Anal. Chem. 2000, 72, 1781-1787
A High-Throughput Screening Method for the Determination of Aqueous Drug Solubility Using Laser Nephelometry in Microtiter Plates Christopher D. Bevan
Physical Sciences, GlaxoWellcome Research and Development, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY U.K. Richard S. Lloyd*
Pharmaceutical Sciences, GlaxoWellcome Research and Development, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY U.K.
High-throughput screening (HTS) is revolutionising the process of drug discovery. Drug activity screening has been successfully automated and is able to test very large numbers of compounds. It is only after leads have been identified from these screens that the physical properties of the compound (e.g., solubility, pKa and lipophilicity) are determined, due in part to the lower throughput of these methods. Solubility and pKa are among the most important features in selecting drugs that are to be well absorbed after oral dosage. The availability of physicochemical property screens is invaluable in order to direct the selection of the most promising potential drug compounds produced by combinatorial chemistry synthesis methods Insoluble drugs often prove difficult to get to market and repay their development costs. Developing low-solubility/low-bioavailability drugs is more time-consuming and expensive than for a compound with more suitable properties. Therefore, a HTS to determine solubility early in the discovery process is a most valuable tool. Traditionally, “equilibrium” solubility has been determined by shaking the compound with the solvent of choice for at least 24 h or until no more will dissolve, then filtering, and determining the concentration of dissolved compound by a suitable analytical method. This approach is inappropriate in a modern drug
discovery setting for a number of reasons. The weighing of hundreds of solid samples at submilligram quantities is no longer a viable proposition due to the time and manpower requirements. Samples are routinely supplied at 10 mM in dimethyl sulfoxide (DMSO) solution for activity screening; therefore, use of these same solutions for physicochemical property screening can save labor and time. Compound stability issues can often compromise the results of methods that require the solutions to stand for several hours. Finally, a method that is integrated within the activity screening laboratory has the attraction of being readily available to the screener to resolve problems in interpretation of suspect or anomalous screen results. We describe a method based on laser nephelometry that can overcome all these problems and utilize the DMSO solutions. The method is novel, rapid, and simple and can be integrated into a high-throughput biological screening process. Principle of Determining Solubility by Nephelometry. When visible light is passed through a suspension, part of the incident radiant energy is dissipated by absorption, reflection, and scattering while the remainder is transmitted. The measurement of the intensity of the scattered light (at right angles to the direction of the incident light) as a function of the concentration of the dispersed phase is the basis of nephelometric analysis.1 Nephelometric analysis can be employed to determine either the point at which a solute begins to precipitate out of a true solution to form a suspension or the concentration at which a suspension when diluted further becomes a solution. The scattered light will remain at a constant intensity until precipitation occurs, at which point it will increase sharply. For example, if a DMSO solution of a water-soluble compound is introduced into an aqueous buffer, the mixture will remain clear unless the aqueous solubility limit is reached, at which point precipitation will occur. Progressive incremental dilution of the suspension with aqueous buffer is then performed until the solute redissolves. Nephelometric scanning of the suspension to solution
* Corresponding author: (e-mail)
[email protected]; (fax) +44(0)1438 763352.
(1) Vogel, A. J. A TextBook of Quantitative Inorganic Analysis, 5th ed.; Longman: London, 1998.
The determination of aqueous solubility in a highthroughput screening environment is invaluable in the selection of the most promising potential drug candidates. We describe a fast method based on laser nephelometry that can determine the solubility of potential drug candidates (usually from combinatorial chemistry) supplied as dimethyl sulfoxide (DMSO) solutions in 96-well plates. In the sample, the percent DMSO is kept constant allowing direct comparison of results. The nephelometric method has been shown to produce results equivalent to those produced by an HPLC method and to be largely unaffected by colored solutions.
10.1021/ac9912247 CCC: $19.00 Published on Web 03/10/2000
© 2000 American Chemical Society
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In this study, we have attempted to keep the concentration of DMSO cosolvent constant at 5% so that solubility enhancement by a gradual increase in DMSO is avoided. Furthermore, the use of a 1-5% DMSO-enriched aqueous buffer is the concentration range adopted by activity screeners at GlaxoWellcome. Pharmaceutical companies typically employ DMSO in low percentage amounts as a cosolvent with water for preparing samples for screening.
Figure 1. Effect of plate type on signal intensity.
