Fast Monolithic Micellar Liquid Chromatography: An Alternative Drug

An Alternative Drug Permeability Assessing. Method for High-Throughput ... potential oral drug molecule is an important research issue, since this cha...
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Anal. Chem. 2004, 76, 7304-7309

Fast Monolithic Micellar Liquid Chromatography: An Alternative Drug Permeability Assessing Method for High-Throughput Screening A. Detroyer,† S. Stokbroekx,‡ H. Bohets,‡ W. Lorreyne,‡ P. Timmerman,‡ P. Verboven,‡ D. L. Massart,† and Y. Vander Heyden*,†

Department of Pharmaceutical and Biomedical Analysis, Pharmaceutical Institute, Vrije Universiteit BrusselsVUB, Laarbeeklaan 103, B-1090 Brussels, Belgium, and Johnson and Johnson Pharmaceutical Research and Development, a Division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, 2340 Beerse, Belgium

Several methods estimating the partitioning over biological membranes and thus the biological activity of potential oral drug molecules have been developed and are described in the literature. A previous study suggested that fast micellar liquid chromatography on a monolithic column could be one of them. For a set of diverse pharmaceuticals, retention by this fast chromatographic method was determined, besides other parameters also thought or established to describe oral permeability or absorption, e.g., from the Caco-2 permeability method. In view of a high-throughput determination of membrane permeability, a study was made of which information fast micellar liquid chromatography is providing and to what degree this system can replace other methods, i.e., deliver similar information. The retention with this fast method, which is mainly based on hydrophobic interactions, proved useful to sort substances into classes of Caco-2 and percent intestinal absorption. Estimating the partitioning over biological membranes of a potential oral drug molecule is an important research issue, since this characteristic influences its biological activity. Ignoring these absorption studies during drug discovery blocked drug development pipeline in the pharmaceutical industry;1,2 thus, in vitro models of intestinal membranes, e.g., the Caco-2 method, were introduced.3,4 They express various drug transport mechanisms, which makes them good predictive methods. However, they are laborious so less suited as a high-throughput screening (HTS) method in early discovery stages.5 HTS methods for permeability measurements usually just mimic the main transport mechanism, i.e., passive diffusion.5 They * To whom correspondence should be addressed. E-mail: [email protected]. Phone: (+32) 2 477 47 34. Fax: (+32) 2 477 47 35. † Vrije Universiteit BrusselsVUB. ‡ Janssen Pharmaceutica N.V. (1) Bohets, H.; Annaert, P.; Mannens, G.; Van Beijsterveldt, L.; Anciaux, K.; Verboven, P.; Meuldermans, W.; Lavrijsen, K. Curr. Top. Med. Chem. 2001, 1, 367-83. (2) Bleicher, K. H.; Bohm, H. J.; Muller, K.; Alanine, A. I. Nat. Rev. Drug Discovery 2003, 2, 369-378. (3) Artursson, P.; Karlsson, J. Biochem. Biophys. Res. Commun. 1991, 175, 880885. (4) Lenneras, H. J. Pharm. Pharmacol. 1997, 49, 7, 627-638. (5) Hidalgo, I. J. Curr. Top. Med. Chem. 2001, 1, 385-401.

