TL neuro

June 3, 2016

Inhalation delivery of psychostimulants to rats using e-vape technology

Filed under: 4-MMC/Mephedrone, Cathinones, E-cigarettes, MDPV, Methamphetamine — mtaffe @ 4:03 pm

Although inhaled exposure of drugs is a prevalent route of administration for human substance abusers, animal models of inhaled exposure to psychomotor stimulants (cocaine, methamphetamine, synthetic cathinones, etc) are not commonly available. Inhaled use of methamphetamine is more common than other routes of administration in habitual and dependent users (Das-Douglas et al. 2008; Heinzerling et al. 2010; Wood et al. 2008) and the SAMHSA/TEDS treatment admission database for 2012 shows 4.7% of treatment seekers in the USA were admitted for smoked cocaine vs 2.2% for other routes of cocaine administration. There is limited evidence that people are using e-cigarettes for inhalation of methamphetamine (Evans 2014; Rass et al. 2015), “bath salts” (Johnson and Johnson 2014; Rass et al. 2015) and “flakka” (presumptively α-pyrrolidinopentiophenone; alpha-PVP) as reported (Anderson 2015).

We have therefore developed a method for the delivery of psychostimulant drugs to rats and evaluated the impact of methamphetamine (MA), 3,4-methylenedioxypyrovalerone (MDPV; “bath salts”) and 4-methylmethcathinone (4-MMC; mephedrone). The following paper describing our initial studies has been recently accepted for publication in Neuropsychopharmacology:

Locomotor stimulant and rewarding effects of inhaling methamphetamine, MDPV and mephedrone via electronic cigarette-type technology. Jacques D. Nguyen1, Shawn M. Aarde1, Maury Cole2, Sophia A. Vandewater1, Yanabel Grant1 and Michael A. Taffe1
1Committee on the Neurobiology of Addictive Disorders; The Scripps Research Institute; La Jolla, CA, USA
2La Jolla Alcohol Research, Inc, La Jolla, CA, USA

Schematic of the inhalation chamber

Schematic of the inhalation chamber

Our exposure model for this study involved a standard sized rat housing chamber with a sealed lid- these are commercially available for a variety of purposes. The chamber was plumbed for regulated airflow and incorporated the ability to deliver and exhaust the vapor from an e-cigarette type device. The overall approach for delivery to rodents is under patent to La Jolla Alcohol Research, Inc which has been instrumental in developing the equipment for our studies. This collaboration has resulted in a number of studies so far, this one is the second one to be published. The first paper described the effects of THC inhalation (blogpost). The company has also recently been awarded an SBIR Phase II Grant (R44 DA041967) to further develop and enhance commercialization of the device.

Control of the dose administered to the rat in this system is a key initial topic of investigation. We determined in this paper whether the dose can be altered with the manipulation of a number of variables. The concentration off the drug may be altered in the propylene glycol (PG) vehicle (aka “e-juice”)- our standard condition for this study was 100 mg/mL but effects from 12.5-200 mg/mL were also explored for different drugs. For the most part this study found concentration-dependent effects only across drugs (4-MMC was much less potent than MA or MDPV) when the puffing and inhalation duration was held constant. The puffing regimen and duration of inhalation exposure can be altered as well. In most of our studies we delivered 10-s vapor puffs with 2-s intervals between them every 5 minutes for durations of 10-40 min (approximately 0.125 ml was used in a 40 min exposure session). Varying the total duration from 10 to 30 min resulted in dose dependent effects of inhaling MA (12.5 mg/mL) or MA (12.5 mg/mL).

Vape decreases ICSS thresholds

A decrease in ICSS threshold was produced by inhalation exposure to 4MMC (200 mg/mL), MA (100 mg/mL) and MDPV (100 mg/mL). Similar effects were produced by i.p. administration of 4MMC (1.0mg/kg), MA (0.5 mg/kg) or MDPV (0.5 mg/kg). Significant differences from the respective Vehicle condition are indicated by *.

We present data on the intracranial self-stimulation reward paradigm in this paper. This is a model in which electrodes are implanted into the medial forebrain bundle of the rat and it is trained to respond for small deliveries of electrical current which has a rewarding or reinforcing effect. The procedure used for this study ramps the stimulation up and down during a session until the threshold necessary for the individual to experience a reinforcing effect is determined. Once the animals are trained to generate stable thresholds, they can be tested by administering drugs before the session. If a drug has a rewarding or reinforcing effect, it tends to lower the threshold below the baseline level. Here we show that all three drugs decrease reward thresholds in male rats. The reduction in the reward threshold was of a similar magnitude when drug was administered by injection or by vapor inhalation. This is a key indication that this procedure can generate reinforcing or rewarding levels of drug in the rats.

