07 06

Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas
Volumen 7, Nmero 6, 2008
2008 Los Autores Derechos de Publicacin 2008 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 7 (6), 281 - 294 BLACPMA ISSN 0717 7917 Revisin | Review
Especial sobre Interaccin de Productos Naturales y Frmacos / Special Issue on Natural Products and Drug Interactions
Kidney proximal human tubule HK-2 cell line as a tool for the investigation of P-glycoprotein modulation by natural compounds
[La lnea celular de tbulo proximal de rin humano HK-2 como una herramienta para la investigacin de la modulacin de P-glicoprotena por compuestos naturales]
Elisabetta CHIELI*and Nadia ROMITI Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Sezione di Patologia Generale e Clinica. Universit degli Studi di Pisa. Scuola Medica, via Roma 55, I-56126 Pisa, Italy.
*Contact: e-mail: chieli@biomed.unipi.it Submitted February 13, 2008; Accepted February 29, 2008; Published Online 15 October 2008
Abstract ABCB1 (MDR1/P-glycoprotein), the best characterized multidrug transporter belonging to the ABC transporter superfamily, is nowadays acknowledged to have a major impact on drug resistance to chemotherapy and drug bioavailability and disposition. A number of natural compounds, dietary phytochemicals and herbal remedies have the ability to modulate P-glycoprotein function and /or expression and therefore the potentiality to cause food-drug, or herb-drug interactions. The elucidation of these interactions may be important not only to predict possible undesirable effects deriving from the concomitant intake of herbal constituents and conventional drugs, but also for further studies of positive uses of these interactions as a way to increase the bioavailability of drugs that are P-gp substrates. In particular, the study of P-gp inhibition by herbal constituents may provide an approach for the identification of lead compounds for the design of news chemosensitizers to reverse multidrug resistance in tumor cells. This review describes our recent investigations on the potential herb-drug interactions involving P-glycoprotein, using as a model the HK-2 cells, an immortalized line of proximal tubule cell derived from human normal kidney.
Keywords: HK-2 cell line, P-glycoprotein, Calcein-AM, Rhodamine-123, phytochemicals.
Resumen El ABCB1 (MDR1/P-glicoprotena), el transportador multidroga mejor caracterizado que pertenece a la superfamilia de transportadores ABC, se reconoce hoy da por tener un gran impacto en la resistencia de los frmacos a la quimioterapia y a la biodisponibilidad y disposicin de los medicamentos. Varios compuestos naturales, fitoqumicos dietticos y remedios herbarios tienen la capacidad de modular la funcin y/o expresin de la glicoprotena P (P-gp) y, por consiguiente, la potencialidad para causar interacciones alimento-frmaco o hierba-frmaco. La elucidacin de estas interacciones no slo es importante para predecir posibles efectos indeseables que derivan de la ingestin concomitante de componentes herbarios y los frmacos convencionales, tambin se usa para los estudios sobre los usos positivos de estas interacciones como una manera de aumentar el biodisponibilidad de frmacos que son substratos de P-gp. En particular, el estudio de la inhibicin de P-gp por los componentes herbarios provee un acercamiento a la identificacin de compuestos para el diseo de quimiosensibilizadores para invertir la resistencia multidroga en clulas tumorales. Esta revisin describe nuestras recientes investigaciones sobre las interacciones potenciales hierba-frmaco que involucran P-gp mediante un modelo de clulas HK-2, una lnea celular inmortalizada de tbulo proximal derivada del rin humano normal.
Palabras clave: Lnea celular HK-2, P-glicoprotena, Calcena-AM, Rodamina-123, fitoqumicos.
Abbreviation list: ABC- ATP-binding cassette CYP- cytochrome P450 MRP- Multidrug resistance-associated protein ADME- absorption, distribution, metabolism and excretion MDR- Multidrug resistance P-gp- P-glycoprotein
Chieli and Romiti
P-glycoprotein modulation by natural compounds in HK-2 cells
INTRODUCTION Pharmacological interactions possibly derive from the modulation in expression and/or activity of two major pharmacokinetic disposition systems, namely cytochrome P450 (CYP) and the multidrug transporter P-glycoprotein (Aszalos, 2007a and 2007b; Liu et al., 2007; Marchetti et al., 2007; Pal and Mitra, 2006). The transmembrane efflux pump P-glycoprotein (P-gp) is a member of the ATP-binding cassette (ABC) superfamily transporters, membrane-bound proteins, able to export from cells a variety of chemicals, including drugs, xenobiotics, natural products and peptides. These efflux pumps, which are powered by the energy of ATP hydrolysis, have important physiological, pharmacological and toxicological functions (Linton, 2007; Schinkel and Jonker, 2003). P-gp, encoded by the MDR1 gene (now ABCB1), is the best known member of the ABC transporters. P-gp was initially discovered in 1970s in tumor cells as responsible for the emergence of the multidrug resistant (MDR) phenotype. The early years of P-gp research have been described in detail in a recent review by Gottesman and Ling (2006). P-gp has been retrieved also in many normal cells and tissues where, as well as many others ABC transporters, is believed to play a significant role in detoxification and protection against xenobiotics. In fact, P-gp is physiologically expressed at the apical surface of epithelial cells of several organs, like liver, intestine and kidney, devoted to secretion/excretion of a variety of metabolites, xenobiotics and drugs. Other strategical locations are the brain blood-barrier, bloodtestis and bloodovarian barriers, and placenta (Fromm, 2004; Marchetti et al., 2007). In the normal liver, P-gp is believed to play a significant role in the secretory processes of endoand xenobiotics across the canalicular membrane into the bile and, like other transport systems localized to the canalicular membrane, it may be considered as one of the complex multistep process which ensures the vectorial transport of a variety of chemicals across the hepatocyte (LeBlanc, 1994). Accordingly, P-gp located at the luminal side of renal proximal tubule appears involved in the active secretion of many drugs and nephrotoxins (Ernest and BelloReuss, 1998), while enterocyte P-gp activity is important in the absorption process of a series of chemicals (refs. in Berggren et al., 2007).
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Nowadays, P-gp is considered a crucial molecule involved in all the pharmacokinetic processes: absorption, distribution, metabolism and excretion (ADME) (Tanigawara, 2000; Varma et al. 2003) and a number of single-nucleotide polymorphisms (SNPs) in ABCB1 gene has been correlated with different responsiveness to many drugs (Marzolini et al., 2004). P-gp shows broad substrate specificity with a prevalent affinity for hydrophobic compounds; so many natural and synthetic xenobiotics and endogenous molecules are substrates/modulators of its activity (Chan et al., 2004). It is generally accepted that coadministration of compounds that interact with this transporter (as a substrate, inhibitor, or inducer) can result in interactions that affect the pharmacokinetics and pharmacodynamics of the coadministered drugs (Aszalos, 2007a; Aszalos, 2007b; Endres et al., 2006; Keogh and Kunta, 2006; Lin, 2007; Lin, 2003; Yu, 1999). Among these chemicals a number of vegetables and related phytochemicals have been reported to inhibit P-gp including garlic, black pepper, green tea polyphenols, flavonoids, curcumin, genistein, naringin, capsaicin, and others (Han et al., 2006; Hu et al., 2005; Izzo and Ernst, 2001; Izzo, 2005; Jodoin et al., 2002; Meijerman et al., 2006; Molnar et al., 2006; Nabekura et al., 2005; Nabekura et al., 2007; Pal and Mitra, 2006; Rodriguez-Proteau et al., 2006; Zhou et al., 2004; Zhou et al., 2007). It is therefore crucial to rapidly identify chemical entities -including herbal remedies, nutraceuticals, and food derived compounds- able to modulate P-gp, in order to predict possible herb-drug interactions. This review will primarily focus on the recent efforts of our laboratory devoted to investigate in vitro these interactions by using, as a model, the human cell line HK-2 derived from the proximal convoluted tubule of the kidney. Assessment of P-gp modulation by chemicals Various screening models and methodologies have been evolved for the identification and characterization of P-gp modulators (Perloff et al., 2003; Rautio et al., 2006; Shirasaka et al., 2006; Taub et al., 2005; Varma et al., 2003). Each technique has its own advantages and limitations. The most commonly in vitro methods used to evaluate whether a chemical compound is a modulator of the P-gp efflux transporter are three: i) Bi-directional transport assay with a probe P-gp substrate in functionally polarized epithelial cell monolayer. The preferred cells lines include Caco-2, transfected LLC-PK1-
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MDR1, and transfected MDCK-MDR1. LLC-PK1 and MDCK wild type cells are used as negative controls; ii) ATPase activity assay that measure substrate stimulated-ATPase activity in the presence of substrates, which interact on different P-gp sites, with and without test compounds, allowing identification of P-gp inhibitors. ATPase activity can be determined using cell membranes, cultured cells and membrane vesicles; iii) Drug accumulation and efflux assays where the amount of a substrate, generally a fluorescent probe, taken up by cells expressing P-gp, is compared in the presence and absence of inhibitors. This last method, also if cannot distinguish substrate from inhibitors, can screen compounds rapidly and could suggest further investigations employing more complex methodologies. In our laboratory, for the study of possible interactions between chemicals and P-gp, we utilized a cell line from human kidney proximal tubule and uptake/efflux assays. Furthermore, because the expression of P-gp may be influenced by chemicals, we used this cell line also for the in vitro evaluation of P-gp modulation at protein (immunoblotting) and at mRNA (RT-PCR) levels.
Renal P-glycoprotein Together with the liver, the kidney is the most important detoxifying organ (Inui et al., 2000). The kidney tubular cell has a large spectrum of functions, which modify the ultrafiltrate before its turning into final urine. The modification regards solutes secretion or solutes absorption. Many of these movements of substances between blood and tubular duct occur by different carrier work. The proximal tubular cells have a transport activity higher than all the other renal segments and, furthermore, are exposed to high concentrations of xenobiotics. These cells express a lot of membrane transporters, some of which belonging to the ABC superfamily, in particular P-gp (ABCB1) (Kruidering et al., 1994; Lee and Kim, 2004; Schlatter et al., 2006). P-gp is localized together other multidrug resistanceassociated proteins (MRPs) on apical surface (brush border) and is implicated in endogenous substrate transport and tubular secretion of toxic substances (Ernest and Bello-Reuss, 1998). A scheme of the main ABC transporters present in the proximal tubular cell is shown in Fig. 1.
Figure 1. Localization of P-gp and other ABC transporters in the kidney proximal tubule.
BASOLATERAL MEMBRANE
TUBULE LUMEN
Left: immunohistochemical localization of P-gp in human kidney (polyclonal anti mdr Ab-1). Right: P-gp/MDR1 (ABCB1), MRP2 (ABCC2), MRP4 (ABCC4) and BCRP (ABCG2) are expressed on the apical (luminal) membrane of renal epithelial cells and export compounds from the cytoplasm of tubule cell to the urine. MRP1 (ABCC1) and MRP6 (ABCC6) are expressed on the basolateral membrane. (Inui et al., 2000; Huls et al., 2008; Lee and Kim, 2004).
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The HK-2 cell line HK-2 cell line is an immortalized line derived from the human epithelial renal proximal tubule. Zager and coll. utilized a kidney from a male normal adult considered unsuitable for transplantation (Ryan et al., 1994). The cortical proximal tubule segment was isolated, cultured and exposed to a recombinant virus containing the E6 and E7 genes of HPV16. A cell clone, in which PCR analysis confirmed the incorporation of HPV16 E6/E7 construct in genomic DNA, was designed as HK-2 and was able to continuously grow for more than a year. At the molecular level the products of E6 and E7 genes bind to the DNA regulatory proteins, resulting in a cell facilitated proliferation. Phenotypically, the cell line HK-2 has the same characteristics of adult normal tubular cells, as demonstrated by a series of biochemical tests. In particular, it was shown that the HK-2 cells maintain the brush border typical enzymatic activities (acid and alkaline phosphatase, leucine aminopeptidase, gammaglutamyltranspeptidase). Therefore, this new line has been proposed as a valuable tool for the study of physiological and
physiopathological human renal tubule, as well as the mechanisms of damage and repair at the level of tubular cell and has been widely employed in research. In our laboratory we showed, for the first time, the presence of a functional MDR1 (ABCB1)encoded P-gp in HK-2 cells (Tramonti et al., 2001) and thereafter we characterized such a line for a series of parameters regarding this transporter, including the presence of MDR1, but not MDR3 (ABCB4) transcripts, expression at protein level on cell membranes, immunocytochemical localization and the responsiveness to a series of modulators including acknowledged P-gp inhibitors, like verapamil and cyclosporin A, as well as some drugs, like dexamethasone and calcitriol (Romiti et al., 2002). On the whole, the results suggested that HK-2 cells could be suitable as a tool to investigate the role of P-gp in renal physiopathology. Further characterization have indicated that HK-2 line, while expressing a robust amount of P-gp, that resulted not only quite stable in the time, passage number, culture conditions, but also responsive to chemical modulation, apparently lacks expression of the apical ABC transporter MRP2 (Fig. 2, Chieli and Romiti, unpublished results).
Figure 2. RT-PCR and Western blot analysis of ABCB1/P-gp and ABCC2/MRP2 in HK-2 or Caco-2 cell lines.
ac o C -2 ac o2
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Left: RT-PCR analysis shows that HK-2 express MDR1 (ABCB1) mRNA only, while Caco-2 cell line expresses both MDR1 (ABCB1) and MRP2 (ABCC2) mRNAs. M.W.,123-bp DNA ladder. Right: Western blot analysis of crude membranes from HK-2 or Caco-2 cells shows that both cell lines express, also if to a different degree, P-gp, while the band corresponding to MRP2, well represented in Caco-2, is undetectable in HK-2 cell membranes (lane 3). For immunoblotting monoclonal anti-P-gp or polyclonal anti-MRP2 antibodies and peroxydase-conjugated anti-mouse or anti-rabbit secondary antibodies were used. Blots were developed with the ECL detection system.
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MRP2 together with P-gp are known as the main apical transporters in tubule cell, devoted to excretion of drugs and xenobiotics in the tubule lumen (refs. in Huls et al., 2008). The absence of MRP2 expression and, in parallel, the observed lack of effect of probenecid, an inhibitor of MRPs, in P-gp functional tests (Nieri et al., 2006), suggest the HK-2 cells suitable to assay chemical effects selectively on P-gp, rather than cells in which also MRP2 is appreciably expressed, e.g. Caco-2 (Prime-Chapman et al., 2004). Assessment of chemical-P-gp interactions: methodological notes The HK-2 model has been used by us to check if chemical compounds (in particular natural compounds from vegetables) present in food or employed in phytotherapy, may modify the in vitro expression and function of P-gp. While P-gp expression at protein level (immunoblotting) or at mRNA level (RT-PCR) doesnt pose difficulties in interpretation, functional assays for Pg-p merit some comments. Throughout our studies we have used two different tests to evaluate the influence of chemicals
on P-gp activity, i. e. the Calcein-AM and the Rhodamine-123 accumulation assays. The principle of these assays are schematically shown in Figs. 3 and 4.
Figure 3. Scheme of the Calcein-AM assay for the assessment of Pglycoprotein activity.
The P-gp substrate nonfluorescent Calcein-AM is hydrolysed by cellular esterase to the fluorescent Calcein. Calcein is not a substrate for P-gp and it cannot leave the cell via the plasma membrane, whereas the nonfluorescent Calcein-AM is extruded from the MDR-1 expressing cells. The intracellular accumulation of fluorescent Calcein is inversely proportional to the transport capacity of P-gp. A low expression of P-gp or the presence of P-gp inhibitors produces a rise in cell fluorescence.
Figure 4. Scheme of the rhodamine-123 assay for the assessment of P-glycoprotein activity.
Intracellular fluorescence ng Rho-123/mg protein
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Left: scheme of the rhodamine-123 test (Rho-123) for the measurement of P-gp activity. The fluorescent mitochondrial probe Rho-123 enters into the cells and accumulates in cytoplasma and mitochondria. Because Rho-123 is a substrate for P-gp and it is extruded actively form the cells, the intracellular amount of Rho-123 is inversely proportional to P-gp activity. Inhibitors of P-gp cause an increase in cell fluorescence. Right: effects of two acknowledged inhibitors of P-gp, Verapamil and Cyclosporine A on Rho-123 accumulation by HK-2 cells. Cells were incubated in medium containing 5 g/ml Rho-123 for two hours; thereafter cell plates were washed and extracted with n-butanol. Rho-123 was quantified by spectrophotofluorimetry. At the top: the relative fluorescence microscope images.
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Calcein-AM test: This assay is largely employed (Eneroth et al., 2001; Holl et al., 1994; Tiberghien and Loor, 1996). At least two main cautions in the measurement of P-gp activity by this method must be considered. First, it is known that whereas CalceinAM is a substrate of P-gp, the fluorescent calcein is transported, out of the cell, by MRPs, therefore, this test couldnt be strictly specific for P-gp. Anyway, in HK-2 this possibility may be reasonably excluded, due to the fact that, as yet recalled, the major apical MRP transporter, MRP2, is apparently not expressed; furthermore, the MRPs inhibitor probenecid did not influence, in HK-2 cells, the results of Calcein-AM test (Nieri et al., 2006). Secondly, it must be taken into account that, independently from P-gp activity, the fluorescent calcein derives from the action of esterase on the nonfluorescent precursor Calcein-AM (Fig. 3). This occurrence -also reflected by the fact that Calcein-AM test is also employed as an index of cell viability- alerts on possible interferences by compounds able to influence cell esterase activity. In this regard it is suggestive that some flavonoids have been described recently to inhibit esterase activity (Li et al., 2007). Effects of this type (or even opposite) could influence the results of Calcein-AM assay causing under/overestimation of the test compound influence on the P-gp activity. Rhodamine-123 test: This test is also widely used (Perloff et al., 2003; Wang et al., 2006). However, this also requires some cautions. First, Rho-123 is a fluorescent mitochondrial dye, therefore it accumulates in cell and mitochondrial uptake accumulation may depend on mitochondrial transmenbrane potential (MTP) and cell membrane potential. Moreover, even if Rho-123 has always been considered to enter into the cells in a passive way, Troutman and Thakker (2003) have suggested the presence of a membrane influx carrier for this fluorescent probe. These things must be taken into account especially when a lack of correlation is observed between the results obtained with different methods, making difficult the interpretation. From some time, routinely, in our laboratory, always we use both Calcein-AM and Rho-123 tests, which in most situations gave congruent results. Table 1 summarize our studies about the interactions between herbal constituents and P-gp in the HK-2 model.
Flavonoids The plant flavonoids are a wide class of natural compounds commonly present in fruits, vegetables and beverages obtained from plants, like tea, red wine, and beer. Consumption of a diet rich in flavonoids is believed to produce a variety of health benefits, including antioxidant, anti-inflammatory, antiallergic effects, as well as protection in cardiovascular diseases and cancer (Yao et al., 2004). Flavonoids have been recognized as Pglycoprotein modulators for a long time, also if contradictory effects were reported in studies using different multidrug-resistant cell lines. Anyway, because flavonoids were shown to be modulators with bi-functional interactions at vicinal ATP-binding site and steroid-interacting region of P-gp, the controversial reports about flavonoid-P-gp interactions are now generally ascribed to the different binding of the model substrates to multiple binding sites of P-gp (Shapiro and Ling, 1997; Conseil et al., 1998). The effects of flavonoids on ABC transporters, including P-gp, have been recently reviewed and, from the majority of the more recent studies, the indication emerges that flavonoids are on average inhibitors of P-gp-mediated transport (Di Pietro et al., 2002; Morris and Zhang, 2006). In our previous studies employing primary rat hepatocytes as a model of cells expressing mdr1b Pgp we found that the flavonols quercetin, kaempferol and galangin were able, also if at a different degree, and with differences related to the used substrate, to modulate P-gp activity (Chieli et al., 1995). Other flavonoids, like the flavone apigenin, were also shown to inhibit P-gp activity, and, more consistently, to significantly downregulate P-gp expression in primary rat hepatocytes (Chieli et al., 1998). These results have been confirmed also in the HK-2 model (Fig. 5, unpublished results). The effects of apigenin appear very interesting by considering that it is a common flavonoid, present in a variety of vegetables, as well as in their extracts, marketed as diet and herbal supplements. Apigenin has demonstrated anticancer activities and chemopreventive potential towards prostate cancer (Patel et al., 2007).
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Chieli and Romiti
Figure 5 Influence of some phenolics on P-gp expression
HK-2 cells
Rat hepatocytes
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Apigenin, and to a minor extent, kaempferol, downregulate Pgp amounts (top) and mdr1 transcripts (bottom) in HK-2 cells and in rat hepatocytes. Cont- Control , Kaem- Kaempferol, Apig- Apigenin, Curc- Curcumin, Resv- Resveratrol
Like many other flavonoids, apigenin is poorly bioavailable and little is known about its absorption and metabolism. Apigenin disposition has been studied in Caco-2 cells by Hu et al. (2003). The downregulation of P-gp expression, observed by us (in two different models) coupled with the inhibition of transport function, could theoretically lead to pharmacokinetic interactions with drugs that are transported by P-gp by enhancing their oral bioavailability. Of course, the relative contribute of CYP3A4 ought to be investigated. Our results are in agreement with a recent study of Lohner et al. (2007) in which apigenin, unlike many other flavonoids, was found unable to induce P-gp expression in Caco-2 cells and intestinal cells in vivo. Some flavonoids belonging to different subclasses, i.e. epigallocatechin gallate (EGCG) the most abundant flavanol found in green tea, the flavanone naringenin, abundant in grapefruit juice and the chalcone xanthohumol, a flavonoid abundant in hop (Humulus lupulus), a beer ingredient, were also tested in HK-2 cells. All of three phytochemicals were able, with different potencies, to decrease the activity of Pgp (Table 1), according to literature data obtained in the Caco-2 transport model (Jodoin et al., 2002; Rodriguez-Proteau, 2006) or in other cell line, like the MDR overexpressing KB-C2 cells in which P-gp activity inhibition was assessed by the Rho-123 accumulation test (Kitagawa et al., 2005). Furthermore, besides the inhibition of P-gp function, we observed also a significant decrease of P-gp expression in immunoblots of HK-2 cells cultured in the presence of these compounds.
