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Capillary electrophoresis technologies for screening in drug discovery
Drug Discovery Today: Technologies
Vol. 2, No. 2 2005
Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Screening technology
Capillary electrophoresis technologies for screening in drug discovery Hong Wan*, Richard A. Thompson DMPK & Bioanalytical Chemistry, AstraZeneca R&D Mo¨lndal, S-43183 Mo¨lndal, Sweden
The high separation efficiency of capillary electrophoresis (CE), combined with the high selectivity and sensitivity of mass spectrometry (MS) detection offers the potential of unique resolving power and high-throughput capacity to the analysis and structural identification of complex mixtures. Recent advances in CE-MS interfaces and commercially available 96-capillary instruments have made the implementation of routine CE methods for drug screening feasible.
Introduction Capillary electrophoresis (CE) is a miniaturized and high efficiency separation technique with many application areas in the pharmaceutical community. Recent advances in interfacing CE to MS have significantly increased the potential of this technique [1–3]. The technique has shown great success in high-throughput pKa screening in particular. However, other areas of physicochemical and biological screening, such as lipophilicity (log P) determination by microemulsion electrokinetic chromatography (MEEKC), drug–protein binding measurement and ligand–receptor interaction by high performance frontal analysis-capillary electrophoresis (HPFACE) and affinity capillary electrophoresis (ACE) are of increased interest. CE technologies have particular advantages in chiral drug screening and applications. Finally, CE in combination with microchip technologies shows great potential in drug screening.
*Corresponding author: H. Wan ([email protected]) 1740-6749/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2005.05.010
Section Editors: Jeff Brockman – Psychiatric Genomics Inc., Gaithersburg, MD, USA Mark Divers – AstraZeneca, Mo¨lndal, Sweden High-throughput screening pKa by CE and CE-MS Several applications of pKa determination by CE have been reported, and the state of the art of this technique has recently been summarized [4–6]. A 96-capillary format instrument has become commercially available from CombiSep (http://www.combisep.com/flashindex.html), which is suitable for high-throughput pKa screening as well as other applications [6]. A schematic work-flow of pKa screening is depicted in Fig. 1. In our opinion, the main drawback of UV absorbance detection is its relatively low sensitivity. Typically, it requires a minimal compound concentration of 20 mM and therefore has limitations in measuring the poorly soluble compounds often encountered in drug discovery. Although UV sensitivity can be improved by on-line sample stacking [7], an optimal injection has to be applied. Alternatively, poorly soluble compounds can be measured by titration method in a cosolvent buffer system followed by Yasuda–Shedlovsky extrapolation [8]. However, this requires a series of methanol containing solutions and is therefore time-consuming and can lead to inaccurate data. As a consequence of the above limitations, we have developed a highly selective and sensitive CE-MS assay for pKa screening [9]. Up to now, more than 500 new chemical entities from different classes and lead series have been successfully screened including many poorly soluble or sparingly soluble compounds (aqueous solubility <1 mM). Typically, poorly soluble compounds can be dissolved in 50% acetonitrile (ACN) or methanol and directly measured in nonsolvent www.drugdiscoverytoday.com
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Figure 1. Schematic work flow of pKa screening by CE-UV and CE-MS. meff is the effective mobility of the compound; positive value indicating positive charge, whereas negative value indicating negative charge; N is a neutral marker (DMSO); 1 and 2 are basic and acidic compounds separated from the neutral marker; pressure reduces the migration times dramatically without affecting pKa measurement. Compared with UV detection, CE-MS pKa-screening method offers a higher throughput capacity based on sample pooling, and it is more suitable for poorly and sparingly soluble compounds. In addition, the compounds can be measured simultaneously with several reference standards, ensuring more accurate pKa data.
buffers. So far, the only main drawback of the CE pKa assay that we have experienced is the poor electrospray ionization (ESI) signal associated with lipophilic compounds in some instances. However, this can be improved by using either higher compound concentrations or atmospheric pressure photoionization (APPI) or more sensitive mass spectrometers. Recently, our pKa assay has been extended to the application of 14 volatile background electrolyte (BGE) buffers, and a fully automated screening assay including rapid data evaluation has been set up. Because many compounds can be pooled and measured simultaneously together with several reference standards, this technique not only provides high throughput but also ensures high quality data with accuracy and reproducibility greater than 0.2 pKa units. In addition, as shown in Fig. 1, the effective mobility versus pH can provide the charge distribution and ionization profile of a compound in a wide pH range, thus facilitating better understanding of other physicochemical properties such as pH-dependent lipophilicity, transport mechanisms and protein binding, among others.