Activity rates after inhalation of  3,4-methylenedioxypyrovalerone (MDPV; 25,50,100mg/mL) or 4-methylmethcathinone (4MMC/mephedrone; 100, 200mg/mL).

Mean (N=13; + SEM) activity rates after inhalation of 3,4-methylenedioxypyrovalerone (MDPV; 25,50,100mg/mL) or 4-methylmethcathinone (4MMC/mephedrone; 100, 200mg/mL). Gray shaded symbols indicate a significant difference from PG vehicle at the corresponding time point. Base = pre-inhalation baseline.

Locomotor activity was measured after vapor inhalation using a radiotelemetry system that generates activity rates as counts per minute. In this figure we show the activity before and after inhalation of the PG vehicle and then three concentrations of MDPV and two concentrations of 4-methylmethcathinone (4-MMC, mephedrone) for 40 min. Locomotor activity was increased for 2-3 h after the initation of vapor for all three MDPV concentrations and for the 200 mg/mL concentration of 4-MMC. Similar effects were observed for MA and we went on to show that the dopamine D1-like receptor antagonist SCH23390 (10 ug/kg, i.p., prior to inhalation) blocked locomotor increases caused by inhalation of each drug. This is as would be expected, similar to the effect of SCH23390 on locomotor stimulant effects of these drugs when injected in rodents.

Wheel Activity after MDPV or MA

Mean (N=7; ± SEM) wheel activity of male rats after inhalation of methamphetamine (100 mg/mL in PG), MDPV (100 mg/mL in PG) or the PG alone for 40 minutes. Gray shaded symbols indicate a significant difference from PG. A significant difference from the 30 min time point within an inhalation condition is indicated with *, and a difference from MA with #, for corresponding time points.

Another study in the paper investigated effects of vapor inhalation of the MA and MDPV on wheel activity. Under vehicle treatment, rats run more on the wheel in the first 30 minutes and then run significantly less for the subsequent 90 min of a 2 h session. When allowed to use the wheel after vapor exposure to MA or MDPV, activity is initially suppressed and then rebounds as the session continues. Presumably, the initial suppression of wheel activity is related to the increase in chamber locomotor activity found in the radiotelemetry study- if the rats were running around the cage they might be unlikely to enter the wheels. In the study depicted, MDPV caused significantly more activity than vehicle inhalation 60-90 min after finishing the vapor inhalation. MA in this experiment did not increase activity compared with vehicle, however activity was significantly higher in the last thirty minutes than the first 30 min after MA inhalation. Additional data found no significant effects of 40 min of inhalation of either MDPV or MA at a 25 mg/mL concentration and a third study found elevations of wheel activity 90-120 min after a 20 min inhalation of MA (100 mg/mL). In total, the wheel activity data confirm dose-dependent effects on a second measure of locomotion.

Overall, this study is the first to demonstrate behavioral effects of e-cigarette type inhalation delivery of psychostimulants to rats. This further validates our model and encourages additional study of the risks of e-cigarette delivery of psychoactive substances in laboratory animal models.
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J. D. Nguyen, S. M. Aarde, M. Cole, S. A. Vandewater, Y. Grant and M. A. Taffe. Locomotor stimulant and rewarding effects of inhaling methamphetamine, MDPV and mephedrone via electronic cigarette-type technology, 2016, accepted article preview 9 June 2016; doi: 10.1038/npp.2016.88 [ PublisherSite ][ PubMed ]

Funding and Disclosures for this paper: This work was funded by support from the United States Public Health Service National Institutes of Health (R01 DA024105, R01 DA024705, R01 DA035281 and R44 DA041967) which had no direct input on the design, conduct, analysis or publication of the findings. Subsets of these data were first presented at the Experimental Biology meeting in 2015 and the Annual Meeting of the Society for Neuroscience 2015. Development of the apparatus was supported by La Jolla Alcohol Research, Inc and MC is inventor on a patent for this device. SAV consults for La Jolla Alcohol Research, Inc.

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May 31, 2016

Inhalation model for evaluation of e-cigarette based delivery of THC

Filed under: Cannabis, E-cigarettes, Vape inhalation — mtaffe @ 11:13 am

Our interest in developing inhalation techniques for delivering cannabinoids, most especially the primary active constituent Δ9-tetrahydrocannabinol (THC), to rats arose from the realization that increasing numbers of people were using non-combusted methods for inhalation. When we started this project there were no studies using a Volcano type or e-cigarette type of system to deliver THC to rodents. Of course the majority of cannabis consumption has always been via smoke inhalation and there have been a few prior studies in laboratory models, primarily from the Lichtman laboratory. Our focus was therefore on the non-combustible techniques stemming from the evidence of personal acquaintance reports, a plethora of Web sites advertising methods, an emerging literature showing human practices (Giroud et al, 2015, Morean et al, 2015) and from suggestions that e-cigarette delivery may offer a safer alternative for medical cannabis consumers (Varlet et al, 2016).

