Introduction

Chinese traditional medicine has provided for millennia a basis for disease treatment and health care to the Chinese nation and for China’s social stability. Particularly, this system of medicine has played a key role in management of various life-threatening epidemics in China [1] and is officially recommended for COVID-19 [2,3,4]. Since the foundation of the People’s Republic of China in 1949, the Chinese government has attached much significance to the practice and growth of Chinese traditional medicine, with herbal medicines being regulated as drug products. Chinese herbal medicines are often formulated as tablet, capsule, and droplet pill for oral administration and as injectable formulation for parenteral administration; they are required to be manufactured in compliance with good manufacturing practice as for drugs by licensed pharmaceutical companies [5]. Many Chinese herbal medicines are extensively used and prescribed by physicians of Western medicine, as well as physicians of traditional medicine. Since 1996, an ambitious plan has been underway in China to develop herbal medicines in line with contemporary standards of pharmaceutical sciences [6]; the Chinese National Medical Products Administration (NMPA) requires herbal medicines to be proved safe, effective, and quality-consistent before marketing [7]. Historically, herbal medicines were not extensively tested before they were approved in China, owing to paucity of the requisite technology at the time. Recently, a number of patent herbal medicines, extensively used in clinics, have shown therapeutic benefits in rigorous clinical trials, similar to those for synthetic drugs [8,9,10,10,Differential pharmacokinetics of a class of herbal compounds and interspecies differences

Different classes of constituents may have significantly different compound doses from a dosed herbal medicine. In this case, the levels of systemic exposure to constituents of different classes can be very different. Even for a class of constituents with comparable compound dose levels, the compounds can also exhibit significantly different body exposure, pharmacokinetics, and interactions with drug metabolizing enzymes and transporters. For example, phenolic constituents of Salvia miltiorrhiza roots (Danshen) are caffeic acid derivatives, occurring as monomers (tanshinol), dimers (rosmarinic acid and salvianolic acid D), trimers (salvianolic acid A and lithospermic acid), and tetramers (salvianolic acid B), these Danshen compounds contain one or more catechol moieties with one or more carboxyl groups. In addition, protocatechuic aldehyde is also a major Danshen constituent, which contains a catechol compound without any carboxyl group. After oral administration of compound Danshen droplet pills (a Danshen-containing herbal medicine extensively used to treat angina pectoris) in humans, tanshinol was the only Danshen constituent that exhibited significant systemic exposure [26]. The other Danshen constituents salvianolic acids A, B, and D, rosmarinic acid, lithospermic acid, and protocatechuic aldehyde were either poorly absorbed from the gastrointestinal tract or were extensively metabolized, which resulted in poor detection of their unchanged forms in plasma after dosing. After intravenously dosing DanHong injection (another Danshen-containing herbal medicine extensively used to treat coronary artery disease and ischemic stroke), these Danshen phenolic compounds exhibit different interactions with hepatic and renal uptake transporters, resulting in different profiles of hepatobiliary and renal excretion [43, 69]. Lithospermic acid and salvianolic acid B (both >500 Da) underwent systemic elimination, initiated by OATP1B1/OATP1B3-mediated hepatic uptake; rosmarinic acid and salvianolic acid D (350–450 Da), initiated by OATP1B1/OATP1B3/OAT2-mediated hepatic uptake and by OAT1/2-mediated renal uptake; and protocatechuic acid and tanshinol (both <200 Da), initiated by OAT1/OAT2-mediated renal uptake and OAT2-mediated hepatic uptake. Here, protocatechuic acid is an oxidized metabolite of protocatechuic aldehyde by hepatic aldehyde dehydrogenase. Panax notoginseng roots (Sanqi) is a Chinese medicinal herb extensively used to treat ischemic cardiovascular and cerebrovascular diseases. Triterpene saponins (most of them derived from tetracyclic dammarane), including ginsenoside Rb1 and ginsenoside Rd of 20(S)-protopanaxadiol-type (ppd-type; with O-attached sugar moieties at C3 and/or C20 positions) and ginsenoside Rg1 and notoginsenoside R1 of 20(S)-protopanaxatriol-type (ppt-type; with a free hydroxyl group at the C-3 and O-attached sugar moieties at C6 and/or C20 positions), are the major bioactive constituents of Sanqi. Despite their structural similarity, significantly different elimination kinetics occurs between the ppd-type and ppt-type ginsenosides, as indicated by human t1/2 of 53.5‒175.9 h for the former and 1.3‒1.4 h for the later [39]. The different elimination kinetics results from differences both in hepatobiliary excretion and in renal excretion [36]. Unlike the ppt-type ginsenosides, the human hepatic transporters OATP1B3 and MRP2/BCRP/BSEP/MDR-1 do not mediate the excretion of ppd-type ginsenosides. In addition, extensive plasma protein binding limits glomerular-filtration-based excretion of the ppd-type ginsenosides, while low plasma protein binding facilitates such renal excretion of the ppt-type ginsenosides.

