Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorks
The safety implications of blocking the human cardiac Na + channel (hNav1.5) make it prudent to test for this activity early in the drug discovery process and design-out any potential liability. This needs a method with adequate throughput and a demonstrable predictive value to effects in native car...
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| creator | Harmer, A.R. Abi-Gerges, N. Easter, A. Woods, A. Lawrence, C.L. Small, B.G. Valentin, J.-P. Pollard, C.E. |
| description | The safety implications of blocking the human cardiac Na
+ channel (hNav1.5) make it prudent to test for this activity early in the drug discovery process and design-out any potential liability. This needs a method with adequate throughput and a demonstrable predictive value to effects in native cardiac tissues. Here we describe the validation of a method that combines the ability to screen tens of compounds a day, with direct assessment of channel function.
The electrophysiological and pharmacological properties of hNav1.5 were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks™ HT). A pharmacological comparison was also made between IonWorks™ HT and canine cardiac Purkinje Fibre action potential upstroke data.
Activation curve parameters for hNav1.5 in IonWorks™ HT were not statistically different (
p
>
0.05) from those generated using conventional electrophysiology. IonWorks™ HT
V
1/2
=
−
22
±
0.8 mV, slope
=
6.9
±
0.2 (
n
=
11); conventional electrophysiology
V
1/2
=
−
20
±1.6 mV, slope
=
6.4
±
0.3 (
n
=
11). Potency values for a range of hNav1.5 blockers determined using IonWorks™ HT correlated closely with those obtained using conventional electrophysiology (
R
=
0.967,
p
<
0.001). The assay was able to distinguish between highly use-dependent blockers (e.g. tetracaine) and blockers that do not display strong use-dependence (e.g. quinidine). Comparison of the degree of hNav1.5 inhibition and decrease in canine Purkinje fibre action potential upstroke velocity (
V
max) showed that the IonWorks™ HT assay would have predicted the outcome in Purkinje fibres in the majority of cases, with false negative and positive rates estimated at 8 and 7%, respectively. Finally, hNav1.5 pharmacology was similar when determined using either IonWorks™ HT or IonWorks™ Quattro, although the latter yielded more consistent data.
The assay described combines a functional assessment of hNav1.5 with medium-throughput. Furthermore the assay was able to reveal information on the use-dependency of compound block, as well as predicting Na
+ channel effects in more integrated systems such as the cardiac Purkinje fibre action potential. This makes it possible to determine quantitative potency data, and mechanistic information about use-dependence, in a timeframe short enough to influence medicinal chemistry. |
| doi_str_mv | 10.1016/j.vascn.2007.09.002 |
| format | Article |
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+ channel (hNav1.5) make it prudent to test for this activity early in the drug discovery process and design-out any potential liability. This needs a method with adequate throughput and a demonstrable predictive value to effects in native cardiac tissues. Here we describe the validation of a method that combines the ability to screen tens of compounds a day, with direct assessment of channel function.
The electrophysiological and pharmacological properties of hNav1.5 were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks™ HT). A pharmacological comparison was also made between IonWorks™ HT and canine cardiac Purkinje Fibre action potential upstroke data.
Activation curve parameters for hNav1.5 in IonWorks™ HT were not statistically different (
p
>
0.05) from those generated using conventional electrophysiology. IonWorks™ HT
V
1/2
=
−
22
±
0.8 mV, slope
=
6.9
±
0.2 (
n
=
11); conventional electrophysiology
V
1/2
=
−
20
±1.6 mV, slope
=
6.4
±
0.3 (
n
=
11). Potency values for a range of hNav1.5 blockers determined using IonWorks™ HT correlated closely with those obtained using conventional electrophysiology (
R
=
0.967,
p
<
0.001). The assay was able to distinguish between highly use-dependent blockers (e.g. tetracaine) and blockers that do not display strong use-dependence (e.g. quinidine). Comparison of the degree of hNav1.5 inhibition and decrease in canine Purkinje fibre action potential upstroke velocity (
V
max) showed that the IonWorks™ HT assay would have predicted the outcome in Purkinje fibres in the majority of cases, with false negative and positive rates estimated at 8 and 7%, respectively. Finally, hNav1.5 pharmacology was similar when determined using either IonWorks™ HT or IonWorks™ Quattro, although the latter yielded more consistent data.
