MDR- and CYP3A4-mediated drug–herbal interactions
Introduction
Vitamins and herbal supplements have long been self-administered primarily to manage side effects of drugs and/or to improve overall physical and mental health. According to recent epidemiological reports, almost 40% of American population use complimentary and alternative medicine (CAM) during their lifetime (Eisenberg et al., 2001, Ernst, 1998, Kessler et al., 2001). Herbs are very often self-administered along with therapeutic drugs. Patients diagnosed with HIV or cancer exhibit a higher CAM use, especially various antidepressants and energy pills to cope with their mental and physiological instability. About 70% patients living with HIV and about 55% of the general population use some form of complementary therapy with herbal medicines (Astin, 1998) . The reasons for such herbal consumption may be due the facts that (1) most of herbal users do not inform their allopathic doctors about CAM intake; (2) herbals are generally considered a traditional health aid which do not require stringent preclinical and clinical assessments by appropriate regulatory agencies; (3) currently no proper surveillance procedure exists for quality control and for monitoring adverse effects of herbs or herb–drug combinations; and (4) a general misconception persists among most CAM users that herbal medicines are safe and free from side effects and drug interactions, because these products are of natural origin. Such widely held view is unfortunately not true and often misleading. A single herb generally contains a number of putative biochemicals, each of which may exert some degree of pharmacological effects. Phytochemicals, similar to therapeutic drugs, may be biologically active and capable of modulating physiological actions through synergistic and antagonistic effects. Natural products, as taken by the general population, are usually complex mixtures of many molecular entities. Both the putative active ingredient(s) and other constituents present in that mixture have the potential to interact with various classes of drugs. Conventional pharmacokinetic literature generally deals with drug–drug interactions, but recently such interactions between herbal agents and prescription drugs have drawn attention, because of increasing physician awareness of the widespread adverse effects of undisclosed herbal use by the patients. Useful data about actual and potential drug–herbal interactions are accumulating from various sources, including in vitro and in vivo laboratory studies, clinical and preclinical trials.
Natural products, as used by the general population, are usually complex mixtures of many compounds. Both the putative active ingredient(s) and other constituents present in that mixture have the potential to cause interactions with various classes of drugs. Such interactions include induction or inhibition of metabolizing enzymes and drug efflux proteins. Prokaryotes and eukaryotes cannot distinguish between a natural chemical originating from plants and a chemical synthesized in laboratory. Consequently, physiological, pharmacologicals and toxicologicals effects of these chemicals irrespective of their origins remain the same. Ever-increasing use of herbs with western medicines raises the potential for drug–herbal interactions, which may alter drug bioavailability through altered absorption, metabolism and distribution. Primary mechanisms of drug/herb interaction involve either induction or inhibition of intestinal drug efflux pumps [efflux proteins such as P-glycoprotein (P-gp) and multiple resistance proteins (MRPs)] and intestinal and hepatic metabolism by cytochrome P450s (CYPs) (Evans, 2000, Ioannides, 2002, Wilkinson, 1997). A concerted action by both drug efflux pumps and CYPs lower oral bioavailability of many drugs, i.e., protease inhibitors, macrolides and azoles. Therefore, modulation of intestinal efflux proteins and intestinal and hepatic CYPs (particularly CYP3A4) is the key to altered systemic drug concentration. Therefore, consumption of herbs that are capable of modulation of efflux proteins and CYP3A4 may cause clinically relevant drug–herbal interactions and alter drug bioavailability (Fugh-Berman, 2000, Fugh-Berman and Ernst, 2001, Izzo and Ernst, 2001). Any inhibitory effect of herbs on efflux proteins and CYP3A4 may result in enhanced plasma and tissue concentrations leading to toxicity, while any inductive effect may cause reduced drug concentrations leading to decreased drug efficacy and treatment failure.
Herbal products such as St. John's wort (SJW), garlic, ginkgo biloba, ginseng and milk thistle are increasingly being consumed (Table 1) worldwide for the treatments of many conditions ranging from depression to high blood pressure, from diabetes to general improvement of health outcome. Two reviews have recently been published by Ioannides (2002) and Zhou et al. (2004) on metabolic and pharmacokinetic interactions of drugs with herbal agents.