Significant interspecies differences in body exposure, pharmacokinetics, and interactions with drug metabolizing enzymes and transporters are another factors that need to be considered in multi-compound pharmacokinetic research on herbal medicines. For example, senkyunolides G and I are two phthalide constituents of XueBi**g injection and originate from the injection’s component herbs Ligusticum chuanxiong rhizomes (Chuanxiong) and Angelica sinensis roots (Danggui). After intravenously dosing XueBi**g, both the phthalides are major circulating compounds in humans, but only senkyunolide I is the major circulating phthalide in rats with senkyunolide G limitedly detected [23]. Terpene lactones are a class of bioactive constituents of ShuXueNing injection (a botanical drug product of Ginkgo biloba leaf extract, used as add-on therapies in patients with ischemic cardiovascular and cerebrovascular diseases). In humans, the terpene lactones ginkgolides A (molecular mass, 408 Da) and B (424 Da) were eliminated from the systemic circulation predominantly via glomerular-filtration-based renal excretion [45]. However, these two terpene lactones were eliminated both via renal and hepatobiliary excretion in rats [41]. After orally dosing Sanqi extract in humans, ppt-type ginsenosides are extensively deglycosylated by the colonic microbiota into 20(S)-protopanaxatriol, which can be absorbed in the colon and oxidized into several circulating metabolites by the host enterohepatic CYP3A. [42] However, such microbial deglycosylation occurs poorly in rats [27].

Research example: pharmacokinetics-based identification of pseudoaldosterogenic compounds of Glycyrrhiza uralensis roots (Gancao)

Glycyrrhiza uralensis root (Gancao) is a widely used medicinal herb in Chinese traditional medicine. However, Gancao-induced pseudoaldosteronism (characterized by hypokalaemia, hypertension, and peripheral edema) could be an adverse effect of Gancao-containing medicines and dietary products, via inhibition of renal 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) [70]. Given that Gancao is widely used as a component in Chinese herbal medicines, uncovering the compounds that are together responsible for the Gancao-induced adverse effect has implications for precisely defining conditions for safe use of Gancao-containing medicines, such as LianhuaQingwen capsule (an herbal medicine officially recommended as treatment for COVID-19 in China). Although glycyrrhizin is a major Gancao constituent with bioactivities such as inhibiting the replication of the SARS-associated virus, [71] this saponin and its several metabolites have been documented to inhibit 11β-HSD2 [72,73,74,75,76,77,78,79,80]. We identified pseudoaldosterogenic Gancao compounds from circulating Gancao compounds of dosed LianhuaQingwen, based on the compounds’ in vivo access to and in vitro inhibition of human renal 11β-HSD2 [47]. This renal enzyme is highly expressed in the epithelia of the distal tubule and collecting duct [81, 82]. After composition analysis of LianhuaQingwen for constituents originating from Gancao, we performed a multi-compound pharmacokinetic investigation by dosing the capsule in human volunteers, as well as in rats, to identify the major circulating Gancao compounds and to assess their in vivo access to renal 11β-HSD2. The identified potentially important Gancao compounds were then assessed in vitro for their inhibition of human renal 11β-HSD2. Among the 41 Gancao constituents detected in LianhuaQingwen, glycyrrhizin (1) and licorice saponin G2 (2) are the major Gancao saponin constituents. Poor intestinal absorption of 1 and 2 and their access to colonic microbiota result in significant levels of their respective deglycosylated metabolites glycyrrhetic acid (8) and 24-hydroxyglycyrrhetic acid (M2D) occurring in the systemic circulation after colonic absorption. These circulating metabolites are glucuronized/sulfated in the liver and then excreted into bile. Hepatic oxidation of 8 yields M2D. Due to their good membrane permeability, circulating 8 and M2D gain access to (via glomerular filtration and then passive tubular reabsorption) and potently inhibit renal 11β-HSD2. Collectively, 1 and 2 are metabolically activated to the pseudoaldosterogenic compounds 8 and M2D (Fig. 2). Although the microbial metabolites 8 and M2D are not the only Gancao-related compounds exhibiting inhibitory activities on 11β-HSD2, they are the only ones that are bioaccessible for the renal enzyme. Both 8 and M2D are extensively bound to plasma albumin (fu, <1%), limiting the renal glomerular filtration of 8 and M2D, with such binding acting as a ‘safety belt’ that allows the herb Gancao and Gancao-containing medicines to be widely used in Chinese traditional medicine. This finding allows us watch out for people who may susceptible to Gancao-induced pseudoaldosteronism, i.e., patients with hypoalbuminemia.

