TL neuro

July 19, 2019

Cannabidiol by vapor inhalation in rats

Filed under: Cannabidiol, Cannabis, E-cigarettes, Tobacco/Nicotine, Vapor Inhalation — mtaffe @ 2:37 pm

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.

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 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 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. When male or female Wistar rats experience CBD by vapor inhalation, their body temperature did 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 prolonged the activity of THC. Another thing that was slightly strange was that 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 [followup 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. There is growing evidence that CBD works in part by activating serotonin 1a (5-HT1a) receptors and activation of this receptor (e.g., by injecting the agonist 8-OH-DPAT) drops the body temperature of rats precipitously. We 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 seemed to be slightly greater than the response of male Sprague-Dawley rats in that paper.

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

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, in press. [ Publisher Site ]

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 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 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 male Wistar rats.

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

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.

This figure 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 with the 5-HT1a antagonist WAY 100,635. This shows that the 5-HT1a receptor is 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. As you can see, the effect of nicotine alone is not obvious (although it did enhance locomotor activity in this group) but it slightly increases the effect of CBD when the two are co-administered. Interestingly CBD did suppress 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.

 

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March 19, 2017

Vaccination against methamphetamine works in female rats

Filed under: Methamphetamine, Vaccines, Vape inhalation, Vapor Inhalation — mtaffe @ 9:32 am

We have shown that a vaccine designed to blunt the effects of methamphetamine works in male rats in two prior publications, summarized here and here. We have also had success showing that vaccines directed against the synthetic cathinones MDPV (“bathsalts”) and alpha-PVP (“flakka”) work to reduce the effects of those drugs. A brief video outlining the approach to generating vaccines that might be helpful for drug abused created by NIDA can be found here.

The following has recently been accepted for publication:

Nguyen, J.D., Bremer, P.T., Hwang, C.S., Vandewater, S.A., Collins, K.C., Creehan, K.M., Janda, K.D. and Taffe, M.A. Effective active vaccination against methamphetamine in female rats, Drug Alcohol Depend, 2017, 175:179-186. [Publisher Site] [PubMed]

In this study we show that an increase in the amount that female rats move around their cages after an injection of methamphetamine is reduced in the MH6-KLH vaccinated rats.

As you can see in the explainer video, the main principle of anti-drug vaccination is that antibodies can bind some of the drug molecules (methamphetamine in this case) in the bloodstream, thereby preventing them from getting into the brain. This capacity to retain methamphetamine is relatively fixed at a given point in the vaccine sequence, thus administering a sufficiently high dose can (should) overcome the protection.

In our data, the effects of the vaccine were dose dependent. This is Figure 4 from the paper which depicts locomotor activity rates (counts per minute) in the MH6-KLH and KLH groups in the first and second hours after injection of methamphetamine in three doses [Significant differences from the Vehicle and 0.25 mg/kg within Group and Hour are indicated by §, from Vehicle (only) by # and from the 0.5 mg/kg condition by &. ]. There is a dose-dependent increase in activity rate compared with the vehicle injection condition. With respect to the active vaccination group, complete protection was found at the 0.25 mg/kg dose and partial protection at 0.5 mg/kg compared with the KLH group; the two groups were about the same after 1.0 mg/kg was injected. This further enhances our ability to interpret these data as a specific effect of the vaccination and to determine where the threshold for effective protection may lie.

There was another finding in this study which was slightly disappointing in terms of the vaccine study but greatly enhanced our understanding of another thing that we have been working on, namely vapor inhalation techniques to deliver drugs to rats for various research purposes. Most specifically we showed that e-cigarette type vapor inhalation of methamphetamine (and MDPV and mephedrone) increases the activity of male rats to a similar extent as it does when injected (blogpost overview). We used this model in the present study as well and confirmed that just as with male rats, the female rats activity in the cage was increased after vapor inhalation of methamphetamine to about the same extent as after the injected doses. Therefore up to this point in time we were assuming that the dose delivered to the rat was approximately similar when similar behavioral results were produced.

Unfortunately there was no difference in the effects of inhaled methamphetamine across the vaccinated and control groups of rats. We originally interpreted this as potentially a difference in the rate of drug penetration into the brain which minimized the ability of the vaccine-generated antibodies to prevent locomotor effects.

Upon reviewer request we then examined the blood levels of methamphetamine after injection (0.25, 1.0 mg/kg, i.p.) and the inhalation condition in a different group of unvaccinated female rats. We found that methamphetamine was about ten times higher in the blood after inhalation versus injection in this new study. This of course explains why the vaccinated group was not protected, i.e., the dose under inhalation was far past the ability of the antibodies to sequester in the bloodstream.

The curious thing is still why a similar level of locomotor activity was produced at the 10-fold difference in methamphetamine levels. Very likely this is due to the rate at which drug is delivered to the animal- in our inhalation model this takes place over 30 minutes whereas an injection takes seconds. Obviously one of our next avenues of research is to better determine the way that drug levels increase in the blood during vapor inhalation.

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