**3.6 Robustness and reproducibility of the new method**

To determine robustness and reproducibility or the assay, we generated 6 independent series of FBMEs (1-2 24-well plates each, total number 192 FBMEs) and analysed them under standard conditions (culture medium with insulin, aprotinin and tranexamic acid; measurements done by video-optical recordings). Two FBMEs could not be transferred from the casting moulds, 4 FBMEs were not recognized by the software and 17 did not beat during the recording time (60 s) at day 15. Thus, the total success rate was 89% (169/192; Figure 8). Contraction parameters were examined and compared among different series. These results showed that the average force per series (day 15) was between 0.11 and 0.22 mN (series SD 7.6%), the contraction time (T1=time to peak) ranged between 66 and 81 ms (series SD 41%), relaxation time (T2=time to 80% relaxation) between 67 and 88 ms (series SD 25%), frequency between 162 and 20 beats per minute (series SD 109%), construct diameter between 1.3 and 1.4 mm (series SD 9.9%) and length between 5.6 and 6.7 mm (series SD 16.7%). The relatively large size in FBME diameter in these examinations could be attributed to the use of tranexamic acid.

Multi-Well Engineered Heart Tissue for Drug Screening and Predictive Toxicology 83

**Series 1**

**Frequency (bpm)**

**Series 2**

**<sup>250</sup>** \*

**Series 3**

**7.5** \* \*

**Series 4**

**Series 5**

**Series 6**

Fig. 8. Reproducibility of the assay. FBMEs were generated at 6 different time points

n=18, series 5: n=39, series 6: n=47 (Hansen et al. 2010).

**3.7 Cardiotoxic and proarrhythmic effects of drugs** 

FBMEs stopped to beat after 3 days.

**Series 1**

**0.00 0.05 0.10 0.15 0.20**

**T1, series 1**

**0.00**

**0.05**

**0.10**

**Time (seconds)**

**0.15**

**T1, series 2**

**T1, series 3**

**T1, series 4**

**T1, series 5**

**T1, series 6**

**T2, series 1**

**T2, series 2**

**T2, series 3**

**T2, series 4**

**T2, series 5**

**T2, series 6**

**Force (mN)**

**Series 2**

\*

**0.25** \*

**A B** 

**Series 3**

**Series 4**

**C D** 

**Series 5**

\*

**Series 6**

(series 1-6) and spontaneous activity was recorded on day 15. Parameters of contractility (A: force, B: frequency, C: contraction time T1, relaxation time T2) and construct dimensions (D) were averaged and compared. Minimal and maximal values were used to test for significant differences and are indicated with \* p<0.05 (Student's t test). Bars show means +/- SD. Analysed FBMEs for each series were: Series 1: n=21, series 2: n=24, series 3: n=20, series 4:

**DM, series 1**

**DM, series 2**

**0.0**

**2.5**

**5.0**

**DM, lenghth (mm)**

**DM, series 3**

**DM, series 5**

**DM, series 6**

**length, series 1**

**length, series**

**length, series 3**

 **2**

**length, series 4**

**length, series**

**length, series 6**

 **5**

**DM, series 4**

To determine whether the new method could be used for the detection of cardiotoxic and proarrhythmic drug effects, well characterized compounds with known cardiotoxic and repolarization-inhibitory effects were tested. The cardiotoxic drug doxorubicin was applied in different concentrations (0.1-1.000 nmol/L) for up to 96 h. Doxorubicin-treated FBMEs showed time- and concentration-dependent changes in contractile force. Very low concentrations of doxorubicin (1-10 nmol/L) led to a trend towards an increase in contractile force, 100 nmol/L induced a transient increase in contractile force after 24 h which was followed by a decrease at 72 and 96 h (Figure 9). In the presence of 1 µmol/L doxorubicin all

To examine a repolarization-inhibitory effect on FBMEs, the experimental IKs-blocker chromanol 293B as well as the clinically used drugs quinidine and erythromycin were



(minK-related peptide 1; KvLQT1; ERG; function: IKs or IKr)

Q1a IKs producing slow voltage-gated channel subunit alpha (KvLQT1)

Family Abbre-

viation

Description

KCN A5 Voltage-gated channel subunit Kv1.5

D3 Voltage-gated channel subunit Kv4.3

E1 Potential voltage-gated channel subunit beta (KvLQT1; ERG; function: IKs or Kr)

