CB1 contributions to THC-induced hypothermia after vapor inhalation

By this point the laboratory has published a number of papers showing that the vapor inhalation of Δ⁹-tetrahydrocannabinol (THC) reduces body temperature of rats. This was mostly done to validate our e-cigarette based method of drug delivery since hypothermia, along with anti-nociception, hypolocomotion and catalepsy, is a key marker of cannabinoid action in the rat (or mouse). This tetrad of signs was developed as a laboratory readout of THC-like effects before it was known where THC acted in the brain, before the endocannabinoid receptors were identified and before specific pharmacological tools were available to target the cannabinoid type 1 (CB₁) receptor.  Since we are a behavioral pharmacological lab one of the primary ways to validate is to show that effects depend on the dose of the drug that is administered, and we have done that using both time of exposure and concentration of the drug in the vapor to control dose. The pharmacological tools are available now so the secondary go-to validation for  a behavioral pharmacological laboratory is to determine if antagonist drugs which block the ability of THC to interact with, e.g., the CB₁ receptor, inhibit or block the effects of the inhaled drug.

A new paper (a pre-print version is available here) from the laboratory describes a several year journey of confusion about why a very simple experiment that “should” work, did not.

Nguyen, J.D., Creehan, K.M., Grant, Y., Vandewater, S.A., Kerr, T.M. and Taffe, M.A. Explication of CB₁ receptor contributions to the hypothermic effects of Δ⁹-tetrahydrocannabinol (THC) when delivered by vapor inhalation or parenteral injection in rats. Drug Alcohol Depend, 2020, in press.

The compound SR141716 (it was approved as a treatment medication as “Rimonabant” but pulled from the market for suicidal ideation reasons) interacts with the CB₁ receptor, both preventing THC and other agonists from working (i.e., as an antagonist) and potentially reducing constitutive activity of that receptor (called an “inverse agonist” effect). SR141716 works fine for the prevention of some THC-induced effects including the aforementioned tetrad of signs in rats. Most specifically including hypothermia. So long as the THC is injected. It also blocks anti-nociceptive effects of THC when injected OR when inhaled.

Figure 1 from this new paper shows the speed with which the rat tail is withdrawn from a 52 degree C water bath after a 30 minute inhalation session. When the vapor is from the PG vehicle, the tail is withdrawn within about 3 sec (open bar). After THC (grey bars) inhalation this slows to over 6 seconds in male and female rats. This is the anti-nociceptive effect of THC when delivered by inhalation but the magnitude is similar to what is produced by THC injection. Pretreatment with SR141716 (black bars) before the inhalation session entirely prevents the anti-nociceptive effect of THC in male rats entirely and greatly reduces it in female rats.  So far, so good, and actually we showed something similar in our very first paper, Nguyen et al 2016 (see Figure 4B).

Dating back to our very original studies, this is not what happens with the temperature response. Figure 2 from the new paper shows (Panels A, B, D) that SR141716 administered prior to THC inhalation does not affect the initial drop in body temperature, observed immediately after the 20-30 minute session.

What SR does do is slightly accelerate the return of body temperature to the normal range. To walk through the logic of these first panels, the study in A suggested perhaps the SR simply wasn’t effective until 90 minutes later so in B we moved the pre-treatment interval to 90 minutes before inhalation. No difference. There’s always a concern about dose so in Panel D we increased from 4 mg/kg SR141716 to a 10 mg/kg dose, and had no change. In Panel C, we injected the SR after the vapor session and again, no change in the overall picture, suggesting that even with pre-vapor administration of the SR141716, essentially the same level of activity was present in the post-vapor monitoring period.

The rest of the paper describes more experiments along the same theme- trying to give the antagonist the best chance to “work” and further confirming that it does work to prevent body temperature changes…..just so long as the THC is injected (either intraperitoneally or intravenously).

None of the usual pharmacological explanations related to antagonist/agonist dose, speed of uptake into the brain or competition for binding sites seem to be at work here. We’ve altered dose and injection route. We’ve evaluated both sexes and two different rat strains. Studies range across animal age (and therefore size) and their treatment history, including a chronic THC dosing group that exhibited partial tolerance to the THC inhalation. We have conducted vehicle inhalation controls, which do not alter body temperature, and thus there cannot be any role of the vapor exposure by itself. We used an alternate antagonist/inverse agonst drug and produced similar qualitative results.

Most convincingly, of course, the effects of the antagonist differ between the temperature and the nociceptive effects of THC inhalation.