St. John's wort (Hypericum perforatum) is the most commonly used herbal product, which causes severe drug–herbal interaction. SJW has been implicated in a number of clinically relevant drug interactions reducing the therapeutic efficacy of many therapies, i.e., transplantation, AIDS, cancer, etc.). SJW is commonly consumed for the relief of depression, anxiety, inflammation of the skin and blunt injuries, to name a few. The crude extract is a complex mixture of several active compounds such as hypericin, quercetin, isoquercitin, biflavonoids, hyperforin, naphthodanthrones, procyanidines, catechin tannins, chlorogenic acid, etc. (PDR—herbal). Among these, hyperforin is the main constituent responsible for its antidepressant action, which is primarily mediated through inhibition of synaptic reuptake of neurotransmitters (serotonin, norepinephrine and dopamine) (Moore et al., 2000). Pharmacokinetic interactions of SJW with other medicinal agents are summarized in Table 2. A number of clinical studies have indicated that SJW lowered steady state plasma concentrations of amitriptyline, cyclosporin, digoxin, fexofenadine, indanavir, methadone, midazolam, nevirapine, phenprocoumon, saquinavir, simvastatin, tacrolimus, theophyline and warfarin (Zhou et al., 2004) Conversely, simultaneous administration of SJW extract with ritonavir caused 100-fold elevation of ritonavir uptake by MDCK-MDR1 cells (Fig. 1). Serum concentration of the active metabolite SN-38 in cancer patients receiving irinotecan was also reduced by SJW consumption. However, it did not alter the pharmacokinetics of carbamazepine, detromethorpan, mycophenolic acid, pravastatin and tolbutamide (Table 2). Several case reports have indicated that SJW consumption subsequent to transplantation resulted in subtherapeutic plasma cyclosporin levels leading to organ rejection (Breidenbach et al., 2000a, Breidenbach et al., 2000b, Karliova et al., 2000, Ruschitzka et al., 2000, Zhou et al., 2004). Intermenstrual bleeding and unplanned pregnancies were also reported following concomitant use of SJW and oral contraceptives (Yue et al., 2000, Zhou et al., 2004). Development of serotonin syndrome occurred when SJW and selective serotonin-reuptake inhibitors (sertaline and paroxetine) were coadministered. A detailed analysis of such pharmacokinetic interactions is available in recent reviews (Ioannides, 2002, Zhou et al., 2004).
Garlic (Allium sativum) is generally taken for the remedy of arteriosclerosis, hypertension, high cholesterol, respiratory inflammation, hooping cough, bronchitis and joint pains. The active compounds include organosulfur, allicin, fructosans and saponins. Compared to SJW, very limited information is available on drug–garlic interactions. A substantial decrease in plasma levels resulting in 51% and 17% reduction in AUC of saquinavir and ritonavir respectively in the presence of garlic was reported in healthy volunteers (Gallicano et al., 2003, Piscitelli et al., 2002a, Piscitelli et al., 2002b). Garlic altered pharmacokinetic parameters of acetaminophen and also produced hypoglycemia with chlorpropamide (Izzo and Ernst, 2001). Concomitant administration of garlic with anticoagulants such as cumadin and antiplatelets (aspirin and dipyridamole) may increase the risk of bleeding due to garlic's effect on fibrinogen and platelet aggregation (Bordia et al., 1998, Harenberg et al., 1988, Legnani et al., 1993) (Table 2).
Ginseng (Panax ginseng): Varieties of ginseng including Chinese ginseng (Panax ginseng) and Siberian ginseng (Eleutheroccus senticosus) are widely available. It is commonly used for alleviation of many aliments such as lack of stamina, fatigue and debility, lack of concentration, impotence and anxiety. Limited information is available on drug interactions with ginseng. Although Siberian ginseng did not exhibit any effect on plasma levels of alprazolan or dextromethorphan, Chinese ginseng produced adverse effects with phenelzine, warfarin and alcohol (Janetzky and Morreale, 1997).
Ginkgo (Ginkgo biloba): This herb is frequently taken for the enhancement of memory functions. It is also used for symptomatic relief of organic brain dysfunction, intermittent claudication, vertigo and tinnitus. The extract contains various flavonoids (quercetin, kaempferol, isorhamnetis, p-coumaric acid), biflavonoids and proanthocyanidins. This herbal agent has been found to produce adverse effects with several drugs such as aspirin, acetaminophen and warfarin (Chavez and Chavez, 1998, Rosenblatt and Mindel, 1997).
