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Pharmacokinetics and Bioanalysis of Chiral Drugs

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Stereoisomers are compounds in which the structural groups in the molecule are arranged differently in three dimensions without changing the atomic composition, atom-atom connection mode, and bonding sequence. Stereoisomers are divided into two categories: Enantiomers and Diastereoisomers. Enantiomers (ie, enantiomers) refer to stereoisomers that cannot overlap in space and are mirror images of each other, just like human left and right hands, which are called “chiral” [1]. A compound containing two enantiomers of each other is called a chiral compound, and a compound containing only one enantiomer is called an optically pure chiral compound. The equimolar mixture of left-handed and right-handed enantiomers is called racemate.

chiral drugs
Chiral enantiomers

Biological macromolecules such as proteins, polysaccharides, nucleic acids, etc. all have chirality. Except for bacteria and other organisms, proteins are composed of L-amino acids; polysaccharides and nucleic acids are in the D-configuration. They form a chiral environment in the organism. After the drug enters the human body, its pharmacological effects are mostly related to the chiral matching and molecular recognition ability between it and the target molecule in the body. Therefore, there are significant differences in the pharmacological activity, metabolic process and toxicity of the enantiomers of chiral chemical drugs in the human body. The use of the principle and technology of “chiral” to develop new drugs has become one of the new directions of the international medical community [ 2].

Chiral drug classification

Chiral drugs can be divided into the following 6 situations according to their effects [3]:

One enantiomer is a competitive antagonist of the other, such as (-)-isoproterenol and (+)-isoproterenol.
Enantiomers have opposite effects, such as dihydropyridine antagonists, S-type enantiomers are strong activators of L-type voltage-dependent calcium channels, and R-type enantiomers are mostly blockers.
One enantiomer mainly has a therapeutic effect, and the other enantiomer mainly produces side effects. For example, the S-type enantiomer of ketamine exerts analgesic and analgesic effects, while the R-type enantiomer has a central excitatory effect; thalidomide (Thalidomide) embryo toxicity and teratogenic effects are mainly produced by the S-type enantiomer.
The two enantiomers produce different types of pharmacological effects and can be used as therapeutic drugs. For example, dextropropoxyphene is an analgesic, while levpropoxyphene enantiomer is a cough medicine.
The mutual benefit of enantiomers, such as: the S-type enantiomer of propranel mainly blocks β receptors, while the R-type enantiomer has an inhibitory effect on sodium channels, and it is anti-arrhythmic when administered with the exosome Abnormal effect is good.
One enantiomer has pharmacological activity, and the other enantiomer is inactive or very weak. For example, the in vitro antibacterial activity of levofloxacin is 8-128 times that of dexofloxacin.

The importance of research and development of chiral drugs

In the past, chiral drugs were often sold as racemates. In 1961, the racemic sedative Thalidomide (Thalidomide) was taken by pregnant women to produce teratogenic events. It was not until 1965 that the R-enantiomer of thalidomide was found to be a sedative, and the other S-enantiomer was not only There is no sedation and teratogenic effects [2].

Subsequent studies have shown that this is not an isolated phenomenon, and many chiral drugs have similar conditions. Although the two enantiomers of chiral drugs have similar physical and chemical properties, they have a high degree of stereoselectivity in the chiral environment in vivo and exhibit different Pharmacokinetics and Pharmacology & Pharmacodynamics. Therefore, the clinical efficacy of chiral drugs is The combined result of the stereoselectivity of the biological activity of the drug and the stereoselectivity of the process in vivo. The examples in Table 1 show that the difference between many drugs and poisons is only two enantiomers with different stereoconfigurations, which may even directly endanger the life and health of the human body. Therefore, in the process of drug research and development, each one should be chiral. The center’s drugs conduct enantiomeric pharmacological research.

ADME for chiral drugs

In the chiral environment of the human body, the stereoselectivity of the mutual recognition and interaction between the enantiomers of chiral drugs and biological macromolecules leads to the pharmacological difference of chiral drugs, that is, the pharmacological stereoselectivity. Pharmacological stereoselectivity is divided into pharmacodynamics stereoselectivity (stereo-selectivity in pharmacodynamics) and pharmacokinetics stereoselectivity (stereo-selectivity in pharmacokinetics). The pharmacodynamic stereoselectivity refers to the differences in the pharmacodynamic effects and mechanisms between the enantiomers of chiral drugs. The pharmacokinetic stereoselectivity refers to the difference in the absorption, distribution, metabolism and elimination of the enantiomers of chiral drugs. 90% of the commonly used chiral drugs are administered with racemates. The problems caused by this method of administration have attracted more and more attention and attention.