Figure 2. Effect of sample depth on forward-scattered signal
transition point can be used as a measure of the solubility of the solute by reference to the solute concentration at the transition point. Lipinski2,3 and Quartermain4 have published protocols for the determination of drug solubility by nephelometric and turbidimetric methods, but neither author claims quantitative performance at a constant percentage of DMSO cosolvent in static microtiter plate wells. Furer and Geiger5 adopted a stepwise dilution of a fine suspension approach using UV spectrophotometry to determine the concentration at which the turbidity just disappears. These authors produced some satisfactory results when comparing their test set results to those from the Pesticide Manual reference methods. Goodwin et al.6have recently published protocols for measuring solubility using flow cytometry. If the precipitated drug settles out, flow cytometry will not detect the precipitated drug in the flowing stream. Nephelometry has the advantage that it detects drug that has precipitated. Neither method is sensitive to purity/ stability/identity of compound whereas HPLC-UV and HPLCMS are. (2) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev. 1997, 23, 3-25. (3) Lipinski, C. A. IBC 6th Annual Conference on Rational Drug Design, 1997. (4) Quartermain, C. P.; Bonham, N. M.; Irwin, A. K. Eur. Pharm. Rev. 1998, 18 (4), 27-32. (5) Furer, R.; Geiger, M. Pestic. Sci. 1977, 8, 337-344. (6) Goodwin, J.; Merrill, S.; Asa, D. A New Rapid Technique for Sensitive Solubility Measurements: a Flow Cytometric Approach. A poster presented at the 5th Annual Society for Biomolecular Screening (SBS) Conference, Edinburgh, September,13-16, 1999.
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EXPERIMENTAL SECTION Instrumentation. The laser nephelometer used in this study is the NEPHELOstar (BMG LabTechnologies, Offenburg, Germany). This instrument is based around a laser-nephelometer employing a polarized helium-neon laser that lases in the red at 632.8 nm. The laser beam is passed through the well in a vertical and concentric path. If the laser beam is passed through an empty well or a clear liquid within the well, then the light beam is not scattered and passes through unchanged. In turbid suspensions, scattered light is detected by a photodetector at right angles to the incident laser beam. The NEPHELOstar will only measure the light that is “forward” scattered. The energy of the scattered light is directly proportional to the particle concentration in the suspension for up to 3 orders of magnitude. High concentrations of particles can exhibit a quenching effect on the scattered signal. Chemicals and Materials. 96-Well Microtiter Plates. Four types of 96-well microtiter plates were tested from the following suppliers: (1) White plate is an Isoplate, EG & G Wallac, Catalog No. 1450-515 (EG & G Wallac, Milton Keynes, U.K.). (2) Black plate is an Optiplate 2000, Porvair Sciences, Catalog No. 301002. (3) Clear plate is a Porvair Sciences, Catalog No. a9203 (Porvair Advanced Materials, Unit 6, Shepperton Business Park, Govett Lane, Shepperton, Middlesex,TW17 8RA, U.K.). (4) Nunc plates are Catalog No. 269620 (Nalge Nunc International Corp.). The U.K. distributor is Fisher Scientific UK (Bishop Meadow Rd., Loughborough, LE11 0RG, U.K.). Phosphate-Buffered Saline (PBS) Concentrate. Sodium phosphate (0.1 M)/9% sodium chloride, pH 6.9, Fluka Catalog No. 79383, is diluted 1:10 with water to give a PBS buffer at a pH of 7.4 (Fluka, The Old Brickyard-New Road, Gillingham, Dorset, SP8 4JL U.K.). Ludox Silica Suspension. A 34% suspension of silica in water, pH ∼3.6, surface area 110-150 m2/g, containing aluminum stabilizing counterion, Catalog No. 42,085-9 (Aldrich Chemical Co, Inc., Milwaukee, WI 53233). This stock suspension was diluted arbitrarily with water to make a suspension at a convenient concentration to measure on the nephelometer. An aliquot (10 mL) of this solution (in duplicate) was evaporated to dryness under vacuum and the weight of silica per 10 mL determined. The silica suspension used was found to contain 0.85 mg silica/mL. Ondansetron Hydrochloride. This is dissolved in DMSO to give a 5 mg/mL solution and is a GlaxoWellcome compound obtained from the Reference Substance Unit, GlaxoWellcome Operations U.K. Ltd. (Barnard Castle, County Durham, DL12 8DT, U.K.). NEPHELOstar Instrument Settings. All the sample wells were scanned with an integration time of 0.1 s, so that a plate (96 samples) could be scanned in ∼68 s.
Figure 3. Effect of color on signal in clear aqueous solutions and Ludox suspensions
Figure 4. Determination of LOD for silica.