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try to estimate the membrane partition coefficient of the drug, Km, representing the partition between water and the lipid phase of a membrane. Accordng to Fick’s law,6 Km is directly related to the diffusion of the compound since passing any biomembrane essentially means passing bimolecular phospholipid layers. A first method is to determine the log P of a substance as Km estimator. Originally, P is defined as the experimental partition coefficient of a substance between two nonmiscible phases (octanol/water) using the shake-flask method.7 Later on, estimations of classical RP-HPLC systems were made8-10 and calculation methods based on fractionation of the molecular structure were developed.11,12 However, log P only gives a rough approximation of Km due to the thermodynamic differences in partition principles between the octanol/water system, a bulk phase, and the biomembrane, an interfacial phase.13 Later on, highly automated methods, where membranelike structures are inserted, were developed. One such method is the parallel artificial membrane permeability assay. This robotized method developed by Kansy14 determines the diffusion of substances over phosphatidylcholine bilayers placed on a filter. Another option is to use micellar liquid chromatography (MLC). This type of RP-HPLC, described by Armstrong,15 uses a mobile phase in which a surfactant above its critical micellar concentration is present. The micelles formed and the monomers adsorbed to the stationary phase show a structural resemblance to biomembranes,16,17 and retention on given MLC systems was found directly related to log P values.18,19 (6) Flyn, G. L.; Yalkowski, S. H. J. Pharm. Sci. 1972, 63, 838-852. (7) OECD guideline to test chemical products 107, partition coefficient (noctanol/water), shake flask method; OECD, Paris, France, 1981. (8) Harnisch, M.; Mo ¨ckel, H. J.; Schulze, G. J. Chromatogr. 1983, 282, 315332. (9) Valko´, K. J. Liq. Chromatogr. 1984, 7, 1405-1424. (10) Nasal, A.; Siluk, D.; Kaliszan, R. Curr. Med. Chem., 2003, 10, 381-426. (11) Rekker, R. F. The hydrofobic fragmental constant; Pharmacochemistry Library; Elsevier: New York, 1977. (12) Hansch, C.; Leo, A. Exploring QSAR, Fundamentals and Applications in Chemistry and Biology; American Chemical Society: Washington, DC, 1995. (13) Woodrow, B. N.; Dorsey, J. G. Environ. Sci. Technol. 1997, 31, 28122820. (14) Kansy, M.; Senner, F.; Gubernator, K. J. Med. Chem. 1998, 41, 1007-1010. (15) Armstrong, D. W.; Nome, F. Anal. Chem. 1981, 53, 1662-1666. (16) Berthod, A.; Garcı´a-Alvarez-Coque, M. C. Micellar Liquid Chromatography; Marcel Dekker, New York, 2000. 10.1021/ac048944k CCC: $27.50

© 2004 American Chemical Society Published on Web 11/06/2004

In a previous paper,20 a very fast MLC system was developed on a monolithic column. It allowed the analysis of substances with various log P values, using only one mobile phase, in a time span of less than 10 min. Because of its good quantitative structureretention relationships with log P and its membrane characteristics, it was thought this method could be an alternative HTS membrane passage estimation technique for oral drugs. Since this remained to be evaluated, the retention characteristics of a number of substances were determined with fast monolithic MLC and compared to in vivo partitioning measured by both Caco-2 and oral intestinal absorption methods. Besides the predictive capability, differences in analysis time and labor intensity were considered for the different techniques. EXPERIMENTAL SECTION Substances. Acebutolol-HCl, alprenolol-HCl, atenolol, carbamazepine, chlorpheniramine maleate, clonidine-HCl, desipramineHCl, diphenhydramine-HCl, imipramine-HCl, labetalol-HCl, oxprenolol-HCl, pindolol, promazine-HCl, propranolol-HCl, ranitidineHCl, and trifluoperazine-HCl were obtained from Sigma (St. Louis, MO or Steinheim, Germany). Thioridazine and timolol maleate were from Merck (Darmstadt, Germany). Alniditan, astemizole, enilconazole, flubenazole, itraconazole, ketoconazole, levocabastine-HCl, mebenazole, terconazole, and radiolabeled 3H-mannitol, 14C-mannitol, 14C-alniditan, 3H-itraconazole, and 3H-levocabastine were supplied by Johnson & Johnson Pharmaceutical Research & Development, a division of Janssen Pharmaceutica N.V. (Beerse, Belgium). Chlorpromazine-HCl came from Fluka Chemie (Buchs, Switzerland). Cimetidine was a gift from Smith-Kline Beecham (Herts, U.K.) and esmolol-HCl from Du Pont De Nemours (Le Grand Saconnex, Switzerland). Metoprolol came from Ciba-Geigy (Groot-Bijgaarden, Belgium). Fast Monolithic MLC. The fast MLC measurements were based on a protocol described earlier.20 The chromatograph was composed of an isocratic L-6000 pump, an L-7400 spectrophotometric detector, and a D-7500 integrator, all Merck-Hitachi (Tokyo, Japan). A Chromolith Performance RP-18e analytical column (100 × 4.6 mm i.d.) from Merck was used. The flow rate was 7 mL/ min, the injection volume 20 µL, and the detection wavelength 225 nm. Stock solutions containing 100 µg/mL of the drugs were prepared in an ethanol/0.05 M sodium dodecyl sulfate (SDS) (99% purity, Merck) aqueous solution (10/90 v/v). The mobile phase (MP) consisted of SDS (0.1125 M)-1-propanol (10% v/v) in a 0.01 M sodium dihydrogen phosphate buffer (pH 7.4). After dissolving the SDS and before adding 1-propanol, the pH was adjusted to 7.4 with NaOH. The MP was filtered through a Super R-450 membrane of 0.45-µm pore size and 47-mm diameter (Gelman Sciences, Ann Arbor, MI) and degassed. The MLC retention factor k was calculated using the dead time measured from a potassium iodide (4.2 µg/mL aqueous solution) peak. Caco-2 Technique. For the Caco-2 permeability studies, the Caco-2 cells at passage 80 were seeded on 24-well cell culture (17) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; Wiley: New York, 1980. (18) Detroyer, A.; Vander Heyden, Y.; Carda-Broch, S.; Garcı´a-Alvarez-Coque, M. C.; Massart, D. L. J. Chromatogr., A 2001, 912, 211-221. (19) Garcı´a-Alvarez-Coque, M. C.; Torres Lapasio´, J. R. Trends Anal. Chem. 1999, 18, 8, 533-543. (20) Detroyer, A.; Vander Heyden, Y.; Reynaert, K.; Massart, D. L. Anal. Chem. 2004, 76, 1903-1908.

