correctly., Objective: Assessment of the basophil activation test in the diagnosis of IgE-mediated allergy to the antitussive pholcodine and associated sensitizations to neuromuscular blocking agents (NMBA)., Methods: Three patients with a suspected IgE-mediated allergy to pholcodine were investigated using skin tests, quantification of specific IgE, and flow cytometric activation of basophils., Results and Conclusion: Flow cytometric activation of basophils, with simultaneous analysis of CD63 appearance and median histamine content per cell, is the only technique capable to correctly document pholcodine allergy. The negative predictive value of basophil activation tests might help to elucidate on the controversial putative cross-reactivity between pholcodine and NMBA., (Copyright © 2013 International Clinical Cytometry Society.)

Morphine conjugate vaccines have effectively reduced behavioral effects of heroin in rodents and primates. To better understand how these effects are mediated, heroin and metabolite distribution studies were performed in rats in the presence and absence of vaccination. In non-vaccinated rats 6-monoacetylmorphine (6-MAM) was the predominant opioid in plasma and brain as early as 1 minute after i.v. administration of heroin and for up to 14 minutes. Vaccination with morphine conjugated to keyhole limpet hemocyanin (M-KLH) elicited high titers and concentrations of antibodies with high affinity for heroin, 6-MAM, and morphine. Four minutes after heroin administration vaccinated rats showed substantial retention of all three opioids in plasma compared to controls and reduced 6-MAM and morphine, but not heroin, distribution to brain. Administration of 6-MAM rather than heroin in M-KLH vaccinated rats showed a similar drug distribution pattern. Vaccination reduced heroin-induced analgesia and blocked heroin-induced locomotor activity throughout 2 weeks of repeated testing. Higher serum opioid-specific antibody concentrations were associated with higher plasma opioid concentrations, lower brain 6-MAM and morphine concentrations, and lower heroin-induced locomotor activity. Serum antibody concentrations over 0.2 mg/ml were associated with substantial effects on these measures. These data support a critical role for 6-MAM in mediating the early effects of i.v. heroin and suggest that reducing 6-MAM concentration in brain is essential to the efficacy of morphine conjugate vaccines.

Background: The diagnosis of anaphylactic reactions due to opiates during anaesthesia can be difficult, since in most cases various drugs may have been administered. Detection of specific IgE to poppy seed might be a marker for sensitisation to opiates in allergic people and heroin-abusers. This study assessed the clinical value of morphine, pholcodine and poppy seed skin-prick and IgE determination in people suffering hypersensitivity reactions during anaesthesia or analgesia and drug-abusers with allergic symptoms., Methods: We selected heroin abusers and patients who suffered severe reactions during anaesthesia and analgesia from a database of 23,873 patients. The diagnostic yield (sensitivity, specificity and predictive value) of prick and IgE tests in determining opiate allergy was analysed., Results: Overall, 149 patients and 200 controls, mean age 32.9 ± 14.7 years, were included. All patients with positive prick to opiates showed positive prick and IgE to poppy seeds, but not to morphine or pholcodine IgE. Among drug-abusers, 13/42 patients (31%) presented opium hypersensitivity confirmed by challenge tests. Among non-drug abusers, sensitisation to opiates was higher in people allergic to tobacco (25%), P<.001. Prick tests and IgE against poppy seed had a good sensitivity (95.6% and 82.6%, respectively) and specificity (98.5% and 100%, respectively) in the diagnosis of opiate allergy., Conclusions: Opiates may be significant allergens. Drug-abusers and people sensitised to tobacco are at risk. Both the prick and specific IgE tests efficiently detected sensitisation to opiates. The highest levels were related to more-severe clinical profiles., (Copyright © 2011 SEICAP. Published by Elsevier Espana. All rights reserved.)