Determination of lipophilicity log P by MEEKC Microemulsion electrokinetic chromatography is an electrodriven separation technique. Separations are typically 172
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achieved by partitioning between water and oil-in-water microemulsions consisting of surfactant-coated nanometersized droplets of oil suspended in aqueous buffer. A cosurfactant, such as a short-chain alcohol is generally employed to stabilize the microemulsion. This novel technique with various microemulsion systems has been extensively investigated and recently reviewed [10–13]. Basically, by using a series of standard literature log Po/w values (octanol–water partition coefficient), log P can be indirectly calculated by a linear relationship between separation capacity k0 and log Po/w (log P = A log k0 + B). As the microemulsion separation is rather similar to octanol–water partitioning, the retention of MEEKC (k0 ) is shown to correlate highly with the log Po/w achieved by the conventional shake flask method, and correlates better than log P obtained by HPLC [13]. Several groups have demonstrated that both MEEKC (SDS based) and VEKC (CTAB-SDS vesicles or AOT vesicles) systems (abbreviations in Table 1) are suitable for rapid, highthroughput determination of log P for neutral, weakly acidic and weakly basic compounds with a wide range of log P [14– 17]. Validation and a long-term assessment of MEEKC for high-throughput determination of log P has been conducted on a 96-capillary instrument [17], showing acceptable accura-
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Table 1. Specific CE technologies used for drug screening Screen assay
pKa
Name of specific CE-MS technologies CE-UV
Lipophilicity (log P)
Protein binding (fu%) drug–protein interaction
Chiral separation
Microchip-based screening assays
MEEKC
ACE HPFA-CE
Chiral CE Chiral MCE
CE-UV CE-LIF CE-MS
Chiral impurity or ee determination by ratio of two enantiomer peak areas
Depending on specific assay, quantitative peak area or other parameters
VEKC
Effective mobility by migration time relative to a neutral marker
Capacity factor k0 by migration time relative to a neutral marker and microemulsion
Binding constant Ka by migration time shift (ACE) Unbound free fraction (fu%) by ratio of standard and analyte peak heights (HPFA-CE)
Pros
High throughput (sample pooling) by CE-MS or capillary array by UV High sensitivity (MS) Insensitive to impurities Minute sample required Ease of automation
Throughput comparable to or higher than HPLC and shake flask Assay not limited by solubility Suitable for a wide range of log P from 1 to 6.6 Applicable for full pH range
One to two orders of magnitude Simple and rapid chiral resolution and method development smaller sample size than ED Lower cost than PHLC High-throughput screening Possibility of reversing EMO as by MS detection compared to chiral HPLC Simpler and faster than ED Potential for in vivo protein binding measurement
Small sample size Possibility for direct analysis of complex matrices Ability to work with complex assay designs Ability to work in parallel
Cons
Poor signals of ESI in a few cases Requires neutral samples (could be improved by APPI interface) Less suitable for log D (7.4) determination (but can be Low sensitivity (UV), minimal indirectly calculated compound concentration 20 mM using log P and pKa)
Low sensitivity with UV Protein adsorption to capillary inner wall (could be reduced) Requires further validation of applications (HPFA-CE-MS)
Unsuitable for chiral preparative scales Chiral selector could interfere with MS detection
Separation channels necessary for many assays Interface to MS is under development Issues with binding to surfaces
Refs
[4–7,9]
[20–30]
[2,31–41]
[42–46]
[10–17]
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MEEKC: microemulsion electrokinetic chromatography; HPFA: high performance frontal analysis; ACE: affinity capillary electrophoresis; MCE: microchip electrophoresis; ee: enantiomeric excess; LIF: laser induced fluorescence; ED: equilibrium dialysis; VEKC: vesicle electrokinetic chromatography (CTAB-SOS visicle, AOT vesicle); CTAB: cetyltrimethylammonium bromide; SOS: sodium octyl sulfate; AOT: bis(2-ethylhexyl) sodium sulfosuccinate.
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Parameter measured
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cies (0.5 log P unit), particularly when considering the variations of reported values in the literature. Obviously, MEEKC can offer many advantages compared with HPLC and the shake flask method. MEEKC can be used in the full pH range suitable for a diversity of structures, can be automated, requires only small amounts of samples and short analysis times. Another distinct advantage of the MEEKC technique is that the separation system is not limited by solubility because poorly soluble compounds can be dissolved in cosolvent or micelles-mediated buffer, ensuring good signals in almost all cases. Most MEEKC applications are performed with UV detection. To the best of our knowledge, no MS detection has been used in a MEEKC system for log P determination. In principle, similar to CE-MS pKa screening, the log P values of many compounds can be simultaneously measured in such a high separation efficient microemulsion system if combined with a highly selective MS detection. More accurate data should be obtained by MS detection than by UV detection because on-line calibration is possible with MS detection. It can be expected that the high micellar concentration (SDS) present in the BGE could result in ion suppression, which can be a major limitation when using ESI-MS detection. However, the sensitivity of MS, if the signals are suppressed, can be compensated by using higher compound concentrations. Alternatively, the application of APPI-MS can dramatically improve MS sensitivity in nonvolatile buffer system such as that used in MEEKC [18,19].
