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Sunday, May 29, 2011

Information from the FDA - subscription services

The FDA of course has to be accessible and transparent, and they have several services a physician should subscribe to in order to remain up to date. I am using a service (https://mail.google.com/mail/?shva=1#inbox/1303ba92ab78b523) where I get information on new releases and approvals - but most of these turn out issues pertaining to foods. But today I received info on a newly approved antibiotic, and this information goes straight into one of my lectures..
This reference: http://www.fda.gov/Drugs/ResourcesForYou/Consumers/ucm054420.htm informs about drug approval procedural topics which also contains a link to information on currently approved drugs. Sometimes we hear about a "new drug" which often is just an extended use of a drug approved for other indications - and so I can check whether in fact the new indication is already official (viz. sildenafil and altitude sickness!). RSS services also are available - you may wish to make use of this for a while or may find it interesting for times to come.

Saturday, May 28, 2011

Sildenafil metabolism and clearance

Here is a follow-up on sildenafil – after a question in class. Some of this will make perfect sense to you after the coming drug metabolism lecture.

Firstly, I just discovered that the drug now has a third medical indication. I will have to check whether this indication is officially sanctioned by the FDA, but the drug apparently is useful for the prevention and treatment of high-altitude pulmonary edema associated with altitude sickness such as that suffered by mountain climbers. While this effect has only recently been discovered, sildenafil is already becoming an accepted treatment for this condition, in particular in situations where the standard treatment of rapid descent has been delayed for some reason. I recently read some blogs about climbing Everest (an 80-year old died when trying to set an age record!), but in none of the altitude sickness cases the drug was mentioned.
Metabolism:
Sildenafil is rapidly absorbed and distributed throughout tissues following oral ingestion. Maximum plasma concentration occurs between 30 minutes to 2 hours following oral administration. Absorption and plasma concentration can be reduced if taken with food.
Following absorption in the gut, sildenafil enters the hepatic vein in high concentration. As sildenafil makes its first pass through the liver, much of it is metabolized by the cytochrome P450 enzyme system. The P450 enzyme system breaks sildenafil into a number of metabolites. One of these metabolites is UK-103,320 which retains some PDE-5 inhibiting capacity. The sildenafil which is not metabolized and the UK-103,320 are then distributed throughout the body having the primary effect of blocking PDE-5 activity in the corpus cavernosum. Sildenafil and metabolites are cleared primarily through the biliary system with about 80% excreted in feces. The half-life of sildenafil is about 4 hours.
The P450 enzyme responsible for most sildenafil metabolism is CYP3A4. This enzyme is known to be inhibited by a number of substances. Some of the substances which have been demonstrated to interfere with the CYP3A4 metabolism of sildenafil include: ketoconazole, erythromycin, cimetidine and ritonavir. Inhibiting the action of CYP3A4 results in increased plasma sildenafil concentration and an extended half-life.
Grapefruit is a known intestinal CYP3A4 inhibitor. Research indicates that grapefruit can increase the bioavailability of sildenafil. While the effects are modest it may be wise to avoid concomitant use.
Protease inhibitors used in treatment of AIDS also are metabolized by the above CYP enzyme(s) and thus also give rise to higher plasma levels of sildenafil based on competition. I am sure that there may be issues sometimes also regarding drugs like warfarin used for blood coagulation control.
(NB: Some of the above was copied straight from the source such as Wikipedia.)

Wednesday, May 25, 2011

Want to know how we measured adenyly cyclase activity?