A simple, rapid competitive immunochromatography (ICG) strip test was developed to detect morphine in urine samples using a monoclonal antibody produced in-house and conjugated to gold nanoparticles. Hybridoma cells were cultured and the Amor-HK16 monoclonal antibody against morphine was obtained from the supernatant after purification by salting out and passing through a Protein G-Agarose affinity column. Morphine was obtained from morphine sulfate and a C6-hemisuccinate derivative of morphine was prepared, conjugated to bovine serum albumin, and immobilized to a nitrocellulose membrane as the test line. Goat anti-mouse antibody was used as a binder in the control line in the detection zone of the strip. Colloidal gold particles of diameter approximately 20 nm were prepared and conjugated to the monoclonal antibody. The detection limit of the test strip was found to be 2000 ng/mL of morphine in urine samples. Reliability was determined by performing the ICG test on 103 urine samples and comparing the results with those obtained by thin-layer chromatography. The sensitivity of the test was 100%, and the analysis time for the assay was approximately 5 min. The new ICG method was adequately sensitive and accurate for the rapid screening of morphine in urine.

Opioid conjugate vaccines have shown promise in animal models as a potential treatment for opioid addiction. Individual vaccines are quite specific and each targets only a limited number of structurally similar opioids. Since opioid users can switch or transition between opioids, we studied a bivalent immunization strategy of combining 2 vaccines that could target several of the most commonly abused opioids; heroin, oxycodone and their active metabolites. Morphine (M) and oxycodone (OXY) haptens were conjugated to keyhole limpet hemocyanin (KLH) through tetraglycine (Gly)(4) linkers at the C6 position. Immunization of rats with M-KLH alone produced high titers of antibodies directed against heroin, 6-monoacetylmorphine (6-MAM) and morphine. Immunization with OXY-KLH produced high titers of antibodies against oxycodone and oxymorphone. Immunization with the bivalent vaccine produced consistently high antibody titers against both immunogens. Bivalent vaccine antibody titers against the individual immunogens were higher than with the monovalent vaccines alone owing, at least in part, to cross-reactivity of the antibodies. Administration of a single concurrent intravenous dose of 6-MAM and oxycodone to rats immunized with the bivalent vaccine increased 6-MAM, morphine and oxycodone retention in serum and reduced the distribution of 6-MAM and oxycodone to brain. Vaccine efficacy correlated with serum antibody titers for both monovalent vaccines, alone or in combination. Efficacy of the individual vaccines was not compromised by their combined use. Consistent with the enhanced titers in the bivalent group, a trend toward enhanced pharmacokinetic efficacy with the bivalent vaccine was observed. These data support the possibility of co-administering two or more opioid vaccines concurrently to target multiple abusable opioids without compromising the immunogenicity or efficacy of the individual components., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In a previous study, the authors have shown that herpes simplex virus type 1 (HSV-1) glycoprotein B DNA vaccine but not live vaccine (non-virulent KOS strain) failed to induce protective immunity against acute HSV-1 challenge in morphine-dependent mice. The present study reports the effect of morphine withdrawal on protective immunity induced by live HSV-1 immunization. BALB/c mice were vaccinated with KOS strain as a live vaccine. Three weeks later, they were exposed to morphine for 14 days. On day 14, withdrawal was induced by administration of normal saline instead of morphine. One day later, immune responses against HSV-1 were assessed by measuring cytotoxicity, lymphocyte proliferation and interferon-γ production. Protection against HSV-1 was assessed by measuring the mortality rate after acute HSV-1 challenge. The results showed that withdrawal from morphine reduces protective immunity against acute HSV-1 challenge. These findings raise the possibility that withdrawal from morphine may increase the susceptibility of drug addicts to infectious diseases., (Copyright © 2012 S. Karger AG, Basel.)