Scientifically this is a nagging mystery. We’ve thrown effort at these studies over several years now, and have tried other manipulations that fail to resolve the question to our satisfaction. The literature on injected THC is reasonably robust but there are few investigations reported for inhaled exposure. What exists is limited by the experimental design. For example, Wilson and colleagues (2002) showed that the temperature response to inhaled THC could be attenuated by SR141716 pre-treatment in mice, but they only measured temperature at a single time-point after inhalation, with locomotor, nociception and catalepsy tests being performed first. It cannot be determined, therefore, if the temperature response at some earlier timepoint was unaffected by the SR141716 pre-treatment. This partial effect also conflicts with a complete blockade of the temperature response to inhaled THC in a different strain of mice reported by Marshell and colleagues (2014).

Sadly, this is the sort of mystery that doesn’t really justify a lot more effort and money to resolve. While important and useful for validation purposes, the hypothermic response to THC does not appear to have much translational value. It is not clear that human body temperature is affected by THC ingestion at all, nor what health implications this might have even if humans do experience temperature reductions.

This paper is one that I feared might not ever be publishable. It presents a bit of a mystery and does not actually solve it. This is not typically what is found to be publishable in academic science reporting. But from a conduct of science perspective it is really important to get out there. Just as we followed this frustrating path after starting from a expectation of rapid pharmacological validation of our method, others might likewise wish to validate their inhalation models. E-cigarette use for delivering cannabinoids continues to be very popular with both medical and recreational consumers of cannabis (via extracts). This encourages researchers to try to adopt similar methods to explore any possible implications for health or well being. There are other labs doing similar work already and they are, in many cases, rooted in behavioral pharmacological thinking as much as we are. At the very least, our paper serves as a warning that things may not be simple.

More usefully, perhaps someone will come up with a brilliant insight about where our thinking has led us astray. We could be missing something incredibly simple that explains all of this in a satisfying way. That is the absolute brilliance of the grand enterprise of science, i.e., that publishing work leads to someone else either confirming, contradicting or explaining our current state of knowledge.

Cannabidiol by vapor inhalation in rats

Cannabidiol is increasingly popular, occurring in a dizzying array of products in a highly unregulated retail market. This includes creams, oils, lotions, capsules and e-cigarette liquids, among many other items. A simple search for CBD on google will give you a taste of what I mean, if this is new to you. Just about every single senior person I talk to, it seems, is using CBD or knows another person who is using CBD  for various ailments.

The following has just been accepted for publication:

Javadi-Paydar, M.,  Creehan, K.M., Kerr, T.M.  and Taffe, M.A. Vapor inhalation of cannabidiol (CBD) in rats.  Pharmacol Biochem Behav, 2019 Jul 20:172741. doi: 10.1016/j.pbb.2019.172741. [ Publisher Site ][ PubMed ]

Figure 1: This figure has been adapted from Taffe et al. 2015. Click to enlarge.

We have been interested in studying the effects of CBD ever since reading a paper [Morgan et al., 2010] that appeared to show that the presence of CBD in cannabis protected users against the memory impairing effects of acute THC intoxication, subsequent to smoking their preferred cannabis. This led to our interest in the potentially interactive effects of CBD and THC and, in particular, tests of the hypothesis that CBD would reduce the effects of THC. Our initial papers on this were Wright et al 2013 and Taffe et al 2015. Of primary relevance for the discussion of our new work, the latter paper showed that CBD did not alter the body temperature (see Figure 1 C, D; blue bars) or activity of rats when injected at doses of 30 or 60 mg/kg, i.p.. Our new work confirms our prior finding that this may be due to the route of administration since, when male or female Wistar rats experience CBD by vapor inhalation, their body temperature does go down, albeit not as severely as when exposed to THC [Javadi-Paydar et al, 2018].