Fig. 2: A schematic overview of pharmacokinetics-based identification of the pseudoaldosterogenic compounds glycyrrhetic acid (8) and 24-hydroxyglycyrrhetic acid (M2D).
figure 2

The Gancao constituents glycyrrhizin (1) and licorice saponin G2 (2) are metabolically activated by glucuronidase of the colonic microbiota to the metabolites 8 and M2D, respectively, which can access (via passive tubular reabsorption) and inhibit renal 11β-HSD2. (Reprinted from Lan et al., 2021 [47] with permission of Acta Pharmacol Sin).

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Excessive and prolonged use of Gancao-containing herbal medicines and dietary products, as well as glycyrrhizin formulations, is a main cause of pseudoaldosteronism [72]. To ensure safe use of Gancao-containing medicines like LianhuaQingwen, (1) it is pivotal to increase awareness of Gancao-induced pseudoaldosteronism; (2) caution should be exercised when a Gancao-containing medicine is administered concurrently with other Gancao-containing medicines and dietary products; (3) a Gancao-containing medicine should not be administered to patients with decreased 11β-HSD2 activity, hypokalemia, hypertension, impaired liver function, or hypoalbuminemia; and (4) once pseudoaldosteronism occurs, the Gancao-containing medicine and co-administered Gancao-containing products should be withdrawn, and diuretics (such as spironolactone and eplerenone) and drugs (that can alkalinize pH of tubular fluid) may be administered to reduce tubular reabsorption of 8 and M2D.

Drug-combination pharmacokinetic research on Chinese herbal medicines

Pharmacokinetic compatibility (PKC) of a combination therapy

In the practice of Chinese traditional medicine, multi-herb combination therapies (FangjiPeiwu) have been extensively used to achieve therapeutic advantages of enhanced efficacy and safety. Two principles, i.e., QiqingHehe and JunChenZuoShi, guide how to select and combine herbs for the multi-herb combination therapy [83]. QiqingHehe sums up seven ways of using herbs, i.e., using an herb alone (Danxing) and using herbs in combination (to achieve beneficial combination effects and to avoid adverse combination effects). The beneficial combination effects are ** pills for the secondary prevention of myocardial infarction: a randomised clinical trial. Evid Based Complement Altern Med. 2013;2013:738391." href="#ref-CR10" id="ref-link-section-d266226150e1013_1">10,112]. However, another human DDI investigation reported that 28-day administration of the same P. ginseng capsule (500 mg, t.i.d.; Vitamer, CA, USA) did not significantly alter midazolam metabolism [113]. In addition to the approach of midazolam phenoty** being different between the two investigations, information was not provided on interaction-related human pharmacokinetics of the test botanical products and the quality consistency between the product lots used in the two investigations. Ginsenosides are the main pharmacologically active constituents of XueShuanTong, a lyophilized extract of Sanqi for intravenous administration. Recently, ginsenosides, particularly those of ppd-type, were found to potently inhibit hepatic OATP1B3 and, to a lesser extent, OATP1B1 [36, 38]. Given that co-medication of XueShuanTong with synthetic drugs is common for treatment of ischemic cardiovascular or cerebrovascular diseases and that levels of systemic exposure to ginsenosides via intravenous administration route are much higher than the respective ones via oral administration route [39, 42], DDI information is essential for making clinical decision on therapies co-administering XueShuanTong with synthetic drugs. To this end, XueShuanTong-drug interaction potential was evaluated and the investigation focused on assessment of CYP3A induction and OATP1B inhibition.

