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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20

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

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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