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

FANTASTIC trip, interesting ending...

            After many weeks in DC, I had the impulse decision to fly to Kansas to see my boyfriend.  Come to think of it, I’m not even sure I mentioned it to my parents because it was so impulse (surprise, I just got back from Kansas!).  Charlie and his cousin’s were playing a mini concert at a bar/restaurant, so with the help of his parents, I decided to surprise him.  I bought the ticket earlier this week on a whim, and got on a plane on Friday morning.  I went down to Union Station and caught the MARC train, thinking it would be faster.  I think I accidently purchased a ticket on the Amtrack, so it was $14 and I had to wait (this is a common trend in my life, trial and error).  The night before leaving for Kansas, I thought it was a brilliant idea to go out to Cactus Cantina with my volleyball team after playing… lets just say a few too many margaritas made it not a pleasant morning. 

The margaritas came back to haunt me around 8 am when I got on my first flight from Baltimore to Philadelphia. Small plane + hungover + I get motion sick = nausea at its best.  When I got to Philadelphia, I decided to eat my hangover away.  I ate an insane amount of food.  My layover in Philly was a couple hours and instead of doing my homework the night before, I  had gone out with the volleyball team. So, during my layover, I started working to get my online homework done.  Thankfully, I got most of it done before boarding my plane to Kansas City.  On the plane I wrote some of the paper that was due, but I needed the internet for some of it, so I stopped and went to sleep.  Once in Kansas City, I was picked up by the “Seaport Airlines” and they took me to a different airport that was about 10 minutes away.  While in the van going there, I asked the driver how big the plane was.  The response, “9 passenger.”  UM WHAT!?!  All I know about planes that tiny, is that they tend to crash and EVERYONE DIES.  When I got in the little building to check in, I was amazed by the procedure.  It went as follows-

Worker: “do you have any hazardous materials, firearms, or anything else that should not be on a plane?”

Me: “um, no”

Worker: “ok please place your bags on this scale.”

***Notice there was ZERO security.  I did not even walk through a metal detector.  So not only am I getting on a TINY plane, I could be getting on a plane with a psycho that has a gun, or a bomb, or something crazy in their suitcase because NO ONE LOOKED!!!!!!!

            I got in the plane and said a prayer that was something along the lines of “please God, do not let me die.”  In addition to the utter lack of security, you are allowed to use your cell phone and electronic devices during all times during the flight.  Um, what?!  The flight went fairly smooth until we began our decent.  We hit turbulence that was frightening.  Once again, I said a little prayer.  I have never been to thrilled to reach Salina, Kansas.  Charlie’s mom picked me up from the airport and took me to a hotel.

 While at the hotel I got the rest of my homework done. Around 8 PM, Charlie’s cousin picked me up and we went to grab some food before surprising Charlie.  This whole time I was texting him to “break a leg.”  I’m so sneaky. They began playing at 10 PM, so around 10:15 PM I walked into the restaurant/bar.  IT WAS PACKED.  It was awesome to see how many people came out to watch them play! I didn’t want him to see me before I was closer, and I didn’t want him to see me while he was in the middle of singing a song, so I hid behind some tall guy for a little bit.  During a pause during a song, I popped out from behind the tall guy and tried to make eye contact.  When he saw me, his jaw literally dropped.  He took a double take and sat there dumb-founded. J He got up from his chair and made his way through the crowd to give me a hug.  It was adorable!

That night is when life threw me another curve ball… around 4 am, the hotel fire alarm began going off.  It was not your average loud fire alarm, it was BLASTING.  I FLEW from my bed.  Once I opened the door to my hotel room, I glanced around and everyone on my floor was also in the hallway.  Then the alarm went off.  I went back to bed… only to be awoken AGAIN anywhere from 30 minutes to an hour.  It went off for maybe 10 seconds, then turned off.  After about the 6^th time that this happened, I called the front desk, FURIOUS!  I asked them if the alarm was going to stop going off.  I also said that it was ridiculous and that I should not have to pay full price for the hotel room.  The front desk woman took half off my hotel room, and I went back to bed.  Pam, Charlie’s mom had booked the hotel for 3 nights for me, but after the fire alarm fiasco, I decided to check out.  I went down to the front desk and told them that I should not have to pay for breakfast either (Pam had bought me breakfast in advance).  So, the hotel took half off the hotel room and I got free breakfast.