The assay described combines a functional assessment of hNav1.5 with medium-throughput. Furthermore the assay was able to reveal information on the use-dependency of compound block, as well as predicting Na
+ channel effects in more integrated systems such as the cardiac Purkinje fibre action potential. This makes it possible to determine quantitative potency data, and mechanistic information about use-dependence, in a timeframe short enough to influence medicinal chemistry.</description><identifier>ISSN: 1056-8719</identifier><identifier>EISSN: 1873-488X</identifier><identifier>DOI: 10.1016/j.vascn.2007.09.002</identifier><identifier>PMID: 17980627</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Animals ; Biophysical Phenomena ; Biophysics ; Canine ; CHO Cells ; Cricetinae ; Cricetulus ; Dogs ; Dose-Response Relationship, Drug ; Drug Evaluation, Preclinical - methods ; Electrophysiology ; Female ; hNav1.5 ; Human ; Humans ; Inhibitory Concentration 50 ; IonWorks ; Male ; Membrane Potentials ; Methods ; Muscle Proteins - antagonists & inhibitors ; Muscle Proteins - metabolism ; NAV1.5 Voltage-Gated Sodium Channel ; Predictive Value of Tests ; Purkinje Fibers - drug effects ; Reproducibility of Results ; SCN5a ; Sodium Channel Blockers - pharmacology ; Sodium Channels - metabolism</subject><ispartof>Journal of pharmacological and toxicological methods, 2008, Vol.57 (1), p.30-41</ispartof><rights>2007 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c303t-9a2c6372c78c591a59f50a9915731068b313651e29ea0712928da8edc4690ee83</citedby><cites>FETCH-LOGICAL-c303t-9a2c6372c78c591a59f50a9915731068b313651e29ea0712928da8edc4690ee83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S1056871907002493$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,4010,27900,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17980627$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Harmer, A.R.</creatorcontrib><creatorcontrib>Abi-Gerges, N.</creatorcontrib><creatorcontrib>Easter, A.</creatorcontrib><creatorcontrib>Woods, A.</creatorcontrib><creatorcontrib>Lawrence, C.L.</creatorcontrib><creatorcontrib>Small, B.G.</creatorcontrib><creatorcontrib>Valentin, J.-P.</creatorcontrib><creatorcontrib>Pollard, C.E.</creatorcontrib><title>Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorks</title><title>Journal of pharmacological and toxicological methods</title><addtitle>J Pharmacol Toxicol Methods</addtitle><description>The safety implications of blocking the human cardiac Na
+ channel (hNav1.5) make it prudent to test for this activity early in the drug discovery process and design-out any potential liability. This needs a method with adequate throughput and a demonstrable predictive value to effects in native cardiac tissues. Here we describe the validation of a method that combines the ability to screen tens of compounds a day, with direct assessment of channel function.
The electrophysiological and pharmacological properties of hNav1.5 were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks™ HT). A pharmacological comparison was also made between IonWorks™ HT and canine cardiac Purkinje Fibre action potential upstroke data.
Activation curve parameters for hNav1.5 in IonWorks™ HT were not statistically different (
p
>
0.05) from those generated using conventional electrophysiology. IonWorks™ HT
V
1/2
=
−
22
±
0.8 mV, slope
=
6.9
±
0.2 (
n
=
11); conventional electrophysiology
V
1/2
=
−
20
±1.6 mV, slope
=
6.4
±
0.3 (
n
=
11). Potency values for a range of hNav1.5 blockers determined using IonWorks™ HT correlated closely with those obtained using conventional electrophysiology (
R
=
0.967,
p
<
0.001). The assay was able to distinguish between highly use-dependent blockers (e.g. tetracaine) and blockers that do not display strong use-dependence (e.g. quinidine). Comparison of the degree of hNav1.5 inhibition and decrease in canine Purkinje fibre action potential upstroke velocity (
V
max) showed that the IonWorks™ HT assay would have predicted the outcome in Purkinje fibres in the majority of cases, with false negative and positive rates estimated at 8 and 7%, respectively. Finally, hNav1.5 pharmacology was similar when determined using either IonWorks™ HT or IonWorks™ Quattro, although the latter yielded more consistent data.