This finding required some follow-up, extraordinary claims requiring extraordinary evidence and all that. Although in the Taffe et al 2015 paper, CBD did appear to increase the magnitude of the hypothermia associated with THC when each were injected, i.p., (red trace and summary bars in Figure 1) there are data suggesting that this may be due to metobolic interference whereby CBD merely prolongs the activity of THC. Another thing that was slightly strange was the fact we observed that CBD reduces temperature of Wistar rats. We used Sprague-Dawley rats for the Taffe et al 2015 paper because initial pilot experiments suggested that perhaps Wistar (male) rats were less sensitive to the body temperature lowering effects of THC [a follow up to that is available in a pre-print]. Yet Javadi-Paydar et al (2018) found effects of vaporized CBD in male and female Wistar rats. Perhaps this is due to the difference between CB1 receptor mediated effects and serotonin 1a (5-HT1a) receptor mediated effects. There is growing evidence that CBD works in part by activating 5-HT1a receptors, and activation of this receptor (e.g., by injecting the agonist 8-OH-DPAT) drops the body temperature of rats precipitously. As an example we had published a figure on this as a positive control in the Wright et al 2012 paper focused on the activity of the cathinone mephedrone [blog post summary]. To further complicate matters, the response of male Wistar rats to 8-OH-DPAT in that paper seemed to be slightly greater than the response of male Sprague-Dawley rats.

Figure 2: Plasma CBD in male and female Wistar rats after vapor inhalation (top panels) or injection (bottom panels).Click to enlarge.

An earlier version of this manuscript was posted as a pre-print on June 04, 2019, and updated with a version almost identical to the final submitted manuscript on Jul 18, 2019.

The first critical thing in this new paper was to get a point of reference for the doses the animals were getting through vaporized CBD versus i.p. injection. This figure shows the plasma levels experienced at the end of vapor sessions are within the range of plasma levels observed 35 minutes after an injection. This was in male and female Wistar rats, making it a follow-up to the thermoregulatory data in the Javadi-Paydar et al (2018) paper. One of the major ways that we control dose with our inhalation model is to alter the concentration of the drug in the e-liquid vehicle (we use propylene glycol; PG), while holding other parameter fixed. So for CBD we have used concentrations of 100 and 400 mg per mL of the PG. Now admittedly we have only published the effects of 30 mg/kg CBD when injected, at the lower end of the dose range. But based on some pilot work I doubt that we’ll find out that lower dose of CBD are causing hypothermia when injected- but it could still be about dose. Our time-point here for injection was designed for comparison with the inhalation model but levels were likely much higher at 5 minutes after injection whereas they were increasing essentially linearly across the inhalation interval. Nevertheless, we are clearly not getting much, much higher plasma loads of CBD via inhalation, at least not in the blood.

The next step for this paper was to replicate the body temperature effect, which we did in a group of male Wistar rats.

Figure 3: Temperature responses to vapor inhalation of CBD and nicotine in male Sprague-Dawley rats. Open and grey symbols depict statistical differences summarized in the manuscript. Adapted from Javadi-Paydar et al 2019. Click to enlarge.

We then went on to evaluate the effect of CBD inhalation on body temperature in male Sprague-Dawley rats and found a similar (perhaps slightly increased relative to the Wistar male rats) degree of hypothermia under identical vaping conditions.

Figure 3 shows that CBD concentration-dependent reductions in body temperature are found in male Sprague-Dawley rats (blue data series), thereby replicating and extending to an additional rat strain. The next experiment showed that the effect of CBD (at the 100 mg/mL concentration) is attenuated when animals are pre-treated with the 5-HT1a antagonist WAY 100,635. This shows that the 5-HT1a receptor is very likely involved in the hypothermic response to vaporized CBD, further adding to the growing evidence that CBD acts at this receptor.

You may have noticed that the top two panels of Figure 3 include a nicotine inhalation condition and a CBD + nicotine inhalation condition. There are a couple of reasons for this. Most generally, CBD has been shown to attenuate relapse to alcohol and cocaine self-administration in rats and may reduce the salience of cigarette-associated cues in humans. Reviews of the potential of CBD as an anti-drug abuse treatment can be found here and here. The second rationale is that human substance users often use more than one drug at a time. THC and CBD co-occur in cannabis. People frequently use cannabis along with tobacco and/or alcohol. Our work in Javadi-Paydar et al 2019 examined potential interactive effects of THC with nicotine thus it was an obvious followup to see if CBD interacted with nicotine. As you can see in Figure 3, the effect of nicotine alone on body temperature is not obvious in this group (although it did enhance locomotor activity). Nicotine did, however, increase the effect of CBD when the two were co-administered. Interestingly CBD also suppressed the locomotor activating effects of vaporized nicotine inhalation in this study. So the combined effect appears to be independent, not interactive- i.e., an opposition when the two independent drug effects are in the opposite direction (locomotor activity) and add together when the two independent drug effects are in the same direction (see Javadi-Paydar et al 2019 for more on this interactive drug logic and on the hypothermia caused by nicotine inhalation).