Acinetobacter baumannii is an important nosocomial pathogen in civilian intensive care units. Recently the incidence has increased in wounded military personnel. Morphine is documented in numerous animal studies to be immunosuppressive and to sensitize to infection. The hypotheses were tested that morphine, administered for analgesia in the battlefield, predisposes to Acinetobacter infection, and that the opioid may have an additive or synergistic effect with trauma. To test these hypotheses, an intraperitoneal infection model was established in mice using several Acinetobacter strains. Morphine administered for 48 h by implantation of a slow-release morphine pellet increased mortality compared to animals receiving a placebo pellet, an effect that was blocked by the mu-opioid receptor antagonist, naltrexone. Acinetobacter burdens in the blood, spleens, livers, and lungs of morphine-treated mice, were significantly higher than those in placebo-treated animals, confirming that mortality was due to potentiated growth of the bacteria. There were also elevated levels of pro-inflammatory cytokines in morphine-treated versus placebo-treated mice. Morphine caused a reduction in the total number of cells in the peritoneal cavity, a decrease in the percentage and total numbers of neutrophils, and a decrease in the total number of macrophages. Morphine treatment also suppressed levels of the neutrophil-inducing molecules, IL-17A and KC/CXCL1. However, IL-17A(-/-) mice given morphine were not sensitized to Acintobacter infection to a greater degree than similarly treated wild-type mice. Trauma alone did not sensitize to Acinetobacter infection, and there was no additive effect between morphine and trauma. These results support the hypothesis that morphine potentiates Acinetobacter infection.

Background: Neuromuscular blocking agents (NMBA) are responsible for most immediate hypersensitivity reactions during anesthesia, as a result of the presence of a quaternary ammonium ion. The aim of this study was to evaluate the diagnostic performance of a commercial immunoglobulin E (IgE) test (quaternary ammonium morphine [QAM]) for diagnosing sensitivity to NMBA., Methods: We tested 168 patients exposed to NMBAs during anesthesia. Of those patients, 54 had an uneventful procedure and 114 had immediate hypersensitivity reactions, and 57 patients had positive skin tests to the administered NMBA, whereas 57 had negative skin tests. Specific IgE concentrations determined with the QAM method based on a morphine solid phase were compared with those obtained with a recommended experimental method with a choline solid phase., Results: For the QAM test, a 0.35 kUA/l positivity cutoff was chosen from the receiver operating characteristics curve. QAM-specific IgE was found in 84.2% of skin test-positive reactors (80.7% with the recommended method; no significant difference), and binding was inhibited by the culprit NMBA in 80% of cases. The frequency of QAM-specific IgE positivity was significantly higher in skin test-negative reactors (24.6%) than in controls (9.3%), suggesting NMBA sensitivity., Conclusion: Sensitivity of the QAM test (84.2%), together with its simplicity and suitability for routine laboratory use, makes it a valuable tool, in conjunction with skin tests, for diagnosing NMBA sensitivity in patients who react after NMBA injection. The QAM test is of particular interest when skin tests are not available or not reliable or give results poorly compatible with mediator release or clinical features.

One of the sensitive and standard tests to control the safety of a vaccine is the inoculation of such vaccine to the air pocket of Lohmann specific pathogen free eggs. The aim of this study is to control the safety of morphine vaccine. This study reveals the safety of morphine vaccine by employing Lohmann specific pathogen free embryo eggs. The changeable parameters in this test were: weight of eggs, safety of eggs embryo, vaccine concentration, normal saline and temperature of the incubator. To study, the safety of morphine vaccine, we used 30 eggs (after controlling the safety of eggs and their embryos) which were divided into two groups of control (15 eggs) and test (15 eggs). After weighing the eggs, the eggs under experiment were inoculated with morphine vaccine and the control group was inoculated with physiological solution. Both groups were incubated and weight of the eggs and chickens were determined accordingly. The eggs of each group were controlled by their weights showing healthy, normal growth and evolution. The comparison between the weights of control and experimental groups did not show any significant changes. Exactly growth and evolution of each group were found equally to be balancing for three weeks after injection. Finally all eggs were observed to be safe, alive and in evolutionary form. By comparing the growth and evolution amongst each egg in the group under experiment, after injection, the eggs did not show any adverse reaction after inoculation with therapeutic human morphine vaccine.