There were 2 problems to be overcome: Tinyest amounts of cyclic AMP had to be detected and quantitated, and ATPases had to be "neutralized" because they would eat up ATP so fast, there was nothing left for the adenylyl cyclase.
Enzyme preparation: We worked with fat cells. Homogenizing cells often destroyed the enzyme activity - so we treated fat pads with collagenase to obtain free cells that could then be lysed in hypotonic buffer, yielding membrane "ghosts" that proved to retain activity of membrane-associated enzymes like the cyclase.
Radioactive substrate - p32-labeled  ATP: This was obtained from a manufacturer that was able to custom produce ATP that had radioactive P32 only in the alpha postition, the one that of course was retained in the cAMP product. It had to have highest specific radioactivity to produce the sensitivity we needed. This was a new market for the manufacturer, and we managed to make them improve their product in terms of purity - initial 2-dimensional chromatography studies we did showed them and us that the preps contained radioactive impurities exceeding what we expected our enzyme product, P32 labeled cyclic AMP, to be.
New enzyme technology: To date (1967) enzyme studies always involved incubates of 1-10 ml - but that would waste radioactive substrates immensely - so we developed microassays in volumes of 50 uL. Fortuitously, the Eppendorf company was in the process of pioneering pushbutton pipetting devices with disposable tips for pipetting and transferring amounts down to 5 microliter to special propylene incubation vessels (before that we had to use tiny test tubes that needed siliconizing the glass surface so that the tiny volumes would mix and form one volume rather than scattering inside the tube). These pipettes were intended for hospital labs - and we may have been the first to use them for enzyme assays in research.
The assay: Now we combined, in 5-10 uL increments to a total of 50 uL, a Mg ion containing buffer solution, ATP-alpha-P32, various other components to be tested (hormone solutions), high concentrations of creatinine kinase and creatinine phosphate (supposed to regenerate any ATP hydrolyzed by ATPases)  and enzyme preparations (fat cell ghosts) - the latter added last to start the reaction. To stop the reaction we initially used immersion in boiling water - but then we just added  10 uL of a mix of high concentrations of Assays and evaluate ATP/ADP/AMP and unlabeled cyclic AMP plus EDTA (a Mg complexing agent). In  the next step we used a chromatographic separation method on thin-layer cellulose plates with poly-imine that served as an ion exchange system. After chromatographic separation we identified the location of cyclic AMP and of the other nucleotides uinder UV light, marked them with pencil and then cut out the areas containing these. The cut out strips were immersed in scintillation vials and counted in a common scintillation medium to count the radioactivity levels. This way we could determine how much of the radioactive ATP had been transformed into cyclic AMP and calculate the enzyme rates in proper units - pmol/min/mg protein.
The exciting thing was then that indeed the enzyme rates proved to be affected by various hormones such as epinephrine, glucagon, TSH, ACTH, FSH and others produced dose-dependent activation of the enzyme.
I cannot believe today that we went through all this trouble to study this enzyme, needing such an elaborate setup. Once you are able to develop a technology to measure something new and reproducibly so, you can crank out data and generate publications because every enzyme assay produces new findings and results!

And here is a little question for you: How come fat cells seem to respond to so many different hormones with lipolysis (mediated always by cyclic AMP? This is not seen in any other tissue or cell.

How to determine AUC (and then bioavailability)

