Where to get jwh 018 usa

When administered to mice, JWH produces effects consistent with other CB 1 receptor agonists, including hypothermia, analgesia, reduced motor activity, and catalepsy 5 , 6. Several lines of clinical evidence support a relationship between heavy cannabis use and risk for development of psychosis and schizophrenia 10 — Although the precise underpinnings of schizophrenia are not fully understood, dysregulation of brain serotonin 5-HT systems has been implicated in certain psychotic symptoms, such as paranoia and hallucinations 14 — Preclinical studies in rodents show that exposure to CB 1 receptor agonists can influence 5-HT receptor responsiveness in vivo.

By contrast, Hill et al. Franklin et al. Despite the continued misuse of synthetic cannabinoids by humans, little is known about the functional consequences of repeated administration of JWH or related substances found in spice products. Given the emerging evidence for interactions between cannabinoid and 5-HT systems in the brain, we sought to determine the effects of repeated treatment with JWH on the behavioral responsiveness to selective 5-HT receptor agonists.

Body temperatures and catalepsy scores were determined during the repeated dosing regimen of JWH, while body temperatures and agonist-induced behaviors were measured following challenge doses of 5-HT drugs.

We hypothesized that repeated exposure to JWH would enhance subsequent behavioral responsiveness to DOI in rats [e. JWH and rimonabant were dissolved into a mix of dimethyl sulfoxide:Tween sterile saline, whereas other drugs were dissolved in sterile saline. All injections were administered at a volume of 1. After 2 weeks of acclimation to the vivarium, rats were subjected to surgical procedures and subsequently used for experiments.

Rats were rapidly anesthetized with isoflurane using a drop jar which contained a raised floor above a gauze pad saturated with 5 mL of isoflurane. Once fully anesthetized, each rat received a surgically implanted IPTT transponder Bio Medic Data Systems, Seaford, DE, USA to facilitate the non-invasive measurement of body temperature via a portable radio frequency reader system handheld reader.

Animals were individually housed postoperatively and allowed 7—10 days for recovery. As a first step in our study, we examined the dose—response effects of acute JWH administration in a cohort of 12 rats. Rats were tested once per week for three consecutive weeks.

On test day, rats were moved to the testing room in their home cages and given 1 h to acclimate. Feeding trays were removed, and wire lids were placed atop the cages. Rats received sc injections of JWH 0. Immediately before injection, and at various times thereafter 0. Observers were not blind to the drug treatment condition. Rats were assigned a catalepsy score based on three behaviors: immobility absence of movement , flattened body posture, and splayed limbs limbs spread out away from the center of the body.

Once dose—response experiments were completed, we next tested the effect of pretreatment with the CB 1 receptor antagonist rimonabant on the responses induced by JWH in a cohort of 12 rats. Rats were pretreated with either 1. Body temperature measurements and behavior scoring were carried out as described previously for acute dose—response experiments.

Results from the acute dose—response experiments demonstrated that 1. Thus, this dose was used for the repeated injection experiments carried out in a group of 32 rats. The repeated dosing with JWH or its vehicle was carried out in the vivarium. Rats fitted with surgically implanted sc temperature transponders received a single sc injection of either 1. Immediately before injection, and at 1, 2, and 4 h post-injection, body temperature was measured using the handheld reader, and animals were observed for 90 s.

One day after the last repeated treatment with JWH or vehicle i. One cohort of 16 rats received 0. The doses of DOI and 8-OH-DPAT were based on preliminary dose—response experiments, which identified drug doses evoking robust behavioral changes that were less than maximal data not shown. The specific non-contingent behaviors induced by DOI were wet dog shakes and back muscle contractions i. Both behaviors are known to be mediated by 5-HT 2A receptors in rats 21 — The numbers of wet dog shakes and skin jerks present during the observation period were tallied.

Wet dog shakes were defined as a rapid and sudden rotation of the head, neck, and shoulders from one side to the other, analogous to the way a wet dog may shake to dry itself. Skin jerks were defined as brief paraspinal muscle contractions of the back muscles in a tail to head direction. Possible scores for each behavior were 0 behavior absent , 1 behavior present , or 2 behavior intense or continuous.