The following has been recently accepted for publication in Neuropharmacology.
Inhaled delivery of Δ9-tetrahydrocannabinol (THC) to rats by e-cigarette vapor technology. Jacques D. Nguyen1, Shawn M. Aarde1, Sophia A. Vandewater1, Yanabel Grant1, David G. Stouffer1, Loren H. Parsons1, Maury Cole2 and Michael A. Taffe1
1Committee on the Neurobiology of Addictive Disorders; The Scripps Research Institute; La Jolla, CA, USA
2La Jolla Alcohol Research, Inc; La Jolla CA, USA

Schematic of the inhalation chamber

Schematic of the inhalation chamber


Our exposure model for this study involved a standard sized rat housing chamber with a sealed lid- these are commercially available for a variety of purposes. The chamber was plumbed for regulated airflow and incorporated the ability to deliver and exhaust the vapor from an e-cigarette type device. We tried a number of commercial tanks in this study, one specific example is the Protank 3 Atomizer by Kanger Tech. The overall approach for delivery to rodents is under patent to La Jolla Alcohol Research, Inc which has been instrumental in developing the equipment for our studies. This collaboration has resulted in a number of studies so far, this one is the first to be published. The company has also recently been awarded an SBIR Phase II Grant (R44 DA041967) to further develop and enhance commercialization of the device.

Dosing control was managed in this system with the manipulation of a number of variables. One of the major goals of this study was to determine how the dose delivered to the animal might be regulated by altering these vaping parameters. The concentration off the drug (in this case Δ9-tetrahydrocannabinol (THC) may be altered in the propylene glycol (PG) vehicle (aka “e-juice”)- our standard condition for this study was 200 mg/mL but effects from 25-100 mg/mL were also explored and showed a concentration-dependent effect when the puffing and inhalation duration was held constant. The puffing regimen and duration of inhalation exposure can be altered as well. In most of our studies we delivered 10-s vapor puffs with 2-s intervals between them every 5 minutes for durations of 10-40 min (approximately 0.125 ml was used in a 40 min exposure session). This study established that for a given THC concentration in the vehicle, the duration over which animals were exposed could produce graded effects consistent with a dose-dependent pattern.

Mean (N=8; ±SEM) temperature response to THC inhalation for 10, 20 or 30 min in 5 min intervals. A significant difference from both the baseline and the other exposure conditions is indicated by the open symbols and from the 10 min condition by the shaded symbols.

Mean (N=8; ±SEM) temperature response to THC vapor inhalation for 10, 20 or 30 min in 5 min intervals. A significant difference from both the baseline and the other exposure conditions is indicated by the open symbols and from the 10 min condition by shaded symbols.


This first figure depicts THC-induced reductions in body temperature produced by THC inhalation for 10-30 minutes in male rats, using a radiotelemetry system for reporting temperature every 5 minutes. The figure depicting this experiment in the paper depicts 30 min averages but I really like this version so I’m including it here. [For those concerned with statistics, see below.] The points to the left indicate a pre-inhalation baseline interval in the telemetry recording chambers. There is a break in the series because we didn’t record them during vapor inhalation (see our SFN 2014 poster presentation for a pilot study recording during inhalation). The main point here is that 10 min of inhalation doesn’t change body temperature, 30 min has a major hypothermic effect and 20 min produces an intermediate effect. Thus, this system is able to produce dose-dependent effects that are so helpful for interpretation of behavioral pharmacology studies. We show in the paper that i.p. injection of 10-20 mg/kg THC produces a temperature nadir similar to that produced by 20-30 min of inhalation (see a blog post on our 2015 paper on temperature responses to injected THC for comparison). Our telemetry measure of locomotion did not show any suppression in this experiment but we do show a suppression of activity in both males and females in Figure 2 of the paper. There was some evidence that female rats are more sensitive to the hypothermia induced by, e.g., THC 50 mg/mL for 30 min in this study, likely because of their lower bodyweight compared with the male rats.

Mean tail-flick latency measured following 20 min of exposure with pre-treatment with SR141716 (SR; 4 mg/kg, i.p.) or Vehicle (N=8). Significant differences compared with respective vehicle condition are indicated by *, differences from SR+THC vapor by #.

Mean tail-flick latency measured following 20 min of exposure with pre-treatment with SR141716 (SR; 4 mg/kg, i.p.) or Vehicle (N=8). Significant differences compared with respective vehicle condition are indicated by *, differences from SR+THC vapor by #.