E2 Potential voltage-gated channel subunit beta

H2a IKr producing rapid voltage-gated channel subunit beta (ether-a-go-go-related gene (ERG) channel 1)

J3 G protein-activated inward rectifier channel 1 (Kir3.1)

J5 G protein-activated inward rectifier channel 4 (Kir3.4)

J8 ATP-sensitive inward rectifier channel 8 (Kir6.1)

J12 ATP-sensitive inward rectifier channel 12 (Kir2.2)

A1H Voltage-dependent subunit alpha-1H (T-type)

A1I Voltage-dependent subunit alpha-1I (T-type)

B1 Voltage-dependent subunit beta-1 (L-type)

B2 Voltage-dependent subunit beta-2 (L-type)

B3 Voltage-dependent subunit beta-3 (L-type)

B4 Voltage-dependent subunit beta-4 (L-type)

SCN 1A Voltage-gated channel protein type-1 subunit alpha

1B Voltage-gated channel subunit beta-1

4B Voltage-gated channel subunit beta-4

Table 1. Overview of the analysed ion channels shown in figure 7.

3A Voltage-gated channel protein type-3 subunit alpha

4A Voltage-gated channel protein type 4 subunit alpha

5A Voltage-gated channel protein type-5 subunit alpha

CACN A1C Voltage-dependent subunit alpha-1C (L-type)

Fig. 8. Reproducibility of the assay. FBMEs were generated at 6 different time points (series 1-6) and spontaneous activity was recorded on day 15. Parameters of contractility (A: force, B: frequency, C: contraction time T1, relaxation time T2) and construct dimensions (D) were averaged and compared. Minimal and maximal values were used to test for significant differences and are indicated with \* p<0.05 (Student's t test). Bars show means +/- SD. Analysed FBMEs for each series were: Series 1: n=21, series 2: n=24, series 3: n=20, series 4: n=18, series 5: n=39, series 6: n=47 (Hansen et al. 2010).

#### **3.7 Cardiotoxic and proarrhythmic effects of drugs**

To determine whether the new method could be used for the detection of cardiotoxic and proarrhythmic drug effects, well characterized compounds with known cardiotoxic and repolarization-inhibitory effects were tested. The cardiotoxic drug doxorubicin was applied in different concentrations (0.1-1.000 nmol/L) for up to 96 h. Doxorubicin-treated FBMEs showed time- and concentration-dependent changes in contractile force. Very low concentrations of doxorubicin (1-10 nmol/L) led to a trend towards an increase in contractile force, 100 nmol/L induced a transient increase in contractile force after 24 h which was followed by a decrease at 72 and 96 h (Figure 9). In the presence of 1 µmol/L doxorubicin all FBMEs stopped to beat after 3 days.

To examine a repolarization-inhibitory effect on FBMEs, the experimental IKs-blocker chromanol 293B as well as the clinically used drugs quinidine and erythromycin were

Multi-Well Engineered Heart Tissue for Drug Screening and Predictive Toxicology 85

**T1, baseline**

**T1, baseline**

**0.0 0.1 0.2 0.3 0.4 0.5 0.6**

**T1, T2 (sec)**

**T1, 1**

**T1, 10**

**T1, 100**

**T1, 1000**

**T2, baseline**

Erythromycin (µM)

**T2, 1**

**T2, 10**

**T2, 100**

**T2, 1000**

\*

**0.0 0.1 0.2**

**0.4 0.6 0.8 1.0**

**T1, T2 (sec)**

 **T1, 0.1**

**T1, 1**

**T1, 10**

**T1, 100**

**T2, baseline**

Chromanol 293B (µM)

**T2, 0.1**

**T2, 1**

**T2, 10**

**T2, 100**

\*

Fig. 11. Effect of repolarisation inhibitors on FBME contraction (T1) and relaxation time (T2). FBMEs were incubated with increasing concentrations of the indicated compounds (1-2 h) and evaluated before application of drug (baseline) and after each concentration. Note the absence of effect of all compounds on T1 and the concentration-dependent increase in T2 with chromanol, quinidine and erythromycin (at 1000 µM of erythromycin FBMEs

discontinued contractile activity). A typical alteration of contraction peak morphology with