inserts (Millicell-PCF, 0.4-µm pore size, 12-mm i.d., surface 0.6 cm2 from Millipore (Billerica, MA)) at 63 000 cells/cm2. The cell culture medium consisted of Dulbecco’s modified Eagle medium supplemented with 5% fetal calf serum (FCS), 1% nonessential amino acids, 1% L-glutamine, and 100 units/mL penicillin/streptomycin (all Life Technologies, Paisley, Scotland). It was replaced 1 day after seeding and every 2-3 days thereafter. At the day of the experiments, i.e., 21 days postseeding, the culture medium was replaced with the transport medium (TM) () Hank’s balanced salt solution) (Life Technologies) containing 10% FCS, pH 7.4 at 37°C. The integrity of the cell monolayer in each insert was evaluated by the transepithelial electrical resistance, measured before the experiment and determined by leakage of 14C- or 3H-mannitol during the transport experiment. In addition, the Caco-2 cell batch was validated by measuring transport of 14C-alniditan and 3Hlevocabastine, which are control compounds for low and high absorption, respectively. Ethanolic solutions of the authentic radiolabeled substances were evaporated to dryness; the residues were reconstituted in dimethyl sulfoxide (DMSO) (all J.T. Baker, Deventer, Holland) containing an appropriate amount of the unlabeled substance to obtain 8 mM stock solutions. These stock solutions needed to be diluted 400-fold in TM to obtain a final concentration in the dosing solutions of 20 µM (radioactive concentration ∼10 kBq/mL). Stock solutions of all compounds under investigation (8 mM) were prepared in DMSO and diluted to a final concentration of 20 µM with TM. To initiate permeability experiments, the apical side of the monolayers received 0.4 mL of drug solution, the basolateral side 0.6 mL of TM. The experiments were executed in triplicate in an incubator at 37°C and an atmosphere of 5% CO2. The amount of solute permeated (apical to basolateral direction) was determined from samples (100 µL) taken at the apical side both at the time of initiation and termination (0 and 120 min, respectively). Moreover 100-µL samples were taken at the basolateral side 15, 60, and 120 min after dose administration. The removed volumes were replaced with fresh TM medium. The radiolabeled samples were analyzed by liquid scintillation counting with the Tri-Carb, 1900TR (PerkinElmer, Boston, MA). The samples of other compounds were analyzed using individual qualified LC-MS/MS methods on API 3000 and API 4000 (SciexApplied Biosystems, Foster City, CA). Sample preparation consisted of a semiautomated protein precipitation step. To obtain protein precipitation, 50 µL of DMSO and 250 µL of acetonile (J.T. Baker) were added to each of the 100-µL samples. After centrifugation, 150 µL of the supernatant was used for injection in the LC-MS/MS system. Each investigated substance required an optimization in the LC and MS/MS step to increase sensitivity. The different LC methods were however all performed on a monolithic column (Chromolith Speed rod C18, 50 × 4.6 mm i.d., Merck). All Caco-2 experiments were performed under “sink” conditions; i.e., the concentration of the solute in the receiver side was at all times less than 10% of the dose applied, thus minimizing the back-diffusion of the solute to the donor side. The apparent permeability coefficient, Pa caco-2 was calculated as

