Petr Česla

Jan Fischer

Pavel Jandera
email: pavel.jandera@upce.cz

Prof Pavel Jandera

Separation of Phenolic Acids and Flavone Natural Antioxidants by Two-Dimensional 1 Method Combining Liquid Chromatography and Micellar Electrokinetic Capillary 2 Chromatography 3 4

Keywords: Micellar electrokinetic chromatography, Two-dimensional separation, Flavones, Phenolic compounds MEKC -Micellar Electrokinetic Chromatography, LC -Liquid Chromatography, SDS -15 Sodium dodecyl sulphate, 2D -two-dimensional, CE -capillary electrophoresis, CD -16 cyclodextrins, EOF -electroosmotic flow, C18 -octadecyl, RP -reversed phase, LC-LC two-17 22 Flavones, Micellar electrokinetic chromatography, Phenolic compounds, Two-dimensional 23 separations

dimensional liquid chromatography, LCxLC -comprehensive two-dimensional 18 chromatography, LC-MEKC -two-dimensional combination of liquid

on a phenyl column in the first dimension and on a C18 column in the second dimension [29]. 72 Acidic compounds were separated using a cation-exchange column in the first dimension and 73 a C18 column in the second dimension with a gradient of acetonitrile in acetate buffer [30]. 74 Recently, porous-shell fused-core C18 columns were combined in the second dimension with 75 a microbore column packed with phenyl-silica [31] or with PEG-silica [32] particles in the 76 first dimension for separation of phenolic antioxidants in wine, beverages and other food 77 samples in two-dimensional reversed-phase liquid chromatography. 78

The best improvement in the number of separated compounds, i.e. the highest increase 79 in the peak capacity with respect to single-dimension separations is achieved in highly 80 orthogonal two-dimensional systems with non-correlated selectivities in the first and in the 81 second dimension. Two reversed-phase systems are easy to connect on-line, but show more or 82 less correlated separation selectivities and consequently lower than maximum peak capacity 83 can be achieved in practice. On the other hand, combinations of reversed-phase and ion-84 exchange systems are not very suitable for two-dimensional separations of non-ionic 85 compounds, for which ion-exchange columns usually show low retention and separation 86 selectivity. Finally, on-line two-dimensional combinations of reversed-phase and normal-87 phase (adsorption) systems show high degree of orthogonality, but poor compatibility of 88 aqueous-organic RP mobile phases with purely organic solvents used as the mobile phase in 89 normal-phase chromatography. Therefore, combinations of HPLC in one dimension with 90 electromigration separation systems in the other one offer attractive possibilities of highly 91 orthogonal separations, complementary to LC-LC two-dimensional systems. Moreover, 92 electromigration methods are generally like to provide narrow peaks and hence high peak 93 capacity. 94 Two-dimensional capillary electrophoresis has been applied to separation of proteins 95 [33,34]. 2D combinations of liquid chromatography and capillary electrophoresis offer 96 possibility for increasing the peak capacity for separation of multi-component samples 97 containing ionic compounds. The LC-CE methods have been applied mainly in the analysis of 98 peptides in protein digests and of proteins in various biological samples [31,[35][36][37][38][39][40][41][42][43][44]. On-line 99 connection of RPLC to capillary zone electrophoresis (CZE) has been introduced by Bushey 100 and Jorgenson [35] and further applied by Jorgenson's group [31,36,37]. Off-line connections 101 of LC and CE have been described by several research groups [38,39,45]. The off-line samples [45]. Selected fractions containing phenolic compounds were isolated for 115 quantification and in vitro studies of anti-carcinogenic capacity. Like ion-exchange liquid 116 chromatography, capillary electrophoresis is not suitable for separations of non-ionic 117 compounds, which can be better separated by micellar electrokinetic chromatography, usually 118 in buffers containing sodium dodecylsulphate (SDS). Zhang et al. [46] employed 119 comprehensive 2-D HPLC-MEKC for the separation of natural compounds in traditional 120 Chinese medicines. The HPLC instrument was connected on-line to the CE system through 121 interface based on effluent stacking-injection of fractions collected from a micro-LC column 122 in a 0.25 µl chamber during 50-60 s, which was the time for the second-dimension MEKC 123

analysis. 124

In the present work, the effects of the addition of substituted cyclodextrins to the 125 background electrolyte on the separation selectivity of phenolic acids and flavone related 126 compounds in MEKC were investigated. A 2-D method combining liquid chromatography 127 with MEKC using automated off-line coupling with CE autosampler as the fraction collector 128 interface was developed, to avoid time limitations in the second, MEKC dimension and to 129 allow using a previously optimized HPLC separation on a polyethylene glycol (PEG) column, 130 whose dimensions are not on-line compatible with the MEKC separation in the second 131 dimension [29,47,48]. The main objective was to investigate possible improvement of the 132 overall performance of the LC-MEKC method with earlier reported LCxLC 2D separations of 133 phenolic and flavone natural antioxidants [32,49]. The HPLC-MEKC 2-D method was 134 applied for the analysis of green tea samples. 135 The standards of flavone related compounds and phenolic acids (Figure 1) were 143 obtained from Fluka (Buchs, Switzerland) and Sigma-Aldrich (St. Louis, MO, USA). 144 Ammonium acetate, boric acid, sodium tetraborate, sodium dodecyl sulphate and substituted 145 cyclodextrins, methyl-β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin, 146 heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin), and heptakis(6-O-sulfo)-β-cyclodextrin, were 147 obtained from Fluka (Buchs, Switzerland), acetonitrile was from Sigma-Aldrich (St. Louis, 148 MO, USA), methanol from Merck (Darmstadt, Germany) and formic acid from Lachema 149 (Brno, Czech Republic). 150

The extract of Jasmine Green Tea, (Twining & Company Ltd., London, England) was 151 prepared by keeping the tea bag in 100 mL of 95°C hot water for 3 minutes and cooling to the 152 laboratory temperature. Before injection, the extract was filtrated using Millipore 0.45 µm 153 syringe filter (Bedford, MA, USA). 154 155

Equipment 156

All MEKC experiments were carried out using Agilent 3D CE (Agilent, Palo Alto, CA, 157 USA) instrument at 20 kV. pH of the background electrolyte solutions was measured using an 158 Orion 3 Start pH meter (Thermo Scientific, Waltham, MA, USA). Water was purified on an 159 Ultra Clear UV apparatus (SG, Hamburg, Germany). The LC step of the 2-D method was 160 carried out using an Agilent 1200 series Rapid Resolution liquid chromatograph in the first 161 dimension. The outlet of the LC column was connected through a switching valve with the 162 CE autosampler for automated collection of the fractions from first dimension. Figure 2 The LC separations were carried out on a Discovery HS PEG column at 40°C using 167 gradient elution with 10 mmol/L ammonium acetate in water (pH = 3.0 adjusted by the 168 addition of formic acid) as the solvent A and acetonitrile as the solvent B. The mobile phase 169 gradient program was from 1% to 30%B in 15 min at 0.4 mL/min. After the end of each 2-D 170 run, the column was equilibrated with the initial gradient mobile phase for 10 min. The 171 injection sample volume was 5 µL. 172

The detection wavelengths of the CE instruments were set to 220, 254, 280 and 350 173 nm. All electropherograms were extracted at wavelength 280 nm being the most general for 174 the different classes of studied compounds. The capillary temperature was kept at 25 °C. 175 Samples were injected by applying overpressure of 50 mBar on the inlet sample vial for 10 s 176 in single CE analyses. New capillaries were preconditioned by rinsing subsequently with 0.5 177 mol/L sodium hydroxide for 10 min, with water for the next 10 min and finally with the 178 background electrolyte for 10 min. Further, the capillaries were rinsed prior to every injection 179 for 1 min with the background electrolyte. In the LC-MEKC 2D experiments, 25 mmol/L 180 borate buffer pH 9.05 with addition of 10 g/L SDS and 1.85 g/L heptakis(6-O-sulfo)-β-181 cyclodextrin was used as the background electrolyte in the second, MEKC dimension. The 182 background electrolytes and mobile phases were filtered using a Millipore (Bedford, MA, 183 USA) 0.45 µm filter and degassed by ultrasonication before use. 184

In the two-dimensional setup, an Agilent 3D CE autosampler was used as the interface 185 between the LC and the MEKC separation systems (Figure 2). The outlet of the LC column 186 was connected to the autosampler via a six-port switching valve, which was programmed to 187 collect the fractions of the effluent from the LC column during the analysis in the first 188 dimension (Figure 2A) into the vials equipped with 250 µL glass inserts. The CE autosampler 189 was rotated each 15 s, thus the volume of the fractions was 100 µL. In total, 42 1 st dimension 190 fractions containing analytes were collected during the gradient elution, from 0.5 min to 191 11min, for the analysis in the MEKC 2 nd D system. 192 The fractions collected from the LC dimension were stored in the autosampler vials 193 until the LC analysis run has been finished. After the end of the LC step, the valve was 194 switched into the second position and the effluent from the first dimension was redirected to 195 the waste while the LC column was re-equilibrated with the mobile phase used at the start of 196 the gradient. Meanwhile, the collected fractions were subsequently analyzed in the MEKC 197 second dimension (Figure 2B). 198 In continuation of our earlier studies of the applications of two-dimensional LCxLC 211 methods for separation of flavones and phenolic acids [26,29,32,47], we investigated 212 possibilities of using electromigration separation instead of LC separation in the second 213 dimension of a two-dimensional system. Most flavone compounds are not-ionized in 214 background electrolytes commonly used in CZE separations. To obtain successful separation 215 of these compounds, we used MEKC with sodium dodecyl sulphate additive (SDS). For this 216 purpose, we first optimized the MEKC step for best separation of 25 selected representative 217 standard compounds (Fig. 1). Based on the previously published results [14], we used 25 218 mmol/L borate background electrolyte with pH adjusted to 9.05 to obtain appropriate 219 electroosmotic flow velocity. On the basis of the experimental migration times, t mi , we 220 calculated the MEKC retention factors, k i , of sample compounds using the well-known Eq. 221

(1): 222

        - - = MIC i m EOF EOF i m t t t t t k , , 1 (1

) 223

Here, t EOF is the migration time of the electroosmotic flow, measured as the migration 224 time of thiourea and t MIC is the migration time of the SDS micelles, measured using the Sudan 225 III dye as the marker compound, which is completely retained in the SDS micellar pseudo-226 stationary phase. All sample compounds appear between t EOF and t mic . Table 1 shows the 227 experimental t mi of the standard compounds in various working electrolytes tested. 228

The mobility and hence the migration times and the retention factors of flavones and 229 phenolic compounds in MEKC depend on their structures. Generally, the mobility increases 230 with increasing number of -OH groups (myricetin, 6 < quercetin, morin, 5 < apigenin, 3 < 7-231 hydroxyflavone, 1). Further, catechins with five hydroxy groups ((+)-catechin and (-)-232 epicatechin) migrate faster than quercetin with the same number and position of hydroxy-233 groups, but flavone structure (Fig. 3A). Retention of flavonoid glycosides is affected by the 234 position of glycosylation: hesperidin and naringin (glycosylated at the position 7) elute later 235 than corresponding aglycones hesperetin and naringenin, but rutin (glycosylated at the 236 position 3) migrates faster than quercetin. First, we investigated the effect of the SDS on the 237 separation in the concentration range 4.3 -20.2 g/L. Generally, the migration times and the 238 resolution increase at higher concentrations of the SDS micelles in the background 239 electrolyte. This effect is stronger for the compounds with lower migration velocities. The 240 electrolytes with 10 g/L SDS provided the best compromise between the resolution of the 241 standards and the time of analysis, hence we used this concentration throughout this work 242 with various electrolytes (Fig. 3A). 243

