CytoPatch Study: Effect of Cisapride, (d-l)-sotalol and Terfenadine on herg channel currents of transfected CHO cells

Transcription

1 CytoPatch Study: Effect of Cisapride, (d-l)-sotalol and Terfenadine on herg channel currents of transfected CHO cells

2 1. Conclusion In this study, the operating efficiency of the CytoPatch instrument was compared to conventional patch clamping. We investigated the effect of Cisapride, (d-l) Sotalol and Terfenadine on the herg channel in transfected CHO cells. There was a good agreement between IC 50 values obtained with conventional patch clamping and IC 50 values obtained with the CytoPatch instrument (values for conventional Patch-Clamping/ Cyto- Patch : Cisapride 6,2/ 8,6 nm, (d-l)-sotalol 281/ 361 μm, Terfenadine 8,4 / 29,6 nm). When compared to values from the literature, our obtained data are clustered in the lower range. This reflects the high quality of the data generation and the good performance of the micro fluidic compound application. A detailed analysis of the efficiency (with a total success rate of 75%) as well as the seal rate (median of seal resistance is 2.26 GOhm) was evaluated with Cisapride as an example. 2. Introduction herg The human Ether-a`-go-go-Related-Gene (herg) is a potassium channel expressed in human heart. herg encodes for a component of the rapidly repolarising cardiac potassium current (I Kr ) which contributes to the repolarisation of the myocardial cell at the end of the action potential (Ekins et al 2002). Pharmaceuticals that block herg channels cause QT prolongation. This may lead to life-threatening arrhythmias in patients. Cisapride (Prepulsid ) is a prokinetic agent which is commonly used to treat gastroesophageal reflux. This drug has been withdrawn from the US market because it has been associated with serious cardiovascular side effects. It is recognized that Cisapride may lead to life-threatening arrhythmias and sudden death in patients with electrolyte disturbances or when taken in conjunction with other drugs. The reason of these severe side effects is the inhibition of the cardiac herg channel, which may lead to QT prolongation (Mohammad et al., 1997), (Rampe et al., 1997),(Redfern et al., 2003). The beta-adrenergic blocker Sotalol (Betapace ) is used to treat certain types of arrhythmias and arterial hypertension and is indicated as secondary prophylaxis after cardiac infection. Only patients with life-threatening ventricular arrhythmias are allowed to take Sotalol in the United States. Sotalol is marketed as a racemic mixture ((d-l)-sotalol.) The beta-adrenergic blocking effect is solely executed by the l-form. However, the d-form has a scientifically proven effect on the herg ion channel (Numaguchi et al 2000). A clinical trial showed that Sotalol is associated with increased mortality after myocardial infarction (Waldo et al 1996). The H1 receptor antagonist Terfenadine (Seldane ) is an antihistamine. After the discovery that the drug has unwanted side effects on the herg channel (Roy et al 1996), it has been withdrawn from the US market.

3 Cisapride, Sotalol and Terfenadine are examples of marketed pharmaceutical substances causing dangerous side effects. Consequently, they are a high risk for the pharmaceutical industry. The International Committee on Harmonization (ICH), a federation of the pharmaceutical regulatory authorities of Japan, Europe and the US claim for an efficient screening of marketed drugs for determing the effect on the herg channel. Today, the pharmaceutical industry minimizes this risk by screening their drugs for direct interaction with the herg channel. There is thus a new demand for efficient screening methods for pharmaceuticals affecting the herg channel. 3. Aim of the Study Considering the effect of Cisapride, (d-l)-sotalol and Terfenadine on the herg channel in transfected CHO cells as an example, we compared the performance of the CytoPatch instrument with the conventional patch clamp technique. Our obtained IC 50 values were compared with literature data. Furthermore, we determined the success rate of CytoPatch and evaluated seal resistance. 4. Materials Cell culture Chinese Hamster Ovary (CHO) cells expressing the herg channel were purchased from Cytomyx Inc. The cells were grown in adherent culture in T-flakes. 2 to 3 days prior to experiment, the cells were cultivated in petri dishes. Prior to an experiment, the cells were detached by incubation in a calcium-free buffer. Buffer The same buffer was used for the CytoPatch instrument and for manual patch clamp. The extracellular solution consists of (in mm): 140 NaCl, 2.5 KCl, 2 MgCl 2, 2 CaCl 2, 10 HEPES, 10 Glucose, ph 7.4. Osmolarity was adjusted to 320 mosm with sucrose. The intracellular solution consists of (in mm): 100 K-Gluconat, 20 KCl, 1 CaCl 2, 1 MgCl 2, 10 HEPES, 11 EGTA-KOH, 5 ATP-Na 2, 2 GSH, ph 7.2 CytoPatch chips and conventional patch pipettes CytoPatch chips, filled with intra- and extracellular solutions, had a pipette resistance of 8-9 MOhm. For the classic patch clamp experiments we used standard patch pipettes with a pipette resistance of 4-6 MOhm. Data acquisition Conventional patch clamp data were generated with a classic patch clamp setup and analyzed with the patch clamp amplifier EPC10 from HEKA and the corresponding software. Data generated with the CytoPatch instrument were analyzed with the Cytocentrics amplifier and software technology. The sampling frequency of the data analysis hardware inside the CytoPatch