CBD is often described as non-psychoactive constituent of cannabis because it does not appear to have the same dramatic subjective properties as delta-9-tetrahydrocannabinol. Also because there are a lot of studies where it does not appear to do much to a rodent when administered by itself. There are exceptions, but I think a fair take away is that often enough it has been found inactive. This may very well be due to investigating CBD in assays that are tuned to detect THC-like effects that are presumably mediated by the CB1 or CB2 receptors. Our thermoregulatory assay, fortunately, is sensitive to both CB1 and 5-HT1a agonists. It may also be the case that the route of administration is a fundamental contributor to observing or not observing effects of CBD in a rat. There are several pharmacokinetic possibilities that may explain this. Our plasma data are fairly limited in a temporal sense and we don’t know from plasma levels what the kinetics look like in the brain. It could be that there is a much different blood/brain ratio associated with the two routes of administration. It may be that the speed of initial brain entry of a threshold amount of drug varies as well. Additional work will be necessary to full determine how the route of administration alters the effects of CBD in rats and how this might translate to the human condition.

 

Current Topics in Behavioral Neurosciences on Novel Psychoactive Substances

There is a new Current Topics in Behavioral Neuroscience book on New and Emerging Psychoactive Substances that has been organized by Michael H. Baumann, Ph.D., of the Intramural Research Program of the National Institute on Drub Abuse. This editorial effort resulted in 18 chapters on various topics of interest which are now available online.

Chapter 1: Madras, B. The Growing Problem of New Psychoactive Substances (NPS) [link]

Chapter 2: Glennon, R.A. and Dukat, M. Structure-Activity Relationships of Synthetic Cathinones [link]

Chapter 3: Simmler, L.D. and Liechti, M.E. Interactions of Cathinone NPS with Human Transporters and Receptors in Transfected Cells [link]

Chapter 4: Solis, E. Electrophysiological Actions of Synthetic Cathinones on Monoamine Transporters [link]

Chapter 5: Baumann, M.H., Bukhari, M.O., Lehner, K.R., Anizan, S., Rice, K.C., Concheiro, M. and Huestis, M.A. Neuropharmacology of 3,4-Methylenedioxypyrovalerone (MDPV), its Metabolites, and Related Analogs [link]

Chapter 6: Negus, S.S. and Banks, M.L. Decoding the Structure of Abuse Potential for New Psychoactive Substances: Structure-Activity Relationships for Abuse-Related Effects of 4-Substituted Methcathinone Analogs [link]

Chapter 7: Watterson, L.R. and Olive, M.F. Reinforcing Effects of Cathinone NPS in the Intravenous Drug Self-Administration Paradigm [link]

Chapter 8: Aarde, S.M. and Taffe, M.A. Predicting the Abuse Liability of Entactogen-Class, New and Emerging Psychoactive Substances via Preclinical Models of Drug Self-administration.[link]

Chapter 9: King, H.E. and Riley, A.L. The Affective Properties of Synthetic Cathinones: Role of Reward and Aversion in Their Abuse [link]

Chapter 10: Kiyatkin, E.A. and Ren, S.E. MDMA, Methylone, and MDPV: Drug-induced Brain Hyperthermia and its Modulation by Activity State and Environment [link]

Chapter 11: Angoa-Pérez, M., Anneken, J.H., Kuhn, D.M. Neurotoxicology of Synthetic Cathinone Analogs [link]

Chapter 12: Wiley, J.L, Marusich, J.A. and Thomas, B.F. Combination Chemistry: Structure–Activity Relationships of Novel Psychoactive Cannabinoids [link]

Chapter 13: Tai, S. and Fantegrossi, W.E. Pharmacological and Toxicological Effects of Synthetic Cannabinoids and Their Metabolites [link]

Chapter 14: Järbe, T.U.C. and Raghav, J.G. Tripping with Synthetic Cannabinoids (‘Spice’): Anecdotal and Experimental Observations in Animals and Man [link]

Chapter 15:Halberstadt, A.L. Pharmacology and Toxicology of N-Benzylphenethylamine (“NBOMe”) Hallucinogens [link]

Chapter 16: Papaseit, E., Molto, J., Muga, R., Torrens, M., de la Torre, R. and Farre, M. Clinical Pharmacology of the Synthetic
Cathinone Mephedrone [link]