At the end of the observation period, the scores for the three behaviors were summed to produce a 5-HT syndrome score for each time point. After acute serotonergic drug challenge, body temperatures were measured using the handheld reader at 0.

Data were tabulated, analyzed, and graphically depicted using GraphPad Prism version 5. Statistical analyses were performed on data from all 7 days of the JWH repeated administration experiment, however, Figure 3 only shows data from selected days to make the graphs easier to interpret. The left panel of Figure 1 illustrates the effect of acute JWH administration on core body temperature in male rats. It is worth noting that 0. As seen in the right panel of Figure 1 , JWH dose-dependently increased the summed catalepsy behavioral score Kruskal—Wallis statistic Figure 1.

Core temperature measures and summed catalepsy scores for rats receiving acute subcutaneous injections of 0. Core temperature and behavioral score were recorded at 0, 0. The left panel of Figure 2 shows that pretreatment with 1. Figure 2. Core temperature measures and summed catalepsy scores for rats receiving either subcutaneous sc vehicle VEH or 1.

The left panel of Figure 3 depicts the effects of 1. Because vehicle administration did not affect body temperature over the course of repeated injections, we compared the effects of JWH treatments across days to those of vehicle treatment on day 1. On day 1 of JWH exposure, temperature was significantly reduced from vehicle at the 1, 2, and 4 h timepoints.

By day 3 of treatment, hypothermia was observed only at the 1 h timepoint, and on days 6 and 7, no reduction in temperature was observed. Figure 3. Core temperature measures and summed catalepsy scores for rats receiving either subcutaneous vehicle VEH or 1. The right panel of Figure 3 depicts the effects of 1. Vehicle administration did not significantly alter summed catalepsy scores on day 1 of treatment, and there was no change in scores for vehicle-treated rats over the 7-day treatment regimen.

Since vehicle administration did not change catalepsy scores over the course of treatment, we compared the effects of JWH treatment across days to the effects of vehicle treatment on day 1. Using this analysis, JWH increased catalepsy scores compared to vehicle Kruskal—Wallis statistic Rats received 0. Similar non-significant effects between pretreatment groups were observed at day 7. DOI did not significantly affect core body temperature in rats pretreated with JWH or vehicle at either test day data not shown.

Figure 4. Summed scores for wet dog shakes and back muscle crawls skin jerks induced by a subcutaneous challenge injection of 0. Behavioral scores were recorded at 0, 0. A separate cohort of rats was given 0. Figure 5. Summed scores for serotonin syndrome behaviors and mean temperature recordings induced by a subcutaneous challenge injection of 0. Behavioral scores and core temperatures were recorded at 0, 0.

Post hoc tests revealed that temperature was significantly decreased in the JWH group compared to the vehicle group at 1.

Figure 6. Time-course of core body temperature changes induced by a subcutaneous challenge injection of 0. Temperatures were recorded at 0, 0. The psychiatric literature supports a strong relationship between heavy cannabis use and risk for subsequent psychosis and schizophrenia In addition, misuse of synthetic cannabinoids such as JWH and its analogs is associated with induction of more severe psychotic symptoms when compared to the effects of marijuana 26 , The aim of the present study was to use the popular synthetic cannabinoid JWH to further explore the relationship between repeated cannabinoid exposure and serotonergic dysregulation.

JWH is a potent non-selective cannabinoid receptor agonist that was found in the first generation of spice products 1 , 2. The present experiments yielded three primary findings.

First, in contrast to the results of others [e. Second, we found a modest and significant enhancement of sensitivity to behavioral and hypothermic effects induced by 8-OH-DPAT in rats exposed to repeated injections of JWH Finally, our data show that rats receiving daily injections of JWH develop profound tolerance to its hypothermic and cataleptic effects, such that these effects are nearly absent after 7 days of treatment.

Typical behavioral responses to DOI administration in rats are wet dog shakes analogous to the head twitch response in mice and back muscle contractions, also known as skin jerks 21 — These responses are accepted as specific indicators of 5-HT 2A receptor activation since the effects are blocked by selective 5-HT 2A receptor antagonists. We found no significant difference in the number of wet dog shakes or skin jerks induced by DOI between the cannabinoid-treated and vehicle-treated groups at either time point.