Milk thistle (Silybum marianum): This herb is generally recommended for dyspepsia, liver and gallbladder problems. Several active compounds including apigenin, luteolin, kaempferol sterols, fumaric acids and silymarins (silybinin, silydianin and silychristin) are present in this preparation. Milk thistle caused a slight decrease in plasma indinavir concentration in healthy volunteers (Piscitelli et al., 2002a, Piscitelli et al., 2002b). Silymarin compounds have reduced the toxic effects of cisplatin on kidney without compromising the drug's antitumor activity. Studies involving adriamycin have also reported similar results (Bokemeyer et al., 1996, Gaedeke et al., 1996). Silymarin helped to prevent liver damage from hepatotoxic drugs including butyrophenones, phenothiazines, acetaminophen, halothane, dilantin and alcohol (Brinker, 1998).
Pure herbal constituents such as quercetin, hypericin, hyperforin, kaempferol, silibinin and allicin from SJW, ginseng, milk thistle and garlic can cause inhibition and/or induction of both P-gp-mediated efflux and CYP-mediated metabolism. Quercetin, hypericin and kaempferol exhibited remarkable inhibition of P-gp-mediated ritonavir efflux by accelerating cellular uptake in Caco-2 cells (Fig. 2). Similar results were also obtained with allicin in MDCK-MDR1 cells, which express high amounts of P-gp (Patel et al., 2004). Chemical structures of active herbal components significantly involved in altering drug efflux and metabolism are depicted in Fig. 3.
Many drug substances along with a variety of naturally occurring dietary or herbal components are capable of interacting with the CYP enzyme system and P-gp efflux pump in several ways:
- (1)
A herbal component can be a substrate of one or several isoforms of CYP enzymes and/or efflux systems (P-gp, MRP and BCRP). Therefore, one substrate can compete for another substrate for either metabolism by the same CYP isozyme and/or efflux system resulting in higher plasma concentrations due to competitive inhibition.
- (2)
A herbal constituent can also be an inducer of one or several CYP isoforms and/or efflux systems, thereby lowering plasma concentrations due to either higher metabolism and/or higher efflux. Such interactions may produce subtherapeutic plasma drug concentrations.
- (3)
A compound can also be an inhibitor of CYP450 enzymes resulting in reduced activity of one or several isoforms of CYPs. If a compound is an inhibitor of efflux system, it will reduce drug efflux resulting in improved absorption. However, induction is a slow process, dependent on the rate of protein synthesis. Expression of specific mRNA may be possible within a few hours, but functional expression and maturation of such proteins may require longer duration. In contrast, inhibition is more rapid and can produce results within a very short period of time, particularly if the inhibition is competitive in nature.
The most versatile enzyme system involved in the metabolism of xenobiotics is cytochrome P450. The CYP3A family of enzymes constitutes the most predominant phase-I drug metabolizing enzymes and accounts for approximately 30% of hepatic CYP and more than 70% of intestinal CYP activity. Moreover, CYP3A is estimated to metabolize between 50% and 70% of currently administered drugs (Watkins et al., 1987). A congener of CYP family is CYP3A4, the most abundant form (Kolars et al., 1992). This CYP3A4 enzyme is present primarily in the hepatocytes and enterocytes (Guengerich et al., 1986, Parkinson, 1996). It is now fairly established that naturally occurring dietary supplements can modulate hepatic and entrocytic CYP activity. Perhaps the best documented clinically relevant drug interaction is observed with grapefruit juice. Simultaneous consumption of grapefruit juice with a number of therapeutic agents that are subject to first pass intestinal/hepatic metabolism, resulted in higher plasma levels with subsequent adverse effects (Bailey et al., 1998, Fuhr, 1998). Grapefruit juice acts through inhibition of intestinal CYP3A4, which regulate pre-systemic metabolism (Guo et al., 2000). Although hepatic biotransformation can make a major contribution to systemic drug elimination, a combination of hepatic and intestinal drug metabolism may cause significant pre-systemic or first-pass drug loss.