The absorption of most drugs is a passive diffusion process, and the rate and extent of absorption depend on the fat solubility of the drug. Since there is no obvious difference in the fat-solubility and water-solubility of the two chiral enantiomers, there is no stereoselectivity in the absorption through passive diffusion through biological membranes. When chiral drugs are absorbed through active transport or facilitated transport, the spatial structure of the drug can be recognized by cell membrane carriers or enzymes, and stereoselectivity will occur, resulting in differences in the absorption of enantiomers. When chiral drugs are absorbed through active transport in the gastrointestinal tract, the absorption characteristics of the two enantiomers may be significantly different, and many chiral drugs exhibit stereoselectivity when the first pass elimination reaction occurs. The D-enantiomer of the β-lactam antibiotic cephalexin is actively absorbed by the dipeptide transport system of the intestinal tract, and the dipeptide transport system has specificity and saturation for the transport of the D-enantiomer, while L- Enantiomers can inhibit the absorption of D-enantiomers. The β-receptor blocker Pryolol, the R-isomer is more prone to first-pass effects, while the pharmacologically active S-isomer is less likely to occur. In addition, different drug concentrations, pH values, co-solvents and dosing intervals also have a certain effect on the stereoselective absorption of drugs. For example, when the β-receptor blocker propranolol is used for transdermal absorption of its lipophilic prodrug, the stereoselectivity of skin esterase tends to hydrolyze R-propranolol and absorb S-propranolol [4].


The distribution degree of the drug depends on the lipid solubility of the drug and the binding ability of the drug with plasma proteins and tissues. The partition coefficient of a drug through a membrane is usually not affected by chirality, but the protein binding rate of drug enantiomers may vary greatly. Its stereoselectivity is mainly reflected in the process of binding to plasma proteins or tissues.

Plasma protein binding

In plasma, the plasma proteins that bind to free drugs are mainly albumin and β-acid glycoprotein. The former is usually combined with acidic drugs, while the latter is mainly combined with basic drugs. The difference in binding ability between chiral enantiomers and these two types of proteins leads to differences in plasma protein binding. In humans, the AUC ratio of (+)-enantiomer to (-)-enantiomer of reboxetine is 0.15, because the protein binding rate of (+)-enantiomer with higher activity is lower, And the elimination from the body is relatively fast [5].

Tissue distribution

The distribution of chiral drugs in tissues also has stereoselectivity. This selectivity is not only related to the free fraction of drugs in plasma proteins, but also to the binding of drugs to tissues and transmembrane transport. According to reports by MIRANDA et al., fenoprofen has stereoselectivity in tissue distribution. For example, in the joint cavity membrane fluid of arthritis patients, the AUC of the active S-enantiomer is approximately 10 times that of the R-enantiomer [6 ].


Enantiomeric metabolic pathway

Among the chiral drugs that have been studied, most drug metabolism exhibits different degrees of substrate stereoselectivity. Research on this aspect mainly focuses on the redox reaction involved in the CYP450 enzyme system. The difference in the stereoselective direction of different metabolic pathways of a drug is closely related to being catalyzed by different CYP450 isoenzymes. On the other hand, although some enantiomers are catalyzed and metabolized by the same metabolic enzymes, due to the different affinity of the same enzyme for the isomers, or the different ratios of several metabolic enzymes involved in the metabolism of the enantiomeric drugs, all It will lead to the difference in the metabolic rate and the amount of metabolism between the two in the body [4]. The β-receptor blocker propranolol is oxidized and metabolized by CYP2D enzyme system in the body, and mainly produces 4-OH-propranolol, 5-OH-propranolol and N-despropyl-propranolol. In its pharmacokinetic studies, the Km and Vm values of the three metabolites are all expressed as R-enantiomer

Interconversion between enantiomers

Chiral transformation refers to the conformational transformation of enantiomers in the metabolic process, which complicates the study of the metabolism and kinetics of chiral drugs. The main organ where enantiomers undergo chiral transformation is the liver, followed by the kidney and gastrointestinal tract. By studying the interconversion of enantiomers in vivo, it is possible to understand whether an enantiomer can accumulate by slowing down the elimination of the other enantiomer through conversion to another enantiomer. Thalidomide is because of its rapid racemization in the body, and the teratogenic effect of its S isomer has produced tens of thousands of seal fetuses, which has become a catastrophic drug toxicity in human history [8] .

The molecular structure and mutual transformation of thalidomide

Iami T et al. studied the stereoselective disposal of pranoprofen in hunting dogs and found that the conversion of R-(-)-enantiomer to S-(+)-enantiomer can reach 14 %, this chiral transformation plays an important role in slowing down the elimination of S-(+)-enantiomer in dogs [9].