4 instrument is 10 khz. All data presented within this document were filtered at 5 khz with the built-in Bessel Filter of the Cytopatch TM Amplifier. Voltage protocol and data analysis The pulse protocol as displayed below was used for CytoPatch - and for conventional patch clamp experiments. Initially, the cells maintained at a holding potential of 80 mv. Then they were clamped to 50 mv for 200 ms ( baseline step ). Subsequently, the cells were depolarized to +40 mv for 2 s ( inactivation phase ) and finally repolarized to 50 mv ( tail current phase ). The protocol was executed every 30s. Graph 1. Pulse protocol for the evaluation of herg currents. All Whole Cell Recordings show a leak current. This means that the ion channel current even at a total inhibition of the channel is not 0 pa. An established baseline step protocol accounts for changes in leak current. The cell is clamped to the same potential in the baseline step and in the tail current phase. The former resting potential of 80 mv prevents the opening of the herg channel during the baseline step. The values from these measurements were then subtracted from the current at the tail current phase. This leads to an exact evaluation of the leak current. According to this, all data evaluations were performed by subtracting the maximal current amplitude during the tail current phase from the medium current amplitude of the baseline step. The current amplitude after the correction is called Peak Tail current. Series resistance compensation also leads to an improvement of data quality. The series resistance was only compensated at the manual patch clamp setup (compensation of the HEKA amplifier is 70%). Because of a missing software module, we were not able to compensate the series resistance of the CytoPatch instrument. This is planned for the near future and will lead to further improvement of data quality. Compound application The sensitivity of the herg channel was determined by testing different Cisapride concentrations. Cisapride and Terfenadine were purchased from Sigma Aldrich. 1 mm stock solution of Cisapride and 10 nm stock solution of Terfenadine were dissolved in DMSO and stored in 100 μl aliquots at 20 C. Immediately prior to experiment, this stock was melted and diluted in extracellular buffer (1:1000) to a final concentration of 1 μm for Cisapride and 10 nm for Terfenadine. Concentrations of 1 nm, 10 nm and 100 nm were obtained by a step-by-step dilution (1:10) of the stock solution.

5 Terfenadine was additionally tested with concentrations of 3 nm, 30 nm and 300 nm. The maximum concentration of DMSO in solution was 0.1%. (d-l)-sotalol was diluted in extracellular solution to a final concentration of 60 mm. The stock solutions were stored in aliquots at 20 C. immediately prior to experiment, one Sotalol stock solution was melted and diluted in extracellular buffer to a final concentration of 10 mm, 3 mm, 300 und 30 μm. Immediately after going whole cell, the tail current was recorded under continuous perfusion of buffer for 5 min. After this control phase, the application with up to 3 concentrations per compounds starts. The measurement started with the lowest and ended with the highest concentration. Duration of compound application was 5 min. It was extended when the effect of the compound (plateau phase) was not obtained after 5 min. In conventional as well as in automated patch clamp experiments, whole cell configurations last from 20 min up to 1 h. Some cells were stable for as long as 30 min after the third compound application and we were able to perform a washout after this time. IC 50 curve generation The dose-response data were fitted by the equation: 100 y = 1 + m1 m0 ( ) m2 mo: compound concentration, m1: IC 50 value m2: Hill coefficient.