Many flavone and phenolic acid standards could be separated by MEKC with 10 g/L SDS in 244 the background electrolyte, however some critical pairs could not be resolved in this system 245 and some standards were only partially separated (e.g., hesperetin/4-hydroxyphenylacetic 246 acid, syringic acid/myricetin, chlorogenic acid/vanilic acid/salicylic acid, 247 quercetin/morin/hesperidin, p-hydroxybenzoic acid/apigenin, 7-hydroxyflavone/biochanin A) 248

. To improve the resolution, we tested the effects of the organic solvents and various 249 substituted cyclodextrin additives (CD) to the background electrolyte. The addition of 250 methanol or acetonitrile up to 20% (v/v) did not improve the separation, but only increased 251 the time of analysis. The increase in the separation time was due to the effect of the organic 252 solvent on decreased experimental EOF mobility, which was 36.0x10 -8 m 2 V -1 s -1 , in purely 253 aqueous electrolyte without an organic solvent additive; 23.8x10 -8 m 2 V -1 s -1 in the electrolyte 254 with 15% methanol and 27.9x10 -8 m 2 V -1 s -1 .in the electrolyte with 15% acetonitrile. 255

In the background electrolytes with cyclodextrins, the partitioning equilibrium in 256 MEKC is affected by the formation of complexes of samples with cyclodextrins in the 257 aqueous phase, which compete for the analytes with the micellar pseudostationary phase. We 258 compared three non-charged cyclodextrins (methyl-β-cyclodextrin, heptakis ( of phenolic compounds, which are more or less ionized in the running buffers at pH = 9. This 266 applies mainly to phenolic acids, but even other compounds may be present in the form of 267 anions. Taking into account all these complex equilibria, it is not possible to predict a-priori 268 general effects of the CD additives on the migration times, separation selectivity and 269 resolution, which strongly depend on the sample structures. 270

Table 1 shows the retention factors of the test standards in running buffers with 1.3 -271 7.2 g/L cyclodextrins and 10 g/L SDS. The values and even the order of migration times of 272 flavones and phenolic acids depend significantly on the type and concentration of 273 cyclodextrin in the running buffer. Cyclodextrin additives affect also the bandwidths and the 274 separation efficiency characterized by the number of theoretical plates per meter, N (Table 1). 275

The methyl-β-cyclodextrin additive decreases the plate number by 20-25% with respect to the 276 buffer without the CD additive. The other CD additives moderately decrease the separation 277 efficiency only at the concentrations of 4 g/L or more. 278

To compare the performance of various running buffers, we selected the "critical 279 pairs" of standard phenolic and flavone compounds with adjacent peaks, i and j, which are 280 most difficult to resolve in various MEKC systems. For these "critical pairs" we determined 281 the separation selectivity, α i,j , defined as the ratio of the corresponding retention factors, k i (of 282 earlier migrating compound i) and k j (of later migrating compound j), respectively: 283

MIC j m MIC i m EOF i m EOF j m i j j i t t t t t t t t k k , , , , , 1 1 -
- ⋅ - - = = α (2) 284 285
The separation selectivity α i,j = 1 means that the analytes i and j co-elute in a single peak; the 286 higher is α i,j , the better is the separation selectivity and the resolution of analytes.

287

The separation selectivity for "critical pairs" of flavones and phenolic acids 288 significantly depends on the type of cyclodextrin (Table 2). The number of "critical pairs" 289 with resolution R S,i,j < 1 significantly decreases in working buffers containing CD additives ( 290 there are only three "critical pairs" with R S,i,j < 1. The concentration of the CDs also strongly 295 affects the selectivity of separation and the resolution. It should be noted that the change in 296 the type and concentration of the CD additive to the running buffer may cause change in the 297 order of migration times of "critical pairs". The reversed migration order is marked by asterisk 298 in Table 2. 299 Fig. 4 shows the plots of resolution of some critical pairs of flavones and phenolic 300 acids versus the concentration of heptakis(6-O-sulfo)-β-cyclodextrin in background 301 electrolyte. Most significant improvement in resolution was achieved in the background 302 electrolyte with 1.85 g/L anionic heptakis(6-O-sulfo)-β-cyclodextrin. 303

The separation of 25 test compounds in this background electrolyte shown in Fig. 3B 304 is only slightly longer than in the buffer without CD (Fig. 3A), but the resolution of the pairs 305 of compounds 1/2, 11/21, 6/13 improves considerably and there are 20 resolved peaks in the 306 sample in comparison to 18 in the buffer without CD. The addition of 1.85 g/L heptakis(6-O-307 sulfo)-β-cyclodextrin does not affect the efficiency (the number of theoretical plates, Table 1). 308

Hence, this optimized running buffer composition was applied for the separation of flavones 309 and phenolic acids in the two-dimensional LC-MEKC system. 310 311

Correlations between the LC and MEKC methods 312

The best improvement of resolution and peak capacity in 2D systems is achieved in 313 so-called "orthogonal" systems, where the separation principles in both dimensions are 314 completely different. The degree of orthogonality of a two-dimensional separation system can 315 be described by the correlation coefficient of the retention (migration) times, or of the 316 retention factors in the two dimensions [49]. We compared the retention times of flavones and 317 phenolic acids on four HPLC columns with different stationary phases and dimensions with 318 the MEKC migration times in the running buffer with 1.85 g/L heptakis(6-O-sulfo)-β-319 cyclodextrin (Table 3). Other MEKC separation systems showed similar correlations with the 320 HPLC separations and are not shown here. There are generally low correlations between the 321 MEKC and HPLC characteristics. The lowest correlations of the migration times for both 322 flavone compounds and phenolic acids was observed for the column 2 (Discovery HS PEG), 323 with a polyethyleneglycol bonded stationary phase, higher for the column 3 (Novapak 324 Phenyl) with a phenyl bonded stationary phase and for the columns 1 and 4 with C18 bonded 325 stationary phases, which can be possibly attributed to some similarities in the separation 326 This trend in system correlations agrees with our previously published work [48] -330 [50], where the combinations of a PEG column with a C18 column provide highly different 331 chromatographic selectivity and high degree of orthogonality in comprehensive two-332 dimensional LCxLC. The correlation coefficients between the retention data of flavone and 333 phenolic acid standards on a PEG column and on various C18 columns, including monolithic, 334 fully porous particle and superficially porous solid core particles, R 2 = 0.07 -0.43, higher 335 than in the LC-MEKC two-dimensional setup described in the present work (0.049, Table 3), 336 which therefore shows higher orthogonality. Moreover, a lower concentration of acetonitrile 337 in the mobile phase used for the separation of flavones and phenolic acids on the PEG column 338 in comparison to the C18 column is beneficial for the MEKC separation in the second 339 dimension, where the injection of sample dissolved in mobile phase with high concentration 340 of organic solvent can decrease the concentration of micelles and impair the resolution of 341 sample compounds. 342 343

Two-dimensional LC-MEKC separations 344

To match the column dimensions with the separation capillary in the MEKC system 345 and to minimize the dilution of the transferred fractions, a narrow-bore polyethyleneglycol 346 column was selected for the first dimension in the LC-MEKC separations (Discovery HS 347 PEG, 50x2.1 mm i.d., 5 µm particles), with a gradient of acetonitrile in aqueous ammonium 348 acetate buffer. Fig. 5 shows the two-dimensional separation of the standard compounds (as a 349 contour plot) with the HPLC elution times on the X-axis and the MEKC migration times on 350 the Y-axis in the optimized 2D off-line separation system. All 25 standards are resolved and 351 there are some spots of minor impurities in the two-dimensional retention plane. The 352 estimated maximum theoretical peak capacity, based on the average experimental bandwidths 353 is approximately 450 in the present automated off-line 2D LC-MEKC system, in comparison 354 to 30 in the MEKC and 20 in the HPLC one-dimensional systems. The overall time necessary 355 for the subsequent MEKC analysis of all HPLC fractions is long, 10.5 hours, but the analysis 356 can run unattended overnight. In the recently reported comprehensive LCxLC 2D method 357 with a PEG column in the first dimension and a porous-shell fused core Ascentis C18 column 358 in the second dimension [32], we could completely resolve 24 compounds of 27 phenolic F o r P e e r R e v i e w 13 peak capacity was only 12 and the total two-dimension peak capacity was cca 280, slightly 361 more than half of the peak capacity in the present work. We are presently working on the 362 reduction of the second dimension MEKC separation time. Fig. 6 shows an example of 363 practical applications of the present two-dimensional LC-MEKC system for the separation of 364 a sample of green tea extract. 365 366 4 Concluding remarks 367 MEKC separation of 25 flavone related compounds and phenolic acid standards was 368 achieved in 15 min using an optimized MEKC system employing the background electrolyte 369 containing 25 mmol/L borate buffer (pH 9.05) with the addition of 10 g/L SDS and 1.85 g/L 370 heptakis(6-O-sulfo)-β-cyclodextrin. The presence of anionic cyclodextrin additive allows 371 improving the resolution of the MEKC method. An automated off-line two-dimensional 372 separation system was developed coupling a HPLC gradient separation on a bonded 373 polyethylene glycol column in the first dimension and a MEKC separation in a borate buffer 374 with SDS and cyclodextrin additives. 375

The experimental setup developed in this work has several advantages over the 376 comprehensive on-line real-time LC-MEKC 2D system. The off-line system does not require 377 that the analysis of the collected fraction in the second dimension should be accomplished in 378 the time of collection of the next fraction. Less stringent limitations of the second-dimension 379 separation time considerably increase the available second-dimension peak capacity and the 380 number of peaks that can be resolved during a single analysis run, of course at the cost of a 381 longer total analysis time. Further, using an external fraction collector to store the fractions of 382 the HPLC column effluent in vials, matching the lengths and inner diameters of the HPLC 383 column and the MEKC capillary and the flow rates in both separation systems is not that 384 critical as in the comprehensive setup. The whole 2D system is closed and fully automated, so 385 that there is less risk of loosing some sample during the manipulation with the collected 386 fractions, which improves the reliability of the system. Last but not least, the analysis of the 387 individual fractions in the second dimension can be repeated and (or) the on-line pre-388 concentration step of the sample in the capillary before the MEKC analysis can be employed 389 to improve the sensitivity and the detection limits. 390 Further research is in progress to investigate the possibilities of reducing the overall 391 analysis time in a high peak capacity two-dimensional LC/MEKC system. 392 Separation selectivities, α i,j , calculated according to the Eq. ( 2) and resolution, Rs i,j , of the critical pairs of flavones and phenolic acids in

  and does not impose restrictions on the dimension of HPLC column in the 106 first dimension. On-line (comprehensive) methods analyze the whole applied amount of 107 sample, offer better automation possibilities and higher sample throughput, however they 108 require special instrument modification, micro-or capillary HPLC columns that can 109 efficiently handle only limited sample volumes and put severe time limitations on the second-110 dimension separation, which must be accomplished in collection time of the next HPLC 111 fraction from the first dimension. 112 Applications of 2D LC-CE for the analysis of naturally occurring compounds are less 113 common. Garcia-Villalba et al. used HPLC-CE for the analysis of extra-virgin olive oil 114

  capillary with extended 125 µm light path cell (25 µm I.D., 48 cm total 139 length, 40 cm to the detector) was obtained from Agilent (Palo Alto, CA, USA). The 140 Discovery HS PEG, 5 µm, 50 mm x 2.1 mm i.d. column was obtained from Supelco 141 (Bellafonte, PA, USA). 142

  163shows the experimental setup used for the 2D LC-MEKC setup.