Pa caco-2 ) J/AC0 Analytical Chemistry, Vol. 76, No. 24, December 15, 2004

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where J is the rate of appearance of the drug in the receiver side (mg/s), C0 is the initial concentration of the solute (mg/L) at the donor side, and A is the surface area of the filter (cm2). The effective permeability Pe Caco-2 was determined as

Pe caco-2 )

Vd ∆% A ∆t

Table 1. Data Matrix Containing the Substances; Their Permeability Assessing Measurements: Pe Caco-2, log P, k from MLC, and Percent Intestinal Absorption no.

(2)

where Vd is the volume in the donor side (cm3), A the surface area of the filter (cm2), and ∆%/∆t the percent mass transported per time interval (s). The permeability coefficients were determined, at least in triplicate, and the mean and standard deviations calculated for evaluation. Log P. The log P values of the substances were calculated from their molecular structure by applying the freely available on-line interactive LOGKOW program21 of the Environmental Science Center of Syracuse Research Corp. (Syracuse, NY). The acid-base dissociation constants, pKa, were obtained with the ACD/pKa database 4.06 (1999) of the Advanced Chemistry Development Corp. (Toronto, Canada). RESULTS AND DISCUSSION Fast MLC: An Introduction. During previous investigations, the influential factors for an MLC system were optimized to fit the needs of a HTS permeability estimating method for oral drugs.18,20 The factors now mimic as much as possible the physiological circumstances, while the system is still practical and driven to its maximum speed by working on a monolithic column with a flow rate of 7 mL/min. The technique is easy to set up in any laboratory and can easily be automated. It is fast, needing only an elution time of at the most 10 min per substance. It allows determination of substances from a large log P range (0-6.5, Table 1) with one MP setting, which is impossible with classical RPHPLC. Because of the use of the amphiphilic SDS, samples do not show dissolution problems. Moreover, since it is a chromatographic method, any degradation or impurity will be detected making the use of pure samples unnecessary. Fast MLC: Relationship with Caco-2. To evaluate this fast MLC system as a permeability assessing method for oral drugs, its results had to be compared to the Caco-2 results for a diverse set of pharmaceutical substances from various pharmacological families (Table 1). To situate fast MLC among HTS permeability methods, log P results also had to be obtained. In analogy with most commercialized pharmaceuticals, the test substances were bases (high pKa) and their molecular weights were within a normal range for drugs (MM between 236 and 533). Nevertheless, the substances covered broad ranges in expected log P, permeability values and transport mechanisms. Once the different methods were applied, the resulting data matrix contained structural, retention, and activity parameters all thought to describe the membrane passage of the substances (Table 1). For Caco-2, two permeability parameters, both expressed in centimeters per second, could be calculated: an apparent permeability Pa at each time point and an effective permeability Pe determined from the permeability at all time points. As their results were closely related (results not shown), Pa was removed from further discussion. Because a low correlation (21) http://esc_plaza.syrres.com/interkow/kowdemo.htm.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

substance namea acebutolol-HCl C alprenolol-HCl C astemizole F atenolol C carbamazepine A chlorpheniramine maleate (() D chlorpromazine-HCl A cimetidine E clonidine-HCl B desipramine-HCl A diphenhydramine-HCl D enilconazole F esmolol-HCl C imipramine-HCl A ketoconazole F labetalol-HCl C mebenazole F metoprolol C oxprenolol-HCl C pindolol C promazine-HCl A propanolol-HCl C ranitidine-HCl E terconazole F thioridazine A timolol maleate C trifluoperazine-HCl A flubenazole F