6 5. Results Conventional patch clamp technique The manual patch clamp technique acts as reference for the CytoPatch instrument. The success rate of the conventional patch clamp data showed a good agreement with the general information of the cell supplier (70-80% seal rate, 70% stable whole cells; Cytomyx, 2004). We determined an IC 50 for Cisapride of 6.15 nm, for Sotalol of 281 μm and for Terfenadine of 8.3 nm. The data are in good agreement with the values known from literature. 168 pa pa pa 55. 1k s 56. 5ks 57.9 lks Graph 2. Inhibitory effect of 10 nm Cisapride on the herg channel, measured with the manual patch clamp technique. Blue trace presents the peak tail current, green trace presents the leak current. Graph 3. Inhibitory effect of 10 nm Cisapride on the herg channel, measured with the manual patch clamp technique. Figures present examples of ion channel recordings. Left channel: control phase. Middle channel: effect of Cisapride. Right channel: wash-out. The inhibition of the ion channel is approximately 60%. CytoPatch technique The success rate for stable whole cell recordings performed with the CytoPatch instrument is comparable to data obtained in manual patch clamp experiments (Graph 3). Particularly the cytocentering process, which positions a cell directly above the patch pipette of the chip, leads to a 100% success rate of exactly positioned cells. This is an excellent starting point for seal formation. The high seal rate of the CytoPatch chips (92%) leads to a high rate of stable whole cell recordings. Thus, a total of 75% Successfully Completed Experiments were obtained.

7 Seal resistance and current amplitudes The median seal resistance for data obtained with the CytoPatch instrument (n=11) is 2.26 GOhm. Thus the median value is much higher than the minimal acceptance criteria of 1GΩ. The success rate for stable Whole Cell Recordings (n=9) is in the characteristic range for the CHO cell line (75%). The current amplitude of the herg tail currents is pa (Median 488 pa). The Baseline Step protocol is for data adjustment (see materials). Due to the good data quality, an adjustment of only 5% was necessary. Cumulative Success Rate Seal Resistance Median Seal Resistance: 2.26 GOhm Graph 4 (on the left). Success rates of the CytoPatch instrument. 12 CytoPatch chips were used for this study. A successful giga seal formation was obtained with 11 CytoPatch chips (Seal rate92%). 9 of these 11 chips (82%) showed stable Whole Cell Recording (WCR) lasting longer than 15 min. At least the application of one compound concentration occurs (normally up to 3 compound concentrations = Successfully Completed Experiment, SCE). Graph 5 (on the right) displays the distribution of the seal resistance obtained with the CytoPatch instrument. Examples of whole cell recordings pa Buffer 10nM 100nM Buffer 0, minutes Graph 6. CytoPatch per-formance: Raw data trace shows current of a herg-cho cell. Tail current Tail current (leak corrected) Leak Graph 7. CytoPatch per-formance: Time response during the application of 10 nm and 100 nm Cisapride, followed by a wash-out.

8 Dose response curve Conventional patch clamp data are in a very good agreement with data obtained with the CytoPatch instrument (graph 7,8,9). Sensitivit y to Cisapride Sensitivity to Sotalol Sensitivity to Terfenadine Graph 8: Dose-response curve above show the blocking effect of Cisapride, Sotalol and Terfenadine (from left to right). Red traces show data obtained with the CytoPatch, blue traces show data obtained with the manual patch clamp technique 6. Literature and Discussion IC 50 values for Cisapride, Sotalol and Terfenadine differ considerable from lab to lab (Ekins et al 2002), (Kirsch et al 2004). These differences are due to the use of different cell lines and stimulation protocols. The effect of Sotalol, for example, also depends on the temperature during measurement (Ekins et al., 2002). The table below summarizes the IC 50 values for the three compounds used in this study and displays a comparison of our data with values from literature. 6 5 Conventional Patch Clamp: Blue vertical line: : literature range log c (nm) Blue horizontal line: own data Automated Patch Clamp: 1 Red vertical line : literature range 0 Cisapride Sotalol Terfenadine Red horizontal line : CytoPatch Graph 9. Automatically and manually obtained IC50 values in relation to the literature. Blue vertical line: Literature range for conventional patch clamp, blue horizontal line: Manually obtained data within this study. Red vertical line: Literature range for automated patch clamping, red horizontal line: Data obtained with CytoPatch.

9 When compared to values from literature, CytoPatch IC 50 and our manual obtained IC 50 values are clustered in a lower range. This reflects the sensitivity of our system and is due to the innovative design of the CytoPatch compound application. It permanently perfuses the cell, which leads to an optimal compound concentration in the cell s surrounding. Furthermore, there was a good agreement between IC 50 value obtained with CytoPatch and IC 50 values obtained with the manual Patch clamp technique. Terfenadine is known as a sticky compound which adsorbs to some kinds of materials. This is a high request to the perfusion system and compound handling. Some automated patch clamp systems have no perfusion and perform the patch clamp process on other materials than quartz glass. This leads to false IC 50 values, which tend to be too high and unrealistic. When compared to values from literature, the CytoPatch IC 50 for Terfenadine is within the literature range, with a clear trend to the lowest regions. In summary, CytoPatch combines high quality data generation with a high success rate. This is due to the high quality of the recording system, excellent compound handling and the instrument s rapid perfusion system, which allows exact determination even of sticky compounds.

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