8 The 1

 81 UV detector signal of the CE instrument was processed using the export function 199 of the CE Chemstation software. The exported ASCII data were converted into a matrix with 200 the rows corresponding to the fraction cycle periods. The 2D chromatograms were plotted as 201 the contour plots with the first dimension times on the x-axis and the second dimension times 202 on the y-axis coordinates, respectively, using proprietary software developed for the 203 evaluation of 2D LCxLC chromatograms[32]. The identification of the sample compounds 204 was based on the comparison of the LC elution times and the CE migration times in the 205 individual dimensions and the signal ratio at the four pre-set wavelengths (220, 254, Optimization of the MEKC separation of phenolic and flavone compounds. 210

  2,6-di-O-259 methyl)-β-cyclodextrin, heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin) and negatively charged 260 heptakis(6-O-sulfo)-β-cyclodextrin) additives to the background electrolyte. The neutral CDs 261 migrate along the capillary towards the detector (cathode) at the electroosmotic flow (EOF) 262 migration velocity, whereas the negatively charged CD moves in the same direction, but at 263Deleted: (e.g., hesperetin/4hydroxyphenylacetic acid, myricetin/pcoumaric acid, hesperidin/phydroxybenzoic acid) as the SDS micelles, i.e., against the direction of EOF. The 264 resulting migration velocities are further contributed to by the own electrophoretic mobilities 265

  buffers containing negatively charged heptakis(6-O-sulfo)-β-cyclodextrin), where 294

  non-charged compounds in the MEKC systems with SDS micelles in the 327 background electrolyte and the non-polar stationary phases in reversed-phase liquid 328 chromatography. 329

Figure 3 Figure 4 501Figure 1

 341 Figure captions 480481Figure 1 482 Structures of flavone compound and phenolic acid standards. 483 484 Figure 2 485 Experimental setup used in the automated off-line LC-MEKC two-dimensional method. 486 LC separation and fraction collecting step (A), MEKC analysis of collected fractions (B). 1 -487 HPLC separation column, 2 -switching valve, 3 -fused silica capillary, 50 µm i.d., 200 µm 488 o.d., distance approx. 1 mm between the end of the capillary and the cap of the vial, 4 -CE 489 autosampler, vials with 100 µL glass insert and with adapted caps with 4 mm hole, 5 -CE 490 separation capillary, 6 -background electrolyte vials, 7 -sample inject vial. 491 492 Figure 3 493 MEKC separations of 25 flavones and phenolic acids in background electrolyte containing 10 494 g/L SDS (A) and with addition of 1.85 g/L heptakis(6-O-sulfo)-β-cyclodextrin (B). 495 Capillary 48 cm (40 cm to the detector) x 25 µm I.D. with bubble cell extended to 125 µm 496 optical path length. Background electrolyte: 25 mmol/L borate pH 9.05 containing 10 g/L 497 SDS; applied potential 20 kV, overpressure injection at 30 mBar/10 s, detection: UV, 280 nm, 498 25°C. The numbers of compounds are as in Figure 1. 499 500 Figure 4 501 The effect of heptakis(6-O-sulfo)-β-cyclodextrin concentration, c CD , in background 502 electrolyte on resolution, R s , of some critical pairs of flavones. 503 Capillary, background electrolyte and other separation conditions as in Figure 3, except for 0 504 -5.6 g/L of heptakis(6-O-sulfo)-β-cyclodextrin, 20 kV. The numbers of compounds are as in 505 Figure 1. 506 507 Figure 5 508 2-D separation of 25 flavone and phenolic acid standards. The numbers of compounds are as 509 in Figure 1. See text for experimental conditions. 510 511 Figure 6 512

Table 1

 1 Retention factors, k, calculated using Eq. (1), electroosmotic flow time, t EOF , migration time of micelles, t MIC , and efficiencies of the separation of flavones and phenolic acids in the 25 mmol/L borate background electrolyte pH 9.05 with 10 g/L SDS and addition of various substituted cyclodextrins.

	Page 19 of 27					ELECTROPHORESIS
	Compound	Without CD	methyl-β-CD			heptakis(6-di-O-methyl)-β-CD	heptakis(6-tri-O-methyl)-β-CD	heptakis(6-O-sulfo)-β-CD
	Cyclodextrin concentration [g/L]	0	1.31	3.93	6.6	1.33	4.00	6.66	1.43	4.29	7.15	1.85	5.55
	t EOF [min]	4.96	5.10	5.19	5.30	5.22	5.29	5.37	5.20	5.26	5.35	5.12	5.54
	t MIC [min]	15.29	15.25	15.21	15.26	16.09	16.16	16.15	15.69	15.71	15.55	18.08	20.19
	1	(-)-Epicatechin	3.99	4.23	4.41	4.64	4.53	4.69	4.90	4.60	4.80	5.13	4.15	5.57
	2	(+)-Catechin	4.29	4.54	4.73	4.93	4.86	5.05	5.27	4.95	5.19	5.57	5.15	6.34
	7	Rutin	5.30	5.80	6.10	6.44	6.22	6.51	6.76	6.39	6.70	7.14	5.76	9.00
	10	Naringenin	6.22	6.70	6.88	6.98	7.29	7.57	7.47	7.32	7.16	7.68	6.35	8.77
	11	Hesperetin	6.85	7.22	6.88	6.72	7.83	7.57	7.73	7.77	7.34	7.14	7.11	11.06
	21	4-Hydroxyphenylacetic acid	6.85	7.59	8.12	8.72	8.18	8.67	9.18	8.43	8.98	9.85	6.68	8.58
	24	Ferulic acid	8.02	8.64	9.22	9.94	9.33	9.86	10.46	9.58	10.22	11.22	7.76	9.60
	20	Syringic acid	8.46	8.64	9.22	9.94	9.33	9.86	10.46	9.58	10.22	11.22	7.76	9.60
	14	Myricetin	8.46	9.48	10.22	10.81	10.23	11.20	11.39	10.72	11.42	12.36	12.42	16.21
	23	p-Coumaric acid	9.58	10.41	12.28	12.21	11.27	13.16	12.83	11.60	12.48	14.27	9.26	12.57
	25	Chlorogenic acid	10.43	8.64	10.22	10.81	10.23	11.20	11.39	10.58	11.26	12.36	8.06	10.39
	19	Vanilic acid	10.43	10.41	12.28	13.95	12.33	13.16	14.09	12.70	13.71	15.45	9.95	12.57
	18	Salicylic acid	10.43	11.82	12.87	13.95	12.78	13.81	15.14	13.41	14.44	16.31	9.95	13.46
	6	Quercetin	13.12	14.50	16.76	16.99	14.05	13.81	11.39	13.41	16.51	13.81	14.89	11.06
	13	Morin	13.12	17.94	17.48	16.99	22.74	23.62	25.39	22.72	24.95	29.02	15.54	16.21
	12	Hesperidin	13.12	14.50	16.42	14.59	16.72	17.16	12.83	17.47	14.44	16.31	17.79	21.50
	17	p-Hydroxybenzoic acid	15.95	14.50	17.48	17.73	15.81	19.11	18.84	16.39	17.85	21.48	12.06	17.07
	5	Apigenin	15.95	20.33	22.07	23.22	20.66	19.11	19.48	20.72	17.85	20.42	16.15	16.21
	4	7-Hydroxyflavone	17.26	12.69	10.22	8.72	14.05	12.00	9.18	13.41	9.45	9.85	12.42	17.07
	9	Biochanin A	17.26	17.94	16.42	12.21	20.05	20.03	19.48	20.72	24.95	29.02	16.15	27.71
	8	Naringin	18.99	17.94	16.42	14.59	21.50	17.90	15.51	22.72	16.51	13.81	15.54	27.71
	22	Caffeic acid	25.57	34.18	36.19	47.57	36.82	46.92	49.55	40.02	45.81	62.57	22.03	33.08
	15	Gallic acid	32.10	45.00	58.30	76.40	59.44	57.26	78.46	63.82	76.47	107.92	29.69	49.06
	16	Protocatechuic acid	52.68	77.95	106.28	179.83	96.12	113.83	180.66	112.56	160.52	499.58	39.77	78.79
	3	Flavone	84.97	121.71	106.28	97.90	180.65	152.06	118.93	220.57	160.52	123.38	64.50	1408.6
	Average number of theoretical	196 000	153 000	147 000	160 000	190 000	170 000	136 000	202 000	186 000	172 000	191 000	151 000
	plates per meter of capillary					Wiley-VCH

Table 2

 2

  25 mmol/L borate background electrolyte pH 9.05 with 10 g/L SDS and addition of various cyclodextrins.

											Page 20 of 27
	Critical	Without CD	3.93 g/L		4.0 g/L		4.29 g/L		1.85 g/L
	pairs i,j			methyl-β-CD	heptakis(6-di-O-methyl)-β-CD	heptakis(6-tri-O-methyl)-β-CD	heptakis(6-O-sulfo)-β-CD
		α i,j	Rs i,j	α i,j	Rs i,j	α i,j	Rs i,j	α i,j	Rs i,j	α i,j	Rs i,j
	4/9	1	0	1.61	5.19	1.67	10.63	2.64	21.38	1.30	5.22
	5/9	1.08	1.98	*1.34	5.90	1.05	0.92	1.40	4.72	1	0
	6/12	1	0	*1.02	0.41	1.24	3.16 *1.14	2.36	1.20	4.63
	6/13	1	0	1.04	0.83	1.71	8.48	1.51	8.84	1.04	1.08
	8/9	*1.10	2.41	1	0	1.12	1.43	1.51	8.84	1.04	1.13
	10/11	1.10	2.69	1	0	1	0	1.03	0.71	1.12	3.65
	11/21	1	0	1.18	4.14	1.15	3.81	1.22	3.68	*1.06	1.93
	12/13	1	0	1.07	1.24	1.38	5.42	1.73	7.98	*1.15	3.58
	13/17	1.22	2.73	1	0	*1.24	3.19 *1.40	4.72	1.29	6.57
	14/25	1.23	4.45	1	0	1	0 * 1.01	0.49	*1.54	7.25
	17/5	1	0	1.26	4.34	1	0	1	0	1.34	8.67
	18/25	1	0	*1.26	2.20	*1.23	1.92 *1.28	3.61	1.23	5.00
	19/18	1	0	1.05	0.83	1.05	0.97	1.05	1.05	1	0
	19/25	1	0	*1.20	1.91	*1.17	1.61 *1.22	3.34	*1.23	5.00
	20/14	1	0	1.11	1.12	1.14	1.34	1.12	2.86	1.60	7.55
	20/24	1.06*	1.04	1	0	1	0	1	0	1	0
	23/19	1.09	2.19	1	0	1	0	1.10	1.96	1.08	2.03
							Wiley-VCH

* -reversed migration order of the i,j compounds in critical pair

ELECTROPHORESIS F o r P e e r R e v i e w

Table 3 .