Literature-mined information, as well as our earlier related study findings [39, 42], was used to facilitate the DDI investigation by avoiding missing potentially important XueShuanTong compounds. After thorough composition analysis of XueShuanTong for ginsenosides and evaluation of associated lot-to-lot quality variability, a multi-compound pharmacokinetic study was performed in healthy volunteers (intravenously receiving XueShuanTong) to identify circulating ginsenosides, unchanged and metabolized, with significant levels of systemic exposure and their pharmacokinetics that was important for perpetrating the DDI. Two types of XueShuanTong-perpetrated DDI study were performed: (1) evaluation of potential of XueShuanTong for inducing CYP3A in healthy volunteers and the results were confirmed by a cell-based induction study with respect to mRNA level and enzyme activity, using the major circulating ginsenosides and (2) prediction of XueShuanTong’s potential for inhibiting OATP1B3 by the major circulating ginsenosides using their time-dependent unbound plasma concentrations in humans. In the CYP3A induction study, possible influence of enzyme inhibition on the results was assessed in vivo and in vitro. In the OATP1B inhibition study, inhibition of OATP1B together by multiple XueShuanTong ginsenosides was assessed in vitro, and the data were processed using the Chou-Talalay method. Samples were analyzed by liquid chromatography/mass spectrometry.

A total of 50 ginsenosides with compound doses of >0.01 μmol/day were detected and characterized in XueShuanTong: 14 ppd-type ginsenosides, 18 ppt-type ginsenosides, and 18 ginsenosides of other types. As shown in Fig. 3, ginsenosides Rb1 and Rd  of ppd-type and ginsenoside Rg1 and notoginsenoside R1 of ppt-type were the major circulating XueShuanTong compounds; their DDI-related pharmacokinetics comprised compound dose-dependent levels of systemic exposure and, for ginsenosides Rb1 and Rd, long terminal half-lives (32‒57 and 58‒307 h, respectively) and low unbound fractions in plasma (0.8%‒2.9% and 0.4%‒3.0%, respectively). Repeatedly dosing intravenous XueShuanTong does not induce human CYP3A4/3A5. Based on human pharmacokinetics and overall inhibitory potency of its bioavailable ginsenosides, intravenously dosed XueShuanTong is found to have high potential for OATP1B3-mediated drug interactions, which is attributed chiefly to ginsenoside Rb1. This finding informs a need for further PBPK model-based prediction of the interaction potential for XueShuanTong and, if necessary, clinical DDI studies of XueShuanTong. Because the ginsenosides selectively inhibit OATP1B3, XueShuanTong is likely to alter pharmacokinetics of selective substrates of OATP1B3 when co-administered, rather than selective substrates of OATP1B1 or dual substrates of OATP1B1/1B3. Several selective OATP1B3 substrates, such as cholecystokinin-8, telmisartan, amanitin, dioscin, convallatoxin, ouabain, dihydroouabain, ouabagenin, and ppt-type ginsenosides, have been reported [36, 114,115,116,117,118]. Information on potential DDIs should be an important part of the patient information leaflet of herbal medicines. However, most Chinese herbal medicines currently lack such information. Increased awareness of ginsenosides’ pharmacokinetics and XueShuanTong-drug interaction potential will help ensure safe use and effective combination therapies of XueShuanTong and synthetic drugs.

Fig. 3: Chemical composition, circulating compounds, and DDI potential of XueShuanTong.
figure 3

Ginsenosides present in XueShuanTong (a), their systemic exposure and renal excretion in human volunteers who received a 2.5-h infusion of XueShuanTong at 500 mg/person (b), and estimated total DDI indexes on OATP1B1 and OATP1B3 for a single 2.5-h intravenous infusion of XueShuanTong at 500 mg/person and for repeated doses of XueShuanTong at 500 mg/person every day on day 18 (c). Chemical structures of the major circulating ginsenosides after dosing XueShuanTong are shown in panel (d). The XueshuanTong constituents 1‒14 are ppd-type ginsenosides; 31‒48 are ppt-type ginsenosides; and 51‒68 are other type ginsenosides. M4, M6, M8, and M11‒M14 are metabolites of the ppt-type ginsenosides (see Ref. [42] for more information about these metabolites). 1, ginsenoside Rb1; 2, ginsenoside Rd; 31, ginsenoside Rg1; 32, notoginsenoside R1; PPD, 20(S)-protopanaxadiol; and PPT, 20(S)-protopanaxatriol. Glc glucopyranosyl, Xyl xylopyranosyl. (Reprinted from Pintusophon et al., 2019 [39] with permission of Acta Pharmacol Sin).