Drug–protein interaction by affinity capillary electrophoresis-mass spectrometry (ACE-MS) and by high performance frontal analysis-capillary electrophoresis (HPFA-CE) Recent advances in ACE-MS is allowing rapid and selective identification of both synthetic and biological molecules and screening combinatorial libraries for drug leads or candidate ligands. Affinity capillary electrophoresis was initially employed for the determination of the binding constants of small molecules to proteins, and this technique has been extended for the examination of ligand–receptor affinities or interactions between drugs and various biomolecules. Several comprehensive reviews of this technique have been published [20,21]. The advantage of ACE-MS technique was exemplified as an on-line approach for the selection and identification of active ligands from a combinatorial mixture, for example screening and determining interacting structural moieties of D-tri and tetrapeptide libraries containing up to 361 compounds for ligands of vancomycin [22]. The potential of screening large libraries of up to 1000 peptides was also demonstrated directly by ACE-MS incorporating an affinity solid-phase extraction step before ACE-MS [22]. The library components that showed the strongest affinity were identified. The principle of ACE is to measure the electrophoretic mobility of a ligand as a function of receptor concentrations 174
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in the BGE. Consequently, the dissociation constant Kd can be obtained. Alternatively, the binding constant can also be measured directly by so-called bioaffinity ESI-MS [23]. However, bioaffinity ESI-MS requires quantitative measurements of the concentrations or responses of free ligand, free solute and their complex, respectively. Although the latter approach is even more rapid and straightforward and with higher capacity, it suffers from difficulties in obtaining accurate concentration determinations for complexed forms because of low ionization efficiencies of the complexes resulting from ion suppression. Currently, it seems that ACE rather than the bioaffinity approach is more suitable for rapid ranking and estimation of the binding of drugs to proteins, and in particular, identification of the selective binding of drugs to specific proteins as a high-throughput assay. In principle, several drugs belonging to the same or different series of new chemical entities can be simultaneously screened on the basis of 1:1 isotherm model. Lewis et al. [24] applied ACE for the screening of novel antimicrobial targets from a small molecular library of 44,000 compounds that possessed drug-like properties and antimicrobial activity against drug-resistant clinical isolates and concluded that ACE is a valuable tool for the fast, efficient detection of specific binding molecules that possess biological activity. It can be expected that ACE coupled with MS will provide a promising method for screening. The drug–protein binding can also be evaluated by another new technique, high performance frontal analysis and capillary electrophoresis (HPFA-CE), by quantitatively measuring the peak height ratio of a standard drug concentration to the drug resolved from the drug–protein complex. The principles of this technique and applications are found in a recent review [25]. The unique feature of this technique is that a large volume of sample is injected onto the capillary to generate a plateau, which linearly corresponds to the drug concentration. The advantages of HPFA-CE are that only small sample volumes of protein and drug are required, and it offers the possibility of rapid protein binding measurements during equilibrium without disturbing the binding conditions. Moreover, the measurements are typically done in one step at near-physiological conditions. This approach turns out to be much less labor-intensive than conventional equilibrium and ultrafiltration method where follow-up separation steps, such as HPLC are usually required. HPFACE is suitable for measurements of diverse structures including basic, neutral and acidic compounds with a wide range of binding constants from low to high binding affinities. Ishihama et al. [26] determined the binding extents of six anionic drugs to human serum albumin (HSA) solution-based combinatorial libraries and found that the measured bound fractions ranging from 78.7 to 99.9% were consistent with the results obtained by ultrafiltration. Østergaard et al. [27] employed HPFA-CE for the determination of the binding
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sites of drugs binding to HSA and applied this to drug displacement studies. Jia et al. [28] reported a pressureassisted HPFA-CE approach for rapid determination of binding constants of 17 drugs to HSA and of 4 drugs to AGP. In a similar way, Martinez-Pla et al. [29] used a short-end injection (back vacuum) for rapid evaluation of drug binding to HSA and human plasma. With analysis times shorter than 3 min for each injection, the measured binding constants are still in good accordance with much longer migration times, indicating that the HPFA-CE technique is applicable to high-throughput screening of drug–protein binding [29]. Reproducibility of measurements for this method is typically less than 10% coefficient of variation [28,29], which is comparable with the conventional equilibrium technique. One major limitation of the technique is its low sensitivity with UV detection, which limits its use to screening in vitro drug–protein binding. As a result, a drug concentration higher than 20 mM has to be used. In addition, if the drug (free ligand) is not separated from the receptor (protein), or suffers from low sensitivity, it is difficult to evaluate binding. Nevertheless, these drawbacks can be overcome by the application of MS detection. Wan et al. [30] have recently illustrated a novel single run measurement of protein binding by HPFA-CE coupled with MS detection. The measurements of in vitro plasma protein binding using drug concentrations less than 10 mM were achieved by MS detection. Another benefit of MS detection is that the drug molecules can be detected even without complete resolution from the protein. Although the current use of HPFA-CE has not been popularly recognized, in our opinion, this technique has potential to become a valuable tool in screening drug–protein binding or characterizing the interaction between the drug and other receptors as well, and would be especially beneficial for biological samples that are only available in minute quantities.
Chiral drug screening One of the most widespread uses of CE in drug discovery is for chiral separations. Drug enantiomers can have different pharmacokinetic properties and potential biological activities, and these parameters are included as an essential property in the description of a chiral drug by pharmaceutical regulatory authorities. Chiral separation of two enantiomers and monitoring the enantiomeric excess (ee) are of crucial importance for discriminating their efficacies in various in vitro and in vivo assays. CE has become one of the most efficient chiral separation techniques in this area in terms of rapid method development, low cost, selectivity and capability for highthroughput screening. Various chiral selectors have been investigated and several chiral separation modes and widespread applications are found in numerous recent reviews [2,31–33]. The numerous applications indicate that cyclodextrins (CDs) as chiral selectors in CE buffer are the most
Drug Discovery Today: Technologies | Screening technology
popularly used and simplest approach to achieve enantioselectivity. Charged CDs in particular showed more efficient and enhanced chiral selectivity as compared with natural neutral CDs for a wide diversity of structures [34,35]. Perrin et al. [35] have demonstrated rapid screening for chiral separations by short-end injection capillary electrophoresis using highly sulfated cyclodextrins as chiral selectors. The use of short-end injection dramatically reduces analysis times. Results were very satisfying because almost all compounds (62 out of 67) could be baseline-resolved. To avoid trial and error in chiral method development, a screening strategy for the development of enantiomeric separation methods was presented by Johnson & Johnson-Pharmaceutical R&D [36]. Using this screening approach, Jimidar et al. [37] could baseline separate all eight stereoisomers of a compound containing three chiral centers. Alternatively, high-throughput screening of chiral drugs can be achieved by a 96-capillary array instrument allowing the simultaneous screening of different selectors and drug candidates [17]. In addition to fast separation and high enantioselectivity, another unique feature of chiral CE is that the migration elution order (MEO) of enantiomers can be reversed by use of different chiral selectors [38]. A versatile scheme for reversing the MEO has been suggested using acidic ibuprofen and basic propranolol as model compounds [39]. The use of MEO can facilitate trace enantiomeric excess (ee) determination, as highlighted by Blomberg and Wan [40]. Recent applications of CE for determination of 0.015–0.3% chiral impurities of drugs were summarized by Scriba [31]. It should be pointed out that the performance of MEO could not be readily achieved by other chiral methods, such as supercritical fluid chromatography or HPLC. Different types of chiral columns require intensive testing to achieve trace enantiomeric determination for both enantiomers. Chiral CE, as a complementary or superior technique to HPLC in many circumstances, could be suitable for therapeutic monitoring of a drug and its metabolites in drug screening. Bonato [41] has summarized a list of numerous enantioselective analyses of drugs and metabolites in biological fluids.
