Carboxylesterases Mediated Herb-drug Interactions: A Systematic Review

For more information: emily.li@wecistanche.com

Dan-Dan Wang, Yun-Qing Song, Ya-Di Zhu, Yi-Nan Wang, Hai-Feng Li, Guang-Bo Ge , Ling Yang

1 Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

2 School of basic medical science, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

Highlights

This review summarized recent progress in human carboxylesterases (hCEs) mediated herb-drug interactions (HDIs). The key roles of hCEs in drug metabolism, the inhibitory capacities, and the inhibition mechanism of a variety of herbal extract and herbal constitutes against hCEs have been well summarized. Furthermore, the challenges and future perspectives in this field are highlighted by the authors. All information and knowledge presented here will be very helpful for the pharmacologists to deeper understand the interactions between herbal constituents and hCEs, as well as for clinical clinicians to reasonably use herbal medicines for alleviating hCEs-associated drug toxicity or avoiding the occurrence of clinically relevant hCEs-mediated HDIs.

Cistanche is a kind of Herba medicine and has many functions

Abstract

Esterases participate in the metabolism of~10% of the clinical drugs that contain ester or amide bonds, but the esterases mediated drug/herb-drug interactions(DDIs or HDIs) have not been reviewed in-depth, Carboxylesterases(CEs), the most abundant esterases expressed in the metabolic organ of mammals, play a pivotal role in the hydrolysis of a variety of endogenous and xenobiotic esters, In the human body, two predominant carboxylesterases including hCE1 and hCF2 have been identified and extensively studied over the past decade. These two enzymes have been found with hydrolytic activity towards a variety of endogenous esters and ester-containing drugs. Recent studies have demonstrated that strong inhibition on hCEs may slow down the hydrolysis of CEs substrates. which may affect their pharmacokinetic properties and thus trigger potential DDLs or HDls. Over the past decade, many herbal extracts and herbal constitutes have been found with strong inhibitory effects against CEs, and their potential riSks on herb-drug interactions(HDls) have also attracted much attention. This review focused on recent progress in hCEs mediated herb-drug interactions. The roles of hCEs in drug metabolism, the inhibitory capacities, and the inhibition mechanism of a variety of herbal extracts and herbal constitutes against hCEs have been well summarized. Furthermore, the challenges and future perspectives in this field are highlighted by the authors, All information and knowledge presented in this review will be very helpful for the pharmacologists to deeper understand the metabolic interactions between herbal constituents and hCEs, as well as for clinical clinicians to reasonable use herbal medicines for alleviating hCEs-associated drug toxicity or avoiding the occurrence of clinically relevant hCEs-mediated HDIs.

Keywords: Human carboxylesterases(CEs), hCE1. hCE2,herb-drug interactions. Natural inhibitors

Background

Drug metabolizing enzymes (DMEs)play a pivotal role in the metabolic clearance of drugs or other xenobiotic compounds by converting lipophilic molecules to more water-soluble metabolites, which can be readily excreted through the kidney or biliary clearance. Inhibition or induction of DMEs may affect the pharmacokinetic properties of therapeutic drugs and thus trigger clinically relevant drug/herb-drug interactions (DDIs or HDIs)[1-4]. The regulatory agencies, such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), have issued guidelines for industry on evaluation of the inhibition potentials of drugs under development on the key human DMEs prior to approval [5,6]. Drug metabolism is divided into phase I and phase II reactions. In phase I reactions, polar groups are introduced to the molecules through oxidation, reduction, and hydrolysis. In phase II reactions, phase I metabolites or the parental compounds themselves undergo conjugation reactions with hydrophilic moieties including glucuronic acid, sulfate, glutathione, or amino acids. Among all known DMEs involved in phase I reactions, cytochrome P450 enzymes(CYPs)play a crucial role in drug metabolism, followed by esterases, which contributed to the metabolism of ~10% of the clinical drugs that contain ester or amide bonds. Over the past decade, CYPs mediated DDIs or HDIs have been well-summarized in several reviews, but the esterases-mediated drug/herb-drug interactions have not been reviewed in depth [5].