 3 1Correlation coefficients, R 2 , between the LC retention times and the MEKC migration times of flavones, related compounds and phenolic acids. 2For LC separation conditions see the references[26,29,32,49]; MEKC conditions as in Figure3B.3

	4
	LC column	LC mobile phase	Without CD		1.85 g/L heptakis-(6-O-sulfo)-β-	LC
						CD			conditions
			Flavones Phenolic	All	Flavones Phenolic	All
				acids	compounds		acids	compounds
	1 Ascentis Express C18,	15% (v/v) ACN /	0.564	0.232	0.196	0.425	0.188	0.189	[26,32]
	(Supelco, Bellefonte, PA, USA),	water,
	30 x 3 mm, 3 µm	0.5 ml/min, 40°C
	2 Discovery HS PEG,	10% (v/v) ACN /	0.162	0.049	0.049	0.153	0.037	0.047	[29]
	(Merck, Darmstadt, Germany), 50	water,
	x 2.1 mm, 5 µm	0.4 ml/min, 40°C
	3 Novapak Phenyl,	15% (v/v) ACN /	0.417	0.002	0.155	0.332	0.001	0.140	[29,49]
	(Waters, Milford, MA, USA), 50	water,
	x 3.9 mm, 5 µm	1 ml/min, 40°C
	4 Purospher STAR,	30% (v/v) ACN /	0.506	0.045	0.200	0.480	0.034	0.178	[49]
	(Merck, Darmstadt, Germany),	water,
	125 x 4 mm, 5 µm	1 ml/min, 40°C

in the buffer without CD, 9 in the buffer with methyl-β-cyclodextrin, 7 in the buffer with

heptakis(2,6-di-O-methyl)-β-cyclodextrin and 4 in the buffer with heptakis(2,3,6-tri-O-

methyl)-β-cyclodextrin). The improvement of separation selectivity and resolution is even

acids and flavone standards in 30 min total separation time. However, the second dimension

Deleted: in the form of the

Wiley-VCH ELECTROPHORESIS

2-D separation of a green tea sample. The numbers of compounds are as in Figure1.

Acknowledgements 394

This work was supported by the Ministry of Education, Youth and Sports of the Czech


Kajs-Marie Schützer
email: kajs-marie.schutzer@astrazeneca.com

K-M Schützer

M K Svensson

S Zetterstrand

U G Eriksson

K Wåhlander

Kajs-Marie Schützer

K-M Schützer

Kajs-Marie Schützer

on glomerular filtration during treatment with the direct thrombin inhibitor AZD0837

Keywords: thrombin inhibitor, renal function, glomerular filtration rate, creatinine, tubular secretion, tubular transporter proteins thrombin inhibitor, renal function, glomerular filtration rate, creatinine, tubular secretion, tubular transporter proteins

Reversible elevations of serum creatinine but no effect

INTRODUCTION

Creatinine is a non-protein waste product of creatine phosphate metabolism by skeletal muscle tissue. Creatinine production is continuous and is proportional to muscle mass. Creatinine is freely filtered in the kidney and therefore the serum creatinine (s-creatinine) level is dependent on glomerular filtration rate (GFR), but is also influenced by active renal tubular excretion of creatinine, diet and muscular mass [1;2]. S-creatinine is often used as an indicator of renal function both in clinical practice and during drug development [3].

During drug development, elevations in s-creatinine, raising the question of nephrotoxicity have occasionally been found. Sometimes this may indeed be a reflection of adverse renal effects and in other cases due to a beneficial renal functional effect, such as the renal haemodynamic alterations of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers [4], or due to reversible benign inhibition of the renal tubular secretion of creatinine as seen with cimetidine [5][6][7].

AZD0837 is an oral direct thrombin inhibitor currently under clinical development for stroke prevention in atrial fibrillation [8;9]. AZD0837 is rapidly absorbed and bioconverted into its active form, AR-H067637, via an intermediate metabolite, AR-H069927 [10][11][12]. AZD0837 is only to a minor degree excreted into the urine [13]. During clinical development of AZD0837 a reversible increase in mean s-creatinine of approximately 10% has been found. The objective of the present study was to investigate potential mechanisms for this increase in s-creatinine and to evaluate whether it may be due to a decrease in renal GFR or inhibition of tubular secretion of creatinine.

METHODS

Subjects

Healthy men and women, aged 60-75 years, with normal clinical and laboratory findings including a normal renal function (> 70 ml/min), as estimated by creatinine clearance (CrCl) according to the Cockcroft-Gault formula [START_REF] Cockcroft | Prediction of creatinine clearance from serum creatinine[END_REF], a body mass index of 18-30 kg/m 2 and who gave written, informed consent prior to enrolment were eligible to participate in the study. Exclusion criteria included significant recurrent illness, a history of bleeding or thrombotic disorder, a history of, or ongoing, severe allergy or hypersensitivity, hypersensitivity to X-ray contrast medium, cardiovascular, hepatic or significant gastrointestinal disease, or a positive HIV or hepatitis B or C test. The use of anticoagulant, antiplatelet (including aspirin), fibrinolytic or nonsteroidal anti-inflammatory drugs was not permitted from 2 weeks prior to the study until completion. The study was approved by the independent Institutional Review Board/Research Ethics Committee Berlin, Germany and the national medical agency BfArm, Germany. The study was performed in compliance with the Declaration of Helsinki.

Study design

This was an open-label, randomised, placebo-controlled, two-way crossover Phase I study (D1250C00033) with two treatment periods of 8 days each, separated by a washout period of 10-21 days as shown in Figure 1. Repeated doses of AZD0837 450 mg extended-release formulation (three 150 mg tablets) were given as a once-daily administration in the morning for 8 days in one period and corresponding placebo tablets were given during the other period.

Cimetidine (Tagamet ® , GlaxoSmithKline) was co-administered on Days 6-8 in both periods in a three-times-daily regimen of 400+400+800 mg as a positive control for inhibition of tubular secretion of creatinine. A pre-entry screening visit, including an overall health examination, was conducted within 14 days prior to the first study day. This comprised a medical history, history of bleeding and thrombotic occurrences, physical examination, 12-lead electrocardiogram and measurement of blood pressure, heart rate and laboratory status (haematology, clinical chemistry, urinalysis and coagulation analysis [activated partial thromboplastin time (APTT)]).

Laboratory measurements were repeated during both treatment periods: before administration of study drug on Day 1, on Day 5 and Day 8 before administration, and 7-10 days after completion of last treatment period. Subjects whose 24-hour APTT values after last dose were more than 10 seconds greater than their pre-dose values were required to remain at the study site until the values had normalised.

Assessments of renal function

Serum creatinine

S-creatinine was measured before every AZD0837/placebo dose on Days 1-8 and in the mornings of Days 9 and 10. In addition, s-creatinine was measured 12 hours after AZD0837/placebo dose on Days 5 and 8. S-creatinine was analysed by routine spectrophotometry methods (Architect c8000, Abbott GmbH & Co. KG, Wiesbaden-Delkenheim, Germany).

Serum cystatin C

Serum cystatin C (s-cystatin C), an additional glomerular filtration marker, was determined before every AZD0837/placebo dose on Days 1, 5 and 8 and in the morning of Day 10 [15]. Scystatin C was analysed by routine methods (Image immunochemistry system, Beckman GFR was determined by plasma clearance of iohexol (iCl) (four-sample method) on Day 5 in each treatment period. Iohexol 300 mg/ml was given as a 5 ml intravenous injection 3 hours after the AZD0837/placebo dose. Blood (7.5 ml in heparinised tubes) for determination of the plasma concentration of iohexol was taken at pre-dose and at 1, 2 and 4 hours after the injection of iohexol. A standardised high water intake (> 1000 ml) was given before injection of iohexol (Omnipaque ® , Nycomed Amersham Health, Cork, Ireland) to ensure an adequate urine production. The plasma samples for iCl were analysed using high-performance liquid chromatography (Renalyzer PRX 90, Provalid AB, Lund, Sweden) at the Department of Clinical Chemistry, Sahlgrenska University Hospital, Sweden.

Endogenous creatinine clearance

Endogenous CrCl was measured by analysis of 24-hour urine collections on Days 5 and 8. The bladder was emptied before the urine collection period started. A 10 ml sample was taken from all urine from the total collection period (0-24 hours). A blood sample for analysis of creatinine (2.7 ml serum separating tube) was drawn after 12 hours (mid-period). The urine and serum analyses of creatinine were carried out using routine spectrophotometry methods (Architect c8000, Abbott GmbH & Co. KG, Wiesbaden-Delkenheim, Germany).

Urinary markers of renal damage

Urinary albumin, immunoglobulin G, α 1 -microglobulin and β 2 -microglobulin were determined in a 12-hour urine collection starting in the evening before the AZD0837/placebo dose on Days 1 and 5. Urinary albumin and creatinine (albumin/creatinine ratio; ACR) were determined in the 24-hour urine collection on Days 5 and 8. Urinary markers were analysed by standard methods at the Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden.

Pharmacokinetics

Plasma samples for measurement of AZD0837, AR-H069927 (the intermediary metabolite) and AR-H067637 (the active form of AZD0837) concentrations were analysed by Analytico Medinet (AM) B.V., the Netherlands. For determination of plasma concentration, reversed-phase liquid chromatography-tandem mass spectrometry after solid-phase extraction was used. The limit of quantification (LOQ) was 10 nmol/l for all analytes. The method was developed for 250 µl human citrate plasma, and plasma concentrations were adjusted for the dilution of blood with citrate buffer in the sampling tubes. AZD0837, AR-H069927 and AR-H067637 were extracted from human plasma by means of an automated solid-phase extraction (extraction columns: 1 ml

Statistical analysis

This trial set out to test whether AZD0837 would affect GFR. A clinically relevant effect on GFR was considered to be 10% (for example, a change in GFR of 7 ml/min/1.73m 2 in a population with a mean GFR of 70 ml/min/1.73m 2 ), and the "no effect limit" was set to a difference of -5 ml/min/1.73m 2 . With 24 evaluable subjects the power to conclude "no effect" was 90%, given that AZD0837 did not affect GFR, at the significance level of 5%, and assuming a standard deviation of 8 ml/min of the difference in GFR. Since the previously observed elevation in screatinine with AZD0837 could correspond to a potential reduction in GFR, only a reduction in GFR was considered clinically relevant, and a one-sided test of significance was therefore used.

GFR was analysed in an analysis of variance (ANOVA) model with fixed factors for treatment, sequence and period, and a random factor for subject. To test for "no effect" a two-sided 90% confidence interval (CI) for differences in treatment means was constructed. If the lower bound of this CI (corresponding to the lower bound of the one-sided 95% CI) was no less than -5 ml/min it was to be concluded that there was no clinically relevant effect on GFR by AZD0837. Endogenous CrCl, s-creatinine and s-cystatin C were analysed using descriptive statistics.

Pharmacokinetic parameters for AZD0837 and its metabolites (AR-H069927 and AR-H067637) as well as for cimetidine were calculated with non-compartmental analysis using WinNonlin Professional version 4.1 (Pharsight Corporation, Mountain View, USA), and included the maximum plasma concentration (C max ) and area under the plasma concentration versus time curve during the dosing interval (AUC τ ). Ratios of the true geometric means for the pharmacokinetic parameters were estimated to evaluate changes for the combination of AZD0837 and cimetidine versus when given alone. For AZD0837 and its metabolites, a mixed effect analysis of variance model (ANOVA) was used with the logarithm of C max or AUC τ as the response variable, period and day as fixed factors and subject as a random factor. For cimetidine pharmacokinetics, descriptive statistics were used.

RESULTS

Demographics

Thirty subjects (21 men and 9 women) with a mean age of 64.1 ± 3.2 years and a mean body mass index of 26.6 ± 2.2 kg/m 2 were randomised. All subjects had a creatinine clearance > 70 ml/min and only one subject was on chronic medication. In this subject, treatment with an angiotensin-converting enzyme inhibitor (enalapril) was recorded at study start and was continued during the study. All subjects completed the study within a 3-month period (first subject in 14 August and last subject out 14 November 2006).