Microchip electrophoresis (MCE) Concurrent with the development of CE applications has been the emergence of electrophoresis in the microchip format. The basis for all chip-based systems is either pressure-driven or electro-osmotic flow and has been reviewed elsewhere [42]. The main attractions of the microchip platform are the ability to execute high resolution separations in a few hundred seconds and the capability for parallel processing of complex assays which include separations and integrated sample preparation in the same device. This not only gives the potential of high capacity but also of moving away from the low complexity assays used today for screening in drug discovery [43]. www.drugdiscoverytoday.com
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Figure 2. High-speed chiral separation of three basic drugs in 11 s on a microchip. The separation was performed on a commercial Shimadzu MCE-2010 system with a diode array detector (UV imagine detection, 200 nm) located along the separation channel, using charged CD as the chiral selector (HS-g-CD). BGE: 25 mM triethylammonium phosphate, pH 2.5, 5% HS-g-CD. The separation channel is 25 mm long 20 mm deep and 50 mm wide at the top. Although relatively small micochips with a separation length of only 25 mm are used, it is possible to perform the chiral separation of a mixture of three drugs in a single run. This shows an example of the great potential of using MCH for fast screening and enantiomeric separation of chiral compounds and suitable for ee determination as well. Reproduced, with permission, from Ref. [45], copyright (2003) Wiley-VCH Verlag GmbH & Co. KgaA, Weinheim.
Although various degrees of miniaturized CE systems have been available for several years, MCE in micromachined devices has only been recently reported. As with CE in general in the pharmaceutical industry, the largest number of applications are chiral separations, which have recently been reviewed [44]. Ludwig et al. [45] demonstrated an impressively fast enantiomeric separation of norephedrine on the commercial Shimadzu MCE microchip system in less than 2.5 s, and other 18 basic chiral drugs in less than 1 min with high reproducibility. An example of ultra fast chiral separation of three basic drugs in less than 11 s is shown in Fig. 2. A recent advancement is the development of ACE on microchips [46]. The method used chips with both UV and electrochemical detection. Studies were performed with model solutions of neurotransmitters using sulfated b-cyclodextrin as a substrate. The resolving power on the chips was not as high as in the more traditional capillary technique, resulting in less precise data. This was compensated for by the use of an internal standard.
Conclusions CE technologies have found unique applications in physicochemical profiling in drug discovery, such as high-throughput screening of pKa, log P determination, and drug–protein interaction studies. CE has also been successfully applied to enantioseparations of various chiral drugs, screening the 176
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enantioselective binding of chiral drugs to specific proteins and monitoring chiral drugs and metabolites in biological fluids. In view of the methodology and practical applicability, the mobility or migration time measurement-based approaches in CE such as pKa, log P, affinity CE and ee determination are more robust and reproducible than those pertaining to concentration quantification (Table 1). In addition, introduction of an online calibration approach can significantly improve measurement accuracy. It can be anticipated that with advances in new MS interfaces and more sensitive mass spectrometers, the microchip-based miniaturized technologies will have a profound impact on drug screening. This will be especially true in dealing with a large number of small bioanalytical samples, in particular from in vitro and in vivo screening because of ultra fast measurements and minute sample consumptions with fully automated performance. With its many advantages, CE has become a recognized and established technique with very promising
Links Aigilent, CE-UV, CE-MS-ESI-ion trap: http://www.chem.agilent.com CombiSep, A 96-capillary array instrument with UV absorbence detection: http://www.combisep.com/flashindex.html Beckman Coulter, CE-UV, CE-LIF: http://www.beckman.com Caliper Technologies, Microfluidic device: http://www.caliperls.com http://www.lab-on-a-chip.com
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and interesting applications in many areas of microscale analysis and high-throughput screening, and its use will continue to increase in the pharmaceutical industry.
Outstanding issues Awareness of the inherent potentials and advantages of CE technologies over conventional assays. Introduction and implementation of new miniaturized CE methods are not a technical challenge in drug screening. CE is in general considered to be less robust and reproducible than established HPLC methods. This practical issue, however, should not limit implementing new CE methods based on relative migration or mobility measurements, such as pKa, log P and so on. Currently available CE-MS interfaces are based on sheath liquid performance, which dilutes analyte concentration eluted from CE separation column before being transferred to the MS analyzer and consequently reduces MS signals by ten to 100-fold. Further development and validation of rugged sheathless or nanoflow CE-MS interfaces for routine use will dramatically improve MS sensitivity and open new applications. Further evaluation and cross-validation of ACE and HPFA-CE coupled with MS detection is expected to give a new microscale assay for screening in vivo drug–protein binding, for example. Microchips fabricated by computer-aided design tools allow highly reproducible analysis. Integration of separation channels with mass spectrometers will bring microchip-based technologies into drug screening.