The assay described combines a functional assessment of hNav1.5 with medium-throughput. Furthermore the assay was able to reveal information on the use-dependency of compound block, as well as predicting Na
+ channel effects in more integrated systems such as the cardiac Purkinje fibre action potential. This makes it possible to determine quantitative potency data, and mechanistic information about use-dependence, in a timeframe short enough to influence medicinal chemistry.</description><subject>Animals</subject><subject>Biophysical Phenomena</subject><subject>Biophysics</subject><subject>Canine</subject><subject>CHO Cells</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>Dogs</subject><subject>Dose-Response Relationship, Drug</subject><subject>Drug Evaluation, Preclinical - methods</subject><subject>Electrophysiology</subject><subject>Female</subject><subject>hNav1.5</subject><subject>Human</subject><subject>Humans</subject><subject>Inhibitory Concentration 50</subject><subject>IonWorks</subject><subject>Male</subject><subject>Membrane Potentials</subject><subject>Methods</subject><subject>Muscle Proteins - antagonists & inhibitors</subject><subject>Muscle Proteins - metabolism</subject><subject>NAV1.5 Voltage-Gated Sodium Channel</subject><subject>Predictive Value of Tests</subject><subject>Purkinje Fibers - drug effects</subject><subject>Reproducibility of Results</subject><subject>SCN5a</subject><subject>Sodium Channel Blockers - pharmacology</subject><subject>Sodium Channels - metabolism</subject><issn>1056-8719</issn><issn>1873-488X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kE9vEzEQxS3Uiv6BT4BU-dTbLmM7u7YPHFAFtFLVXkBwsybeSeJ0s17s3Uj59rgkEjdOMyO990bvx9gHAbUA0X7c1nvMfqglgK7B1gDyDbsURqtqYcyvs7JD01ZGC3vBrnLeAoCyYvGWXQhtDbRSX7L18ziFXcg4hThwHDq-xz50xzOuOPIddWHeVdMmxXm9GeeJU09-SnHcHHKIfVwfqiVm6vjmCfeibjjmjAc-5zCs-UMcfsb0kt-x8xX2md6f5jX78fXL97v76vH528Pd58fKK1BTZVH6VmnptfGNFdjYVQNorWi0EtCapRKqbQRJSwhaSCtNh4Y6v2gtEBl1zW6PuWOKv2fKkyvlPPU9DhTn7IRVxhoti1AdhT7FnBOt3JjCDtPBCXCvfN3W_eXrXvk6sK7wLa6bU_y8LGD-eU5Ai-DTUUCl5D5QctkHGnyBmAo118Xw3wd_AAynjeE</recordid><startdate>2008</startdate><enddate>2008</enddate><creator>Harmer, A.R.</creator><creator>Abi-Gerges, N.</creator><creator>Easter, A.</creator><creator>Woods, A.</creator><creator>Lawrence, C.L.</creator><creator>Small, B.G.</creator><creator>Valentin, J.-P.</creator><creator>Pollard, C.E.</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7U7</scope><scope>C1K</scope></search><sort><creationdate>2008</creationdate><title>Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorks</title><author>Harmer, A.R. ; Abi-Gerges, N. ; Easter, A. ; Woods, A. ; Lawrence, C.L. ; Small, B.G. ; Valentin, J.-P. ; Pollard, C.E.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c303t-9a2c6372c78c591a59f50a9915731068b313651e29ea0712928da8edc4690ee83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Animals</topic><topic>Biophysical Phenomena</topic><topic>Biophysics</topic><topic>Canine</topic><topic>CHO Cells</topic><topic>Cricetinae</topic><topic>Cricetulus</topic><topic>Dogs</topic><topic>Dose-Response Relationship, Drug</topic><topic>Drug Evaluation, Preclinical - methods</topic><topic>Electrophysiology</topic><topic>Female</topic><topic>hNav1.5</topic><topic>Human</topic><topic>Humans</topic><topic>Inhibitory Concentration 50</topic><topic>IonWorks</topic><topic>Male</topic><topic>Membrane Potentials</topic><topic>Methods</topic><topic>Muscle Proteins - antagonists & inhibitors</topic><topic>Muscle Proteins - metabolism</topic><topic>NAV1.5 Voltage-Gated Sodium Channel</topic><topic>Predictive Value of Tests</topic><topic>Purkinje Fibers - drug effects</topic><topic>Reproducibility of Results</topic><topic>SCN5a</topic><topic>Sodium Channel Blockers - pharmacology</topic><topic>Sodium Channels - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Harmer, A.R.</creatorcontrib><creatorcontrib>Abi-Gerges, N.</creatorcontrib><creatorcontrib>Easter, A.</creatorcontrib><creatorcontrib>Woods, A.</creatorcontrib><creatorcontrib>Lawrence, C.L.</creatorcontrib><creatorcontrib>Small, B.G.</creatorcontrib><creatorcontrib>Valentin, J.-P.</creatorcontrib><creatorcontrib>Pollard, C.E.