Saturday and Sunday I spent time with Charlie.  As usual, we ate a lot of food (I have been informed he gained 5 lbs while I was in town).  It was a really good time.

Monday… things became interesting.  The day started off normal.  I woke up and watched a tv show while Charlie prepared for a video interview.  After his interview (which lasted 1.5 hrs!!!), we began our drive to Kansas City for me to catch my flight.  All seemed well, it was a sunny day and traffic wasn’t bad… until we hit a detour that had us pull off onto a one lane country road.  I 70, basically the ONLY way to get across Kansas, was closed!!!!!!  Closed for 20 miles!!!! We were only going 5 miles an hour because traffic was horrendous on the detour.  I started freaking out at this point because we had planned exactly as much time as we needed to get me to KC.  I called the airlines asking what happens if I missed my flight.  They informed me that it would be minimum $175 to change my ticket.  We realize there is no way that I am getting there in time for my flight.  So we turn around and I start calling airlines to find out if anyone has a flight to the DC area before 9 am Tuesday morning (I needed to get to work).  I find out that the reason I70 is closed is because some INSANE Arkansas man decided it would be a fantastic idea to leave chemicals in a hotel room in Junction City, Kansas and call 911 to say that he left them there.  The highway was closed because they pulled him over on the interstate and brought in the FBI, robots, and the bomb squad to make sure he didn’t have anything hazardous in his truck.  IDIOT.

Anyway, we end up stopping at a McDonalds to try to figure out what to do.  Should I fly to KC from Salina?  Nope, I would still miss my flight.  Should I fly out of Wichita?  Well, there was a flight to DC, but it would take 12 hrs to get there.  Charlie calls his dad who is amazing at travel planning.  He looks up flights for me to fly into DC, which is funny because he was already in DC for a medical convention.  He finds me a flight out of Kansas City at 6 AM Tuesday morning to get me into Baltimore at 9:30 AM (NOT CHEAP).  So I stay at a hotel and fly out at 6 AM.

Chris, Charlie’s cousin, picks me up from the Baltimore airport and drives me directly to Rockville to work.  So I rolled into work wearing Capri sweats and a t shirt looking like hell.  I had not showered, hadn’t put in my contacts, or put on makeup.  I wanted to go to sleep, but went into work and stayed until 7:30 PM.

Overall, it was a fantastic trip with a very interesting ending….

Holocaust Museum and work update

HOLOCAUST MUSEUM
Since last time a I posted, I have been busy!  I had a couple friends come to visit so we hit up some of the historic things in DC.  My favorite thing we went to was the Holocaust Museum.  It is unbelievable.  The building has a dark, solemn feeling.  People do not speak loudly and everyone acted in a very respectful way.  We spent about 2 hours in there reading everything.  The room of shoes is what most people find the most moving.  There is a room that has piles of shoes that were taken from a concentration camp.  Before people were killed, they had to strip down and take everything off so the clothing and shoes could be reused.  It's a very disturbing room and it still has a funny smell to the room.  There is a train car from one of the trains that took people to the concentration camps in the museum.  The train car is very disturbing.  I stood in it and tried to picture what it was like to be cramped into a tiny car with very little air, no food, and people dying from the heat and exhaustion.  Towards the end of the museum there is a wall that has people who helped Jewish people hide and escape Nazis.  I respect those people so much to stand up for what is right even though they were often murdered for doing the right thing. I'm glad that I took time to go the museum, even though it was depressing to see what humans are capable of.

