Figure 1: Researchers Dr. Kazunobu Tsunemoto and Takashi Yoshikado use EVOLUTE® EXPRESS, a hydrophilic polymer-based solid-phase extraction plate, for plasma sample analysis in clinical trials at Yuichi Sugiyama’s laboratory at RIKEN.
The laboratory directed by Professor Yuichi Sugiyama at RIKEN analyses clinical trial data for establishing quantitative interactions between pharmacokinetics and drugs. By using EVOLUTE® EXPRESS solid-phase extraction plates to pre-process the plasma samples for testing, the researchers were able to conduct the study efficiently at a low cost. We spoke with researchers Dr. Kazunobu Tsunemoto and Takashi Yoshikado about their work.
Yoshikado explains: “Yuichi Sugiyama retired from his position as professor at the Graduate School of Pharmaceutical Sciences, University of Tokyo, and when he transferred to RIKEN, I was assigned to work together with him. The laboratory was originally founded on molecular pharmacokinetics.”
Yoshikado: “Pharmacokinetics examines the changes in the drug concentration in the body after ingestion. In our laboratory, we conduct research on pharmacodynamics, up to and including whether a drug becomes an antagonist to depress the target function, and the mechanisms that strengthen its function as an agonist. Our ultimate goal is to create highly effective medicines with no side effects for patients. In addition, drug development must go through many processes. In particular, since clinical trials are very costly, appropriate steps must be taken in advance. For this reason, it is very important to build a system to quantitatively predict pharmacokinetics, efficacy, and side effects. Our research topic is the building of this system.
Yoshikado: “That’s right, there are individual differences in the effects of drugs, and drug combinations also cause new interactions, affecting the drugs’ effects as well as side effects. We can predict their behaviour by using a mathematical model called PBPK, a physiological pharmacokinetic model. By entering the parameters of essentially any drug in this model, it is possible to quantitatively predict the various pharmacokinetic outcomes. However, the predicted results must be verified to determine if they match the pharmacokinetic data from actual human clinical trials, so we also conducted clinical trials with groups of healthy people for verification. We used EVOLUTE® EXPRESS CX for pre-processing of this analysis.”
Yoshikado: “This clinical trial combined the cassette dose and semi-micro dose. This provides benefits of administering multiple drugs at one time, the cassette doses, while controlling the costs of actual trials and also enabling the administration of low concentrations of drugs – that’s the semi-micro doses – which maintains a high level of safety for the research subjects. In fact, the micro-dose is a technique that Professor Sugiyama established in the NEDO project. Last year, it won an award from the Ministry of Finance, Trade and Industry for development or commercialization of an innovative drug discovery support technology utilizing micro-dose clinical trials.
The dosage in this study ranges from 1 to 10% of the drug concentration that would be used for medical purposes. Therefore, quantitative sensitivity is very important for the analysis. In addition, several kinds of drugs must be analysed at the same time because cassette doses are used. Pre-processing is very important in order to ensure quantitative sensitivity for trace amounts of multiple target substances.
In this study, two types of atorvastatin and grapefruit juice were used as a digestive tract transporter OATP2B1 inhibitor. We then tested combinations of five types of interacting agents such as celiprolol and sumatriptan, and we use EVOLUTE® EXPRESS CX to pre-process the samples for targets celiprolol and sumatriptan, which have similar physical properties to cationics.”
Figure 2: EVOLUTE® EXPRESS CX
Yoshikado: “Pre-processing was necessary because the clinical plasma samples are not always clean. The load on the measurement instruments is also considered. Earlier, we had been using a solid-phase extraction system made by another company, not specific to cationics, and the cost was high. For this study we were introduced to EVOLUTE® EXPRESS, and we compared several sorbents within the series. The recovery rate was best in the CX type.”
Tsunemoto: “The sensitivity is better, and because the steps are not difficult, it also has the benefit of being easy to use. A detailed protocol was also prepared for us to use in this study, and it was very helpful as we simply followed it.”
Yoshikado: “Since our work is not only quantitative, it is important to us to work very efficiently. The current clinical trial required the processing of around 1,200 samples, it goes without saying that speed and reproducibility were concerns,
but also the effort and the cost. The actual cost was cut down to around two-thirds.”
Tsunemoto: “No, not particularly. We are very satisfied.”
Yoshikado: “I would be grateful if we could have a consultation when we are working with a new compound. In our case, the measured drugs change with each clinical trial, so we would welcome support relating to the Biotage application aspects.”
Yoshikado: “That’s right. The type of pre-processing must properly fit the target. We are using ISOLUTE PLD+ protein and phospholipid removal plates instead of EVOLUTE® EXPRESS CX for the new clinical trials we are conducting.”
Tsunemoto: “Protein and phospholipids are nasty problems. In particular, plasma contains high quantities of phospholipids, and this causes ion suppression that reduces the sensitivity of LC/MS. They must absolutely be removed.”
Yoshikado: “In our tests, we sometimes use several hundred microliters of plasma in order to ensure quantitative sensitivity, and phospholipids and extra protein are inevitably going to be present. So pre-processing is important. The situation with semi-micro-dose and micro-dose is unique.”
Yoshikado: “Like I said, ISOLUTE PLD+ is planned to be used in clinical trials on the contribution rate of drug-metabolizing enzymes and transporters in humans. We need to understand how to provide accurate predictions for the pharmacokinetics of these compounds. We use a technique for quantification we call ‘probes’. For example, we have this drug-metabolizing enzyme CYP3A. Certain drugs like midazolam will be specific to that probe – that is, they bind to it specifically as a substrate. So we can track the midazolam pharmacokinetics by monitoring their CYP3A activity to quantify the metabolism rate.
In addition, we are planning a clinical trial on drug-drug interactions to be able to establish more high-precision predictions. There are medicines which inhibit enzymes, and raising the blood levels of other drugs can cause various side effects.
We want to get data for a qualitative analysis to determine the dangers of such side effects when used in combination with other drugs. In this clinical trial, we look at the interaction between drugs together with the probe. The pharmacokinetics and drug interactions are complex, but can still be measured quantitatively. For this test, ISOLUTE SLE+ is also a candidate we are considering specifically for the analysis method. We are happy for your continued support and consultation.”
RIKEN
(National Research and Development Institute)
Employees: 3,397 (2012) Budget: ¥3 billion
RIKEN is Japan’s largest and most comprehensive research organization for basic and applied science and a world leader in a diverse array of scientific disciplines. Since its foundation in 1917, RIKEN has fostered pioneering research in the entire range of the natural sciences, from developmental biology and neuroscience to quantum physics and computer science.
Today, RIKEN encompasses a network of world-class research centres across Japan, with main campuses in Wako, Tsukuba, Yokohama, Kobe and Harima offering state-of-the-art facilities that rank among the best in the world.
Literature number: PPS409