Preliminary studies that have directly investigated SJW interactions with CYPs indicate that it may modulate CYP, particularly the 3A4 isoform. Several in vitro studies revealed that crude extract of SJW inhibits CYP3A4 and such inhibition is of a competitive nature (Obach, 2000, Patel et al., 2004). These studies also identified hyperforin, hypericin, quercetin and 13,118-biapigenin as components primarily responsible such inhibitory interactions.
Since HIV protease inhibitors, macrolide antibiotics and azole antifungals along with many herbal agents are substrates of the same CYP3A4, these compounds can affect oral bioavailability of therapeutic agents indicated in the treatment of immunosuppression, cancer, AIDS and other opportunistic infections. Depending on the mechanisms of herbal interactions with therapeutic agents, these substances could lower blood levels of the anti-HIV drugs, thus possibly putting people at risk for the development of resistance. Even though many clinical studies reported that St. John's wort had an inductive action on CYP3A4, one study suggested that SJW had no statistically significant effect on CYP3A4 induction (Markowitz et al., 2000). This latter report is consistent with another recent study describing CYP3A4 metabolic interaction (Bray et al., 2002). This study reported that 4-day treatment of SJW extract or its constituents, hypericin and hyperforin, in mice did not result in any CYP3A4 induction. In contrast, an inhibitory effect of the major constituents of St. John's wort was also reported in the CYP transfected cells (Obach, 2000). In line with this observation, quercetin one of the major constituents in St. John's wort was reported to have an inhibitory effect on CYP3A4 (Zou et al., 2002). The discrepancy so far remains unresolved.
Since the herbal products can also competitively inhibit CYP, simultaneous intake of these agents may raise blood levels of therapeutic agents, thus exposing patients to a greater risk of serious side effects. A recent report from our laboratory have revealed pure herbal constituents (quercetin, hypericin and kaempferol) inhibit CYP3A4-mediated cortisol metabolism (Patel et al., 2004). However, silibinin did not inhibit any cortisol metabolism (Fig. 4). In another study, SJW extracts and its major constituents have been shown to diminish enzyme activities of recombinant CYP1A2, 2C9, 2C19, 2D6 and CYP3A4 (Obach, 2000). Several isolated constituents of SJW were found to be capable of competitively reducing CYP activities, with the biflavone I3,II8-biapigenin being the most potent inhibitor of CYP3A4. A drug interaction is expected to occur in vivo when [I]/Ki is > 0.2, where [I] is the maximum unbound plasma concentration (Ito et al., 1998). After single dose administration of standard SJW extract (300 mg) containing 5% hyperforin, plasma concentration of hyperforin in human reach a maximum of 0.17–0.5 μM (Agrosi et al., 2000, Biber et al., 1998). So, a [I]/Ki value of 0.35–1.04 for CYP3A4, 0.11–0.33 for CYP2C9 and 0.09–0.28 for CYP2D6 can occur, which may cause potential drug interactions with several CYP substrates. Under such conditions, a number of drugs such as amitriptyline and dextromethorphan (CYP2D6), warfarin, tolbutamide and phenprocoumon (CYP2C9), HIV protease inhibitors, midazolan, oral contraceptives and cyclosporin (CYP3A4) are expected to interact with SJW. A recent review has described such pharmacokinetic interactions in details (Zhou et al., 2004).
We have experimentally proved that prolonged exposure of quercetin, hyperforin and kaempferol cause significant increase of CYP mRNA expression levels (Fig. 5) in Caco-2 cells (Patel et al., 2004). Also, SJW is capable of inducing CYP3A4 in healthy volunteers as demonstrated by significant increase in 6-β-hydroxy cortisol/cortisol ratio in the urine (Roby et al., 2000) and midazolan clearance (Dresser et al., 2003, Wang et al., 2001). Therefore, it appears that higher levels of CYP enzymes are expressed upon prolonged exposure to herbals. Other in vitro studies have also indicated that crude extract of SJW is a potent inducer of CYP2B6 and 3A4. Hyperforin not hypericum caused marked induction of CYP3A4 expression in primary human hepatocytes (Goodwin et al., 2001, Moore et al., 2000, Wentworth et al., 2000). Several animal (Bray et al., 2002, Durr et al., 2000a, Durr et al., 2000b) and human studies (Dresser et al., 2003, Roby et al., 2000, Wang et al., 2001) indicate that SJW after 4 to 14 days of administration resulted in higher activity of CYPs along with increased clearance of fexofenadine and cyclosporine. All these in vitro and in vivo studies suggest that SJW possesses both CYP inducing and inhibitory properties. However, the induction for CYP activity requires appropriate dose and duration of herbal exposure. This long-term effect in turn can elevate metabolism of xenobiotics, consequently reducing their bioavailability, whereas in short-term CYP may actually raise steady state drug levels.