A gain of 30 was set and used throughout to allow direct comparison of all results. Optical Quality of Plates. This type of nephelometry relies on the forward scattering of laser light (referred to as “the signal” in this paper). Imperfections on the bases of the plate wells such as scratches, foreign matter, or fingerprints that could scatter light produce a false positive signal. A Porvair Sciences clear plate was scanned and cleaned with a blast from a Tech-spray (tetrafluoroethane) to remove dust and rescanned. To compare the nephelometric signal, three commonly used plates were scanned empty and then scanned containing a series of 10 “double dilutions” of the silica suspension. All wells had a final volume of 200 µL. Effect of Changing Sample Volume. As the depth of liquid the light has to pass through increases, one might expect an increase in forward-scattered light as well as absorption of the incident laser light. To investigate the effect of path length on the forward-scattered light, a series of 96-well plates were prepared. Each plate contained 96 wells filled with different volumes of colloidal silica suspension. All 96 wells were scanned with the nephelometer. Volumes used were 100, 200, 300, 350, and 400 µL of colloidal silica aqueous suspension. When the well was filled with 400 µL, a meniscus above the well was evident. Effect of Solution Color on Signal. The color of solutions assayed may affect the signal by absorption of the incident red laser light beam at 632.8 nm.
To investigate this, rows of 12 wells were prepared containing colored water: Row A contained 100 µL of water plus 100 µL of red Whatman buffer (Catalog No. 6602 3184). Row B contained 100 µL of water plus 100 µL of blue Whatman buffer (Catalog No. 6602 3190). Row C contained 100 µL of water plus 100 µL of yellow Whatman buffer (Catalog No. 6602 3187). Row D contained 200 µL of water. The plate was scanned nephelometrically. To investigate whether color affects the scattered signal from a silica suspension (a model insoluble compound), rows of 12 wells were prepared containing colored silica suspension: Row A contained 100 mL of silica suspension plus 100 µL of red Whatman buffer (Catalog No. 6602 3184). Row B contained 100 µL of silica suspension plus 100 µL of blue Whatman buffer (Catalog No. 6602 3190). Row C contained 100 µL of silica suspension plus 100 µL of yellow Whatman buffer (Catalog No. 6602 3187). Row D contained 100 µL of silica suspension plus 100 µL of water. The plate was scanned nephelometrically. To quantitate the absorption of each solution at the laser wavelength, the red, blue, and yellow solutions were diluted 1:1 with water and scanned from 400 to 800 nm in a Varian, Carey 500, UV-visible-NIR spectrophotometer. Determination of Limit of Detection (LOD). The silica suspension was used as a model insoluble compound to determine the LOD of this method. Eight silica suspension samples were serially diluted to prepare 10 double dilutions and then scanned nephelometrically. Ondansetron HCl was selected as a stable, high-purity drug compound. Its solubility lies in the range of interest. Eight ondansetron samples were serially diluted to prepare 10 double dilutions and then scanned nephelometrically. The solubility of ondansetron was measured nephelometrically and the result compared with that determined by comparator methods based on quantitative HPLC.7 A range of concentrations of ondansetron HCl was prepared by serial dilution of a stock solution with 5% DMSO/PBS buffer. A series of 10 double dilutions was prepared and scanned by nephelometry. (7) Precipitation from DMSO method: Drug solubility determination is based on the dilution of a sample supplied as a DMSO solution (10 mM) diluted with buffer. The measurement of concentration after filtration is achieved by comparison of the UV absorbance of the saturated solution with that of a known standard following an HPLC separation. The standard solution is prepared by dissolving a known weight of compound in a suitable solvent such as methanol or DMSO.
Analytical Chemistry, Vol. 72, No. 8, April 15, 2000
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Table 1. Calculation of Student t-Test on Results from a Silica LOD Experiment row
silica concn (as µg/mL)
A
B
C
D
E
F
G
H
850 425 213 106 53 27 13 7 3 2 1 0
409 271 143 88 62 49 40 35 35 36 56 31
422 291 146 91 94 65 46 40 38 35 35 33
427 285 158 90 64 89 47 35 39 36 37 33
425 292 148 98 68 48 47 40 38 34 36 35
431 289 147 96 72 49 46 40 38 35 37 37
429 288 151 96 69 51 42 38 42 35 35 39
434 298 152 96 71 51 46 41 69 35 34 35
431 358 147 95 66 55 42 40 48 38 40 35
one-sided p value
verdict
0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0558 0.4473 0.0460 0.6121 0.2155
signif at 1% signif at 1% signif at 1% signif at 1% signif at 1% signif at 1% not signif not signif not signif not signif not signif
Table 2. Calculation of Student t-Test on Results from Ondansetron LOD Experiment row
silica concn (as µg/mL)
A
B
C
D
E
F
G
H
250 125 63 31 16 8 4 2 1 0.5 0.2 0
869 122 85 43 39 36 46 36 36 92 36 33
972 184 101 44 55 39 39 37 37 39 41 37
396 106 40 41 44 36 40 39 39 45 38 39
316 94 70 47 39 36 90 37 37 43 46 36
640 130 82 43 38 43 40 38 38 39 38 42
1082 104 77 46 50 43 49 42 40 40 40 40
377 155 118 51 39 43 43 178 42 41 38 nda
2090 207 83 46 43 45 42 40 40 43 41 39
a
one-sided p value
verdict
0.0000 0.0000 0.0000 0.2834 0.4242 0.6997 0.1617 0.1257 0.7004 0.1185 0.4260
signif at 1% signif at 1% signif at 1% not signif not signif not signif not signif not signif not signif not signif not signif
Not determined.