In this book chapter we describe a recently developed method (Hansen et al. 2010) to generate miniaturized, fibrin-based EHTs (FBMEs) in a 24-well format and determine their contractile activity in an automated manner. This technique turned out to be robust and highly reproducible. Its main advantages are its simplicity in terms of handling, the standard 24-well format, its robustness and the high content automated readout of contractile activity. Compared to previously EHT-protocols (Eschenhagen et al. 1997, Zimmermann et al. 2002), three major changes were introduced. (i) Collagen I was replaced by fibrinogen and thrombin. Due to the fast fibrin-polymerisation, the heart cells were homogenously distributed throughout the entire hydrogel. Polymerisation occurs in minutes and allows transfer of the constructs from the casting moulds to a new mediumfilled culture dish after two hours. Moreover, fast solidification allowed 50% higher cell concentration (0.6x106/150 µl versus 2.5x106/900 µl), because it prevents accumulation of

increasing concentrations of quinidine, chromanol and erythromycin is shown in supplementary figure 5. \* p<0.05 (Student's t test). Bars show means +/- SEM, each spot

represents one analysed FBME (Hansen et al. 2010).

**4. Conclusion** 

**T1 baseline**

**baseline**

**0.0 0.1 0.2 0.3 0.4 0.5 0.6**

**T1, T2 (sec)**

**T1 0.1**

**T1 1.0**

**T1 10**

**T1 100**

**baseline**

Quinidine (µM)

**T2 0.1**

**T2 1.0**

**T2 10**

**T2 100**

\*

**0.0 0.1 0.2 0.3 0.4 0.5 0.6**

**T1, T2 (sec)**

**T1 d1**

**T1 d2**

**T1 d3**

**T1 d4**

**T2 baseline**

Control

**T2 d1**

**T2 d2**

**T2 d3**

**T2 d4**

Fig. 9. Doxorubicin toxicity on FBMEs. FBMEs were incubated in the presence of doxorubicin (0.1-1000 nM, starting at day 13 of culture), average forces were determined daily. While doxorubicin at 0.1 µM increased force after 24 hours, higher concentrations (1 µM) lead to a time-dependent reduction in force development. \* p<0.05 (Student's t test). Bars show means +/- SEM, number of evaluated (beating) constructs as indicated (Hansen et al. 2010).

tested. All three compounds induced a concentration-dependent delay in relaxation time (T2; Figure 10). In the presence of chromanol 293B, FBMEs already showed a prolongation of T2 at a concentration of 1 µmol/L. At 100 µmol/L chromanol, T2 was 7-fold longer than control, resulting in a "church-like" configuration of the twith (Figure 10). Quinidine and erythromycin, both associated with arrhythmias in clinical applications, also extended the relaxation time at high concentrations (100 µmol/L). Time of contraction was not affected by any of the compounds.

Fig. 10. Chromanol 293B-induced "church-like" contraction pattern. Cutout of the original contraction recordings in the presence and absence of Chromanol 293B (100 µM; modified from: Hansen et al. 2010).

Fig. 11. Effect of repolarisation inhibitors on FBME contraction (T1) and relaxation time (T2). FBMEs were incubated with increasing concentrations of the indicated compounds (1-2 h) and evaluated before application of drug (baseline) and after each concentration. Note the absence of effect of all compounds on T1 and the concentration-dependent increase in T2 with chromanol, quinidine and erythromycin (at 1000 µM of erythromycin FBMEs discontinued contractile activity). A typical alteration of contraction peak morphology with increasing concentrations of quinidine, chromanol and erythromycin is shown in supplementary figure 5. \* p<0.05 (Student's t test). Bars show means +/- SEM, each spot represents one analysed FBME (Hansen et al. 2010).

#### **4. Conclusion**

84 Toxicity and Drug Testing

**Baseline 24 h 48 h 72 h 96 h**

\*

Doxorubicin (0.1 nM)

44444

**Baseline 24 h 48 h 72 h 96 h**

**Baseline 24 h 48 h 72 h 96 h**

441

Doxorubicin (1000 nM)

44444

Doxorubicin (1 nM)

**Force (% of baseline)**

**Force (% of baseline)**

**Force (% of baseline)**

**Force (% of baseline)**

Fig. 9. Doxorubicin toxicity on FBMEs. FBMEs were incubated in the presence of doxorubicin (0.1-1000 nM, starting at day 13 of culture), average forces were determined daily. While doxorubicin at 0.1 µM increased force after 24 hours, higher concentrations (1 µM) lead to a time-dependent reduction in force development. \* p<0.05 (Student's t test). Bars show means