Our findings differ from those of Hill et al. It is noteworthy that we observed trends for augmented wet dog shakes and attenuated skin jerks in rats exposed to JWH, but these effects did not reach significance, perhaps due to variability in the behavioral data. We also administered a submaximal dose of 0. Hill et al. This hypothesis was later supported by the work of Franklin et al. It is well known that HU displays a much longer time course of action when compared to other synthetic cannabinoids, including JWH, and may bind pseudo-irreversibly to the CB 1 receptor.

Hruba and McMahon found that rhesus monkeys trained to discriminate THC from vehicle continued to emit drug-appropriate responses for 48 h after administration of HU, while such responses to THC and CP 55, ceased after 5 h. Thus, the discrepancies between our results and those of Hill et al.

Our study used a repeated cannabinoid administration paradigm followed by the administration of DOI after 1 and 7 days of withdrawal, so this may help to explain the differences between our results and those of Darmani. The present findings in rats show that administration of CB 1 agonists causes considerable catalepsy see Figures 1 — 3 , so it seems possible that suppression of motor activity caused by acute cannabinoids could influence subsequent behavioral effects of 5-HT 2A receptor agonists.

We purposefully designed our experiments to examine the responsiveness to 5-HT agonists at 1 and 7 days after the acute effects of cannabinoid administration had subsided. We found a modest yet significant increase in the behavioral and hypothermic effects induced by 8-OH-DPAT in rats receiving repeated JWH treatments when compared to those receiving repeated vehicle treatments.

In a previous study, Hill et al. Both hypothermia and corticosterone release are presumably mediated by 5-HT 1A receptors in the brain 31 , thus Hill et al. It seems possible that discrepancies between our results and those of Hill et al.

On the other hand, Zavitsanou et al. Our data demonstrating an increase in 5-HT 1A receptor sensitivity after exposure to JWH is a unique finding, and its relationship to the development of psychiatric symptoms following cannabinoid exposure warrants further study. Future research should determine whether 5-HT 1A upregulation occurs after repeated exposure to other synthetic cannabinoids.

While the present study reveals the ability of these JWH derivatives to act at CB1Rs, pharmacokinetic analysis will be required to definitively determine the presence of these metabolites in target tissues in collective concentrations high enough to elicit adverse effects in vivo.

The recent discovery of detectable nanomolar concentrations of M2, M3, and M5, the most efficacious of the JWH derivatives examined in this study, in human urine [24] , supports the current proposal that metabolites contribute to the effects of K2 and should thus be pursued further.

Moreover, the main foci of the current K2 literature are only clinical case studies reported from emergency departments [12] , [13] , [14] , [15] , [16] , [18] and methods detailing the analytical detection of synthetic K2 cannabinoids in body fluids [4] , [6] , [8] , [23] , [24] , [33]. For these reasons, coupled with the rapidly growing use and dangerous adverse effect profile of K2 products, it is critical that clinicians and basic scientists obtain a strong mechanistic understanding of the cannabinoid constituents in K2, so these drugs can be identified, regulated and therapies designed to address the adverse effects.

Currently, little is known regarding the structure-activity relationships SARs at CB1R of aminoalkyindole cannabimimetics that are substituted around the indole nucleus, especially at positions 4—7. Although Eissenstat et al. To our knowledge, the present work is the first to show that hydroxylation at positions 5 and 6 metabolites M2 and M3 retain significantly higher activity, with little difference in affinity, compared to hydroxylation at positions 4 and 7 M1 and M4.

The importance of the substituent at the end of the pentyl chain is also underscored in the present work by the observation that addition of a carboxylic acid M6 , but not a hydroxyl group M5 totally eliminates affinity for CB1R. Detailed characterization of the specific enzymes responsible for biotransformation of JWH and structurally similar synthetic cannabinoids would be an important step to fully understand the consequences of JWH use in humans.