Figure 5. Determination of solubility of ondansetron in 5% DMSO/ PBS
Effect of Standing, Shaking, and Sonication. The effect of physically shaking the plate on the stability of the readings was tested to establish whether plate shaking is a necessary prerequisite to scanning the plate. A plate of samples of ondansetron precipitates was allowed to stand for 20 h and then repeat scanned after a 20-s, 3-mm linear shaking period within the nephelometer. No results were culled from this data set because we did not want to mask any effects that could have been caused by shaking the plate. The effect on the solubility determination of ultrasonicating (which is often employed to disperse particles in suspension) the plates while the ondansetron is being precipitated and after it has 1784 Analytical Chemistry, Vol. 72, No. 8, April 15, 2000
Figure 6. Effect of standing and sonication on signal.
precipitated was also investigated. The previous experiment was repeated and the plate scanned nephelometrically. Then the plate was immersed in an ultrasonication bath for 10 min and rescanned. The plate was allowed to stand for 3 h and scanned again. The plate was then immersed in an ultrasonication bath for 10 min and scanned again. Validation of the Nephelometric Method against HPLC. A set of compounds was selected to validate the reference HPLC solubility method from DMSO solution (unpublished). This set
Table 3. Solubility of Validation Set As Determined by the “from Solid”, “from DMSO Solution”, and “Nephelometric” Methods solubility (µg/mL) no.
from solid by HPLC
from DMSO soln by HPLC
from nephelometry
commenta x x x x x x x x x red soln, insoluble by eye x x x x x x x with DMSO soln by HPLC result x
1 2 3 4 5 6 7 8 9 10
acetazolamide allopurinol bendroflumethiazide benzocaine benzthiazide betamethasone butamben butylparaben chlorpropamide clofazimine
>500 337 12 >500 11 63 125 139 >500 111 >68 33 >83 20 >196 85 88 >138 30
>111 >68 13 >83 27 >196 100 97 >138 1
11 12 13 14 15 16 17
flurbiprofen gemfibrozil hydrocortisone hydroflumethiazide iodipamide nitrofurazone oxyphenbutazone
>500 >500 258 187 >500 163 >500
>122 >125 175 >166 >57 >99 122 >125 >181 >166 57 >99 40
18 19 20 21 22 23 24 25 26 27 28 29
phenacemide phenylbutazone prednisone propylparaben tolazamide trimethoprim tyrosine hydroquinine morin hydrate phenyl salicylate theobromine 2-hydroxy-3-isopropyl-6methylbenzoic acid progesterone
182 320 98 273 350 >500 no result >500 192 16 >500 >500
>89 >154 >179 89 >174 >145 151 90 >97
>89 77 89 >89 90 145 91 120 ndb >107 90 >97
6
15
20
30 a
compound
x x x x x red soln x x x
x indicates acceptable agreement.
of compounds also had solubility determined from the original solids by HPLC.8 A series of eight replicate double dilutions was prepared so that each compound occupied the whole of one 96well plate. The plates were scanned nephelometrically. The solubility has been assigned as the point at which the signal increases significantly above that established for a clear solution. However, many members of this selection of compounds are soluble and we wished to test the method further with real research compounds that are less soluble. A set of six compounds (A-F) was selected from a development project that had a history of insoluble compounds. This set of compounds had solubility determined directly from the solids and from DMSO solution by HPLC comparator reference methods. A series of double dilutions was prepared for each compound and nephelometry performed. The solubility has been assigned as the point at which the signal increases significantly above that established for a clear solution. Reproducibilty from Site to Site. At a different GlaxoWellcome site, five plates containing eight serial double dilutions (8) Dissolution from the solid method: A saturated solution is prepared from the solid (up to 2.0 mg/mL), and its concentration after filtration is measured by comparison with a standard of known concentration. The measurement of concentration is achieved by comparison of the UV absorbance of the saturated solution and that of a known standard following an HPLC separation. The standard solution is prepared by dissolving a known weight of compound in a suitable solvent such as methanol or DMSO.
Table 4. Solubility of Sparingly Soluble Development Compounds by “from Solids”, “from DMSO Solution”, and “Nephelometric” Methods solubilitya (µg/mL) compd A B C D E F
from solid
from DMSO soln by HPLC
from nephelometry
