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Solubility Screening Measurements at Organon Newhouse. Darren Edwards Organon Laboratories, Newhouse. Outline. Brief introduction to Organon Newhouse Requirements for a solubility screen How we screen for solubility – SolKin Validation of SolKin Description of what was done

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solubility screening measurements at organon newhouse
Solubility Screening Measurements at Organon Newhouse

Darren Edwards

Organon Laboratories, Newhouse

outline
Outline
  • 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
organon
Organon
  • 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 newhouse
Physico Chemical Analysis at Newhouse

Organon Scotland

Lead Finding Teams

Proof of Concept Teams

Venture Teams

Pre-clinical / CMC

Marketing &

Lead

Pre-clinical / CMC

Hit

Target

Registration

Development

Sales

Optimization

Development

Optimization

I

II

Clinical

Clinical

b

II

III

Development

Development

a

Exploratory

Full Development

Discovery

Development

and Launch

Research

Development

Physchem assays in support of HO and LO projects

physicochemical tests at newhouse
Physicochemical 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 newhouse1
Physicochemical 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 requirements
Solubility 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 assay
SolKin assay
  • Our solution – the “SolKin” assay
  • Based upon shake-flask technique
  • HPLC analysis using conventional equipment
  • Developed and validated in-house
schematic of solkin method

Add 6 µl DMSO stock to 300 µlbuffer (2% DMSO)

Mix 24 hoursin 96-well plate

10 mMDMSO

Filter (Millipore PCTE)

1:10 dilution with DMSO

1:20 dilution with DMSO

Inject 3 different volumes(4, 8, 12 µl) to give calibration curve

Schematic of SolKin method

Sample

Preparation

(in duplicate)

Standardpreparation

Inject two different volumes(5 and 50 µl) and quantify

All done in two 96-well platesOne for samples, one for standards

schematic hplc instrumentation
Schematic HPLC instrumentation

Agilent 1100 system

WATER + 0.1% Formic acid

VacuumDe-gasser

1 ml/min

Binary pump

Thermostatted

autosampler

VacuumDe-gasser

MeCN

+ 0.1% Formic acid

HPLC column, 5 cm Xterra C18, 3.5 µm

UV/vis PDA

Dionex Chromeleon and Excel

hplc conditions
HPLC conditions

Gradient program

Typical Chromatogram

95

% Acetonitrile

5

1

2

3

4

solkin method key points
SolKin 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 work
Validation 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 work1
Validation Work
  • Comparison of SolKin data with literature
  • Recovery work
  • Effects of DMSO on solubility
  • Effects of agitation time
validation work2
Validation Work
  • Comparison of SolKin data with literature
  • Recovery work
  • Effects of DMSO on solubility
  • Effects of agitation time
comparison with literature data
Comparison 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 selection
Compound 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 compounds
Selected Compounds

haloperidol

progesterone

griseofulvin

estradiol

phenytoin

testosterone

Hydrocortisone-21-acetate

spironolactone

digoxin

triamcinolone

diazepam

thiamylal

dexamethasone

lorazepam

prednisolone

solkin data versus literature
SolKin data versus Literature

Is this comparison useful?

How good is literature data?

Highlights lack of published data

validation work3
Validation Work
  • Comparison of SolKin data with literature
  • Recovery work
  • Effects of DMSO on solubility
  • Effects of agitation time
validation recovery
Validation - 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:

Methyl parabens

Salicylic acid

Caffeine

recoveries summary
Recoveries : Summary
  • 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
validation work4
Validation Work
  • Comparison of SolKin data with literature
  • Recovery work
  • Effects of DMSO on solubility
  • Effects of agitation time
dmso solubility
DMSO/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 solubility1
DMSO/solubility

2% DMSO chosen100 mg/l upper limit considered acceptableLimited dataset!

validation work5
Validation Work
  • Comparison of SolKin data with literature
  • Recovery work
  • Effects of DMSO on solubility
  • Effects of agitation time
agitation time
Agitation 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 time1
Agitation Time

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 control1
Quality 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*

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

*Minitab software

Special cause – 9 points below mean

issues identified from validation and qc work
Issues 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 work
Future 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
summary
Summary
  • 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
acknowledgements
Acknowledgements

Organon Labs, Newhouse

Wullie Arbuckle

Yvonne Lamont

Strathclyde University

George Gettinby, Professor of Statistics