**6.1** *In vitro* **model for CYP3A and P-gp studies**

The *in vitro* CYP inhibition study is usually performed in human liver microsomes (HLMs) or human liver hepatocytes (HLHs), Caco-2 cell lines, or recombinant CYP (rCYP) enzymes [56]. The technology used for CYP inhibition includes luminescence, florescence, radiometric, and HPLC or LC-MS/MS assay. In florescence and luminescence assays, the metabolism of profluorescent or proluminescent substrate by CYP enzyme to give their fluorescent or luminescent product, respectively, is measured in rCYP enzyme. In radiometric method, the release of radiolabel on metabolism of substrate is measured in HLM. In HPLC or LC-MS/MS assay, the concentration of substrate/probe drugs is measured in HLM, HLH, and rCYP enzymes [57].

The *in vitro* analyses used for P-gp-mediated efflux studies include cytotoxicity assay, accumulation/efflux assays, transport assays, and ATPase assays. In cytotoxicity assays, the IC50 end point (concentration of P-gp substrates or inhibitors) that inhibits the growth of P-gp expressing cells is measured. In accumulation/efflux assay, the accumulation of drugs in P-gp expressing cells is measured. In transport assays, the permeation of drugs from apical-to-basolateral and basolateral-to-apical compartment in polarized epithelial cells is measured, whereas in ATPase assay, the stimulation or inhibition of P-gp ATPase enzyme activity in membranes of P-gp expressed cells is measured [58, 59].

## **6.2** *In vivo* **models for CYP3A and P-gp-mediated DDI studies**

Various strains of mice (including transgenic and knockout), rats, and nonhuman primate (monkey) have been used for CYP3A, P-gp, and dual CYP3A/P-gpmediated DDI studies [60].

Since midazolam and triazolam are specifically extensively metabolized by CYP3A4 and ketoconazole is a potent inhibitor of CYP3A4, several groups of studies used as model for experimental CYP3A-mediated DDI studies. Moreover, midazolam is not a substrate for P-gp, whereas the ketoconazole can effectively inhibit both CYP3A and P-gp, which can confirm that the interaction between them can be sole attributed to effects on CYP3A. CYP gene knockout and humanized mice have been established for CYP3A-mediated drug interaction using midazolam as substrate [61]. Several strains of rats are most commonly used for CYP3A-mediated drug interaction using midazolam, diltiazem, nifedipine, and doxorubicin as substrates [62, 63]. In addition, cynomolgus monkey was also used as model for

*Flavonoid-Mediated Modulation of CYP3A Enzyme and P-Glycoprotein Transporter: Potential… DOI: http://dx.doi.org/10.5772/intechopen.92712*


#### **Table 1.**

In vitro *effects of some common flavonoids on CYP3A4 and P-gp activity.*

CYP3A-mediated DDIs; however, its choice is limited due to its high cost, handling, and ethical concerns [64].

Valspodar, elacridar, and zosuquidar are more selective P-gp inhibitors and thought to be low affinity for CYP enzymes, and other drug transporter proteins are commonly used for *in vivo* DDI studies in mice and rats. The drugs such as paclitaxel, cyclosporine, and digoxin are most commonly used as substrates for P-gpmediated inhibition. Moreover, Rho123 is attractive probe and has been widely used as marker to evaluate P-gp functions because it is not a substrate for CYP3A enzyme [65–67]. The cynomolgus monkey has been also used as model to evaluate effect of P-gp-mediated DDI using erythromycin and fexofenadine as substrate [64, 68].

The evaluation of flavonoid-mediated DDI by using dual substrates of CYP3A and P-gp, inhibitors, and species has been discussed in detail in Section 9 and summarized in **Table 1**.

### **7. Flavonoids as P-gp and CYP3A modulators**

Flavonoids (a group of polyphenolic compounds) are mainly abundant in vegetables and fruits and routinely consume through our common diet and in the form of beverages (plant-derived), for example, wine and tea [37]. In addition, they are the main constituents of many herbal products/formulations. Structurally, these compounds possess a framework consisting of a chromane ring together with an aromatic ring that is attached at different positions. Based on various substitutions and the oxidation status of the ring C atoms, flavonoids can be categorized into various subclasses, including flavones, flavonols, flavonones, flavanols, isoflavones, and chalcones. Since the last decade, there has been a drastically increase in scientific work on flavonoids, with >2000 publications/year containing "flavonoids" as a keyword found in different literature sources. Flavonoids display antioxidant, anticarcinogenic, antiviral, anti-inflammatory, and antiesterogenic properties, and high intake of flavonoids has been linked with a reduced risk of cancer, cardiovascular disease, osteoporosis, and other age-related degenerative diseases [37, 69].

