november 30 th 2005 perkin elmer seer green uk
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November 30 th 2005 Perkin Elmer, Seer Green UK. “….(these permeability) values are not equivalent (in value) and vary among labs, model compounds must be analysed by (both) methods to establish the correlation model for a given laboratory.”

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slide2
“….(these permeability) values are not equivalent (in value) and vary among labs, model compounds must be analysed by (both) methods to establish the correlation model for a given laboratory.”

ie. don’t try to correlate your data with anyone else’s data and expect it to fit nicely. It has to be relevant to your work, using your compounds under your conditions.

“……correlation refers to the relationship established in a given lab using model compounds having well characterised (permeation) mechanisms.”

Kerns et al J. Pharm Sci 93 6 1440-1453 2004

herein lies the crux of all the problems that we have within the field of physical profiling
Herein lies the crux of all the problems that we have within the field of physical profiling
  • Literature is awash with high quality data and superb science
    • On well documented, historical compounds
    • Behave well (mostly) and lots known about them
  • Not a common occurrence in the pharmaceutical industry
    • Compounds are novel (we hope!)
    • Don’t know much about them
  • No-one is going to put their company databases into the public domain
so how do we know that we are comparing like with like
So how do we know that we are comparing like with like?
  • Most of the time we probably aren’t
    • Units are different
      • Permeability: log, 10-6cms-1, 10-7cms-1
    • Incubation time is different
    • Stirring speeds
    • Temperature
    • Buffer conditions
    • Ionic strengths
    • Who grew the cells
    • Etc etc
  • That’s just for permeability, for all the other parameters its just as bad
not quite 505 000 definitions but
Not quite 505,000 definitions but
  • Kinetic solubility
  • Thermodynamic solubility
  • Intrinsic solubility
  • Unbuffered solubility
  • Buffered solubility
  • Pseudothermodynamic solubility
    • Co-solvents
    • Incubation time
    • Buffer conditions
    • Temperature
    • Ionic strength
    • …….
  • Etc 504,989 times
or how about permeability
Literature has infinite details on mechanism of PAMPA assay

People such as Avdeef have written prolifically on the subject

As for the theory, we were up to speed

But for more practical issues there are things like:

The size of the UWL does not depend on the volume flow. This prediction is correct only for vigorously stirred systems (Pohl et al 1997)

There is a UWL thickness corresponding to each molecule diffusing in the solution (Pohl et al 1998)

Thickness of the UWL does depend on the extent of stirring of the bulk solution, the layer becoming thinner with increased stirring (Karlsson and Arturrson 1991)

Or how about Permeability
  • So how fast do we stir, for our unknown samples in an assay we are unfamiliar with?
but it s not just stirring there are also things like
The resulting sandwich was incubated at room temperature under constant light shaking (50-100rpm for 5 hours (Faller et al 2000)

Donor and acceptor plates are incubated together for 12 – 16 hours (Millipore 2002)

….plates were incubated at 30oC for 2 hours or 15 hours (Sugano et al 2001)

…plates were incubated at room temperature for 2 hours with gentle shaking (Zhu et al 2002)

….6 – 15 hours (Ruell et al 2003)

….30 mins at 23oC (Avdeef et al 2004)

….4 hours incubation (Avdeef et al 2004)

The same goes for %DMSO, membrane constituents, sink conditions, buffers etc.

Millipore application note shows that they have looked into some of these variables

Not a luxury we can all afford

But it’s not just stirring, there are also things like…..
final couple of quotes
Final couple of quotes:
  • “As with any assay, there is an initial period of trial and error before reproducible results are obtained. Experience and familiarity with an assay helps in spotting potential problems and making adjustments accordingly. Some potential pitfalls are not obvious…. (Millipore 2002)
  • And one which was interesting to interpret:
    • Replace plate lid and incubate at room temperature for 16 hours. Note: place the assembled plate into a sealed container with wet paper towels to avoid evaporation (Millipore 2002)
slide14
So how are we supposed to talk to each other

Without giving away all our proprietary information

Bumbling about in the dark

To find out if we are on the right lines

That’s why we are here

I sincerely hope that this will be the first of many opportunities to get together and talk science

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