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Toxicology Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><jtitle>Journal of pharmacological and toxicological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Harmer, A.R.</au><au>Abi-Gerges, N.</au><au>Easter, A.</au><au>Woods, A.</au><au>Lawrence, C.L.</au><au>Small, B.G.</au><au>Valentin, J.-P.</au><au>Pollard, C.E.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorks</atitle><jtitle>Journal of pharmacological and toxicological methods</jtitle><addtitle>J Pharmacol Toxicol Methods</addtitle><date>2008</date><risdate>2008</risdate><volume>57</volume><issue>1</issue><spage>30</spage><epage>41</epage><pages>30-41</pages><issn>1056-8719</issn><eissn>1873-488X</eissn><abstract>The safety implications of blocking the human cardiac Na
+ channel (hNav1.5) make it prudent to test for this activity early in the drug discovery process and design-out any potential liability. This needs a method with adequate throughput and a demonstrable predictive value to effects in native cardiac tissues. Here we describe the validation of a method that combines the ability to screen tens of compounds a day, with direct assessment of channel function.
The electrophysiological and pharmacological properties of hNav1.5 were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks™ HT). A pharmacological comparison was also made between IonWorks™ HT and canine cardiac Purkinje Fibre action potential upstroke data.
Activation curve parameters for hNav1.5 in IonWorks™ HT were not statistically different (
p
>
0.05) from those generated using conventional electrophysiology. IonWorks™ HT
V
1/2
=
−
22
±
0.8 mV, slope
=
6.9
±
0.2 (
n
=
11); conventional electrophysiology
V
1/2
=
−
20
±1.6 mV, slope
=
6.4
±
0.3 (
n
=
11). Potency values for a range of hNav1.5 blockers determined using IonWorks™ HT correlated closely with those obtained using conventional electrophysiology (
R
=
0.967,
p
<
0.001). The assay was able to distinguish between highly use-dependent blockers (e.g. tetracaine) and blockers that do not display strong use-dependence (e.g. quinidine). Comparison of the degree of hNav1.5 inhibition and decrease in canine Purkinje fibre action potential upstroke velocity (
V
max) showed that the IonWorks™ HT assay would have predicted the outcome in Purkinje fibres in the majority of cases, with false negative and positive rates estimated at 8 and 7%, respectively. Finally, hNav1.5 pharmacology was similar when determined using either IonWorks™ HT or IonWorks™ Quattro, although the latter yielded more consistent data.
The assay described combines a functional assessment of hNav1.5 with medium-throughput. Furthermore the assay was able to reveal information on the use-dependency of compound block, as well as predicting Na
+ channel effects in more integrated systems such as the cardiac Purkinje fibre action potential. This makes it possible to determine quantitative potency data, and mechanistic information about use-dependence, in a timeframe short enough to influence medicinal chemistry.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>17980627</pmid><doi>10.1016/j.vascn.2007.09.002</doi><tpages>12</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1056-8719 |
| ispartof | Journal of pharmacological and toxicological methods, 2008, Vol.57 (1), p.30-41 |
| issn | 1056-8719 1873-488X |
| language | eng |
| recordid | cdi_proquest_miscellaneous_19389872 |
| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Animals Biophysical Phenomena Biophysics Canine CHO Cells Cricetinae Cricetulus Dogs Dose-Response Relationship, Drug Drug Evaluation, Preclinical - methods Electrophysiology Female hNav1.5 Human Humans Inhibitory Concentration 50 IonWorks Male Membrane Potentials Methods Muscle Proteins - antagonists & inhibitors Muscle Proteins - metabolism NAV1.5 Voltage-Gated Sodium Channel Predictive Value of Tests Purkinje Fibers - drug effects Reproducibility of Results SCN5a Sodium Channel Blockers - pharmacology Sodium Channels - metabolism |
| title | Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorks |
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