Due to the wide range of health benefits of flavonoids and their remarkable safety record, numerous herbal preparations containing these compounds are marketed in various formulations as dietary supplements. The total daily intake of flavonoids in the average US diet has been estimated to be more than 1 g [37]. Therefore, the concentration of flavonoid expected to be present is sufficient after the ingestion of flavonoids and/or flavonoid-containing supplements, suggesting a potential herb-drug interaction.

Thus, the consumption of higher doses of flavonoids is common in daily life, and it may increase the risk of pharmacokinetic interactions with clinically used medicine. This concern is also confirmed by increasing evidence, which showed significant or even life-threatening interactions between flavonoids or flavonoid containing products and prescription drugs [70, 71].

Initial publications cite various examples of flavonoids as P-gp transport inhibitors, thereby affecting the bioavailability and cellular uptake of anticancer drugs. These experiments include *in vitro* analyses of the effects of flavonoids on the intracellular accumulation of P-gp substrates using P-gp-overexpressing cells and a variety of clinical and animal model studies, especially involving P-gp knockout animals [37]. For example, concomitant administration of quercetin increased moxidectin oral bioavailability in lambs [72]; oral bioavailability of quinine was increased by naringin [73]; cyclosporine by baicalein and its aglycone [74]; and paclitaxel by flavones in rats [75]. Similarly, quercetin increased the oral bioavailability of paclitaxel and tamoxifen in rats [76, 77] and digoxin in pigs, which results in high toxicity [78]. All of these studies indicate that flavonoid-P-gp interactions can occur *in vivo*, resulting in pharmacokinetic interactions. In contrast to the above results, several flavonoids appear to induce P-gp transport, resulting in a decrease in the bioavailability of substrate drugs. For instance, *in vitro* studies, kaempferol and quercetin, produced inductive effects on P-gp efflux [79, 80], and therefore, the consumption of pure herbal constituents, which contains hypericin, kaempferol, quercetin, and silibinin for 10 days, may produce a significant increase in the expression of P-gp mRNA [81]. *In vivo* studies have also indicated that long-term exposure (14 days) to St. John's wort (a flavonoid-containing herbal product) leads to higher expression of MDR1 in the rat intestine [82]. Based on these findings, it has been concluded that chronic exposure to some flavonoids induces intestinal expression of P-gp, resulting in reduced intestinal drug absorption, possibly due to enhanced drug efflux; however, the inhibitory effects on P-gp-mediated efflux are based on short-term exposure. Meanwhile, subsequent *in vitro* and *in vivo* studies indicate that the pharmacokinetic interactions of drugs with flavonoids may result in the modulation not only of drug transporters (P-gp) but also of metabolizing enzymes, especially CYP3A, that is, dual inhibition of P-gp and CYP 3A. *In vitro* studies confirmed that quercetin, myricetin, baicalein, and silibinin were found to produce dose-dependent inhibition of CYP 3A4 activity in CYP inhibition assay and increased the cellular accumulation of rhodamine (MCF-7/ADR cell) in P-gp transport studies [83–87]. However, in another study, quercetin and rutin were found to induce the function of CYP 3A4 and P-gp, which may lead to increase the bioavailability of substrate drugs [88]. *In vitro* effects of some common flavonoids on CYP3A4 and P-gp activity are summarized in **Table 1**. In *in vivo* studies, coadministration of quercetin, baicalein, silibinin, epigallocatechein, and kaempeferol increased the bioavailability of oral tamoxifen dose dependently through inhibition of P-gp efflux and reduction in the first-pass metabolism through inhibition of CYP3A metabolism in the small intestine and/or liver [78, 84, 89–91]. Similarly, quercetin, silibinin, naringin, flavone (2-phenyl-4H-1-benzopyran-4-one), genistein, and morin also increased the oral bioavailability of paclitaxel mainly through inhibition of CYP3A4-mediated metabolism in the small intestine and/or liver and inhibition of the P-gp efflux in the small intestine [75–77, 92–94]. Additionally, quercetin, myricetin, and baicalein also reduced the bioavailability of doxorubicin by similar mechanism [83, 84, 95]. Similar results were also produced by morin, quercetin, and niringin with diltiazem and baicalein with nimodipine [85, 96–98]. In contrast, reduced oral bioavailability of tamoxifen by biochanin A and cyclosporine by quercetin and rutin was also reported in rats [88, 99]. *In vivo* interaction of flavonoids with dual substrates of CYP3A4 and P-gp is summarized in **Table 2**.

*Flavonoid-Mediated Modulation of CYP3A Enzyme and P-Glycoprotein Transporter: Potential… DOI: http://dx.doi.org/10.5772/intechopen.92712*


**Table 2.**

In vivo *interaction of some common flavonoids with dual substrates of CYP3A and P-gp.*