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Other polyphenols Another phenolic compound, which has received much attention in recent years, resveratrol, has been investigated in the HK-2 model. Resveratrol (trans3,5,4'-trihydroxystilbene), a natural product derived from grapes, has been shown to have a series of beneficial properties, including anti-inflammatory, antioxidant and chemopreventive activities (Das and Maulik, 2006; Holme and Pervaiz, 2007; Shankar et al., 2007). Resveratrol was found to inhibit significantly P-gp-dependent Rho-123 efflux from rat hepatocytes (Chieli et al., 1998) with the same potency of the P-gp inhibitor verapamil, but in the HK-2 cells it was unable to produce significant effects on P-gp activity, also if able to slightly increase the P-gp immunoblottable amount in cells cultured for three days in its presence (Table 1). This result suggests that pharmacokinetic interactions of resveratrol with P-gp substrates are improbable. On the other hand, red wine, assayed in our model, produced a substantial increase in P-gp expression at protein level, possibly due to the phenolic mix present in this beverage. Curcumin, (diferuloylmethane), is a natural phenolic yellow pigment present in the roots of Curcuma longa that has attracted great interest for its biological activities, potentially useful for clinical use, including antioxidant, anticarcinogenic and hepatoprotective properties (refs. in Romiti et al., 1998). Our previous studies employing the model of primary rat hepatocytes showed that curcumin was able to lower the increase of mdr1b P-gp that spontaneously occurs during culture, and to inhibit
Chieli and Romiti
Table 1. Interactions of various chemicals with P-glycoprotein investigated on the HK-2 cell model. Inhibition of P-gp activity Rho-123 Pure phytochemical compounds Apigenin Crocin Curcumin Resveratrol Kaempferol Naringenin Azadirachtin EGCG Xanthohumol Polyacetylenes & polyenes from Echinacea pallida Crude extracts Hypericum p. Humulus l. Neem oil Saffron Propolis Echinacea spp. n-hexane extracts Beverages Grapefruit Red wine Drugs Calcitriol Cimetidine Cyclosporin A Dexamethasone Verapamil Simvastatin Cannabinoids ++++ none ++++ ++ ++ + +++ ++ + ++ + +++ +++ +++ increased increased increased increased increased increased increased n.d. n.d. Nieri et al., 2006 Tramonti et al., 2001 Romiti et al., 2001 Chieli et al., 2002 Romiti et al., 2002 + n.d. ++ n.d. decreased increased decreased n.d. Romiti et al., 2004 unpublished results +++ ++ + n.d. ++ ++ + ++ n.d. ++ decreased decreased n.d. increased decreased n.d decreased n.d. n.d. n.d. Romiti et al., 2008 Chieli and Romiti, in preparation unpublished results ++ none + none ++ n.d. n.d. +++ +++ ++ + none n.d. n.d. ++ ++ none + + ++ decreased n.d. decreased decreased n.d. decreased decreased n.d. decreased n.d. n.d. n.d. n.d. decreased n.d. Romiti et al., 2004 unpublished results Calcein-AM P-gp expression WB MDR-1 expression RT-PCR References
Chieli and Romiti, in preparation
Romiti et al., 2008
EGCG-epigallocatechin gallate; n.d.- non determined; WB- Western blot
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the P-gp-dependent efflux of Rho-123 from hepatocytes in a dose-dependent manner. These observations suggested curcumin as a compound endowed with chemosensitizing properties on MDR phenotype. Recently, such properties of curcumin have been confirmed by various studies (Chearwae et al., 2004; Limtrakul, 2007; Zhang et al., 2007) and the mechanism by which curcumin downregulates the multidrug-resistance mdr1b gene of rat, i.e. the inhibition of PI3K/Akt/NfkappaB pathway, has been identified (Choi et al., 2008). In spite of these results in the HK-2 model curcumin did not modify P-gp expression (Table 1). Furthermore, also interference on Rho-123 accumulation, as an index of P-gp activity inhibition, was very weak. However, these negative results coming from few and preliminary studies need confirmation. St. Johns wort The traditional herb St. Johns wort (Hypericum perforatum) has been used since ancient times as a medicinal herb. Today St. Johns wort is widely used for the treatment of mild/moderate depressive disorders (Schulz, 2006). Self-medication with commercial preparations of this herb is acknowledged to cause important interactions with
prescribed drugs (Dasgupta et al., 2007; Zhou et al., 2004). The mechanism behind these adverse reactions is believed to be the induction of drug metabolizing enzymes, in particular CYP3A4, the CYP isozyme most abundant and/or the multidrug transporter P-gp (Drr et al., 2000). We have tested a crude extract of a commercial preparation of St. Johns wort in our cell model. Western blot of HK-2 cell membranes cultured for three days in the presence of various concentrations of the extract showed a weak, but significant increase in P-gp immunoblottable amount, suggesting P-gp induction (Fig. 6). This result is in agreement with the hypothesis that P-gp induction by St. Johns wort may lead to the decrease in the bioavailability of drugs that are P-gp substrates by increasing their efflux. On the other hand, both Rho-123 and CalceinAM functional assays indicated a significant inhibition of P-gp pump. The reason for this result is unknown, yet, but it could be ascribed to the effects of hypericum active principles e.g. hypericin, acting either as inducer as well as inhibitor of P-gp (Pal and Mitra, 2006). Furthermore, the commercial preparations of hypericum contain a wide range of flavonoid amount (Gdtel-Armbrust et al., 2007) that could contribute to the observed inhibition of P-gp activity.
Figure 6. Effects of Hypericum perforatum extracts on HK-2 P-gp expression and P-gp activity.
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Cells were cultured in the presence of various concentrations of hypericum extract (Hyp) in DMSO as vehicle. Western blot (A) and RT-PCR (B) both show a little, but significant increase of P-gp expression at protein and at mRNA level. Both functional tests, the Calcein-AM and, to a greater extent, the Rho-123 assays (C), showed a dose-dependent ability of hypericum extracts to inhibit P-gp activity. Verapamil (Vp) was used as internal control. Bars are mean SD of a representative experiment.
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2.
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40 0
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Grapefruit juice Grapefruit juice (GFJ)-drug interactions, firstly reported in 1990s, have been demonstrated by many in vitro and in vivo studies (Kane and Lipsky, 2000). GFJ has proven to alter the pharmacokinetics of a variety of drugs, including calcium channel blockers, immunomodulators, statins and others. The major mechanism of GFJ-drug interactions appeared to be the inhibition of CYP enzymes, in particular the CYP3A4 isoform, causing the reduction of the firstpass metabolism. However, other mechanisms have been demonstrated, in particular modulation by GFJ of P-gp-mediated efflux transport in intestinal cells. Since CYP3A4 and P-gp share a variety of substrates, their function could modulate in concert the bioavailability of drugs, by affecting their presystemic clearance (Kiani and Imam, 2007; Mertens Talcott et al., 2006). Although the literature findings about GFJ-P-gp interactions are sometimes in conflict, reporting both activation and inhibition of P-gp transporter by GFJ, recent in vivo and in vitro observations suggest that GFJ is able to inhibit the efflux of P-gp substrates (de Castro et al., 2007). In our investigations on possible interactions between grapefruit and P-gp in the HK-2 model, we showed that GFJ, as well as its main phenolic constituents, kaempferol and naringenin, were able to inhibit P-gp activity in HK-2 cells in a concentrationdependent fashion (Romiti et al., 2004). In the same year, Honda et al., (2004), observed the same inhibitory effect of GFJ and some GFJ components, on P-gp activity of Caco-2 cells and demonstrated that GFJ was able to interact with not only P-gp but also with MRP2. Because HK-2 cells apparently lack MRP2 (as yet mentioned), the transport inhibitory effects observed in this cell line may be referred more selectively to P-gp. Besides the Calcein-AM assay, employed in our study, the Rho-123 test, even if to a lesser extent, also suggests a modulation of P-gp activity by GFJ (Fig. 7). In the same study, we observed also a downregulation of P-gp in cells cultured for few days in presence of GFJ and its main phenolics, in the absence of any toxicity. As a functional consequence, the decreased amount of P-gp protein made cells more susceptible to toxic effects of P-gp drug substrates, like cyclosporine A or vinblastine. These findings suggested to us that GFJ could alter the handling of P-gp substrates not only by modulating P-gp
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dependent transport, but also P-gp amount. Such interactions might play additive roles in modify pharmacokinetic of P-gp substrates.
Figure 7. Effects of grapefruit juice on HK-2 P-gp activity
300 Intracellular fluorescence (% of control) 250 200 150 100 50 0

l tro con M 20 Vp % J5 GF 5% GF
Calcein-AM Rho-123
HK-2 P-gp activity modulation by concentrated grapefruit juice obtained by a commercial source (GFJ), or juice obtained by hand-squeezed fresh fruit (GF). Final concentration of both juices in culture medium was 5%. Verapamil (Vp) was used as internal control. Bars are mean SD of a representative experiment with five replicates. Either the Calcein-AM assay or - also if to a lesser extent - the Rho-123 test, show an increase in cell fluorescence in treated cells as compared with controls, suggesting an inhibition of P-gp pump function.
Echinacea One of the most frequently used medicinal plants both in the United States and in Europe, is today represented by echinacea, a popular herb used for centuries by Native Americans primarily to reduce the symptoms and duration of colds and flu-like illness. A variety of health benefits have been attributed to echinacea, including the increase in non specific activity of the immune system, facilitation of wound healing, anti-inflammatory and antioxidant effects due to the presence in the medicinal echinacea species (E. angustifolia, E. purpurea, E. pallida) of several classes of active constituents in particular polyphenols, polysaccharides and alkylamides (Barnes et al., 2005, Woelkart et al., 2006; Woelkart and Bauer, 2007). In spite of the impressive record of laboratory and clinical research, many aspects of echinacea properties are still lacking. In particular, very few studies have been devoted to investigate possible interactions between echinacea and the major pharmacokinetic disposition systems. While the effects of echinacea on CYP have been recognized (Yale and Glurich, 2005; Modarai et al., 2007), the
Chieli and Romiti
influence of this plant on P-gp is largely unknown. A very recent in vivo study (Gurley et al., 2008) suggests that echinacea supplementation probably should not be a cause of drug interactions dependent on P-gp, being unable to significantly modify the pharmacokinetic profile of digoxin, a P-gp substrate. However, at the same time, in our in vitro model, we have shown for the first time that lipophilic extracts of echinacea roots are able to inhibit P-gp activity in HK2 cells (Romiti et al., 2008). Furthermore, isolated components from E. pallida, like poly-acetylenes and polyenes (Pellati et al., 2006), were also able to inhibit P-gp-dependent transport (Table1 and Fig. 8). The observed inhibitory effects on HK-2 P-gp by echinacea extracts or by the lipophilic components of E. pallida are not, on the whole, dramatical. However, by considering the widespread and increasing use of echinacea preparations in complementary and alternative medicine, this feature of the plant constituents requires additional investigation and suggests some caution in the use of echinacea herbal products for conventional therapy. On the other hand, also the potentiality of echinacea phytochemicals as chemosensitizers under P-gp mediated resistance in cancer therapy deserves further consideration.
Figure 8. Influence of Echinacea n-hexane root extracts on HK-2 P-gp activity.
500 Intracellular fluorescence (% of control) 400 300 Calcein-AM 200 100 0
co nt ro l Vp 20 M l Pu r3 g /m ol ye l ne 10 0 M g /m l 3 g/ m
CONCLUSION Recently, many important advances have been made in the knowledge of the roles played by drug metabolizing enzymes (phase I, like CYP), phase II (various conjugating enzymes), or phase III (several ABC transporters, including P-gp), in the ADME process involving xenobiotics, drugs and natural compounds. In parallel, evidences for the likely interactions drug-drug, drug-herb, herb-herb, are also dramatically increased. Against this very complex background, the importance emerges of a rapid individualization of chemicals potentially able to cause such adverse reactions. In addition to sophisticated methods, expensive, time-consuming, and sometimes not well reproducible, it may be useful to have simple in vitro assays to screen compounds for interactions with the major biological determinants of drug disposition. Our results show that HK-2 cell line may be conveniently employed as a sensitive, versatile, reproducible tool to investigate the effects of various chemicals on P-gp, providing indications that can represent a starting point for more refined and detailed studies. REFERENCES
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Rho-123
Pa l3
An g
*Pentadeca-(8Z, 13Z)-dien-11-yn-2-one
P-gp activity, assessed by the calcein-AM or the Rho-123 test, in cells exposed to n-hexane extracts of different Echinacea species or to the polyene pentadeca-(8Z,13Z)-dien-11-yn-2-one, isolated from the nhexane extract of E. pallida roots by a bioassay-guided fractionation. Verapamil (20 M) was used as internal control. Bars are mean S.D. of a representative experiment with five replicates. Both tests show that the extracts of E. pallida at 3g/ml and the polyene pentadeca-(8Z,13Z)-dien11-yn-2-one at 100 M are particularly able to decrease P-gp activity. Vp- verapamil; Ang- E. angustifolia; Pal- E. pallida; Pur- E. purpurea.
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*P
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2008 The authors, licensee BLACPMA ISSN 0717 7917
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Kidney proximal human tubule HK-2 cell line as a tool for the investigation of P-glycoprotein modulation by natural compounds
[La lnea celular de tbulo proximal de rin humano HK-2 como una herramienta para la investigacin de la modulacin de P-glicoprotena por compuestos naturales]
Elisabetta CHIELI*and Nadia ROMITI Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Sezione di Patologia Generale e Clinica. Universit degli Studi di Pisa. Scuola Medica, via Roma 55, I-56126 Pisa, Italy.
*Contact: e-mail: chieli@biomed.unipi.it Submitted February 13, 2008; Accepted February 29, 2008; Published Online 15 October 2008
Abstract ABCB1 (MDR1/P-glycoprotein), the best characterized multidrug transporter belonging to the ABC transporter superfamily, is nowadays acknowledged to have a major impact on drug resistance to chemotherapy and drug bioavailability and disposition. A number of natural compounds, dietary phytochemicals and herbal remedies have the ability to modulate P-glycoprotein function and /or expression and therefore the potentiality to cause food-drug, or herb-drug interactions. The elucidation of these interactions may be important not only to predict possible undesirable effects deriving from the concomitant intake of herbal constituents and conventional drugs, but also for further studies of positive uses of these interactions as a way to increase the bioavailability of drugs that are P-gp substrates. In particular, the study of P-gp inhibition by herbal constituents may provide an approach for the identification of lead compounds for the design of news chemosensitizers to reverse multidrug resistance in tumor cells. This review describes our recent investigations on the potential herb-drug interactions involving P-glycoprotein, using as a model the HK-2 cells, an immortalized line of proximal tubule cell derived from human normal kidney.
Keywords: HK-2 cell line, P-glycoprotein, Calcein-AM, Rhodamine-123, phytochemicals.
Resumen El ABCB1 (MDR1/P-glicoprotena), el transportador multidroga mejor caracterizado que pertenece a la superfamilia de transportadores ABC, se reconoce hoy da por tener un gran impacto en la resistencia de los frmacos a la quimioterapia y a la biodisponibilidad y disposicin de los medicamentos. Varios compuestos naturales, fitoqumicos dietticos y remedios herbarios tienen la capacidad de modular la funcin y/o expresin de la glicoprotena P (P-gp) y, por consiguiente, la potencialidad para causar interacciones alimento-frmaco o hierba-frmaco. La elucidacin de estas interacciones no slo es importante para predecir posibles efectos indeseables que derivan de la ingestin concomitante de componentes herbarios y los frmacos convencionales, tambin se usa para los estudios sobre los usos positivos de estas interacciones como una manera de aumentar el biodisponibilidad de frmacos que son substratos de P-gp. En particular, el estudio de la inhibicin de P-gp por los componentes herbarios provee un acercamiento a la identificacin de compuestos para el diseo de quimiosensibilizadores para invertir la resistencia multidroga en clulas tumorales. Esta revisin describe nuestras recientes investigaciones sobre las interacciones potenciales hierba-frmaco que involucran P-gp mediante un modelo de clulas HK-2, una lnea celular inmortalizada de tbulo proximal derivada del rin humano normal.
Palabras clave: Lnea celular HK-2, P-glicoprotena, Calcena-AM, Rodamina-123, fitoqumicos.
Abbreviation list: ABC- ATP-binding cassette CYP- cytochrome P450 MRP- Multidrug resistance-associated protein ADME- absorption, distribution, metabolism and excretion MDR- Multidrug resistance P-gp- P-glycoprotein
Chieli and Romiti
INTRODUCTION Pharmacological interactions possibly derive from the modulation in expression and/or activity of two major pharmacokinetic disposition systems, namely cytochrome P450 (CYP) and the multidrug transporter P-glycoprotein (Aszalos, 2007a and 2007b; Liu et al., 2007; Marchetti et al., 2007; Pal and Mitra, 2006). The transmembrane efflux pump P-glycoprotein (P-gp) is a member of the ATP-binding cassette (ABC) superfamily transporters, membrane-bound proteins, able to export from cells a variety of chemicals, including drugs, xenobiotics, natural products and peptides. These efflux pumps, which are powered by the energy of ATP hydrolysis, have important physiological, pharmacological and toxicological functions (Linton, 2007; Schinkel and Jonker, 2003). P-gp, encoded by the MDR1 gene (now ABCB1), is the best known member of the ABC transporters. P-gp was initially discovered in 1970s in tumor cells as responsible for the emergence of the multidrug resistant (MDR) phenotype. The early years of P-gp research have been described in detail in a recent review by Gottesman and Ling (2006). P-gp has been retrieved also in many normal cells and tissues where, as well as many others ABC transporters, is believed to play a significant role in detoxification and protection against xenobiotics. In fact, P-gp is physiologically expressed at the apical surface of epithelial cells of several organs, like liver, intestine and kidney, devoted to secretion/excretion of a variety of metabolites, xenobiotics and drugs. Other strategical locations are the brain blood-barrier, bloodtestis and bloodovarian barriers, and placenta (Fromm, 2004; Marchetti et al., 2007). In the normal liver, P-gp is believed to play a significant role in the secretory processes of endoand xenobiotics across the canalicular membrane into the bile and, like other transport systems localized to the canalicular membrane, it may be considered as one of the complex multistep process which ensures the vectorial transport of a variety of chemicals across the hepatocyte (LeBlanc, 1994). Accordingly, P-gp located at the luminal side of renal proximal tubule appears involved in the active secretion of many drugs and nephrotoxins (Ernest and BelloReuss, 1998), while enterocyte P-gp activity is important in the absorption process of a series of chemicals (refs. in Berggren et al., 2007).
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Nowadays, P-gp is considered a crucial molecule involved in all the pharmacokinetic processes: absorption, distribution, metabolism and excretion (ADME) (Tanigawara, 2000; Varma et al. 2003) and a number of single-nucleotide polymorphisms (SNPs) in ABCB1 gene has been correlated with different responsiveness to many drugs (Marzolini et al., 2004). P-gp shows broad substrate specificity with a prevalent affinity for hydrophobic compounds; so many natural and synthetic xenobiotics and endogenous molecules are substrates/modulators of its activity (Chan et al., 2004). It is generally accepted that coadministration of compounds that interact with this transporter (as a substrate, inhibitor, or inducer) can result in interactions that affect the pharmacokinetics and pharmacodynamics of the coadministered drugs (Aszalos, 2007a; Aszalos, 2007b; Endres et al., 2006; Keogh and Kunta, 2006; Lin, 2007; Lin, 2003; Yu, 1999). Among these chemicals a number of vegetables and related phytochemicals have been reported to inhibit P-gp including garlic, black pepper, green tea polyphenols, flavonoids, curcumin, genistein, naringin, capsaicin, and others (Han et al., 2006; Hu et al., 2005; Izzo and Ernst, 2001; Izzo, 2005; Jodoin et al., 2002; Meijerman et al., 2006; Molnar et al., 2006; Nabekura et al., 2005; Nabekura et al., 2007; Pal and Mitra, 2006; Rodriguez-Proteau et al., 2006; Zhou et al., 2004; Zhou et al., 2007). It is therefore crucial to rapidly identify chemical entities -including herbal remedies, nutraceuticals, and food derived compounds- able to modulate P-gp, in order to predict possible herb-drug interactions. This review will primarily focus on the recent efforts of our laboratory devoted to investigate in vitro these interactions by using, as a model, the human cell line HK-2 derived from the proximal convoluted tubule of the kidney. Assessment of P-gp modulation by chemicals Various screening models and methodologies have been evolved for the identification and characterization of P-gp modulators (Perloff et al., 2003; Rautio et al., 2006; Shirasaka et al., 2006; Taub et al., 2005; Varma et al., 2003). Each technique has its own advantages and limitations. The most commonly in vitro methods used to evaluate whether a chemical compound is a modulator of the P-gp efflux transporter are three: i) Bi-directional transport assay with a probe P-gp substrate in functionally polarized epithelial cell monolayer. The preferred cells lines include Caco-2, transfected LLC-PK1-
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MDR1, and transfected MDCK-MDR1. LLC-PK1 and MDCK wild type cells are used as negative controls; ii) ATPase activity assay that measure substrate stimulated-ATPase activity in the presence of substrates, which interact on different P-gp sites, with and without test compounds, allowing identification of P-gp inhibitors. ATPase activity can be determined using cell membranes, cultured cells and membrane vesicles; iii) Drug accumulation and efflux assays where the amount of a substrate, generally a fluorescent probe, taken up by cells expressing P-gp, is compared in the presence and absence of inhibitors. This last method, also if cannot distinguish substrate from inhibitors, can screen compounds rapidly and could suggest further investigations employing more complex methodologies. In our laboratory, for the study of possible interactions between chemicals and P-gp, we utilized a cell line from human kidney proximal tubule and uptake/efflux assays. Furthermore, because the expression of P-gp may be influenced by chemicals, we used this cell line also for the in vitro evaluation of P-gp modulation at protein (immunoblotting) and at mRNA (RT-PCR) levels.
Renal P-glycoprotein Together with the liver, the kidney is the most important detoxifying organ (Inui et al., 2000). The kidney tubular cell has a large spectrum of functions, which modify the ultrafiltrate before its turning into final urine. The modification regards solutes secretion or solutes absorption. Many of these movements of substances between blood and tubular duct occur by different carrier work. The proximal tubular cells have a transport activity higher than all the other renal segments and, furthermore, are exposed to high concentrations of xenobiotics. These cells express a lot of membrane transporters, some of which belonging to the ABC superfamily, in particular P-gp (ABCB1) (Kruidering et al., 1994; Lee and Kim, 2004; Schlatter et al., 2006). P-gp is localized together other multidrug resistanceassociated proteins (MRPs) on apical surface (brush border) and is implicated in endogenous substrate transport and tubular secretion of toxic substances (Ernest and Bello-Reuss, 1998). A scheme of the main ABC transporters present in the proximal tubular cell is shown in Fig. 1.
Figure 1. Localization of P-gp and other ABC transporters in the kidney proximal tubule.
BASOLATERAL MEMBRANE
TUBULE LUMEN
Left: immunohistochemical localization of P-gp in human kidney (polyclonal anti mdr Ab-1). Right: P-gp/MDR1 (ABCB1), MRP2 (ABCC2), MRP4 (ABCC4) and BCRP (ABCG2) are expressed on the apical (luminal) membrane of renal epithelial cells and export compounds from the cytoplasm of tubule cell to the urine. MRP1 (ABCC1) and MRP6 (ABCC6) are expressed on the basolateral membrane. (Inui et al., 2000; Huls et al., 2008; Lee and Kim, 2004).
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The HK-2 cell line HK-2 cell line is an immortalized line derived from the human epithelial renal proximal tubule. Zager and coll. utilized a kidney from a male normal adult considered unsuitable for transplantation (Ryan et al., 1994). The cortical proximal tubule segment was isolated, cultured and exposed to a recombinant virus containing the E6 and E7 genes of HPV16. A cell clone, in which PCR analysis confirmed the incorporation of HPV16 E6/E7 construct in genomic DNA, was designed as HK-2 and was able to continuously grow for more than a year. At the molecular level the products of E6 and E7 genes bind to the DNA regulatory proteins, resulting in a cell facilitated proliferation. Phenotypically, the cell line HK-2 has the same characteristics of adult normal tubular cells, as demonstrated by a series of biochemical tests. In particular, it was shown that the HK-2 cells maintain the brush border typical enzymatic activities (acid and alkaline phosphatase, leucine aminopeptidase, gammaglutamyltranspeptidase). Therefore, this new line has been proposed as a valuable tool for the study of physiological and
physiopathological human renal tubule, as well as the mechanisms of damage and repair at the level of tubular cell and has been widely employed in research. In our laboratory we showed, for the first time, the presence of a functional MDR1 (ABCB1)encoded P-gp in HK-2 cells (Tramonti et al., 2001) and thereafter we characterized such a line for a series of parameters regarding this transporter, including the presence of MDR1, but not MDR3 (ABCB4) transcripts, expression at protein level on cell membranes, immunocytochemical localization and the responsiveness to a series of modulators including acknowledged P-gp inhibitors, like verapamil and cyclosporin A, as well as some drugs, like dexamethasone and calcitriol (Romiti et al., 2002). On the whole, the results suggested that HK-2 cells could be suitable as a tool to investigate the role of P-gp in renal physiopathology. Further characterization have indicated that HK-2 line, while expressing a robust amount of P-gp, that resulted not only quite stable in the time, passage number, culture conditions, but also responsive to chemical modulation, apparently lacks expression of the apical ABC transporter MRP2 (Fig. 2, Chieli and Romiti, unpublished results).
Figure 2. RT-PCR and Western blot analysis of ABCB1/P-gp and ABCC2/MRP2 in HK-2 or Caco-2 cell lines.
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Left: RT-PCR analysis shows that HK-2 express MDR1 (ABCB1) mRNA only, while Caco-2 cell line expresses both MDR1 (ABCB1) and MRP2 (ABCC2) mRNAs. M.W.,123-bp DNA ladder. Right: Western blot analysis of crude membranes from HK-2 or Caco-2 cells shows that both cell lines express, also if to a different degree, P-gp, while the band corresponding to MRP2, well represented in Caco-2, is undetectable in HK-2 cell membranes (lane 3). For immunoblotting monoclonal anti-P-gp or polyclonal anti-MRP2 antibodies and peroxydase-conjugated anti-mouse or anti-rabbit secondary antibodies were used. Blots were developed with the ECL detection system.
