Solubility Screening Measurements at Organon Newhouse
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Transcript of Solubility Screening Measurements at Organon Newhouse
Solubility Screening Measurements at Organon
Newhouse
Solubility Screening Measurements at Organon
Newhouse
Darren Edwards
Organon Laboratories, Newhouse
OutlineOutline
Brief introduction to Organon Newhouse Requirements for a solubility screen How we screen for solubility – SolKin Validation of SolKin
Description of what was done Issues and problems
Quality control Future work Summary
OrganonOrganon
Founded 1923 in the Netherlands Part of AKZO Nobel Organon has approx 13,000 employees, 2000 in
R&D Opened site at Newhouse in Scotland in 1947 Over 300 scientists of all disciplines Responsible for Cardiovascular, CNS and
Analgesia research at Organon
Physico Chemical Analysis at NewhousePhysico Chemical Analysis at Newhouse
Lead Finding Teams Proof of Concept Teams Venture Teams
Research Development
DiscoveryExploratory
DevelopmentFull Development
and Launch
Target RegistrationLeadOptimization
Pre-clinical / CMCDevelopment
Pre-clinical / CMCDevelopment
Marketing & Sales
ClinicalDevelopment
ClinicalDevelopment
IIIa
IIbIII
HitOptimization
Physchem assays in support of HO and LO projects
Organon Scotland
Physicochemical tests at NewhousePhysicochemical tests at Newhouse
HO and LO Solubility
Screens (from DMSO) Solids (miniaturised shake-flask) Potentiometric (pSOL)
Lipophilicity HPLC Miniaturised shake-flask
pKa Sirius GLpKa
PAMPA Via NV Organon, Oss
Physicochemical tests at NewhousePhysicochemical tests at Newhouse
HO and LO Solubility
Screens (from DMSO) Solids (miniaturised shake-flask) Potentiometric (pSOL)
Lipophilicity HPLC Miniaturised shake-flask
pKa Sirius GLpKa
PAMPA Via NV Organon, Oss
Methodology
Validation
Quality control
Solubility screen - requirementsSolubility screen - requirements
Not high throughput Low 100’s of compounds prepared per week Screen all for solubility
Wanted to assay from 10 mM DMSO solution Automatically prepared for all compounds Maximum volume ca. 50 µl
Chemists wanted a solubility value, rather than a range
Sensitive (down to 1 mg/l) Reasonably accurate and precise Specific
SolKin assaySolKin assay
Our solution – the “SolKin” assay Based upon shake-flask technique HPLC analysis using conventional equipment Developed and validated in-house
Schematic of SolKin methodSchematic of SolKin method
0
100
200
300
400
500
600
700
0 0.2 0.4 0.6 0.8 1
Amount inj. on column (µg)
Pea
k ar
ea
Standardpreparation
SamplePreparation (in duplicate)
10 mMDMSO
1:10 dilution with DMSO
1:20 dilution with DMSO
Inject 3 different volumes(4, 8, 12 µl) to give calibration curve
Mix 24 hoursin 96-well plate
Add 6 µl DMSO stock to 300 µlbuffer (2% DMSO)
Filter (Millipore PCTE)
Inject two different volumes(5 and 50 µl) and quantify
All done in two 96-well platesOne for samples, one for standards
Schematic HPLC instrumentationSchematic HPLC instrumentation
VacuumDe-gasser
Binary pump
Thermostattedautosampler
UV/vis PDA
1 ml/min
WATER + 0.1% Formic
acid
MeCN + 0.1% Formic
acid
VacuumDe-gasser
HPLC column, 5 cm Xterra C18, 3.5 µm
Dionex Chromeleon and Excel
Agilent 1100 system
HPLC conditionsHPLC conditions
1 2 3 4
% A
ceto
nit
rile
95
5
Gradient program Typical Chromatogram
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00-100
200
400
600
800
1,000 SOLKIN 051005_LF 40 #138 [modified by lamonty] UV_VIS_1mAU
min
Abso
rban
ce [m
AU]
Retention Time [min]
2.693
WVL:230 nm
SolKin Method - Key PointsSolKin Method - Key Points
Based upon a shake-flask approach Uses standard HPLC equipment and a generic reversed-
phase gradient method with UV detection 230 nm usually used for quantitation
50 µl of 10 mM DMSO solution needed (<0.5 mg) 12 µl for samples 25 µl for standard
Specific (chromatographic) 2% DMSO in final solution
Upper limit of 100 mg/l for MW 500.