We used to discuss in a small group some of the ways in which pharmacokinetic parameters were determined for a given drug. In the absence of such a time slot, let me explain here the issue of determining the AUC from experimental data (plasma concentration versus time, after giving a certain single dose of drug to a patient) – this being necessary of course for determining the bioavailability of a drug.
How can we determine the AUC for a curve obtained by experimental data points, not being linear but rather representing a Bateman function (first order exponential drug absorption plus first order exponential elimination...)?
Usually you would plot the data to see whether they look reasonable and represent a curve that you expect. Then there are several options.
  1. Draw the curve by hand or computer, using a grid as a background and count (estimate) the grid squares “under the curve”. The number of grids is all you need usually, but knowing the area for each grid, the grid count gives you the total area in the exact dimensions (say, [mg/mL times time]).
  2. After drawing a curve as above, cut out the area to be measured and weigh the paper (so-called “integration paper used to be available with a rather constant weight/area. The nice thing is that this method can take care of any shape of a cirver for which you otherwise may not have a descriptive formula. Of course, the weight is just something that is proportional to the AUC, but weiging a cutout of a rectangle or square for which you can calculate the exact are in [mg/L x time] can give you an absolute value.
  3. I have seen engineers using some x/y tracing gadgets that run up a counter number for you as you trace around an area.
  4. Mathematically, use the Batement equation to fit the data points to and calculate the parameters by iterative procedures (rate constants in and out): And, after inserting the parameter estimates into the formula, integrate it between the limits of time zero to somewhere when the curve almost touches the x-axis or maybe to infinity. This would be an enormous task without computers!The plotting program called “Sigmaplot” is tremendous for such a task. The obtained integral of course is the AUC.
  5. Fit the data point sto a polynomial (y = a + bx + cx 2 + dx3 .......) and after insertion of the parameter estimates integrate the function between the same limits as above.
  6. Use the experimental data and generate rectangles of time intervals times PC values that approximate the AUC, and this can be easily done by using a spreadsheet calculator. That is what I would be using nowadays where I do no longer have available Sigmaplot! You can try it out, it is fun: The spreadsheet is included in your ancillary files and available on the Student Drive under my name following the Basics lectures
  7. After drawing a curve as above, cut out the area to be measured and weigh the paper (so-called “integration paper used to be available with a rather constant weight/area). The nice thing is that this method can take care of any shape of a curve for which you otherwise may not have a descriptive formula.
  8. Mathematically, use the Batement equation to fit the data points to and calculate the parameters (rate constants in and out): And, after inserting the parameter estimates into the formula, integrate it between the limits of time zero to somewhere when the curve almost touches the x-axis or maybe to infinity. This would be an enormous task without computers!The plotting program called “Sigmaplot” is tremendous for such a task. The obtained integral of course is the AUC.
  9. Fit the data point to a polynomial (y = a + bx + cx 2 + dx3 .......) and after insertion of the parameter estimates integrate the function between the same limits.
  10. Use the experimental data and generate rectangles of time intervals times PC values that approximate the AUC, and this can be easily done by using a spreadsheet calculator. That is what I would be using nowadays where i do no longer have available Sigmaplot! You can try it out, it is fun: The spreadsheet is included in your ancillary files and available on the Student Drive under my name following the basics lectures. File name: “CP and AUC Calculations Spreadsheet”.
One you have values for AUC, exact or proportionally, you then of course take the AUC for the i.v. administration as 100% and calculate the bioavailability for your drug in relation to that. How many times in one patient, and how many patients total? I have no idea - ask the clinical pharmacologist of a company! I know one and will let you know what he says...

Monday, May 23, 2011

Treatment switch

A patient has been on the antibiotic ampicillin for some time (E. coli upper urinary tract infection; this would not be the correct therapy - i.e. one would not use ampicillin alone, and pl. ignore this point here). He now is stable enough to be switched to a drug that has the same mechanism of action and antibacterial spectrum, but it is commonly used orally. 
The infusion rate of ampicillin was 500 mg/kg/day.
You make the switch on day zero at 8 am, stopping ampicillin infusion and starting with amoxicillin oral 4 times a day.
How much amoxicillin should you give orally every 4 hours, and how long would it take for the switch to be complete, i.e. the patient being on full therapy with amoxicillin? The half life of ampicillin is about 1 hour.

Saturday, May 21, 2011

How much needed to get a desired plasma concentration?

Suppose you want to achieve a plasma concentration for a drug, say penicillin, of 1 mg/L. How much of the drug would you have to administer, say i.v., in a 70 kg person?


(Ignore the fact that as soon as you give something elimination kicks in right away so that you would be chasing an elusive point in time...).


If you feel that you cannot calculate or estimate this and need some more information to do so, what information do you need - or what assumptions are you making to make your estimate?

Determining the bioavailability of a drug

Below I am inserting a hand-drawn graph.  It represents two plasma concentration (PC) versus time curves, using in one case an oral dose of a drug X and in the other an i.v. dose of the drug (same amount in each case).