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Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Screening technology
Capillary electrophoresis technologies for screening in drug discovery Hong Wan*, Richard A. Thompson DMPK & Bioanalytical Chemistry, AstraZeneca R&D Mo¨lndal, S-43183 Mo¨lndal, Sweden
The high separation efficiency of capillary electrophoresis (CE), combined with the high selectivity and sensitivity of mass spectrometry (MS) detection offers the potential of unique resolving power and high-throughput capacity to the analysis and structural identification of complex mixtures. Recent advances in CE-MS interfaces and commercially available 96-capillary instruments have made the implementation of routine CE methods for drug screening feasible.
Introduction Capillary electrophoresis (CE) is a miniaturized and high efficiency separation technique with many application areas in the pharmaceutical community. Recent advances in interfacing CE to MS have significantly increased the potential of this technique [1–3]. The technique has shown great success in high-throughput pKa screening in particular. However, other areas of physicochemical and biological screening, such as lipophilicity (log P) determination by microemulsion electrokinetic chromatography (MEEKC), drug–protein binding measurement and ligand–receptor interaction by high performance frontal analysis-capillary electrophoresis (HPFACE) and affinity capillary electrophoresis (ACE) are of increased interest. CE technologies have particular advantages in chiral drug screening and applications. Finally, CE in combination with microchip technologies shows great potential in drug screening.
*Corresponding author: H. Wan ([email protected]) 1740-6749/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2005.05.010
Section Editors: Jeff Brockman – Psychiatric Genomics Inc., Gaithersburg, MD, USA Mark Divers – AstraZeneca, Mo¨lndal, Sweden High-throughput screening pKa by CE and CE-MS Several applications of pKa determination by CE have been reported, and the state of the art of this technique has recently been summarized [4–6]. A 96-capillary format instrument has become commercially available from CombiSep (http://www.combisep.com/flashindex.html), which is suitable for high-throughput pKa screening as well as other applications [6]. A schematic work-flow of pKa screening is depicted in Fig. 1. In our opinion, the main drawback of UV absorbance detection is its relatively low sensitivity. Typically, it requires a minimal compound concentration of 20 mM and therefore has limitations in measuring the poorly soluble compounds often encountered in drug discovery. Although UV sensitivity can be improved by on-line sample stacking [7], an optimal injection has to be applied. Alternatively, poorly soluble compounds can be measured by titration method in a cosolvent buffer system followed by Yasuda–Shedlovsky extrapolation [8]. However, this requires a series of methanol containing solutions and is therefore time-consuming and can lead to inaccurate data. As a consequence of the above limitations, we have developed a highly selective and sensitive CE-MS assay for pKa screening [9]. Up to now, more than 500 new chemical entities from different classes and lead series have been successfully screened including many poorly soluble or sparingly soluble compounds (aqueous solubility <1 mM). Typically, poorly soluble compounds can be dissolved in 50% acetonitrile (ACN) or methanol and directly measured in nonsolvent www.drugdiscoverytoday.com
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Figure 1. Schematic work flow of pKa screening by CE-UV and CE-MS. meff is the effective mobility of the compound; positive value indicating positive charge, whereas negative value indicating negative charge; N is a neutral marker (DMSO); 1 and 2 are basic and acidic compounds separated from the neutral marker; pressure reduces the migration times dramatically without affecting pKa measurement. Compared with UV detection, CE-MS pKa-screening method offers a higher throughput capacity based on sample pooling, and it is more suitable for poorly and sparingly soluble compounds. In addition, the compounds can be measured simultaneously with several reference standards, ensuring more accurate pKa data.
buffers. So far, the only main drawback of the CE pKa assay that we have experienced is the poor electrospray ionization (ESI) signal associated with lipophilic compounds in some instances. However, this can be improved by using either higher compound concentrations or atmospheric pressure photoionization (APPI) or more sensitive mass spectrometers. Recently, our pKa assay has been extended to the application of 14 volatile background electrolyte (BGE) buffers, and a fully automated screening assay including rapid data evaluation has been set up. Because many compounds can be pooled and measured simultaneously together with several reference standards, this technique not only provides high throughput but also ensures high quality data with accuracy and reproducibility greater than 0.2 pKa units. In addition, as shown in Fig. 1, the effective mobility versus pH can provide the charge distribution and ionization profile of a compound in a wide pH range, thus facilitating better understanding of other physicochemical properties such as pH-dependent lipophilicity, transport mechanisms and protein binding, among others.