Pe Caco-2, 10-6 cm/s

log P

MLC k

0.45 29.05 1.39 0.21 28.19 37.29

1.19 2.81 6.43 -0.03 2.25 3.82

2.59 12.06 53.65 1.33 1.56 7.29

9.32 0.91 30.28 17.41 31.72 27.11 3.00 29.72 21.53 12.86 24.96 27.48 28.28 22.91 28.53 25.64 0.84 18.81 5.76 15.45 4.05 19.47

5.20 0.57 1.89 4.80 3.11 4.10 2.00 5.01 4.45 2.41 2.71 1.69 1.83 1.48 4.56 2.60 0.29 3.48 6.45 1.75 5.11 2.91

18.00 1.06 3.78 17.68 11.29 10.65 4.47 15.09 4.02 7.47 1.56 4.68 7.25 2.39 13.18 9.92 1.39 6.41 19.06 4.82 22.24 1.86

int abs %b 89.5 93.0 9.0 50.0

85.0 95.0 100

95.0 95.0 95.0 90.0 90.0 90.0 50.0 90.0

a A, B, C, D, E, and F are different pharmacological families. b Data retrieved from ref 24.

coefficient between parameters does not imply no relationship is present, next, the results for each of the systems were plotted opposed to one another. From previous findings18,20 it appeared that the log k MLC and log P are usually linearly related, which was confirmed in this study (r ) 0.83). It is thus possible to estimate log P directly from the log k measured with fast MLC. Since for log P as well as for Caco-2 it is known that they are linked to membrane passage,22 good relationships were expected between both. From Figure 1a it appeared that there is indeed a relationship between log P and Pe Caco-2. However, it could not easily be modeled (r ) 0.10). The displayed results indicated more or less a yes/no situation for membrane passage, depending on the log P of the substances. A minimum cutoff value (log P ) 1.40) could be defined below which no Caco-2 passage was seen, followed by a log P interval at which Pe Caco-2 was high (log P ) 1.40-5.10), and finally a more gradual decrease in permeability with increasing log P (for log P > 5.10). These observations can be explained by the fact that highly water soluble compounds (low log P) will not be able to cross the lipid layer of the Caco-2 cells, unless massive active transport takes place. Compounds with a more lipophilic nature (intermediate log P of 1.40-5.10) will easily cross the lipid bilayer of the Caco-2 cells resulting in a high Pe. Compounds with a log P > 5.10 will tend to precipitate in the cell culture medium resulting in a low permeability. (22) Artursson, P.; Karlsson, J. Biochem. Biophys. Res. Commun. 1991, 175, 3, 880-885.

Table 2. Permeability Classification Limits for the Pe Caco-2, Percent Intestinal Absorption, log P, and log k MLC permeability

low

Pe Caco-2 (cm/s) % int absb permeability log MLC log kc

high

10-6

low

Pc

c

intermediate 1.46 × 50

a

10.00 × 89 high

1.40 0.15

10-6

intermediate 5.10 1.25

a Defined at Johnson & Johnson Pharmaceutical Research & Development, a division of Janssen Pharmaceutica N.V. b Defined by the FDA. Deduced from the cutoff values when comparing with the Caco-2 system.

Figure 1. Relationship plots: (a) Pe Caco-2 versus log P; (b) Pe Caco-2 versus log k MLC. The lines mark arbitrarily defined cutoffs.