The comparison of biomacromolecular crystal structures is traditionally based on the root-mean-square distance between corresponding atoms. This measure is sensitive to the presence of outliers, which inflate it disproportionately to their fraction. An alternative measure, the percentile-based spread (p.b.s.), is proposed and is shown to represent the average variation in atomic positions more adequately. It is discussed in the context of isomorphous crystal structures, conformational changes and model ensembles generated by repetitive automated rebuilding.

A surface-sensitive fluorescence measurement platform is utilised in the detection of morphine. The platform is based on a polystyrene parabolic lens that enables the simultaneous application of total internal reflection excitation and supercritical angle fluorescence detection in the measurements. The molecular recognition of morphine is based on two antibodies, one against morphine and the other against the immune complex formed between the anti-morphine antibody and a morphine molecule. The antibodies are applied in a sandwich-like format in a one-step test, where the molecular binding onto a liquid-solid-interface is monitored in real time. Morphine concentrations between 0.6 and 18.2 ng/mL were reliably determined in 60 s, while concentrations down to 2.7 ng/mL were detected already in 20 s. With appropriate recognition molecules the technique is applicable also to other drugs and small analytes.

Previous studies have shown that tolerance to the antinociceptive effect of morphine develops after a prolonged exposure, but its mechanisms remain unclear. In the present study, we examined whether anti-morphine antibody produced by chronic morphine exposure would contribute to the development of morphine antinociceptive tolerance in rats. Our results showed that anti-morphine antibody was present in rats rendered tolerance to antinociception after intrathecal morphine exposure for seven consecutive days. Superfusion of anti-morphine antibody onto spinal cord slice dose-dependently produced an inward excitatory current in spinal cord dorsal horn neurons using whole-cell patch-clamp recording, which surpassed morphine-induced outward inhibiting current. Co-administration of morphine with a monoclonal antibody (2.4G(2)) against Fc receptors for seven days significantly attenuated the production of anti-morphine antibody as well as the behavioral manifestation of morphine tolerance in same rats. These results indicate that anti-morphine antibody produced by morphine exposure may contribute to the development of morphine tolerance possibly through counteracting the inhibitory morphine effect on spinal cord dorsal horn neurons., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: The aim of this study was to test, on a multinational level, the pholcodine (PHO) hypothesis, i.e. that the consumption of PHO-containing cough mixtures could cause higher prevalence of IgE antibodies to PHO, morphine (MOR) and suxamethonium (SUX). As a consequence the risk of anaphylaxis to neuromuscular blocking agents (NMBA) will be increased., Methods: National PHO consumptions were derived from the United Nations International Narcotics Control Board (INCB) database. IgE and IgE antibodies to PHO, MOR, SUX and P-aminophenyl-phosphoryl choline (PAPPC) were measured in sera from atopic individuals, defined by a positive Phadiatop test (>0.35 kU(A)/l), collected in nine countries representing high and low PHO-consuming nations., Results: There was a significant positive association between PHO consumption and prevalences of IgE-sensitization to PHO and MOR, but not to SUX and PAPPC, as calculated both by exposure group comparisons and linear regression analysis. The Netherlands and the USA, did not have PHO-containing drugs on the markets, although the former had a considerable PHO consumption. Both countries had high figures of IgE-sensitization., Conclusion: This international prevalence study lends additional support to the PHO hypothesis and, consequently, that continued use of drugs containing this substance should be seriously questioned. The results also indicate that other, yet unknown, substances may lead to IgE-sensitization towards NMBAs.