Chapter 17: Mayer, F.P., Luf, A., Nagy, C., Holy, M., Schmid, R., Freissmuth, M., Sitte, H.H. Application of a Combined Approach to Identify New Psychoactive Street Drugs and Decipher Their Mechanisms at Monoamine Transporters [link]

Chapter 18: Schifano, F., Orsolini, L., Papanti, D., Corkery, J. NPS: Medical Consequences Associated with Their Intake [link]

 

Thoughts on Proposition 64 to Legalize Recreational Marijuana in California

I wrote a brief note on Facebook the other day to outline what I thought were several points that come up when people in the community ask me about the upcoming vote on recreational marijuana (link to ballotpedia summary of Prop 64). This was picked up in a post at Forbes by David Kroll (a handy summary video is here)

This piece was noticed by Sasha Foo at KUSI and she was kind enough to film a news segment which aired on 2 November, 2016. This links to the 6 pm broadcast version.

My Facebook remarks (with a few key links to data sources added):

I’m in California which will be voting on Proposition 64 which legalizes recreational marijuana. As many of my friends, neighbors and acquaintances are aware that I work in the substance-abuse fields of science, they have questions. So I thought I would put some of my usual responses/points down on a Fb post.

First, some background on my opinions. I work for you, the taxpayer of the US. This is because my work is funded by grants from the National Institutes of Health. Because these are primarily from the National Institute on Drug Abuse, my role is to investigate the effects of recreational drugs on the brain (and the rest of the body) with some attention paid to how this might affect the health of humans.

This is most emphatically not a policy role. I have no special expertise on public policy and my comments are not meant in that way. I do hope that science can be used to inform policy and, frankly, I wish that public policy across the board paid a lot more attention to facts and data. This is not to say, however, that I believe that the facts necessarily lead all interested people to the same *policy* decision. Because policy requires the weighing of factors and pitting positives and negatives of various kinds against each other.

As far as legalizing recreational marijuana goes, I do think that the epidemiological, human laboratory and animal laboratory data has some relevance to the Prop 64 issues. So, I’m going to list a few facts.

1) Marijuana is addictive. Full stop. The conditional probability of dependence is about 9% where like-to-like comparisons put cocaine and methamphetamine at 15%, heroin at 25-45% (data are terrible) and alcohol at 4%. Alcohol is a huge problem because 85%+ of people consume it at least annually. In contrast, less than 1% of people have ever tried heroin, 0.4% in the past year. Marijuana comes in at about 32% annual prevalence for ages 19-28. The scope of the addiction issue depends on how many people are using it, obviously. This will go up with legalization- but we don’t have any idea how much.

2) 5-6% of high-school seniors use Marijuana daily. Daily. That’s the US average. I don’t have numbers for California. http://www.monitoringthefuture.org/data/15data/15drfig4.pdf

3) Marijuana addiction is as “real” as any other. Frequency of withdrawal symptoms and severity of those symptoms were compared between marijuana and tobacco smokers and the data were nearly indistinguishable. Most people are much more familiar with nicotine dependency (which is a higher rate, btw, probably 33%+) since it is more common, not embarrassing to discuss in public and is conventionally recognized. A lack of personal familiarity with the scope of withdrawal in the people who are marijuana dependent doesn’t mean that it doesn’t exist.

4) There is no such thing as “psychological” versus “physical” dependence since the brain is part of the body and the mind is the functioning of the brain. Keep in mind that people can be months to years out from their last use of any drug and still relapse severely. This is not being driven by the withdrawal symptoms that most everyone recognizes when they talk about “physical” dependence.

5) Marijuana acutely impairs cognitive and other behavioral functions.

6) Behavioral tolerance with chronic exposure is substantial. Blood levels of THC in animals or humans are a poorer proxy for impairment (versus other drugs) if you do not know anything about the prior exposure history.

7) THC is detectable in the body for a very long time compared with many other drugs of abuse. One study found detectable THC, or one of the main metabolites, for 30 days of in patient study (chronic users).

8) Trying to make specific predictions about an individual who uses marijuana from general findings (there is always a central tendency or average around which the distribution of data points or individual outcomes varies) is a fools’ errand. We can only predict general trends. Conversely, and this is important for your personal introspection, the evidence from one given data point or individual doesn’t tell us much that is informative about the average trend. The fact that it is your personal experience does not make it more valid.

Finally, there is much we simply don’t know. Any given scientific study or data set is limited by how it was generated. This doesn’t mean we throw up our hands and say it is all bunk or uninterpretable but it means one does have to think about it a bit.