Determination of lipophilicity log P by MEEKC Microemulsion electrokinetic chromatography is an electrodriven separation technique. Separations are typically 172
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achieved by partitioning between water and oil-in-water microemulsions consisting of surfactant-coated nanometersized droplets of oil suspended in aqueous buffer. A cosurfactant, such as a short-chain alcohol is generally employed to stabilize the microemulsion. This novel technique with various microemulsion systems has been extensively investigated and recently reviewed [10–13]. Basically, by using a series of standard literature log Po/w values (octanol–water partition coefficient), log P can be indirectly calculated by a linear relationship between separation capacity k0 and log Po/w (log P = A log k0 + B). As the microemulsion separation is rather similar to octanol–water partitioning, the retention of MEEKC (k0 ) is shown to correlate highly with the log Po/w achieved by the conventional shake flask method, and correlates better than log P obtained by HPLC [13]. Several groups have demonstrated that both MEEKC (SDS based) and VEKC (CTAB-SDS vesicles or AOT vesicles) systems (abbreviations in Table 1) are suitable for rapid, highthroughput determination of log P for neutral, weakly acidic and weakly basic compounds with a wide range of log P [14– 17]. Validation and a long-term assessment of MEEKC for high-throughput determination of log P has been conducted on a 96-capillary instrument [17], showing acceptable accura-
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Table 1. Specific CE technologies used for drug screening Screen assay
pKa
Name of specific CE-MS technologies CE-UV
Lipophilicity (log P)
Protein binding (fu%) drug–protein interaction
Chiral separation
Microchip-based screening assays
MEEKC
ACE HPFA-CE
Chiral CE Chiral MCE
CE-UV CE-LIF CE-MS
Chiral impurity or ee determination by ratio of two enantiomer peak areas
Depending on specific assay, quantitative peak area or other parameters
VEKC
Effective mobility by migration time relative to a neutral marker
Capacity factor k0 by migration time relative to a neutral marker and microemulsion
Binding constant Ka by migration time shift (ACE) Unbound free fraction (fu%) by ratio of standard and analyte peak heights (HPFA-CE)
Pros
High throughput (sample pooling) by CE-MS or capillary array by UV High sensitivity (MS) Insensitive to impurities Minute sample required Ease of automation
Throughput comparable to or higher than HPLC and shake flask Assay not limited by solubility Suitable for a wide range of log P from 1 to 6.6 Applicable for full pH range
One to two orders of magnitude Simple and rapid chiral resolution and method development smaller sample size than ED Lower cost than PHLC High-throughput screening Possibility of reversing EMO as by MS detection compared to chiral HPLC Simpler and faster than ED Potential for in vivo protein binding measurement
Small sample size Possibility for direct analysis of complex matrices Ability to work with complex assay designs Ability to work in parallel
Cons
Poor signals of ESI in a few cases Requires neutral samples (could be improved by APPI interface) Less suitable for log D (7.4) determination (but can be Low sensitivity (UV), minimal indirectly calculated compound concentration 20 mM using log P and pKa)
Low sensitivity with UV Protein adsorption to capillary inner wall (could be reduced) Requires further validation of applications (HPFA-CE-MS)
Unsuitable for chiral preparative scales Chiral selector could interfere with MS detection
Separation channels necessary for many assays Interface to MS is under development Issues with binding to surfaces
Refs
[4–7,9]
[20–30]
[2,31–41]
[42–46]
[10–17]
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MEEKC: microemulsion electrokinetic chromatography; HPFA: high performance frontal analysis; ACE: affinity capillary electrophoresis; MCE: microchip electrophoresis; ee: enantiomeric excess; LIF: laser induced fluorescence; ED: equilibrium dialysis; VEKC: vesicle electrokinetic chromatography (CTAB-SOS visicle, AOT vesicle); CTAB: cetyltrimethylammonium bromide; SOS: sodium octyl sulfate; AOT: bis(2-ethylhexyl) sodium sulfosuccinate.
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Parameter measured
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cies (0.5 log P unit), particularly when considering the variations of reported values in the literature. Obviously, MEEKC can offer many advantages compared with HPLC and the shake flask method. MEEKC can be used in the full pH range suitable for a diversity of structures, can be automated, requires only small amounts of samples and short analysis times. Another distinct advantage of the MEEKC technique is that the separation system is not limited by solubility because poorly soluble compounds can be dissolved in cosolvent or micelles-mediated buffer, ensuring good signals in almost all cases. Most MEEKC applications are performed with UV detection. To the best of our knowledge, no MS detection has been used in a MEEKC system for log P determination. In principle, similar to CE-MS pKa screening, the log P values of many compounds can be simultaneously measured in such a high separation efficient microemulsion system if combined with a highly selective MS detection. More accurate data should be obtained by MS detection than by UV detection because on-line calibration is possible with MS detection. It can be expected that the high micellar concentration (SDS) present in the BGE could result in ion suppression, which can be a major limitation when using ESI-MS detection. However, the sensitivity of MS, if the signals are suppressed, can be compensated by using higher compound concentrations. Alternatively, the application of APPI-MS can dramatically improve MS sensitivity in nonvolatile buffer system such as that used in MEEKC [18,19].