Laboratory testing of oral fluid for drugs of abuse continues to expand in the workplace, legal, treatment, and health settings. In this study, we assessed recently developed homogeneous Roche DAT screening assays for amphetamines, cocaine metabolite [benzoylecgonine (BZE)], methamphetamines, and opiates in oral fluid. Precision and accuracy were assessed using control samples at +/-25% of cutoff. Sensitivity, specificity, and agreement compared to liquid chromatography-tandem mass spectrometry (LC-MS-MS) was assessed by analysis of oral fluid specimens collected from 994 subjects enrolled in a drug treatment or probation and parole drug-testing program. An additional 180 research specimens from Kroll Laboratories were analyzed for amphetamine and methamphetamine. Screening cutoff concentrations (ng/mL) were as follows: amphetamines, 40; cocaine metabolite, 3; methamphetamines, 40; and opiates, 10. LC-MS-MS analyses were performed with the following cutoff concentrations (ng/mL): amphetamine, 40; BZE, 2.0; methamphetamine, 40; and codeine or morphine, 10. The percent coefficient of variation ranged from 3.4% to 7.3%. Sensitivity and specificity of the Roche DAT assays compared to LC-MS-MS were > 94%, and agreement was > 96% for the four assays. The performance of the Roche DAT assays suggests these new homogeneous screening assays will be an attractive alternative to existing more labor-intensive enzyme immunoassays.

Using specific egg yolk antibodies (IgY), a strip-based immunochromatographic assay was developed for rapid detection of morphine in urine samples. IgY type antibody against morphine was generated by immunizing chickens with well-characterized monoacetyl morphine-protein conjugate. The antibody was labeled with gold nanoparticles and used as an immunoprobe in the dipstick format for the visual detection of morphine in urine samples. The dipstick was developed using three membranes: an application pad made of glass fiber membrane to hold the tracer, a signal generation test line on nitrocellulose membrane (detection zone) and a cellulose membrane used as an absorption pad. Analytes of interest (morphine and its analogues) added to the sample well, dissolved the labeled antibody (tracer), and the antigen-antibody complex formed was transported by the flow caused by capillary action to the test line. The color signal of test line in proportion to the morphine concentration in urine samples was measured using a detector. The developed dipstick assay format was optimized, showing the average IC(50) values of morphine as low as 9.45 ng/mL, the detection range of 1-1000 ng/mL and the lowest detection limit 2.5 ng/mL under optimal conditions of analysis. The correlation between the developed dipstick and ELISA was 0.948 in the analysis of urine samples spiked with morphine. The developed dipstick could be a highly sensitive and convenient tool for rapid detection of opiate drugs in samples with high degree of stability.

Substance abuse is a growing world-wide problem. The big four drugs of abuse that might lend themselves to immunotherapy are nicotine, cocaine, morphine/heroin and methamphetamine. Tobacco abuse has a well-known enormous impact on major chronic cardiovascular and pulmonary diseases, while the last three, aside from their neuropsychological effects, are illegal, leading to crime and incarceration as well as the transmission of viral diseases. Having an efficient vaccine that would generate antibodies to sequester the drug and prevent its access to the brain could go a long way toward helping a motivated addict quit the addiction. This review will discuss what has been done to bring such vaccines to human use, and what the challenges are for the future of this promising intervention.

Current pharmacotherapies for treating morphine/heroin dependence are designed to substitute or block addiction by targeting the drug itself rather than the brain. The heroin addict is still being exposed to addictive opiates, and consequently may develop tolerance to and experience withdrawal and drug's toxic effects from the treatment with high incidence of relapse to addictive drug consumption. As for other drugs of abuse, an alternative approach for morphine/heroin addiction is an antibody-based antagonism of heroin's brain entry. This review summarizes the literature examining important aspects of neurobiological and pharmacological processes involved in opiate dependence. Thereafter, classical pharmacological interventions for opiate dependence treatment and its major clinical limitations are reviewed. Finally, relevant preclinical studies are examined for comparisons in the design, use, immunogenic profile and efficacy of several models of morphine/heroin vaccine as immunologic interventions on the pharmacokinetics and behavioral of morphine/heroin in the rat as animal model.

In this study, we prepared monoclonal antibodies against morphine, methadone, babital, methamphetamine, and phencyclidine, then developed a dot-ELISA method by such antibodies to test their efficacy in clinical application and the screening of urine samples. It was found that there were 36 narcotics-positive drug users, including 28 morphine positive, six methamphetamine positive, and two positive for both. All the results were confirmed by commercial drug testing kits.