Esterases belong to the serine hydrolase enzyme family, which share a conserved catalytic mechanism that enlists a key serine nucleophile within a catalytic triad. As their name implies, esterases catalyze the hydrolysis of numerous compounds with ester/amide bonds into the corresponding alcohol and carboxylic acid and thus play crucial roles in a wide range of physiological and pathological processes, such as xenobiotic metabolism, lipid homeostasis, cancer, diabetes and obesity [7,8]. In mammals, carboxylesterases(CEs) are the most abundant esterases in the metabolic organ (such as liver, intestine, and kidney), which play a pivotal role in the hydrolysis of a variety of endogenous and xenobiotic esters and have been extensively studied over the past decade [9]. In the human body, human carboxylesterase 1 (hCE1)and human carboxylesterase 2(hCE2)are two key mediators responsible for the hydrolytic metabolism of various ester xenobiotics including ester drugs (such as oseltamivir, clopidogrel, irinotecan, and capecitabine) and environmental toxicants (such as pyrethroids)[9, 10]. Human CE1 and human CE2 share 47% amino acid sequence identity, but these two enzymes exhibit extremely different substrate distribution and specificity. Generally, hCE1 is abundantly expressed in the human hepatocytes and adipocytes, with lesser amounts in the kidney, monocytes, lung, intestine, testis, heart, and macrophages. By contrast. hCE2 is expressed mainly in the small intestine and colon and is also detectable in the kidney, liver, heart, brain, and testis. Human CEl and CE2 also exhibit distinct substrate specificities. Generally, hCEl prefers to hydrolyze the ester substrates with a small alcoholic group and a large, bulky acyl group, such as enalapril, oseltamivir, imidapril, clopidogrel, meperidine, D-luciferin methyl ester, and the illegal drugs heroin and cocaine [9]. By contrast, CE2 prefers to hydrolyze esters with a relatively large alcohol group and a small acyl group, such as irinotecan, prasugrel, capecitabine, flutamide, and fluorescein diacetate [8].

Inhibition on hCEs may slow down the hydrolysis of hCEs substrate drugs in vivo, and thus modulate their pharmacological and toxicological effects. For instance, clopidogrel, one of the most frequently prescribed antiplatelet agents, the majority of which can be rapidly hydrolyzed to an inactive metabolite by hepatic hCE1, only a small proportion of which can be activated by CYPs to form 2-oxo-clopidogrel, followed by conversion to the active metabolite [11-14]. Co-administration with hCE1 inhibitors may partially block the hydrolytic pathway of clopidogrel, while the formation rates of the active metabolite via CYP-mediated bioactivation will be increased, which may increase the exposure to clopidogrel active metabolite and enhance its antiplatelet effects. Furthermore, irinotecan, a hCE2 substrate drug, could trigger severe delayed diarrhea due to the overproduction of SN-38(the hydrolytic metabolite of irinotecan) in the small intestine, co-administration with potent hCE2 inhibitors may ameliorate CPT-11 associated life-threatening diarrhea in patients and thus improve the patient's quality of life [15-18]. With this goal in mind, many hCE2 inhibitors have been developed for alleviating irinotecan-induced toxicity or prolonging the half-lives of hCE2 substrate drugs.

