Physicochemical Profiling for Early Drug Discovery at UCB Barbara Mason UCB, Slough, UK ACDLabs 2 nd Annual PhysChem Symposium Obernai, 20 th October 2005
PhysicochemicalProfiling for Early Drug Discovery at
UCB
Barbara MasonUCB, Slough, UK
ACDLabs 2nd AnnualPhysChem Symposium
Obernai, 20th October 2005
Target ID Target Validation HTS Hit 2 Lead Lead
Optimisation
Later stage research is supported by recognised, industry standard methods which are capable of generating gold standard data but on a much reduced number of compounds, to assist in candidate selection
Physical Chemistry work in collaboration with DMPK colleagues to provide a full data package for every compound using systems which are directly relevant to the assay system used to generate potency and selectivity data.
Discovery PhysChem, UCB Slough
• The assays we routinely carry out
• Some that we are developing
• Data
GI PAMPA(all NCEs)
BBB PAMPA(all NCEs)
Caco-2(request)
Permeability
SnapSol(request)
BioSol(all NCEs)
AKAS(all NCEs)
QSol(request)
TSol(request)
Solubility
HT Log D7.4(all NCEs)
BIO-PAMPA(all NCEs)
HT pKa(all NCEs)
In Progress
10 mM DMSO Stock
PPB(request)
Log P(request)
pKa(request)
Discovery PhysChem (Slh)
PhysChem (Brne + Cam)
DMPK (Slh)
Solubility1. Intrinsic solubility – solubility of the neutral species regardless
of physiological relevance of pH (similar to Log P)
2. Unbuffered solubility – solubility of a saturated solution at whatever pH the solution ends up at (self – buffering)
3. Buffered solubility – solubility at a specific pH, 5 or 7.4 for example (similar to Log D – takes pKa into account)
4. Kinetic solubility – solubility of the fastest dissolving or fastest precipitating species
5. Thermodynamic solubility – equilibrium solubility of all species
Solubility – A Cascade of Assays Needed
• Greater Confidence in in-vitro and in-vivo assay results to avoid false negatives
• Poorly soluble compounds may undergo non-specific binding with proteins leading to false positives
• Measurement at multiple pH levels is more useful than single point in some circumstances
• Solubility – pH profile provides a better understanding of absorption through the pH gradient of the GI tract
• Kinetics, thermodynamics and DMSO content
GI PAMPA(all NCEs)
BBB PAMPA(all NCEs)
Caco-2(request)
Permeability
SnapSol(request)
BioSol(all NCEs)
AKAS(all NCEs)
QSol(request)
TSol(request)
Solubility
HT Log D7.4(all NCEs)
BIO-PAMPA(all NCEs)
HT pKa(all NCEs)
In Progress
10 mM DMSO Stock
PPB(request)
Log P(request)
pKa(request)
Discovery PhysChem (Slh)
PhysChem (Brne + Cam)
DMPK (Slh)
AKAS – Automated Kinetic Aqueous Solubility• All novel NCEs pass through this assay (~100/week)
• 10mM DMSO stock solutions are diluted in buffer at 4 pHs– 5% final DMSO concentration
• Samples are shaken for 90 minutes then filtered
• Concentration of sample in the filtrate is determined by UV plate reader against a calibration curve- Spectra measured from 240-400nm− λmax / isosbestic point
• Measuring at 4 pHs gives us a number of advantages– Any pH instability is flagged early in the discovery process– Approximate pKa values can be estimated by effects on solubility
ND 0 - 30 uM 30 - 150 uM 150 - 350 uM >350 uMpoor modest good
BioSol – Solubility under in-vitro conditions
• In a high throughput drug discovery screen, primary assay data are generated in enzyme or protein based assays
• Solubility of the compounds may be compromised by the presence of these proteins and incubation media
• Maximum quantity of sample present is very low• For cellular systems DMSO content has to be kept to a minimum
• This assay gives a handle on sample solubility at relevant DMSO concentration and also flags potential protein binding issues in a high throughput method which can then be studied using more traditional PPB methods by the DMPK group.
