Graduated from the faculty of pharmaceutical sciences, School of Pharmacy, Kanazawa University, in 1983. Engaged in in vivo safety pharmacology and in vitro safety research at Dalichi Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Ltd., and Daiichi Sankyo Rd Novare Co., Ltd. Assumed current position since March 2019. (Integrated & Translational Science Research, Principal ResearcherDirector, PhDDoctor of pharmacology.)
- hERG assay having a significant impact on the development Go/No go decision
- Support for cardiotoxicity assessment with a high probability of success even with a small quantity of the compound in the early stage of research! What’s the secret?
- Quick and low cost hERG assay Axcelead DDP offers! Data are available within a week after receiving the compound!
- A gemstone that might actually be a good medicine is overlooked?! Potential of NaV1.5 and CaV1.2 assays
- To be the best partner for cardiac toxicity assessment
1hERG assay having a significant impact on the development Go/No go decision
Dr. Takasuna, thank you for the opportunity today! Let me begin by asking about the importance of hERG assay, which is positioned as an essential GLP toxicity study to be conducted prior to clinical phase I study.
This is deeply related with drug-induced sudden cardiac death, the cause of which remains unknown for almost half a century. It turned out that 98% of the drugs that cause this sudden cardiac death bind to K+ channels encoded by a gene named hERG in cardiomyocytes, and inhibit K+ currents (hERG currents). More specifically, when hERG currents are inhibited, sudden cardiac death (lethal arrhythmia) is triggered by prolongation of the ECG QT interval as a starting point. The hERG assay to evaluate the interaction between hERG K+ channel and the candidate compound was proposed as an essential study prior to human study. Although various methods are available to investigate whether the compound inhibits K+ channels, the “hERG current assay” is chosen as the gold standard as it can evaluate the effect on actually flowing hERG currents under the closest physiological conditions.
I see. If the risk of inducing lethal arrhythmia is found just before commencing a human clinical study, all the efforts might be ruined…
Exactly. In safety assessment, this toxicity profile is a development killer. It is important to eliminate this risk as early as possible in the process of research and development. We have established, this time, an assay that is capable of high quality screening at high speed and low cost for our clients in the initial exploratory researches. I am in charge of in vitro toxicity screening, not limited to hERG assay, to be performed at an early stage of research and development. “Quick, cheap, but good” is our motto. We are willing to offer proactive support for risk assessment on the study result and subsequent planning of the research and development strategy, as well as measurement and data provision, by using our experience in drug discovery research.
2Support for cardiotoxicity assessment with a high probability of success even with a small quantity of the compound in the early stage of research!
What’s the secret?
You mentioned, “Quick, cheap, but good.” Actually, how do you know whether the test went well or not?
To provide high quality data for our clients, we have established various, strict criteria in the test condition and data adoption standards. The criteria for seal resistance value*, magnitude of the current flow, or stability, for example. We use plates with 384 wells. Since the bottom tier is the? most prone to pressure, resulting in damage to the cells, we use 360 wells, excluding the wells in the bottom tier, for testing. Among these wells, we consider the wells that have met all the strict criteria we have established and provide them for our clients as a result of a “successful testing.”
*: Seal resistance value: The cell membrane and the electrode must be tightly bonded without any gap. If not, the current leaks out, and the accurate current value cannot be measured. The seal resistance value is an indicator how well the cell membrane is bonded to the electrode.
What are the important factors to meet such strict criteria?
One of the factors is the quality and handling of cells in cultivation, and the other one? is SyncroPatch 384PE, introduced this time. Regarding the former, we take the robustness and adaptability of the cells, as well as the test condition with the least burden on the cells, into consideration. Although cells have high growth ability, they are tiny, fragile creatures. We “usually treat them gently with affection but grab them tightly when it is necessary in the test.” Such a flexible handling is important, just like in a love affair (laughter). For now, four female researchers are in charge of this test, and they are very good at handling the cells. Their careful work with love may make the cells gentle and strong, I guess.