Assessments of renal function

Serum creatinine S-creatinine levels over time in the two treatment periods are shown in Figure 2. A 6% increase in mean s-creatinine (5 µmol/l) compared with baseline was observed already during Day 1 of AZD0837 treatment, then reached a plateau and was stable during the subsequent days of administration of AZD0837. After co-administration of cimetidine with AZD0837, a 21% increase (17 µmol/l) compared with baseline was observed. During placebo treatment s-creatinine remained stable until the start of cimetidine treatment (Day 6) when a rapid 17% increase (14 µmol/l) was observed, reaching a plateau 2 days later. At follow up, s-creatinine returned to baseline and all subjects had values within the normal range.

Serum cystatin C

No increase in s-cystatin C during the treatment period with AZD0837 was observed (Figure 3).

Glomerular filtration rate

Individual GFR values on Day 5 of the AZD0837/placebo treatment period are shown in Figure 4. The diagonal reference lines indicate a change from placebo by ± 10%. Descriptive statistics for GFR on Day 5 by treatment period and differences between treatments are displayed in Table 1. A mean difference in GFR of -1.6 ml/min /1.73 m 2 (90% CI: -3.7 to 0.5) was estimated at Day 5 of the AZD0837 treatment period compared with the placebo period.

One subject had an unreliable GFR measurement when on AZD0837; upon excluding this subject the mean difference in GFR between treatments was -0.7 ml/min/1.73m 2 (90% CI: -2.3 to 0.9). Since the lower bound of the CI was no less than -5 ml/min it could be concluded that AZD0837 had no clinically relevant effect on GFR.

Endogenous creatinine clearance

Endogenous CrCl was measured on Day 5 (without cimetidine) and Day 8 (with cimetidine) in the placebo and AZD0837 treatment periods, respectively. Treatment with AZD0837 resulted in a significant decrease in CrCl of -5.7 ml/min/1.73 m 2 (95% CI: -11.3 to -0.12) compared with placebo treatment. Three days of cimetidine treatment resulted in a significant decrease in CrCl of -15.1 ml/min/1.73 m 2 (95% CI: -20.9 to -9.2) during the placebo treatment period and -13.5 ml/min/1.73 m 2 (95% CI: -19.2 to -7.7) during treatment with AZD0837. The numerically lowest CrCl was seen after co-administration of cimetidine and AZD0837, the estimated mean difference between placebo plus cimetidine and AZD0837 plus cimetidine being -4.2 ml/min/1.73 m 2 (95% CI: -10.1 to 1.8).

Urinary markers of renal damage

None of the urinary markers of renal damage (albumin, immunoglobulin G, α 1 -microglobulin, β 2 -microglobulin and ACR) showed any treatment-related changes and no abnormal values were observed during the treatment periods (data not shown).

Pharmacokinetics

Co-administration of AZD0837 with cimetidine did not alter the exposure (AUC τ and C max ) of AZD0837, AR-H069927 and AR-H067637. With respect to AR-H067637, the estimated ratios (with vs. without cimetidine) of the geometric means for AUC (0-24) and C max were 0.971 (95% CI: 0.846 to 1.114) and 0.911 (95% CI: 0.781 to 1.062), respectively. Furthermore, there was no relevant difference in the mean ± standard deviation of AUC τ and C max of cimetidine when coadministered with AZD0837 (11.5 ± 2.84 µg.h/ml and 2.98 ± 0.750 µg/ml) versus placebo (12.0 ± 2.84 µg.h/ml and 3.11 ± 0.819 µg/ml).

Safety evaluation

Overall, there were few adverse events reported in this study, indicating that the study treatments were well tolerated (data not shown). Most adverse events had single occurrences and there was no apparent difference in the adverse event profile between the treatments.

Diarrhoea was reported by one subject during the treatment period with AZD0837 alone, and this has previously been identified as a possible side effect of AZD0837 [8;9] . Due to the pharmacodynamic effect, it cannot be ruled out if AZD0837 contributed to one event of haematochezia; however, one bleeding event was also noted with placebo. One event of a thrombophlebitis can reasonably be attributed to the indwelling cannula and the repeated puncture of the vessels of the forearm.

DISCUSSION

In this study an overall mean increase of approximately 6% in s-creatinine but no change in scystatin C was found in elderly healthy subjects treated with AZD0837. The increase in screatinine was not accompanied by a decrease in GFR, and, since a simultaneous reduction in CrCl was found, this suggests that inhibition of renal tubular secretion of creatinine by AZD0837 is the likely mechanism to explain the observed increase in s-creatinine during treatment with AZD0837.

The increase in s-creatinine seen during AZD0837 treatment had a relatively rapid onset, similar to that found during cimetidine treatment, and s-creatinine concentrations reached a steady state within 24 hours. The decline in s-creatinine after withdrawal of AZD0837 and/or cimetidine also occurred rapidly and s-creatinine concentrations were close to pre-treatment values at 24 hours after stopping treatment. Accounting for the time for wash-out of AZD0837 and cimetidine, the rates of onset and offset appeared similar. The observed time pattern is consistent with an inhibition of tubular secretion of creatinine since s-creatinine has a half-life of 6 hours and a new steady-state thus should be reached by 24 hours. Similar time patterns for s-creatinine have also been found in previous studies with AZD0837 [8;9]. Cystatin C is produced steadily by all types of nucleated cells in the body and is freely filtered in the glomeruli and then absorbed and degraded in the proximal tubuli. Cystatin C is therefore used as a marker of glomerular filtration [START_REF] Dharnidharka | Serum cystatin C is superior to serum creatinine as a marker of kidney function: a meta-analysis[END_REF]. In addition, it has been suggested that s-cystatin C might be an earlier and more rapid indicator of change in GFR than s-creatinine [START_REF] Herget-Rosenthal | Serum cystatin C--a superior marker of rapidly reduced glomerular filtration after uninephrectomy in kidney donors compared to creatinine[END_REF]. In the present study no change in s-cystatin C over time was found in any of the treatment groups. An increase in s-creatinine but not in s-cystatin C has also been shown in a Phase II study with AZD0837 [8]. These findings taken together indicate that the increase in s-creatinine is the result of an effect on tubular handling of creatinine instead of a direct effect on GFR.

To evaluate a potential effect of AZD0837 on renal function in this study the GFR was measured by plasma iCl (four-sample method). A clinically relevant effect of GFR was set to 10% taking into account the previously described within-individual coefficient of variation for iCl measurements [START_REF] Sterner | Iohexol clearance for GFRdetermination in renal failure--single or multiple plasma sampling?[END_REF]. No significant change in GFR was found during treatment with AZD0837 as compared with treatment with placebo.

Since an effect on tubular handling of creatinine had been suggested in previous studies with AZD0837 we tried to further evaluate this by co-administrating cimetidine. Cimetidine is a wellknown competitive inhibitor of the tubular secretion of creatinine via effects on the human organic cation transporter-2 (OCT2) and does not have any direct effect on GFR [5;7]. Maximum inhibition of tubular secretion of creatinine has been reported within 24 hours after start of three-times-daily dosing with 800 mg cimetidine in young healthy subjects [START_REF] Van Acker | Creatinine clearance during cimetidine administration for measurement of glomerular filtration rate[END_REF]. A slightly lower dose (400+400+800 mg) was chosen in the present study and was given for 3 days to ensure inhibition of renal tubular secretion of creatinine. An expected reduction in endogenous CrCl by cimetidine was found both during treatment with placebo and AZD0837. The additive effect on tubular creatinine secretion by cimetidine on top of AZD0837 suggests that secretion was only partially blocked by 450 mg of AZD0837 and that AZD0837 is a weaker inhibitor of tubular creatinine secretion than cimetidine. The numerically lowest CrCl was seen after coadministration of cimetidine and AZD0837, thus indicating that the cimetidine dose used in this study by itself did not completely block tubular creatinine secretion. A pharmacokinetic drugdrug interaction between AZD0837 and cimetidine is unlikely since no effect on the AUC and Cmax of AZD0837 or its metabolites was found in this study.

An effect of AZD0837 on inhibition of tubular secretion of creatinine is further supported by in vitro data from wild-type and OCT2-transfected HEK293 cells showing that AZD0837 is an inhibitor of basolateral OCT2-mediated transport of creatinine. AZD0837, AR-H069927 and AR-H067637 inhibited OCT2-mediated transport of creatinine (10 µM) with IC 50 values of 37, 27 and 0.7 µM, respectively. Cimetidine, used as a positive OCT2 inhibitor, inhibited the OCT2 transport of creatinine with an IC 50 value of 21 µM (unpublished data). A benign effect on renal handling of creatinine was further supported in the present study by the fact that no treatmentrelated changes in renal damage markers were found during the treatment periods.

Recently, the apical transporter system multidrug and toxin extrusion-1 (MATE1) protein has been reported to potentially be involved in the tubular secretion of creatinine [START_REF] Sato | Transcellular transport of organic cations in double-transfected MDCK cells expressing human organic cation transporters hOCT1/hMATE1 and hOCT2/hMATE1[END_REF]. AZD0837 and its metabolites were identified as MATE1 inhibitors of varying strength (with IC 50 values of 70.6, 29.0 and 0.28 µM for AZD0837, AR-H069927 and AR-H067637, respectively [unpublished data]). Inhibition of MATE1 could thus be an additional benign mechanism for inhibition of creatinine secretion in the kidney. Another potential explanation to elevations in s-creatinine is an increased production of creatinine. Unfortunately, no markers of muscle metabolism were measured in this study; however, none of the participating patients reported any muscular symptoms. In addition, no significant mean difference in total excreted amount of creatinine was found at Day 5 between AZD0837 and placebo (-0.13 mmol [95% CI: -0.90 to -0.63]), arguing against a change in creatinine production.

This study was conducted in healthy elderly subjects in order to assess markers of renal function in a population similar to the population that potentially will be treated with AZD0837 in the future. To minimise the influence of other medications the use of concomitant medications in the study was restricted. A placebo-controlled cross-over design was chosen to enable intraindividual subject evaluation and to minimise the time effects and increase the power to detect minimise the potential carry-over effect between the treatment periods. The lengths of the treatment periods were selected to ensure that steady-state conditions were achieved with regard to the plasma levels of AZD0837 and its metabolites on Day 5, the first day of renal assessments.

In conclusion, an overall increase of approximately 6% in s-creatinine but no change in scystatin C was found in elderly healthy subjects treated with AZD0837. The increase in screatinine was not accompanied by a decrease in GFR, as measured by plasma clearance of iohexol. These findings, together with unpublished in vitro data, suggest that inhibition of renal tubular secretion of creatinine is a likely mechanism resulting in the observed increase in screatinine during treatment with AZD0837. During drug development, elevations in s-creatinine, raising the question of nephrotoxicity have occasionally been found. Sometimes this may indeed be a reflection of adverse renal effects and in other cases due to a beneficial renal functional effect, such as the renal haemodynamic alterations of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers [4], or due to reversible benign inhibition of the renal tubular secretion of creatinine as seen with cimetidine [5][6][7].

F

AZD0837 is an oral direct thrombin inhibitor currently under clinical development for stroke prevention in atrial fibrillation [8;9]. AZD0837 is rapidly absorbed and bioconverted into its active form, AR-H067637, via an intermediate metabolite, AR-H069927 [10][11][12]. AZD0837 is only to a minor degree excreted into the urine [13]. During clinical development of AZD0837 a reversible increase in mean s-creatinine of approximately 10% has been found. The objective of the present study was to investigate potential mechanisms for this increase in s-creatinine and to evaluate whether it may be due to a decrease in renal GFR or inhibition of tubular secretion of creatinine. The study was approved by the independent Institutional Review Board/Research Ethics Committee Berlin, Germany and the national medical agency BfArm, Germany. The study was performed in compliance with the Declaration of Helsinki.