The key roles of CEs in both human health and xenobiotic metabolism arouse great interest in the discovery of CEs inhibitors to modulate endogenous metabolism or to improve the outcomes of patients administrated ester drugs, as well as to avoid potential risks of DDIs or HDIs. Over the past decade, a panel of isoform-specific optical probe substrates has been developed, which strongly facilitated high-throughput screening and characterization of CEs modulators and the investigations on hCEs associated DDIs or HDIs [19-22]. With the help of these newly developed optical probe substrates, the inhibitory effects of herbal extracts and their constituents on hCEs have been well investigated [9]. Considering that herbal medicines are widely used in Asia countries for the treatment of various diseases in clinics, it is necessary to investigate the metabolic interactions of herbal constitutes with hCEs before the combination use of herbal medicines and clinical drugs. With the intention of improving the reader's knowledge of the HDIs associated with hCEs, the roles of hCEs in drug disposition, the inhibitory effects of herbal medicines, the inhibition potentials, and the action mechanism of herbal constitutes against hCEs have been well-summarized in this review. All information and knowledge presented in this review will be very helpful for the deep understanding of the interactions between herbal constituents and hCEs, as well as for clinical clinicians to reasonable use herbal medicines for alleviating hCEs-associated drug toxicity or avoiding the occurrence of clinically relevant hCEs-mediated HDIs.

Human CEs substrate drugs

Human CEs are key enzymes from the serine hydrolase superfamily, which efficiently catalyze the hydrolysis of a variety of ester/amide-containing pharmaceutical products [23-25]. It is widely recognized that the function of hCEs can influence drug metabolism and clinical outcomes. In this review, we outline the known substrate drugs of hCE1 and hCE2 and highlight the relevance of hCEs functions to contemporary pharmacotherapy [26, 27].

As one of the most important phase I drug-metabolizing enzymes, hCE1 is involved in toxin detoxication and drug metabolism (Table 1).On one hand, hCE1 mediates the metabolic activation of many prodrugs(such as temocapril, oseltamivir, sacubitril, etc.)[27. On the other hand, hCE1 promotes the metabolic inactivation and clearance of some esterified drugs(such as clopidogrel, methylphenidate, and cocaine, etc.). A recent study reported that a new class of promising anticancer

phospho-nonsteroidal compounds, anti-inflammatory drugs (phosphor-NSAIDs), are also inactivated by hCEl and hCEl inhibitors will improve the efficacy of these phospho-NSAIDs both in vitro and in vivo. As for hCE2, it has been reported responsible for the activation of several anti-tumor prodrugs, for instance, CPT-11 and LY2334737(Table 1)[28]. Actually, many factors including drugs, genetic factors, and disease status, have been reported can cause individuals and tissues differences in both expression and function of hCE1 and hCE2, and further influence the clinical outcomes of hCEs substrate drugs [29].

The genetic factor was one of the extensively studied factors affecting the clinical outcomes of CEs substrate drugs [44, 45]. Over the past decade, a vast number of single-nucleotide polymorphisms (SNPs) have been reported in the NCBI SNP database. Notably, the allele and haplotype frequencies of known SNPs showed significant differences among different ethnic groups. For instance, the D260fs and the G143E variants were two important functional SNPs in Caucasian populations, while these two CES1 genetic polymorphisms were not found in a Korean population. Up to now, many functional genetic variants of CES1 and CES2 have been reported, which may be associated with the individual difference in the responses to contemporary pharmacotherapy [10,46-49]. Clopidogrel is a prodrug that has been widely used to inhibit platelet aggregation. Following oral administration, more than 85% of clopidogrel can be rapidly hydrolyzed to its carboxylic acid (an inactive metabolite)by hCE1.Zhu et al. Reported that the CES1 variants G143E and D260fs diminished the hCE1 activity, which impaired the metabolism of clopidogrel [46][10]. Aspirin is an antiplatelet agent that is frequently used for the prevention of cerebrovascular and cardiovascular events. Aspirin is also a CEs substrate drug that is mainly hydrolyzed by gastrointestinal CE2 to form its active hydrolytic metabolite. Tang et al. reported that the CES2 variant A139T decreased human CES2 activity and thus decreased aspirin hydrolysis [46]. The association between SNPs in the human CES2 gene and CPT-11 hydrolysis has also been reported [48,50]. Among Japanese volunteers, the CES2 variants rs72547531 and rs72547532 were associated with decreased human CE2 activity and reduced CPT-11 hydrolysis activity in vivo. [48] Moreover, disease status also can affect the expression or function of CEs and drug response. Xu et al collected and analyzed 18 types of tumors, found 2 types (gallbladder tumor and lymphoma) did not express hCE2, 5 types expressed weak hCE2, and 11 types expressed moderate to high hCE2 levels. Moreover, CE2 protein was highly variable among liver samples, with a 15-fold range in the cytosol and a 3-fold range in microsome fractions. More importantly. liver microsomal hCE2 protein expression was significantly correlated with irinotecan activation to SN-38 [51]. LY2334737 is an oral prodrug of the clinically efficacious anticancer agent, gemcitabine. The hydrolysis of LY2334737 to gemcitabine is mediated by hCE2.A recent study exhibited the cellular hCE2 expression confers prodrug sensitivity [43]. Since these two enzymes play crucial roles in the hydrolysis of a variety of endogenous esters and ester-containing drugs, the strong inhibition on human CEs may slow down the hydrolysis of CEs substrates, which may affect their pharmacokinetic properties and thus trigger potential drug/herb-drug interactions.