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Chinese herbal medicine as potential victim of pharmacokinetic DDI

In China, herbal medicines are regarded as therapeutic and regulated as drug products. This requires evaluating potential of Chinese herbal medicines not only for perpetrating DDIs but also for acting as victim in DDIs. Natural products such as dietary supplements in many Western countries are not drug products; their therapeutic effects and adverse effects do not need to be and are normally not well defined, like those of conventional drugs. So, little is known which compounds are responsible for these effects of the natural products. Unlike natural product–drug interactions, drug-natural product interactions have been limitedly investigated and documented for their potential.

As a prerequisite for evaluating drug–herbal medicine interaction potential, it should be understood which compounds are responsible for therapeutic or adverse effect of the test herbal medicine and whether altered levels of exposure to these active compounds are associated with reduced efficacy or increased toxicity of the herbal medicine. An herbal medicine is deemed a potential victim of DDI if the enzyme(s) or transporter(s) mediating the intestinal absorption or systemic elimination of its active compounds can be inhibited without other route(s) that can significantly compensate the impaired route or if the interacting protein(s) can be induced.

Inflammation in the liver precedes and promotes the progression towards liver cirrhosis and hepatocellular carcinoma. Intravenous glycyrrhizin has been incorporated into the management of liver diseases (including cause-specific treatment) due to its additional value of anti-inflammation and hepatoprotection [119,120,121,122,123]. Glycyrrhizin, triterpene saponin, is a major constituent present in Glycyrrhiza uralensis root (Gancao) and other medicinal Gancao species (G. inflate root and G. glabra root). Despite the therapeutic effects, high dose and prolonged use of intravenous glycyrrhizin formulations, as specified on the patient information leaflets, possibly induce pseudoaldosteronism by inhibiting 11β-HSD2. Pseudoaldosteronism can result from increased AUC0-∞ of glycyrrhizin. Unlike oral administration, intravenous administration yields unchanged glycyrrhizin as the major circulating form. Unchanged glycyrrhizin is eliminated mainly via hepatobiliary excretion, rather than renal excretion and metabolism. Therefore, level of systemic exposure to and hepatobiliary excretion of unchanged glycyrrhizin are factors influencing intravenous glycyrrhizin-induced pseudoaldosteronism. To this end, human transporters mediating hepatobiliary excretion of glycyrrhizin were characterized at the cellular and vesicular levels and compared with rat hepatic transporters. The role of Oatp1b2 in glycyrrhizin’s elimination and pharmacokinetics was evaluated in rats using the inhibitor rifampin. A PBPK model for glycyrrhizin, incorporating transporter-mediated hepatobiliary excretion, was established and applied to predict human systemic exposure to glycyrrhizin and its potential as the victim in DDI [37].

Glycyrrhizin is of poor membrane permeability. Hepatobiliary excretion of glycyrrhizin involves human OATP1B1/1B3 (Oatp1b2 in rats)-mediated hepatic uptake from blood and human MRP2/BCRP/BSEP/MDR1 (Mrp2/Bcrp/Bsep in rats)-mediated hepatic efflux into bile. No other hepatic transporters exhibit such hepatic uptake activity. In rats, rifampin-impaired hepatic uptake of glycyrrhizin can significantly increase its systemic exposure to glycyrrhizin (Fig. 4). Glomerular-filtration-based renal excretion of glycyrrhizin is slow due to extensive protein binding in plasma. Quantitative analysis using the PBPK model suggests a high likelihood for glycyrrhizin to be a victim of hepatic DDIs when co-medicated with potent dual inhibitors of OATP1B1/1B3 (Fig. 4).

Fig. 4: Potential of intravenous glycyrrhizin as the victim in DDIs.
figure 4

Observed (dots) and PBPK model-simulated (lines) plasma concentration-time profiles of glycyrrhizin in rifampin-untreated (control) and rifampin-treated rats that intravenously received glycyrrhizin at 2.6 mg/kg (a), such plasma concentration-time profiles in humans who received a 12-min intravenous infusion of glycyrrhizin at 40 (in green), 80 (in blue), and 120 (in red) mg/person (b), and PBPK model-predicted influence of impaired hepatic OATP1B1/1B3 activities on levels of systemic exposure to glycyrrhizin (c and d). Chemical structure and comparative elimination routes of glycyrrhizin are shown in panel (e). The plasma levels of glycyrrhizin were prospectively predicted under a 12-min i.v. infusion of glycyrrhizin at 120 mg/person. The assumption, used as a worst-case estimate, was that the inhibition of OATP1B1/1B3 was constant over time. Sustaining the impairment of OATP1B1/1B3 activities by the inhibitory perpetrator depends on the perpetrator’s inhibition potency and pharmacokinetics (the unbound plasma concentrations and elimination t1/2). The observed human plasma concentration data of glycyrrhizin were digitalized from a publication by Yamamura et al. [148] with permission of Elsevier and APhA. Glu glucuronosyl. (Reprinted from Dong et al., 2018 [37] with permission of John Wiley and Sons).