METHODS

Subjects

Study design

This was an open-label, randomised, placebo-controlled, two-way crossover Phase I study (D1250C00033) with two treatment periods of 8 days each, separated by a washout period of 10-21 days as shown in Figure 1. Repeated doses of AZD0837 450 mg extended-release formulation (three 150 mg tablets) were given as a once-daily administration in the morning for 8 days in one period and corresponding placebo tablets were given during the other period.

Cimetidine (Tagamet ® , GlaxoSmithKline) was co-administered on Days 6-8 in both periods in a three-times-daily regimen of 400+400+800 mg as a positive control for inhibition of tubular secretion of creatinine. A pre-entry screening visit, including an overall health examination, was conducted within 14 days prior to the first study day. This comprised a medical history, history of GFR was determined by plasma clearance of iohexol (iCl) (four-sample method) on Day 5 in each treatment period. Iohexol 300 mg/ml was given as a 5 ml intravenous injection 3 hours after the AZD0837/placebo dose. Blood (7.5 ml in heparinised tubes) for determination of the plasma concentration of iohexol was taken at pre-dose and at 1, 2 and 4 hours after the injection of iohexol. A standardised high water intake (> 1000 ml) was given before injection of iohexol (Omnipaque ® , Nycomed Amersham Health, Cork, Ireland) to ensure an adequate urine production. The plasma samples for iCl were analysed using high-performance liquid chromatography (Renalyzer PRX 90, Provalid AB, Lund, Sweden) at the Department of Clinical Chemistry, Sahlgrenska University Hospital, Sweden.

Assessments of renal function

Endogenous creatinine clearance

Endogenous CrCl was measured by analysis of 24-hour urine collections on Days 5 and 8. The bladder was emptied before the urine collection period started. A 10 ml sample was taken from all urine from the total collection period (0-24 hours). A blood sample for analysis of creatinine (2.7 ml serum separating tube) was drawn after 12 hours (mid-period). The urine and serum analyses of creatinine were carried out using routine spectrophotometry methods (Architect c8000, Abbott GmbH & Co. KG, Wiesbaden-Delkenheim, Germany).

Urinary markers of renal damage

Urinary albumin, immunoglobulin G, α 1 -microglobulin and β 2 -microglobulin were determined in a

12-hour urine collection starting in the evening before the AZD0837/placebo dose on Days 1 and 5. Urinary albumin and creatinine (albumin/creatinine ratio; ACR) were determined in the 24-hour urine collection on Days 5 and 8. Urinary markers were analysed by standard methods at the Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden.

Pharmacokinetics

Plasma samples for measurement of AZD0837, AR-H069927 (the intermediary metabolite) and AR-H067637 (the active form of AZD0837) concentrations were analysed by Analytico Medinet (AM) B.V., the Netherlands. For determination of plasma concentration, reversed-phase liquid chromatography-tandem mass spectrometry after solid-phase extraction was used. The limit of quantification (LOQ) was 10 nmol/l for all analytes. The method was developed for 250 µl human citrate plasma, and plasma concentrations were adjusted for the dilution of blood with citrate buffer in the sampling tubes. AZD0837, AR-H069927 and AR-H067637 were extracted from human plasma by means of an automated solid-phase extraction (extraction columns: 1 ml

Statistical analysis

This trial set out to test whether AZD0837 would affect GFR. A clinically relevant effect on GFR was considered to be 10% (for example, a change in GFR of 7 ml/min/1.73m 2 in a population with a mean GFR of 70 ml/min/1.73m 2 ), and the "no effect limit" was set to a difference of -5 ml/min/1.73m 2 . With 24 evaluable subjects the power to conclude "no effect" was 90%, given that AZD0837 did not affect GFR, at the significance level of 5%, and assuming a standard deviation of 8 ml/min of the difference in GFR. Since the previously observed elevation in screatinine with AZD0837 could correspond to a potential reduction in GFR, only a reduction in GFR was considered clinically relevant, and a one-sided test of significance was therefore used.

GFR was analysed in an analysis of variance (ANOVA) model with fixed factors for treatment, sequence and period, and a random factor for subject. To test for "no effect" a two-sided 90% confidence interval (CI) for differences in treatment means was constructed. If the lower bound of this CI (corresponding to the lower bound of the one-sided 95% CI) was no less than -5 ml/min it was to be concluded that there was no clinically relevant effect on GFR by AZD0837.

Endogenous CrCl, s-creatinine and s-cystatin C were analysed using descriptive statistics.

Pharmacokinetic parameters for AZD0837 and its metabolites (AR-H069927 and AR-H067637) as well as for cimetidine were calculated with non-compartmental analysis using WinNonlin Professional version 4.1 (Pharsight Corporation, Mountain View, USA), and included the maximum plasma concentration (C max ) and area under the plasma concentration versus time curve during the dosing interval (AUC τ ). Ratios of the true geometric means for the pharmacokinetic parameters were estimated to evaluate changes for the combination of AZD0837 and cimetidine versus when given alone. For AZD0837 and its metabolites, a mixed effect analysis of variance model (ANOVA) was used with the logarithm of C max or AUC τ as the response variable, period and day as fixed factors and subject as a random factor. For cimetidine pharmacokinetics, descriptive statistics were used.

RESULTS

Demographics

Thirty subjects (21 men and 9 women) with a mean age of 64.1 ± 3.2 years and a mean body mass index of 26.6 ± 2.2 kg/m 2 were randomised. All subjects had a creatinine clearance > 70 ml/min and only one subject was on chronic medication. In this subject, treatment with an angiotensin-converting enzyme inhibitor (enalapril) was recorded at study start and was continued during the study. All subjects completed the study within a 3-month period (first subject in 14 August and last subject out 14 November 2006).

Assessments of renal function

Serum creatinine S-creatinine levels over time in the two treatment periods are shown in Figure 2. A 6% increase in mean s-creatinine (5 µmol/l) compared with baseline was observed already during Day 1 of AZD0837 treatment, then reached a plateau and was stable during the subsequent days of administration of AZD0837. After co-administration of cimetidine with AZD0837, a 21% increase (17 µmol/l) compared with baseline was observed. During placebo treatment s-creatinine remained stable until the start of cimetidine treatment (Day 6) when a rapid 17% increase (14 µmol/l) was observed, reaching a plateau 2 days later. At follow up, s-creatinine returned to baseline and all subjects had values within the normal range.

Serum cystatin C

No increase in s-cystatin C during the treatment period with AZD0837 was observed (Figure 3).

Glomerular filtration rate

Individual GFR values on Day 5 of the AZD0837/placebo treatment period are shown in statistics for GFR on Day 5 by treatment period and differences between treatments are displayed in Table 1. A mean difference in GFR of -1.6 ml/min /1.73 m 2 (90% CI: -3.7 to 0.5) was estimated at Day 5 of the AZD0837 treatment period compared with the placebo period.

One subject had an unreliable GFR measurement when on AZD0837; upon excluding this subject the mean difference in GFR between treatments was -0.7 ml/min/1.73m 2 (90% CI: -2.3 to 0.9). Since the lower bound of the CI was no less than -5 ml/min it could be concluded that AZD0837 had no clinically relevant effect on GFR.

Endogenous creatinine clearance

Endogenous CrCl was measured on Day 5 (without cimetidine) and Day 8 (with cimetidine) in the placebo and AZD0837 treatment periods, respectively. Treatment with AZD0837 resulted in

Urinary markers of renal damage

None of the urinary markers of renal damage (albumin, immunoglobulin G, α 1 -microglobulin, β 2 -microglobulin and ACR) showed any treatment-related changes and no abnormal values were observed during the treatment periods (data not shown).

Pharmacokinetics

Co-administration of AZD0837 with cimetidine did not alter the exposure (AUC τ and C max ) of AZD0837, AR-H069927 and AR-H067637. With respect to AR-H067637, the estimated ratios (with vs. without cimetidine) of the geometric means for AUC (0-24) and C max were 0.971 (95% CI: 0.846 to 1.114) and 0.911 (95% CI: 0.781 to 1.062), respectively. Furthermore, there was no relevant difference in the mean ± standard deviation of AUC τ and C max of cimetidine when coadministered with AZD0837 (11.5 ± 2.84 µg.h/ml and 2.98 ± 0.750 µg/ml) versus placebo (12.0 ± 2.84 µg.h/ml and 3.11 ± 0.819 µg/ml).

Safety evaluation

Overall, there were few adverse events reported in this study, indicating that the study treatments were well tolerated (data not shown). Most adverse events had single occurrences and there was no apparent difference in the adverse event profile between the treatments.

Diarrhoea was reported by one subject during the treatment period with AZD0837 alone, and this has previously been identified as a possible side effect of AZD0837 [8;9] . Due to the pharmacodynamic effect, it cannot be ruled out if AZD0837 contributed to one event of haematochezia; however, one bleeding event was also noted with placebo. One event of a thrombophlebitis can reasonably be attributed to the indwelling cannula and the repeated puncture of the vessels of the forearm.

DISCUSSION

In this study an overall mean increase of approximately 6% in s-creatinine but no change in scystatin C was found in elderly healthy subjects treated with AZD0837. The increase in screatinine was not accompanied by a decrease in GFR, and, since a simultaneous reduction in CrCl was found, this suggests that inhibition of renal tubular secretion of creatinine by AZD0837

is the likely mechanism to explain the observed increase in s-creatinine during treatment with AZD0837.

The increase in s-creatinine seen during AZD0837 treatment had a relatively rapid onset, similar to that found during cimetidine treatment, and s-creatinine concentrations reached a steady state within 24 hours. The decline in s-creatinine after withdrawal of AZD0837 and/or cimetidine also occurred rapidly and s-creatinine concentrations were close to pre-treatment values at 24 hours after stopping treatment. Accounting for the time for wash-out of AZD0837 and cimetidine, the rates of onset and offset appeared similar. The observed time pattern is consistent with an inhibition of tubular secretion of creatinine since s-creatinine has a half-life of 6 hours and a new steady-state thus should be reached by 24 hours. Similar time patterns for s-creatinine have also been found in previous studies with AZD0837 [8;9].

Cystatin C is produced steadily by all types of nucleated cells in the body and is freely filtered in the glomeruli and then absorbed and degraded in the proximal tubuli. Cystatin C is therefore used as a marker of glomerular filtration [START_REF] Dharnidharka | Serum cystatin C is superior to serum creatinine as a marker of kidney function: a meta-analysis[END_REF]. In addition, it has been suggested that s-cystatin C might be an earlier and more rapid indicator of change in GFR than s-creatinine [START_REF] Herget-Rosenthal | Serum cystatin C--a superior marker of rapidly reduced glomerular filtration after uninephrectomy in kidney donors compared to creatinine[END_REF]. In the present study no change in s-cystatin C over time was found in any of the treatment groups. An increase in s-creatinine but not in s-cystatin C has also been shown in a Phase II study with AZD0837 [8]. These findings taken together indicate that the increase in s-creatinine is the result of an effect on tubular handling of creatinine instead of a direct effect on GFR.

To evaluate a potential effect of AZD0837 on renal function in this study the GFR was measured by plasma iCl (four-sample method). A clinically relevant effect of GFR was set to 10% taking into account the previously described within-individual coefficient of variation for iCl measurements [START_REF] Sterner | Iohexol clearance for GFRdetermination in renal failure--single or multiple plasma sampling?[END_REF]. No significant change in GFR was found during treatment with AZD0837 as compared with treatment with placebo.

Since an effect on tubular handling of creatinine had been suggested in previous studies with AZD0837 we tried to further evaluate this by co-administrating cimetidine. Cimetidine is a wellknown competitive inhibitor of the tubular secretion of creatinine via effects on the human organic cation transporter-2 (OCT2) and does not have any direct effect on GFR [5;7].