Compared to the log k results determined with the fast MLC, Pe Caco-2 expressed a relationship similar to that with log P (Figure 1b). This was due, of course, to the close linear relationship between log k of fast MLC and log P. New cutoff values, now for retention in MLC, could be defined. Below log k ) 0.15, no Caco-2 passage was seen, between log k ) 0.15-1.25, permeability was high, and once log k > 1.25, Pe Caco-2 gradually decreased. Since both log P and retention on fast MLC systems allow setting cutoffs, they might be considered rough estimators of Caco2. Prediction Possibilities for Caco-2 with Fast MLC. Compared to MLC determinations and log P calculations, a wide variability in results can be seen for Caco-2. The Caco-2 reproducibility is low, and large interlaboratory changes can be observed as there is no uniform procedure described.23 Consequently, the numeric Caco-2 permeability values are often just used for classification. The substances are grouped in classes with low, middle, and high permeability according to classification limits defined within the investigating laboratory. For Caco-2, classification limits defined at Johnson & Johnson Pharmaceutical Research & Development, a division of Janssen Pharmaceutica N.V, are given (23) Ren, S.; Lien, E. J. Prog. Drug Res. 2000, 54, 1-23.

in Table 2. The Pe Caco-2 upper and lower limits were the measured values for alniditan (low) and levocabastine (high permeability). For MLC and log P, such limits were not defined. However, these systems seemed to allow defining cutoff values classifying the permeability similar to the Pe Caco-2 (Figure 1). Thus, we decided to introduce log P and log k MLC limits to predict the corresponding Caco-2 permeability class from log P and log k MLC measurements, respectively. Compared to Pe Caco-2, a log P or log k MLC below the first cutoff value in Figure 1 was considered to predict a low Caco-2 permeability. For the values between the cutoff limits, intermediate to high permeability, and beyond the second cutoff, intermediate to low permeability could be defined (Table 2). Each substance was classified into a permeability class based on its log P or MLC values and the limits set in Table 2. Compared to the classification based on the measured Pe Caco-2 values using log P, 26 out of 28 substances, and using MLC retention, 25 out of 28 were classified similarly (Table 3). The Caco-2 technique uses intestinal wall cells, and in theory, several transport mechanisms are involved simultaneously.2 However, it takes three weeks of care to grow the monolayer followed by a tedious sampling and the analysis of 18 samples per substance. The fast MLC and log P methods predict Pe Caco-2, in much shorter analysis times. Thus, these methods can be proposed as a HTS alternative for Caco-2. When using log P, it should be known that, to estimate log P values a “fragmental” calculation method is used.21 This in-silico method is very fast and accurate for simple molecules. However, the structure has to be known. It is also less accurate for complex molecules or molecules that have no close resemblance to any of these in the original reference data matrix.11 Although these HTS methods for membrane permeability estimation only mimic passive diffusion, we did not separately study the substances known to have different transport mechanisms. When new molecules are submitted to a HTS permeability method, their diffusion principles/mechanisms are not known. The objective is to classify as many substances correctly as possible, regardless of their transport mechanism. Prediction Possibilities for Oral Absorption with Pe Caco-2, log P, and Fast MLC. Percent intestinal absorption data were retrieved from the literature (Table 1).24 These figures are the result of the actual absorption process over the biomembranes of the intestines and the blood vessels; they are not to be confused (24) Wessel, M. D.; Jurs, P. C.; Tolan, J. W.; Muskal, S. M. J. Chem. Inf. Comput. Sci. 1998, 38, 726-735.

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Table 3. Classification of the Substances According to the Limits Set in Table 2a no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

substance name acebutolol-HCl C alprenolol-HCl C astemizole F atenolol C carbamazepine A chlorpheniramine maleate (() D chlorpromazine-HCl A cimetidine E clonidine-HCl B desipramine-HCl A diphenhydramine-HCl D enilconazole F esmolol-HCl C imipramine-HCl A ketoconazole F labetalol-HCl C mebenazole F metoprolol C oxprenolol-HCl C pindolol C promazine-HCl A propanolol-HCl C ranitidine-HCl E terconazole F thioridazine A timolol maleate C trifluoperazine-HCl A flubenazole F