Interesting! I, as an amateur, thought SyncroPatch was important through my preparatory survey, but now I know how to handle the cells could be the key as well!
Cell handling is also important, and of course, SyncroPatch is a wonderful machine.When it comes to SyncroPatch 384PE, it adopts borosilicate glass chip substrate (plate), so that the assay wells are hard to adsorb the compound. The material of the plate used to prepare the compound is also important. Some data indicate that in the use of polypropylene plates, the hERG current inhibitory concentration changes significantly with time after the preparation of the drug solution (presumably as a result of the decrease in drug concentration due to drug adsorption). Although glass plates are expensive, Axcelead DDP is willing to spend money to use glass plates so as to maximize the original appeal of SyncroPatch 384PE and provide high quality data constantly.
I guess your clients have only a small amount of the compound in the early phases of research. Is measurement still possible during such stage?
The more wells in a plate, the less amount of drug solution we use to put in each well. A method like manual patch where a few milliliters are perfused per minute requires a certain amount of compound. SyncroPatch with 384 plates can quickly provide high quality data comparable to those by the manual patch clamp method, even with a small amount of compound. Usually, we usually ask to provide 26 μL of 10 mM DMSO solution and evaluate in four concentrations ranging from 1 to 30 μM.
3Quick and low cost hERG assay Axcelead DDP offers!
Data are available within a week after receiving the compound!
Tell me about your current assay system.
We perform regular assays every week and basically report the result within 7 days after receiving the compound, unless re-measurement is required. Some of our clients have their own automated patch clamp systems, but ask us the assay due to internal congestion and efficiency. In my past experience, it took at least 1 month to receive the data after sending the compound to the lab overseas.
1 week is very fast!
One of the keys to quick assessment is the “assessment by cumulative drug exposure.” A laboratory usually uses one well to dispense one concentration for assessment. We, Axcelead DDP, use one well to dispense four different concentrations cumulatively, one concentration at a time starting with the lowest through the highest in a 5-minute interval. This method allows to evaluate more compounds on one plate. For example, if we use 1 well to dispense only 1 concentration for assessment, we evaluate only 17 compounds by using the 384 well plate. If we dispense different 4 concentrations cumulatively, we can evaluate 70 compounds. This method also decreases the number of expensive plates in use, resulting in higher throughput and cost reduction.
Is the method by cumulative four concentrations dosing technically more difficult?
We had a hard time finding a condition that stabilizes the current for a longer period as this method requires a longer time for measurement. The evaluation takes 5 min per one concentration of the drug. In the case of single administration, a condition that stabilizes the current only for 5 min is enough for the testing. For the assessment by administering four concentrations cumulatively, we needed to find a condition that stabilizes the current for 20 min. We have overcome the task through our knowledge, experience, patience, and teamwork of the members and have become able to obtain stable data!
This has only been achieved with such experienced people!
Actually, Axcelead DDP have another specialty! We use “frozen cells stably expressing hERG” to enable quick assessment after a long vacation. Usually, when frozen cells are thawed and immediately patched, the channel expression is not stable, and we need to wait for some time before testing. After repeating the procedures of cultivation and passaging until the expression of the channel has been stabilized, testing can be finally performed. For example, when the staff takes a long vacation during summer and winter seasons, cultivation and passaging are stopped. After they return from vacation, it takes a week or two to start testing. However, Axcelead DDP use the frozen cells we made by ourselves so that we can start testing the day after the long vacation! Honestly, I have no idea why we can produce such excellent cells. It may be my responsibility to elucidate where the know-how lies in this assay system established and maintained by the researchers in charge (laughter). I was transferred from another company and was honestly surprised to see this quite unusual laboratory that performs this assay with ready-to-use frozen cells. This is a skill that a middle-aged man like me cannot imitate (laughter).