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MRP2 together with P-gp are known as the main apical transporters in tubule cell, devoted to excretion of drugs and xenobiotics in the tubule lumen (refs. in Huls et al., 2008). The absence of MRP2 expression and, in parallel, the observed lack of effect of probenecid, an inhibitor of MRPs, in P-gp functional tests (Nieri et al., 2006), suggest the HK-2 cells suitable to assay chemical effects selectively on P-gp, rather than cells in which also MRP2 is appreciably expressed, e.g. Caco-2 (Prime-Chapman et al., 2004). Assessment of chemical-P-gp interactions: methodological notes The HK-2 model has been used by us to check if chemical compounds (in particular natural compounds from vegetables) present in food or employed in phytotherapy, may modify the in vitro expression and function of P-gp. While P-gp expression at protein level (immunoblotting) or at mRNA level (RT-PCR) doesnt pose difficulties in interpretation, functional assays for Pg-p merit some comments. Throughout our studies we have used two different tests to evaluate the influence of chemicals
on P-gp activity, i. e. the Calcein-AM and the Rhodamine-123 accumulation assays. The principle of these assays are schematically shown in Figs. 3 and 4.
Figure 3. Scheme of the Calcein-AM assay for the assessment of Pglycoprotein activity.
The P-gp substrate nonfluorescent Calcein-AM is hydrolysed by cellular esterase to the fluorescent Calcein. Calcein is not a substrate for P-gp and it cannot leave the cell via the plasma membrane, whereas the nonfluorescent Calcein-AM is extruded from the MDR-1 expressing cells. The intracellular accumulation of fluorescent Calcein is inversely proportional to the transport capacity of P-gp. A low expression of P-gp or the presence of P-gp inhibitors produces a rise in cell fluorescence.
Figure 4. Scheme of the rhodamine-123 assay for the assessment of P-glycoprotein activity.
Intracellular fluorescence ng Rho-123/mg protein
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Left: scheme of the rhodamine-123 test (Rho-123) for the measurement of P-gp activity. The fluorescent mitochondrial probe Rho-123 enters into the cells and accumulates in cytoplasma and mitochondria. Because Rho-123 is a substrate for P-gp and it is extruded actively form the cells, the intracellular amount of Rho-123 is inversely proportional to P-gp activity. Inhibitors of P-gp cause an increase in cell fluorescence. Right: effects of two acknowledged inhibitors of P-gp, Verapamil and Cyclosporine A on Rho-123 accumulation by HK-2 cells. Cells were incubated in medium containing 5 g/ml Rho-123 for two hours; thereafter cell plates were washed and extracted with n-butanol. Rho-123 was quantified by spectrophotofluorimetry. At the top: the relative fluorescence microscope images.
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Calcein-AM test: This assay is largely employed (Eneroth et al., 2001; Holl et al., 1994; Tiberghien and Loor, 1996). At least two main cautions in the measurement of P-gp activity by this method must be considered. First, it is known that whereas CalceinAM is a substrate of P-gp, the fluorescent calcein is transported, out of the cell, by MRPs, therefore, this test couldnt be strictly specific for P-gp. Anyway, in HK-2 this possibility may be reasonably excluded, due to the fact that, as yet recalled, the major apical MRP transporter, MRP2, is apparently not expressed; furthermore, the MRPs inhibitor probenecid did not influence, in HK-2 cells, the results of Calcein-AM test (Nieri et al., 2006). Secondly, it must be taken into account that, independently from P-gp activity, the fluorescent calcein derives from the action of esterase on the nonfluorescent precursor Calcein-AM (Fig. 3). This occurrence -also reflected by the fact that Calcein-AM test is also employed as an index of cell viability- alerts on possible interferences by compounds able to influence cell esterase activity. In this regard it is suggestive that some flavonoids have been described recently to inhibit esterase activity (Li et al., 2007). Effects of this type (or even opposite) could influence the results of Calcein-AM assay causing under/overestimation of the test compound influence on the P-gp activity. Rhodamine-123 test: This test is also widely used (Perloff et al., 2003; Wang et al., 2006). However, this also requires some cautions. First, Rho-123 is a fluorescent mitochondrial dye, therefore it accumulates in cell and mitochondrial uptake accumulation may depend on mitochondrial transmenbrane potential (MTP) and cell membrane potential. Moreover, even if Rho-123 has always been considered to enter into the cells in a passive way, Troutman and Thakker (2003) have suggested the presence of a membrane influx carrier for this fluorescent probe. These things must be taken into account especially when a lack of correlation is observed between the results obtained with different methods, making difficult the interpretation. From some time, routinely, in our laboratory, always we use both Calcein-AM and Rho-123 tests, which in most situations gave congruent results. Table 1 summarize our studies about the interactions between herbal constituents and P-gp in the HK-2 model.
Flavonoids The plant flavonoids are a wide class of natural compounds commonly present in fruits, vegetables and beverages obtained from plants, like tea, red wine, and beer. Consumption of a diet rich in flavonoids is believed to produce a variety of health benefits, including antioxidant, anti-inflammatory, antiallergic effects, as well as protection in cardiovascular diseases and cancer (Yao et al., 2004). Flavonoids have been recognized as Pglycoprotein modulators for a long time, also if contradictory effects were reported in studies using different multidrug-resistant cell lines. Anyway, because flavonoids were shown to be modulators with bi-functional interactions at vicinal ATP-binding site and steroid-interacting region of P-gp, the controversial reports about flavonoid-P-gp interactions are now generally ascribed to the different binding of the model substrates to multiple binding sites of P-gp (Shapiro and Ling, 1997; Conseil et al., 1998). The effects of flavonoids on ABC transporters, including P-gp, have been recently reviewed and, from the majority of the more recent studies, the indication emerges that flavonoids are on average inhibitors of P-gp-mediated transport (Di Pietro et al., 2002; Morris and Zhang, 2006). In our previous studies employing primary rat hepatocytes as a model of cells expressing mdr1b Pgp we found that the flavonols quercetin, kaempferol and galangin were able, also if at a different degree, and with differences related to the used substrate, to modulate P-gp activity (Chieli et al., 1995). Other flavonoids, like the flavone apigenin, were also shown to inhibit P-gp activity, and, more consistently, to significantly downregulate P-gp expression in primary rat hepatocytes (Chieli et al., 1998). These results have been confirmed also in the HK-2 model (Fig. 5, unpublished results). The effects of apigenin appear very interesting by considering that it is a common flavonoid, present in a variety of vegetables, as well as in their extracts, marketed as diet and herbal supplements. Apigenin has demonstrated anticancer activities and chemopreventive potential towards prostate cancer (Patel et al., 2007).
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Chieli and Romiti
Figure 5 Influence of some phenolics on P-gp expression
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Apigenin, and to a minor extent, kaempferol, downregulate Pgp amounts (top) and mdr1 transcripts (bottom) in HK-2 cells and in rat hepatocytes. Cont- Control , Kaem- Kaempferol, Apig- Apigenin, Curc- Curcumin, Resv- Resveratrol
Like many other flavonoids, apigenin is poorly bioavailable and little is known about its absorption and metabolism. Apigenin disposition has been studied in Caco-2 cells by Hu et al. (2003). The downregulation of P-gp expression, observed by us (in two different models) coupled with the inhibition of transport function, could theoretically lead to pharmacokinetic interactions with drugs that are transported by P-gp by enhancing their oral bioavailability. Of course, the relative contribute of CYP3A4 ought to be investigated. Our results are in agreement with a recent study of Lohner et al. (2007) in which apigenin, unlike many other flavonoids, was found unable to induce P-gp expression in Caco-2 cells and intestinal cells in vivo. Some flavonoids belonging to different subclasses, i.e. epigallocatechin gallate (EGCG) the most abundant flavanol found in green tea, the flavanone naringenin, abundant in grapefruit juice and the chalcone xanthohumol, a flavonoid abundant in hop (Humulus lupulus), a beer ingredient, were also tested in HK-2 cells. All of three phytochemicals were able, with different potencies, to decrease the activity of Pgp (Table 1), according to literature data obtained in the Caco-2 transport model (Jodoin et al., 2002; Rodriguez-Proteau, 2006) or in other cell line, like the MDR overexpressing KB-C2 cells in which P-gp activity inhibition was assessed by the Rho-123 accumulation test (Kitagawa et al., 2005). Furthermore, besides the inhibition of P-gp function, we observed also a significant decrease of P-gp expression in immunoblots of HK-2 cells cultured in the presence of these compounds.
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Other polyphenols Another phenolic compound, which has received much attention in recent years, resveratrol, has been investigated in the HK-2 model. Resveratrol (trans3,5,4'-trihydroxystilbene), a natural product derived from grapes, has been shown to have a series of beneficial properties, including anti-inflammatory, antioxidant and chemopreventive activities (Das and Maulik, 2006; Holme and Pervaiz, 2007; Shankar et al., 2007). Resveratrol was found to inhibit significantly P-gp-dependent Rho-123 efflux from rat hepatocytes (Chieli et al., 1998) with the same potency of the P-gp inhibitor verapamil, but in the HK-2 cells it was unable to produce significant effects on P-gp activity, also if able to slightly increase the P-gp immunoblottable amount in cells cultured for three days in its presence (Table 1). This result suggests that pharmacokinetic interactions of resveratrol with P-gp substrates are improbable. On the other hand, red wine, assayed in our model, produced a substantial increase in P-gp expression at protein level, possibly due to the phenolic mix present in this beverage. Curcumin, (diferuloylmethane), is a natural phenolic yellow pigment present in the roots of Curcuma longa that has attracted great interest for its biological activities, potentially useful for clinical use, including antioxidant, anticarcinogenic and hepatoprotective properties (refs. in Romiti et al., 1998). Our previous studies employing the model of primary rat hepatocytes showed that curcumin was able to lower the increase of mdr1b P-gp that spontaneously occurs during culture, and to inhibit
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Table 1. Interactions of various chemicals with P-glycoprotein investigated on the HK-2 cell model. Inhibition of P-gp activity Rho-123 Pure phytochemical compounds Apigenin Crocin Curcumin Resveratrol Kaempferol Naringenin Azadirachtin EGCG Xanthohumol Polyacetylenes & polyenes from Echinacea pallida Crude extracts Hypericum p. Humulus l. Neem oil Saffron Propolis Echinacea spp. n-hexane extracts Beverages Grapefruit Red wine Drugs Calcitriol Cimetidine Cyclosporin A Dexamethasone Verapamil Simvastatin Cannabinoids ++++ none ++++ ++ ++ + +++ ++ + ++ + +++ +++ +++ increased increased increased increased increased increased increased n.d. n.d. Nieri et al., 2006 Tramonti et al., 2001 Romiti et al., 2001 Chieli et al., 2002 Romiti et al., 2002 + n.d. ++ n.d. decreased increased decreased n.d. Romiti et al., 2004 unpublished results +++ ++ + n.d. ++ ++ + ++ n.d. ++ decreased decreased n.d. increased decreased n.d decreased n.d. n.d. n.d. Romiti et al., 2008 Chieli and Romiti, in preparation unpublished results ++ none + none ++ n.d. n.d. +++ +++ ++ + none n.d. n.d. ++ ++ none + + ++ decreased n.d. decreased decreased n.d. decreased decreased n.d. decreased n.d. n.d. n.d. n.d. decreased n.d. Romiti et al., 2004 unpublished results Calcein-AM P-gp expression WB MDR-1 expression RT-PCR References
Chieli and Romiti, in preparation
Romiti et al., 2008
EGCG-epigallocatechin gallate; n.d.- non determined; WB- Western blot
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the P-gp-dependent efflux of Rho-123 from hepatocytes in a dose-dependent manner. These observations suggested curcumin as a compound endowed with chemosensitizing properties on MDR phenotype. Recently, such properties of curcumin have been confirmed by various studies (Chearwae et al., 2004; Limtrakul, 2007; Zhang et al., 2007) and the mechanism by which curcumin downregulates the multidrug-resistance mdr1b gene of rat, i.e. the inhibition of PI3K/Akt/NfkappaB pathway, has been identified (Choi et al., 2008). In spite of these results in the HK-2 model curcumin did not modify P-gp expression (Table 1). Furthermore, also interference on Rho-123 accumulation, as an index of P-gp activity inhibition, was very weak. However, these negative results coming from few and preliminary studies need confirmation. St. Johns wort The traditional herb St. Johns wort (Hypericum perforatum) has been used since ancient times as a medicinal herb. Today St. Johns wort is widely used for the treatment of mild/moderate depressive disorders (Schulz, 2006). Self-medication with commercial preparations of this herb is acknowledged to cause important interactions with
prescribed drugs (Dasgupta et al., 2007; Zhou et al., 2004). The mechanism behind these adverse reactions is believed to be the induction of drug metabolizing enzymes, in particular CYP3A4, the CYP isozyme most abundant and/or the multidrug transporter P-gp (Drr et al., 2000). We have tested a crude extract of a commercial preparation of St. Johns wort in our cell model. Western blot of HK-2 cell membranes cultured for three days in the presence of various concentrations of the extract showed a weak, but significant increase in P-gp immunoblottable amount, suggesting P-gp induction (Fig. 6). This result is in agreement with the hypothesis that P-gp induction by St. Johns wort may lead to the decrease in the bioavailability of drugs that are P-gp substrates by increasing their efflux. On the other hand, both Rho-123 and CalceinAM functional assays indicated a significant inhibition of P-gp pump. The reason for this result is unknown, yet, but it could be ascribed to the effects of hypericum active principles e.g. hypericin, acting either as inducer as well as inhibitor of P-gp (Pal and Mitra, 2006). Furthermore, the commercial preparations of hypericum contain a wide range of flavonoid amount (Gdtel-Armbrust et al., 2007) that could contribute to the observed inhibition of P-gp activity.
Figure 6. Effects of Hypericum perforatum extracts on HK-2 P-gp expression and P-gp activity.
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Cells were cultured in the presence of various concentrations of hypericum extract (Hyp) in DMSO as vehicle. Western blot (A) and RT-PCR (B) both show a little, but significant increase of P-gp expression at protein and at mRNA level. Both functional tests, the Calcein-AM and, to a greater extent, the Rho-123 assays (C), showed a dose-dependent ability of hypericum extracts to inhibit P-gp activity. Verapamil (Vp) was used as internal control. Bars are mean SD of a representative experiment.
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Grapefruit juice Grapefruit juice (GFJ)-drug interactions, firstly reported in 1990s, have been demonstrated by many in vitro and in vivo studies (Kane and Lipsky, 2000). GFJ has proven to alter the pharmacokinetics of a variety of drugs, including calcium channel blockers, immunomodulators, statins and others. The major mechanism of GFJ-drug interactions appeared to be the inhibition of CYP enzymes, in particular the CYP3A4 isoform, causing the reduction of the firstpass metabolism. However, other mechanisms have been demonstrated, in particular modulation by GFJ of P-gp-mediated efflux transport in intestinal cells. Since CYP3A4 and P-gp share a variety of substrates, their function could modulate in concert the bioavailability of drugs, by affecting their presystemic clearance (Kiani and Imam, 2007; Mertens Talcott et al., 2006). Although the literature findings about GFJ-P-gp interactions are sometimes in conflict, reporting both activation and inhibition of P-gp transporter by GFJ, recent in vivo and in vitro observations suggest that GFJ is able to inhibit the efflux of P-gp substrates (de Castro et al., 2007). In our investigations on possible interactions between grapefruit and P-gp in the HK-2 model, we showed that GFJ, as well as its main phenolic constituents, kaempferol and naringenin, were able to inhibit P-gp activity in HK-2 cells in a concentrationdependent fashion (Romiti et al., 2004). In the same year, Honda et al., (2004), observed the same inhibitory effect of GFJ and some GFJ components, on P-gp activity of Caco-2 cells and demonstrated that GFJ was able to interact with not only P-gp but also with MRP2. Because HK-2 cells apparently lack MRP2 (as yet mentioned), the transport inhibitory effects observed in this cell line may be referred more selectively to P-gp. Besides the Calcein-AM assay, employed in our study, the Rho-123 test, even if to a lesser extent, also suggests a modulation of P-gp activity by GFJ (Fig. 7). In the same study, we observed also a downregulation of P-gp in cells cultured for few days in presence of GFJ and its main phenolics, in the absence of any toxicity. As a functional consequence, the decreased amount of P-gp protein made cells more susceptible to toxic effects of P-gp drug substrates, like cyclosporine A or vinblastine. These findings suggested to us that GFJ could alter the handling of P-gp substrates not only by modulating P-gp
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dependent transport, but also P-gp amount. Such interactions might play additive roles in modify pharmacokinetic of P-gp substrates.
Figure 7. Effects of grapefruit juice on HK-2 P-gp activity
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HK-2 P-gp activity modulation by concentrated grapefruit juice obtained by a commercial source (GFJ), or juice obtained by hand-squeezed fresh fruit (GF). Final concentration of both juices in culture medium was 5%. Verapamil (Vp) was used as internal control. Bars are mean SD of a representative experiment with five replicates. Either the Calcein-AM assay or - also if to a lesser extent - the Rho-123 test, show an increase in cell fluorescence in treated cells as compared with controls, suggesting an inhibition of P-gp pump function.
Echinacea One of the most frequently used medicinal plants both in the United States and in Europe, is today represented by echinacea, a popular herb used for centuries by Native Americans primarily to reduce the symptoms and duration of colds and flu-like illness. A variety of health benefits have been attributed to echinacea, including the increase in non specific activity of the immune system, facilitation of wound healing, anti-inflammatory and antioxidant effects due to the presence in the medicinal echinacea species (E. angustifolia, E. purpurea, E. pallida) of several classes of active constituents in particular polyphenols, polysaccharides and alkylamides (Barnes et al., 2005, Woelkart et al., 2006; Woelkart and Bauer, 2007). In spite of the impressive record of laboratory and clinical research, many aspects of echinacea properties are still lacking. In particular, very few studies have been devoted to investigate possible interactions between echinacea and the major pharmacokinetic disposition systems. While the effects of echinacea on CYP have been recognized (Yale and Glurich, 2005; Modarai et al., 2007), the
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influence of this plant on P-gp is largely unknown. A very recent in vivo study (Gurley et al., 2008) suggests that echinacea supplementation probably should not be a cause of drug interactions dependent on P-gp, being unable to significantly modify the pharmacokinetic profile of digoxin, a P-gp substrate. However, at the same time, in our in vitro model, we have shown for the first time that lipophilic extracts of echinacea roots are able to inhibit P-gp activity in HK2 cells (Romiti et al., 2008). Furthermore, isolated components from E. pallida, like poly-acetylenes and polyenes (Pellati et al., 2006), were also able to inhibit P-gp-dependent transport (Table1 and Fig. 8). The observed inhibitory effects on HK-2 P-gp by echinacea extracts or by the lipophilic components of E. pallida are not, on the whole, dramatical. However, by considering the widespread and increasing use of echinacea preparations in complementary and alternative medicine, this feature of the plant constituents requires additional investigation and suggests some caution in the use of echinacea herbal products for conventional therapy. On the other hand, also the potentiality of echinacea phytochemicals as chemosensitizers under P-gp mediated resistance in cancer therapy deserves further consideration.
Figure 8. Influence of Echinacea n-hexane root extracts on HK-2 P-gp activity.
500 Intracellular fluorescence (% of control) 400 300 Calcein-AM 200 100 0
co nt ro l Vp 20 M l Pu r3 g /m ol ye l ne 10 0 M g /m l 3 g/ m
CONCLUSION Recently, many important advances have been made in the knowledge of the roles played by drug metabolizing enzymes (phase I, like CYP), phase II (various conjugating enzymes), or phase III (several ABC transporters, including P-gp), in the ADME process involving xenobiotics, drugs and natural compounds. In parallel, evidences for the likely interactions drug-drug, drug-herb, herb-herb, are also dramatically increased. Against this very complex background, the importance emerges of a rapid individualization of chemicals potentially able to cause such adverse reactions. In addition to sophisticated methods, expensive, time-consuming, and sometimes not well reproducible, it may be useful to have simple in vitro assays to screen compounds for interactions with the major biological determinants of drug disposition. Our results show that HK-2 cell line may be conveniently employed as a sensitive, versatile, reproducible tool to investigate the effects of various chemicals on P-gp, providing indications that can represent a starting point for more refined and detailed studies. REFERENCES
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Rho-123
Pa l3
An g
*Pentadeca-(8Z, 13Z)-dien-11-yn-2-one
P-gp activity, assessed by the calcein-AM or the Rho-123 test, in cells exposed to n-hexane extracts of different Echinacea species or to the polyene pentadeca-(8Z,13Z)-dien-11-yn-2-one, isolated from the nhexane extract of E. pallida roots by a bioassay-guided fractionation. Verapamil (20 M) was used as internal control. Bars are mean S.D. of a representative experiment with five replicates. Both tests show that the extracts of E. pallida at 3g/ml and the polyene pentadeca-(8Z,13Z)-dien11-yn-2-one at 100 M are particularly able to decrease P-gp activity. Vp- verapamil; Ang- E. angustifolia; Pal- E. pallida; Pur- E. purpurea.
www.blacpma.org
*P
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Transportadores de tipo ABC: consecuencias de su interaccin con flavonoides

[ABC transporters: consequences of interaction with flavonoids]
Ana I. LVAREZ DE FELIPE* , Mivis M. PULIDO DUARTE Depto. de Ciencias Biomdicas, Fisiologa. Facultad de Veterinaria .Universidad de Len. 24071. Espaa.
*Contacto: e-mail: ana.alvarez@unileon.es
Recibido Febrero 18, 2008; Aceptado Marzo 8, 2008; Published Online 15 October 2008
Abstract The ATP-binding cassette (ABC) transporters constitute one of the largest protein families widely distributed and evolutionarily conserved which are involved in detoxifying and transport processes. Mainly they efflux metabolites and toxins and protect and benefit the cell function. They are characterized by the wide substrate specificity being responsible for the active secretion of several drugs some of them with therapeutic relevance in cell targets. This transport could be the origin of multidrug resistance (MDR) in some cases. The knowledge of these proteins is relevant in order to optimize cancer therapies or toxin elimination. Flavonoids are an integral component of our common diet. They are found in fruits, vegetables, teas, wine and beer. Their intake has been associated with lesser risk of some types of cancer, cardiovascular diseases, osteoporosis and age-related degenerative diseases. All these health promoting effects had increased dramatically its consumption. Flavonoids interact with ABC transporters as substrate or inhibitor compounds; in addition they could be involved in controlling the gene expression of some of them. This interaction might have important consequences affecting drug treatment itself and multidrug resistance with special relevance regarding toxin removal. This review will show and discuss the current literature regarding flavonoids and ABC transporters such as P-glycoprotein, MRPs and BCRP/ABCG2
Keywords: P-glycoprotein, Breast Cancer Resistance Protein (BCRP), Multidrug Resistance Protein 1 (MRP1), Multidrug Resistance Protein 2 (MRP2), Flavonoids, Efflux.
Resumen Los transportadores de membrana dependientes de ATP (ABC) son protenas ampliamente distribuidas y conservadas evolutivamente que participan en procesos de transporte y detoxificacin celular. De forma fundamental transportan metabolitos y txicos cuya expulsin beneficia y protege las funciones celulares. Se caracterizan por tener amplia especificidad de sustratos pudiendo transportar de forma activa compuestos de distinta naturaleza, muchos de ellos con importantes acciones teraputicas en dianas celulares. Este transporte puede ser responsable de la multirresistencia a frmacos (MDR). El conocimiento y modulacin de estas protenas es necesario para optimizar distintos tipos de terapias o para promover o controlar la eliminacin de txicos. Los flavonoides forman parte de nuestra dieta siendo especialmente abundantes en frutas, verduras y bebidas de origen vegetal. Son conocidos sus efectos beneficiosos en la salud. Su presencia se asocia a un menor riesgo de cncer, enfermedades cardiovasculares, osteoporosis y otras enfermedades degenerativas propias del envejecimiento. Estas cualidades pro-saludables, han hecho que aumente extraordinariamente su consumo. Los flavonoides interaccionan con los transportadores de tipo ABC, porque actan tanto como sustratos e inhibidores de los mismos e incluso inducen su transcripcin gnica. Esta interaccin puede tener importantes consecuencias en las terapias convencionales de los frmacos y en la presencia de resistencias, y, se puede constituir en una posible forma de modulacin de la presencia de txicos. Esta revisin intentar exponer y discutir las aportaciones que existen relativas a flavonoides y transportadores de tipo ABC, del tipo glicoprotena P, MRPs, y BCRP/ABCG2.