A recent report, employing crude human liver microsomes, suggests that specific isoforms of the cytochromes P system are essential for metabolism of JWH to various mono- and dihydroxylated metabolites [9] , including the metabolites examined in the present study. Likewise, a second in vitro study, using rat liver microsomes [35] , has identified the cytochrome P system responsible for metabolism of the structurally similar CB2 selective agonist JWH However, to our knowledge, no study has reported the specific enzymes responsible for production of the primary and secondary metabolites of JWH When delineated, SARs, mutagenesis and polymorphism studies of these specific enzymes might reveal inter-individual differences for production of active metabolites and thus provide support for why some individuals exhibit greater degrees of severe adverse reactions to K2 exposure.

If correlated to clinical observations, this information could provide a model to predict severe adverse effects in susceptible individuals.

For example, different enzymatic polymorphisms may yield distinct metabolic rates that vary from person to person and produce a preference for formation of certain metabolites relative to others. As predicted by the present study, because different metabolites exhibit various degrees of activity, a bias towards production of more active metabolites that could increase the net activation of CB1R is possible.

Alternatively, it might be predicted that production of less active metabolites by certain individuals would result in antagonistic effects with concurrently administered cannabinoids, potentially leading to greater use of synthetic cannabinoids in an attempt to overcome the reduction in effects.

Such complex effects could have unique and potentially harmful consequences on the delicate balance of the endocannabinoid system, which plays important roles in modulating mood [36] , appetite and energy homeostasis [37] , [38] , pain sensation [39] , immune function [40] , fertility [41] and possibly bone homeostasis [42].

Although several explanations are possible, cannabinoid receptor-independent GPCR activation in response to K2 use is probable. The possibility of CB1R-independence, both GPCR and non-GPCR mediated, is supported by the clinical observations of seizures, hallucinations, anxiety, agitation, panic attacks, and hypertension, which are not typically observed following CB1R activation.

The mechanisms behind these atypical adverse effects, although not completely understood themselves, nevertheless give additional information as to how K2 is acting in vivo. For example, grand mal seizures, which can occur with K2 use, are the result of excessive, aberrant neural synaptic firing that leads to involuntary tonic-clonic spasms. The mechanisms behind grand mal seizures are complex and diverse, but ultimately involve disinhibition of excitatory neurons [43].

Retrograde activation of CB1Rs by cannabinoids and endocannabinoids hyperpolarizes presynaptic neurons and thus inhibits synaptic transmission [44] , and several cannabinoids have even been shown to exhibit anticonvulsive activity [45] , [46] , [47] , [48].

Theories underlying the neurobiological mechanisms of hallucinations and psychosis include abnormal dopaminergeric neurotransmission, as described in the dopamine hypothesis of schizophrenia [52] , serotonergic transmission, as seen with the serotonergic classical hallucinogens [53] , and NMDA glutamate receptor blockade [54].

Alteration of the receptor networks mentioned here are just a few examples of many possible that may result in severe adverse effects seen in an alarmingly large proportion of K2 users.

The present study investigates some previously unknown actions of oxidized products of JWH produced by using the relatively new and increasingly common drug of abuse, K2. Although JWH is a predominant component of K2, it is unfortunately only one of a whole host of cannabimimetic compounds found in varying, unregulated concentrations from brand-to-brand and, even within brands, batch-to-batch of K2.

This reality presents a challenge to researchers and clinicians in their attempts to better understand and predict the biological consequences of K2 use and thus accurately warn the general public about its risks, as well as advise legislators, who are currently working to determine the appropriate legal status of K2. The uncontrolled and heterogeneous nature of K2 also presents a danger to even its more experienced users who may unknowingly use K2 containing particular synthetic cannabinoid blends to which they may have an adverse reaction.

Nonetheless, this work represents an important initial step toward understanding K2 by uncovering significant CB1R affinities and intrinsic activities of five potential metabolites of JWH Since JWH produces metabolites with partial to full agonist activity at CB1Rs, it is justified to posit that similar K2 synthetic cannabinoids can also be biotransformed into molecules with various levels of affinity and activity at CB1Rs, as well as at other receptor systems as discussed above.