I would invite you to read over the Prop 64 provisions. Personally, I see a fair bit of investment of the tax revenue in state sponsored activities to answer some of these issues better, to address some of the obvious concerns, etc. To me this is a positive. The extent to which this will happen, the extent to which actionable information will result, the extent to which activities intended to head off or ameliorate obvious negatives is, however, an unknown.

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

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. Taffe^1
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

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 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 #.

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.
__

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.]

Daily Marijuana Use In Adolescents

The Monitoring the Future Study of longitudinal drug trends releases the latest updates in December each year. The website has links to the updated tables and a few selected Figures.
This graph depicts the percentage of 8th, 10th and 12th grade students in the US who indicate that they have used marijuana at least 25 days out of the past 30 (their definition of “Daily” in the survey). For those who want precision, the 2015 numbers are 1.1% for 8th graders, 3.0% for 10th graders and 6% for 12th graders. You may be inclined to view single digit percentages as no big deal. It seems like a small number. One percent? Hardly worth talking about, right?

Except if, as I do, you have children in one or more of these age ranges. And you go so far as to become acquainted with some of your children’s friends and schoolmates. And acquainted with some of their parents. What you quickly realize is that you know at least 50 kids within your child’s circle at least a little bit. Enough to know their name and something about them. Maybe 100. And your kid probably knows at least 200 fairly well.

So look around? Which 1 of these kids is already smoking pot every day in 8th grade? Which 6 are at the end of high school?

EVERY day. Smoking pot. And the odds are very good that this kid is smoking multiple times a day. Staying high for extended periods.

Remember this when you think dismissively to yourself that “that 7th grader looks stoned, hahaha” as I once did before catching myself. I should know better. And even I don’t really think specific kids are the ones smoking pot every day. Until I think about it.

But the stats say they are. Some of them.

 

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It is also the case that 6.5% of 8th graders, 15% of 10th graders and 21% of 12th graders have used marijuana at least once in the past month. 35% of 12th graders in the past year. This means the daily use rate is 17% of 12th graders who have tried marijuana at least once in the past year.

Smokers have to adapt to e-cigarettes to maximize nicotine yield

One of the reasons that smoked/inhaled drug delivery is highly associated with addiction is that this route allows humans to exquisitely titrate their dosing. Thus for drugs like nicotine that become aversive at higher doses, smoking tobacco in several punctate inhalations over a short interval of time permits the user to avoid unpleasant dose levels.

This contrasts, for example, with buccal administration. If anyone recalls sampling chewing tobacco as a youth, you will understand what I mean. The relatively slowed onset and the larger available dose of the wad of tobacco or snuff packed up against the gums is frequently associated with severe nausea in the naive user.

A similar situation obtains with cannabis for which smoking has been the preferred route of administration. There is, however, relatively familiar use of cannabis via the oral route- think pot brownies. Increasingly, the medical marijuana entities are also selling a variety of edibles for oral administration of marijuana. Again, it is relatively common for naive consumers of edible products to overdose because the subjective effects hit long after a ballistic, irreversible drug administration has been accomplished.

A recent paper on the use of e-cigarettes for cannabis delivery (Etter, 2015) piqued my interest because it suggested that experienced cannabis smokers did not really like the e-cigarette delivery all that much.

Presentations at the recent CPDD meeting referred to the fact that nicotine seekers who use e-cigarette devices have to learn to adjust their inhalation behavior relative to their tobacco smoking. This is described in a paper that I located:

Farsalinos KE, Spyrou A, Stefopoulos C, Tsimopoulou K, Kourkoveli P, Tsiapras D, Kyrzopoulos S, Poulas K, Voudris V. Nicotine absorption from electronic cigarette use: comparison between experienced consumers (vapers) and naïve users (smokers). Sci Rep. 2015 Jun 17;5:11269. doi: 10.1038/srep11269.

The authors examined e-cigarette (EC) use in groups of ex-smokers who had quit and had been using ECs for at least a month and another group of smokers who were not EC users (available for free at PMC here). Subjects were asked to take 10 puffs from a standardized EC device in the first five minutes and then use it at their own discretion for another hour. The study sampled their blood for nicotine levels that were achieved across the study and the key figure from this paper is depicted here. As you can see, the experienced EC users (vapers) reached higher plasma nicotine levels than did the EC-inexperienced smokers. Each group averaged the same number of puffs, around 85-90, but the experience vapers took longer puffs (3.5 vs 2.3 seconds).

