Saturday, August 6, 2011

Pharmacokinetics of a novel FAAH inhibtitor, AM 3506

Before I explain my experiment, I need to explain my coworker's experiment (all the references are on the poster above :) ).

The psycoactive ingredient of marijuana is THC. THC is a cannabinoid and binds the CB1 and CB2 receptors. The body has its own natural cannabinoids, called endocannabinoids. A couple major endocannabinoids are anandamide (AEA) and 2-AG. The only pathway known to break down AEA is the enzyme FAAH (fatty acid amide hydrolase). It has been found that inhibiting the enzyme FAAH leads to anxiolytic (anti-anxiety) and antidepressive effects (along with a bunch of other things).So, a laboratory in the NIAAA synthesized a drug, AM3506 to inhibit FAAH.

Lots of different experiments have been done with the drug, but the lab I work in looked at the drug from a fear perspective. A post-doc in my lab did experiments to see if AM3506 increased fear extinction learning. In PTSD people have impaired fear extinction learning. So think of a solider who associates a certain noise with a horrible outcome, when he/she returns to the US, that noise should not be scary anymore. But, the noise still causes them to freak out because they are unable to learn that the noise is no longer associated with a horrible outcome. There is a strain of mice that also has impaired fear extinction learning (129S1) so they were used for the fear experiments and my experiment.

Fear Conditioning: An hour before the experiment, the mice are injected with either the drug or a control (no drug). After an hour, a mouse is placed into a box (I think the experiment lasts a little over 3 minutes) and a tone is placed 3 times, at the end of the tone, the mouse is given a foot shock. So, the mouse associates the tone with a foot shock. A measurement of fear in mice is freezing. When a mouse freezes, they stop moving completely except for breathing. So during this experiment we record every 5 sec if a mouse is freezing.

The next day we do fear extinction. This is a horribly boring experiment. It takes around 30 minutes PER MOUSE. So we place the mouse in a new environment and play the tone (I think it's 30 times, I can't remember) but there is no shock. While this is going on, we record freezing.

TEN DAYS from the first fear conditioning experiment, the mice are put through "retrieval". For this, the tone is played 3 times, WITHOUT a foot shock and freezing is measured. My coworker found that the mice that were given drug were statistically freezing less, so it appears that the drug facilitates fear extinction learning.

OK, now MY part of the experiment for my poster.... since this drug potentially has therapeutic uses, we wanted to do some preliminary pharmacokinetics. Pharmacokinetics in easiest definition is what the body does to the drug. So, I used groups of 3 mice at 6 different time points (2 for zero hour though). Zero hour vehicle (a control where the mice were only given the liquid that drug goes in, NO drug), 0 hour, 1 hour, 2 hour, 3 hour, 6 hour, and 24 hour. We administered the drug IV (through the mouses tail into a lateral vein = NOT easy) and orally (I used a needle looking thing that had a round ball at the end. So I went in the mouses mouth and down to the stomach to directly deliver it to the stomach).

So at the time points that I listed, we sacrificed the mice. So for example we would give the drug and 1 hour later sacrifice the mouse (1 hour time point). Now, in humans this is normally done by drawing blood at multiple timepoints in 1 human, but I needed the brain and plasma at each timepoint, so we had to use lots of mice). Once all the mice were sacrificed, I extracted the plasma from the collected trunk blood and cut the brain in half because I was using half the brain for part of the experiment. I did lipid extraction (takes FOREVER) and my coworker's husband (also a pharmacist who works for the NIAAA in a lab, cute huh?) ran my samples in mass spectometry.

RESULTS of that part: The drug is only detectable in the brain and plasma at the zero time point for IV delivered dose. So, we think this is because the drug when given orally is highly metabolized by the liver so it is probably in the body as a undetectable metabolite. Also, the drug irreversibly binds FAAH, so it is probably undetectable because it is bound. So rewind back to anandamide (AEA), we measured the AEA brain levels. Basically the results are very interesting because the AEA levels increase (as hypothesized because FAAH is inhibited) but then decrease around 4-6 hours. This is interesting because the effects of the drug (fear extinction learning) are still working, so there must be either a metabolite or downstream effects that are affecting behavior! 

My coworker did some radiolabeled experiments with the plasma and brain as well (I would do it, but I'm not on the protocol to handle radioactive materials) to measure FAAH activity. Long story short (I'm getting sick of typing this), we measured FAAH activity to see if it was inhibited, look at my graphs if you can see it.

Further studies: we are doing intraperitoneal injections and Monday I'll be doing lipid extraction again. We also have lung tissue to do lipid extraction to see if we can detect any drug in the periphery.... Hope I didn't confuse you all too much, I tried to break it down as much as possible.

Oh, and I presented my poster at the NIH on 8/4/11