The absorption rate of the drug in the gastrointestinal tract also affects the stereoselective pharmacokinetics of the drug enantiomer. For example, after oral administration of racemic ibuprofen, the inactive R-enantiomer in the gastrointestinal tract can be transformed into the active S-enantiomer. The longer the drug stays in the gastrointestinal tract, the greater the degree of conversion. Sattaris et al. conducted in vivo kinetic studies on different dosage forms of ibuprofen. After taking ibuprofen racemic sustained-release granules, the ratio of S-enantiomer to R-enantiomer AUC (7.3±1.5) was significantly higher. For suspension (3.6±1.1) and solution (3.5±0.2). The results show that the ratio of the two enantiomers in vivo has a significant correlation with the release rate of the drug from the formulation [10].


Renal clearance

The kidney is the main organ for drug excretion. Renal excretion involves processes such as glomerular filtration, active renal tubular transport, and renal metabolism. The latter two processes involve the active transport and metabolism of the kidney, so the elimination of chiral enantiomers may exist. Stereoselectivity. After administration of sotalol racemate in rats, the drug-time curve of its enantiomers was similar, while after administration of S-sotalol alone, renal clearance decreased from (33.7±6.0) to (28.9±5.6) mL /(Min·kg) (P<0.05); After administration of R-sotalol, the clearance rate did not change significantly [11]. The renal clearance rate of S-sotalol is greatly affected by renal blood flow, but less affected by renal excretion. Therefore, after administration of racemate, the β-blocking effect of R-sotalol is caused by Changes in renal blood flow may be the main cause of changes in the renal clearance of S-sotalol.

Bile excretion

Bile excretion is one of the main excretion pathways of drugs and their metabolites. The excretion of chiral drugs and their metabolites in bile involves active and passive processes. It is known that there are three transport systems in the bile duct, namely, organic acid, organic base and neutral compound transport system. The drug delivery mediated by these delivery systems often has stereoselectivity. The experiment found that after intravenous injection of tramadol hydrochloride and trans-oxydemethyltramadol in rats, (-)-trans-tramadol and (-)-trans-oxydemethyltramadol preferentially from bile Excretion in [12].

Separation methods of chiral drugs

It can be seen from the above content that the enantiomeric separation of chiral drugs is extremely necessary. The current methods of enantiomer separation include: biotransformation asymmetric catalysis method, liquid-liquid extraction method, sensor method, permeable membrane method, recrystallization method, capillary electrophoresis, and chromatography. Among them, chromatography can be further divided into: Supercritical Fluid Chromatography (SFC), Gas Chromatography (GC), Capillary Electrochromatography (CEC), High Performance Liquid Chromatography (HPLC). Among them, the fastest growing and most widely used HPLC has high specificity, high sensitivity, high resolution enantiomer resolution and determination methods, which can improve the activity of chiral drugs, reduce side effects, and in-depth study of the mechanism of action. Important theoretical and practical significance [13].

HPLC resolution of enantiomers can use indirect and direct methods. The indirect method is also known as the chiral derivatization reagent method (CDR), which uses a chiral reagent and the resolved product to perform off-column derivatization to generate diastereomers, which can be resolved by traditional chiral HPLC. The direct method is divided into the chiral stationary phase method (CSP) direct separation and the chiral mobile phase additive method (CMPA). Because CMPA adds a chiral additive to the mobile phase to produce diastereomeric ion pairs or complexes, it is separated on the chromatogram. However, due to the non-volatility of the chiral additive, it is not suitable for LC-MS, so it is the most The chiral analysis commonly used for quantitative analysis is the chiral derivatization reagent method and the chiral stationary phase method.

The chiral derivatization reagent method can improve the resolution, selectivity and sensitivity, but if the chiral derivatization reagent is impure or there is degradation of metabolites (such as degradation of phase II metabolites) during the derivatization process, it will cause quantitative deviation. Moreover, the derivatization process is generally complicated and lengthy, and excessive reagents can also cause damage to the analytical column.

The chiral stationary phase method is still the first choice for chiral analysis. Its advantages are fast, simple, efficient, with a wide variety of types, and a wide range of applications. At present, there are many chiral stationary phases for high performance liquid chromatography developed internationally, mainly including the following categories:

Pikle-type chiral stationary phase (also known as brush-type chiral stationary phase): mainly includes π-base type (with electron-pushing substituents), π-acid type (with electron-withdrawing substituents) and amino acids.

Ligand exchange chiral stationary phase: a stationary phase based on the ligand exchange of metal complexes.

Macrocyclic chiral stationary phases: mainly include macrocyclic antibiotic chiral stationary phases, cyclodextrin chiral stationary phases and crown ether chiral stationary phases.

Packed imprinted chiral stationary phase: organic polymer generated by molecular template.