**Baseline 24 h 48 h 72 h 96 h**

4 3 4 4

Doxorubicin (100 nM)

tested. All three compounds induced a concentration-dependent delay in relaxation time (T2; Figure 10). In the presence of chromanol 293B, FBMEs already showed a prolongation of T2 at a concentration of 1 µmol/L. At 100 µmol/L chromanol, T2 was 7-fold longer than control, resulting in a "church-like" configuration of the twith (Figure 10). Quinidine and erythromycin, both associated with arrhythmias in clinical applications, also extended the relaxation time at high concentrations (100 µmol/L). Time of contraction was not affected by

Fig. 10. Chromanol 293B-induced "church-like" contraction pattern. Cutout of the original contraction recordings in the presence and absence of Chromanol 293B (100 µM; modified

+/- SEM, number of evaluated (beating) constructs as indicated (Hansen et al. 2010).

any of the compounds.

**Baseline 24 h 48 h 72 h 96 h**

3 3333

Control

**Baseline 24 h 48 h 72 h 96 h**

Doxorubicin (10 nM)

44444

**Force (% of baseline)**

**Force (% of baseline)**

from: Hansen et al. 2010).

In this book chapter we describe a recently developed method (Hansen et al. 2010) to generate miniaturized, fibrin-based EHTs (FBMEs) in a 24-well format and determine their contractile activity in an automated manner. This technique turned out to be robust and highly reproducible. Its main advantages are its simplicity in terms of handling, the standard 24-well format, its robustness and the high content automated readout of contractile activity. Compared to previously EHT-protocols (Eschenhagen et al. 1997, Zimmermann et al. 2002), three major changes were introduced. (i) Collagen I was replaced by fibrinogen and thrombin. Due to the fast fibrin-polymerisation, the heart cells were homogenously distributed throughout the entire hydrogel. Polymerisation occurs in minutes and allows transfer of the constructs from the casting moulds to a new mediumfilled culture dish after two hours. Moreover, fast solidification allowed 50% higher cell concentration (0.6x106/150 µl versus 2.5x106/900 µl), because it prevents accumulation of

Multi-Well Engineered Heart Tissue for Drug Screening and Predictive Toxicology 87

~4. Additionally, pilot experiments could show that a further miniaturization to a 96-well format would be possible. At this point the 24-well format turned out to be a good compromise between miniaturization and ease of handling. FBMEs do not longer need to be manually transferred as single tissues because the entire silicon racks were handled. In contrast to that, circular EHTs needed to be manually transferred from the casting mould to a motorized stretcher and finally from the stretcher to the organ bath for the measurements. Compared to this method, the video-optical recordings were simple and robust. To confirm the calculated forces, isometric measurements were exemplarily done in the organ bath in parallel and showed that the calculated forces were roughly in the range of the measured ones (0.3 vs. 0.9 mN). The threefold difference could indicate a systematic error, but more likely reflects optimized conditions in the organ bath (preload optimization, electrical pacing and isometric versus auxotonic contraction). A force of 0.9 mN (organ bath) and a diameter of 0.2 mm (Figure 4A; without tranexamic acid) results in a relative force development of 28.7 mN/mm2. This is still lower than values in an intact muscle (50 mN/mm2), but point in the right direction. The development of a software which works by automated figure recognition was important in two aspects. On the one hand, it is timesaving since it calculates all important contractile parameters automatically. On the other

Initial experiments to determine the suitability of FBMEs to detect either cardiotoxic or proarrhythmic effects looked promising. The known cardiotoxic drug doxorubicin suppressed contractile activity at the highest concentration (1.000 nmol/L) and increased force at lower concentrations (100 nmol/L) and earlier time points (24 h). This may be due to the reactive oxygen species-generating effect of this compound, which at later time points

