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Official websites use. Share sensitive information only on official, secure websites. For commercial re-use, please contact journals. 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. 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. 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. Opioids are some of the most widely abused illicit drugs worldwide, resulting in health problems, criminal activity, and economic burdens UNODC, The prevention of relapse is one of the most challenging problems in addiction treatment. Currently, some pharmaceutical agents are available for the maintenance of opiate abstinence. Opioid receptor agonists, such as methadone, and partial agonists, such as buprenorphine, are used as substitution therapies to reduce opioid craving and improve physical health and social functioning, whereas opioid antagonists, such as naloxone and naltrexone, are effective in detoxification and reversal of the acute adverse effects of opioid Fareed et al. However, several disadvantages overshadow the benefits of these medications. For example, methadone and buprenorphine have abuse potential Fareed et al. Naltrexone causes side effects because of long-term opioid receptor blockade Kosten et al. Therefore, ideal therapies with lasting treatment effects and few side effects are needed to enhance treatment adherence and prevent relapse Fareed et al. Immunotherapy has a mechanism of action that is different from the above therapeutic agents and is a promising alternative for relapse prevention Anton et al. A drug-carrier protein conjugate vaccine stimulates the immune response to generate antibodies that are specific to the target drug. The antibodies restrict the abused drug to the periphery and thus prevent its entry into and actions in the central nervous system Kosten and Owens, ; Anton et al. Several clinical trials have shown that higher antibody levels that are triggered by vaccines for cocaine Martell et al. Studies of methamphetamine vaccines have focused mainly on hapten designs to trigger a sufficiently high antibody level Duryee et al. To our knowledge, the published opioid vaccine studies are mainly preclinical research. Rabbits that were immunized with morphinehemisuccinate conjugated to bovine serum albumin BSA produced antibodies 8 weeks later Wainer et al. Monkeys that were immunized with morphinehemisuccinate-BSA exhibited a reduction of heroin intake in a self-administration model Bonese et al. Another morphine-keyhole limpet hemocyanin KLH vaccine with the same hapten triggered a sustained antibody response that lasted at least 8 weeks Kosten et al. However, the hapten in these studies was the same as the very first morphine vaccine introduced in An M-KLH vaccine with a atom linker altered the distribution of heroin and its metabolites and attenuated heroin-induced behavior in rats Raleigh et al. However, the acquisition of heroin self-administration was prevented only in rats that were vaccinated with heroin-KLH but not morphine-KLH. Our previous study presented a vaccine with a novel hapten, 6-glutarylmorphine, conjugated to KLH without a linker arm Li et al. Higher antibody concentrations and affinity would result in better antagonism of abused drugs. Therefore, vaccines with a higher antibody response and specificity need to be developed. In the present study, we hypothesized that a novel opioid vaccine with 6-glutarylmorphine as the hapten conjugated to KLH via a linker arm of 6 carbon atoms leads to a stronger immune response, higher antibody affinity, and thus better opioid blockade. The hapten 6-glutarylmorphine MG was synthesized similarly to our previous work Li et al. Briefly, 1. The resultant morphine base 1g and excess glutaric anhydride 1. The mixture then underwent reduced pressure distillation for solvent removal. The target product and excess morphine were separated by thin-layer chromatography on silica gel with ethanol, dioxane, benzene, and ammonia water volume ratio as the solvent system. The residues were then dissolved in 50mL distilled water and adjusted to pH 9. After filtration, the filtrate of the mixture was adjusted to pH 6. NaOH solution 1M was then added to adjust the pH to 8. Louis, MO were dispersed in mL distilled water, pH 5. The reaction mixture was purified by dialysis in PBS pH 7. During the active immunization procedure, the rats received vaccine administrations 4 times. On day 0, each rat was injected subcutaneously s. Louis, MO and 0. Rats that received KLH emulsified with the same solvent were used as controls. Subsequently, 3 booster injections were s. For the detection of antibody accumulation, lingual vein blood was collected on day 10 after each immunization. To assess the stability of the antibodies, blood was collected every 10 days until 2 months after the last vaccination. The primary antigen, morphine-BSA conjugate, for plate coating was synthesized similarly to a previous report Li et al. Absorbance was spectrophotometrically read at nm and is expressed as optical density OD; Keyler et al. The ODs on day 0 were used as controls. Briefly, sera were in a dilution of Serum samples were collected on day 10 after the fourth vaccination. The competitor compounds included morphine, heroin, buprenorphine, naloxone, nalorphine all from Qinghai Pharmaceutical Co. Dopamine levels in the nucleus accumbens NAc induced by morphine were measured by high-performance liquid chromatography HPLC with electrochemical detection. Rats were divided into 4 groups. Five minutes after