Palabras clave: Glicoprotena P, BCRP, MRP1, MRP2, Flavonoides, Bombas exportadoras.
lvarez de Felipe y Pulido Duarte
Transportadores ABC y flavonoides
INTRODUCCIN Transportadores de membrana dependientes de ATP (transportadores ABC) Los transportadores de membrana dependientes de ATP, tambin conocidos como transportadores ABC, debido a su denominacin en ingls: ATP-Binding Cassette, constituyen una superfamilia de protenas que actan como transportadores activos primarios o bombas exportadoras. La amplia presencia de estas protenas, prcticamente en todos los organismos vivos (Glavinas et al., 2004) con una estructura muy conservada, sugiere un papel importante en la funcin celular, as, estas protenas juegan un papel esencial en la proteccin de los organismos frente a metabolitos txicos y compuestos presentes en la dieta estando implicadas en transporte de compuestos en el intestino, barrera hemato-enceflica, placenta, etc. (Borst y Elferink, 2002, Eisenblatter y Galla, 2002). Tambin es importante considerar que estos transportadores participan, en el fenmeno de multirresistencia a frmacos antitumorales y anti-retrovirales (Mizuno et al., 2003; Sugimoto et al., 2005). La estructura bsica que define a los miembros de esta familia de protenas es la combinacin de un lugar de unin al ATP y diferentes dominios transmembrana. En mamferos, las protenas ABC funcionalmente activas contienen doce dominios transmembrana distribuidos en dos mitades homlogas, dos zonas de unin al ATP ubicadas en la parte citoplasmtica y un sitio de glicosilacin. Esta estructura bsica puede estar presente en una nica protena de una cadena polipptida (full-transporters), o en dos protenas separadas (half-transporters), en este ltimo caso los transportadores ABC necesitan una dimerizacin especfica para su funcin (Glavinas et al., 2004). La secuenciacin del genoma humano ha permitido caracterizar, hasta el momento, 48 genes ABC y ms de 50 tipos distintos de transportadores. Estos 48 genes ABC en el genoma humano se dividen en 7 subfamilias basndose en la estructura gnica, alineamiento de los amino cidos y anlisis filogentico (Dean, 2002) Las mutaciones en los transportadores ABC (Dean y Allikmets, 2001) causan o contribuyen a diferentes desrdenes de tipo Mendeliano tales como adrenoleucodistrofia, fibrosis qustica, degeneracin retiniana y alteraciones en el transporte de colesterol y cidos biliares. Estas mutaciones tambin intervienen en deswww.blacpma.org
rdenes complejos considerados como multifactoriales. Entre todos ellos y, debido a su implicacin en estrategias teraputicas antitumorales, desrdenes lipdicos, trastornos metablicos, e incluso terapia gnica, existe un grupo de tres protenas que ha sido estudiado en mayor profundidad: glicoprotena P (Pgp), las protenas asociadas a la resistencia de frmacos MRPs y la protena asociada al cncer de mama BCRP/ABCG2 . Todos ellos se localizan en la membrana plasmtica, en zonas basolaterales y apicales y son capaces de transportar compuestos estructuralmente diferentes, as como sus metabolitos y derivados conjugados de una amplia variedad de compuestos. La salida (efflux) de estos compuestos ocurre de una manera activa y dependiente de ATP y puede tener lugar en contra de gradiente de concentracin, ya que la hidrlisis del ATP proporciona la energa suficiente para este proceso. La estructura de los transportadores ABC (P-gp, MRP1, MRP2, BCRP) se muestra en la Fig. 1.
Figura 1. Estructura de los transportadores ABC.
Glicoprotena P
Ext.
BCRP
Int.
NBD NBD
N
NBD
N N
MRP 1,2
Ext.
Int.
NBD NBD
La P-gp, primer transportador de tipo ABC descubierto, codificada por el gen ABCB1, MDR1 en humanos o mdr1a y mdr1b en roedores, consta de 1280 aminocidos, con 12 dominios transmembrana y dos sitios de unin para el ATP. Una estructura similar encontramos en MRP1, MRP2, pero adems tienen una extensin que contiene 5 dominios transmembrana y un grupo amino terminal localizado extracelularmente. Esta familia de protenas est codificada por los genes ABCC1-ABCC2, respectivamente. La BCRP, codificada por el gen ABCG2, consta de 655 aminocidos y, pero a diferencia del resto de los transportadores, solo tiene 6 dominios
transmembrana con un sitio de unin para el ATP (half-transporter). La P-gp ejerce una funcin fisiolgica crucial, concretamente protege a las clulas y rganos frente a compuestos txicos y metabolitos (Glavinas et al., 2004), y tambin parece estar involucrada en el transporte de algunos fosfolpidos endgenos (Smit et al., 1993; Schinkel, 1997). Est localizada en la membrana plasmtica, especficamente en la porcin apicalluminal de las clulas epiteliales pertenecientes al borde en cepillo en el intestino, tambin en la membrana de los canalculos biliares del hepatocito, la membrana luminal de las clulas del tubo proximal del rin y en las clulas endoteliales de la barrera hematoenceflica, adems se ha localizado en ovario, testculo, clulas de la mdula espinal, etc. (Zhu, 1999). La mayora de sus sustratos tienen tres caractersticas comunes: son hidrfobos con elevados pesos moleculares y presentan un nitrgeno cargado positivamente a pH neutro (Di Pietro et al., 2002). Los MRPs son una familia formada por 12 protenas de estructura similar. Sin embargo, solamente seis son transportadores ABC (MRP1-6) (Borst et al., 2000). Esta revisin se centrar en MRP1 y MRP2 porque son los transportadores que estn ms estrechamente relacionados con los flavonoides. La caracterizacin funcional del MRP1 revela que los sustratos preferentes de este transportador son aniones orgnicos que incluyen frmacos conjugados con glutatin (GSH), glucuronatos o sulfatos. Adems participa en el transporte de aniones anfiflicos no conjugados, junto con el glutatin libre. La principal funcin fisiolgica del MRP1 es el transporte de leucotrieno C4 (LTC4) (Borst et al., 2000). Esta protena es responsable del transporte de un amplio rango de frmacos hidrofbicos y del fenmeno de multiresistencia en clulas tumorales. MRP1 presenta una amplia distribucin tisular (Borst et al., 2000, 2002) que incluye a las clulas epiteliales aunque su localizacin es basolateral. MRP2 (cMOAT) es otro miembro de la familia de los MRPs, muy relacionado con MRP1, su secuencia de amino cidos presenta hasta un 49% de homologa con MRP1 (Leslie et al., 2001). Acta como transportador de aniones orgnicos localizado en la membrana canalicular de los hepatocitos y en las membranas apicales del epitelio intestinal y renal donde puede participar en el transporte de bilirrubina y otros aniones orgnicos, y en la detoxificacin de muchos compuestos endgenos y xenobiticos. MRP1 y MRP2 pueden actuar sobre los mismos sustratos
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aunque con distintas eficiencias en el transporte, se desconocen las consecuencias clnicas de estos procesos. En pacientes con el sndrome DubinJohnson, la mutacin inherente al MRP2 provoca problemas en la secrecin hepatobiliar de conjugados anfiflicos aninicos y se produce una hiper-bilirrubinemia conjugada severa (Paulusma et al., 1997; Toh et al., 1999). Ambos, transportadores MRP1 y MRP2 se asocian con la resistencia a frmacos en las clulas tumorales (Prez-Toms, 2006). Referente a la protena asociada al cncer de mama (BCRP/ABCG2) es importante destacar que, al igual que la P-gp, esta protena protege a las clulas y rganos frente a compuesto txicos y metabolitos, est implicada en el fenmeno de multirresistencia a frmacos (Glavinas et al., 2004) y en el transporte de hormonas (Eisenblatter y Galla, 2002). En humanos, esta protena se localiza en la membrana apical de las clulas epiteliales de intestino, colon, hgado, glndula mamaria, placenta, mdula sea, cerebro, pulmn, y rin (Malieepard et al., 2001; Doyle y Ross, 2003). Los estudios realizados en ratn (m-Bcrp1) tambin demuestran una amplia distribucin (Tanaka et al., 2004). Se ha demostrado su presencia en clulas pluripotenciales hematopoyticas y en el epitelio mamario (Krishnamurthy y Schuetz; 2006; Jonker et al., 2005) Transportadores ABC y multirresistencia a frmacos El fenmeno de multirresistencia a frmacos (MDR) constituye una de las principales causas de mayor fracaso en la quimioterapia del cncer. Los transportadores de tipo ABC se encuentran presentes en tejidos normales del organismo humano y en los tumores que de ellos se derivan. La resistencia a frmacos es un fenmeno asociado con una disminucin en la acumulacin intracelular de frmacos, hecho atribuido a la expresin de transportadores, principalmente del tipo ABC en la membrana celular (Prez-Toms, 2006; Calcagno et al., 2007). Adems, la sobre-expresin de estos transportadores da lugar a fenmenos de multiresistencia a un amplio espectro de agentes antitumorales (Kuo, 2007). En el caso de los tumores cerebrales la quimioterapia es menos efectiva que en otros tipos de cncer, debido a que la llegada de molculas al cerebro es finamente regulada por las clulas endoteliales de la microvasculatura cerebral que conforman la barrera hematoenceflica (BHE). Los transportadores ABC tambin forman parte de la
BHE al expresarse en la membrana apical de las clulas endoteliales (Rao et al., 1999; Eisenblatter y Galla, 2002; Potschka et al., 2003) De los transportadores implicados en la MDR el ms estudiado es P-gp, responsable de la resistencia a diversos compuestos naturales como alcaloides de la vinca, taxanos, epipodofilotoxinas y antraciclinas, entre otros. Se ha detectado P-gp en varias neoplasias hematolgicas y slidas (Godstein, 1996) y puede aparecer de novo o ser inducida por el tratamiento con quimioterapia, hormonoterapia o radioterapia (Trock et al., 1997). BCRP fue aislada inicialmente en lneas celulares multirresistentes seleccionadas por exposicin a la mitoxantrona, se relaciona con la elevada resistencia a las antraciclinas (mitoxantrona, daunorrubicina, doxorrubicina) y a los inhibidores de la topoisomerasa I, de hecho es un eficiente transportador de topotecan, pero no parece afectar a los taxanos, alcaloides de la vinca ni al cisplatino (Maliepaard et al., 1999). Se ha demostrado expresin de BCRP en leucemias y tumores slidos, a veces asociada con recadas o recidivas, o como factor pronstico de supervivencia (Doyle y Ross, 2003; Benderra et al., 2004) Existen diversos tipos de moduladores que pueden revertir la MDR in vitro tales como bloqueadores de los canales de calcio, antagonistas de la calmodulina, pptidos hidrofbicos, inhibidores de las protena cinasas, antibiticos derivados hormonales y flavonoides (Schinkel y Jonker 2003; Sugimoto et al., 2005; Choi 2005; Perez-Toms, 2006). La estructura de la mayora de estos moduladores presenta un tomo de nitrgeno y dos anillos planares aromticos. Muchos de estos moduladores son transportados por transportadores ABC (P-gp y otros). La inhibicin se consigue mediante mecanismos de competicin en los sitios de unin, por lo que son necesarias elevadas concentraciones de los inhibidores. Algunos de estos compuestos a estas concentraciones pueden provocar efectos nocivos in vivo, tales como cardiotoxicidad (verapamilo) o alteraciones inmunolgicas (ciclosporina). Por lo tanto, uno de los mayores retos en la actualidad en este campo es la bsqueda de moduladores seguros que reviertan la MDR. Los flavonoides presentan una serie de caractersticas estructurales y funcionales, que sern descritas a continuacin, que les permiten ser unos excelentes moduladores de los ABC y por lo tanto bloquear o revertir la MDR. Adems, la seguridad de estos compuestos est ampliamente comprobada.
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Flavonoides Los flavonoides constituyen un amplio grupo de productos naturales presentes en los vegetales, y de los que se han descrito ms de 6500 compuestos diferentes. Su estructura est caracterizada por la presencia de un anillo de cromano al que se une un anillo aromtico en posicin 2, 3 o 4. La clasificacin de los flavonoides est basada en las diferentes sustituciones y en el estado de oxidacin del anillo C, por lo que se consideran las siguientes subclases: flavonas, flavonoles, flavanonas, flavanoles, isoflavonas y chalconas (Fig. 2). Los flavonoides forman parte de nuestra dieta y son especialmente abundantes en frutas, verduras y bebidas de origen vegetal tales como el vino y el t. Por ejemplo, el mosto (zumo de uva) presenta un contenido total de flavonoides de 200850 mg/L de los cuales de la naringina es el ms abundante (145638 mg/L) (Ross et al., 2000), mientras que el zumo de naranja contiene hesperidina en una concentracin de 200450 mg/L (Erlund et al., 2001; Manach et al., 2003). Si consideramos que 100 g de soja contienen 100200 mg de isoflavonas, (genistena, daidzena, glicitena, y sus correspondientes glucsidos), se calcula que en un da la asimilacin de flavonoides procedentes de la dieta en un adulto, pueden ser 200300 mg (Hollman et al., 1999). Setchell et al., (2001) han descrito que mujeres premenopusicas sanas presentan concentraciones plasmticas de genistena de 1 M en el caso de la ingesta diaria de 50 mg de genistena. Existen excelentes revisiones centradas en diversos aspectos de los flavonoides tales como nutricionales, mdicos e interacciones con frmacos (Havsteen, 2002; Morris y Zang, 2006; Moon et al., 2006), aunque los procesos sobre absorcin y biodisponibilidad de flavonoides, todava no se conocen en profundidad (Hendrich, 2002). La mayora de los flavonoides se presentan en forma de glucsidos, pero su carcter hidrofbico hace que se absorban en el tracto gastrointestinal, como agliconas. Los glucsidos son hidrolizados por las glucosidasas presentes en la mucosa o secretadas por la flora presente en el colon o en el hgado (Day et al., 1998). El tiempo necesario para obtener concentraciones mximas plasmticas tras administraciones orales de genistina o daidzina (que son glucsidos de genistena y daidzena) es mucho mayor que el obtenido cuando se administran las correspondientes agliconas (9 h vs. 2 h) sin detectarse concentraciones de glucsidos en plasma (Setchell et al., 2001). En el caso de quercetina ocurre el mismo proceso, este compuesto est presente en la
cebolla como glucsido. Estos procesos no estn totalmente dilucidados ya que existen trabajos que muestran que la biodisponibilidad de los glucsidos procedentes de la cebolla, fu significativamente mayor que la de sus agliconas (Hollman et al., 1995, 1996), lo que sugiere que tambin los glucsidos son absorbidos en cierta medida. Los derivados glucsidos de la quercetina son sustratos del transportador de glucosa dependiente de Na (SGLT-1) (Wolffram et al., 2002), lo que implica la absorcin de estos glucsidos mediada por SGLT-1.
Figura 2. Estructura de los flavonoides
Los flavonoides sufren extensos procesos de glucuronidacin y sulfacin intestinal y heptica (Chen et al., 2003; Liu and Hu, 2002, Wang et al., 2006) y son los derivados conjugados las especies predominantes en el plasma tras administracin oral. El compuesto original (parental) esta presente en la circulacin en un rango de concentracin muy bajo aunque su presencia es persistente debido, en la mayora de las casos, a la circulacin enteroheptica de los metabolitos conjugados. (Setchell et al., 2001; Chen et al., 2005). Cuando se administran de forma endovenosa, las concentraciones de estos compuestos
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alcanzan concentraciones superiores a 100 M (Ferry et al., 1996). Los flavonoides pueden ser metabolizados por el citocromo P450 como se ha demostrado en estudios in vitro mediante microsomas hepticos humanos y de ratas (Nielsen et al., 1998; Hu et al., 2003; Moon et al., 2006), aunque se desconoce la importancia de dichos procesos in vivo. Estos compuestos, considerados como fitoestrgenos, despiertan mucho inters no solo entre la comunidad cientfica, sino tambin entre los consumidores debido a sus propiedades antioxidantes, anticarcinognicas, antivirales, anti-inflamatorias y capacidad pro y anti-estrognica (Middleton et al., 2000; Havsteen, 2002). Son numerosos los trabajos que relacionan la presencia de flavonoides con un menor riesgo de cncer, enfermedad cardiovascular, osteoporosis y enfermedades degenerativas (Lee et al., 1991; Potter et al., 1998; Middleton et al., 2000; Havsteen, 2002; Huxley y Neil, 2003). El binomio -descenso de cncer/presencia de flavonoides- se demuestra en estudios epidemiolgicos. En mujeres orientales, se ha asociado la mayor presencia de soja en la alimentacin con una menor incidencia del cncer de mama (Lee et al., 1991, Messina et al. 1994). Linseisen et al. (2004) comprueban como una mayor presencia de genistena y daidzena en dietas occidentales (Alemania) tambin puede reducir el cncer de mama. Los estudios con animales han demostrado que los flavonoides son capaces de prevenir el crecimiento y desarrollo de tumores inducidos por agentes qumicos o transplantados quirrgicamente. (Bchler et al., 2003; Kohno et al., 2002; Rice et al., 2002). Las propiedades antitumorales de los flavonoides se deben a sus distintas posibilidades de interaccin con dianas celulares (cellular targets) gracias a su estructura polifenlica y a los diversos sustituyentes (Williams et al., 2004). Los polifenoles tienen capacidad de absorber la radiacin UV por lo que se consideran anti-mutagnicos. Asimismo los flavonoides, debido a su estructura, presentan capacidad anti-estrognica al unirse a los receptores estrognicos responsables de la proliferacin celular por lo que son capaces de inhibir a diversas cinasas y ATPasas (Middleton et al., 2000; Havsteen, 2002). Los flavonoides presentan capacidad antioxidante y pueden controlar diversos factores angiognicos, as como la sntesis de estrgenos mediante la actividad aromatasa (Williams et al., 2004). Ellos actan sobre enzimas requeridas para la activacin de pro-carcingenos e intervienen en la detoxificacin de agentes cancergenos (Kellis and
Vickery, 1984; Moon et al., 2006). Estas propiedades han permitido utilizar con xito algunos derivados sintticos de flavonoides, del tipo del flavopiridol, en terapias antitumorales (Siegenthaler et al., 1992; Aklilu et al., 2003). Por otra parte, el consumo de este tipo de compuestos tambin tiene a su favor su escasa toxicidad (Middleton et al., 2000), por lo que se han descrito resultados favorables en modelos animales tras administraciones de dosis elevadas (superiores a 500 mg/kg). La concentracin de flavonoides en intestino puede ser alta tras la ingestin de algunos alimentos, especialmente si estn suplementados, y causar interacciones con frmacos. Se estima que un 10% de la poblacin puede consumir este tipo de productos y aumentar hasta un 30-70% en las personas con enfermedades especficas (Duggan et al., 2001; Ni et al., 2002). En estos grupos, el consumo podra ser an mayor, lo que aumentara la probabilidad de interaccin de los flavonoides con los frmacos prescritos en cada terapia. Interaccin de frmacos con flavonoides mediados por transportadores ABC: efectos sobre la biodisponibilidad oral Los frmacos que son sustratos de dichas protenas pueden sufrir procesos de secrecin activa en hgado, rin, y, si son administrados oralmente, sufrir una menor captacin intestinal (Sugimoto et al. 2005). La excrecin intestinal de frmacos es un mecanismo importante de eliminacin, que afecta a la biodisponibilidad de una gran variedad de compuestos. La diversidad que existe en estos mecanismos no puede ser explicada a travs de un nico sistema de eliminacin y de la accin unilateral (Prez-Toms 2006; Calcagno et al., 2007). Las interacciones entre protenas exportadoras de frmacos y los sistemas de metabolismo intestinal aaden una nueva dimensin a la ecuacin que describe la biodisponibilidad (Schinkel y Jonker 2003; Mizuno et al., 2003). Los procesos de captacin, metabolismo y la eliminacin intestinal de frmacos tras una administracin oral, mediados por transportadores ABC, tienen consecuencias directas en la reduccin de la absorcin, que puede ser una causa generalmente no reconocida de baja biodisponibilidad oral (Taipalensuu et al., 2001), tambin intervienen en procesos de absorcin dependiente de la dosis y en las interacciones entre frmacos, que afectan a la tasa de absorcin (Mizuno et al., 2003; Calcagno et al., 2007). En este sentido, diversos estudios farmacocinticos (Soldner et al.,
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1999; Becquemont et al., 2001) y en cultivos celulares (Rodrguez-Proteau et al., 2006) describen modificaciones de la biodisponibilidad de distintos frmacos al coadministrar flavonoides. Morris y Zhang (2006), sealan especficamente en su revisin que la co-administracin de mosto, que presenta cantidades elevadas de naringina, aumenta significativamente la biodisponibilidad oral de la felodipina (Bailey et al., 1993), nimodipina (Fuhr et al., 1998), ciclosporina (Ducharme et al., 1995), saquinavir (Kupferschmidt et al., 1998) y decrece la biodisponibilidad oral de fexofenadine (Dresser et al., 2002) en humanos. La silimarina aumenta la eliminacin del metronidazol y de su principal metabolito el hidroxi-metronidazol (Rajnarayana et al., 2004). En animales, diversos estudios muestran la interaccion de frmacos con los flavonoides que afectan la biodisponibilidad as como la eliminacin biliar. Por lo que la naringina modifica la biodisponilidad oral de la quinina (Zhang et al., 2000), la crisina aumenta la biodisponibilidad de la nitrofurantoina (Wang y Morris, 2007), la baicalina y su aglicona la baicaleina incrementan la biodisponibilidad de la ciclosporina en ratas (Lai et al., 2004), flavona y quercetina aumentan las concentraciones de paclitaxel tras administracin oral en ratas (Choi et al., 2004a,b). Por otra parte, felamurina y quercetina disminuyen la biodisposicin oral de la ciclosporina en cerdos y ratas (Chen et al., 2002; Hsiu et al., 2002). Transcripcin de transportadores ABC (BCRP) y flavonoides Otro aspecto que merece ser revisado son los recientes hallazgos que relacionan factores de transcripcin de determinados transportadores con algunos flavonoides (Wang, 2007). Ebert et al. (2005) mostraron que la activacin del receptor AhR podra inducir la expresin de BCRP. Este factor de transcripcin que regula la induccin de la familia de los citocromos CYP1, tambin induce algn miembro de los MRPs pero no induce a la P-gp (Colombo et al., 2003). Considerando que tanto BCRP como P-gp se localizan en la membrana apical de clulas epiteliales y que comparten un amplio rango de sustratos, el que los dos transportadores sean regulados por diferentes mecanismos de transcripcin, puede ser un mecanismo compensatorio para la defensa celular, en el caso de que alguno pueda ser bloqueado. Por otra parte, se ha demostrado que los flavonoides pueden inducir la expresin gnica a travs de diversos factores de transcripcin, lo que incluye el AhR (Hankinson,
1995). Flavonoides tales como chrisina, flavona, quercetina y genistena actan como agonistas de AhR. La relacin entre la induccin de BCRP, mediada por estos compuestos y su papel en la eliminacin de txicos, suscita inters en la comunidad cientfica. Ebert et al. (2007) han unido estos importantes aspectos al demostrar que el transporte mediado por BCRP del benzopireno y de sus metabolitos de fase II (sulfoderivados) es inducido por fitoestrgenos. El benzopireno es un agente txico que acta como carcingeno celular. Este hallazgo subraya la importancia de los transportadores ABC en la disposicin de los txicos y de forma especial el papel de los fitoqumicos como posibles protectores de las acciones inducidas por benzopirenos y sus metabolitos pro-mutagnicos. Interaccin de la glicoprotena P (P-gp) con flavonoides P-gp presenta una distribucin tisular muy relacionada con la disposicin de xenobiticos al estar localizada en la membrana apical de las clulas epiteliales de intestino, hgado, rin y clulas endoteliales de la barrera hematoencefalica lo que influye notablemente en los procesos de absorcin, distribucin y eliminacin de frmacos, ya que limita la biodisponibilidad oral y la distribucin cerebral, por lo que facilita la excrecin biliar y la eliminacin renal de los frmacos (Cordon-Cardo et al., 1989; Thiebaut et al., 1987). Esto ha sido demostrado tanto en estudios clnicos como en modelos animales, especialmente los que usan animales knockout (mdr1 -/-) para dicha protena (Schinkel et al., 1995, 1997; Sparreboom et al., 1997). Tambin es importante el amplio espectro de los sustratos de P-gp. Son sustratos de P-gp los frmacos antitumorales (antraciclinas, alcaloides de la vinca, epipodopilotoxinas y taxol); los cardiovasculares como digoxina y quinidina; los anti-retrovirales como saquinavir, indinavir y ritonavir; los inmunosupresores como ciclosporina; los antibiticos como actinomycin D; los esteroides como cortisol, aldosterona y dexametasona (Mizuno et al., 2003) y las citocinas como IL-2, IL-4, IFN- (Drach et al., 1996). Ambos factores, distribucin y espectro de sustratos, inciden en el que sea el transportador ms estudiado, implicado en la multirresistencia a frmacos. Como se ha comentado, se ha detectado P-gp en varias neoplasias hematolgicas y slidas (Godstein, 1996). En las clulas neoplsicas, la sobre-expresin de P-gp puede contribuir a la quimiorresistencia en
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diversos tipos de cncer y leucemias, lo que explica la aparicin del fenotipo MDR despus de una recidiva (Trock et al., 1997). Existen excelentes revisiones acerca de la interaccin de la P-gp con flavonoides sobre todo centradas en procesos de MDR (Di Pietro et al., 2002; Zhang y Morris, 2003). Los primeros flavonoides descritos que interaccionaron con P-gp fueron los flavonoles kaempferol, galangina y quercetina que estimulaban el transporte de 7,12-dimetilbenz(a)antraceno, un carcingeno que induce tumores de mama (Phang et al., 1993) y tambin estimulaban el transporte de adriamicina en clulas HCT-15 procedentes de cncer de colon (Critchfield et al., 1994). Sin embargo, otros trabajos muestran resultados opuestos, la quercetina inhibe la salida de rodamina 123 en cultivos MCF-7 de cncer de mama, y de ese modo el fenotipo MDR (Scambia et al., 1994) y en hepatocitos que sobre-expresan P-gp, los flavonoles son capaces de inhibir la salida de los frmacos (Chieli et al., 1995). La ventaja que suponen las diversas posibilidades de interaccin de este tipo de molculas, debido a su particular estructura, con las dianas celulares, es un inconveniente a la hora de poder interpretar estas acciones antagnicas. En la interaccin transportador-flavonoide pueden estar implicados diversos sitios de unin (Choi, 2005). Shapiro y Ling (1997) han propuesto la existencia en P-gp de diferentes lugares de unin para frmacos con efecto de cooperacin positiva. La accin del flavonoide depende del lugar de unin del frmaco. La complejidad de esta interaccin ha sido tambin puesta de manifiesto por Mitsunaga et al. (2000), quienes demostraron que quercetina y kaempferol, a 10 M, disminuan la acumulacin de vincristina en clulas endoteliales de cerebro de ratn (MBEC4) que expresaban P-gp, mientras que a 50 M, la aumentaban. Resultados similares se obtenan in vivo en ratones, medidos a travs del ratio de concentraciones cerebro/plasma, mediante la administracin de dosis de quercetina de 0,1 mg/kg y 1 mg/kg, respectivamente. Los autores explican que concentraciones bajas de quercetina estimulan indirectamente la actividad P-gp a travs de la fosforilacin. Aunque existen resultados controvertidos, Morris y Zhang (2006) en su revisin consideran que la mayora de los flavonoides (agliconas) tienen un efecto inhibidor en el transporte mediado por P-gp. Estos flavonoides incluyen genistena, biochanina A, morina, floretina, silimarina, crisina, flavona, hesperetina, naringenina, 3,3,4,5,6,7,8-heptametoxiflavo-
na, nobiletina, tangeretina y t verde; y los polifenoles epicatequina-galato, catequina-galato y epigalocatequina (Castro and Altenberg, 1997; Jodoin et al., 2002; Mitsunaga et al., 2000; Zhang y Morris, 2003). Las interacciones de los flavonoides (glucsidos) tales como naringina, hesperidina y rutina, con P-gp es menor (Mitsunaga et al., 2000). Los mecanismos subyacentes a la interaccin entre P-gp y flavonoides radican en la posibilidad de unin de los flavonoides a mltiples lugares en el transportador. Genistena, epicatequina-galato, catequina-galato, epigalocatequina y silimarina pueden unirse directamente al sitio de unin de los sustratos del transportador e impedir la unin de sustratos fotoactivos de P-gp (Castro and Altenberg, 1997; Jodoin et al., 2002; Zhang and Morris, 2003). Conseil et al. (1998) establecieron que la interaccin de los flavonoides era bifuncional (sitio de unin al ATP y sitio de unin de los esteroides), ambos dentro del dominio citoslico de la P-gp (Fig. 3).