Maximum inhibition of tubular secretion of creatinine has been reported within 24 hours after start of three-times-daily dosing with 800 mg cimetidine in young healthy subjects [START_REF] Van Acker | Creatinine clearance during cimetidine administration for measurement of glomerular filtration rate[END_REF]. A slightly lower dose (400+400+800 mg) was chosen in the present study and was given for 3 days to ensure inhibition of renal tubular secretion of creatinine. An expected reduction in endogenous CrCl by cimetidine was found both during treatment with placebo and AZD0837. The additive effect on tubular creatinine secretion by cimetidine on top of AZD0837 suggests that secretion was only partially blocked by 450 mg of AZD0837 and that AZD0837 is a weaker inhibitor of tubular creatinine secretion than cimetidine. The numerically lowest CrCl was seen after coadministration of cimetidine and AZD0837, thus indicating that the cimetidine dose used in this study by itself did not completely block tubular creatinine secretion. A pharmacokinetic drugdrug interaction between AZD0837 and cimetidine is unlikely since no effect on the AUC and Cmax of AZD0837 or its metabolites was found in this study.

An effect of AZD0837 on inhibition of tubular secretion of creatinine is further supported by in vitro data from wild-type and OCT2-transfected HEK293 cells showing that AZD0837 is an inhibitor of basolateral OCT2-mediated transport of creatinine. AZD0837, AR-H069927 and AR-H067637 inhibited OCT2-mediated transport of creatinine (10 µM) with IC 50 values of 37, 27 and 0.7 µM, respectively. Cimetidine, used as a positive OCT2 inhibitor, inhibited the OCT2 transport of creatinine with an IC 50 value of 21 µM (unpublished data). A benign effect on renal handling of creatinine was further supported in the present study by the fact that no treatmentrelated changes in renal damage markers were found during the treatment periods.

Recently, the apical transporter system multidrug and toxin extrusion-1 (MATE1) protein has been reported to potentially be involved in the tubular secretion of creatinine [START_REF] Sato | Transcellular transport of organic cations in double-transfected MDCK cells expressing human organic cation transporters hOCT1/hMATE1 and hOCT2/hMATE1[END_REF]. AZD0837 and its metabolites were identified as MATE1 inhibitors of varying strength (with IC 50 values of 70.6, 29.0 and 0.28 µM for AZD0837, AR-H069927 and AR-H067637, respectively [unpublished data]). Inhibition of MATE1 could thus be an additional benign mechanism for inhibition of creatinine secretion in the kidney.

Another potential explanation to elevations in s-creatinine is an increased production of creatinine. Unfortunately, no markers of muscle metabolism were measured in this study; however, none of the participating patients reported any muscular symptoms. In addition, no significant mean difference in total excreted amount of creatinine was found at Day 5 between AZD0837 and placebo (-0.13 mmol [95% CI: -0.90 to -0.63]), arguing against a change in creatinine production.

This study was conducted in healthy elderly subjects in order to assess markers of renal function in a population similar to the population that potentially will be treated with AZD0837 in the future. To minimise the influence of other medications the use of concomitant medications in the study was restricted. A placebo-controlled cross-over design was chosen to enable intra- minimise the potential carry-over effect between the treatment periods. The lengths of the treatment periods were selected to ensure that steady-state conditions were achieved with regard to the plasma levels of AZD0837 and its metabolites on Day 5, the first day of renal assessments.

In conclusion, an overall increase of approximately 6% in s-creatinine but no change in scystatin C was found in elderly healthy subjects treated with AZD0837. The increase in screatinine was not accompanied by a decrease in GFR, as measured by plasma clearance of iohexol. These findings, together with unpublished in vitro data, suggest that inhibition of renal tubular secretion of creatinine is a likely mechanism resulting in the observed increase in screatinine during treatment with AZD0837.

F

6 ISOLUTE

 6 C6-column 25 mg). Chromatography was performed on an Hypersil HyPurity C18, 5 µm (100 mm x 2.1 mm) protected by a C18 pre column at a flow of 0.75 ml/min. The mass spectrometer used was an Applied Biosystems/MDS Sciex API-3000 system with turbo-ion spray interface and Analyst Software. Cimetidine concentrations in plasma were determined by PRA International -Early Development Services, Bioanalytical Laboratory, the Netherlands, using liquid chromatography with ultraviolet detection after solid-phase extraction. The LOQ was 0.10 µg/ml.

  glomerular filtration. The length of the washout period was chosen to

  Healthy men and women, aged 60-75 years, with normal clinical and laboratory findings including a normal renal function (> 70 ml/min), as estimated by creatinine clearance (CrCl) according to the Cockcroft-Gault formula[START_REF] Cockcroft | Prediction of creatinine clearance from serum creatinine[END_REF], a body mass index of 18-30 kg/m 2 and who gave written, informed consent prior to enrolment were eligible to participate in the study. Exclusion criteria included significant recurrent illness, a history of bleeding or thrombotic disorder, a history of, or ongoing, severe allergy or hypersensitivity, hypersensitivity to X-ray contrast medium, cardiovascular, hepatic or significant gastrointestinal disease, or a positive HIV or hepatitis B or C test. The use of anticoagulant, antiplatelet (including aspirin), fibrinolytic or nonsteroidal anti-inflammatory drugs was not permitted from 2 weeks prior to the study until completion.

  bleeding and thrombotic occurrences, physical examination, 12-lead electrocardiogram and measurement of blood pressure, heart rate and laboratory status (haematology, clinical chemistry, urinalysis and coagulation analysis [activated partial thromboplastin time (APTT)]). Laboratory measurements were repeated during both treatment periods: before administration of study drug on Day 1, on Day 5 and Day 8 before administration, and 7-10 days after completion of last treatment period. Subjects whose 24-hour APTT values after last dose were more than 10 seconds greater than their pre-dose values were required to remain at the study site until the values had normalised.

  was measured before every AZD0837/placebo dose on Days 1-8 and in the mornings of Days 9 and 10. In addition, s-creatinine was measured 12 hours after AZD0837/placebo dose on Days 5 and 8. S-creatinine was analysed by routine spectrophotometry methods (Architect c8000, Abbott GmbH & Co. KG, Wiesbaden-Delkenheim, Germany).Serum cystatin CSerum cystatin C (s-cystatin C), an additional glomerular filtration marker, was determined before every AZD0837/placebo dose on Days 1, 5 and 8 and in the morning ofDay 10 [15]. Scystatin C was analysed by routine methods (Image immunochemistry system, Beckman

6 ISOLUTE

 6 C6-column 25 mg). Chromatography was performed on an Hypersil HyPurity C18, 5 µm (100 mm x 2.1 mm) protected by a C18 pre column at a flow of 0.75 ml/min. The mass spectrometer used was an Applied Biosystems/MDS Sciex API-3000 system with turbo-ion spray interface and Analyst Software. Cimetidine concentrations in plasma were determined by PRA International -Early Development Services, Bioanalytical Laboratory, the Netherlands, using liquid chromatography with ultraviolet detection after solid-phase extraction. The LOQ was 0.10 µg/ml.

Figure 4 .

 4 Figure 4. The diagonal reference lines indicate a change from placebo by ± 10%. Descriptive

  in CrCl of -5.7 ml/min/1.73 m 2 (95% CI: -11.3 to -0.12) compared with placebo treatment. Three days of cimetidine treatment resulted in a significant decrease in CrCl of -15.1 ml/min/1.73 m 2 (95% CI: -20.9 to -9.2) during the placebo treatment period and -13.5 ml/min/1.73 m 2 (95% CI: -19.2 to -7.7) during treatment with AZD0837. The numerically lowest CrCl was seen after co-administration of cimetidine and AZD0837, the estimated mean difference between placebo plus cimetidine and AZD0837 plus cimetidine being -4.2 ml/min/1.73 m 2 (95% CI: -10.1 to 1.8).

  glomerular filtration. The length of the washout period was chosen to

Table 1 . Descriptive statistics for glomerular filtration rate (GFR) (ml/min/1.73m 2 ) on Day 5, and absolute and relative differences between treatments

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Table 1 . Descriptive statistics for glomerular filtration rate (GFR) (ml/min/1.73m 2 ) on Day 5, and absolute and relative differences between treatments

 1 This subject most likely had an erroneous analysis result during AZD0837 administration, according to the laboratory; the next higher GFR value in a subject was 74.

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a

European Journal of Clinical Pharmacology

ACKNOWLEDGEMENTS

We would like to thank Bengt Hamrén and Johan E Karlsson at AstraZeneca R&D Mölndal, Sweden, for their scientific advice in the planning of this study. The study was funded by AstraZeneca. Editorial assistance for the compilation of this manuscript was provided by Lars Elvelin, AstraZeneca R&D Mölndal, Sweden, and by Lee Kempster, MediTech Media (UK), and was funded by AstraZeneca.

ACKNOWLEDGEMENTS

We would like to thank Bengt Hamrén and Johan E Karlsson at AstraZeneca R&D Mölndal, Sweden, for their scientific advice in the planning of this study. The study was funded by AstraZeneca. Editorial assistance for the compilation of this manuscript was provided by Lars Elvelin, AstraZeneca R&D Mölndal, Sweden, and by Lee Kempster, MediTech Media (UK), and was funded by AstraZeneca.

CONFLICTS OF INTEREST

Results: A 6% increase in mean s-creatinine, but no increase in s-cystatin C, was observed during treatment with AZD0837. Co-administration of cimetidine resulted in a 21% increase in screatinine. A significant decrease in CrCl was found during AZD0837 treatment compared with placebo (-5.73 ml/min; 95% CI -11.3 to -0.12). No significant difference in GFR (-1.6 ml/min/1.73 m 2 ; 90% CI -3.7 to 0.5) was seen during treatment with AZD0837 versus placebo.

No changes in renal damage markers were found during the treatment periods.

Conclusions: An increase in s-creatinine, a decrease in CrCl, but no decrease in GFR, were found during treatment with AZD0837. Findings suggest that inhibition of renal tubular secretion of creatinine is the likely cause of the observed increase in s-creatinine. .


Chaitanya Dongre
email: c.dongre@ewi.utwente.nl

Jasper Van Weerd

Rob Van Weeghel

Rebeca Martinez Vazquez

Roberto Osellame

Giulio Cerullo

Marina Cretich

Marcella Chiari

Hugo J W M Hoekstra

Markus Pollnau

Martinez Vazquez

High-resolution electrophoretic separation and integrated-waveguide excitation of fluorescent DNA molecules in a lab on a chip

Keywords: integrated optofluidics, lab on a chip, fluorescence, ultrasensitive Laser induced fluorescence (LIF), Microfluidic fluorescence, integrated optofluidics, lab-on-a-chip, ultrasensitive

teaching and research institutions in France or abroad, or from public or private research centers.

photomultiplier tube, the limit of detection is as low as 2.1 picomolar. In the diagnostically relevant size range (~150-1000 base-pairs) the molecules are separated with reproducibly high sizing accuracy (> 99%) and the plug broadening follows Poissonian statistics. Variation of the power dependence of migration time on base-pair size -probably with temperature and condition of the sieving gel matrix -indicates that the capillary migration cannot be described by a simple physical law. Integrated waveguide excitation of a 12-µm narrow microfluidic segment provides a spatiotemporal resolution that would, in principle, allow for a 20-fold better accuracy than is currently supported by state-of-the-art electrophoretic separation in microchips, thereby demonstrating the potential of this integrated optical approach to fulfill the resolution demands of future electrophoretic microchips.