Monoclonal antimorphine antibodies both free and conjugated with horse- radish peroxidase have been raised and used to develop an assay kit for the detection of narcotic opiate-based drugs by an immuno-enzyme assay (IEA). The kit contains all ingredients necessary for the enzymatic reaction. A total of 215 urine and blood samples were analysed using the new kit. The results were compared with the data obtained by thin layer chromatography and high-performance liquid chromatography. False negative results were absent while false positive (inconclusive) results were recorded in three cases, probably due to the fact that sensitivity of IEA is higher than that of control methods. It is concluded that the kit may be used in laboratory screening studies for detecting opiates in biological fluids.

A novel carboxylic acid derivative of monoacetylmorphine (MAM-COOH) was synthesized and conjugated with bovine serum albumin (BSA) for generating polyclonal antibodies against the target molecule heroin and its major metabolites. The conjugate was characterized by fluorescence spectroscopy, polyacrylamide gel electrophoresis, and mass spectrometry to confirm the extent of haptenization of the carrier protein. A high titer (1:64,0000) of antibody was obtained by using the conjugate with an optimum protein/hapten molar ratio of 1:100. The generated antibody showed good binding affinity with heroin and its metabolites monoacetylmorphine (MAM) and morphine. The relative affinity constant (K (aff)) of the antibody was 3.1 x 10(7) l mol(-1), and the IC(50) values obtained for heroin, MAM, morphine, and codeine were 0.01, 0.013, 0.012, and 0.014 ng ml(-1), respectively. A fluorescence-based competitive inhibition immunoassay procedure was developed for the estimation of heroin and its major metabolites in standard and biofludic samples over a concentration range up to 0.01 ng ml(-1) with good signal reproducibility (p < 0.05). The method can be used as a convenient quantitative tool for the sensitive screening of major metabolites of heroin in biological samples.

Background: Neuromuscular blocking agents (NMBAs) can cause anaphylaxis through immunoglobulin E (IgE) antibodies that bind quaternary ammonium ion epitopes. These epitopes are present in numerous common chemicals and drugs, exposure to which, theoretically, could be of importance in the development and maintenance of the IgE sensitization promoting allergic reactions. Pholcodine is one such drug, which in a recent pilot study was shown to induce a remarkable increase in serum IgE levels in two IgE-sensitized individuals. The present study explores the effect of pholcodine exposure on IgE in a population with previously diagnosed IgE-mediated anaphylaxis towards NMBAs., Methods: Seventeen patients were randomized to 1 week's exposure with cough syrup containing either pholcodine or guaifenesin. The primary variables serum IgE and IgE antibodies towards pholcodine, morphine and suxamethonium were measured before and 4 and 8 weeks after start of exposure., Results: Patients exposed to pholcodine had a sharp rise in levels of IgE antibodies towards pholcodine, morphine and suxamethonium, the median proportional increases 4 weeks after exposure reaching 39.0, 38.6 and 93.0 times that of the base levels respectively. Median proportional increase of IgE was 19.0. No changes were observed in the guaifenesin group., Conclusion: Serum levels of IgE antibodies associated with allergy towards NMBAs increase significantly in sensitized patients after exposure to cough syrup containing pholcodine. Availability of pholcodine should be restricted by medical authorities because of the potential risk of future allergic reactions to muscle relaxants.

