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Application of Stable Isotope-Labeled Drugs in Clinical Pharmacokinetic Research

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Clinical pharmacokinetics (Clinical Phamacokinetics) research has revealed the mystery of the fate of many drugs in the body, and has enriched the knowledge of human clinical rational use of drugs. It undoubtedly plays an important guiding role in avoiding the occurrence of adverse drug reactions and improving the level of drug treatment. The key to clinical pharmacokinetic research is whether to obtain accurate and objectively law-reflecting concentrations (contents) of drugs and their metabolites in biological samples. ), selectivity (Selecivity) and specificity (Specficity) are closely related. In vivo drug analysis includes high performance liquid chromatography (HPLC), gas chromatography (GC), immunoassay (IA), radioisotope tracer method (RIT), gas chromatography-mass spectrometry (GC-MS), liquid phase Chromatography-mass spectrometry (LC-MS) and so on. These methods have high sensitivity and specificity, especially GC-MS and LC-MS, which have the unique advantages of GC, LC separation and MS detection, and the detection limit can reach the level of pg/ml. Due to the availability of these two methods, stable isotope-labelling (SIL) tracer technology has been greatly developed in the field of clinical pharmacokinetic research in the past two decades. This article reviews stable isotope-labelling. Principles and methods of drug application in clinical pharmacokinetic research.

1 Knowledge about stable isotopes and their markers

1.1 Introduction to Isotope Chemistry

Isotopes are atoms of the same chemical element, which have different masses due to the different numbers of neutrons in the nucleus. There are light and heavy isotopes. According to physical properties, isotopes are divided into two forms: radioactivity and stability. Radioactive isotope (radioactive isotope) such as: 3H, 14C undergoes its own decay process and emits radiant energy, which is unstable and has a physical half-life. Although radioisotopes are still used in the analysis of biological samples (radioimmune analysis), their application is strictly limited due to the potential adverse effects on the human body due to their radiation effects, and they are often used in radiotherapy medicine and imaging medicine. Stable isotope (stable isotope) is non-radioactive and has stable physical properties. It exists in nature in a certain proportion and is harmless to the human body. It can be labeled into the drug component by chemical synthesis. The labeled drugs and unlabeled drugs in biological samples The concentration of the labeled drug can be detected simultaneously using GC-MS or LC-MS methods. There are four commonly used stable isotopes: 2H, 13C, 15N and 18O (see Tab).

The natural abundance in the table indicates the percentage of stable isotopes present in nature. Taking carbon as an example, there are two forms of stable isotope, 12C and 13C, which account for 98.893% and 1.107% of the total content respectively (100% in total). In each drug molecule, carbon atoms naturally exist in the ratio of the above two isotopes. Each organic matter is a mixed molecule composed of different isotope nuclides (Nulide). For example, the molecular formula of verapamil is C27H38N2O4, the molecular weight is 454, and the average amount of the stable isotopes is 454.27. In a drug molecule, the presence of a natural 13C atom has a molecular weight of 455. Therefore, when using MS to detect drugs, an isotope group peak appears at a mass-to-charge ratio (m/e) of 455, and its intensity is consistent with the content of the molecule. The number of atoms of this element and the natural abundance of its heavy isotopes are closely related. For a certain organic compound CWHXNYOZ, the relative intensity of the M+1 (molecular weight+1) and M+2 peaks caused by the natural existence of heavy isotopes can be calculated as follows:

(M+1) Peak relative intensity (%)=(1.1×W)+(0.015×X)+(0.037×Y)+(0.09×Z)

(M+2) Peak relative intensity (%)=(1.1×W)2×(0.2×Z)/200

If the drug isotope 13C, 15N or 2H (deuteriun, deuteriun) is recorded as the d amount label, a strong labeled drug peak will appear at M+1. The simultaneous detection of labeled drugs by GC-MS depends on the difference between them. Existing interference, (ie: the joint contribution of the isotope peaks of the labeled drug at the M+1 mass-to-charge ratio). This test interference can be eliminated by the isotope distribution calculation method. The best way to avoid the isotope family peak interference is to label three The above atoms (such as labeling three hydrogen atoms, denoted as d3), make the quality difference of the labeled drug ≥3. At this time, the isotope peak interference will be small or non-existent.