the challenge injections, the animals were decapitated. Briefly, the mobile phase 0. The dopamine concentrations were calculated from the peak heights of the chromatographic data according to the standard curve BAS, West Lafayette, IN. Ltd and expressed as the total distance traveled in millimeters. The procedure for locomotor sensitization, which was the same as previously described Lu et al. In the adaptation phase, locomotor activity was measured after a daily injection of normal saline 0. In the withdrawal phase for the next 43 days, locomotor activity was measured without morphine injections. The rats were actively immunized with the vaccine 4 times on days 15, 29, 43, and The duration of each measurement was 2 hours. Intravenous cannulation surgery was performed as previously described Lu et al. Briefly, catheters were connected to modified cannulae and inserted into the right jugular vein, with the tip terminating at the opening of the right atrium. The cannulae were anchored to the skull with stainless-steel screws and dental cement. A stainless-steel stylet blocker was inserted into each cannula to maintain patency and prevent infection. All of the rats were allowed to recover for 5 to 7 days after surgery. Each chamber was placed in a light- and sound-controlled box equipped with an exhaust fan to ensure air renewal, a white illumination house light, and a tone stimulator. Each chamber was equipped with a green cue light located 2cm below the top, 2 nosepoke holes located 9cm above the floor, and a pump-driven syringe located on the top. The heroin self-administration procedure was performed according to our previous studies Wang et al. The acquisition of heroin self-administration began during the dark cycle and lasted 10 days 0. Daily training consisted of 3 sessions separated by 5-minute intervals. Each session lasted 1 hour and began with illumination of the house light, which remained on for the entire session. During the 5-minute intervals, the house light was turned off. A fixed ratio 1 reinforcement schedule was used, in which a nosepoke in the active hole led to an infusion of heroin accompanied by a 5-second tone-light cue. Nosepokes in the inactive hole were also recorded but had no scheduled consequences. A second timeout period occurred after each infusion, during which nosepoke responses were recorded but did not result in infusions. To avoid drug overdose, after a rat earned 20 infusions in 1 hour, active nosepokes led to neither drug infusions nor the accompanying tone-light cue. After acquisition, the rats were divided into 2 groups that were matched for the number of heroin infusions. The extinction conditions were the same as those during acquisition, with the exception that heroin was unavailable. The rats were subjected to extinction training every 2 days during the following 52 days of immunization. During the extinction procedure, one group of rats received standard active immunization with morphine-TFCS-KLH, and the other group received immunization with KLH on days 11, 25, 39, and The reinstatement test was conducted after extinction training. The test conditions were the same as those during extinction. Briefly, the rats received a single injection of heroin 0. Active nosepokes during the test resulted in contingent presentation of the tone-light cues, which were paired with heroin infusions during acquisition. The reinstatement tests were conducted on days 10, 14, and 25 after the fourth immunization, and the rats were housed in their homecages between tests. After active immunization, anti-morphine antibody titers reached a peak after the third and fourth vaccinations. The maximal antibody titers were after the third injection Figure 2b. Isotype-specific antibody detection revealed that anti-morphine IgM rapidly reached a peak on day 24 10 days after the second immunization and dropped below the level of detection on day 52 10 days after the fourth immunization. IgG1, IgG2a, and IgG2b increased more slowly, reaching a peak after the third immunization and decreasing after the last booster injection, but the levels remained detectable for at least 70 days on day Figure 2c. Serum samples were collected 10 days after each immunization days 10, 24, 38, and 53 , and the antibody titers reached detectable levels of after the third and fourth immunizations. Antibody titers were also examined every 10 days after the fourth immunization for 2 months. Because booster injections were no longer administered, antibody titers declined but remained detectable for at least 2 months. Seventy days after the last vaccination, antibody titers were still detectable Figure 2d. These results demonstrate that the triggered antibodies possessed high and comparable affinity with both morphine and heroin, but not buprenorphine, naloxone, or nalorphine. The antibodies showed equivalent specificities for morphine, 6-acetylmorphine, and heroin but no cross-reactivity with buprenorphine, naloxone, or nalorphine. B 0 refers to absorbance without competitors, and B refers to absorbance in the presence of competitors at each concentration. Locomotor activity on day 0 served as baseline. A heroin self-administration model was used to determine whether the morphine-TFCS-KLH vaccine prevents the reinstatement of drug-seeking behavior induced by heroin priming Figure 6a. The baselines defined as the average number of heroin infusions over the last 3 days of acquisition were equivalent between groups KLH group: After acquisition, the animals were subjected to 52 days of discontinuous extinction