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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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August 29, 2008

Sticky Containers, Vanishing Drugs

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Posted by Derek

There’s no end to the variables that can kick your data around in drug discovery. If you concentrate completely on all the things that could go wrong, though, you’ll be too terrified to run any useful experiments. You have to push on, but stay alert. It’s like medical practice: most of the time you don’t have to worry about most of the possibilities, but you need to recognize the odd ones when they show up.

One particular effect gets rediscovered from time to time, and I’ve just recently had to take it into account myself: the material that your vials and wells are made out of. That’s generally not a consideration for organic chemists, since we work mostly in glass, and on comparatively large scale. There are some cases where glass (specifically the free OH groups on its surface) can mess up really sensitive compounds, but in drug discovery we try not to work with things that are that temperamental.

But when you move to the chemistry/biology interface, things change. Material effects are pretty well-known among pharmacokinetics people, for example, although not all medicinal chemists are aware of them. The reason is that PK samples (blood, plasma, tissue) tend to have very small amounts of the desired analyte in them, inside a sea of proteins and other gunk. If you’re going down to nanograms (or less) of the substance of interest, it doesn’t take much to mess up your data.

And as it turns out, different sorts of plastics will bind various compounds to widely varying degrees. Taxol (OK, taxotere) is a notorious example, sticking to the sides of various containers like crazy. And you never know when you're going to run into one of those yourself. I know of a drug discovery project whose PK numbers were driving everyone crazy (weirdly variable, and mostly suggesting physically impossible levels of drug clearance) until they figured out that this was the problem. If you took a stock solution of the compound and ran it though a couple of dilutions while standing in the standard plastic vials, nothing was left. Wash the suckers out with methanol, though, and voila.

Here's a paper which suggests that polystyrene can be a real offender, and from past experience I can tell you to look out for polypropylene, especially the cheap stuff. You won't notice anything until you get way down there to the tiny amounts - but if that's where you're working, you'd better keep it in mind.

Comments (24) + TrackBacks (0) | Category: Drug Development | Pharmacokinetics


COMMENTS

1. HelicalZz on August 29, 2008 8:47 AM writes...

Two words - method validation.

Not full blown, but linearity and limit of detection just gotta be done. First.

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2. juniorprof on August 29, 2008 9:37 AM writes...

One more word - cannabinoids. Damn sticky compounds made the PhD work on pharmacology of cannabinoids a real pain in the arse. I would love to be able to go through the lit and put an "apparent" stamp next to nearly every Kd or EC50 value...

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3. CMC guy on August 29, 2008 9:48 AM writes...

I wonder how many possible good candidate compound are "missed" because of such behavior. There is probably a "natural selection" inherent in the discovery assays that would typically eliminate molecules that are very sensitive or contact absorbers because the result is no or low activity.

Certainly this is a bigger problem when dealing with small amounts. A few such bad players do make it into development stream although there the quantities become available and usually do some early profiling that will hint at these issues. There still can be surprise effects encountered when conducting Stability and materials compatibility studies. Without all these headaches our jobs could be boring right? at times would be nice place to be.

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4. Sili on August 29, 2008 9:59 AM writes...

Heh - reverse homoeopathy.

I've been told that the organic (non-medicinal) group next door had a problem like that years ago. A post-grad (or -doc, I forget) liked to seal his flasks with ... damn, I've already forgotten what that stretchable film/wrap/thingie is called ...

Anyway, he couldn't get his compounds NMR clean. Then a coupla years later the boss and a new student redid his work, but made sure to use cork-stoppers.

Turned out that the plastic softeners/phthalates in the wrap had Rfs smack in the middle of his compounds when he ran prep-TLC on them. Without they were nicely crystalline.

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5. Anonymous on August 29, 2008 10:09 AM writes...

Most of the times, the compound's clogP and aqueous solubility give an indication about its potential sticking nature, which we call it as 'non-specific binding'. There are various methods available to tackle it not to affect our study results. Most prominent among them is using silicone oil coating on the surfaces to avoid contact between the drug compound and the plastic material.

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6. RadioChemist on August 29, 2008 10:12 AM writes...

I had an amusing time once working with xenon difluoride. I had been advised to run some fluorinations in PTFE vessels to avoid etching our conventional glass round bottom flasks. The reactions were a flop, until I switched back to glass, whereupon the silanol groups at the surface catalysed the reaction and gave a positive result.

A bit too positive mind... the fluorine went in all over the place. There's a reason XeF2 gets shipped in well reinforced, chunky plastic containers!

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7. Dye Hard on August 29, 2008 10:44 AM writes...

Maybe twenty years ago I read a paper by someone from Stanford (as I recall) who was doing some standard binding assays with tritium-labeled drugs. They ran a blank set of assays, of course, but when they analyzed the data they found that the Scatchard plot from the blank showed a saturable, high-affinity binding site. On the glass fiber filters. The paper's conclusion was: make sure when doing binding assays you run a blank series so that you do not report a "new receptor".

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8. Cellbio on August 29, 2008 10:55 AM writes...