Another issue that should be considered is which heavy isotope labeling and labeling position are used, which mainly depends on the structural characteristics of the drug and the nature of metabolism in the body. The pharmacokinetics and protein binding rate of the labeled and unlabeled substances must be basically the same, and there must be no isotope effect (see section 1.3), and the labeled part cannot be placed in an important position of the drug’s metabolic inactivation or the drug’s action function Basically. Therefore, before using this technology, it is necessary to have knowledge and understanding of the in vivo metabolic characteristics of the drug being studied and the ionization fragmentation formula on MS.

1.2 Use safety

Since Ason et al. first discovered the isotope in 1927, researchers in pharmacology and toxicology have discussed its toxicity, teratogenicity and mutagenicity. The 13C content in mice increased to 15%-20% of the total carbon content, and no teratogenic reactions were observed. 90% of the oxygen in the water and air used to raise the mice was replaced by 18(), and no toxicity and teratogenic reactions were observed for three generations. Only high levels of 2H in the body (accounting for 15% of body weight) will cause significant toxicity to mammals. The human body is mainly composed of hydrogen, carbon, nitrogen, and oxygen. Based on the weight of 70kg of human body, it contains approximately 270g of heavy isotopes. The amount of stable heavy isotopes contained in the human body and daily intake is much greater than the dosage of the labeled drug test (see Tab), it is safe to give constant doses of stable isotope-labeled drugs for human experiments.

1.3 Isotope effect (isotope effect)

Although stable heavy isotopes are almost non-toxic, there may be changes in the metabolic characteristics of the body, that is, the metabolic isotope effect. Due to the difference in quality between light and heavy isotopes, compared with light isotopes, the binding force between heavy isotopes and other atoms is stronger. Larger binding force of heavy isotopes will change some characteristics of drug molecules, such as polarity, molar volume, electron donation, van der Waals attraction, bipolar moment, fat solubility, protein binding rate, etc. When drugs are metabolized in the body, breaking this bonding force is the rate-limiting process of metabolism, and it may appear that the metabolism of heavy isotope-containing drug molecules is slower. The quality of 2H is 1H-labeled drugs, and this effect should be stronger. Someone used rodents to test the isotope metabolism effects of d3-caffeine and d9-caffeine. The half-life of the former and d0-caffeine is no different, while the half-life of the latter is twice as long as d0-caffeine. It shows that the more hydrogen is replaced, the greater the possibility of isotopic metabolism. The quality difference between light and heavy isotopes of carbon, nitrogen, and oxygen is relatively small, and the possibility of isotope metabolism effects is also very small. However, this effect may be more significant when using heavy isotope-labeled drugs for a long time, and attention should be paid to confirm it The basic method for the presence or absence of isotope effects is to conduct a preliminary experiment, and give a subject the same amount of labeled and labeled drugs at the same time, and test the drug concentration in the blood of the two. If the two have the same pharmacokinetic properties, It shows that there is no isotope effect.

2 Application in clinical pharmacokinetic research

The success of the on-line chromatography-mass spectrometry technology enables the quantitative analysis of trace drugs in biological samples. Chromatography separates a variety of substances in the sample, and then ionizes, scans, and detects by a mass spectrometer, which can play a dual role of qualitative and quantitative. In particular, the derivatization separation technology, capillary separation technology, soft ionization technology, selective ion detection rapid scanning, tandem mass spectrometry, etc. developed in the past two decades have greatly improved the specificity and sensitivity of drug detection, making SIL tracer technology It has become an important and promising method in clinical pharmacokinetic research.