until all of the rats presented extinction-like levels of responding, during which they received 4 active immunizations. On days 10, 14, and 25 after the last immunization, the rats were subjected to reinstatement tests 5 minutes after heroin priming 0. Inactive nosepokes were not significantly different between groups during any of the reinstatement tests Figure 6c. The numbers of active nosepokes during acquisition and extinction were not significantly different between groups. Heroin-primed 0. As expected, the vaccine triggered a robust and sustained immunological response, reflected by high and long-lasting serum antibody titers. The antibodies showed equivalent specificities to morphine, 6-acetylmorphine, and heroin, reflecting its efficacy for heroin and its active metabolites. The vaccine possibly reduced the central entry of morphine, reflected by a significant decline in dopamine levels induced by morphine challenge. Vaccination also decreased the expression of behavioral sensitization and heroin-primed drug-seeking behavior. These results indicate that the present vaccine might be a potential approach to prevent the relapse of opioid use, including both heroin and morphine. The hapten MG, which was first designed and reported in our previous work Li et al. First, MG is possibly more stable than the commonly used 6-hemiglutarate hapten, because aflatoxin B2a-hemisuccinate hydrolyzes more rapidly than aflatoxin B2a-hemiglutarate in aqueous solution Lau et al. Slower hydrolysis is speculated to result in a higher sustained immunoconjugate concentration and thus a higher antibody titer Tew et al. Second, the 6-glutarate hapten contained one more carbon atom than 6-hemiglutarate, which makes the morphine structure less likely to be fettered by the carrier protein, possibly leading to better antibody affinity to morphine. In addition to hapten design, we also introduced a longer linker arm between the hapten and carrier protein as reported for a morphine-tetanus toxoid vaccine Anton and Leff, , which probably allows further exposure of the morphine structure to the vaccine and contributes to higher recognition affinity to exogenous morphine by the antibodies. A small-molecule drug per se does not possess immunoreactivity, but when conjugated to a large carrier, the exposed drug structure becomes an epitope that is able to trigger an immune reaction Kosten and Owens, According to studies of cocaine and methamphetamine vaccines, the space between the hapten, carrier, and heteroatoms determines the titers of triggered antibodies Carrera et al. Enhanced stability and sufficient epitope exposure are highly predictive of a higher antibody titer and affinity Carrera et al. Compared with our previous vaccine Li et al. Moreover, the antibody titers reached a peak more rapidly after the third immunization compared with our previous vaccine after the fourth immunization and decayed more slowly. Although both vaccines induced antibodies with comparable affinity to morphine, the present vaccine robustly increased the antibody affinity to heroin 1. Although the active immunization procedures and adjuvants were not the same among studies, our present vaccine showed an advantage of maximal antibody titers that were comparable with other morphine or heroin vaccines Anton et al. The dopamine system, particularly the mesolimbic system, underlies drug reward processing Di Chiara and Imperato, ; Le Moal and Simon, Extracellular dopamine levels in the NAc core are increased by acute heroin administration Gerasimov et al. Typically, as large proteins, antibodies bind to drugs and retain them in peripheral regions, thus blocking their permeation of the blood-brain barrier and rewarding effects Orson et al. One limitation of the present study is that we measured total dopamine concentrations rather than dopamine release by microdialysis. We found that the vaccine blocked the increase in total dopamine levels in the NAc after a single morphine injection. Previous studies found that NAc lesion with 6-hydroxydopamine attenuated the increase in total dopamine induced by heroin Spyraki et al. Total dopamine levels in the NAc homogenate reflect dopamine in synaptic vesicles, synaptic cleft, and cell bodies, and we speculate that NAc undergoes a general increase in dopamine transmission after an acute morphine injection Lammel et al. The locomotor-stimulating effect of opiates is presumed to be modulated by dopamine transmission Le Moal and Simon, Our novel vaccine inhibited the expression of morphine-induced locomotor sensitization, suggesting that it is able to attenuate the psychoactive effects of morphine and heroin. Reexposure to an abused drug provokes relapse in addicts, which is the most difficult problem encountered in drug addiction treatment Shaham et al. Notably, the results of the heroin reinstatement tests indicated that heroin-primed heroin-seeking behavior was inhibited at least 25 days after the last immunization, reflecting a longer-lasting effect than the vaccine in our previous work Li et al. This is consistent with our antibody detection results. The higher affinity of the present vaccine to heroin may also indicate better relapse prevention in a heroin self-administration task. Because the vaccine does not target opioid receptors and the antibodies persisted sufficiently long in the body, such an approach could minimize the side effects related to opioid receptor activation and provide long-lasting protection against the psychoactive effects of specific opioid drugs. Consequently, less effort would be