Overlapping substrate specificities of these proteins result in complex and sometimes perplexing pharmacokinetic profiles of multidrug regimens. Saquinavir undergoes extensive first pass metabolism by the major metabolizing isozyme CYP3A4. Ketoconazole (a selective CYP3A4 inhibitor) inhibited the formation of all saquinavir metabolites. Also, saquinavir lowers the metabolism of terfenadine and causes formation of 6-β-hydroxylation products of testosterone, indicating its specificity towards CYP3A4 (Vella and Floridia, 1998). Metabolism of ritonavir on the other hand is caused by both CYP3A4 and CYP2D6. It significantly inhibits the metabolism of CYP3A4 and CYP2D6 substrates like nifedipine and dextromethorphan, respectively, when administered in combination (Hsu et al., 1998). The major isozyme responsible for indinavir metabolism is CYP3A4. However, metabolism of nelfinavir is caused by several isozymes including CYP3A4 followed by CYP2C19, CYP2D6 and possibly CYP2C9 and CYP2E1 (Li and Chan, 1999, Malaty and Kuper, 1999, Williams and Sinko, 1999).
Multidrug resistance (MDR) proteins play an important role in protecting cells against cytotoxic drugs (Borst et al., 2000). It has recently become apparent that MDR gene products also need to be considered in drug absorption. The multidrug resistance phenotype in tumors is associated with over expression of ATP binding cassette (ABC) efflux pumps termed MDR proteins. P-glycoprotein (P-gp, MDR1, ABCB1) (Chen et al., 1986, Juliano and Ling, 1976, Ueda et al., 1987) is considered a versatile xenobiotic pump. Two other ABC transporters have also been demonstrated to participate in the multidrug resistance of tumors. These are multidrug resistance protein 1 (MRP1, ABCC1) and mitoxantrone resistance protein (MXR) or breast cancer resistance protein (BCRP or ABCG2) (Borst et al., 2000, Cole et al., 1992, Deeley and Cole, 1997, Gottesman and Pastan, 1993, Litman et al., 2001). Recently, many other multidrug resistance proteins have also been identified, i.e., ABCB11 (sister P-gp/BSEP), ABCB4 (MDR3) and five MRP1 homologues (MRP2-MRP6/ABCC2-ABCC6) (Lecureur et al., 2000, Paulusma et al., 1996, Paulusma et al., 1997).
Substrate specificity and tissue distribution of MDR proteins vary widely. MRP1 is almost ubiquitously expressed, while the expression of P-gp is more restricted to tissues involved in absorption and secretion. Although P-gp was initially discovered in cancer cells, it was later observed that a number of normal tissues such as intestine, liver, kidney, pancreas and adrenal gland constitutively express P-gp. High levels of P-gp is expressed in blood–brain barrier and the choroid plexus (Ambudkar et al., 1999, Gottesman and Pastan, 1993, Lin and Yamazaki, 2003, Yu, 1999). BCRP is highly expressed in the placenta, liver and, most interestingly, in various stem cells (Bates et al., 2001, Zhou et al., 2001). All multidrug transporters are localized predominantly in the plasma membrane. In polarized cells, P-gp is localized in the apical (luminal) membrane surface (e.g., in the epithelial cells of the intestine and the proximal tubules of kidney, and in the biliary canalicular membrane of hepatocytes). In contrast, MRP1 expression in polarized cells is restricted to the basolateral membrane. The expression of MRP2, MDR3 and bile salt export pump (BSEP) are predominantly present in the canalicular membrane of hepatocytes, while MRP3 and MRP5 are expressed in the basolateral membranes of these cells. MRP2 is also abundant in the apical membranes of kidney proximal tubules. In polarized cells, BCRP expression was found to be mostly apical (Jonker et al., 2000, Kipp and Arias, 2000, Thiebaut et al., 1987, van Helvoort et al., 1996, Zhou et al., 2001).