Validation WorkValidation Work
No guidelines for validating this sort of test Some very good examples in literature, but
procedures used generally variable This is what we did………
Validation WorkValidation Work
Comparison of SolKin data with literature Recovery work Effects of DMSO on solubility Effects of agitation time
Validation WorkValidation Work
Comparison of SolKin data with literature Recovery work Effects of DMSO on solubility Effects of agitation time
Comparison with Literature DataComparison with Literature Data
Compound Selection Literature solubility values variable
Often no conditions quoted (purity, test media, ionic strength, temperature)
Different amounts of DMSO used, different techniques gave different values
Considered that literature solubility values for neutral compounds would be more reliable no influence of pH upon solubility
Difficult to find poorly soluble (< 100 mg/l?) neutral, available drug compounds with literature values most drugs higher than this
Compound SelectionCompound Selection
15 marketed drugs (all except two effectively neutral) Structures and purity checked Only used compounds for which several literature sources agreed Values as measured from solid material – no DMSO
Triplicate measurements (3 different occasions,3 analysts) in phosphate buffered saline (PBS) pH 7.4 0.05 M (no consensus in literature as to which buffer strength to use –
0.001 M to 0.1M!) 0.15M NaCl used in buffer (again no consensus in literature, but most
common) Ambient temperature (measured as 21±1°C)
Selected CompoundsSelected CompoundsOH
H
H H
OH
O
Cl
O O
O
O
O
H
H
H
O
O
F
O
N
OH
Cl
OH
S
H
H O
O
O
OH
H
H
OH
NH
NH
O
O
NH
O
NH
S
O N
N
Cl
O
H
H
H
OH
OH
O
H
O
O
OH
O
OO
OH
OO
OH
OH
F
OH
H
H
O
OH
OH
O
OH
NH
N
Cl
O
OH
Cl O
F
H
OH
H
OH
O
OH
OH
HOH
H
OH
O
OH
estradiolprogesterone
griseofulvin
haloperidol
Hydrocortisone-21-acetate spironolactonetestosterone phenytoin
thiamylal diazepam digoxin triamcinolone
lorazepam dexamethasone prednisolone
O
OH
H
H H
OH
OO
O
SolKin data versus LiteratureSolKin data versus Literature
Is this comparison useful? How good is literature data? Highlights lack of published data
y = 0.728x + 2.4205
R2 = 0.8186
0
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30
40
50
60
70
80
90
100
0 20 40 60 80 100Literature solubility (mg/l)
So
lKin
so
lub
ility
(m
g/l)
Validation WorkValidation Work
Comparison of SolKin data with literature Recovery work Effects of DMSO on solubility Effects of agitation time
Validation - RecoveryValidation - Recovery
Question – Is a three point calibration curve produced by an autosampler sufficient to give the required accuracy?
Our solution – analyse aqueous solutions of three water soluble drug compounds prepared at a range of different concentrations
Check concentration found against prepared concentration (in duplicate)
Also, check concentrations after filtering Compounds used:
N
N N
N
O
O
O
OH
OH
OH
O
O
Caffeine Salicylic acid Methyl parabens
Validation: - Recovery ResultsValidation: - Recovery Results
Caffeine
70.0%
80.0%
90.0%
100.0%
17575251552.5Prepared concentration (mg/l)
Re
co
ve
ry
Unfiltered
Filtered
Salicylic acid
70.0%
80.0%
90.0%
100.0%
17575251552.5Prepared concentration (mg/l)
Re
co
ve
ry
Unfiltered
Filtered
Methyl parabens
70.0%
80.0%
90.0%
100.0%
17575251552.5Prepared concentration (mg/l)
Re
co
ve
ry
Unfiltered
Filtered
Limited experiment….. Generally recoveries ca. 90% Some reduction in recoveries at high concentrations (175 mg/l)
Probably due to large extrapolations from calibrated range Upper limit 100 mg/l (MW 500)
Methyl parabens shows evidence of membrane retention Filter plates chosen after discussions with manufacturer (Millipore) Interested to hear what plates other groups use….. Lipophilic compounds more of a problem?
