Solubility Screening Measurements at Organon Newhouse

1. Solubility Screening Measurements at Organon Newhouse Darren Edwards Organon Laboratories, Newhouse

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. SolKin assay • Our solution – the “SolKin” assay • Based upon shake-flask technique • HPLC analysis using conventional equipment • Developed and validated in-house

9. 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

10. 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

11. HPLC conditions Gradient program Typical Chromatogram 95 % Acetonitrile 5 1 2 3 4

12. 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.

13. 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………

14. Validation Work • Comparison of SolKin data with literature • Recovery work • Effects of DMSO on solubility • Effects of agitation time

15. Validation Work • Comparison of SolKin data with literature • Recovery work • Effects of DMSO on solubility • Effects of agitation time

16. 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

17. 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)

18. Selected Compounds haloperidol progesterone griseofulvin estradiol phenytoin testosterone Hydrocortisone-21-acetate spironolactone digoxin triamcinolone diazepam thiamylal dexamethasone lorazepam prednisolone

19. SolKin data versus Literature Is this comparison useful? How good is literature data? Highlights lack of published data

20. Validation Work • Comparison of SolKin data with literature • Recovery work • Effects of DMSO on solubility • Effects of agitation time

21. 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

22. Validation: - Recovery Results

23. 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

24. Validation Work • Comparison of SolKin data with literature • Recovery work • Effects of DMSO on solubility • Effects of agitation time

25. 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

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

27. Validation Work • Comparison of SolKin data with literature • Recovery work • Effects of DMSO on solubility • Effects of agitation time

28. 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.

29. 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?

30. Quality Control

31. 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

32. 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

33. 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?

34. 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

35. 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

36. Acknowledgements Organon Labs, Newhouse Wullie Arbuckle Yvonne Lamont Strathclyde University George Gettinby, Professor of Statistics