Not all opioids employed in clinical practice share the same immunosuppressive properties. The potent partial micro-agonist buprenorphine appears to exhibit a neutral effect on the immune responses. Surgery stress is associated with decreased natural killer cell activity (NK) and enhancement of tumor metastasis in rats. We analyzed the ability of buprenorphine to prevent the effects of experimental surgery on HPA activation (plasma corticosterone levels), NK activity and lung diffusion of the NK sensitive tumor MADB106. Buprenorphine (0.1mg/kg) was compared with equianalgesic doses of fentanyl (0.1mg/kg) and morphine (10mg/kg) in this animal model. In normal animals morphine and fentanyl stimulate the HPA axis, decrease NK activity and augment tumor metastasis, while buprenorphine is devoid of these effects. Surgery significantly raised corticosterone levels, suppressed NK activity and increased MADB106 metastasis. Only buprenorphine was able to prevent the neuroendocrine and immune system alterations and ameliorate the increase of tumor metastasis induced by surgical stress. These preclinical findings suggest that an adequate treatment of surgically induced stress immunosuppression with an opioid drug devoid of immunosuppressive effects may also play a protective role against the metastatic diffusion following cancer surgery.

Background: Diagnosis of allergy from neuromuscular blocking agents is not always straightforward. The objectives of the current study were to investigate the value of quantification of immunoglobulin E (IgE) by ImmunoCAP (Phadia AB, Uppsala, Sweden) in the diagnosis of rocuronium allergy and to study whether IgE inhibition tests can predict clinical cross-reactivity between neuromuscular blocking agents., Methods: Twenty-five rocuronium-allergic patients and 30 control individuals exposed to rocuronium during uneventful anesthesia were included. Thirty-two sera (total IgE > 1,500 kU/l) were analyzed for potential interference of elevated total IgE titers. Results were compared with quantification of IgE for suxamethonium, morphine, and pholcodine. Cross-reactivity between drugs was assessed by IgE inhibition and skin tests., Results: Sensitivity of IgE for rocuronium, suxamethonium, morphine, and pholcodine was 68, 60, 88, and 86%, respectively. Specificity was 100% for suxamethonium, morphine, and pholcodine IgE and 93% for rocuronium IgE. ROC analysis between patients and control individuals changed the threshold to 0.13 kUa/l for rocuronium, 0.11 kUa/l for suxamethonium, 0.36 kUa/l for morphine, and 0.43 kUa/l for pholcodine. Corresponding sensitivity was 92, 72, 88, and 86%, respectively. Specificity was unaltered. Interference of elevated total IgE with quantification of IgE was demonstrated by the analysis in sera with a total IgE greater than 1,500 kU/l. IgE inhibition did not predict clinical relevant cross-reactivity., Conclusions: The rocuronium ImmunoCAP constitutes a reliable technique to diagnose rocuronium allergy, provided an assay-specific decision threshold is applied. IgE assays based on compounds bearing ammonium epitopes are confirmed to represent reliable tools to diagnose rocuronium allergy. High total IgE titers were observed to affect specificity of the assays.

Chronic opioid administration modulates lymphocytes' functional capabilities increasing susceptibility to infectious diseases. Bacille-Calmette-Guérin (BCG) vaccination initiates a non-specific and specific cell-mediated immunity orchestrated by T lymphocytes including gammadelta T lymphocytes. gammadelta T lymphocytes increase in natural killer and antigen-directed cytolytic response following BCG vaccination. The objective of this study was to determine morphine effects on gammadelta T lymphocytes' cytolytic activity. Pigs were chronically administered morphine and subsequently vaccinated with Mycobacterium bovis BCG. By administering morphine prior to BCG vaccination, natural killer response was significantly suppressed (p=.034). Furthermore, innate cytolytic response against M. bovis-infected monocytes (p=.002) as well as antigen specific cytolytic functions (p=.04) were significantly altered due to morphine administration. It was concluded that administering morphine prior to BCG vaccination significantly altered gammadelta T lymphocyte cytolytic responses.