Three known proarrhythmic drugs caused a concentration-dependent prolongation of relaxation time in FBMEs. Chromanol 293B is an experimental IKs-blocker with an IC50-value of 8 µmol/L in H9c2 cells (Lo et al. 2005). A concentration of 100 µmol/L was needed to fully block IKs in these cells. This correspond well to the relaxation-prolonging effect in FBMEs, which was visible at 10 µmol/L and marked at 100 µmol/L (Figure 10) matching quite well the published data (Lo et al. 2005). Quinidine, known as a class IA antiarrhythmic drug according to Vaughan-Williams classification (Vaughan-Williams 1975), also blocks IKs and IKr with EC50 values of 10 µmol/L and 300 nmol/L, respectively (Lo et al. 2005; Redfern et al. 2003). In FBMEs, quinidine prolonged relaxation time at 100 µmol/L. This is not an entirely different range in comparison with the determined IC50 for quinidine on IKs but far away from IKr-IC50. This argues again for the role of IKs in FBME-repolarisation. Erythromycin, a clinically used antibiotic, which is associated with *Torsades-de-Pointes* arrhythmias and increased lethality in men (Ray et al. 2004), inhibits IKr but not IKs in dogs (Antzelevitch et al. 1996) and increases action potential duration in rat ventricular cardiomyocytes (IC50 60 µmol/L; Hanada et al. 2003). In FBMEs erythromycin caused an increase in relaxation time at concentrations of 100 µmol/L and above. Even though the channel subunits for both IKr and IKs are expressed on mRNA-level in FBMEs (Figure 7), their role in repolarisation of rat cardiomyocytes is still controversial (Regan et al. 2005). In any case, the observation that the prototype proarrhythmic drugs specifically affected relaxation time in FBMEs indicates that this parameter may be useful as a surrogate of repolarisation. However, the detailed mechanisms behind the cardiotoxic and proarrhythmic effects are still unclear and need to be further investigated in follow up

hand, it provides an objective readout.

studies.

and/or higher concentrations turned into toxicity.

cells at the bottom of the casting moulds with detrimental consequences. Fibrin has additional advantages for future applications, such as transplantations. It is available from autologoues sources and has the ability to couple covalently growth-promoting, angiogenic or other interesting factors (Hubble 2003). (ii) The original ring-format was changed to a stripe-format. This step was very important because it allowed miniaturization (volume reduced from 900 to 150 µl), the use of standard 24-well plates and automation. The silicone racks with 4 pairs of posts each allow simple transfer of FBMEs from one plate to another and thus reduce the number of nonstandardized handling steps to a minimum. Moreover, the silicone posts subject the growing muscle construct to an individually optimized preload and allow them to perform contractile work against the elastic properties of the post. (iii) Video-optical measurements of contractile parameters further improved the whole technique. They superseded the manual transfer for measurements as it was required in former EHT-protocols. Additionally, video-optical recordings allowed simple, reproducible, standardized measurements of large series in a short spell.

The new stripe-format was inspired by a previously described method for generating skeletal muscle tissues (Vandenburgh et al. 2008). Turning the silicone posts up side down was the major difference to this method and simplified handling. In this way the quality of the constructs could be determine with an ordinary microscope or even by eye and opened the possibility for automated video-optical recordings from above the plate. This experimental setup, in combination with fibrin, has several important features. (i) It is simple and not longer addicted to some kind of special dish because standard 24-well cell culture plates can be used. In the beginning silicone posts racks were self-made, which turned out to relevantly limit reproducibility. We therefore have the racks industrial made now, which makes the method robust and highly reproducible (Figure 8). (ii) FBMEs exhibit an excellent cardiac tissue development (Figure 4). Several factors are likely to contribute to these beneficial effects. As outlined above, the fast polymerisation of fibrin allowed higher cell concentrations and lead to homogenous cell distribution throughout the hydrogel. Another reason was that cell survival appeared very high in the current fibrin-based format. Moreover, proliferative activity (of non-myocytes) was very low (Figure 5), making the system stable over prolonged periods. The stable mRNA expression pattern of cardiac markers (α-actinin, α-MHC, titin, calsequestrin 2 and SERCA) may be an additional hint for a relatively stable system. Interestingly, the fetal gene marker β-MHC showed a several fold decrease within in the first six days. This phase was associated with cell spreading and contractions on a cellular level. Later, between day 6 and day 9 there was a ~5-fold increase of the β-MHC mRNA expression, coinciding with the beginning of macroscopic contractions. A third positive effect of the described new technique is the optimized mechanical load on the cardiomyocytes. The mechanical load is produced by the resistance of the silicon posts and adapted individually for each FBME. If the tonic (diastolic force) of the construct is high, post deflection by the FBMEs is stronger and *vice versa*. Furthermore, the FBMEs can perform contractile work against an elastic resistance. These circumstances mimic in some ways the *in vivo* situation in which the beating heart needs to work against the afterload induced by the total peripheric resistance. This kind of optimized "auxotonic" load in contrast to motorized phasic stretch improves tissue development as shown earlier in other EHT-studies (Zimmermann et al. 2006).