Figura 3. Modelo de interaccin propuesto para los flavonoides y P-gp
ATP
E ST E R O I DE S
F L A V O N O ID E S
El estudio realizado por Boumendjel et al. (2002) mediante medidas basadas en la unin directa de los flavonoides con el dominio C- terminal del nucletido de P-gp (NBD2) puso de manifiesto las caractersticas estructurales de los flavonoides que permitan una mayor interaccin con P-gp, lo que consideraba la posicin de grupos hidroxilo as como de los anillos aromticos. Los flavonoides prenilados se presentan como potentes inhibidores de la P-gp ya que son capaces de inhibir la unin y el transporte del sustrato as como la hidrlisis del ATP (Di Pietro et al., 2002). La concentracin necesaria para que los flavonoides puedan modular a la P-gp parece ser alrededor
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de 10 M o superior y se puede obtener tras la ingesta de alimentos. La interaccin se realiza a nivel intestinal con las agliconas. La presencia sistmica de ellas es menor y, adicionalmente, los principales metabolitos de los flavonoides (conjugados glucurnidos y sulfatos) no interaccionan con P-gp debido a que son aniones orgnicos. Los sistemas de biotransformacin intestinales relacionados con el citocromo P450 (CYP) comparten sustratos e inhibidores con P-gp, incluso pueden ser regulados por compuestos similares (Evans 2000; Moon et al., 2006). Esto enfatiza la cuestin de que si algunos componentes de la dieta pueden actuar sobre ambos sistemas, la separacin de ambos efectos puede ser difcil de evaluar. Por ejemplo el mosto, que contiene elevadas cantidades de flavonoides, aumenta la biodisponibilidad de felodipina, nimodipina, ciclosporina y saquinavir (Bailey et al., 1993; Ducharme et al., 1995; Fuhr et al., 1998; Kupferschmidt et al., 1998); todos estos compuestos son sustratos de P-gp y de CYP3A4. La inhibicin de P-gp puede contribuir a la interaccin frmacocintica, pero la modulacin a travs del transportador no puede ser establecida. Muchos de los estudios in vivo confirman las interacciones de P-gp con flavonoides obtenidas in vitro. Choi et al. (2004 a,b) demostraron que flavonas y quercetina incrementan la biodisponibilidad del paclitaxel (sustrato de CYP3A4 y P-gp) en ratas de manera dependiente de la dosis. La baicalena y su aglicona pueden aumentar la biodisponibilidad oral de la ciclosporina en ratas (Lai et al., 2004). Sin embargo, esta correlacin in vitro e in vivo no se observ cuando el flavonoide quercetina se administr con ciclosporina A, (conocido sustrato de P-gp y de CYP3A). As, los estudios de Chen et al. (2002) y Hsiu et al. (2002) mostraron que la biodisponibilidad de la ciclosporina, en cerdos y/o ratas, disminuye significativamente con la co-administracin de los flavonoides. Finalmente, la quercetina aumenta la biodisponibilidad oral de la digoxina en cerdos con resultados txicos (Wang et al., 2004). Interaccin de MRP1 con flavonoides MRP1 es un transportador de aniones orgnicos que incluyen frmacos conjugados con glutatin (GSH), glucuronatos o sulfatos. Adems, participa en el transporte de aniones anfiflicos no conjugados, junto con el glutatin libre (GSH). Tiene una amplia distribucin tisular, que incluye la membrana basolateral del plexo coroideo de las clulas epiteliales (Rao et al., 1999) y contribuye a las funciones de
defensa de la barrera hematoenceflica al evitar el paso de aniones anfiflicos y frmacos antitumorales hacia el lquido cefalorraqudeo (Wijnholds et al., 2000). Su expresin en los hepatocitos y en intestino es menor que la P-gp, por lo que su contribucin total a la disposicin de frmacos es limitada. Versantvoort et al. (1994, 1993) fueron los primeros que demostraron que flavonoides como genistena, biochanina A, apigenina y quercetina actuaban como inhibidores del transporte mediado por MRP1. Los autores proponen que la genistena se utilice como prueba para distinguir el transporte mediado por P-gp en procesos MDR ya que no puede inhibir al transportador por debajo de 200 M (Versantvoort et al., 1994, 1993). En la actualidad se conocen numerosos flavonoides capaces de inhibir el transporte mediado por MRP1 (Leslie et al., 2001; Nguyen et al., 2003; van Zanden et al., 2005). Muchos de estos compuestos, tales como diosmetina, crisoeriol y tamarixetina, presentan valores de concentracin inhibitoria 50 (CI50) por debajo de 50 M, incluso algunos presentan valores por debajo de 10 M (van Zanden et al., 2005). La interaccin de ciertos derivados conjugados con MRP1 ha sido demostrada (Deeley et al., 2006) por lo que la interaccin de los metabolitos de los flavonoides (conjugados glucurnidos y sulfatos) con MRP1 no puede descartarse del mismo modo que existe con otros transportadores ABC (Zhang et al., 2007). Los estudios de Leslie et al. (2001) se realizaron en vesculas, en la que solo esta presente el compuesto en forma de aglicona y los autores establecieron que los glucsidos como genistina y naringina presentaron menores inhibiciones para la actividad MRP1. Los sitios de interaccin en el flavonoide, de la misma forma que en P-gp, pueden incluir dominios NBD y/o sitios de unin para el sustrato (Leslie et al., 2001; Trompier et al., 2003). van Zanden et al. (2004, 2005) han estudiado las caractersticas estructurales necesarias de los flavonoides para su interaccin con MRP1 y establecen como requisitos, adems de la estructura planar debida a la presencia de un doble enlace 2,3 en el anillo C, diversas sustituciones de grupos metoxi e hidroxi en el compuesto. Flavonoides como quercetina, miricetina, apigenina y naringenina son capaces de estimular el transporte de GSH mediado por MRP1 (Leslie et al., 2001, 2003), por lo que se establece un posible mecanismo de control del MRP1 a travs del control de la disponibilidad celular de GSH. Nguyen et al. (2003) han demostrado en clulas Panc-1 que la prewww.blacpma.org
incubacin de las clulas con flavonoides disminuye las concentraciones intracelulares de GSH. Las interacciones in vivo de los flavonoides con MRP1 no estn totalmente establecidas, aunque se sabe que pueden alcanzar en el intestino CI50 tras la ingestin oral, su expresin es limitada y previsiblemente la inhibicin sistmica puede ser de poca importancia (Setchell et al., 2001; Hendrich, 2002). Por otra parte, si existe interaccin con los conjugados, estos si que pueden alcanzar elevadas concentraciones a nivel sistmico y de esta forma alterar la distribucin de frmacos y sustratos de MRP1. Interaccin de MRP2 con flavonoides MRP2 se localiza en la membrana apical de clulas epiteliales en hgado, intestino y en los tbulos proximales del rin (Buchler et al., 1996; Schaub et al., 1999), por lo que puede alterar la biodisponibilidad oral y facilitar la excrecin biliar de sus sustratos. De la misma forma que P-gp se puede presentar en la superficie luminal de las clulas endoteliales de los capilares cerebrales, por lo que contribuye a la funcin de la barrera hematoenceflica (Potschka et al., 2003). A pesar de las similitudes estructurales y de sustratos entre MRP1 y MRP2, los flavonoides pueden inhibir diferencialmente a los dos transportadores. La quercetina que acta como inhibidor del sustrato de MRP1 dinitrofenil glucurnido, no inhibe el citado transporte en el caso de MRP2 (van Zanden et al., 2004); sin embargo, se ha publicado recientemente que los principales metabolitos de fase II de la quercetina son potentes inhibidores tanto de MRP1 como de MRP2 (van Zanden et al., 2007), as como de la interaccin posicional de ismeros de la quercetina con MRP2 (Williamson et al., 2007). Un estudio de van Zanden et al. (2005) evala la capacidad inhibitoria de una serie de flavonoides a travs de la determinacin del valor de CI50 en clulas transfectadas (MCDKII) que expresaban ambos transportadores MRP1 y MRP2. Los mejores inhibidores fueron miricetina y robinetina, pero MRP2 mostr una mayor selectividad para la inhibicin que MRP1. Para MRP2 los valores de CI50 15,0 y 22,2 M para estos flavonoides, respectivamente (van Zanden et al., 2005). Si se considera que miricetina y robinetina se administran en dosis de 100 mg como suplementos dietticos, se podran alcanzar en los lquidos corporales concentraciones inhibitorias para el transporte mediado por MRP2. El transportador MRP2 tambin puede transportar glucsidos aunque con menor
afinidad (Vaidyanathan y Walle, 2001, 2003;), as como derivados glucurnidos y sulfatos (Walle et al., 1999; van Zanden et al., 2007). Todos estos resultados apoyan la posibilidad de que la suplementacin con flavonoides puede alterar la excrecin biliar o la eliminacin renal de los sustratos de MRP2. Es especialmente relevante la hiper-bilirrubinemia inducida por la presencia de flavopiridol y sus derivados conjugados ya que la excrecin de la bilirrubina conjugada, sustrato de MRP2, se ve gravemente alterada (Jager et al., 2003). Interaccin de BCRP con flavonoides De la familia de transportadores ABC posiblemente la interaccin de BCRP con los flavonoides sea la ms estudiada (Cooray et al., 2004; Imai et al., 2004; Zhang et al., 2004a; Zhang et al., 2004b). La BCRP que presenta una amplia distribucin tisular, confiere resistencia a diversos agentes antitumorales tales como (SN-38), mitoxantrona, topotecan y, transporta mltiples compuestos. Krishnamurthy y Schuetz (2006) sealan que son sustratos de BCRP compuestos tales como flavopiridol, daunomicina, etc. entre antitumorales; lamivudina y zidovudina como derivados nuclesidos; compuestos naturales como los flavonoides, los estrgenos sulfato, las porfirinas; compuestos fluorescentes como rodamina 123 y mitoxantrona, y compuestos como la futremorgrina C y sus derivados que adems actan como inhibidores de BCRP. El importante papel de BCRP en la disposicin de frmacos se pone de manifiesto en estudios con ratones knockout (Bcrp1 -/-) as, tras la administracin oral del antitumoral topotecan el rea bajo la curva de concentracin plasmtica (AUC) fue seis veces mayor en los ratones Bcrp1 -/- que en los silvestres (Jonker et al., 2000). La interaccin de BCRP con derivados estrognicos esta demostrada ya que derivados sulfato de los estrgenos son sustratos naturales del transportador (Imai et al., 2003). La co-administracin de flavonoides con sustratos de la BCRP puede alterar la farmacocintica y consecuentemente incrementar la efectividad o la toxicidad de los sustratos del transportador (Imai et al., 2004; Wang y Morris 2007). Experimentos in vitro, con mitoxantrona como sustrato de BCRP muestran que una amplia mayora de este tipo de compuestos, que incluye genistena y daidzena, son capaces de interactuar con BCRP y actuar tanto como sustratos y como inhibidores (Zhang y Morris, 2004a; Katayama et al., 2007). Imai et al. (2004) destacan que la genistena, un miembro de las isoflavonas, mostr un significativo efecto
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inhibitorio de la BCRP, junto con la daidzena. Asimismo, se han obtenido efectos aditivos de mltiples flavonoides sobre la inhibicin de la BCRP (Zhang et al., 2004b). Estos autores sugieren que la prediccin de las interacciones frmaco-alimento mediadas por la BCRP, deberan tambin tener en consideracin la presencia de mltiples flavonoides y servir este resultado de base para la utilizacin de ccteles de flavonoides en la inhibicin de la BCRP. Katayama et al. (2007) establecen una serie de caractersticas estructurales que permiten una mayor interaccin de BCRP con los flavonoides y demuestran que, de 32 flavonoides estudiados, el 3,4,7trimetoxiflavona es el que presenta mayor inhibicin frente a BCRP. Los autores alertan sobre la necesidad de controlar la dieta en los tratamientos antitumorales. Diversos estudios abordan la posibilidad de utilizar flavonoides en animales de experimentacin con el objetivo de modular la farmacocintica de sustratos de BCRP. En este sentido Zhang et al. (2005) evaluaron la influencia de dos flavonoides, chrisina y 7,8 benzoflavona (BF), inhibidores de la BCRP in vitro, en la farmacocintica del topotecan en ratas y ratones knock out deficientes en P-gp. Los resultados del estudio mostraron que ninguno de los dos flavonoides variaba significativamente la farmacocintica del topotecan. Los autores sealan que la falta de correspondencia entre los resultados obtenidos in vitro e in vivo puede deberse a que la actividad inhibitoria de estos flavonoides no es lo suficientemente fuerte para inhibir la BCRP en estas especies. Los resultados obtenidos por Zhang et al (2005) mostraron que la esperada correlacin de los resultados in vitro con los obtenidos in vivo no se obtuvo en el caso del topotecan utilizando inhibidores especficos de BCRP. Hasta la fecha se puede considerar que BCRP es el principal transportador ABC presente en glndula mamaria, adems se ha demostrado que su expresin aumenta durante la lactacin en el hombre y distintas especies animales, lo que incluye animales de experimentacin y rumiantes (Jonker et al., 2005; Pulido et al., 2006). Su implicacin en el transporte de txicos hacia la leche, como el PhIP, agentes quimioteraputicos tales como topotecan, cimetidina, fluoroquinolonas y vitaminas (Jonker et al 2005; Pulido et al 2006; Merino et al., 2006; van Herwaarden et al., 2007) apoya la importancia de estudiar su posible regulacin a travs de inhibidores o inductores con el objetivo de modular la excrecin de frmacos en la leche. A este respecto, en estudios
realizados por nuestro grupo se demostr in vitro que los flavonoides genistena y daidzena eran capaces de inhibir el transporte de enrofloxacina, una fluoroquinolona de amplio espectro utilizada en teraputica veterinaria, en cultivos celulares que sobre-expresan el transportador BCRP/ABCG2. Posteriormente, los datos farmacocinticos mostraron una reduccin significativa en la presencia del frmaco en la leche de las ovejas cuando se coadministraba enrofloxacina (2,5 mg/kg iv.) con genistena (va im. a dosis de 0,8 mg/kg) (Pulido et al., 2006). Considerando que las isoflavonas, procedentes del alimento, en la leche de vaca se encuentran en concentraciones en el rango de g/L (Antignac et al., 2003), esto podra significar una alta probabilidad de interaccin de los flavonoides con la BCRP a nivel de glndula mamaria y, por lo tanto, una posibilidad de modulacin de la excrecin de frmacos a leche. Finalmente, la interaccin de BCRP con flavonoides no slo se relaciona con el control y modulacin de frmacos y toxinas a nivel sistmico ya que recientemente se ha demostrado que BCRP limita la biodisponibilidad oral de flavonoides como genistena y daidzena in vivo as como la distribucin de los fitoestrgenos en cerebro, testculos, epiddimo y feto (Enokizono et al., 2007). CONCLUSIONES Son muchas las expectativas que se pueden derivar tras el conocimiento y la demostracin de la interaccin de los flavonoides con transportadores de tipo ABC. El poder disponer de mecanismos que eviten la multirresistencia a frmacos, sobre todo en los tratamientos antitumorales, constituye una buena herramienta para el aumento de la eficacia de los frmacos y abre nuevas y esperanzadoras posibilidades teraputicas. Sin embargo, no se puede obviar que la complejidad de los procesos de metabolismo y eliminacin de sustratos y flavonoides interfieren en muchas ocasiones con la funcin de los transportadores ABC, de tal forma que se dificulta la interpretacin de los resultados as como el establecimiento de pautas teraputicas. Tampoco se debe dejar de considerar que cada vez son mayores las evidencias que relacionan la variabilidad gentica y los polimorfismos existentes en los transportadores ABC con la distinta efectividad de los tratamientos. El inters de estos estudios tambin se puede centrar en la importancia que tienen los residuos en la leche destinada a los consumidores como prioridad en la salud con especial inters en la deteccin y el
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control de frmacos, ya que existe la posibilidad de la inhibicin del transporte mediada por isoflavonas cuando se administran frmacos cuya secrecin a la leche est mediada por BCRP. Finalmente, el que los fitoestrgenos puedan inducir factores de transcripcin implicados en la detoxificacin y en la funcin de determinados transportadores de tipo ABC para la eliminacin de carcingenos, constituye un factor adicional que incrementa el uso potencial de estos compuestos y subraya la presencia de estos como parte de una dieta equilibrada que contribuye a mejorar nuestra salud. AGRADECIMIENTOS
Este trabajo forma parte de la investigacin desarrollada dentro del proyecto AGL:2006-13186 del Ministerio de Educacin y Ciencia, Espaa.
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An overview on safety issues of interactions between traditional herbal medicines and pharmaceutical medicines
[Una apreciacin global sobre la seguridad de las interacciones entre las medicinas herbarias tradicionales y los frmacos]
Kelvin CHAN School of Applied Sciences, University of Wolverhampton,Wolverhampton WV1 1LY, United Kingdom
*Contact: e-mail: prof.kchan@wlv.ac.uk Submitted March 17, 2008; Accepted May 20, 2008; Published Online 15 October 2008
Abstract
The increasing popularity world-wide of using herbal medicinal materials (HMM) from ethnic traditional medicine such as the widely used Chinese materia medica (CMM) or other ethnic herbal medicines and related proprietary health products (PHP), functional food and prescription herbal medicines has raised concerns over their concomitant use with pharmaceutical medicines (PHARMED) and the consequential adverse effects. In most cases the alleged causes of adverse effects are linked with herbal substances, although the authoritive information on the interactions between HMM/PHP and PHARMED is not plentiful in the literature. There is an urgent need for such a data base. In the 21st century, the public are more informed, from the Internet, about health and medical products and become more knowledgeable about matters relating to their health conditions and well-being in curing and preventing illnesses. They often self-medicate themselves with various health products as well as those over-the-counter (OTC) PHARMED. Some of theses products may have doubtful quality control and contain harmful additives or unchecked ingredients. The future professionals in health and medical care should be knowledgeable or aware of what their patients have been taking or given. Their combining medications may be involved with possible outcomes of adverse reactions or beneficial effects. In actual practice the patients may receive both treatments intentionally or unintentionally, with or without the awareness of the practitioner. In these situations an available authoritive database for interactions between HMM/PHP or prescribed PHARMED will be extremely useful for consultation when treatment problems appear or during emergency situations. Such a database will be welcomed by both practitioners of herbal medicines and orthodox medicine (OM) practitioners. The author has been involved in various research projects of basic and clinical aspects in mainly Chinese medicines among other herbal and pharmaceutical medicines hence examples will be given largely on those related to these disciplines as illustrations in this overview. It is envisaged that some of the safety issues and all other aspects discussed in the following texts are also relevant to other traditional or ethnic medicines.
Keywords: Principles of interactions, Safety issues, Pharmaceutical medicines, Chinese medicinal materials, Traditional herbal medicines, Adverse reactions, Beneficial effects.