The simple interpretation is that if nicotine amount is a function of vapor cloud volume, and delivery across the lungs depends on retention time within the lungs, then longer puffs would result in greater nicotine delivery. The slightly more complex issue, mentioned at the CPDD annual meeting but not addressed in this paper, is that the rate at which a user inhales can be important. The idea is that if you pull too much of the EC vehicle across the heating element it can cool the element, resulting in lower nicotine yield.

Bottom line, EC inhalation for maximum nicotine yield and tobacco smoke inhalation for maximum nicotine yield may require a different inhalation approach.

This then reminds us that when ECs are adapted for crude cannabis extracts or even other drugs, it will require users to learn to adapt their behavior for idealized drug yield before we truly understand the risks. An initial report like Etter (2015) showing cannabis users don’t like to use ECs to deliver THC as well as they like to smoke cannabis need to be viewed in that light

Review/commentary on likely lasting impacts of increasing marijuana use

From Jerry Wright, published as a commentary in Drug and Alcohol Dependence.

Wright, MJ, Jr. Legalizing marijuana for medical purposes will increase risk of long-term, deleterious consequences for adolescents. Drug Alcohol Depend, Volume 149, 1 April 2015, Pages 298–303

This is actually a fairly good review article, not merely a commentary. Lots of citation to relevant research articles.

The main purpose from the Introduction:

Marijuana use for medical purposes is currently legal in 23 states in the U.S.and Washington,DC.This commentary reviews evidence linking frequent marijuana use in adolescence with risk for mental illness and cognitive impairment,the impact of medical marijuana legalization on increasing rates of adolescent marijuana use, changes in the potency of marijuana overtime,and research on marijuana-based medications to make the case that legalizing medical marijuana will increase health-related risks, particularly among adolescents

and the key points from the Conclusion.

Too many adolescents have access to marijuana currently and there is very little evidence that adolescent access to recreational marijuana can be reduced while simultaneously increasing the availability of medical marijuana for adults.Public health interests would be better served by streamlining the bureaucracy that impedes research on marijuana-based medications and focusing our efforts on identifying compounds in marijuana that confer unique therapeutic benefit.

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Jerry was a postdoctoral fellow in the Taffe Lab from 2009-2012.

Cannabidiol fails to attenuate THC-induced hypothermia

The following has been accepted for publication:

M A Taffe, K M Creehan, S A Vandewater Cannabidiol fails to reverse hypothermia or locomotor suppression induced by ∆9-tetrahydrocannabinol in Sprague-Dawley rats. (2015) British Journal of Pharmacology, in press. [Publisher Site; PubMed]

Cannabidiol (CBD) is a constituent of some strains of recreational cannabis plant material but the content of CBD-enriched strains is highly variable in the market (Morgan et al., 2010; Burgdorf et al., 2011). Cannabidiol has traditionally been viewed as an inactive constituent of cannabis, for example it produces minimal disruption of behavioral tasks in humans, monkeys or rodents (Belgrave et al., 1979; Lichtman et al., 1995; Winsauer et al., 1999). There has been a lot of recent interest in CBD for anti-seizure properties (see this blog post for example)

Morgan and colleagues have shown (blog writeup) that smoking cannabidiol-enriched marijuana does not cause the deficits of immediate and delayed prose recall that were caused by CBD-poor cannabis (Morgan et al., 2010) and users habitually exposed to CBD-containing cannabis may have relatively preserved recognition memory versus CBD-poor cannabis users (Morgan et al., 2011). The limits of human field studies (varying CBD/THC dose, no control of individuals who select CBD-rich vs. CBD-poor cannabis) and human lab studies (limited dosing ranges of CBD vs THC) motivate animal studies to investigate how CBD modulates the effects of THC.

Unfortunately, the available evidence on interactive effects of CBD and THC in rodent models present a more complicated picture. While CBD can reverse a conditioned place aversion produced by 10 mg/kg THC in rats (Vann et al., 2008), it may be the case that CBD potentiates the anxiogenic and locomotor suppressant effects of THC in rats treated chronically (Klein et al., 2011). In addition CBD / THC interactions may depend on the pre-treatment offset, as briefly reviewed (Zuardi et al., 2012). When CBD is administered 30 min (or up to 24 hrs) prior to THC in rats or mice, a potentiation can be observed whereas co-administration results in blockade or amelioration of THC effects. The picture may be complicated even further by a suggestion that CBD/THC ratios on the order of 8 are necessary for antagonistic properties and only 1.8 for potentiation of THC-related effects in rodents (Zuardi et al., 1984).