WORK UPDATE
 So I am the data queen.  I copy and paste into excel like you wouldn't believe.  But, seriously.  Besides doing lots and lots of data analysis, I have been doing more tail snips and PCR (genotyping mice).  Next week I will be starting a new project with a post Doc to do my end of the summer poster on, "the Pharmacokinetics of novel drug AM____".  It's a drug that has potential to be used for PTSD.  I learned earlier this week how to do IV injections.  The problem with mice is that they do not have an arm I can just stick a needle in, you have to find a vein in their tail.  This is unbelievably hard!!!!!!  Every time I got the needle in the vein, the mouse would move and the needle would come out of the tail. I also learned how to do a gastric lavage.  So basically I still a like needle like thing down the mouse's throat into it's stomach so I can directly insert the drug into it's stomach to simulate oral ingestion of the drug.  This is SOOOOOO much easier than IV.  I'll be practicing IV lots today so I can get this down....

Reasons you know you are working in a lab...

1. you stop wearing makeup to work because you realize mice do not care what you look like
2. stop trying to make your hair look good-for the same reason listed above.
3. You've wondered why you can't drink distilled water in the lab - Isn't it clean?
4. You cannot function without your first cup of coffee in the morning
5. You speak to mice. ie: hey little fella, please don't move!
6. When you learn that company reps are coming to the building, you first find out if they have food, then the location
7. You constantly think your hands smell like mice... or smell from your gloves.
8. You find shooting the tips off your pipettes fairly amusing... they do go quite far.
9. You have used Kim Wipes as kleenex
10. You don't take a lunch.  Lunch occurs at your desk while you work on data.

These are just a few things I though of today while doing 9 hours of data work....

Visits

Monday, June 27th
My mom and sister, Alisha came to DC.  They got to my house as I was leaving for work.  Alisha had never been to DC and my mom hadn't been here since she was little.  The plan was that I would go to work and they would do all the tourist stuff during the day.  We ate so much food.  We ate at Floriana in Dupont.  If you are in DC and like Italian... eat there.  We had a Bruchetta appetizer followed by a Mussel/Lobster bisque and some sort of beef pasta stuff.  And a 1/2 liter of Sangria. I had never tried Mussels before, but since I am on a culinary voyage this summer, I tried them.  I like mussels!!! Who knew?!?

Later in the week we ate at Meskerem.  It is a Ethiopian restaurant that is well known in DC. The food is served family style and you eat with your hands by using a pancake like bread.  The first few things we ordered were pretty good.  The last thing... not so much.

To sum up the week, I worked all day.  Mom and Alisha walked all day.  Then we all ate and drank. lots.  It was very nice to see them! :)

*I'm skipping a few days here*

Sunday, July 3rd, 2011

Charlie flew to DC to see me! He got here in the morning so we walked around the National Mall at the Folk Festival.  We tried to go to the dress rehearsal for the concert that plays on the 4th at the mall.  We got in and sat down.  We watched some of the banjo-playin music... but it was unbelievably humid and hot that we left before any of the "stars" performed.  I think we lasted about 20 minutes.  After cooling down we went out to eat at a place called Neisha.  Neisha is a Thai food place that is not far from where I live.  I had the pineapple fried rice.  YUMMMMMM.  The rice had shrimp and chunks of pineapple and it was all placed in a hollowed out pineapple shell.