BioSol• All novel NCEs pass through this assay ~100/week
• 10mM DMSO stock solutions diluted in D-PBS– 0.2% final DMSO concentration– 10% bovine serum albumin– 10% glucose– MgCl2, CaCl2 (1µM)
• Control plate without protein
• Shaken for 90 minutes at 30oC then filtered
• Filtrate centrifuged through 10KDa cut off plates– 20 mins– 2000 rpm
• Analysis by HPLC
• Solubility data is returned for 0.2% DMSO along with an indication of potential protein binding issues
QuickSol – Pseudothermodynamic solubility
• 5% DMSO in AKAS is a rather high quantity of co-solvent.• True thermodynamic assay is expensive with respect to sample
quantity and slow.• QuickSol starts from solid material – 0% DMSO
– Solid material not necessarily present in excess (0.5mg / pH)– Single time point, not equilibrium
• Measured on selected compounds by request achieving >150µM at either pH 5 or pH 7.4 in AKAS
• Buffer added to solid material– gentle shaking for 90 mins– filtered
• Filtrate analysed by HPLC against a calibration curve
SnapSol – fragment screening single point solubility
• This has been developed as an aid to fragment screening to ensure that compounds meet minimum solubility criteria
• Can be used to screen a selection under conditions which mimic closely those of the screening deck
• 10mM DMSO stock solutions are diluted with buffer system used inscreening deck to give final DMSO concentration at a relevant level
• Shaken for 90 minutes then filtered
• Analysis of the filtrate by UV plate reader at fixed wavelength against a calibration curve
GI PAMPA(all NCEs)
BBB PAMPA(all NCEs)
Caco-2(request)
Permeability
SnapSol(request)
BioSol(all NCEs)
AKAS(all NCEs)
QSol(request)
TSol(request)
Solubility
HT Log D7.4(all NCEs)
BIO-PAMPA(all NCEs)
HT pKa(all NCEs)
In Progress
10 mM DMSO Stock
PPB(request)
Log P(request)
pKa(request)
Discovery PhysChem (Slh)
PhysChem (Brne + Cam)
DMPK (Slh)
Permeability• Industry wide, in-vitro cell based assays still extensively used to
predict permeability across phospholipid membranes• Advantages and disadvantages are widely known and debated
+ closer mimic to in-vivo systems+ transport mechanisms can be monitored+ varying cell lines can be used for more applicable assays– relatively low throughput (despite new 96 well format)– experimental errors are high– time consuming and expensive
• We have moved to artificial membranes as a means to measure passive diffusion using the PAMPA methodology+ fast and cheap– no information about active transport or efflux mechanisms
Permeability – GI PAMPA• All novel NCEs pass through this assay (~100/week)
• Phosphatidyl choline artificial membrane on solid filter support• 10mM DMSO stock solutions are diluted in buffer to 5%DMSO • Donor / acceptor sandwich incubated
– for 4 hours at 20oC with gentle shaking
• Ratio of sample in the donor and acceptor wells is measured by UV plate reader
• Membrane retention is calculated as a % from mass balance equations and the permeability is returned at 10-7cms-1
ND 0 - 50 50 - 150 >150poor modest good
PAMPA – Blood Brain Barrier• The Blood Brain Barrier is an important membrane – it has very
tight intercellular boundaries
• CNS active drugs must cross the BBB while to avoid undesirable side effects non-CNS active drugs must not– Particularly important to UCB targeting both CNS+ and CNS- disease
states
• We have therefore developed a PAMPA-BBB assay to indicate whether our compounds are CNS+ or CNS-.– Validated against known CNS active compounds
• Methodology is as for the traditional PAMPA assay, with the phosphatidyl choline membrane being replaced with porcine polar brain lipid
* L.Di, E.Kerns et al Eur. J. Med. Chem 38 (2003)223-232
GI PAMPA(all NCEs)
BBB PAMPA(all NCEs)
Caco-2(request)
Permeability
SnapSol(request)
BioSol(all NCEs)
AKAS(all NCEs)
QSol(request)
TSol(request)
Solubility
HT Log D7.4(all NCEs)
BIO-PAMPA(all NCEs)
HT pKa(all NCEs)
In Progress
10 mM DMSO Stock
PPB(request)
Log P(request)
pKa(request)
Discovery PhysChem (Slh)
PhysChem (Brne + Cam)
DMPK (Slh)
Thermodynamic solubility, pKa and Lipophilicity• No physical chemistry profiling would be complete
without thermodynamic solubility, pKa and Log P
– These are performed by colleagues in Braine (Belgium) and Cambridge (UK) on a request basis using their Gold Standard or traditional shake flask methods as part of the development characterisation
• For discovery screening these methods do not lend themselves easily to high throughput.
