1	“Compound precipitation from DMSO and the synergy between water uptake and freeze / thaw cycles Christopher A. Lipinski Adjunct Senior Research Fellow Pfizer Global R&D Groton New London Labs Lipinskica@groton.pfizer.com
2	Freeze thaw cycles harm compound integrity Why? Chemical degradation Compound precipitation Why should cooling, freezing and modest warming cause a chemical change? Change to single freeze / thaw tube storage from plate based storage is correct But historical reasoning has been wrong
3	Freeze thaw literature precedent
4	New Matrical / Pfizer freeze thaw data
5	Agreement or disagreement? No disagreement Abbott freeze thaw cycles not a problem bone dry DMSO crystalline compounds studied Matrical / Pfizer freeze thaw cycles are a problem wet DMSO amorphous compounds studied
6	Fraction of Pfizer Groton compounds having melting point field information
7	Sonic / Acoustic effects on re-dissolution Expected effects with contact sonication excellent mixing efficient cell lysis speed up equilibration time minor effect - may induce precipitation Unexpected effects 50% dissolving of precipitated samples light scattering data rescue of expired HTS plates similar dissolving with contact less acoustics Theory of re dissolving is speculative
8	What goes wrong with freeze thaw cycles HTS master plate is frozen Primary hit in HTS screen Samples stored in frozen plates Plate is thawed Cherry pick “hits” for retest Water uptake by DMSO Plate is refrozen Cycle could be repeated many times How often nobody knows Registration system does not track freeze thaws
9	Synergy between water uptake and freeze thaw Super saturation in DMSO is high possible to see freeze thaw effect Super saturation in DMSO is low to moderate difficult to see freeze thaw effect Water in DMSO increases super saturation Water in DMSO greatly decreases solubility A little water in DMSO decreases compound solubility a whole lot
10	The symptoms to be concerned about Erratic HTS screening results that seem to bear little or no relationship to the biology and maybe even to the chemistry erratic rates of primary hits in HTS screens erratic re-confirm rates on primary HTS actives differences among screening sites differences among compound collections
11	Evidence for 35-40% poor aqueous solubility 70,000 compounds screened at Pfizer, Groton Capsugel Symposium June 2003 Tokyo solubility formulation, customer feedback Collaborator feedback to Matrical “Sonicman” HTS plate based sonicator
12	Dimension of the solubility problem 40% of compounds have poor aqueous solubility Half the problem due to size / lipophilicity Half the problem due to crystal packing DMSO no better than water for compounds insoluble due to crystal packing Suggests an upper limit of 20% compounds insoluble in dry DMSO at 10uM supported by NCI Frederick, MD studies Will this limit ever be achieved? Depends on whether a nucleation event occurs Depends on number of freeze thaw cycles
13	Compounds differ in aqueous and DMSO solubility based on crystalline form Ostwalds “rule of stages” Sequence of compound batch isolation proceeds towards thermodynamically most stable form 1 - amorphous - highest energy solid form 2 - highest energy crystalline polymorph 3 - lowest energy crystalline polymorph Amorphous is the highest energy form most soluble in water and DMSO
14	Early discovery compound purity Pressure on chemistry to increase output crystallization has disappeared Combinatorial compounds are now being purified by automated procedures 85-95% pure by ELSD or UV Compounds “appear” more soluble amorphous state impurities enhance solubility crystal growth retardation
15	Consequences of amorphous compounds Amorphous DMSO solubility is always higher than when compound is crystalline Amorphous compounds from combichem or medchem initially easily dissolve in DMSO allows preparation of DMSO stocks Sets stage for later precipitation problems
16	Thermodynamic aqueous solubility
17	Metastable supersaturated zone
18	Unstable supersaturated zone
19	Water and DMSO solubility Large, lipophilic compound aqueous insoluble DMSO greatly helps aqueous solubility Very crystalline compound may show no computational problem no “rule of 5” violation aqueous insoluble high melting point strong intermolecular crystal lattice DMSO does not help aqueous solubility
20	DMSO really helps aqueous solvation problems when the problem is size / lipophilicity Compound has to make a “hole” in DMSO to dissolve easier to do this in DMSO than in water no H-bond donor / acceptor networks to disrupt DMSO has a high dielectric constant solvates compound dipoles almost all drugs have dipoles
21	Sample in DMSO lifetime Compound disappears from DMSO solution What is the explanation? Chemical integrity in DMSO keep cold and frozen avoid oxygen keep dry Compound solubility in DMSO cold and / or frozen is the worst choice possible avoid freeze thaw cycles
22	DMSO – water phase diagram
23	DMSO and water, non-ideal behavior DMSO plus water exhibits very non ideal behavior change maximum at 33% by weight water corresponds to molar ratio 1 DMSO : 2 H₂O liquid phase is very structured ice-1 like lattice structure more H-bonding, more order than in pure water Melting point declines from 18 deg C to –73 deg C Viscosity increases as water is added to DMSO Solution gets colder as water is added to DMSO (J Org Chem 2001, 66, 5846-5852)
24	Viscosity and water addition to DMSO
25	Compound solubility in DMSO and H₂O As H₂O is added solvent is more structured More difficult to form a cavity More difficult to dissolve a large lipophilic compound Solubility non ideality is expected to be worst at 33% by weight H₂O
26	DMSO solubility dependence on H₂O
27	Material crystallizing from DMSO Parent compound Hydrate DMSO adduct
28	DMSO is a very good H-bond acceptor
29	DMSO adduct
30	Timing factor in compound DMSO solubility Once a compound crystallizes from DMSO it will not easily re-dissolve crystallized compound is in a lower energy, higher melting point, less DMSO soluble form Narrow working window (time window) for keeping most compounds dissolved in DMSO 1 to 2 days at room temperature explains why compounds are active when freshly made but not when stored Freeze thaw cycles increase the probability of crystallization
31	Summary Crystalline state is important to aqueous solubility Crystalline state is important to DMSO solubility be alert for compound precipitation from DMSO do not store liquid DMSO stocks in the refrigerator minimize time once DMSO stocks are diluted expect erratic HTS screening results dependent on minor compound handling differences minimize freeze thaw cycles Poor DMSO solubility is here to stay
32	Acknowledgements The generous support of Pfizer Global R&D, Groton Labs in my post retirement activities is gratefully acknowledged