Herg safty assay

HERG safty assay methods

1. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY Subject In charge Mrs. BABITHA S. M. Pharm.Ph.D Associate Professor, Dept.of Pharmacology, SSCP Presented by HARISH.J 1 M Pharm, Dept.of Pharmacology, SSCP EVALUATION SEMINOR ON HERG SAFETY ASSAY

2. Introduction Creative Bioarray uses the state-of-the-art automated QPatch - HT system to provide a higher-throughput hERG safety assay with better consistency at a lower cost. For small number of compounds or compounds identified with QPatch , we can also perform conventional whole cell patch clamp assay to get detailed mechanistic information. GLP level hERG safety assay can provide high quality data for registration purposes SREE SIDDAGANGA COLLEGE OF PHARMACY

3. What is HERG Safety Assay ?   HERG is an acronym for human ether-a-go-go-related gene, the gene that encodes the pore forming subunit of the delayed rectifier IKr channel in humans. The hERG channel is one of a family of ion channels first identified in a mutant Drosophila melanogaster fruitfly.s An additional link between hERG and cardiac repolarization was provided by the finding of hERG mutations associated with a congenital long QT 2 syndrome linked to chromosome 7. It is generally appreciated that most drugs that either delay repolarization or are associated with TdP block hERG current (see reviews cited above). Thus, block of hERG current has become an accepted surrogate marker for cardiac proarrhythmia and the evaluation of drug block of hERG current has come to play a pivotal role in the preclinical evaluation of potential proarrhythmic risk. SREE SIDDAGANGA COLLEGE OF PHARMACY

4. HERG SAFTY ASSAY BACKGROUND Cardiac safety is a major concern in the development, approval and prescription of new drugs. A substantial number of drugs have been restricted in their application, or have even been withdrawn from the market due to concerns about cardiac safety. Promising novel cardiac safety assays (shown in bold) aim to bridge the gap between high-throughput assays with limited direct clinical relevance and low-throughput, high-cost assay with more direct clinical relevance. SREE SIDDAGANGA COLLEGE OF PHARMACY

5. WHY HERG SAFTY ASSAY SREE SIDDAGANGA COLLEGE OF PHARMACY 1. A number of drugs have been withdrawn from late stage clinical trials due to these cardiotoxic effects, therefore it is important to identify inhibitors early in drug discovery. 2. Reduce the late failure rate, reduce the cost of new drug research and development 3. Improve the rate of new drugs into the late research, 4. Expensive late stage failure would be avoided if toxic compounds were identified earlier.

6. Automated Patch-Clamp higher-throughput hERG safety assay with better consistency at a lower cost. 2. Conventional Patch-Clamp For small number of compounds or compounds identified with Q-Patch. 3. FluxORTM Thallium Assay Can be tested on 96-well plates or 384- well plates to achieve high throughput requirements for compound screening and lead compound optimization. HERG SAFTY ASSAY METHODS

7. SREE SIDDAGANGA COLLEGE OF PHARMACY HERG SAFTY ASSAY METHOD   Automated Patch-Clamp   Animation showing the gigasealing process. The pipette approaches the cell and a plume of liquid flowing out of the pipette makes a small dimple on the surface of the cell. When the resistance has increased enough, a small amount of suction is applied to the pipette which draws the cell membrane into contact with the pipette tip. This creates the gigaohm seal characteristic of a patch clamp recording.

8. Automated Patch-Clamp QPatch series of automatic patch clamp system has the following advantages:   High throughput QPatch HT (X) can record up to 480 cells at the same time, up to 7,000 data / day, 1,000 to 10,000 times higher than traditional patch clamps. High quality data The cell membrane and the QPlate chip can form a true high- impedance seal (seal impedance>1GΩ). Highly automated On-line automated cell preparation, the prepared cells can be used continuously for at least 4 hr. Some cell lines can be used continuously

9. SREE SIDDAGANGA COLLEGE OF PHARMACY Conventional Patch Clamp   The Conventional Patch-Clamp is the most important technical tool for studying ion channels. It is recognized as the "gold standard" for ion channel research and is the most accurate method for measuring ion channels. It is suitable for the study of compounds and ions Channel mechanism of action, can also be used for the declaration of new drugs in the process of drug toxicity evaluation and optimization of the structure of lead compounds.

10. The FluxOR ™ Potassium Ion   Channel Assay is an optically based, homogenous assay for high throughput screening (HTS) measurements of potassium ion channel and transporter activities. The FluxOR ™ Potassium Ion

11. SREE SIDDAGANGA COLLEGE OF PHARMACY An alternative to either manual or automated electrophysiology is a functional assay that measures ion flux across cell or vesicle membranes. This assay offers higher throughput than the auto-mated voltage clamp protocols and takes advantage of the ability of rb + to permeate through herg channels. Typically, cells are loaded with rb + overnight and herg –dependant rb + efflux is initiated by an addition of high extracellular potassium concentrations to depolarize the cell and open herg The amount of Rb + efflux can be calculated by using 86Rb+ as a radio-active tracer or by flame atomic absorption spectrometry (FAAS).Such flux assays, which can operate in 96- and 384-well formats, afford the testing of several thousand compounds per day. They offer a robust measure of channel activity.