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The OATP1B1/1B3-mediated hepatic uptake governs systemic clearance of and systemic exposure to glycyrrhizin. Given that there are many dual OATP1B1/1B3 inhibitors (including the direct-acting antiviral agents paritaprevir and ritonavir) used in clinics and that substantial impairment of OATP1B1/1B3 activities (by ≥80%) can result in a significantly increased plasma AUC0-∞ and prolonged t1/2 of glycyrrhizin, caution should be exercised in the use of intravenous glycyrrhizin, due to its high likelihood to be a victim in DDIs. This DDI information facilitates ensuring safe glycyrrhizin-including combination therapies for liver diseases. To prevent such drug-glycyrrhizin interactions, plasma concentrations of both glycyrrhizin and co-administered dual inhibitors of OATP1B1/1B3 should be monitored and their dosages optimized accordingly. OATP1B1 and OATP1B3 can compensate for the lack of each other. So, coadministration with a selective inhibitor of OATP1B1 or OATP1B3 may not lead to significant changes in level of systemic exposure to glycyrrhizin.

PKC evaluation for an herbal-synthetic medicine combination

Drugs in a combination therapy, particularly for multifactorial diseases, can have distinct mechanisms of action and exert enhanced pharmacodynamic effect. To ensure such therapeutic benefit, a high degree of PKC within the combination therapy is desired, i.e., absence of serious pharmacokinetic DDI among the co-administered drugs. In China, growing evidence shows that incorporating herbal medicines into synthetic drug-based therapies for multifactorial diseases delivers therapeutic benefits [9,10,24]. To reflect clinical reality, 45 antibiotics commonly used in sepsis care in China and 12 XueBi**g compounds bioavailable for DDIs were selected for the drug-combination pharmacokinetic investigation. No XueBi**g compound could pair, as perpetrator, with the antibiotics. Although some antibiotics could, due to their inhibition of UGT2B15, OAT1/2 and/or OATP1B3, pair with senkyunolide I, tanshinol and salvianolic acid B, the potential interactions (resulting in increased systemic exposure) were likely desirable due to these XueBi**g compounds’ low baseline exposure levels. Inhibition of aldehyde dehydrogenase (ALDH) by seven antibiotics (i.e., imipenem, meropenem, ceftazidime, penicillin, ampicillin, oxacillin, and flucloxacillin) probably led to undesirable reduction of systemic exposure to protocatechuic acid  from XueBi**g. The XueBi**g/antibiotic combination exhibited a high degree of PKC at clinically relevant doses, with a PKC index of 0.94, which supports concurrent use of the two types of medicines in sepsis care. Figure 6 depicts this evaluation of PKC degree of combination therapy of XueBi**g and antibiotics. Although the early identification and treatment of sepsis is desirable, prompt administration of all the necessary medications increases the drug interaction risk. For optimal sepsis care, a high degree of PKC is desirable not only between XueBi**g and antibiotics but also between XueBi**g and other medicines and among different types of synthetic medicine. The preceding drug-combination pharmacokinetic approach can be applied to investigate other drug combination therapies.

Fig. 6: A schematic overview of evaluation of PKC degree of combination therapy of XueBi**g and antibiotics and the high degree of PKC supports concurrent use of the two types of medicines in sepsis care.
figure 6

A1‒A45, 45 antibiotics commonly used in sepsis care in China (see Ref. [24] for the detailed information); X1‒X12, 12 major XueBi**g compounds, unchanged and metabolized, that are bioavailable for drug DDIs and bioactive for sepsis care (see Ref. [24] for the detailed information). (Reprinted from Li et al., 2019 [24] with permission of Acta Pharm Sin B).

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Outlook

Despite the many advances and successes described in this Review, only a limited numbers of Chinese herbal medicines have been extensively investigated and the impact of pharmacokinetic research on the modernization of Chinese traditional medicine is still limited owing to the sophistication, expertise, extensive experimental work, high cost, and long study period required. We believe that this situation is changing as pharmacokinetic research is increasingly recognized as a powerful and versatile tool to help identify therapeutically important constituents of complex Chinese herbal medicines and develop herbal medicines for use as or in combination therapies of multifactorial diseases by acting on multiple targets in concert.