July 4th
Charlie and I went to his cousin's house for a BBQ.  His cousin's girlfriend had flown in from Hong Kong and is an excellent cook.  She made SO much food.  It was awesome.  After hanging out all day we drove to Hain's Point to watch the fireworks.  Unfortunately the fireworks had moved from previous years so our view was obstructed by trees.  It was still a good fourth because I got to spend time with Charlie :)
 
**yes I know I write about food a lot, but I'm good at eating :).  Plus the rest of my day is usually almost the same everyday.**

Lab SUMMARY
I have quite the list of things I need to do at work.  Unfortunately most of those things require PCR tubes (a type of tube) and we are out.  So today I spent all day working on data analysis for one of the post Docs.  I was sectioning some brains for the same post doc, but I broke the tray the brain goes on and had to make another.  OOPS!  So tomorrow I'll be sectioning two brains, doing data analysis, hopefully "digesting" more tails to run PCR, and snipping some mice tails to do more genotyping.    I need to put in some extra hours tomorrow and Thursday because I have friends coming on Thursday evening and I really want to go on a Capital Tour with them on Friday.  Which would require me to leave work early.  Sooo, I need to do work tomorrow!!!  I'm going to try to go in early and stay late if necessary. 

NYC!!!!!!

Once again, I am behind on my blogging.... ((I typed this on my iPad, so sorry if the formatting is odd.))

NYC
Friday, June 24th, 2011
I left work early so i could catch the eastern bus from Chinatown to NYC. I met Shruti
and we began our very long bus trip to the city. The trip was supposed to take 4
hours... so naturally it took 6.5 hours. On the trip I had to use the bathroom and I would
pee my pants, so I gave in and used the bus bathroom. EW. There was no light in the
bathroom, the toilet paper was in a puddle on the floor, and there was some graffiti on
the wall. When i went to flush the toilet, the window of the bathroom like few open!!! I
honestly thought the window was falling off the bus! There is a possibiilty that the car
next to us got a free show. I walked out of the bathroom laughing so hard- partially
because i had a heart attack when the window opened.

When we finally got off the bus at Penn station, my phone was about ready to turn off
(as always), so Shruti and I walked into Starbucks to charge it for a couple minutes.
We parted ways and she went to her cousins. I began walking north. It was so nice
out that I walked for a little bit before looking for a cab. When i started looking for a
cab, i couldn't get one!!! After a few more blocks I finally got a cab and made it to my
cousin Tim's street. He came downstairs to meet me with his daughter, Lulu. It was
good to see him, I'm pretty sure the last time I saw him, was 5+ years ago! One of his
comments was that I was tall.... Haha.

We took the elevator up to the 7th floor to his place. It is AMAZING. I am so used to
seeing 20-somethng's apartments that i was shocked to see how big and nice it was. I
guess I expected it to be a lot smaller since it is in the city. The decorations were super
cute too thanks to his wife, Bernie. Unfortunately Bernie was out of town on business
so I didn't get to see her. When I got to Tim's we ordered takeout and relaxed in the
living room. Lulu watched a cartoon and I caught up on my emails and read the news.
Lulu was nice and let me sleep me sleep in her room.

SATURDAY, June 25th

I woke up at 9 am, which is early for a Saturday! After I showered, Tim, Lulu, and I went
to a cute little diner for breakfast. After eating, we walked to central park. I bought two
photos that i thought were really cool. I saw the famous boat house which was neat to
see. We made a stop in the Central Park zoo for Lulu. It was awesome! We saw seals,
penguins, a polar bear, and other random animals.

Lulu and I in Central Park

Lulu and I in the Central Park Zoo

After the zoo we started walking toward Rockafeller Center. Once there we bought tickets to go to the top floor called, "Top of the Rock.". It is 67 floors up so it gave us a fantastic view of the city.
After taking in the sites, we returned to ground level and we walked to Times Square. I
don't see what the big deal about Times Square is. I thought it was kind of gaudy!

At this point we walked to the Wintergarden which is near Ground Zero. The Winter
Garden has a large glass roof and Tim told me that when the first tower collapsed on 9/
11, all the glass was blown out. I looked out at Ground Zero from there and you could
not tell that it wS were the Twin Towers were, I guess i expected to see statue or
memorial or something. There is a new building being built. When the building is done,
it will be the tallest building in the US. From there we took the subway down to Chinatown. It was my first NYC subway experience! I expected it to be way dirtier. It
was strange how so many lines are connected once underground. Also, their subway
not so underground like DC's is, there was only a couple sets of stairs unlike DC which
has huge escalators for the metro. We walked around in Chinatown, then walked
through Little Italy. In Little Italy I bought a canolli which was yummy! At this point, it
was around 4 pm.  We went back to the apartment because we were all tired.