The present study used inbred, histocompatible Fischer 344 (FIS) and Lewis (LEW) rats to begin to explore the role of the hypothalamic-pituitary-adrenal (HPA) axis in the immune processes and pain behavior associated with the carrageenan model of acute hindpaw inflammation. Because the HPA axis contributes in part to morphine's analgesic and immunomodulatory properties, the present study also assessed the effects of morphine in carrageenan-inflamed LEW and FIS rats. The results showed that carrageenan-induced hindpaw swelling and pain behavior were greater in FIS than in LEW rats. The enhanced hindpaw swelling in FIS rats correlated with an increase in myeloperoxidase (MPO; a measure of neutrophils) in the inflamed hindpaw. FIS rats showed lower circulating levels of TNFalpha, higher IL-6 levels, and similar IL-1beta and nitric oxide levels, when compared to LEW rats. Morphine produced a significant decrease in carrageenan-induced hindpaw swelling and MPO in both strains, but morphine did not significantly alter circulating cytokine/mediator levels. Morphine's analgesic effects were greater in the inflamed than the noninflamed hindpaw, and they did not correlate with morphine's anti-inflammatory effects. In fact, low doses of morphine produced a mechanical allodynia and hyperalgesia in the noninflamed hindpaw of FIS, but not LEW, rats. These results suggest a positive relationship between HPA axis activity and acute inflammation and inflammatory pain. In contrast, little evidence is provided for HPA axis involvement in morphine's anti-inflammatory or analgesic effects.

Background: Analgesics are commonly used to provide pain relief after surgery. These drugs produce some extended depression of immunity. A prospective randomized controlled trial was designed to observe expressions of T-lymphocyte subsets (CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+)), natural-killer cells (CD3(-)CD16(+)CD56(+)), and activated T-lymphocytes (CD3(+)CD25(+)) of patients undergoing gastric cancer surgeries and receiving patient-controlled intravenous analgesia (PCIA)., Methods: Forty-five patients undergoing elective gastric cancer surgeries under general anesthesia were randomly allocated into 3 groups. Group I received PCIA using morphine after surgery, group II using tramadol, and group III using tramadol with lornoxicam. The analgesic efficacy was evaluated by visual analog scale (VAS) and Bruggrmann comfort scale (BCS). Expressions of CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) were measured as percentages of total lymphocytes by flow cytometer at 5 time points., Results: There was no significant difference in analgesic efficacy and the baselines of CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) in all groups. Compared with the baseline, CD3(+)CD8(+) had no changes in all groups at any time point. Ninety minutes after incision, CD3(+), CD3(+)CD4(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) were lower in all groups (P<0.05). 24 h after surgery, CD3(+), CD3(+)CD4(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) were lower in group I and group II (P<0.05); meanwhile CD3(+), CD3(+)CD4(+), and CD3(+)CD25(+) returned to the baseline but CD3(-)CD16(+)CD56(+) was still low (P<0.05) in group III. 48 h after surgery, CD3(+), CD3(+)CD4(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) returned to the baseline in group II and group III, but not in group I (P<0.05). 72 h after surgery, CD3(+), CD3(+)CD4(+), CD3(+)CD4(+)/CD3(+)CD8(+) returned to the baseline, but CD3(+)CD25(+) and CD3(-)CD16(+)CD56(+) were still low in group I (P<0.05)., Conclusion: PCIA using lornoxicam with tramadol has the same good analgesic efficacy and less immunity depression than PCIA using morphine or tramadol.

Both pre-clinical and clinical studies make feasible the use of vaccines as novel therapeutic medications to treat drug addiction. No reports to date have shown the development of structural models of opiate candidate vaccines for treating human addiction to such compounds. Here we report on the initial development of a novel structural formulation of a bi-valent vaccine against morphine/heroin. This vaccine was able to trigger and establish a high titer antibody response to haptenized drug with antibodies displaying equivalent specificities for both morphine and heroin. Such antibodies did not cross-recognize structurally dissimilar opiate medications. Furthermore, the evaluation of the potential therapeutic effectiveness of this vaccine was targeted to relapse prevention using a schedule of heroin delivery in the rat self-administration model. Antibodies against heroin blocked its reinforcing effects in rodents. The type of carrier protein used in this vaccine allows further evaluation of its potential therapeutic value for preventing relapse to heroin addiction in humans.