1) Which curve (A or B) represents the intravenous administration?

2) What is the oral bioavailability of drug X - estimated?
Or, if you like MCQ's better - the oral bioavailability is about
A.  120%
B.   50%
C.   80%
D.   10%

Protein phosphorylation - why?

Here is a small challenge question that will come up in class soon enough, pertaining to the observation that essentially all major intracellular regulation pathways (signaling pathways) for metabolism, cellular functions such as growth and cell cycling as well as typical activities of cells seem to involve the action of protein kinases. Just about any hormonal control mechanism involves either activation or inhibition of protein kinases (as well as phophoprotein phosphatases, the reverse event). So, do you have any idea why nature chose protein phosphorylation as a central and common controlling function?
Additionally, you will see that calcium ion (Ca2+) also is involved in just about any signaling pathway - why did nature choose to muse this ion?

Friday, May 20, 2011

More on cultural sensitivity

I mentioned to you that my daughter found some serious problem with my little "Sugar" video: She said nit was insensitive of me to have the "fat" one portrayed as an Indian (I know, I know:  Ishould not call the First Nations people Indians...more on that later). You did not seem to mind - but here is what happened:
Firstly, the online video program offered that option of characters, and I gave it no thought, just went ahead, no offenses intended. Secondly, I told her, it so happens that indeed our Indians in Canada are the worst affected when it comes to diet, obesity, and other metabolically promoted diseases such as Type 2 diabetes, hypertension and cardiovascular issues. Not to speak of additional factors such as alcohol, water quality etc. So, she says, now I am making the situation even worse.
Oh, my sweet daughter meant well, and I, stupid, old and culturally insensitive father, meant no evil. We left it at that! I am refraining, however, from publishing the clip on YouTube or sending copies to the Indian reserves!

Careful with humor in the office!

Humor is something culture specific: remember this when you deal with your patient, because what entertains one may annoy the other!
You found my little sugar video funny or hilarious. Well, I had sent it to a Vincentian friend as well, and the person asked me what is was about - saw no humor in it. That person would never be entertained by Seinfeld, my favored.
A friend once wanted to contribute to a round of jokes while in the company of a few Saudis in Riyadh. He told the one about the guy who, wanting to join a group of Canadian loggers, proved his ability by chopping down a tree or two in next to no time. "Man, where did you develop this skill?" "In Saudi Arabia". "But there are no tress". "Of course not, I was there long enough!"
The Saudis got up and left, obviously embarrassed or annoyed.
For you: careful with your jokes when dealing with patients! I, for one, would make a bad doctor.

Answer to question:

The answer to the question on the previous blog, briefly is that rectal drug absorption takes place via both the portal and the systemic circulation (50/50 about). Only the portion that goes into the portal one will be subject a first pass loss (via liver metabolism, not of course acid destruction in stomach). So rectal absorption indeed produces a higher plasma concentration. Nice to have had so many good answers, in principle! 
Oh, and to the person who asked whether there was a burp at the end of the preceding Xtra video: yes, indeed there was! Inserted via one of the sound options of the program - Do try the program and send your creations!
HB

Tuesday, May 17, 2011

A question for you...

Here is a question that maybe goes beyond what I covered in class so far, but how about doing some thinking and reasoning: 
What do you think will be the consequence of switching from oral administration of a drug to rectal administration, i.e. via a suppository?
And by "consequence" I mean whether you think it would make a difference to the eventual plasma concentration?
(This cannot easily be formulated in form of a multiple choice question. So your reply in the Comment section needs to be in form of a short statement.)


I like to see an answer from everyone - and you won't be punished if you say "I have no idea"..

Sunday, May 15, 2011

An artificial nose?

After watching this video I gained some new respect for the "complication" of other sciences. Doesn't this sound like having constructed a nose? How would you like it if yours beeped everytime it smells your favored parfume?
Incidentally, olfaction involves some interesting processes, right down the line we will be discussing in the lecture on Signaling.