Resumen
El incremento de la popularidad mundial del uso de los materiales medicinales herbarios (HMM), de la medicina tnica tradicional, tales como la ampliamente usada materia mdica china (CMM) u otras medicinas tnicas herbarias y productos propios de salud (PHP) relacionados, alimento funcional y la prescripcin de las medicinas herbarias, han aumentado las preocupaciones sobre sus usos concomitantes con los frmacos (PHARMED) y los efectos adversos consiguientes. En la mayora de los casos, las causas alegadas de efectos adversos estn vinculadas con sustancias herbarias, aunque la informacin autorizada sobre las interacciones entre HMM/PHP y PHARMED no es abundante en la literatura. Existe una necesidad urgente por semejante base de datos. En el siglo XXI, el pblico est ms informado, del Internet, sobre la salud y los productos medicinales y se ha vuelto ms conocedor sobre las materias que relacionan a sus condiciones de salud y bienestar, curando y previniendo enfermedades. Frecuentemente, ellos se auto-administran con varios productos de salud como tambin con productos PHARMED over-the-counter (OTC). Algunos de estos productos pueden tener dudoso control de la calidad y contener aditivos dainos o ingredientes no chequeados. Los futuros profesionales en salud y el cuidado mdico deben conocer o estar conscientes de lo que han estado tomando o han dado a sus pacientes. La combinacin de sus medicamentos puede estar involucrada con posibles reacciones adversas o efectos beneficiosos. En la prctica real los pacientes pueden recibir ambos tratamientos intencionalmente o involuntariamente, con o sin el conocimiento del practicante. En estas situaciones ser sumamente til para la consulta una base de datos disponible autorizada para analizar las interacciones entre HMM/PHP o PHARMED prescritos cuando aparecen las reacciones al tratamiento o durante las situaciones de emergencia. Semejante base de datos ser bienvenida tanto por practicantes de medicinas herbarias como de la medicina ortodoxa. El autor de esta revisin ha estado involucrado en varios proyectos de investigacin de aspectos bsicos y clnicos en medicinas chinas principalmente, entre otras medicinas herbarias y farmacuticas. En esta apreciacin global se darn amplios ejemplos en aqullos casos relacionados a estas disciplinas. Se enfatiza que algunos de los problemas de seguridad y todos los otros aspectos discutidos en los textos siguientes tambin son pertinentes a otras medicinas tradicionales o tnicas.
Palabras clave: Principios de interacciones, Seguridad, Medicamentos, Materiales medicinales chinos, medicinas herbales tradicionales, Reacciones adversas, Efectos beneficiosos.
Chan
Interactions between traditional herbal medicines & pharmaceutical medicines
Abbreviation list:
ADRAC- Adverse Drug Reactions Advisory Committee ADROIT- Adverse Drug Reactions On-line Information Tracking ADRs- Adverse Drug Reactions CM- Chinese medicine CMM- Chinese materia medica Dis- Drug interactions HMM- Herbal medicinal materials MFC- Methotrexate, fluorouracil combinations MFV- Methotrexate, fluorouracil, vinblastin MHRA- Medicine and Healthcare products Registration Agency OM- Orthodox medicine OTC- Over-the-counter PCM- Proprietary Chinese medicines PHARMED- pharmaceutical medicines PHP- Proprietary health products QSE- Quality, safety and efficacy SMZ- Sulfamethoxazole SXT- Sheng Xue Tang TMP- Trimethoprim
INTRODUCTION Herbs and herbal products, known as botanicals in some regions of the world such as North America, play an important role in the healthcare of nearly 80% of the world population particularly in developing countries (Akerele, 1993). For example, Chinese medicinal materials (CMM) and their manufactured products as well as Ayurvedic medicines have been used for thousands of years (Table 1) for prevention and treatment of diseases in China and in India, respectively, apart from relatively recent introduced synthetic pharmaceuticals in orthodox medical (OM) practice in the late 1800s. Without any doubts, all medicinal products used in human and animals should have proven quality, safety and efficacy (QSE). Problems and difficulties arise in the quality assurance of herbal medicinal products because there are so many unidentified chemical entities in the finished products, and the actual bioactive components are seldom known. The physico-chemical properties and mechanisms of actions of herbal products are quite different from pharmaceutical medicines (Table 2). Most of the herbal products from practice of traditional medicine are prescribed using a number of herbal mixtures. Recent advances in analytical chemistry and bimolecular techniques and related disciplines have helped elucidating the complex chemical compositions and bioactivities of these in natural products research. Thus the safety issues of interactions between traditional herbal medicines, or the newer botanicals, and pharmaceutical medicines can be addressed on according to the following sections in this overview. 1.1. Observation on the simultaneous consummation of Chinese Materia Medica (Chinese medicinal materials, CMM) and Pharmaceutical Medicines (PHARMED) Chinese medicine (CM), one of the world's oldest continuous surviving traditions (Zhen, 1995), has been practiced to maintain good health and treat diseases in the Chinese communities and recently by other ethnic groups worldwide (Chan, 2004). CMM and other natural substances and products, acupuncture and related physical therapies and special life style are often used together in the practice, co-existing with orthodox medicine (OM) in China and some regions in the Far East (Chan, 2005). However in the west increasing uses of CM have created both scepticism and support of CM practice that have been the major debate since the successful randomized clinical trial on the use of 10 CMM prescription on atopic eczema was published in 1992 (Atherton et al., 1992). However available in the market are CMM products adulterated with pharmaceutical drugs and wrongly supplied crude CMM with liver and kidney toxicity. These unprofessional practices from commercial organizations do not give CM the right reputation and recognition. It is emphasized that government regulatory agencies should set up harmonized regulatory control over the import and export of natural or herbal products to ensure safety of the public who consume them.
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Table 1. Medicinal products from natural sources in various ethnic cultures.
Complementary Medicine Ayurvedic Chinese Indusyunic Islamic Kampo Korean Other oriental Aromatherapy Herbalism Homoeopathy Botanicals
Natural Sources P,A,M P,A,M P,A,M P,A,M P,A,M P,A,M P,A,M P P P P
Origins India China Pakistan Middle East Japan Korea Other Asian countries European European European American
Recent Turnover Trade No figure > 1.3 billion US$ No figure No figure Getting competitive No figure No figure 14 m in 1996 (UK figure) 38 m in 1996 (UK figure) 20 m in 1996 (UK figure) 10% annual increase
P- Medicinal plants, A- Animal sources, M- Mineral sources (modified from Chan, 2003) Table 2: Comparisons between properties of Chinese medicinal materials (CMM) and pharmaceutical drugs.
Properties CMM Products Physico-chemical Properties Active ingredients Availability of pure compound Availability of raw material Quality of raw material Stability of preparation Biomedical Properties Mechanism of action Toxicological tests Empirical data Specific adverse effects Tolerance of therapy Therapeutic window Suitability for chronic use Placebo controls Controlled clinical trial Often unknown Often unknown Rare Limited Variable Uncertain
Remarks Pharmaceutical Drugs
Known Yes Yes Good Good
Usually known Mandatory Often meaningless Frequent Limited Usually narrow Not yet tested for new drugs Achievable Mandatory
Usually not available in animals Very important Rare through experience Usually good Wide Often well tested Difficult to achievable Usually not available
(Adopted from ref: Chan, 1995)
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Patients in the Far East intentionally or unintentionally may be prescribed CMM and other OTC tonic products containing CMM and OTCPHARMED for alleviating their illnesses. In the past there were practices of incorporating PHARMED into CMM preparations. The rationale may be that it is hoped to reduce side effects of PHARMED, or to produce synergistic effects for better treatment outcome. In most cases the pharmacological mechanisms of the combinations are not well studied and exaggerated adverse effects or therapeutic failures have been observed, although beneficial effects were noted. Patients may also self-medicate with CMM tonic preparations, consumed as dietary soup during convalescence period, while being treated with PHARMED. It is well documented that when several PHARMED are taken together, drugdrug interactions with detrimental effects occur (Li & Jurima-Romert, 1997) and the situation becomes more complicated when CMM products are taken simultaneously. Outside the Far East, apart from the Chinese communities, non-Chinese ethnic patients will probably be exposed to CMM medications through increasing popularity. The problems of PHARMED-CMM interactions will exist. Therefore adverse reactions consequential to CMM products may not be as simple as those due allegedly to toxicity of the herbs only (Chan, 2000). 1.2. The Integrative Practice of Traditional Chinese Medicine and Orthodox Medicine in China The integrative practice of traditional Chinese medicine (CM) into orthodox medicine (OM) in China since the early 1950s has given the opportunity to look at the advantages and disadvantages of each practice and to investigate the benefit from each discipline in order to encourage improvement of healthcare and possibly save treatment costs. China probably is the only country in the world that has developed a healthcare system that incorporates traditional CM into the healthcare policy for the nation. Within the healthcare system the two forms of medical treatment work along side with each other at every level of the healthcare structure. In particular, patients can benefit from preventive medicine, reducing side effects from OM or CM medications and improved quality of life in terminal cases. To achieve these goals it will take a lot of understanding from professionals of both disciplines. No longer
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should practitioners from traditional CM and OM be working in isolation. Professionals who are supportive of this concept of integration should also work to find out if there is any benefit at all in combination treatments. Augmenting OM with acupuncture has been recognized in several areas of pain relief, drug dependence, etc. in the West. However this overview only concerns with CMMPHARMED interactions relating to beneficial outcomes or adverse reactions as a consequence of co-administration. In China, increasing attention has turned towards organized scientific research on this aspect of interactions with beneficial outcomes. From the diet and nutrition aspects many Chinese patients in the community often self medicate with tonic CMM products after serious illnesses or surgical operations while they are still on OM medications. They believe that the herbs will help them to recover rapidly. It is normal procedure to carry out diagnosis of the same patient using traditional CM procedures and OM modern instruments and techniques in hospital practice in China. Experienced CM and or OM practitioners who are knowledgeable of using both types of medications have prescribed both types for certain diseases in order to get effective treatments. The improvement or deterioration of patients disease conditions is the measurement of success or failure of treatment. Some of the observations have been published, mainly in Chinese, in medical journals available in China. 2. Mechanisms of interactions between herbal medicines and pharmaceutical medicines In general drug interactions (DIs) refer to clinical phenomena during drug therapy when the pharmacological or therapeutic actions of a drug are altered by the co-administration of other drugs or substances. The consequence can be advantageous if used properly. But the clinical outcomes can either be an exaggeration of pharmacological or toxic effects or a diminished efficacy of drug treatment; leading to therapeutic failure and endanger patients conditions. The relevance of drug interactions depends on how clinically significant is the therapeutic outcomes. Thus we can consider different categories of drug interactions. Adverse drug interactions can be defined as a situation in which one drug interferes with the pharmacokinetics (at processes involving absorption, distribution,
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metabolism and excretion) or pharmacodynamics (at receptors, enzyme systems or other sites of actions) of another. The initial drug can modify the effect of the second drug, and lead to an enhanced effect, particularly drug toxicity; or a reduced effect, particularly lack of efficacy. Thus, drug interactions may produce enhanced drug effect that is synergistic; the outcomes of interactions may be beneficial or harmful. Interactions can be classified on basis of their significance as: major when life-threatening or permanent damage is involved; moderate if additional treatment is required; or minor when the therapeutic outcome is unnoticeable or not sufficient to affect the desired therapy goals. Figure 1 (Adopted from Chan & Cheung, 2000) illustrates the possible sites of drug-drug or drug-herb interactions. 2.1. Beneficial interactions between CMMPHARMED The integral treatment of illnesses using traditional CM and OM medications aims to bring together the general concepts of syndrome differentiation of CM with the OM principles of disease differentiation. It is obvious that the two systems vary greatly in approaches of diagnosis and treatment. For instance, OM concerns with
microcosmic differentiation of disease state, quantitative analysis of regional lesions or tissues damages, distinction between different disease based on characteristics of pathogenic factors and pathology of lesions. The CM practice on the other hand, deals with macrocosmic differentiation of syndrome, comprehensive qualitative analysis of whole body, distinction between different syndromes based on complex responses to external and internal pathogenic factors. It is logical to combine observations from disease differentiation (OM) and syndrome differentiation (CM) of the patient in order to draw accurate diagnostic conclusion. Treatment can be derived to target regional lesion or malfunction of particular receptor or organ (OM) and imbalance holistic conditions (CM) of the patient. This is one of the principles for integral treatment based on CM and OM. Modern medical technology and sophistication will help to make OM differential diagnosis while experience and personal approaches is needed for accurate CM diagnosis. It is necessary to relate the relationship of CM principles of Yin and Yang balance of the body to the OM understanding of the inter-play between the bodys nervous-endocrineimmune regulations.
Figure 1. The fate of a reversely acting drug in the
ABSORPTION
DISTRIBUTION
SITE OF ACTION z BOUND
ELIMINATION
(METABOLISM & EXCRETION)
BLOOD STREAM
SITE OF METABOLISM
FREE
FREE DRUG + METABOLITES
ENZYMATIC SYSTEMS IN LIVER AND z OTHER TISSUES
SITE OF ABSORPTION z
DRUG IN DOSAGE FORM BOUND DRUG z PLASMA PROTEIN AND ERYTHROCYTES FREE SITE OF EXCRETION EXAMPLES: KIDNEYS, LUNGS, THE BILIARY z SYSTEMS, SKIN
BOUND z SITE OF OTHER TISSUE DEPOT EXAMPLE: ADIPOSE TISSUE, MUSCLE
z Denotes possible site of drug interactions
body
(BROKEN LINES INDICATE PRESENCE OF BIOLOGICAL MEMBRANES)
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Experience of integrative medical practice in China has observed, through experience of practice and recorded case studies, beneficial treatment observation and outcomes with probable explanation or possible mechanisms of interactions although more experimental research and clinical evidences are needed to confirm such observations. This is partly because the OM gold standard of randomized clinical trials is not entirely applicable in the individualized approaches for treatment in CM practice. The following tentative categories of case studies illustrate synergy effects of treatment consequential to co-administration of OM drugs and CMM medications. These case studies were abstracted from medical journals published in China and have been translated into English and edited by the authors for presentation in the text (Chan & Cheung, 2000). The information is reproduced here with permission from the author for illustration. Yet most of these examples did not show any of the gold standard of RCT indicated in the practice of orthodox medicine. The debating issues will be that these are reported cases as practiced of integrative medical approaches shown in China that illustrate the beneficial treatment effects when CMM and PHARMED are co-administered together. Category 1: Combining antibiotics with CMM products producing added beneficial effects Example 1. Trimethoprim (TMP) and Shui Yang Mei (Adinarubella) in treating typhoid. (Luo, 1982): A group of 33 patients took part in the studies and they were given 30ml of CMM decoction Shui Yang Mei (orally 3 times a day) plus TMP (0.1g, twice daily) for treating the typhoid. In the control group, 21 patients were given sulfamethoxazole (SMZ; 1G) and TMP (0.1g), twice daily for the same infection. Both groups of patients were all fully recovered with no recurrence. The curative effects of the two groups were mostly identical (P>0.05). But the CMM-TMP group had no noticeable side effects. From laboratory experimental evidence, 10% Shui Yang Mei aqueous solution showed bacteriostasis to Shigella dysenteriae. This combined CMM-PHARMED showed synergic effect on Salmonella typhia. Example 2: Treatment of acute bacillary dysentery with traditional Chinese medicine and
western medicine combined on analysis of 117 cases (Wu, 1984): The 117 patients with acute bacillary dysentery were divided into 3 different treatment groups, orthodox medication, PHARMED only, sulfamethoxazole (SMZ 40 patients), Chinese Materia Medica (CMM) prescription 1 (CMM 1, 36 patients), and PHARMED plus CMM prescription 2 (SMZ plus CMM2, 41 patients). CMM prescription 1 consisted of 10 CMM herbs: Bai Shao (Radix Paeoniae Alba), Bai Tou Weng (Radix Pulsatillae), Chi Shao (Radix Paeoniae Rubra), Da Fu Pi (Pericarpium Arecae), Da Huang (Radix et Rhizoma Rhei), Dang Gui (Radix Angelica Sinenis), Huang Lian (Rhizoma Coptidis), Huang Qin (Radix Scutellariae), Mu Xiang (Radix Aucklandiae) and Qin Pi (Cortex Fraxini). CMM prescription 2 consisted of 10 CMM herbs: Bai Zhu (Rhizoma Atractylodis Macrocephalae), Bai Shao (Radix Paeoniae Alba), Bing Ling (Semen Arecae), Chi Shao (Radix Paeoniae Rubra), Dang Gui (Radix Angelica Sinenis), Fu Ling (Poria), Huang Qin (Radix Scutellariae), Mu Xiang (Radix Aucklandiae),Shan Yao (Rhizoma Dioscoreae) and Sheng Jun (Radix et Rhizoma Rhei). Four herbs were common in both CMM prescriptions. Treatment outcomes indicated that the combination medication gave the best results than either of the two groups with single medication treatment (P< 0.005). Comments from the publication are: the SMZ had strong bacteriostatic action that often leads to disproportionate population of intestinal bacteria and dysfunction of the stomach and intestine. The CMM medication is not only free of side effects, it can also increase the body defense mechanism by enhancing the release of immunological factors, phagocytosis of reticuloendothelial system, and by activating the kinase system it increases the amount of bateriophages in acute bacillary dysentery. Example 3: Reduction of side effects of Streptomycin by Gan Cao (Radix Glycyrrhiza). (Xu, 1987): It is well known that streptomycin can cause damage to the VIIIth cranial nerve and lead to sensorineural deafness. This toxicity of streptomycin was reduced when it was co-administered with Gan Cao (liquorices root) extract. About 80% of the patients who previously were not able to tolerate the side effects persisted in streptomycin treatment. Gan Cao is a well-known and widely used CMM, often
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incorporated in composite herbal mixture in CMM prescriptions. Category 2: Combating infection with antibiotics and immune-strengthening CMM products In the Chinese integrative medicine approach, bacterial infection in the body can be eliminated by using PHARMED antibiotics (i.e. the OM region treatment approach) while reinforcing the body immuno-function using CMM herbs to strengthen the body defense system (i.e. traditional CM holistic approach). The bodys immune system recognizes and destroys substances foreign to the body, including bacterial cells, other microbes, and foreign toxic compounds. The principles of these combinational approaches can be interpreted as follows. Cells in the circulatory and the lymphatic systems that recognize and destroy these cells are generated in the bone marrow and the lymphatic tissue (thymus, lymph nodes, spleen and tonsils), respectively. These stem cells when initially produced are featureless and cannot be distinguished as what type of blood cells (erythrocytes or different kinds of white blood cells) they will become. After their release into the blood stream they are delivered to all parts of the body. Some become memory cells that as the name implies, recognize specific foreign cells or chemicals to which they have been exposed, and react immediately on the next encountering of those compounds. Substances, such as vaccines, that effect the memory cells stimulate only to one disease or antigen. In general, most herbs that contain so many different chemical compounds, for the immune system do not affect memory cells, but are general immune system stimulators or immunostimulants. They induce the activities of the immune system but are not specific to a particular disease or antigen (i.e. the protein against which immune cells act). They increase resistance by mobilising effector cells that act against all foreign particles, rather than one specific type. Thus the combination of OM antibiotics with CMM products for treating infectious diseases is a logical approach. This may help to reduce bacterial resistance to antibiotics. Huang Qi (Radix Astragalus), a widely used CMM often prescribed as an important herbal component in composite prescription formulae, alone is used as a tonic herb and as medicinal herb in
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combination with others for strengthening the Lung for frequent colds or shortness of breath. It has no demonstrable anti-bacterial effects but it increases the immune system by increasing the number of stem cell in bone marrow and lymph tissues, promoting immune cells from the resting state into heightened activity and reducing the negative side effects of coadministered steroids on the immune system. Quite a few of the CMM products in the form of established proprietary medications or well-tried prescriptions have been shown to possess immuno-strenghtening or modulating properties. In a review on the role of CMM in chemotherapy of cancer the principles of such integrative approaches was illustrated by Wan in 2002. Category 3: Augmenting cardiovascular PHARMED treatment with CMM products Propranolol, a well used PHARMED belonging to the beta-adrenergic blocker group of therapeutics, has been co-administered with aqueous extract of Dan Shen (Radix Salviae Miltiorrhzae) as intravenous injection for treating patients suffering acute myocardial-infarction in Intensive Care Coronary Unit in some hospitals in China since the mid 1980s. The combination treatment gives significantly better outcomes than propranolol alone. Dan Shen aqueous extracts, among many other pharmacological properties, increase microcirculation, inhibit platelet aggregation and have centrally acting anti-anxiety actions. These may explain the beneficial effects with co-administration of propranolol. Extensive research works have been carried out over the past 20 years on Dan Shen in Shanghai and Hong Kong academic research institutes. It is interesting to note that isolated single chemical entities from Dan Shen roots have not produced any useful and marketable conventional PHARMED. Apart from injectables other oral preparations of single herb or composite formulae of Dan Shen are available to the public as preventive remedies against cardiovascular diseases. If these products are not used properly, however, adverse reactions may result if taken with other PHARMED to produce harmful interactions (See subsequent sections below for details).
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Category 4: Augmenting anti-inflammatory action of PHARMED with CMM Products Example 1: Lupus nephritis treated with impact therapy of cyclophosphamide and traditional Chinese medicine (Ruan & Ye, 1994). The seventy-six patients suffering from lupus nephritis were divided into two treatment groups, PHARMED with cyclophosphamide and steroid (35 patients) and combined PHARMED with CMM decoction of 14 herbs ( 41 patients). The 14 herbs were, Bai Hua She She Cao (Herba Heyotis Diffusae), Ban Zhi Lian (Herba Scutellariae Barbatae), Dan Pi (Cortex Moutan Radicis), Fu Ling (Poria), Han Lian Cao (Herba Eclipitae), Ju Hua (Flos Chrysanthemi), Nui Zhen Zi (Fructus Ligustri Lucidi), Qi Zi (Fructus Lycii), Shan Yao (Rhizoma Dioscororeae), Shan Zhu Yu (Fructus Corni), Shu Di (Radix Rehmanniae Preparata), Wu Gong (Scolopendra), Wu Shao She (Zaocys) and Ze Xie (Rpizoma Alismatis). After a six-month treatment course, the therapeutic efficacy was significantly higher in the combination group than the PHARMED medication only group (P<0.05). The PHARMED cyclophosphamide is itself inactive; after oral administration it is metabolized to active metabolites. In OM practice, prednisone (corticosteroid) is often used together in order to increase the rate of metabolism of cyclophosphamide; although single doses of the steroid will inhibit activation of this potent immunosuppressant. Cycolphosphamide causes liver toxicity and long term steroid treatment also causes systemic side effects. The use of CMM may help to build up beneficial effects by rectifying the imbalance of the body functions according to traditional CM concepts. Lupus nephritis usually manifests itself as Liver-Kidney Yin Xu (deficiency) with symptoms such as, lassitude of the loin and legs, dizziness, tinnitus, dry mouth and throat, deep and small pulse, red tongue with a little coating etc. The 14 CMM in the decoction nourish the Liver and Kidney, Yin and clear away Heat, according to the CM practice principles. Example 2: Observation on 134 patients with adult primary nephrotic syndrome with combined traditional Chinese medicine and Western medicine treatment. (Ye et al., 1993). The 134 patients suffering adult primary nephrotic syndrome were separated randomly into two groups and treated with corticosteroid (66 patients) and
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steroid with a decoction consisting of 10 CMM. These were: Dan Shen (Radix Salviae Miltiorrhzae), Di Gu Pi (Cortex Lycii Radicis) Gui Ban (Plastrum Testudinis), Han Lian Cao (Herba Ecliptae) Hong Hua (Flos Carthami), Nu Zhen Zi (Fructus Ligustri Lucidi),Qi Zi (Fructus Lycii), Sheng Di (Radix Rehmanneae) and Yi Mu Cao (Herba Leonuri). The percentage of success from the corticosteroids treatment and the combined therapy was 56.1% and 85.3% respectively and the corresponding percentages for incidence of side effects were 48% and 14.8%. These observations indicate that corticosteroid plus CMM decoction of the 10 components mixture could enhance curative success of adult nephritic syndromes with fewer side effects. Category 5: Reducing adverse effects due to PHARMED chemotherapy during treatment of cancers by CMM products Treatment of cancers (malignant neoplasm or new growth) using chemotherapeutic agents is often started after not so successful of surgical removal, or in conjunction with, radiation therapy. At this late stage patients become physically weak with quite a few signs of adverse effects as the treatment used often affects normal cells. The most severe toxic effects include bone marrow suppression, and nausea and vomiting apart from impairment of healing, depression of growth, causing sterility and hair loss. Some patients become in tolerable to chemotherapy and their quality of life is much reduced. Their immune system is highly compromised. The cytotoxic groups for cancer treatment using PHARMED of cytotoxic agents, depending on which cancer types and the policy for chemotherapy, often consists of at least three and more different groups of anti-neoplasmic agents. They are anti-metabolites (cytarabine, fluorouracil, methotrexate, and mercaptopurine), cytotoxic antibiotics (bleomycin, dactinomycin, doxurubicin, epirubicin, and mitomycin), plant derivatives (etoposide, vincristine), hormones and their antagonists (glucocorticoid, oetrogens such as fosfestrol, anti-oestrogen such as tamoxifen, progestrogens such as megestrol, antiandrogen antagonists such as cyproterone and flutamide, and gonadotrophin-releasing hormone such as goserelin, radio-isotopes such as 131I for thyroid tumours and inhibitors of DNA and RNA (procarbazine).