Drug–protein interaction by affinity capillary electrophoresis-mass spectrometry (ACE-MS) and by high performance frontal analysis-capillary electrophoresis (HPFA-CE) Recent advances in ACE-MS is allowing rapid and selective identification of both synthetic and biological molecules and screening combinatorial libraries for drug leads or candidate ligands. Affinity capillary electrophoresis was initially employed for the determination of the binding constants of small molecules to proteins, and this technique has been extended for the examination of ligand–receptor affinities or interactions between drugs and various biomolecules. Several comprehensive reviews of this technique have been published [20,21]. The advantage of ACE-MS technique was exemplified as an on-line approach for the selection and identification of active ligands from a combinatorial mixture, for example screening and determining interacting structural moieties of D-tri and tetrapeptide libraries containing up to 361 compounds for ligands of vancomycin [22]. The potential of screening large libraries of up to 1000 peptides was also demonstrated directly by ACE-MS incorporating an affinity solid-phase extraction step before ACE-MS [22]. The library components that showed the strongest affinity were identified. The principle of ACE is to measure the electrophoretic mobility of a ligand as a function of receptor concentrations 174
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in the BGE. Consequently, the dissociation constant Kd can be obtained. Alternatively, the binding constant can also be measured directly by so-called bioaffinity ESI-MS [23]. However, bioaffinity ESI-MS requires quantitative measurements of the concentrations or responses of free ligand, free solute and their complex, respectively. Although the latter approach is even more rapid and straightforward and with higher capacity, it suffers from difficulties in obtaining accurate concentration determinations for complexed forms because of low ionization efficiencies of the complexes resulting from ion suppression. Currently, it seems that ACE rather than the bioaffinity approach is more suitable for rapid ranking and estimation of the binding of drugs to proteins, and in particular, identification of the selective binding of drugs to specific proteins as a high-throughput assay. In principle, several drugs belonging to the same or different series of new chemical entities can be simultaneously screened on the basis of 1:1 isotherm model. Lewis et al. [24] applied ACE for the screening of novel antimicrobial targets from a small molecular library of 44,000 compounds that possessed drug-like properties and antimicrobial activity against drug-resistant clinical isolates and concluded that ACE is a valuable tool for the fast, efficient detection of specific binding molecules that possess biological activity. It can be expected that ACE coupled with MS will provide a promising method for screening. The drug–protein binding can also be evaluated by another new technique, high performance frontal analysis and capillary electrophoresis (HPFA-CE), by quantitatively measuring the peak height ratio of a standard drug concentration to the drug resolved from the drug–protein complex. The principles of this technique and applications are found in a recent review [25]. The unique feature of this technique is that a large volume of sample is injected onto the capillary to generate a plateau, which linearly corresponds to the drug concentration. The advantages of HPFA-CE are that only small sample volumes of protein and drug are required, and it offers the possibility of rapid protein binding measurements during equilibrium without disturbing the binding conditions. Moreover, the measurements are typically done in one step at near-physiological conditions. This approach turns out to be much less labor-intensive than conventional equilibrium and ultrafiltration method where follow-up separation steps, such as HPLC are usually required. HPFACE is suitable for measurements of diverse structures including basic, neutral and acidic compounds with a wide range of binding constants from low to high binding affinities. Ishihama et al. [26] determined the binding extents of six anionic drugs to human serum albumin (HSA) solution-based combinatorial libraries and found that the measured bound fractions ranging from 78.7 to 99.9% were consistent with the results obtained by ultrafiltration. Østergaard et al. [27] employed HPFA-CE for the determination of the binding
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sites of drugs binding to HSA and applied this to drug displacement studies. Jia et al. [28] reported a pressureassisted HPFA-CE approach for rapid determination of binding constants of 17 drugs to HSA and of 4 drugs to AGP. In a similar way, Martinez-Pla et al. [29] used a short-end injection (back vacuum) for rapid evaluation of drug binding to HSA and human plasma. With analysis times shorter than 3 min for each injection, the measured binding constants are still in good accordance with much longer migration times, indicating that the HPFA-CE technique is applicable to high-throughput screening of drug–protein binding [29]. Reproducibility of measurements for this method is typically less than 10% coefficient of variation [28,29], which is comparable with the conventional equilibrium technique. One major limitation of the technique is its low sensitivity with UV detection, which limits its use to screening in vitro drug–protein binding. As a result, a drug concentration higher than 20 mM has to be used. In addition, if the drug (free ligand) is not separated from the receptor (protein), or suffers from low sensitivity, it is difficult to evaluate binding. Nevertheless, these drawbacks can be overcome by the application of MS detection. Wan et al. [30] have recently illustrated a novel single run measurement of protein binding by HPFA-CE coupled with MS detection. The measurements of in vitro plasma protein binding using drug concentrations less than 10 mM were achieved by MS detection. Another benefit of MS detection is that the drug molecules can be detected even without complete resolution from the protein. Although the current use of HPFA-CE has not been popularly recognized, in our opinion, this technique has potential to become a valuable tool in screening drug–protein binding or characterizing the interaction between the drug and other receptors as well, and would be especially beneficial for biological samples that are only available in minute quantities.
Chiral drug screening One of the most widespread uses of CE in drug discovery is for chiral separations. Drug enantiomers can have different pharmacokinetic properties and potential biological activities, and these parameters are included as an essential property in the description of a chiral drug by pharmaceutical regulatory authorities. Chiral separation of two enantiomers and monitoring the enantiomeric excess (ee) are of crucial importance for discriminating their efficacies in various in vitro and in vivo assays. CE has become one of the most efficient chiral separation techniques in this area in terms of rapid method development, low cost, selectivity and capability for highthroughput screening. Various chiral selectors have been investigated and several chiral separation modes and widespread applications are found in numerous recent reviews [2,31–33]. The numerous applications indicate that cyclodextrins (CDs) as chiral selectors in CE buffer are the most
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popularly used and simplest approach to achieve enantioselectivity. Charged CDs in particular showed more efficient and enhanced chiral selectivity as compared with natural neutral CDs for a wide diversity of structures [34,35]. Perrin et al. [35] have demonstrated rapid screening for chiral separations by short-end injection capillary electrophoresis using highly sulfated cyclodextrins as chiral selectors. The use of short-end injection dramatically reduces analysis times. Results were very satisfying because almost all compounds (62 out of 67) could be baseline-resolved. To avoid trial and error in chiral method development, a screening strategy for the development of enantiomeric separation methods was presented by Johnson & Johnson-Pharmaceutical R&D [36]. Using this screening approach, Jimidar et al. [37] could baseline separate all eight stereoisomers of a compound containing three chiral centers. Alternatively, high-throughput screening of chiral drugs can be achieved by a 96-capillary array instrument allowing the simultaneous screening of different selectors and drug candidates [17]. In addition to fast separation and high enantioselectivity, another unique feature of chiral CE is that the migration elution order (MEO) of enantiomers can be reversed by use of different chiral selectors [38]. A versatile scheme for reversing the MEO has been suggested using acidic ibuprofen and basic propranolol as model compounds [39]. The use of MEO can facilitate trace enantiomeric excess (ee) determination, as highlighted by Blomberg and Wan [40]. Recent applications of CE for determination of 0.015–0.3% chiral impurities of drugs were summarized by Scriba [31]. It should be pointed out that the performance of MEO could not be readily achieved by other chiral methods, such as supercritical fluid chromatography or HPLC. Different types of chiral columns require intensive testing to achieve trace enantiomeric determination for both enantiomers. Chiral CE, as a complementary or superior technique to HPLC in many circumstances, could be suitable for therapeutic monitoring of a drug and its metabolites in drug screening. Bonato [41] has summarized a list of numerous enantioselective analyses of drugs and metabolites in biological fluids.