Sequence analysis revealed P-gp as a principal member of ABC super family; hence, it transports drug molecules against a concentration gradient at the expense of ATP. It is an integral membrane protein having two homologous halves, each consisting of one hydrophobic domain with six transmembrane segments and one hydrophilic nucleotide-binding domain (Fig. 6). Two halves are connected by a short flexible linker polypeptide to form the functionally active 1280 amino acid protein (Hyde et al., 1990). P-gp encoding genes are found in hamster, mice, human and other species (Sharom, 1997). This transporter is encoded by a small multi-gene family, described as MDR I, II and III. Mammalian P-gp displays approximately 60–65% homology with other species, suggesting that their role in drug trafficking is highly conserved throughout evolution (Gottesman and Pastan, 1993). This efflux protein is a transporter for large hydrophobic, either neutral or positively charged compounds, while the MRP family primarily transports hydrophobic anionic conjugates and extrudes hydrophobic neutral molecules. MRP1-related neutral drug transport is quite an enigma and is probably linked to the transport or allosteric effect of intracellular free reduced glutathione (Borst et al., 2000). The exact spectrum of the BCRP transported substrates has not yet been delineated. However, flavonoids (apigenin, biochanin A, genistein, kaempferol) have been reported to enhance mitoxantone accumulation in MCF-7 MX100 cells (Zhang et al., 2004). P-gp which was initially discovered in cancer cells as the “primary active” drug efflux pump in many MDR cells. P-gp was first characterized as the ATP-dependent transporter responsible for efflux of chemotherapeutic agents from resistant cancer cells (Leslie et al., 2001). Substrates for P-gp cover a broad range of structures including cyclosporin-A, taxol, dexamethasone, lidocaine, erythromycin and protease inhibitors (Chiou et al., 2000, Katoh et al., 2001, Patel and Mitra, 2001, Wacher et al., 2001, Wacher et al., 1995, Watkins, 1997). P-gp represents the most studied member of the ATP binding cassette family of transporters. This family of transmembrane proteins is characterized by one or more ATP binding sites (Weinstein et al., 1990) and play an important role in drug absorption and elimination from various tissues such as the intestine, liver and brain (Ambudkar et al., 1999, Lin and Yamazaki, 2003, Yu, 1999). Evidence accumulated to date suggests that the transporter interacts directly with non-polar substrates within the membrane environment and may act as a drug filppase, transferring drugs from the inner to the outer leaflet of the bilayer (Fig. 7). However, a clear mechanism of such translocation has not yet been established. As a result of such efflux, drug absorption is reduced and bioavailability of xenobiotics is decreased at the target organs (Sharom, 1997, Wacher et al., 1998). Several chemosensitizers that block the action of P-gp are proposed to act as alternate substrates (Wacher et al., 1998). P-gp is expressed in a broad range of tissues including the adrenals, blood–brain barrier, kidney, liver, lungs, pancreas and the esophagus, stomach, jejunum and colon, thus decreasing the oral absorption and bioavailability of xenobiotics (Sharom, 1997, Wacher et al., 1998). In the intestine, P-gp is located almost exclusively within the brush border on the apical surface of mature enterocytes and thus significantly restricts oral delivery of several xenobiotics.
In the last 5 years, St. John's wort has been on the top of the list of herbal usage. Indinavir and saquinavir concentrations were reduced to 57% by SJW and 51% by garlic, respectively (Piscitelli et al., 2000). However, the fact that its interaction could alter outcome of anti-HIV therapy was realized very recently (Piscitelli et al., 2000). Such reduction in indinavir and saquinavir exposure may lead to the development of drug resistance strains and may cause treatment failure in HIV patients. Such reduction was attributed to the induction of P-gp and CYP3A4 expression by SJW (Roby et al., 2000). Several flavanoids, which constitute one of the primary classes of active constituents in most herbs, appear to be capable of modulating P-gp. SJW was shown to induce P-gp (Durr et al., 2000a, Durr et al., 2000b, Perloff et al., 2001, Ruschitzka et al., 2000). Quercetin and kaempferol were found to induce P-gp (Chieli et al., 1995, Scambia et al., 1994, Shapiro and Ling, 1997). Our laboratory has reported (Fig. 8) that a 10-day treatment with pure herbal constituents (hypericin, kaempferol, quercetin and silibinin) can cause a significant increase in P-gp-mRNA expression (Patel et al., 2004). Quercetin and kaempferol have also been reported to induce P-gp (Chieli et al., 1995, Scambia et al., 1994, Shapiro and Ling, 1997). Also, four- to seven-fold elevation in the expression of P-gp in LS180 intestinal carcinoma cells was caused by hypericin or SJW treatment (Perloff et al., 2001). In vivo studies also indicated that long-term (14 days) exposure of SJW leads to higher expression of MDR1 in rat intestine (Durr et al., 2000a, Durr et al., 2000b). SJW taken as 900 mg/day for 14 days resulted in 1.4-fold increase of P-gp expression in healthy volunteers (Durr et al., 2000a, Durr et al., 2000b). In another clinical study, a 4.2-fold increase of P-gp expression was observed in human peripheral blood lymphocytes after chronic treatment with SJW for 16 days (Hennessy et al., 2002).