Introduction

Microfluidic (MF) lab-on-a-chip (LOC) devices [START_REF] Whitesides | The origins and the future of microfluidics[END_REF][START_REF] Manz | Miniaturized total chemical analysis systems: A novel concept for chemical sensing[END_REF] have enabled integrated DNA sequencing [START_REF] Eid | Real-time DNA sequencing from single polymerase molecules[END_REF] and genetic diagnostics [START_REF] Lagally | Integrated genetic analysis microsystems[END_REF][START_REF] Easley | A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability[END_REF]. Capillary electrophoresis (CE) separation is a powerful method over a broad range of molecular sizes, thanks to flexible MF protocols, exploiting a variety of sieving gel matrices. The inherent advantages of MF CE, high-speed operation and low reagent volumes, can be combined with laser-induced fluorescence (LIF) detection [START_REF] Johnson | Fundamentals and practice for ultrasensitive laserinduced fluorescence detection in microanalytical systems[END_REF][START_REF] Schwarz | Recent developments in detection methods for microfabricated analytical devices[END_REF][START_REF] Kuswandi | Optical sensing systems for microfluidic devices: a review[END_REF], resulting in optofluidic integration [9, 10] toward compact, sensitive on-chip bio-analysis tools [START_REF] Verpoorte | Chip vision -optics for microchips[END_REF] which aim at solving real-life challenges in medicine [START_REF] Yager | Microfluidic diagnostic technologies for global public health[END_REF], e.g. identification of genomic deletions or insertions associated with genetic illnesses.

Fluorescence excitation via an integrated optical waveguide (WG) provides advantages over focusing an external laser beam by lenses or through a microscope, such as stability and reproducibility of the optical excitation and avoidance of the requirement to realign the laser beam from time to time. Besides, it is one step toward full sensor integration in the MF LOC. We apply the technique of femtosecond-laser micromachining [START_REF] Davis | Writing waveguides in glass with a femtosecond laser[END_REF][START_REF] Gattass | Femtosecond laser micromachining in transparent materials[END_REF] to post-process optical WGs into commercially mass-produced LOCs on a fast, flexible, chip-by-chip basis [START_REF] Martinez | Integration of femtosecond laser written optical waveguides in a lab-on-chip[END_REF], thereby exploiting the existing mature MF fabrication infrastructure, while achieving integrated fluorescence excitation. Having pioneered the use of such integrated-WG laser excitation for electrophoretic DNA analysis [START_REF] Dongre | Fluorescence monitoring of microchip capillary electrophoresis separation with monolithically integrated waveguides[END_REF], here we demonstrate excellent DNA-sizing accuracy and low background noise, resulting in a lower limit of detection (LOD) compared to earlier accounts [START_REF] Bliss | Rapid fabrication of a microfluidic device with integrated optical waveguides for DNA fragment analysis[END_REF].

Furthermore, the spatio-temporal resolution provided by integrated waveguide excitation

Optofluidic chip fabrication and characterization

The lay-out of our optofluidic chip is presented in Fig. 1. Chips were fabricated in a twostep procedure. Firstly, the MF channel network and MF reservoirs were patterned photolithographically and wet-etched in fused silica glass and then sealed off by bonding another piece of fused silica glass on top. The chip has dimensions of 55 mm × 5.5 mm × 1 mm and the MF channels have a cross section of ~110 µm width and ~50 µm depth (LioniX BV) [http://www.lionixbv.nl].

In a second step, the optical WG was inscribed into the bulk of such a fused silica chip by femtosecond-laser writing at a translation speed of 20 µm/s, using a Ti:Sapphire laser operating at 800 nm wavelength with 150 fs, 4 µJ pulses at a repetition rate of 1 kHz. Employing astigmatic beam shaping, an elliptical cross section of the written WG was obtained, with a major diameter of ~50 µm in the vertical direction, in order to excite the maximum possible volume of the MF channel, while the minor diameter in the horizontal direction is ~12 µm in order to retain a high spatial resolution along the direction of DNA flow and separation. The WG displays a graded refractive index profile and a maximum refractive index increase of 2×10 -3 . Fiber-to-chip coupling of laser light with coupling losses of < 2 dB was achieved and the WG propagation losses were measured to be in the range of 0.5-0.9 dB/cm at the wavelength of 543 nm. The excitation WG intersects the CE separation channel orthogonally at a distance of ~3.6 cm from the MF crossing junction at which the separation commences, toward the end of the MF CE separation channel, close to reservoir 4, where the plug separation is highest. The fluorescence signal was detected through a small detection window in order to reduce the background noise. Constant, inherent, mutual alignment of the excitation WG with its detection window has the potential to render our system more compact, faster to operate, and more reproducible compared to conventional LIF approaches [START_REF] Johnson | Fundamentals and practice for ultrasensitive laserinduced fluorescence detection in microanalytical systems[END_REF][START_REF] Schwarz | Recent developments in detection methods for microfabricated analytical devices[END_REF][START_REF] Kuswandi | Optical sensing systems for microfluidic devices: a review[END_REF].

Results and discussion

Electrophoretic separation protocol

Prior to the experimental runs the inner walls of the MF channel network were coated with an epoxy-poly-(dimethylacrylamide) (EPDMA)-based polymer in order to suppress the electro-osmotic flow and to minimize adsorption of DNA molecules on the MF channel wall. Subsequently, the channels were filled with a sieving gel matrix consisting of hydroxyethyl-cellulose (HEC) (2% wt./vol.), dissolved in 20 mM MES / 20 mM His buffer (pH 6.2) in order to maximize the resolution of the DNA CE separation. SYBR Green I (Molecular Probes Inc.) was added according to the manufacturer's protocol. The reagents were sterilized, filtered (0.22 µm pore size) and stored at 269 K. The CE sample loading and separation protocol was based on actuation voltages of up to 1.5 kV, delivered by Pt electrodes integrated into the MF reservoirs, with the help of a Labview

Base-pair resolution

In an independent experiment carried out in a different microchip, another set of doublestranded DNA molecules with 12 different sizes, in a relatively smaller size range (150-500 bp), was separated and the individual plugs detected. The resulting electropherogram is shown in Fig. 4.

Optimization of the coating and sieving gel matrix led to excellent CE separation of the DNA molecules under integrated-WG laser excitation in both experiments, see Figs. 3A-B and4. When plotting the temporal occurrence of the fluorescence peaks of the 17 molecules in Fig. 3A-B vs. their a priori known base-pair sizes, the two smallest (insufficient interaction with the sieving matrix) as well as the two largest DNA molecules (tendency to fold) exhibit clear deviations, while the peaks of all DNA molecules with sizes of 150-1000 base-pairs, in the diagnostically relevant region, are resolved with a sizing accuracy higher than 99% (Fig. 5A). This high sizing accuracy is

  a 20-fold improvement in accuracy if better MF CE separation protocols become available.

6 (

 6 National Instruments, Inc.) program to steer the MF control system (CapiliX BV) [http://www.capilix.com]. Application of a high voltage forced the negatively charged DNA molecules to migrate into the CE injection channel from sample reservoir 1 to waste reservoir 2. By switching the voltages at all four reservoirs simultaneously a wellconfined plug of DNA molecules -with a volume of ~605 picoliters at the crossing junction of the two MF channels -was injected into the CE separation channel, from the MF crossing junction toward waste reservoir 4, and the DNA molecules contained in the plug volume were separated according to their size.

3. 2

 2 photomultiplier tube (PMT) (H7421-40, Hamamatsu Photonics K.K.) cooled down to 203

  the result of Fig.4for the 12 DNA molecules in a smaller size range. The high sizing accuracy (> 99%) is comparable to state-of-the-art performance in microchip CE-based DNA sequencing without integrated-WG laser excitation[START_REF] Paegel | High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor[END_REF][START_REF] Landers | Molecular diagnostics on electrophoretic microchips[END_REF].Each measured fluorescence peak in the electropherogram is a convolutionalong the flow axis -of the Gaussian WG mode-field cross-section ∆x WG and the diffusion-induced physical width ∆x MF of the MF plug. The resulting spatial plug widths ∆x are related to the temporal electropherogram peak widths ∆t (provided in Fig.3Das full width at half maximum) L = 3.6 cm and t (data on the abscissa of Fig.3A-C) are the migration length and time, respectively. In our experiment, the minimum electropherogram peak width was found to be ∆x = 248 µm (the very first peak in Fig.3B-C). With ∆x WG = 12 µm, we obtain ∆x MF = 247.7 µm >> ∆x WG , i.e., the current spatial resolution is limited by the MF plug width, while the obtained integrated-WG mode profile would allow for a 20-fold better resolution. MF technological advances, e.g. by improving the MF channel-wall coatings and sieving gel matrices, will lead to smaller MF plug widths, thereby fully exploiting the monitoring resolution achieved with integrated optical WGs. Clearly, integrated optical instrumentation is well suited to handle the resolution demands of future MF-chip generations. migration time t vs. DNA size (Fig.5A) we derive the electrophoretic mobility µ according to the equation the distance between the MF crossing junction and the detection window and E is the electric field strength in the MF CE separation channel. The observed linear decrease (on double logarithmic scale) of the electrophoretic mobility with increasing DNA size (Fig.5B) matches well with observations in conventional bulk capillary-based DNA gel electrophoresis setups[START_REF] Dolnik | Capillary electrophoresis in sieving matrices: selectivity per base, mobility slope, and inflection slope[END_REF].By plotting the results of Fig.5Aon a double-logarithmic scale, see Fig.5C, slopes of 0.24 and 0.25 (indicated by the straight lines) are derived for the two consecutive experiments, respectively, investigating the flow of the 17 DNA molecules.These results are significantly different from the slope of 0.31 derived for the flow of the 12 DNA molecules, despite the fact that in all cases a high sizing accuracy was obtained. This variation of power dependence points toward different flow mechanisms in these experiments, potentially depending on ambient temperature and/or condition of the sieving gel matrix, a result that questions whether a general, simple model of electrophoretic flow in micro-capillaries can be established and universally applied.

12 The

 12 Simple and straight-forward integration of optical waveguides into commercial microfluidic chips by femtosecond-laser post-processing enables separation of fluorescently labeled DNA molecules with low limit of detection of 2.1 pM and high of > 99%, leaving plenty of room for further improvement of accuracy via better electrophoretic separation protocols. This fabrication technique also allows for three-dimensional waveguide structures[START_REF] Crespi | Threedimensional Mach-Zehnder interferometer in a microfluidic chip for spatiallyresolved label-free detection[END_REF], thereby further enhancing the potential of the optofluidic chip technology for monitoring on-chip CE. authors acknowledge fruitful discussions with R. Dekker, G. A. J. Besselink, and R.G. Heideman, technical support from A. J. F. Hollink, H. A. G. M. van Wolferen, N. Bellini, and M. J. Gilde, and provision of LOC platform by H. H. van den Vlekkert.Financial support was provided by the European Commission under FP6 project contract IST-2005-034562 -Hybrid Integrated Biophotonic Sensors Created by Ultrafast Laser Systems (HIBISCUS). Psaltis, D., Quake, S. R., Yang, C., "Developing optofluidic technology through the fusion of microfluidics and optics. Nature 2006, 442, 381-386. [10] Monat, C., Domachuk, P., Eggleton, B. J., "Integrated optofluidics: a new river of light," Nat. Photon. 2007, 1, 106-114.

Figure 1 .

 1 Figure Captions

Figure 2 .

 2 Figure 2. Movie recorded with a CCD camera showing transient fluorescence from

Figure 3 .

 3 Figure 3. CE separation in an optofluidic chip of a DNA ladder consisting of double-

Figure 4 . 17 Figure 5 .

 4175 Figure 4. Fluorescence intensity vs. migration time for 12 double-stranded DNA

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