At 11 PM I met up with a group of people from Drake that are in NYC (living there for the summer or have a job).  We went to the Hudson Terrace Lounge and were on the rooftop.

Drake Girls!

Sunday
I met up with a couple of people that I had met in DC that also happened to be in NYC.  We were in Chelsea.  After only a couple hours I had to run back to Penn Station to catch my bus back to DC.  When getting on my bus, I had to walk over the subway grate thing... and naturally had a Marilyn Monroe moment.  The bus driver seemed to enjoy the free show.

Marines, Falafel, drag queens, and dance clubs

Friday night I went to the Marines Evening Parade in the Eastern Market area with my friend, Shruti and some of her other friends.  In order to go to this you must be a member of congress or work on the Hill (term used for people who work at the capital).  One of Shruti's friends worked at the capital so he got a bunch of tickets for us to go.  It is basically a presentation of the Marine Band and drill team (or whatever you call people who spin guns).  It was cool to see...  but I wish they did more of the gun spinning and a little less music playing.

Marine.  I would rotate this, but I can't figure out how!

AMERICA!

More Marines

After watching the "parade," Shruti and I rode the metro to Adam's Morgan.  This part of town is rowdy!  It's like Court Ave on steroids (Des Moines reference).  After the bar, we went to a place to have a falafel. I had never had one, but EVERYONE kept telling me that I needed to try one.  It was surprisingly good!  Here is a little definition (thanks Wikipedia): ball or patty made from ground chickpeas and/or fava beans. Falafel is usually served in a pita, which acts as a pocket, or wrapped in a flatbread known as lafa. The falafel balls are topped with salads, pickled vegetables, hot sauce, and drizzled with tahini-based sauces.
As strange as that sounds, it was yummy!  After eating my first falafel, I went to catch a cab.  At this point I discovered that cabs in DC do not accept credit cards.  This presented a problem because it was 4 am (bars close at 3 am) and the metro stops running at 3 am.  Luckily, one of the guys in my group lent me $20 so I could catch a cab home instead of walk 45 min or more back to my house.

Saturday
I woke up around 11 am and my housemate and I decided to go to a pool.  First we both needed food, so we went to an Indian restuarant called, "Masala Art."   This was my first time eating Indian food!  It was good!!!! I ordered the Tandoori Chicken and it was the perfect amount of spicy.  After eating we went back to the house to wait for his friend to call him so we could go over there.  I went back to sleep.  I am very good at napping.  Not even napping... going into hibernation.  So, I skipped the pool and slept all day (oops!). 
Later in the day I went to Columbia Heights to meet up with friends.  It was a friend-of-a-friend's birthday, so they ordered bottle service at a club.  A rather large group of us went.  I had a blast!

Sunday
Morning was not pleasant.  I got home around 4 am from going out and woke up at 9 am.  I had told some other people that I would go to brunch at Perry's in Adam's Morgan.  The place is very popular because on Sunday mornings they have Drag Brunch.  Yup, drag brunch.  So it is a buffet of food and after everyone has settled in, drag queens give a performance.  I was with a group of girls so it was a blast!  The drag queens were mostly lip syncing to music and dancing around.  A couple of them used the microphone and told jokes and made fun of people.  They also tend to single out the straight men in the audience because they are usually the most uncomfortable in these situations.

Drag Queen #1

Drag queen #2

Under that drag queen you can see Kendra

This is what happens when straight men go to Drag Brunch!

After Drag Brunch I walked around Dupont for a while....
epic weekend.