Have experienced this from the cell screening side. In one example, an oily drug stuck to plastic so well, that dilutions in serum free media failed to yield cellular impacts. The presence of serum in the dilution buffer revealed a nice sigmoidal dose response with nanomolar potency. A formulation scientist decided to try liposomal formulations, and oops, the potency dropped into the picomolar range. Another compound from a different campaign showed beautiful curves w/ or w/o serum, but IC50s were three logs apart. What's in an IC50 measure? Well, a lot can be the assay methodology.

Also have seen compounds partition to the cells themselves. We could demonstrate equivalent IC50s for compounds that were largely in the media, or partitioned to the cells (>85% in each case). Removal of media saw the drug impact diminish in one case and remain in the other, so the compound was available to reach target. We thought about which one would be the best behavour for a drug, all other things being equal, which of course they never are. We went with reducing the potential for tissue accumulation. Any thoughts?

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9. JSinger on August 29, 2008 12:22 PM writes...

... damn, I've already forgotten what that stretchable film/wrap/thingie is called ...

I guess I've also been out of the lab for too long, given how hard I had to think of the name of .... Parafilm!

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10. emjeff on August 29, 2008 12:34 PM writes...

It's not just the containers you have to worry about. At the company where I previously worked, we had a G2B3A antagonist with sush high affinity for the receptor that, when you spun down the blood samples to get plasma, most of the drug went into the pellet.

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11. john.spevacek@aspenresearch.com on August 29, 2008 12:52 PM writes...

As a polymer guy, sleep well knowing that polymers are even more prone to this behavior. Think about that next time you PEGylate something!

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12. Greg on August 29, 2008 3:11 PM writes...

Are there any actual chemists here? You're supposed to silate your glassware with TMSCL!

Rinse 2% TMSCL/benzene.

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13. processchemist on August 30, 2008 4:06 AM writes...

#12

benzene? really smart

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14. philip on August 30, 2008 8:30 AM writes...

Here's how you silate glassware: Fill a vacuum oven with the glassware that you want to silate. Put it on full vac and heat to 110C or so. Isolate the oven from the pump but leave it under vacuum. Put a septum on the outlet valve and inject about 5-10 mL of HMDS. Let it cook at 110C for a day and then cool and pull out your nicely silated glassware. It's great for doing thousands of small vials, maybe still in the box.

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15. drpcp on August 30, 2008 8:50 PM writes...

Yes, there are all those effects caused by things sticking TO your vessel of choice. Then there's the example from nicotinic receptors of the compound leeching FROM the plasticware: Papke et al., 1994 "Inhibition of nicotinic acetylcholine receptors by bis (2,2,6,6- tetramethyl- 4-piperidinyl) sebacate (Tinuvin 770), an additive to medical plastics" -- http://jpet.aspetjournals.org/cgi/content/abstract/268/2/718 . That one kept us guessing for a while -- when a previously reported compound started working on a subclass of receptors that it wasn't supposed to touch. So, even we biologists have our paranoias.

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16. Anonymous BMS Researcher on August 31, 2008 12:30 PM writes...


JSinger on August 29, 2008 12:22 PM wrote...

> I guess I've also been out of the lab for too
> long, given how hard I had to think of the name of > .... Parafilm!

I was UNABLE to remember it until that posting appeared, but when I tested my wife (who like me has been out of the lab for over a decade) SHE was able to remember Parafilm!!!

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17. anon on August 31, 2008 11:40 PM writes...

I'm sure everyone who's even done synthesis has had a plastic squirt bottle experience!! damn plasticiser...

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18. Giagan on September 1, 2008 7:28 AM writes...

Which is correct: "silate" or "silylate"?

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19. Jose on September 2, 2008 2:43 PM writes...

Derek- I am amazed you haven't written on the Lilly-Covance deal! Aside from the geographic peculiarities, I wonder if that will now become the SOP for the industry?

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20. Pete on September 2, 2008 6:54 PM writes...

I seem to remember that when Pedro Cuatrecasas purified the insulin receptor there was a bit of premature excitement caused by (drumroll please)...glove talc!

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21. RTW on September 3, 2008 8:42 AM writes...

#19 Pete - Wow thats a name I haven't heard in a while. Pedro was Ron Creswells right hand man at Parke-Davis prior to the Pfizer take over. I hated glove talc, and my prefered gloves in the lab became the blue nitrile ones. Liked them much better than latex!

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22. Jonathan on September 3, 2008 9:34 AM writes...

Which is correct: "silate" or "silylate"?

I'm just a pharmacologist, but isn't it silanize? Or is that something different? I know we silanize our glass tissue homogenisers so RNA doesn't stick to them when processing tissue.

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23. aes on September 4, 2008 6:51 PM writes...

I remember years ago a PhD candidate who discovered a new order of kinetics that everyone in the lab was excited about. The chemist was very anal about cleaning his glassware and used potassium dichromate to clean his glassware. thesis was about to be published when another lab member tried to duplicate his work and could not and eventually came upon the catalyst of the unique kinetic reaction. bummer for the PhD candidate

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24. Norepi on September 4, 2008 9:33 PM writes...

#22; I believe you are correct; I've never heard "silylate" refer to anything other than "putting a silyl-something onto a molecule" and I've never heard "silanize" refer to anything other than coating one's glassware with silyl-somethings. Silly, huh?

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