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Chemotherapy treatment of cancers using integral approach of PHARMED and CMM has been practiced in some hospitals in China. The concept is to utilize PHARMED cytotoxic agents to target the cancerous cells and CMM medications for restoring imbalances, as diagnosed from clinical picture, due either to the neoplasm or chemotherapy. The following examples illustrate some of the observations. Example: Clinical and experimental studies on chemotherapy combined with Sheng Xue Tang (SXT) recipe for the treatment of late stage gastric cancer (Rao et al., 1990). Eighty-one patients with late-stage gastric cancer were treated with chemotherapy (MFV, methotrexate, fluorouracil, vinblastin; or MFC, methotrexate, fluorouracil combinations). Among them, 63 patients also took the CMM composite formula, SXT, while other 18 patients were treated with chemotherapy only as control group. The prescription formula included following eight herbs: Radix Astragalus; Radix Pseudostellariae; Caulis Spatholobi; Rhizoma Atyractylodis Macrocephalae; Poria; Fructus Lycii; Fructus Ligustri Lucidi and Semen Cuscutae. Clinical observations showed that the CMM prescription could reduce the side effects caused by chemotherapy with improved body weight (see following summaries on Tables 3 and 4).
The clinical observation was complemented with laboratory experimental studies that indicated the SXT decoction could prolong the life of the S-180 tumour bearing mice undergone chemotherapy. 2.2. Harmful effects of interactions between CMM-PHARMED Information on harmful interactions between CMM and PHARMED medications can be obtained from the literature mainly available in Chinese language. These information were edited and compiled into Tables reported previously (Chan & Cheung, 2000). Some clinical observations have been confirmed with experimental investigation. In general the mechanisms described for drug-drug interactions in for PHARMED are also applicable for the CMMPHARMED interactions as understood from conventional science and medical aspects. Complications arise because of the presence of so many chemical entities in the single herb or in the decoction of the composite formulae, and many of which have not yet be identified. Such observations are also applied to other traditional herbal medicines. The following categories summarize the likely mechanisms of involved.
Table 3. A summary of adverse effects during chemotherapy treatment with and without SXT recipe.
Groups Loss of appetite Nausea and Vomiting Diarrhoea Tiredness Limp numbness
Chemotherapy plus SXT recipe 33 cases (100%) 12 (19%) 12 (19%) 0 20 (31%) 0
Chemotherapy only (control) 12 cases (100%) 6 (50%) 6 (50%) 6 (50%) 8 (67%) 4 (33%)
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Table 4. A summary of changes in patients body weight (kg) during chemotherapy treatment with and without SXT recipe.
Groups Number of cases Before treatment ( X SD) After treatment (X SD) T value P value
Chemotherapy plus SXT recipe 29 58.51 1.85 60.66 3.08 18.22 <0.001
Chemotherapy only (control) 12 58.73 5.53 57.16 5.66 0.688 >0.05
Category 1: Formation of insoluble complexes during absorption phase leading to therapeutic failure Some CMM medications whether single herbs or composite prescription decoction or proprietary herbal products contain metal ions that may form insoluble chelates or complexes with PHARMED Tannic acid in some CMM medications can form insoluble complexes with PHARMED antibiotics and drugs containing tertiary amine-alkaloids and metal ions. Alkaloids in CMM medications form precipitates with metal ions in PHARMED. CMM medications containing quercetin (phenols with 5-OH and 4-keto functional groups) can precipitate CMM drugs containing aluminium, bismuth, calcium, ferrous, and magnesium ions. Gan Cao (Liquorice root, Radix Glycyrrhizae) interacts with tetracycline group of antibiotics by reduction of their oral absorption. Yin Chen (Herba Artemisiae Capillaris) forms precipitates with quinidine and antagonizes chloramphenicol actions. Category 2: Affecting transport of drug molecules in the body by CMM leading to reduced effects Some CMM medications have high contents of acids that will alter the physiological pH and thus affect the transport mechanisms of PHARMED drug molecules leading to reduced actions or physiological precipitation. Similarly some CMM medications contain alkali that affect the physiological solubility of the PHARMED drugs thus influencing their excretion and transport. For examples, the presence of acids in Nu Zhen zi (Fructus Ligustri Lucidi), Shan Zha (Fructus Cartaegi), Shan Zhu Yu (Fructus Corni), Wu Mei (Fructus Mume), Zhi Qiao (Fructus
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Aurantii), etc. will decrease the antimicrobial activities of the aminoglycoside group of antibiotics. (Gong, 1989a;b). Category 3: Affecting function of PHARMED diuretics and body electrolyte balance by CMM medications Some PHARMED diuretics such as the potassiumsparing group (amiloride, spironolactone, etc.; Stockley, 1996) should not be co-administered with some CMM medications. These products (Gong, 1989b; Zhu, 1994) contain potassium ions in various forms. Hyperkalaemia may result due to accumulation of the ion from the herbal products and retention from the diuretics. A large group of CMM containing potassium was given as examples in the reference (Chan & Cheung, 2000). Category 4: Destroying amylase in some CMM medications by PHARMED antibiotics Amylase contents in some CMM products are active principles that can be destroyed by PHARMED such as the antibiotics tertracylines and sulphonamides. Key CMM includes Dan dou Chi (Semen Soyae Preparatum), Gu Ya (Fructus Oryzae Sativae Germinatus), Mai Ya (Fructus Hordei Germinatus), Shan Yao (Rhizoma Dioscoreae) etc. (Gong, 1989a; Editorial, 1987; Lin, 1990) Category 5: Destroying glycosides in some CMM products by acidic PHARMED Glycosides in some CMM products may be the active ingredients that can be destroyed if acidic PHARMED (Ascorbic acid, nicotinic acid, glutamic acid and drugs containing mineral acid components as salts) are administered concurrently. (Gong, 1989a;b).
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Category 6: Releasing toxic cyanide from CMM medications by PHARMED Some CMM herbs, in particular, seeds when coadministered with PHARMED release hydrocyanic acid that inhibits the respiratory centre. PHARMED such as codeine, morphine, should not be coadministered with CMM such as Bai Guo (Semen Ginkgo), Ku Xing Ren (Semen Armeniacae) and Tao Ren (Semen Persicae) (Ou 1989; Leng, 1988; 1991). Category 7: Affecting liver metabolizing enzymes that eliminate PHARMED by CMM CMM single herb or complex mixture products may modify the metabolic elimination of PHARMED leading to reducing activity (enzyme induction) or increasing activity (enzyme inhibition) of PHARMED. The pharmacokinetics of warfarin is compromised during co-administration with Dan Shen (Radix Salvia Miltiorrhiza) leading to uncontrollable steady state of plasma concentration. (Chan et al., 1992; 1995) Warfarin is mainly eliminated by the liver and has a narrow therapeutic window during clinical treatment when chronic anticoagulation administration is needed. If the steady state is affected haemorrhagic or clotting episodes will occur. (Lo et al., 1995). The active ingredient of Gan Cao (Radix Glycyrrhizae), glycyrrhizin, is an inhibitor of 11 betahydroxysteroid dehydrogenase, a major metabolic enzyme of glucocorticoids in the liver. Coadministration of Gan Cao potentates the action of prednisolone due to enzyme inhibition. 3. Approaches on Research into Interactions between herbal medicines and pharmaceutical medicines From the observations listed in the above sections the complexity of interactions between herbal ingredients and pharmaceutical drugs is indeed enormous. It is crucial to review steps that can be planned to investigate such interactions. One appreciates that only the clinically significant interactions are relevant to clinical practice. As a result of the focus on adverse drug interactions over the past decades many of the interactions between drugs of the PHARMED are now predictable and unwanted reactions using drug combinations can be avoided by dosage adjustment of one or more of the
interacting drugs. Unfortunately investigations on interactions between herbal medicines such as CMM with PHARMED become more difficult and documentations in literature are not plentiful. Moreover many drug interactions that have been listed in the literature are at times not meaningful nor helpful for clinical practice if they are not relevant. This is because some reported interactions are theoretically possible on the basis of in vitro investigations or animal experimentation that may not have been studied in patient situation. Some studies are based on healthy volunteer investigations or at non-therapeutic doses. The following headings describe possible steps one can initiate studies on drug-drug interactions 3.1. Criteria for choosing interaction studies of clinical relevance Initially, the project leaders should ascertain how valid the reported interaction is, and whether it is worthwhile to design a study to verify the importance of the reported or suspected interactions. Investigational studies of drug-drug interactions can be of predictive value if they mimic the clinical situation and they can be related to drug combinations and regimen that are practiced in the clinic. PHARMED with problematic disposition and pharmacokinetic characteristics and those required for long-term treatment carry the higher risk of occurrence of possible clinically relevant interactions. Situations when patients self-medicate or are put on multiple drugs regimen also warrant investigations. The criteria summarized in Table 5 will be helpful to make a decision. Specific studies, based on the relevant criteria listed in Table 6, can be carried out during the development of new drugs (or CMM products refer to later headings). These may be designed to measure certain biochemical or physiological processes or functions being affected by drug treatment with narrow therapeutic ratios. These functions or biomarkers consequential to drug or herbal treatment, or a combination of the CMM or PHARMED are useful measurements of adverse, beneficial or synergic effects. Table 6 gives examples of the pharmacological classes of drugs and respective processes involved for monitoring.
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Table 5. Criteria considered as potential risks f or occurrence of drug-drug interactions
Drug-drug interactions of 2 or more drugs observed in clinical practice for confirmations Drugs with steep dose-response curve (a small change in concentration will lead to exaggerated effects) Drugs with small therapeutic range especially in combination dosage forms Drugs with problematic disposition or pharmacokinetics Drugs used for long term treatment having effects on drug accumulation, enzyme induction etc. Drugs prescribed simultaneously by several physicians intentionally or unintentionally Drugs self-medicated by patients Drugs showing genetic polymorphism in metabolic elimination (cytochrome P450 CYP2D6, CYP2C19, etc.)
Table 6. Pharmacological classes of drugs with clinical relevant interactions.
Pharmacological Class Antacids drug Anti-arrhythmics Anti-asthmatics Anti-coagulants Anti-convulsants Anti-diabetics Anti-hypertensives (beta-blockers) Cardiac glycosides Cytotoxic agents H2 receptor blockers Psychotropic agents (particularly lithium, MAOIs)
Functions to be monitored Formation of non-absorbable complexes Cardiac rhythm Respiratory stress Blood clotting or hemorrhagic crisis Uncontrolled epilepsy or over sedation Glucose homeostasis Irregular blood pressure (BP) Heart failure or over-digitalization Over-suppression of immune functions Inhibition of drug metabolic elimination Hypertensive crisis
Indices/Biomarkers Plasma levels ECG monitoring Respiratory test Prothrombin time Seizure frequency Blood glucose levels Monitor BP Monitor arrhythmia Immune biomarkers Plasma levels Monitor BP
3.1. Difficulty and barriers towards research in herbal medicine in developed regions In countries where the healthcare system is run by practice of orthodox medicine, clinical and scientific research into the efficacy of herbal medicines in general using conventional methods has met difficulties (Mills, 1996). Practical obstacles in pursuing good research for herbal medicines under can be considered as follows: 1. To obtain results with sufficient statistical weighting is expensive and laborious. Herbal medicine presently receives little or no funding from teaching hospitals, universities or industries in most organizations in the West. Recently through the Office of Alternative Medicines set up by the National Institute of Health in the USA some efforts have be
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made to fill the gap of information on the quality, efficacy and safety of complementary or herbal medicine. In the UK the Medical Research Council has become more inclined towards funding well designed studies on complementary therapies. 2. Herbal medicinal products are complex mixtures with a vast amount of chemicals that may be pharmacologically active or inert. The composite formulae or prescriptions may have different properties from that of the single constituent acting alone. Acceptable models for investigation of herbal medicines from the orthodox medicine camp are not readily available. Herbal practitioners do not generally consider some orthodox models as relevant
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to assess herbal products that have been shown effective through time. 3. The principles and approaches of using herbal ingredients and their effects on the body is not the same as usually understood for conventional medications. For example, CMM medications are used to holistically evoke healing responses in the body to rebalance body functions rather than to attack symptoms as orthodox medications do. Research of these types on CMM medications in the West is not plentiful. Clinical observations in the Chinese language are plentiful. This gap could be reduced in the near future through more international collaboration and understanding of methodology. In his overview on research strategies of herbal medicines Mills (1996) gave some positive approaches that could help research on herbal medicines in the West. However the progress of official contribution from herbal medicine to the mainstream healthcare in developed regions has been slow over the past decade.
into account and considered the interaction study design. Consideration should include the traditional CM information on the CMM medications and their reported conventional pharmacological group functions. Using this approach it is possible to adopt study design from the drug-drug interaction protocol only for those needed for patients. It is considered unethical to test on healthy volunteers for such study. 4.1. Design for interaction study between CMM and PHARMED Using example of Category 5 under Section 2.1, Reducing adverse effects due to PHARMED chemotherapy during treatment of cancers by CMM products, it is possible to design a reasonable study. In the study reported by Rao et al. (1990), the indications given for significant successful reduction of side effects were the general well being of the patients with stomach cancer on combination of chemotherapy and Sheng Xue Tang, SXT (literally means decoction or recipe producing new blood components). The results as reported could be more significant and convincing if other measure outcomes were explored and included in the trial. This might be due to the limited design and certain facilities that were not available during that study. It was obvious that the chemotherapy with MFV (methotrexate, fluorouracil and vinblastine) should not be stopped throughout treatment as decided by the oncologist; the co-administration of SXT would help to alleviate syndromes, due to adverse effects of the MFV treatment, as diagnosed by the traditional CM methodology. If this trial were to be repeated according the present suggested design here, it will include several entry requirements and other outcome measures with better statistical assessment. The 81 late stage gastric cancer patients could be divided into 3 groups of 21 each for 3 different treatments of MFV chemotherapy, MFV chemotherapy plus SXT decoction and MFV chemotherapy plus placebo decoction respectively. Details of design consideration are summarized below: 4.1.1. Trial conditions and procedure 1. Inclusion of a placebo preparation or decoction should give a better outcome of a controlled trial. A decoction similar in color, smell but without the traditional CM effects could be prepared. The successful controlled trial in the atopic eczema
4. Proposed designs for study of interactions between CMM and PHARMED

Most of the interactions between CMM and PHARMED described in the literature are observations reported from clinical practice. They come from the integral approach of medical practices obtain the beneficial effects of combination treatment and some are adverse effects due to intentional or unintentional combination of the two groups of medications. These observations are published mainly in Chinese language. Information gap exists on herbal medicines and their potential for interaction with PHARMED medications. Evidently research in these areas is urgently needed regions where orthodox medical practice is the main stream of healthcare provider when their patients consume herbal medications. The most important step is to obtain reliable sources of case studies. It may be necessary to accept initially the observed effects as suspected interactions and design studies to ascertain the finding by careful follow-up investigations within the patient group who needs the combination herb-drug treatment. All information on the interacting PHARMED stated in Tables3 and 4 should be taken
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2.
3.
4.
5.
6.
study (Atherton et al., 1992) also included a placebo to increase the trials confidence level of significance. Entry by randomization of number of patients into the three treatment groups was a better design. Thus the Chemotherapy plus Sheng Xue Tang group and the Chemotherapy plus Placebo Tang could be compared with the Chemotherapy group only. Pre-chemotherapy profiles of all patients for clinical biochemistry and hematology and kidney and liver function should be available and recorded accordingly together with other parameters for assessment of well being using Quality of Life instruments specifically for cancer group patients. The well being measurements should be administered by trained persons based on properly designed questionnaires. The person making the assessment should be blinded of both treatment groups. Measurement of adverse effects should also be performed with thoroughly designed protocol and recorded without biased remarks or comments. Same procedures should be administered for every patient. Run-in steady state of basic chemotherapy treatment should be achieved by measuring the pharmacokinetic parameters of the chemotherapy drugs during the first week before giving the SXT decoction or Placebo Decoction. Evaluation of pharmacokinetics of the chemotherapy drugs should commence simultaneously. Determination of pharmacodynamics and pharmacokinetics after steady state has attained should be carried out. Observation of adverse side effects and improvement or deterioration of blood picture and other related. Indices or other related biomarkers for cancer progression should be recorded.
such as prevention of hair loss due to PHARMED, improving overall well being due to the immuno-protective or -stimulant actions of the CMM preparation, the confidence level of the significance will be higher due to the randomization and inclusion of a placebo. The clinical biochemistry and hematology reports and kidney and liver function tests and related biomarkers would be favorable. These data are essential indications how the body reacts to the combination treatment. 2. The pharmacokinetics of the three PHARMED cytotoxic drugs could be altered due to the complexity of the unknown chemicals in the SXT and placebo preparations. This alternation could only be detected if the disposition of these PHARMED were followed during the trial.
4.2. A co-ordinated effort for reporting of Adverse Drug Reactions (ADRs) involving herbal medicines
Medical practitioners in main stream healthcare practice in developed countries or regions have expressed the concern and fear of herbal medicines and related products causing toxicity and adverse effects. Yet reported cases of toxicity often do not provide with comprehensive information such as those of the prescribed herbal materials or related products, pre-treatment clinical profiles of patients bio-chemistry, hematology, liver and kidney functions and PHARMED that were co-administered. This can only lead to difficulty in drawing clear conclusions of the significance and relevance of the interactions described. Data as mentioned in the Section 4 of the present overview on proposed design for study of interactions will be needed for consideration. Some other reported cases of toxic reactions due to herbal medications or related products those including CMM
4.1.2. Assessment of Outcomes The possible outcomes may be as follows: 1. If the SXT decoction is effective to reduce adverse effects of the MFV chemotherapy
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Figure 2. Study design for drug interaction studies in cancer patients
Period 1
Days No treatment
Period 2
Full treatment course
Period 2
Period 2
Treatment
Run-in phase Clinical measurement and observation 81
T1 pharmacodynamics pharmacokinetics of MFV * 27
T2 pharmacodynamics pharmacokinetics of MFV in combination with SXT ** 27 1. Evaluation of pharmacodynamics and pharmacokinetics of combination therapy (MFV + SXT) 2. Measurements of clinical data as for MFV group
T3 pharmacodynamics pharmacokinetics of MFV in combination with placebo *** 27
Patient No. with randomized entry Assessment of clinical conditions and other measurements
1. Run-in phase to obtain demographic data etc.
1. Evaluation of pharmacodynamics and pharmacokinetics of basic treatment of (MFV) at steady state. 2. Measurement of clinical data after steady state of MFV. 3. TCM measurements after MFV steady state. Improvement or deterioration.
1. Evaluation of pharmacodynamics and pharmacokinetics of combination therapy (MFV + Placebo) 2. Measurements of clinical data as for MFV group. 3. TCM measurement after MFV steady state Improvement or deterioration.
2. Obtain clinical data on clinical biochemistry haematology, kidney and liver function. 3. TCM diagnositc measurements and syndromes diagnosis. Possible Outcomes assessment Admission to treatment
3. TCM measurement after MFV steady state
Improvement with less side effects or deterioration.
* MFV = Methotrexate, Fluouracil, Vinblastin; ** SXT= Sheng Xue Tang (a CHM decoction of 8 herbs) *** Placebo = a decoction of similar colour and taste but no SXT activity
have involved unqualified practitioners or the products that were adulterated with PHARMED. These can be related to poor documentation of reporting incidents of adverse reactions. Moreover rarely are reported of the beneficial effects resulting from the intentional co-administration of both CMM and PHARMED in integrative medical practice. Proper evidence-base investigations are essential for further development. Contributions from these practices are urgently needed to enlighten the benefit of complementary medicine towards healthcare worldwide. In handling adverse reactions of PHARMED, wellestablished reporting schemes have been in operation in some developed regions. The author is familiar with schemes operating in the UK and Australia. The intention is to describe briefly here such that by modifying and adopting these two schemes it is possible to use the principles behind for recording and reporting adverse reactions involving CMM or other herbal medicines with PHARMED interactions. A centralized co-ordinating effort will be needed for
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quality information to make decision on all medications regarding restriction in use, reduction in dose, or special warnings and precautions.
4.2.1. The Yellow Card Scheme

Working with the Medicine and Healthcare products Registration Agency (MHRA) the UK Committee on Safety of Medicines has established the Yellow Card Scheme (Rawlins, 1988, 1988a) for reporting adverse reactions of medications since 1964. The system receives reports of suspected ADRs directly from doctors, dentists, coroners, pharmacists and other health professionals and indirectly through pharmaceutical companies. It is the UK Adverse Drug Reactions Reporting Scheme that these received reports are placed on a specialized computer system, Adverse Drug Reactions On-line Information Tracking (ADROIT), to facilitate rapid processing and analysis. The scheme has been critically important in monitoring drug safety for all PHARMED in normal clinical practice, increasing knowledge about known
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adverse reactions, and acting as an early warning system for the identification of previously unrecognized adverse reactions. The organization and operation of the Scheme includes collection of reports of suspected adverse reactions, data processing, verification and confirmation of information, and dispatching of advice etc. The data received from reports of suspected ADRs will be analyzed and presented and dispatched via the Current Problems in Pharmacovigilence which is distributed to all doctors, dentists, coroners and pharmacists periodically throughout the year. Obviously the accuracy of initial source of information of the suspected adverse reactions is extremely important. Guidelines on reporting are given. Table 7 shows a general format of reporting suspected adverse reactions arisen due to CHM medication-OM drug interaction proposed for action by the authors (Chan & Cheung, 2000). It is also a suggestion for traditional CM practitioners and OM practitioners Good Clinical Practice to self-regulate until a similar scheme is accepted by appropriate health organizations in regions that such a scheme is considered for monitoring. It is adopted from the UK Yellow Card Scheme reporting card issued by the Scheme operators. It is understood that the present UK Scheme also welcomes inclusion of suspected adverse reactions due to herbal medications. Table 7 listed categories of Adverse Reactions considered serious for reporting as specified in the UK Yellow Card Scheme. In general, serious reactions include those that are fatal, life-threatening, disabling, incapacitating or that result in or prolong hospitalization.
including herbal, traditional or alternative remedies. Regular Australian Adverse drug Reactions Bulletin is distributed to appropriate healthcare professionals for their information free of charge. A modified Blue Card is proposed to deal with reporting or recording interactions between CMM products and PHARMED for practitioners self-regulatory practice (Table 8). Other countries in developed regions may also have similar establishments who would deal with ADRs reporting. Whatever systems are available, it is possible to modify them for reporting and recording ADRs due to interactions between CMM medications and PHARMED.
5. Establishing a data base for safety issues on interactions between CMM and PHARMED
Most of the CMM used for prevention or treatment of diseases according to traditional CM practice are considered safe, even when potent/poisonous herbs are included. This is because, according to Chinese Medicine Theories, when administered as mixtures of CMM, composite formulae (Fu-Fang, in Chinese), through proper combinations of CMM may interact to enhance the therapeutic efficacy and to eliminate or minimize adverse reactions. It is considered that toxic CMM could be rendered non-toxic or less toxic through such combinations. Fu-Fang is the basic practice in Chinese Medicine prescribing. Yet there is little published scientific data in the west to substantiate such observations that have sustained through test of time. Nevertheless, poisoning cases do occur due, probably, to one of the many reasons such as: lack of understanding on how to use CMM; improperly processed items; over-dosing; use of fake or substituted CMM; contamination especially by heavy metals and/or insecticides; poor quality control during manufacturing and adulteration with pharmaceutical drugs; and interactions between CMM and co-administered pharmaceuticals. Hence it is important to identify where the toxicity comes from and what aspects of toxicity are measured.