Our study was designed to determine if CBD attenuates, potentiates or extends the duration of hypothermia and hypomotility produced by acute THC in rats, using radiotelemetric monitoring.

The investigation found no evidence that cannabidiol can ameliorate the thermoregulatory or hypolocomotor effects of THC when administered either simultaneously (as in Figure 1, below) or prior to THC. Increasing the ratio of CBD:THC from 1:1 to 3:1 had no differential effect. Thus we find no protective effect of CBD against these particular endpoints in the rat. This contrasts with our recent finding that CBD can be protective against memory-impairing effects of THC in the monkey (PubMed, blogpost).

Figure 1: Mean (N=5; ±SEM) telemetered body temperature (left panels) and activity rate (right panels) after treatment with 30 mg/kg THC with 30 mg/kg cannabidiol or the vehicle, i.p., administered simultaneously. A Vehicle-only control condition (Veh) is also depicted. Upper panels display the data as collected (5 min intervals) and the lower panels depict the hourly averages used for analysis. A significant difference from Veh (only) is indicated by * and from both other conditions by #. Significant differences from the first hour (within treatment condition) are indicated by §.

SfN 2014 Presentation: Vape drug delivery

We will present a poster describing our efforts to develop technologies for the intrapulmonary (inhaled) delivery of psychoactive drugs at the 2004 meeting of the Society for Neuroscience.

Abstract 810.04 on Board AA05: Development and validation of a device for the intrapulmonary delivery of cannabinoids and stimulants to rats .
Authors: M. A. TAFFE, S. M. AARDE, M. COLE;
Cmte Neurobio. of Addictive Disorders, The Scripps Res. Inst., LA JOLLA, CA;

The presentation time is Wednesday, Nov 19, 2014, 1:00 PM – 5:00 PM.

Abstract Text:

The recent popularization of non-combustible methods for intrapulmonary delivery of psychoactive drugs to humans (Vape, Volcano, e-cigarette, etc) has stimulated interest in the intrapulmonary administration models for rodent studies. We have designed a sealed rodent chamber, with a well regulated air flow, that is suitable for the controlled exposure of rats to psychoactive substances. Use of e-cigarette type delivery systems was found to afford excellent dosing control for this purpose. Studies were conducted in male rats to verify the in vivo efficacy of drug delivery. Implantable radiotelemetry methods were used to demonstrate that a 20 min exposure to [[unable to display character: ∆]]9-tetrahydrocannabinol (THC), or the CB1 receptor full agonist JWH-018, produces a robust hypothermia. The temperature nadir was reached within 40 min of exposure, was of comparable magnitude to that found after 30 mg/kg THC or 1.1 mg/kg JWH-018, i.p. and had resolved within 3 hours compared with a 6 hour time course following injection. Studies also demonstrated that 30 min of intrapulmonary exposure to methamphetamine (MA) significantly increased home cage locomotor behavior for up to 2 hrs. A final study showed that a 30 min intrapulmonary exposure to MA reduced drug intake during the loading phase of intravenous self-administration of MA. Finally, it is shown that rats will nosepoke for the delivery of MA vapor. These studies show that an electronic cigarette type delivery system can be successfully used to model intrapulmonary drug delivery in rats. These techniques will be of increasing utility as recreational users continue to adopt “vaping” for the administration of psychtropic drugs.

Disclosures: M.A. Taffe: None. S.M. Aarde: None. M. Cole: E. Ownership Interest (stock, stock options, royalty, receipt of intellectual property rights/patent holder, excluding diversified mutual funds); La Jolla Alcohol Research, Inc..

This work was supported by NIH grants DA035281 and DA024105.

This figure is small preview of the data that we will be presenting. The figure depicts body temperature responses to 20 minutes of Vape-exposure to THC and the synthetic cannabinoid JWH-018 (upper panel) and locomotor activity responses to 30 minutes of Vape-exposure to methamphetamine (lower panel) in a group (N=7) male rats. In both panels there are comparison data for a session in which animals were just in normal cages with no drug intervention (No Chamber) and another session in the inhalation chamber in which animals were exposed to the Vape delivery vehicle without any drug in it (Vehicle). As you can see, we were successful in delivering active doses of the drugs, each of which had class-specific effects, i.e. cannabinoid hypothermia and stimulant hyperlocomotion.