Regarding the development of a drug screening, miniaturization, reduction of nonstandardized steps during the procedure and an automated, objective readout were very important. The miniaturization to a 24-well format reduced the cell number by a factor of

cells at the bottom of the casting moulds with detrimental consequences. Fibrin has additional advantages for future applications, such as transplantations. It is available from autologoues sources and has the ability to couple covalently growth-promoting, angiogenic or other interesting factors (Hubble 2003). (ii) The original ring-format was changed to a stripe-format. This step was very important because it allowed miniaturization (volume reduced from 900 to 150 µl), the use of standard 24-well plates and automation. The silicone racks with 4 pairs of posts each allow simple transfer of FBMEs from one plate to another and thus reduce the number of nonstandardized handling steps to a minimum. Moreover, the silicone posts subject the growing muscle construct to an individually optimized preload and allow them to perform contractile work against the elastic properties of the post. (iii) Video-optical measurements of contractile parameters further improved the whole technique. They superseded the manual transfer for measurements as it was required in former EHT-protocols. Additionally, video-optical recordings allowed simple, reproducible,

The new stripe-format was inspired by a previously described method for generating skeletal muscle tissues (Vandenburgh et al. 2008). Turning the silicone posts up side down was the major difference to this method and simplified handling. In this way the quality of the constructs could be determine with an ordinary microscope or even by eye and opened the possibility for automated video-optical recordings from above the plate. This experimental setup, in combination with fibrin, has several important features. (i) It is simple and not longer addicted to some kind of special dish because standard 24-well cell culture plates can be used. In the beginning silicone posts racks were self-made, which turned out to relevantly limit reproducibility. We therefore have the racks industrial made now, which makes the method robust and highly reproducible (Figure 8). (ii) FBMEs exhibit an excellent cardiac tissue development (Figure 4). Several factors are likely to contribute to these beneficial effects. As outlined above, the fast polymerisation of fibrin allowed higher cell concentrations and lead to homogenous cell distribution throughout the hydrogel. Another reason was that cell survival appeared very high in the current fibrin-based format. Moreover, proliferative activity (of non-myocytes) was very low (Figure 5), making the system stable over prolonged periods. The stable mRNA expression pattern of cardiac markers (α-actinin, α-MHC, titin, calsequestrin 2 and SERCA) may be an additional hint for a relatively stable system. Interestingly, the fetal gene marker β-MHC showed a several fold decrease within in the first six days. This phase was associated with cell spreading and contractions on a cellular level. Later, between day 6 and day 9 there was a ~5-fold increase of the β-MHC mRNA expression, coinciding with the beginning of macroscopic contractions. A third positive effect of the described new technique is the optimized mechanical load on the cardiomyocytes. The mechanical load is produced by the resistance of the silicon posts and adapted individually for each FBME. If the tonic (diastolic force) of the construct is high, post deflection by the FBMEs is stronger and *vice versa*. Furthermore, the FBMEs can perform contractile work against an elastic resistance. These circumstances mimic in some ways the *in vivo* situation in which the beating heart needs to work against the afterload induced by the total peripheric resistance. This kind of optimized "auxotonic" load in contrast to motorized phasic stretch improves tissue development as shown earlier

Regarding the development of a drug screening, miniaturization, reduction of nonstandardized steps during the procedure and an automated, objective readout were very important. The miniaturization to a 24-well format reduced the cell number by a factor of

standardized measurements of large series in a short spell.

in other EHT-studies (Zimmermann et al. 2006).

~4. Additionally, pilot experiments could show that a further miniaturization to a 96-well format would be possible. At this point the 24-well format turned out to be a good compromise between miniaturization and ease of handling. FBMEs do not longer need to be manually transferred as single tissues because the entire silicon racks were handled. In contrast to that, circular EHTs needed to be manually transferred from the casting mould to a motorized stretcher and finally from the stretcher to the organ bath for the measurements. Compared to this method, the video-optical recordings were simple and robust. To confirm the calculated forces, isometric measurements were exemplarily done in the organ bath in parallel and showed that the calculated forces were roughly in the range of the measured ones (0.3 vs. 0.9 mN). The threefold difference could indicate a systematic error, but more likely reflects optimized conditions in the organ bath (preload optimization, electrical pacing and isometric versus auxotonic contraction). A force of 0.9 mN (organ bath) and a diameter of 0.2 mm (Figure 4A; without tranexamic acid) results in a relative force development of 28.7 mN/mm2. This is still lower than values in an intact muscle (50 mN/mm2), but point in the right direction. The development of a software which works by automated figure recognition was important in two aspects. On the one hand, it is timesaving since it calculates all important contractile parameters automatically. On the other hand, it provides an objective readout.