Can we address these issues with our current technology?
• Thermodynamic Solubility– While the QuickSol assay does not give a true thermodynamic
solubility, it does give a clear indication of DMSO co-solvent effects
• pKa– AKAS solubility gives a good handle on pKa (allowing for the DMSO
effect) since it is run across a pH range• In-house algorithms have demonstrated that it is possible to take the UV
data and use it to produce a better approximation of the pKa (not fully validated yet)
• ACDLabs pKa prediction software
• Lipophilicity– Higher throughput Log D 7.4 robotics based method is being developed
(with knowledge of pKa we can then determine a lipophilicity profile)
Data Package
• List of standard compounds– All well known drugs commonly used in the literature and well
validated
• Data we have measured in our assays
Impact of our data package on discovery projects
• No single piece of assay data should make or break a project– informed interpretation of physical chemistry data alongside
biological data
• Very fine line– But, must not cloud the issue with too much data– Although, much can be learned from large amounts of historical
data to use in future projects
• So, to demonstrate what information can we get from our cascade of assays…..
QuickSol BBB PAMPApH3 (uM) pH5 (uM) pH7.4 (uM) pH9 (uM) pH7.4 (uM) pH7.4 (uM) PPB? Pa (10-7 cm/sec) %R Pa (10-7 cm/sec)
Alprazolam ND >350 >350 >350 820 18 37 30Alprenolol >350 >350 >350 >350 ND 20 122 26Amiloride >350 >350 >350 >350 >1500 0 0
Astemizole >350 >350 68 13 0 14 125 63 19Atenolol >350 >350 >350 >350 >1500 7 0
Caffeine >350 >350 >350 >350 >1500 10 5Chlorpromazine >350 >350 >350 52 >1500 15 94 12Corticosterone >350 >350 >350 >350 526 4 ND
Desipramine >350 >350 >350 >350 >1500 16 222 28Dexamethasone >350 >350 >350 >350 275 18 0 16
Diazepam ND >350 >350 >350 172 19 175 16 25Diltiazem >350 >350 >350 >350 >1500 12 223 38Enoxacin >350 >350 >350 >350 >1500 1 18
Famotidine >350 >350 >350 244 >1500 0 0Furosemide 283 >350 >350 >350 >1500 13 Yes 0 0
Hydrocortisone >350 >350 >350 >350 1237 0 0Imipramine >350 >350 >350 >350 >1500 16 116 40 17Isoxicam 2 >350 >350 >350 >1500 12 Yes 0 0
Ketoconazole >350 >350 252 236 7 10 63 32 8Ketoprofen >350 >350 >350 >350 >1500 7 Yes 0 1
Labetolol >350 >350 >350 >350 >1500 18 0 78Metolazone >350 >350 >350 >350 109 0 3
Naproxen 164 >350 >350 >350 >1500 9 Yes 0 0Nifedipine >350 >350 >350 >350 20 20 133 12 13
Norfloxacin >350 >350 >350 >350 >1500 0 0Ofloxacin >350 >350 >350 >350 >1500 18 0 0
Oxazepam >350 >350 >350 >350 76 8 14 42Piroxicam >350 >350 >350 >350 >1500 11 Yes 10 6
Promazine >350 >350 >350 >350 >1500 17 193 19 12Propranolol >350 >350 >350 >350 >1500 17 109 32
Quinidine >350 >350 >350 >350 >1500 9 28Ranitidine >350 >350 >350 >350 >1500 0 0
Sulfasalazine 144 >350 >350 >350 >1500 0 0Sulpiride >350 >350 >350 >350 >1500 0 0