Recoveries considered acceptable for solubility screen
Recoveries : SummaryRecoveries : Summary
Validation WorkValidation Work
Comparison of SolKin data with literature Recovery work Effects of DMSO on solubility Effects of agitation time
DMSO/solubilityDMSO/solubility
Initially developed test with 2% DMSO Common value in literature
Wanted to see how much difference 5% DMSO would make
11 compounds in duplicate
2% DMSO – upper limit for MW 500 = 100 mg/l
5% DMSO – upper limit for MW 500 = 250 mg/l
DMSO/solubilityDMSO/solubility
2% DMSO chosen100 mg/l upper limit considered acceptable
Limited dataset!
0
20
40
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80
100
120
140
160
180
200
dexa
met
haso
ne
diaze
pam
estra
diol
grise
ofulv
in
halope
ridol
Hc-21
-Ac
loraze
pam
prog
este
rone
spiro
nolac
tone
testo
stero
ne
triam
cinolo
ne
5% DMSO
2% DMSO
Literature
Validation WorkValidation Work
Comparison of SolKin data with literature Recovery work Effects of DMSO on solubility Effects of agitation time
Agitation TimeAgitation Time
Several differences in literature as to stirring time used
Anything between 1 and 48 hours Investigation made using agitation times of 1.5 and
24 hours 1.5 commonly used 24 longest practical stirring time for solubility screen
Duplicate samples, results averaged.
Agitation TimeAgitation Time
0
20
40
60
80
100
120
140
160
dexa
met
haso
ne
diaz
epam
digo
xin
estra
diol
grise
oful
vin
halo
perid
ol
Hc-21
-Ac
lora
zepa
m
phen
ytoin
pred
niso
lone
prog
este
rone
spiro
nola
ctone
test
oste
rone
thia
myla
l
triam
cinolo
ne
So
lub
ility
(m
g/l)
1.5 hours shaking
24 hours shaking
Results after 1.5 hours consistently higher than those after 24 hours agitation.
24 hours gives better correlation to literature.Chose 24 hours, but 1.5 hours OK for screen?
Quality ControlQuality Control
Quality ControlQuality Control
Samples run in duplicate. 48 per plate 2 standards (estradiol and
haloperidol) run in duplicate for every 22 (maximum) samples.
Estradiol – near lower limit of assay (solubility ca. 1.5 mg/l)
Haloperidol, pKa = 7.7, highlights effects of pH changes
Used to generate process control charts…….. Highlight the presence of “special
causes” – which show if the process is out of control
Quality Control – Process Control Diagram* Quality Control – Process Control Diagram*
Observation
Indiv
idual V
alu
e
403632282420161284
2.0
1.5
1.0
_X=1.388
UCL=2.056
LCL=0.720
Observation
Movin
g R
ange
403632282420161284
0.8
0.6
0.4
0.2
0.0
__MR=0.2512
UCL=0.8208
LCL=02
I-MR Chart of Mean Estradiol values (all data from July 2004)
*Minitab software
Using data to try and generate acceptance criteria for assay and give idea of variability Variation can be detected and data rejected if necessary. Work ongoing
Special cause – 9 points below mean
Issues identified from validation and QC workIssues identified from validation and QC work
Still learning and validation ongoing, several key issues…….. Lack of ‘high quality’ literature data
Often no indication of conditions Could we set up a database of solubility data (+ conditions?) on
marketed drugs via this forum? No consensus on minimum validation for this sort of test
Is it possible to use a forum such as this to come up with guidelines?
Quality control No guidelines What is acceptable variation? Forum – guidelines for this?
Future workFuture work
Investigating the use of very fast and multi-channel LC’s to decrease sample analysis times
Refining quality control procedures Use robotic workstation for sample preparation
Manually with multi-channel pipettes at present Mass Spectrometry as detection Other pH values
e.g. pH 6.5 to allow MAD analysis to be done (with PAMPA data from Organon NV, Oss)
Investigate other media e.g. Pharmacology in-vitro test solutions
SummarySummary
Described our solubility screening assay Modified shake-flask method Uses 10 mM dmso solutions Analysis uses standard HPLC equipment Medium throughput
Described our validation/quality control work Highlighted several issues that we found
developing/validating a solubility method
AcknowledgementsAcknowledgements
Organon Labs, Newhouse
Wullie Arbuckle
Yvonne Lamont
Strathclyde University
George Gettinby, Professor of Statistics