More technological advances and broader studies are needed in multi-compound pharmacokinetic research on the Chinese herbal medicine to enhance our ability to identify the medicine’s important constituents that are together responsible for the therapeutic action and/or adverse effect of the medicine. Recently, abundant high-quality data exist and are searchable; understanding of in vitro and in vivo methodology continues to improve; and computational power continues to increase. All these have boosted drug discovery, including the ability to identify drug candidates with appropriate pharmacokinetic properties [139, 140]. By utilizing artificial intelligence/machine learning, the pharmacokinetic properties and overall profiles of numerous constituents can be predicted for an herbal medicine in a more high-throughput and cost-effective manner than traditional methods. Rational and effective integration of the artificial intelligence-based predictions with various experimental measurements can fuel the multi-compound pharmacokinetic research on complex herbal medicines. Initiatives to incorporate artificial intelligence into pharmacokinetic prediction of herbal compounds and medicines are already underway and the main questions are how to best utilize the technique and how to evaluate the benefit of its applications. One of the challenges posed in this field is lack of high-quality experimental data.

In the multi-compound pharmacokinetic research, all types of body exposure that are relevant to the therapeutic action and/or adverse effect of an herbal medicine should be investigated for the medicine’s constituents. Given that the human intestinal microbiota is integral to the physiology of its host [63, 64] and is a major source of variability in pharmacokinetics of and response to many drugs [141, 142], intestine-luminal exposure to the herbal compounds after dosing an herbal medicine should be assessed to determine the compounds’ access to the microbiota. Traditionally, pharmacokinetic investigations have focused on systemic exposure to the drugs and have mostly ignored the intestine-luminal exposure and the significant direct and indirect impacts of the intestinal microbiota on the systemic exposure of drugs. The intestine-luminal exposure to herbal compounds after an orally dosed herbal medicine involves multiple elements: passage of the unchanged constituents through the digestive tract, loss of the unchanged constituents owing to intestinal absorption (particularly in the upper gastrointestinal tract), loss of the unchanged constituents owing to metabolism by the intestinal enzymes (such as lactase-phlorizin hydrolase) or the microbiota, appearance of the metabolites owing to these intestinal metabolism, loss of the metabolites owing to intestinal absorption or further metabolism by the microbiota, and appearance of the metabolites owing to biliary excretion of the conjugated metabolites (most often glucuronides) [143]. After intravenously dosing an herbal injection, the intestine-luminal exposure to herbal compounds is initiated by biliary excretion of the herbal compounds (unchanged and metabolized) and occasionally by intestinal excretion of such compounds. The intestine-luminal exposure via intravenous administration is normally much lower than that via oral administration. In addition, the intestine-luminal exposure to herbal compounds is influenced by many factors, including physicochemical properties of the herbal compounds, expression and function of the intestinal enzymes and transporters and the interplay of these proteins, thickness and pH of mucus, intestinal tissue structure, composition and function of the intestinal microbiota, DDI, gene variants, etc. It is generally thought that drug compounds most accessible to the intestinal microbiota are those that are orally administered but not absorbed in the upper gastrointestinal tract. However, after orally dosing Gancao-containing medicine, the compounds that exhibit significant colon-intestinal exposure are the microbial metabolites glycyrrhetic acid and 24-hydroxyglycyrrhetic acid, rather than the respective unabsorbed parent constituents glycyrrhizin and licorice saponin G2, which appear not to occur in the colon [47]. Assessment of human intestine-luminal exposure to herbal medicines is usually based on analysis of the feces samples, but further investigating the exposure is difficult in healthy volunteer and patient studies. Animal models can be informative but have limited translational potential due to significant interspecies variations in the intestinal microbiota. Therefore, human microbiota-associated animal models have been used widely in intestinal microbiome research [144,145,146]. We have started to utilize human microbiota-associated rats in intestine-luminal exposure studies of herbal medicines. Overall, current understanding of intestine-luminal exposure to herbal medicines is incomplete and there are questions still to be answered.