One of the major tests of cannabinoid activity in a rodent is a decrease in nociception. The ability to sense a noxious stimulus was tested by placing the tail in a 52°C water bath and timing the latency for it to flick it out. The experiment in the figure depicts a study in which the animals were exposed to PG or 200 mg/mL THC for 20 min with and without prior treatment with the cannabinoid 1 receptor antagonist SR141716 (Rimonabant; 4 mg/kg, i.p.). This shows that THC inhalation extends the time for the animal to flick its tail out of the warm water and that this effect is blocked with the antagonist pre-treatment. Although not shown here, the magnitude of the latency change caused by vapor inhalation of THC was the same as that produced by a 10 mg/kg THC i.p. injection. This comparability of the effect of inhaled versus injected THC was also highly consistent with data we generated showing that blood concentrations of THC were very similar when observed 30 min after THC (200 mg/mL) inhalation or 30 min after 10 mg/kg, i.p. injection.

In summary, we’ve created a new model for evaluating inhaled delivery of THC to rats via an e-cigarette type of method. We’ve found significant effects on three of the four traditional measures (Tetrad Test) of cannabinoid activity in a rodent- hypothermia, hypolocomotion and antinociception (the fourth, catalepsy, was not assessed). Effects were of comparable magnitude to those produced by intraperitoneal injection, allowing these data to be placed in context with prior studies using injection delivery of THC. There are several advantages of this model, most pertinently the more rapid timecourse of effects compared to what is produced with an i.p. injection.
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Jacques D. Nguyen, Shawn M. Aarde, Sophia A. Vandewater, Yanabel Grant, David G. Stouffer, Loren H. Parsons, Maury Cole and Michael A. Taffe. Inhaled delivery of Δ9-tetrahydrocannabinol (THC) to rats by e-cigarette vapor technology, 2016, Neuropharmacology, in press. DOI: 10.1016/j.neuropharm.2016.05.021 [PubMed]

Funding and Disclosures for this paper: This work was funded by support from the United States Public Health Service National Institutes of Health (R01 DA024105, R01 DA035281 and R44 DA041967) which had no direct input on the design, conduct, analysis or publication of the findings. Development of the apparatus was supported by La Jolla Alcohol Research, Inc and MC is inventor on a patent for this device. SAV consults for La Jolla Alcohol Research, Inc.

[Stats for body temperature figure: The ANOVA of the five minute temperature intervals (including three baseline samples, -15 to -5, and 40-180 min following initiation of vapor) confirmed main effects of Time post-initiation [F (32, 224) = 38.36; P < 0.0001], Duration of vapor exposure [F (2, 14) = 38.66; P < 0.0001] and the interaction of factors [F (64, 448) = 16.64; P < 0.0001].
The Tukey post-hoc test confirmed significant temperature reductions after 20 (40-70 min post-vapor initiation) or 30 min (40-155 min post-vapor initiation) of vapor exposure to THC compared with each of three baseline samples. Furthermore, the post-hoc test confirmed that temperature after all three exposure durations differed significantly from each other from 40-150 and 160-165 min following vapor initiation. Significant differences in temperature between 10 and 30 min vapor exposures were confirmed for the entire post-vapor duration.]

July 18, 2015

Prevalence of E-cigarette use in 8th-12th graders

Filed under: E-cigarettes, Tobacco/Nicotine, Vape inhalation — mtaffe @ 5:47 pm

The Monitoring the Future survey added electronic cigarettes to its survey for the first time in 2014. The summary tables and figures and full monographs are available for the clicking.

Results show show that 17.1% of high school seniors reported using an E-cigarette at least once in the past 30 days. Rates were almost as high for 10th grade students (16.2%) and somewhat lower for 8th graders (8.7%).

To put this in perspective the 30 day prevalence for cigarettes was 13.6% for 12th graders, 7.2% for 10th and 4.0% for 8th graders. So twice as many 8th and 10th grade students have at least tried an E-cigarette as have tried a regular cigarette.

6.8% of 12th grade students report smoking one or more cigarettes per day. The rate for 10th graders is only 3.3% and 1.4% for 8th graders. One way to put together the E-cigarette/cigarette ratios from the three grade ranges is to observe that daily smoking is more likely with older students. These individuals may either have less need to resort to E-cigarettes for availability reasons or they may reflect the fact that E-cigarettes may not produce good nicotine levels until smokers are motivated to learn to use them.

For those that are unfamiliar with drug use rates in these grade levels, E-cigarette use is high. The percentage of respondents who used any illicit drug other than marijuana in the past 30 days was 7.7%, 5.6% and 3.3% for 12th, 10th and 8th graders respectively. Marijuana rates were 21.2%, 16.6% and 6.5%. The 30 day rates for 12th graders for individual drugs of interest are much lower: 1.0% cocaine or LSD, 1.4% Ecstasy, 0.4% heroin or PCP, 6.4% for any prescription drug.

Since this was only added to the MtF survey recently, we cannot make much from a single point estimate of E-cigarette use. Maybe this was the peak of a new fad, maybe the beginning of a sustained trend.

What we do know is that substantial numbers of adolescents are sampling the use of these devices.

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