Initial experiments to determine the suitability of FBMEs to detect either cardiotoxic or proarrhythmic effects looked promising. The known cardiotoxic drug doxorubicin suppressed contractile activity at the highest concentration (1.000 nmol/L) and increased force at lower concentrations (100 nmol/L) and earlier time points (24 h). This may be due to the reactive oxygen species-generating effect of this compound, which at later time points and/or higher concentrations turned into toxicity.

Three known proarrhythmic drugs caused a concentration-dependent prolongation of relaxation time in FBMEs. Chromanol 293B is an experimental IKs-blocker with an IC50-value of 8 µmol/L in H9c2 cells (Lo et al. 2005). A concentration of 100 µmol/L was needed to fully block IKs in these cells. This correspond well to the relaxation-prolonging effect in FBMEs, which was visible at 10 µmol/L and marked at 100 µmol/L (Figure 10) matching quite well the published data (Lo et al. 2005). Quinidine, known as a class IA antiarrhythmic drug according to Vaughan-Williams classification (Vaughan-Williams 1975), also blocks IKs and IKr with EC50 values of 10 µmol/L and 300 nmol/L, respectively (Lo et al. 2005; Redfern et al. 2003). In FBMEs, quinidine prolonged relaxation time at 100 µmol/L. This is not an entirely different range in comparison with the determined IC50 for quinidine on IKs but far away from IKr-IC50. This argues again for the role of IKs in FBME-repolarisation. Erythromycin, a clinically used antibiotic, which is associated with *Torsades-de-Pointes* arrhythmias and increased lethality in men (Ray et al. 2004), inhibits IKr but not IKs in dogs (Antzelevitch et al. 1996) and increases action potential duration in rat ventricular cardiomyocytes (IC50 60 µmol/L; Hanada et al. 2003). In FBMEs erythromycin caused an increase in relaxation time at concentrations of 100 µmol/L and above. Even though the channel subunits for both IKr and IKs are expressed on mRNA-level in FBMEs (Figure 7), their role in repolarisation of rat cardiomyocytes is still controversial (Regan et al. 2005). In any case, the observation that the prototype proarrhythmic drugs specifically affected relaxation time in FBMEs indicates that this parameter may be useful as a surrogate of repolarisation. However, the detailed mechanisms behind the cardiotoxic and proarrhythmic effects are still unclear and need to be further investigated in follow up studies.

Multi-Well Engineered Heart Tissue for Drug Screening and Predictive Toxicology 89

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stretch of engineered heart tissue induces hypertrophy and functional

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The presented technique still has a number of important limitations with regard to drug screening. (i) The cell preparation cannot be fully standardized. In particular, the age of newborn rats varies from 0-3 days and has significant impact of the quality of FBMEs, most likely explaining part of the variability between series (Figure 6). (ii) The system is not well suited to determine acute positive or negative inotropic effects because measurements are done under spontaneous beating, leading to confounding effects of concomitant negative or positive chronotropic effects. We are working on a system which allows measurements to be done under continuous pacing. (iii) Histological observations showed spindle-shaped cardiomyocytes and a predominantly lateral orientation of connexion-43-postive gap junctions. This suggests that cardiomyocytes, despite functional, molecular and morphological indices of advanced maturation, do not reach an adult phenotype. (iv) Our assay system exclusively monitors alterations in contractile activity and does not directly determine calcium transients or action potential duration. We believe that relaxation time is a good surrogate parameter of action potential, but the direct proof is still lacking. (v) Finally, rodents are known to be poor models for detecting proarrhythmic drug effects because mechanisms governing their action potential differ considerably from that in humans. For example, IKr plays a relatively minor role in rodents, but a major one in humans (Regan et al. 2005). Our present results suggest that proarrhythmic drug effects can still be monitored in this system, but much more work is necessary to determine which ion channel or combination of ion channels have to be blocked to see changes in relaxation time and/or arrhythmias in FBMEs.

