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.