Since HIV protease inhibitors, macrolide antibiotics, azole antifungals and herbals are substrates of same the metabolizing enzymes and transporters, herbal agents can adversely affect the course of HIV treatment and other opportunistic infections. In vitro studies from our laboratory indicated that concomitant administration of erythromycin with SJW and/or ketoconazole can enhance erythromycin oral absorption (Fig. 9). Depending on the mechanism by which herbal compounds interact with CYP450 and efflux proteins, these agents could lower plasma levels of the anti-HIV drugs, thus possibly reducing efficacy and enhancing the risk for the development of drug resistance. Reversibly, these compounds could raise the blood levels of the antiretrovirals, thus placing patients at greater risk of serious side effects. Herbs with reported effects on the CYP3A4 and P-gp include SJW, garlic, ginseng, milk thistle and skullcap.
Employing MDR1 transfected MDCK cells expressing high amount of P-gp, we have recently demonstrated that ritonavir uptake was enhanced by five to eight-fold (Fig. 2) in the presence of 100ìM pure herbal constituents (allicin, kaempferol, quercetin and hypericin) (Patel et al., 2004). In other in vitro studies, we have also observed several fold increase in erythromycin and ritonavir uptake in the presence of SJW extract (Fig. 1, Fig. 4). Inhibitory effect of MRP-mediated ritonavir efflux by SJW was also noted. These results also demonstrate that simultaneous administration of SJW with drugs like erythromycin, saquinavir and ritonavir, can enhance drug absorption due primarily to competitive inhibition of P-gp/MRP-mediated efflux. These in vitro results demonstrated the inhibitory properties of herbs towards P-gp-mediated efflux on short-term exposure. Thus, all these in vitro and in vivo studies have demonstrated that SJW, upon chronic exposure induces intestinal P-gp resulting in reduced intestinal absorption possibly through enhanced drug efflux.
In addition to oxidative metabolism, conjugation reactions may play an important role in the detoxification of xenobiotics from small intestine. Several drug molecules are effluxed into intestinal lumen after being conjugated with a glucuronide or sulfate moiety. The transport system responsible for the cellular extrusion of the conjugated metabolites and organic anions has been characterized recently (Leslie et al., 2001). Herbs can pharmacokinetically act as inhibitors or inducers, when anti-HIV medication or any other conventional therapeutics taken simultaneously. An understanding of the increased/decreased bioavailability of one drug in the presence of herbals may greatly aid in the design of appropriate drug regimen. Coadministration of herbal and therapeutic drugs may lead to increased absorption due to inhibition of P-gp-mediated efflux and CYP-mediated metabolism leading to potential toxic effects. In contrast, chronic administration of certain herbal products (SJW, garlic, etc.) will enhance the production of MDR proteins (P-gp) and CYP enzymes resulting in lower bioavailability and subtherapeutic plasma concentrations of drugs. Finally, this process may lead to the emergence of drug resistance. Effects drugs and herbs on P-gp and CYP3A4 are summarized in Table 3.