The main functions of stable isotope-labeled drugs are:

a. Used as an “analytical internal standard” in the selective ion test of GC-MS analysis. Like the usual internal standard method, the internal standard is added during sample processing to avoid experimental errors and improve the precision of the test.

b. Used together with unlabeled drugs in the human body as a “biological internal standard” to eliminate intra-individual errors caused by different times of administration, and to test the pharmacokinetic characteristics of the drug under specific clinical conditions.

The following is discussed from three aspects. It must be noted that the focus of the review is not to discuss the detailed results of a specific experimental study, but to clarify the principles and methods of applying stable isotope-labeled drugs to this research.

2.1 Bioavailability/bioequivalence and drug absorption research

Due to the similarities in the bioavailability and bioequivalence methods of research drug preparations, the following are considered as the same topic and discussed together. The basic method of studying bioavailability is to use different preparations of the same drug in the body, compare the pharmacokinetic parameters of the two preparations, and evaluate the relative or absolute degree of absorption and utilization of the drug by the body. Generally, random crossover experiments are adopted to feed individual differences. However, the human body has a 24-hour rhythm change, and hemodynamics are not constant. For some drugs with low liver first-pass elimination, human biological factors have little effect on their in vivo disposal, while for some rapid first-pass elimination drugs (high clearance drugs), there will be greater intra-individual differences. Crossover experiments cannot eliminate this difference. A study such as Eichelbaun showed that the intra-individual differences of some high-clearance drugs are even greater than the inter-individual differences. Therefore, when the conventional crossover experiment method is used to explore the bioavailability, it is usually required to have a large enough sample amount in order to obtain credible statistical results. This shortcoming can be overcome if the simultaneous administration of stable isotope-labeled drugs is used. Because the drug and the labeled drug undergo the ADME process in the body at the same time, there is no problem of intra-individual error, and the sample size is required to be small. In a study on the bioequivalence of two imipramine preparations, Heck et al. discussed the sample requirement. If the variation is less than 20% (equivalence probability is 0.8), using the conventional crossover test method, at least 20 cases are required. Subjects, and the application of SIL tracing method only needs 3-4 cases. Eichelbaun et al. used 6 subjects to explore the relative bioavailability of verapamil (ie d0-verapamil) tablets, and gave the same dose of d3-verapamil (biological internal standard) to d7-verapamil was used as an internal standard for analysis, and the relative bioavailability of d0-verapamil tablets was determined to be 108.07%. This result is consistent with the results of the same drug (190 %) is quite different. The researchers believe that the oral liquid administration of the drug has been completely absorbed, and the oral tablet cannot have a relative bioavailability as high as 190%, and explained that it is due to the suspicious data obtained from the cross-test. It is caused by the large difference in first-pass metabolism in individuals with the drug. Hage used labeled drugs to explore the bioavailability of flecainide solution in 6 subjects. Intravenous injection of d0-flecainide and oral administration of d3-flecainide solution were used, using negative ion chemical ionization selective ion detection technology The blood concentration of d0- and d3-flecainide was measured on GC-MS to obtain the absolute bioavailability of the solution.

The gastrointestinal absorption of drugs is affected by many factors. Different drugs have different absorption sites and mechanisms in the gastrointestinal tract. SIL untracked technology can also be used to explore the characteristics of the first gastrointestinal absorption of drugs. Bode et al. used this technology to successfully explore the absorption of nifedipine controlled-release preparations in different parts of the gastrointestinal tract with only 4 subjects. Give the drug 20mg controlled-release preparation to the stomach, jejunum, colon (two parts) and intravenous drip (reference preparation) at 5 times of cross positioning. At the same time, oral administration of the same dose of 13C-labeled drug (13C4-nifedipine) The solution of nitrendipine was used as the analytical internal standard. The plasma concentration of 12C-13C4-nitropyridine at different time after administration was measured on GC-MS. The result was that 4 controlled-release preparations were administered. The jejunum is better for drug absorption at the drug site. With AUC as a parameter, compared with intravenous infusion, the absolute bioavailability of each part of the drug is between 42-56%. The study effectively used 13C4-nitropyridine as the “biological internal standard”, and strictly controlled the errors caused by intra-individual differences in this high-clearance drug.

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