Microchip electrophoresis (MCE) Concurrent with the development of CE applications has been the emergence of electrophoresis in the microchip format. The basis for all chip-based systems is either pressure-driven or electro-osmotic flow and has been reviewed elsewhere [42]. The main attractions of the microchip platform are the ability to execute high resolution separations in a few hundred seconds and the capability for parallel processing of complex assays which include separations and integrated sample preparation in the same device. This not only gives the potential of high capacity but also of moving away from the low complexity assays used today for screening in drug discovery [43]. www.drugdiscoverytoday.com
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Figure 2. High-speed chiral separation of three basic drugs in 11 s on a microchip. The separation was performed on a commercial Shimadzu MCE-2010 system with a diode array detector (UV imagine detection, 200 nm) located along the separation channel, using charged CD as the chiral selector (HS-g-CD). BGE: 25 mM triethylammonium phosphate, pH 2.5, 5% HS-g-CD. The separation channel is 25 mm long 20 mm deep and 50 mm wide at the top. Although relatively small micochips with a separation length of only 25 mm are used, it is possible to perform the chiral separation of a mixture of three drugs in a single run. This shows an example of the great potential of using MCH for fast screening and enantiomeric separation of chiral compounds and suitable for ee determination as well. Reproduced, with permission, from Ref. [45], copyright (2003) Wiley-VCH Verlag GmbH & Co. KgaA, Weinheim.
Although various degrees of miniaturized CE systems have been available for several years, MCE in micromachined devices has only been recently reported. As with CE in general in the pharmaceutical industry, the largest number of applications are chiral separations, which have recently been reviewed [44]. Ludwig et al. [45] demonstrated an impressively fast enantiomeric separation of norephedrine on the commercial Shimadzu MCE microchip system in less than 2.5 s, and other 18 basic chiral drugs in less than 1 min with high reproducibility. An example of ultra fast chiral separation of three basic drugs in less than 11 s is shown in Fig. 2. A recent advancement is the development of ACE on microchips [46]. The method used chips with both UV and electrochemical detection. Studies were performed with model solutions of neurotransmitters using sulfated b-cyclodextrin as a substrate. The resolving power on the chips was not as high as in the more traditional capillary technique, resulting in less precise data. This was compensated for by the use of an internal standard.
Conclusions CE technologies have found unique applications in physicochemical profiling in drug discovery, such as high-throughput screening of pKa, log P determination, and drug–protein interaction studies. CE has also been successfully applied to enantioseparations of various chiral drugs, screening the 176
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enantioselective binding of chiral drugs to specific proteins and monitoring chiral drugs and metabolites in biological fluids. In view of the methodology and practical applicability, the mobility or migration time measurement-based approaches in CE such as pKa, log P, affinity CE and ee determination are more robust and reproducible than those pertaining to concentration quantification (Table 1). In addition, introduction of an online calibration approach can significantly improve measurement accuracy. It can be anticipated that with advances in new MS interfaces and more sensitive mass spectrometers, the microchip-based miniaturized technologies will have a profound impact on drug screening. This will be especially true in dealing with a large number of small bioanalytical samples, in particular from in vitro and in vivo screening because of ultra fast measurements and minute sample consumptions with fully automated performance. With its many advantages, CE has become a recognized and established technique with very promising
Links Aigilent, CE-UV, CE-MS-ESI-ion trap: http://www.chem.agilent.com CombiSep, A 96-capillary array instrument with UV absorbence detection: http://www.combisep.com/flashindex.html Beckman Coulter, CE-UV, CE-LIF: http://www.beckman.com Caliper Technologies, Microfluidic device: http://www.caliperls.com http://www.lab-on-a-chip.com
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and interesting applications in many areas of microscale analysis and high-throughput screening, and its use will continue to increase in the pharmaceutical industry.
Outstanding issues Awareness of the inherent potentials and advantages of CE technologies over conventional assays. Introduction and implementation of new miniaturized CE methods are not a technical challenge in drug screening. CE is in general considered to be less robust and reproducible than established HPLC methods. This practical issue, however, should not limit implementing new CE methods based on relative migration or mobility measurements, such as pKa, log P and so on. Currently available CE-MS interfaces are based on sheath liquid performance, which dilutes analyte concentration eluted from CE separation column before being transferred to the MS analyzer and consequently reduces MS signals by ten to 100-fold. Further development and validation of rugged sheathless or nanoflow CE-MS interfaces for routine use will dramatically improve MS sensitivity and open new applications. Further evaluation and cross-validation of ACE and HPFA-CE coupled with MS detection is expected to give a new microscale assay for screening in vivo drug–protein binding, for example. Microchips fabricated by computer-aided design tools allow highly reproducible analysis. Integration of separation channels with mass spectrometers will bring microchip-based technologies into drug screening.
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