Activation of the mammalian xenobiotic-sensing nuclear receptors PXR and CAR: P-gp/MDR1 is regarded as one of the major factor in the development of cellular drug resistance. Upregulation of CYP enzymes in response to herbals may also play an important role in lowering drug concentration. A close chromosomal location of P-gp (MDR gene locus 7q21–7q21⁎7q21.1–7q21.1) and CYP3A4 (CYP3A4 gene locus 7q21.3–7q22.1⁎) genes, their expression in mature enterocytes and similar substrate specificities suggest that the function of these two proteins may be complementary in nature and may form a coordinated intestinal barrier (Mitra and Patel, 2001). This view was further validated by the stimulatory effect of SJW on pregnane X receptor, which regulates many CYP isoforms in rats (Moore et al., 2000). A recent study also demonstrated that SJW induces CYP3A4 by negatively acting on interleukin-6, which is known to inhibit the pregnane X receptor that in turn may be involved in the expression of the CYP class of enzymes (Fiebich et al., 2001).
Molecular mechanisms of induction of P-gp and drug-metabolizing enzymes are still being investigated by researchers. Recently, the role of two members of nuclear receptor superfamily of transcription factors, the pregname X receptor (PXR) and the constitutive androstane receptor (CAR) have been discovered. These receptors act as sensors for drugs including xenobiotics. Members of nuclear receptor superfamily comprise four modular domains: (1) a highly variable N-terminal region having an activation function (Af-1); (2) a DNA binding domain with two zink-finger motifs, a flexible domain and a ligand binding domain (LBD) that also contains an activation function (AF-2). Following cellular entry, xenobiotics and other activators either (1) trigger cytoplasmic-nuclear translocation of CAR by promoting the release of so far unknown proteins or (2) directly activate PXR in the nucleus. Subsequently, both PXR and CAR heterodimerize with RXR (retinoid-X-receptor) bind to their respective response elements on target gene and increase transcription of those genes. In the flanking regions of several genes, response elements have been found that are activated by both PXR and CAR, and thus allow direct cross communication between these two receptors (Fig. 10). New insights into the transcriptional regulation of CYP3A4 revealed that vitamin D and its derivatives regulate the transcription by interacting with vitamin D response (VDR) element. This regulation of intestinal CYP3A4 by VDR is complementary to the apparent role of PXR in regulating hepatic CYP3A4 (Ambudkar et al., 2003, Handschin and Meyer, 2003, Synold et al., 2001).
High levels of MDR1 expression are often observed in drug-naive tumor cells even though original tissue may not express P-gp (Ogretmen and Safa, 2000). Altered gene expression may be influenced by a variety of components such as heat-shock protein (Chin et al., 1990a, Chin et al., 1990b, Miyazaki et al., 1992), cytokines (Combates et al., 1997), differentiating agents (Bates et al., 1992, Zhou et al., 1993), chemotherapeutics (Chaudhary and Roninson, 1993, Chin et al., 1990a, Chin et al., 1990b) and herbal agents (Patel et al., 2004). Oncogenes and tumor suppressor genes can influence the levels of P-gp in various tissues (Thottassery et al., 1997, Zastawny et al., 1993). Several studies have demonstrated that wild type p53 represses transcription of endogenous MDR1 gene (Thottassery et al., 1997, Zastawny et al., 1993). Such repression is mediated by a direct interaction of p53 with novel binding element within the proximal MDR1 promoter (Scotto and Johnson, 2001). Paradoxically, mutant p53 proteins, instead of repression, activate the MDR1 promoter (Chin et al., 1992, Dittmer et al., 1993). The role herbal agents play in this p53-MDR1 cascade needs further investigation.
Clinical studies have revealed that SJW causes an inductive effect on CYP3A4. One study, however, suggested that SJW had no statistically significant effect on CYP3A4 enhancement (Markowitz et al., 2000). In contrast, an inhibitory effect of the major constituents of SJW on CYP enzymes was also reported in CYP enzyme transfected cells (Obach, 2000). Quercetin, one of the constituents in SJW, was similarly reported to have an inhibitory effect on CYP3A4 (Zou et al., 2002). The discrepancy so far remains unresolved.
Section snippets
Conclusions
In vitro studies, clinical trials and case reports suggest that herbal agents particularly SJW and garlic interact with a number of prescribed medications. It should not be assumed that all prescribed drugs interact with herbals. Xenobiotics which are subjects of P-gp-mediated efflux and CYP-mediated metabolism are likely to be potential candidates for drug/herbal interaction. Most of the clinical studies mentioned in this article report contradictory findings where the duration of exposure of
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