Thus, validation of the new system will require testing of a large number of drugs that are known to cause cardiac arrhythmias in humans and those that are known to be free of arrhythmic side effects, including those that have effects on HERG but are not associated with *Torsades-de-Pointes* arrhythmias. Moreover, a number of randomly chosen new chemical entities should be analysed to obtain an estimate how many non selected compounds give a signal. These studies are currently under way.

#### **5. References**


The presented technique still has a number of important limitations with regard to drug screening. (i) The cell preparation cannot be fully standardized. In particular, the age of newborn rats varies from 0-3 days and has significant impact of the quality of FBMEs, most likely explaining part of the variability between series (Figure 6). (ii) The system is not well suited to determine acute positive or negative inotropic effects because measurements are done under spontaneous beating, leading to confounding effects of concomitant negative or positive chronotropic effects. We are working on a system which allows measurements to be done under continuous pacing. (iii) Histological observations showed spindle-shaped cardiomyocytes and a predominantly lateral orientation of connexion-43-postive gap junctions. This suggests that cardiomyocytes, despite functional, molecular and morphological indices of advanced maturation, do not reach an adult phenotype. (iv) Our assay system exclusively monitors alterations in contractile activity and does not directly determine calcium transients or action potential duration. We believe that relaxation time is a good surrogate parameter of action potential, but the direct proof is still lacking. (v) Finally, rodents are known to be poor models for detecting proarrhythmic drug effects because mechanisms governing their action potential differ considerably from that in humans. For example, IKr plays a relatively minor role in rodents, but a major one in humans (Regan et al. 2005). Our present results suggest that proarrhythmic drug effects can still be monitored in this system, but much more work is necessary to determine which ion channel or combination of ion channels have to be blocked to see changes in relaxation time and/or

Thus, validation of the new system will require testing of a large number of drugs that are known to cause cardiac arrhythmias in humans and those that are known to be free of arrhythmic side effects, including those that have effects on HERG but are not associated with *Torsades-de-Pointes* arrhythmias. Moreover, a number of randomly chosen new chemical entities should be analysed to obtain an estimate how many non selected

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compounds give a signal. These studies are currently under way.

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**5** 

**Prediction of Partition Coefficients** 

**Partition Coefficients** 

*1University of North Texas, 2University College London,* 

> *1United States 2United Kingdom*

**and Permeability of Drug Molecules** 

**in Biological Systems with Abraham Model Solute Descriptors Derived from Measured** 

**Solubilities and Water-to-Organic Solvent** 

William E. Acree, Jr.1, Laura M. Grubbs1 and Michael H. Abraham2

Modern drug testing and design includes experimental *in vivo* and *in vitro* measurements, combined with *in silico* computations that enable prediction of the drug candidate's ADMET (adsorption, distribution, metabolism, elimination and toxicity) properties in the early stages of drug discovery. Recent estimates place the discovery and development cost of a small drug molecule close to US \$1.3 billion, from the time of inception to the time when the drug finally reaches the market place. Only 20 % of conceived drug candidates proceed to clinical trial stage testing, and of the compounds that enter clinical development less than 10 % receive government approval. Reasons for the low success rate include unsatisfactory efficacy, poor solubility, poor bioavailability, unfavorable pharmacokinetic properties, toxicity concerns and drug-drug interactions, degradation and poor shelf-life stability. Unfavorable pharmacokinetic and ADME properties, toxicity and adverse side effects account for up to two-thirds of drug failures. Traditional ADME analyses relied heavily on whole animal assays and the more labor intensive biochemical studies. High throughput screening methods, fast ADMET profiling assays, and computational approaches have allowed the pharmaceutical industry to identify quickly the less promising drug candidates in the very early development stage so that time and valuable resources are not spent

pursuing compounds that have little probability of reaching the general population.

Of the fore-mentioned properties, the drug's aqueous solubility will likely be one of the first properties measured. Aqueous solubility is a major indicator of the drug's solubility in physiological gastrointestinal fluids and is a major indicator of the drug's oral bioavailability. Approximately 40 % of the proposed new pharmaceutical candidates are rejected in the very early stages of drug discovery because of their poor aqueous solubility resulting in bioavailability problems (Lukyanov and Torchilin, 2004; Keck *et al.*, 2008). The

**1. Introduction** 