Tenoxicam >350 >350 >350 >350 >1500 14 Yes 6 2Terfenadine >350 >350 204 0 0 0 0 0Testosterone >350 >350 >350 >350 199 201 21
Theophylline >350 >350 >350 >350 >1500 0 0Timolol >350 >350 >350 >350 >1500 0 13
Trimethoprim >350 >350 >350 >350 1159 0 1Verapamil >350 >350 >350 >350 >1500 16 188 13 25
Warfarin >350 >350 >350 >350 >1500 6 Yes 2 1
GI PAMPAAKAS BioSol
Ketoconazole:
• Solubility assays show clear effect of DMSO
• Distinction between permeability assays and membrane retention also noted
AKASpH7.4 (uM)
252
N
N
O
OO
ON N
Cl
Cl
BioSol QuickSolpH7.4 (uM) pH7.4 (uM)
10 7
BBB PAMPAPa (10-7 cm/sec) %R Pa (10-7 cm/sec)
63 32 8
GI PAMPA
Warfarin:
• Solubility assays show no effect by DMSO (AKAS vs QuickSol)
• BioSol shows drop despite 0.2% DMSO and evidence of protein binding – consistent with literature
O
OH O
AKAS BioSol PPB? QuickSolpH7.4 (uM) pH7.4 (uM) pH7.4 (uM)
>350 6 yes >1500
Astemizole:
• Relatively low solubility while the PAMPA data is relatively high
• Can be explained by membrane retention– Lipophilic compound
N
N NH
N
O
F
AKASpH7.4 (uM) Pa (10-7 cm/sec) %R
68 125 63
GI PAMPA
pKa
pH3 (uM) pH5 (uM) pH7.4 (uM) pH9 (uM)2 >350 >350 >350
AKASIsoxicam
pH3 (uM) pH5 (uM) pH7.4 (uM) pH9 (uM)144 >350 >350 >350
AKASSulfasalazine
pH3 (uM) pH5 (uM) pH7.4 (uM) pH9 (uM)>350 >350 240 0
AKASTerfenadine
NS
O
NH
OH
OO
ON
N NH
SO O
NN
OH
O
OH
NOH
OH
pH Stability issues – from AKAS UV spectra
Alprazolam
pH3pH5
pH7.4pH9
N
NN
N
Cl
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
240 260 280 300 320 340 360 380 400
Wavelength / nm
Abs
orba
nce
0.5uM0.2uM
0.05uM0.01uM
pH3pH9
Abs
orba
nce
Wavelength / nm
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
240 260 280 300 320 340 360 380 400
Calibration Spectra
Wavelength / nm
Sample Spectra
And finally a word of Caution..
N
N N
N
O
O
GI PAMPA PAMPA-BBBPa 10-7cm/sec Pa 10-7cm/sec
10 5
Caffeine
O
O
N O
O
N
GI PAMPA Caco-2Pa 10-7cm/sec Pa 10-7cm/sec
188 263
Verapamil
“high” PAMPA, but “low” Caco-2 due to efflux pump
N
N N
NH
O
O
GI PAMPA Caco-2Pa 10-7cm/sec Pa 10-7cm/sec
0 447
Theophylline
“low” PAMPA, but “high” Caco-2 due to active transport
“low” GI-PAMPA and “low” PAMPA-BBB but known to be CNS+ due to active transport across BBB
Acknowledgements
• PhysChem, Slough, UK– Richard Taylor– Christine Prosser– Emily Freeman
• PhysChem, Cambridge, UK– John Cooper– Benedicte Fau– Dave Sherwood
• PhysChem, Braine, Belgium– Luc Quere– Liliane Ellens– Geraldine Longfils
• DMPK, Slough, UK– Ted Parton– Lloyd King– Hanna Hailu– Mark Baker– Sarah Bartlett– Simon Carter– Judith van Asperen
Bit of a Cheek…….
• Physical Chemistry Symposium
• PerkinElmer, Seer Green, Buckinghamshire • Wednesday 30 November 2005.• No registration fee
– Register at: [email protected]• Bring a poster!!
• Who Should Attend?– Scientists who have been actively involved in
driving forward this area of the industry within their own institutions.