Peptide or protein chiral stationary phase: commonly used human α1-acid glycoprotein (α1-AGP), human serum albumin (HSA), bovine serum albumin (BSA), ovomucoid, cellobiohydrolase (CBH) )Wait.

Glycopeptide chiral stationary phase: There are mainly vancomycin chiral stationary phase and rifamycin B chiral stationary phase.

Polysaccharide chiral stationary phase: mainly includes fiber chiral stationary phase and starch chiral stationary phase.

Study on the Method of Determination of Chiral Drugs in Biological Samples

It is very challenging to establish a robust LC-MS method for determining the content of chiral drugs in biological samples because:
① HPLC-MS/MS method is often used for biological sample analysis. The liquid phase system used is generally a reversed phase system, but the liquid phase system with many chiral separation conditions is a normal phase system [14], see Table 2. Both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are disadvantageous;
②The resolution of chiral chromatographic columns is usually lower than that of achiral columns, and there are very few influencing parameters that can improve the separation effect;
③It is not easy to achieve gradient elution on chiral chromatographic columns;
④In order to meet the requirement of resolution, the analysis running time is generally longer;
⑤The stability and reproducibility of chiral chromatographic columns are worse than ordinary achiral columns;
⑥ Chiral chromatographic columns are expensive, and the cost of purchasing many different types of chiral columns is too high;
⑦The success or failure of chiral separation depends largely on the experience and repeated tests of the chromatographer.

All in all, the development of chiral methods is difficult and time-consuming.

The establishment of chiral chromatographic separation needs to determine the separation system of the method first. If the separation of the optical enantiomers can be achieved on multiple chiral stationary phases, then the chiral chromatographic column can be initially screened according to the separation system, such as: polar organic phase A mixture of acetonitrile and methanol containing organic acids or bases), the stationary phase can be cyclodextrin; reverse-phase mobile phase (a mixture of acetonitrile and water containing volatile acids, alkalis or buffers), and the stationary phase is usually protein or polysaccharide Type; normal phase mobile phase (mixed liquid of n-hexane, ethanol or isopropanol with or without volatile acids and bases), stationary phase is often used for polysaccharide type chiral columns.

The separation of analyte enantiomers is the most difficult part in developing a robust chiral LC-MS/MS bioanalysis method. The ideal separation is that the enantiomers achieve baseline separation on the chromatogram with sharp and symmetrical narrow peaks. It should be noted that the chiral resolution achieved by using racemates during method development represents only the best state and may be misleading, because the concentration ratio of enantiomers in real samples is not necessarily 1:1. When the separation is incomplete and the chromatographic peaks are severely tailed, if the smaller enantiomer elutes after the larger enantiomer, the smaller enantiomer may be masked by the larger enantiomer. Under the peak. In this case, it is necessary to use a chiral column with opposite optical rotation or a different chiral column to achieve complete separation of the two enantiomers under a large concentration difference (CD), or the minor peak is in the main peak Before flowing out.

Our laboratory has quickly established the HPLC-MS/MS method in the “Bioequivalence Study of L-Amlodipine Besylate (2.5mg/tablet) and Amlodipine Besylate (5mg/tablet)” The concentration of L-Amlodipine in human plasma. The chromatographic column ULTRON ES-OVM (4.6×150mm, 5μm) is used to fix the non-volatile ovomucoid on silica gel. It has a wide range of chiral recognition and is suitable for the separation of reversed-phase systems, and the sensitivity can reach ng level. Finally, 16mM ammonium acetate aqueous solution: acetonitrile=75:25 was used as the reverse phase mobile phase for isocratic elution, and the amlodipine racemate was effectively separated on the chromatogram to separate the l-amlodipine and dextro-amlodipine, as shown in the figure 4. And the sensitivity of L-Amlodipine is 0.05 ng/mL, which has been successfully applied to the analysis of biological samples. The drug-time curve is shown in Figure 5.


Although the two enantiomers of chiral drugs have similar physical and chemical properties, they have a high degree of stereoselectivity in the chiral environment in vivo, and exhibit different pharmacokinetics and pharmacodynamics. Therefore, the clinical efficacy of chiral drugs is a drug The combined result of the stereoselectivity of biological activity and the stereoselectivity of in vivo processes. If the active enantiomer is fully utilized to avoid the poorly active or inactive or even toxic other enantiomer to form a single enantiomer drug, it has the advantages of good curative effect, small side effects, and high safety. Therefore, the research and development of single-enantiomer drugs has become the focus of current new drug innovation, and the pharmacokinetic research and biological analysis of chiral drugs will further understand the characteristics of drug absorption, distribution, metabolism and excretion in the body, and maximize the It has important theoretical and practical significance to effectively exert the efficacy and reduce the toxic and side effects, and guide the rational use of drugs in clinical practice.

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