CES mediated herb-drug interactions

As one important class of phase I drug-metabolizing enzymes, hCEs play a key role in toxin detoxication and drug metabolism. Since the catalytic activity of CEs has been reported to affect the efficacy and clinical outcomes of numerous esterified drugs, potent inhibition of the hCEs by herb ingredients may result in herb-drug interactions. Thus, the reported herb extracts or herbal constitutes that display potent inhibition towards CEs are summarized and discussed in the following section.

Herbal extracts with CEs inhibition activity

A number of studies have investigated the inhibitory effects of herb extracts on hCEs activity. The herbal extracts displaying inhibitory effects on hCEs are listed in Table 2. White Mulberry Root-bark(WMR) is an edible Chinese herbal used for the treatment of inflammation, nephritis, and asthma. The ethanolic extract from WMR displayed strong inhibitory effects against hCE2 and the IC50 value 30.32 μg/mL【52】. The crude extract of Fructus Psoraleae (FP)also showed a significant inhibitory effect towards hCE2-mediated FD hydrolysis, and the catalytic activity of hCE2 could be completely inhibited at a concentration of 12 ug/mL while the ethanol extract of FP displayed relatively weak inhibitory effects towards hCEI at the same dose. The inhibitory effects on hCE2 by different extracts of Salvia miltiorrhiza ("Danshen")prepared using hot water, acetone, or 56% ethanol. As summarized in Table 2, organic solvent extracts of "Danshen" roots exhibited the strongest inhibitory towards hCE2 with the IC50 value determined as low as 160 ng/ml [53], suggesting that potent hCE2 inhibitors are present within the acetone or ethanolic"Danshen root" extracts. It is worth noting that the acetone extract of “Danshen root”, we're capable to reduce the sensitivity of U373G cells expressing hCE2 to irinotecan, suggesting that the hCE2 inhibitors from “Danshen root” are cell-permeable and may modulate SN-38 production in vivo. Another study found that St John’s wort, black cohosh, and ginger root extract could potentially inhibit CEs mediated biotransformation of irinotecan. As shown in Table 2, the inhibition ability of these herbal extracts was ranked as black cohosh > Ginger > St John’s wort [54]. Furthermore, Li et al have systematically collected and evaluated the inhibitory effects of 100 herbal extracts on hCE2 using FD as a probe substrate (Table 3), which provide important information for the further study on the herbal constitutes with hCEs inhibition activity [55].