needed to maintain patient compliance. A recently published study showed that a heroin vaccine with the metabolically reactive heroin region exposed for immunization recognition induced antibodies that specifically recognized heroin and its metabolite 6-acetylmorphine but not morphine Schlosburg et al. Our vaccine triggered high and sustained antibody levels, and the design of the lengthened linker, together with the lengthened hapten, might be applied in the synthesis of other more specific vaccines with different epitope exposure. A potential limitation of anti-addiction vaccines, including morphine and heroin vaccines, is that patients may take drug doses that are several-fold higher than their usual doses to overcome the effect of the vaccine, similar to the results in rats subjected to pharmacological opioid receptor blockade Koob et al. However, several clinical trials of cocaine and nicotine vaccines did not show any evidence of compensatory smoking or cocaine use and found that a higher antibody titer indeed resulted in significantly higher abstinence rates Hatsukami et al. Another limitation of drug vaccines is that antibodies gradually decrease after the last vaccine injection, and reexposure to nonconjugated drug is not sufficient to trigger immunological memory. However, long-term immunological memory that is generated during the humoral antibody response facilitates a more rapid response to the future booster injections with the vaccine Kosten and Owens, Therefore, repeated booster injections of the vaccine are required to maintain a sustained antibody level. In summary, we provided a novel KLH-conjugated vaccine that attenuated the behavioral and psychoactive effects of heroin and morphine. The vaccine produced high and sustained antibody titers against both morphine and heroin. After being conjugated with clinically approved carriers, our hapten may be therapeutically applied to morphine and heroin addiction. We thank Dr. Yan-Ping Bao for helpful comments on the manuscript. As a library, NLM provides access to scientific literature. Int J Neuropsychopharmacol. Find articles by Qian-Qian Li. Find articles by Cheng-Yu Sun. Find articles by Yi-Xiao Luo. Find articles by Yan-Xue Xue. Find articles by Shi-Qiu Meng. Find articles by Ling-Zhi Xu. Find articles by Na Chen. Find articles by Jia-Hui Deng. Find articles by Hai-Feng Zhai. Find articles by Thomas R Kosten. Find articles by Jie Shi. Find articles by Lin Lu. Find articles by Hong-Qiang Sun. Li and C. Sun contributed equally to this work. Open in a new tab. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.

A Conjugate Vaccine Attenuates Morphine- and Heroin-Induced Behavior in Rats

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What are the most addictive drugs? This question seems simple, but the answer depends on whom you ask. There are other facets to measuring the addictive potential of a drug, too, and there are even researchers who argue that no drug is always addictive. Given the varied view of researchers, then, one way of ranking addictive drugs is to ask expert panels. In , David Nutt and his colleagues asked addiction experts to do exactly that — with some interesting findings. Nutt et al. In addition to being arguably the most addictive drug, heroin is dangerous, too, because the dose that can cause death is only five times greater than the dose required for a high. Heroin also has been rated as the second most harmful drug in terms of damage to both users and to society. In experiments on animals, cocaine caused dopamine levels to rise more than three times the normal level. Crack cocaine has been ranked by experts as being the third most damaging drug and powdered cocaine, which causes a milder high, as the fifth most damaging. Cocaine is similar to other addictive stimulants, such as methamphetamine — which is becoming more of a problem as it becomes more widely available — and amphetamine. Nicotine is the main addictive ingredient of tobacco. When somebody smokes a cigarette, nicotine is rapidly absorbed by the lungs and delivered to the brain. More than two-thirds of Americans who tried smoking reported becoming dependent during their life. In the WHO estimated there were more than 1 billion smokers and it has been estimated that tobacco will kill more than 8m people annually by Laboratory animals have the good sense not to smoke. Barbiturates — also known as blue bullets, gorillas, nembies, barbs and pink ladies — are a class of drugs that were initially used to treat anxiety and to induce sleep. They interfere with chemical signalling in the brain, the effect of which is to shut down various brain regions. At low doses, barbiturates cause euphoria, but at higher doses they can be lethal because they suppress breathing. Barbiturate dependence was common when the drugs were easily available by prescription, but this has declined dramatically as other drugs have replaced them. This highlights the role that the context plays in addiction: if an addictive drug is not widely available, it can do little harm. The WHO has estimated that 2 billion people used alcohol in and more than 3m people died in due to damage to the body caused by drinking. Alcohol has been ranked as the most damaging drug by other experts, too. This article has been amended to reflect an error in the reading of Nutt et al. Edition: Available editions Europe. Become an author Sign up as a reader Sign in. Eric Bowman , University of St Andrews. Heroin Nutt et al. Events More events.

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