After the major exposed compounds are identified for an herbal medicine, two important aspects need to be further investigated, particularly for those that are pharmacologically and/or toxicologically active, i.e., (1) effective modulation of their body exposure levels and (2) their in vivo access to the associated targets. These aspects are seldom investigated in traditional pharmacokinetic research, but can influence the medicine’s therapeutic outcome. Understanding how the exposure levels of active herbal compounds can be effectively modulated is important for ensuring effectiveness of an herbal medicine, by optimally matching the compounds’ pharmacokinetic concentrations (after dosing the medicine) to their respective therapeutic concentrations, or for avoiding occurrence of adverse effect of the medicine, by containing the pharmacokinetic concentrations not to exceed the respective toxic concentrations. Meanwhile, understanding whether the exposure levels of the major active herbal compounds could be significantly modulated is vital for evaluating potential for the medicine to be a victim in a DDI. Our pioneering investigations have involved modulating levels of systemic exposure to several herbal compounds [36, 37, 147]. In traditional pharmacokinetic investigations, drugs are evaluated for systemic exposure and associated disposition, but little is known about their further access to the therapeutic/toxic targets. There are biological barriers for drug molecules moving from the systemic circulation to the locus of action, so it is important to understanding the physiological structures of the target tissues at cellular level. Revealing how pseudoaldosterogenic glycyrrhetic acid and 24-hydroxyglycyrrhetic acid access the renal 11β-HSD2 allows us to identify the ‘safety belt’ for extensive use of Gancao and Gancao-containing medicines in Chinese traditional medicine and people who are susceptible to Gancao-induced pseudoaldosteronism (such as patients with hypoalbuminemia) [47].

So far, drug-combination pharmacokinetic research on Chinese herbal medicines has focused mainly on identifying risks of unintentional DDI associated with herbal medicines used as or in combination therapies. Safe and effective combination therapies are ensured by avoiding the identified risks. In practice of Chinese traditional medicine, many toxic herbs are used for therapeutic purposes, and FangjiPeiwu is used to minimize their toxicities. This deliberate combination for minimizing herb’s toxicity (‘JianduPeiwu’ in Chinese) is another way of ensuring safe combination therapies. Based on the QiqingHehe principle, JianduPeiwu is hypothesized to involve three mechanisms of two types; they are toxicity antagonism, associated with ‘**angwei’/’**angsha’, that utilizes the combination’s other herb(s) to minimize the toxicity of the toxic herb by counteracting the toxic effect or by reducing the level of exposure to the toxic compounds via pharmacokinetic DDI and efficacy synergism, associated with ‘**angxu’ and’**angshi’, that decreases the dosage of the toxic herb but maintains the same efficacy while minimizing the toxicity. Last year, the National Natural Science Foundation of China approved support for a major project in JianduPeiwu for therapeutic use of toxic Chinese herbs. The funded project (82192910) involves both multi-compound and drug-combination pharmacokinetic research (which works closely with other disciplines) and selects the commonly used toxic Chinese medicinal herbs Euodia rutaecarpa fruit (Wuzhuyu) and Psoralea corylifolia fruit (Buguzhi) and their formulated herb combinations (i.e., Wuzhuyu decoction, Zuo** pill, Eshen pill, and Sishen pill) for investigation. The multi-compound pharmacokinetic approach is used as the forerunner for identification of toxic compounds of the toxic herbs, as well as interacting and/or therapeutically active compounds of the combinations’ other component herbs. The identified compounds are investigated for the disposition factors governing their levels of body exposure, the pharmacokinetic differences among them, and the simultaneous accessibility to their respective targets. The drug-combination pharmacokinetic approach is used to evaluate PKC degrees of combinations for possible toxicity antagonism that counteracts the toxic effect and for possible efficacy synergism that reduces the dosage of the toxic herb; the approach is also used to evaluate the potential for and toxicological outcome of pharmacokinetic DDIs that result in possible toxicity antagonism. The pharmacokinetic research is to support elucidating potential mechanisms of JianduPeiwu and efficacy–toxicity relationship of the herbs in combinations and serves as a translational discipline. The whole project is multidisciplinary and aims to develop rational strategy for safe and therapeutically effective use of toxic Chinese herbs in clinics.

Over the past decade, great advances have been achieved in the theory, methodology, techniques, and requirements of, together with their applications in, pharmacokinetic research on Chinese herbal medicines, which has become an emerging field in pharmacokinetics. This is a great window of opportunity for the modernization of Chinese traditional medicine to capitalize on the pharmacokinetic approaches, with the valuable ultimate goal of ‘more effective and safe herbal medicines available to patients’.