Inhibition of herbal constitutes on human CEs

Flavonoids. Flavonoids are polyphenolic compounds that are widely distributed in vegetables, fruits, and beverages, such as tea and wine, which fulfill pharmacological properties. Recent studies have demonstrated that some natural flavonoids, including 5,6-dihydroxyflavone, hispidulin, eupatilin, isorhamnetin and apigenin 7-O-methyl ether, are strong inhibitors against hCE2 [56], while nevadensin, an abundant natural constitute from Lysionotus pauciflorus Maxim., is a relatively specific inhibitor of hCE1 [57]. Sun et al have found the major ingredients of FP, including neobavaisoflavone, corylifolinin, Corey Olin, psoralen, corylin, and bavachinin showed strong inhibition towards the activity of hCE1 in a dose-dependent manner [58]. Li et al have reported that the major constitutes in Fructus Psoraleae, isobavachalcone, including neobavaiso flavone, bavachinin, cortisol A, and bakuchiol can potently inhibit hCE2-mediated FD hydrolysis in HLM [55]. Both Lineweaver-Burk and Dixon plots demonstrated that these five natural flavonoids against hCE2 in HLM functioned as a non-competitive inhibitor against hCE2-mediated FD hydrolysis in HLM, with the K; values evaluated as 3.89 μM，1.64 μM，1.12μM，0.62μM，and 2.12 μM， respectively. Liu et al have identified and characterized the major flavonoids in White Mulberry Root-bark are naturally occurring hCE2 inhibitors, using chemical fingerprinting analysis combined with hCE2 inhibition assays [52]. On the basis of LC retention times, UV and MS spectral data, three major constitutes in White Mulberry Root-bark are efficiently identified as SD (sanggenone D), KG (kuwanon G), and SC(sanggenone C). The also values of SD, KG, and SC against CE2 in HLM were evaluated as 1.09 uM,1.14uM, and 1.02 uM, respectively 52]. These findings are very helpful for medicinal chemists to design and develop more potent and highly selective flavonoid-type hCE2 inhibitors [64].

Triterpenoids. Triterpenoids are a diverse group of natural products with wide distribution, high chemical diversity, and important pharmacological properties. Zou et al collected a series of natural triterpenoids and tested their inhibitory effects against CEs using D-luciferin methyl

(DME), and ester 6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydropyridine-2-yl benzoate (DDAB) as specific optical substrate for hCE1, and hCE2,respectively. Following the screening of these natural triterpenoids, oleanolic acid(OA), and ursolic acid (UA)were found with strong inhibitory effects on hCEI while showing weak inhibitory effects on hCE2 [59]. Twelve new and ten known protostane triterpenoids were isolated from the rhizome of Alismaorientale, while four of them(Alismanol B,25-O-Ethylalisol A, Alismanol D, Alismanol F)showed moderate inhibitory activities and were selective toward hCE2 enzymes, with ICs values of 8.68，4.72，4.58，and 2.02 μM，respectively 【65】. Moreover, the inhibition kinetics of Alismanol F toward hCE2-mediated4-benzoyl-N-butyl-1,8-naphthalimide (MPN) hydrolysis was established, and the K; value was determined as low as 1.76 μM using a mixed inhibition model.

Fatty acids are present in many herbal extracts. Recent work reported the inhibition of hCEs activity using THP1 monocytes/macrophages and hCEs by fatty acids. Crow et al. found that most naturally occurring fatty acids strongly inhibited the hydrolytic activities of hCE1, with the IC50 values within the micromolar range, and unsaturated fatty acids displayed better inhibitory effects on hCE1 than saturated ones, but they did not display strong inhibition towards hCE2 (Table 4). Among these fatty acids tested, 5Z, 8Z, 11Z, 14Z-Eicosatetraenoic acid (arachidonic acid, C20:4 ω6) showed the strongest inhibitory effects toward hCE1, with IC50 value 2 µM [60].