Altogether, the presence of parent synthetic cannabinoid molecules within a single dose of K2, combined with the respective active metabolites produced, could conceivably act in concert to produce the dynamic range of effects observed following use of various K2 preparations.

The idea that active metabolites are generated from not just one, but several parent molecules found within a single drug of abuse, is novel and exciting, but complicates matters by introducing an intrinsic polypharmacy effect. In conclusion, the discovery that JWH metabolites, and other oxidized products of JWH, partake actively and diversely in the activity of K2 provides a substantial avenue of exploration and thus serves as an essential building block in combating problems associated with an increasingly common drug of abuse.

Louis, MO , respectively. Louis, MO. To prepare crude membrane homogenates, brains were thawed on ice, pooled and suspended in ice-cold homogenization buffer 50 mM HEPES pH 7. Supernatants were discarded and pellets were resuspended in ice cold homogenization buffer, homogenized and centrifuged similarly twice more.

Assays were performed in triplicate, in a final volume of 1 mL, as previously described [59]. Total binding was defined as the amount of radioactivity observed when 0. Specific binding was calculated by subtracting non-specific from total binding. After overnight extraction, bound radioactivity was determined by liquid scintillation spectrophotometry.

Specific binding is expressed as a percentage of binding occurring in vehicle samples e. Louis, MO and water ad libitum until immediately before testing.

Animals were acclimated to the laboratory environment 2 days prior to experiments and were tested in groups of 6 mice per condition. All studies were carried out in accordance with the Declaration of Helsinki and with the Guide for Care and Use of Laboratory animals as adopted and promulgated by the National Institutes of Health.

A rostral-caudal cut approximately 1. At least 7 days were imposed between surgery and experimental observation of drug effects to allow incisions to heal and mice to recover normal body weights.

Following surgery, implanted mice were individually housed in Plexiglas mouse cages Implanted transmitters produced activity- and temperature-modulated signals that were transmitted to a receiver model ER Receiver, Mini Mitter Co. Albans, VT equipped with exhaust fans, which further masked ambient laboratory noise. On experimental days, mice were weighed, marked, and returned to their individual cages during which at least 1 hr of baseline data were collected. Cannabinoid doses were then calculated and drugs prepared for injection.

Mice were then placed into a new cage with fresh bedding to stimulate exploratory behavior. Temperature and locomotor activity data were collected at regular 5-min intervals and processed simultaneously by the Vital View data acquisition system Mini Mitter Co.

Curve fitting and statistical analyses for in vitro experiments were performed using GraphPad Prism version 4. The Cheng-Prusoff equation [61] was used to convert the experimental IC 50 values obtained from competition receptor binding experiments to K i values, a quantitative measure of receptor affinity.

Non-linear regression for one-site competition was used to determine the IC 50 for competition receptor binding. For core body temperature experiments, the area under the curve AUC was calculated using a trapezoidal rule from 0—10 hr.

For locomotor activity, total locomotor counts were summed from 0—10 hr. The authors would like to thank Ms. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Background K2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks.

Download: PPT. Figure 1. Figure 2. Figure 4. Figure 5. Discussion This study importantly demonstrates for the first time that five potential Phase I hydroxylated metabolites of the synthetic cannabinoid JWH bind with high nanomolar affinity to, and very efficaciously activate, CB1Rs in vitro. Statistical Analysis Curve fitting and statistical analyses for in vitro experiments were performed using GraphPad Prism version 4. Acknowledgments The authors would like to thank Ms.

References 1. Toxicol Lett — View Article Google Scholar 2. Bioorg Med Chem Lett 4: — View Article Google Scholar 3. J Pharmacol Exp Ther — View Article Google Scholar 4.

J Mass Spectrom — View Article Google Scholar 5. Lisbon, Portugal. Chem Pharm Bull Tokyo — View Article Google Scholar 7. View Article Google Scholar 8. Drug Test Anal. View Article Google Scholar 9. Anal Bioanal Chem — View Article Google Scholar Br J Pharmacol — Vienna, Austria: United Nations.

Every-Palmer S Warning: legal synthetic cannabinoid-receptor agonists such as JWH may precipitate psychosis in vulnerable individuals. Addiction —