Pe Caco-2, 10-6 cm/s

log P

MLC log k

int abs, %

L H L L H H

L H I L H H

H H I L H H

H H L I

I L H H H H I H H H H H H H H H L H I H I H

I L H H H H H H H H H H H H H H L H I H I H

I L H H H H H H H H H H H H H H L H I H I H

I H H

H H H H H H I H

a L, I, and H indicate substances belonging to the low, intermediate, or high permeability class, respectively.

with oral bioavailability data where a first-pass hepatic metabolism effect can be included. Viable alternatives are interesting since determining percent intestinal absorption is complex in nature and evidently not suitable in a drug discovery HTS environment. Because of the successful classification of Caco-2 permeability with the fast MLC method and the known use of the Caco-2 results to predict oral absorption,22 a good relationship of log k MLC with the intestinal absorption parameter was expected. The percent intestinal absorption is also used more as a quantitative measure, and classification limits for oral absorption were defined by the FDA25 (Table 2). Plotting the results of the intestinal absorption versus the other permeability assessing methods allowed evaluating their relationships (Figure 2). First we considered Pe Caco-2, which is the most frequently used alternative for oral absorption. From Figure 2a, it seemed that a high Caco-2 permeability indicates a high intestinal absorption. Taking the classification limits defined in Table 2 for Caco-2 and for percent intestinal absorption into consideration, it could be observed in Table 3 that only 4 of the 15 oral absorption results would be wrongly predicted from the Pe Caco-2 results (9 in Figure 2a). It should be emphasized that, in the HTS, the detection of the substances with high oral absorption is most important. With Caco-2, only 1 (acebutolol) of 11 substances was not identified as having a high intestinal permeability. A closer investigation of poorly predicted substances might provide a better understanding of the membrane transport (25) Waver of in vivo bioavailability and bioequivalence studies for immediaterelease solid oral dosage froms based on a biopharmaceutics classification system; FDA, CDER, Rockville, MD, 2001.

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Figure 2. Relationship plots: (a) percent intestinal absorption versus Caco-2 permeability, (b) percent intestinal absorption versus log P, and (c) percent intestinal absorption versus log k MLC. Substances with correctly (b) or wrongly (9) predicted percent intestinal absorption class when applying the limits of Table 2 are differentiated.

mechanism. However, such investigation is outside the scope of this paper. Since the Caco-2 technique is not suited for HTS, it was also not further investigated in our context. Considering log P and log k MLC, and taking into account the higher defined cutoff values, i.e., 1.40 and 5.10 for log P and 0.15 and 1.25 for log k MLC, allowed predicting the intestinal absorption for 10 of 15 substances correctly using log P and 11 of 15 using log k (Table 3, b and 9 in Figure 2b,c). Looking only at the high intestinal absorption, only one substance (acebutolol) with log P and none with MLC was misclassified. Predicting high oral absorption is thus not a problem while defining low and intermediate percent intestinal absorption might be, especially

because the intermediately classified substances always were rather borderline cases (Figure 2b,c). Since the number of substances with low percent intestinal absorption is small in the data set, it might be a little hasty to generalize the above conclusions. However, obtaining data of substances with low percent oral absorption is not evident since they are eliminated from the drug development pathway much earlier and usually no percent intestinal absorption measurements are performed on such substances. Still, the use of the log k from fast MLC on monolithic columns seems to be a possible HTS alternative for oral absorption prediction of (potential) drug molecules. CONCLUSIONS The earlier developed fast MLC method appears to give directly correlated results with log P. Modeling the results of the labor-intensive Caco-2 method does not seem feasible using the frequently applied linear modeling techniques. However, both

MLC and log P seem to allow predicting whether a substance will show Caco-2 permeability, making them alternative HTS methods. A comparison with real percent intestinal absorption values confirmed the previous findings. Although the number of molecules studied was limited, high intestinal absorption estimation is possible with MLC, log P, and Caco-2. In all, the use of fast MLC as a HTS possibility for absorption estimation was demonstrated. ACKNOWLEDGMENT Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

Received for review July 19, 2004. Accepted September 20, 2004. AC048944K

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