Others Besides the above-mentioned compounds, other compounds with carboxylesterase inhibition capacity have also been reported. Wang et. al obtained phenolic glycosides and monoterpenoids from the roots of Euphorbia bracteolate, all of them showed the inhibitory effect against hCE2 by MPN-based fluorescence bioassay in vitro, with the strongest inhibitor scopoletin-7-O-β-d-(6’-galloyl)-glucopyranoside (IC50 7.17 μM) [61]. Shikonin, a natural naphthoquinone compound derived from the herb Lithospermumerythrorhizon, is widely used for its various pharmacological activities. A recent study exhibits that shikonin significantly inhibits the activity of CE2 when FD and NCEN are used as substrates [62]. A chemical investigation of the roots of Euphorbia ebracteolate identified eighteen diterpenoids and glycosides and most of them showed moderate inhibitory effects against hCE2 [63]. Recent studies showed that some tanshinones are potent hCEs inhibitors toward both hCE1 and hCE2 in vitro, such as tanshinone IIA and tanshinone I. Meanwhile, their ability to affect intracellular inhibition of hCE2 was assayed using 4-methylumbelliferone acetate (4-MUA) as a substrate. By using cells expressing hCE2, tanshinone IIA and tanshinone I was proved could reduce the sensitivity of cells to CPT-11, due to reducing the production of SN-38 [53]. Recent work demonstrated that tanshinone IIA, tanshinone I, dihydrotanshinone, and cryptotanshinone were all irreversible inhibition of hCEs and can inactivate human CEs both in vitro and in cell culture systems and can modulate the metabolism of the esterified drug oseltamivir [64].

Conclusion and future perspectives

Over the past decade, the key roles of hCEs in the hydrolysis of a variety of endogenous and xenobiotic esters have been well-investigated. Considering the crucial roles of hCEs in both endogenous and xenobiotic metabolism, it is necessary to evaluate the regulatory effects of clinical drugs and herbal medicines on hCEs, and to predict the potential beneficial or undesirable effects of hCEs-associated herb-endobiotic interactions or herb-drug interactions (HDIs). Over the past ten years, biochemists have made a significant breakthrough in the development of practical and specific optical

substrates for sensing hCE1 or hCE2 in complicated biological systems [66-69], which strongly facilitate high-throughput screening and characterization of hCE1 modulators (such as inhibitors, inactivators, simulators, and inducers) and further investigations on hCEs-associated HDIs. With these probe substrates in hands, the inhibition or induction assays of herbal extracts or herbal constitutes on hCEs in tissue preparations or living systems can be conducted in a more convenient and efficient way. Up to now, a variety of herbal extracts and herbal constitutes have been found with hCEs inhibition activity. However, most of the previous investigations on hCEs inhibition were conducted in liver microsomes, and the capability of all reported herbal constitutes targeting intracellular hCEs and their potency against hCEs in living systems have not been well investigated. Thus, it is urgently necessary to construct more practical methods for screening and characterization the inhibitory effects of herbal constitutes targeting intracellular hCEs in living systems or in vivo [70]. For those herbal extracts with strong hCEs inhibition activity, it is necessary to further identify the major natural inhibitors from herbs. In these cases, chemical fingerprinting analysis should be used in combination with fluorescence-based inhibition assays, such a strategy has been successfully used to identify and characterize the naturally occurring inhibitors of hCE2 in several herbal medicines [55]. Furthermore, to better predict the clinically relevant hCEs-associated HDIs, it is very necessary to conduct in vitro-in vivo extrapolation (IVIVE) using reliable data about both human beings and hCEs inhibitors, including the physiological parameters of the particular patients, the pharmacokinetic data, and inhibition constants of major hCEs inhibitors in human tissues. Taken together, currently available data call for more in-depth studies on hCEs-associated herb-endobiotic interactions or herb-drug interactions (HDIs), such as the biological functions of hCEs in endogenous metabolism, the relevance of hCEs to human diseases, the response of hCEs inhibitors on mammalian CEs from various species, as well as the interactions between hCEs and their ligands. All these studies will be very helpful for further investigations on hCEs-associated HDIs and the possible consequences.

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