The Blue Card System

Chan and Cheung (2000) also suggested, depending on convenience and circumstantial situations, to adopt the Blue Card System initiated originally by the Adverse Drug Reactions Advisory Committee (ADRAC), a subcommittee of the Australian Drug Evaluation Committee, who encourages the reporting of all Suspected Adverse Reactions to drugs or other medical substances,
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*Table 7. Form for reporting suspected adverse reactions due to interactions of Chinese herbal medicines and pharmaceutical medicines information is kept as confidential records Do not be put off reporting because some details are not known
REPORTER: HEALTH CARE PROFESSIONALS Name Professional Address OM PRACTITIONERS/TCM PRACTITIONERS Name Professional Address
Telephone Speciality Signature: Date:
Telephone Speciality Has this case been discussed with the consultant / GP
Yes
No
PATIENT'S DETAILS SURNAME _______________________ Other names _______________________
Sex M Weight Kg F Hospital if relevant ________________________________________ Hospital Number ____________ Date of birth (or age)__________
00 SUSPECTED CHINESE HERBAL MEDICINAL PRODUCTS Give brand name of formulae or Route Daily dose herbs ________________________ ___________ ____________ ________________________ ___________ ____________
Date started ___/____/__ ___/___/___
Date stopped ___/___/___ __/___/____
Therapeutic indication ________________ ________________
ORTHODOX DRUGS TAKEN IN THE LAST 3 MONTHS INCLUDING SELF-MEDICATION Give brand if known. Write None if Route Daily dose Date started no other drugs has been taken ________________________ ___________ ____________ ___/____/__ ________________________ ___________ ____________ ___/____/__ ________________________ ___________ ____________ ___/____/__ ________________________ ___________ ____________ ___/____/__ ________________________ ___________ ____________ ___/____/__ SUSPECTED REACTIONS Is the patient hospitalised because of the reaction? ________________________ ________________________ ________________________ ________________________
Date stopped ___/___/___ ___/___/___ ___/___/___ ___/___/___ ___/___/___
Therapeutic indication ________________ ________________ ________________ ________________ ________________
Yes No ___________ ___________ ___________ ___________
____________ ____________ ____________ ____________
Date reaction started ___/____/__ ___/____/__ ___/____/__ ___/____/__
Date reaction ended ___/___/___ ___/___/___ ___/___/___ ___/___/___
Outcome eg. fatal recovered, continuing ________________ ________________ ________________ ________________
Relevant additional information including medical history, investigations, known allergies, suspected drug interactions. For congenital abnormalities state all other drugs taken during pregnancy and the LMP. Please attach additional pages if necessary ______________________________________________________________________________________ ______________________________________ If you would like information about other reports associated ______________________________________ with the suspected drug , tick here * Adopted form the Yellow Card Scheme for Reporting ADRs, UK
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4.2.2.
*Table 8. Form for reporting suspected adverse drug reaction involving interactions of Chinese herbal medicines and pharmaceutical medicine drugs (Note: Identities of Reporter, Patient and Institution will remain Confidential) Patient (Initials or Record No. Only) Age: Sex: Adverse Reaction Description: Date of Onset of Reaction: Daily Dosage and Route / Height: Weight: / Date Begun Date Stopped Reason for Use
All Chinese Herbal Medicinal Products and OM Drugs Prior to Reaction Asterisk Suspected Drug(s) (Please use trade names)
Treatment (of reaction): Outcome: Recovered Not Yet Recovered Sequelae: No Yes Date of Recovery Unknown (describe) / / Fatal Date of Death / /
Comments (eg. relevant history, allergies, previous exposure to this drug): Reporting Doctor Name: Address: Signature Date: / / or Pharmacist Others (Please tick)
Return Forms(s) To University Department on Professional Association of Traditional Chinese Medicine Address: * Fax: Adopted from the Blue Card System of the ADRAC, Australia
5.1. Assessment of Intrinsic Toxicity of CMM

Intrinsic toxicity of properly processed (i.e. Quality Control-checked) potent/poisonous CMM can be assessed using various models available. Specific potent/poisonous CMM included in well-known composite formulae have survived the test of time, but such potent CMM and Fu-Fang containing them, although included in the Chinese Pharmacopoeia, can only be prescribed by qualified traditional CM practitioners. Individual CMM, which are highly
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potent or poisonous are seldom used singly, and when used in clinical settings where the principles of CM are not understood or not followed, their use can be highly dangerous. Experience and incidents of occurrence can be shared via networking. Existing toxicology models can be used to ascertain the toxicity of the Fu-Fang. For examples, cardiac, liver and neurological toxicity can be assessed, using cell culture and in vivo animal models if toxicity is suspected.
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Toxicity of components of individual potent/poisonous or non toxic CMM, if needed for assessment, is far more difficult and may not be relevant to the composite use of CMM. Most of the authenticated CMM, when used properly in the form of decoction, have stood the test of time for their safety issues as Pharmacopoeia composite formulae. Nevertheless, if toxicity assessment is needed in vivo animal models can be used. But other forms of extraction in proprietary Chinese medicines (PCM) of these CMM may show toxicity that has not been tested in human use. The toxicity of these PCM should be assessed according to those animal toxicity tests used for pharmaceuticals. The opportunity of networking to exchange experience in these issues will be beneficial to acquire the present understanding and allow future drafting of guidelines for what toxicological tests are needed.
cytochrome P450 isozymes; interaction with Pglycoprotein and other efflux mechanisms, plasma protein binding and clinical pharmacokinetic interaction trials may be performed. Experimental results can produce an indication of inhibition or induction of various isoforms by CMMs, which can alter the metabolic properties of drugs, thus resulting in changes in the pharmacological and/or toxicological effects of other drugs administered concurrently. To establish a battery of such tests and validate their contribution to the clinical assessment of the potential problems of individual interactions will be an important contribution to healthcare when the two groups of medications are co-administered.
5.3. New methodologies and techniques for quality, safety & efficacy evaluation of CMM
The evaluation of CMM safety is complicated by multiple factors, such as the geographical origin of plant material, different processing techniques, dosage, route of administration and compatibility with other medicines. This variation in chemical constitution can make it difficult to elucidate the toxic component(s) and to explain all the pharmacological mechanisms of CMM. The advent of system biology including metabonomics, which provides a new platform for the study of CMM in the post-genomic era, and has been successfully applied in many other fields such as drug discovery, phytochemistry, and clinical research. Due to the diversity in polarity, molecular weight, and concentrations of active molecules, it is generally accepted that a single analytical technique cannot provide sufficient visualization of the metabolome, and multiple technologies are needed for a comprehensive view. NMR, chromatographic and their hyphenated techniques are the most popular analytical methods used. As with the treatment concept of CMM, the key point of metabonomics is to emphasize holistic effects, especially regarding clinical efficacy and potential toxicity. Metabonomics can identify the principle changes in the chemical components and pharmacological activities of CMM under different conditions. Combined with in vivo and molecular biology protocols, metabonomics can help to explain mechanisms and evaluate the safety and efficacy of CMM.
5.2. In vitro and in vivo methods to assess CMM-PHARMED interactions and clinical safety
As mentioned above, one of the major causes of toxicity of CMM may come from interactions between them and pharmaceutical medicines. Such information in the English text is not abundant; the situation of integrative practice of traditional CM with orthodox medicine has shown both beneficial and adverse effects of CMM when co-administered with pharmaceutical medicines. Data collections from practices within and outside China will be essential to identify such observations. The database will collate all published reports of experimental, theoretical data and clinical reports of toxicity and CMM-PHARMED interactions. This is important as laboratory predictions do not always correlate with significant clinical adverse events, and it is important to validate experimental methods. The lack of reliable data has led to a situation where theoretical and unproven potential interactions are presented as contraindications, despite a lack of clinical reports confirming them. This practice is immensely damaging to the perception of traditional CM by the orthodox medical and pharmacy professions, and is often misplaced or misleading. To ascertain the validity and relevance of CMMPHARMED interactions a series of cell culture and in vivo models can be devised using expertise in China and other regions as appropriate, to test the effect of CMM on the inhibition or induction of a range of
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REFERENCES
Akerele O. 1993. Nature's medicinal bounty: Don't throw it away. World Health Forum 14: 390-395. Atherton DJ, Sheehan MP, Rustin MH, Whittle B and Guy MB. 1992. Treatment of atopic eczema with traditional Chinese medicinal plants. Pediatr Dermatol 9 (4): 373375. Chan K. 1995. Progress in traditional Chinese medicine. Trends Pharmacol 16: 182-187. Chan K. 2000. Observations of the use of Chinese herbal medicinal products and orthodox drugs. In: Chan K & Cheung L, Editors. Interactions between Chinese Herbal Medicinal Products and Orthodox Drugs, Harwood Academic Publishers, The Taylor & Francis Group, June, 2000, Chapter 6, pp. 51-56. Chan K. 2003. Some aspects of toxic contaminants in herbal medicines. Chemosphere 52: 1361-1371. Chan K. 2005. Chinese medicinal materials and their interface with Western medical concepts. J Ethnopharmacol 96: 118. Chan K, Lo ACT, Yeung JHK, & Woo KS. 1995. The effects of Danshen (Salvia miltiorrhiza) on warfarin pharmacodynamics and pharmacokinetics of warfarin enantiomers in rats. J Pharm Pharmacol 47: 402-406. Editorial. 1987. An overview on the combined use of Chinese herbs and orthodox drugs. Chinese J Integr Trad West Med 3(7): 135-138. Gong L-R. 1989a. Interactions of Chinese patent medicine and orthodox drugs (I). Chinese J Integr Trad West Med 5(9): 315-317. Gong L-R, 1989b. Interactions of Chinese patent medicine and orthodox drugs (II). Chinese J Integr Trad West Med 6(9): 375-376. Leng F-N. 1988. Editor, Fundamental of Chinese Patent Traditional Chinese Medicinal Products. (I). Peoples Health Publishers Ltd., Beijing China. Leng. F-N. 1991. Editor, Fundamental of Chinese Patent Traditional Chinese Medicinal Products. (II) Peoples Healthe Publishers Ltd., Beijing China. Li PL, Jurima-Romert M. 1997. Drug-drug Interactions: Scientific and Regulatory Perspectives, International Edition, Academic Press. Lin B. 1990. A discussion on the interactions of Chinese herbs and antibiotics. Shan Dong J Trad Chinese Med 2: 28-29. Lo ACT, Chan K, Yeung JHK, & Woo KS. 1992. The effects of Danshen (Salvia miltiorrhiza) on pharmacokinetics and pharmacodynamics of warfarin in rats. Eur J Drug Metab Ph 17: 257-262 Lo ACT, Chan K, Yeung JHK, & Woo KS. 1995. Danggui (Angelica sinesis) affects the pharmacodynamics not the pharmacokinetics of warfarin in rabbits. Eur J Drug Metab Ph 20:41-46.
Luo R-D. 1982. Trimethoprim (TMP) and Shui Yang Mei (Adinarubella) in treating typhoid. Chinese J Modern Dev Trad Med 2(4): 246. Mills S. 1996. Herbal medicines: research strategies. In: Fundamentals of Complementary and alternative Medicine, Churchill Livingstone, International Edition, pp 394-407. Ou M. 1989. Chinese-English Manual of Commonly Used Prescriptions in Traditional Chinese Medicine. Joint Publication (HK) Ltd. Rao XQ & Yu RC. 1990. Clinical and experimental studies on chemotherapy combined with Sheng Xue Tang" (SXT) recipe for the treatment of late stage gastric cancer. Beijing J Trad Chinese Med (1): 46-49 Ruan J & Ye RG. 1994. Lupus nephritis treated with impact therapy of cyclophosphamide and traditional Chinese medicine. Chinese J Integ Trad West Med 14 (5): 276278. Stockley IH. 1996. Dug Interactions: A source book of adverse interactions, their mechanism, clinical importance and management. The Pharmaceutical Press. London United Kingdom. Wan JMF. 2002. The progress of using Chinese herbal medicines in cancer research. In: Chan K & Lee H, Eds. The Way Forward for Chinese Medicine, Harwood Academic Publishers, The Taylor and Francis Group, Chapter 8, pp. 131-153. Wu YC. 1984. Treatment of acute bacillary dysentery with traditional Chinese medicine and western medicine combined. on analysis of 117 cases. Chinese J Integ West Med 4(9): 525-527. Xu Y-Z. (1987). Reduction of side effects of streptomycin by Gan Cao (Radix Glycyrrhizae). Chinese J Modern Develop Trad Med 3(7): 137. Ye RG. 1993. 134 patients with adult primary nephrotic syndrome treated with combined traditional Chinese medicine and Western medicine. Chinese J Integ Trad West Med 13(2): 84-85. Zhen Z-A. 1995. History of Chinese Medicine, Shanghai Science & Technology Press, China. Zhu J-H 1994. The Interaction of Chinese Herbs and orthodox drugs. Peoples Health Publisher Ltd. Beijing, China.
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Use of natural products in anti-cancer alternative therapy: risk of interactions with conventional anti-cancer drugs
[Uso de productos naturales como terapia alternativa anti-cancerosa: riesgos de interacciones con frmacos anticancerosos convencionales]
Idania RODEIRO*, Yack MAGARIO, Omar OCEJO, Gabino GARRIDO and Ren DELGADO Laboratorio de Farmacologa, Centro de Qumica Farmacutica, 200 y 21, Playa, Ciudad de La Habana, Cuba.
*Contact: e-mail: idania.rodeiro@infomed.sld.cu Submitted April 13, 2008; Accepted July 20, 2008; Published Online 15 October 2008
Abstract Many cultures have used plants to treat medical affections and a percentage of modern medicines are obtained from plants. Today, several herbals have been screened for anticancer activity and many patients with cancer take plant extracts in addition to chemotherapy. This review offers an overview of the knowledge about the use of herbals and derivatives as a viable anticancer alternative therapy and their interactions (particularly, modulation of P-450 system and P-glycoprotein) with conventional anti-cancer drugs. It is suggested that health care professionals and patients should be aware of the potential for adverse interactions of this products with the anti-cancer drugs.
Keywords: Natural products, Anti-cancer drugs, Herbal-drug interaction.
Resumen Muchas culturas han usado las plantas para tratar afecciones mdicas y un porcentaje de las medicinas modernas son obtenidas a partir de las plantas. Hoy en da varias hierbas han sido usadas para el tratamiento del cncer y muchos pacientes con cncer toman extractos de plantas en adicin a al quimioterapia. Esta revisin ofrece una panormica del conocimiento acerca del uso de las hierbas y sus derivados como una terapia alternativa viable para el cncer y sus interacciones (particularmente, en la modulacin del sistema P-450 y la glicoproteina-P). Es recomendado que los profesionales de la salud y los pacientes estn atentos al potencial de interacciones adversas de estos productos con las drogas antitumorales.
Palabras clave: Productos naturales, Frmacos anti-cancergenos, Interaccin frmaco-hierba.
INTRODUCTION Every culture throughout history has used plants to treat medical problems. Originally, the specific utility of herbs was assumed to be based on their shape or colour. Today in most of the developing world, plant remedies are the most prevalent treatments, with recipes handed down from generation to generation. Within the last decade there has been a dramatic increase in the sale and use of herbal and food supplements by Western populations and within the UK (Ritchie, 2007). The use complementary and alternative medicine (CAM) by patients with cancer and survivors has been widely studied. Most patients use CAM to 'complement' the conventional therapies of
radiotherapy, chemotherapy and surgery. Health professionals in general have expressed positive views when CAM is used 'complementarily' and not as an 'Alternative'. Results so far published have shown that CAM can contribute to improving the quality of life of cancer patients and their general well-being (Adams and Jewell, 2007) and CAM modalities play a role in supportive care and cancer (Leis and Millard, 2007). For example, today, this kind of therapy is used in many countries of the world, including development countries (Wolf et al., 2006; Simon et al., 2007; Tovey et al., 2006; Montazeri et al., 2007). Not only adult but paediatric cancer patients have used CAM too (GomezMartinez et al., 2007; Lim et al., 2006; Karadeniz et al., 2007; Gzm et al., 2007).
Rodeiro et al
Natural products - anti-cancer drugs interactions
However, the use of CAM in combination with conventional chemotherapeutics has incremented significantly risk for adverse effects, particularly due the narrow therapeutic index of many anti-cancer drugs. Besides, clinical data about pharmacokinetic (PK) interactions of CAM with chemotherapeutic drugs are few, it is suggested that CAManticancer drug interactions contribute significantly to the interindividual variations in PK and clinical problems of unexpected toxicities and under-treatment seen in cancer patients (Marchetti et al., 2007). Patients taking chemotherapeutic drugs and CAM, at least 27% were at risk for developing clinically relevant CAMdrug interactions (Welder et al., 2006). Thus, the consequences of CAMdrug interactions are, a new focus of attention during the pre-clinical and clinical investigations of the chemotherapeutic agents. The objective of this item is to substantially review some preclinical and clinical data on phytochemicals, in terms of their effects as a potential treatment of cancer and their observed interactions with standard therapies. USE OF NATURAL PRODUCTS BY PATIENTS WITH CANCER AND KNOWN DRUG INTERACTIONS Plants have played an important role as a source of effective anti-cancer agents, and it is significant that over 62% of currently used anti-cancer agents are derived in one way or another from natural sources, including plants, marine organisms and micro-organisms (Fig. 1; Gonzales and Valerio, 2006). Among the promising bioactive food components being investigated at the National Cancer Institute in prevention clinical trials to reduce cancer risk are indole-3-carbinol, sulforaphane, phytoestrogen isoflavones, perillyl alcohol, and green tea polyphenols among others (Table 1; Greenwald, 2004). Sulforaphane (SF) Preclinical and clinical evidences. Epidemiological studies suggest that a diet rich in broccoli can reduce the risk of cancer at several sites, including the prostate, lung, breast and colon (Juge et al., 2007). The anticarcinogenic activity has been largely attributed to the biological activity of SF, the isothiocyanate derived from 4-methylsulphinylbutyl glucosinolate, which accumulates in broccoli (Gasper et al., 2007). SF modulates many cancer-related
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events, including susceptibility to carcinogens, cell death, cell cycle, angiogenesis, invasion and metastasis (Zhang and Tang, 2007; Fimognari and Hrelia, 2007). It has been reported in many studies that SF suppresses the growth of some tumoral cells lines (Chuang et al., 2007; Matsui et al., 2007; Mi et al., 2007; Park et al., 2007; Pledgie-Tracy et al., 2007; Shan et al., 2006; Pappa et al., 2007a). These findings suggest that cell vulnerability to SFmediated apoptosis is subject to regulation by cellcycle-dependent mechanisms (Fimognari et al., 2007). Other study provides that SF also acts to inhibit angiogenesis via suppression of endothelial cell proliferation (Jackson et al., 2007). Although, in clinical studies there were not that advances, a formal phase I study of safety, tolerance and pharmacokinetics was carried out (Shapiro et al., 2006). No recent drug interactions or medical contraindications with the use of SF have been reported. Epigallocatechin-3-gallate (EGCG) Preclinical and clinical evidences. Green tea polyphenols are considered beneficial to human health, especially as cancer chemopreventive agents. Some results in animal and epidemiological studies suggest that EGCG the most abundant polyphenol in green tea, could inhibit the invasion and migration of human oral and colon cancer cells and that the effects may partially because of the decreased productions of MMP-2, MMP-9 and uPA (Ho et al., 2007). It is demonstrated that EGCG can inhibit the growth of some cancer cell lines, including hepatocellular carcinomas cell lines HLE, HepG2, HuH-7 and PLC/PRF/5 through the induction of cell-cycle arrest (Nishikawa et al., 2006), in ovarian carcinoma cell lines HEY and OVCA 433 (Spinella et al., 2006a,b), in human colorectal cancer cell lines HT-29 and HCA-7 (Hwang et al., 2007; Peng et al., 2006). Not only EGCG but other flavonoids such as quercetin and rutin have anticarcinogenicity and antitoxicity properties through inhibition of oxidative activation (Hu et al., 2006). Effects on drug-metabolizing enzymes, Pglycoprotein and pharmacokinetic interactions: Muto et al. (2001) demonstrated that epigallocatechin gallate inhibit the in vitro activity of CYP1A1/2 and CYP3A4. Moreover, a general inhibition of several P-450s (CYP2A6, CYP2C19, CYP2E1) by epigallocatechin gallate has been observed, indicating a non-specific inhibitory effect.
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Figure 1. Anti-tumor drugs approved from 1981-2002 (62% were of natural origin).
Vincristine Irinotecan Actinomicin D Asparaginase Vinorelbine Bleomycin Daunomycin Etoposide Estramustine Mithramycin Megestrol Paclitaxel Vinblastine Streptozocin Leuprolide Idarubicin Epirubicin Mitoxantrone Goserelin acetate Cytosine arabinoside based natural origin on natural compounds Floxuridine Fluorouracil Methotrexate Tamoxifen ANTITUMOR THERAPY Faslodex Aminoglutethemide
Table 1. Preclinical studies with natural products in cancer.
Herbal/Compound Scutellaria baicalensis Garlic (Allium sativum) Saw Palmetto (Serenoa repens) Curcumin Indole-3-carbinol Caffeic acid phenethyl ester Epigalocatechin-3-gallate Genistein Sulforaphane Licopene
Treatment Glioma and colon cancer. Prostate, glioblastoma and colon cancer. Prostate and ovarian cancer. Intestinal, prostate, breast, colon, glioma, prostate and ovarian cancer. Melanoma, colon, prostate, breast and bone metastasis of breast cancer. Glioma and colon cancer. Hepatocellular carcinomas, ovarian carcinoma and colorectal cancer. Breast, prostate, bladder and neuronal cancer. Osteosarcoma, prostate, lung, breast, colon, hepatocarcinoma, ovarian cancer and cervical carcinoma . Prostate, colon, human erythroleukemia and B chronic lymphocytic leukaemia.
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The activation of benzo(a)pyrene, 2-amino-1-methyl6-phenyl-imidazole-(4,5-b)pyridine and aflatoxin B-1 by recombinant CYP1A1/2 and CYP3A4, respectively,was also inhibited by this molecule. These effects have been associated with the chemopreventive properties of catechins observed in rodents. The potential interactions of green tea catechins on the metabolic activation of irinotecan, a prodrug directed to the treatment of metastatic colorectal cancer have been suggested (Mirkov et al., 2007). Recently, a heteroactivation of CYP1A1 by teas and tea polyphenols was observed, which supports the hypothesis that this mechanism of CYP1A1 activation may be part of the explanation for the lack of epidemiological support for cancer prevention observed by tea (Nagle et al., 2006). Genistein (Gen) Preclinical and clinical evidences. Gen, found at high levels in soybeans and soy foods (Meeran and Katiyar, 2008), is a controversial candidate cancer preventive phytochemical whose effects on prostate (Perabo et al., 2008) and breast cancer cells have been the most studied (Lavigne et al., 2007; Moiseeva et al., 2007). The genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone, induced G2-M cell cycle arrest in T47D tumorigenic breast epithelial cells (Vauzour et al., 2007). Genistein has been shown to inhibit human prostate cancer (PCa) cell motility (Craft et al., 2008). Some studies suggest that Gen may be a promising chemopreventive agent against androgen-dependent and independent PCa (Wang et al., 2007). In vitro studies have shown induction of apoptosis and inhibition of cell growth in androgen-sensitive and androgen-independent cell lines (Vaishampayan et al., 2007). But, Gen also in vivo decreases the proliferative potential, retards cancer progression and maintains the integrity of the prostatic epithelial cells (El Touny and Banerjee, 2007). Not only Gen but other bioactive flavonoid (baicalein) (Chao et al., 2007) and dietary isoflavones (biochanin A and daidzein) have been related to anticancer activity on bladder, breast and neuronal cancer respectively (Moon et al., 2007; Lo et al., 2007). Even, a study carried out by Nagata et al. (2007) showed Gen and daidzein were significantly associated with decreased risk of prostate cancer in Japanese men.
Effects on drug-metabolizing enzymes, Pglycoprotein and pharmacokinetic interactions: Genistein and daidzein interact with transporters such as P-gp and the canalicular multispecific organic anion transporter (Evans, 2000). Genistein inhibit Pglycoprotein-mediated efflux of paclitaxel, and its oxidative metabolism catalyzed by CYP 3A4 and 2C8 (Li and Choi, 2007). Biochanin A and formononetin are converted to genistein and daidzein in human microsomes, respectively. Daidzein is a competitive inhibitor of CYP1B1 and genistein exhibited mixed inhibition (Roberts et al., 2004). Curcumin (Cur) Preclinical and clinical evidences: Cur is a polyphenolic compound derived from dietary Asian spice turmeric (Curcuma longa L.), particularly across the Indian subcontinent (Gradisar et al., 2007). It possesses diverse pharmacologic effects including, immunomodulators (Ferguson and Philpott, 2007), antiproliferative (Karunagaran et al., 2007) and antiangiogenic activities (Anand et al., 2007). Cur has been shown to protect against skin, oral, intestinal, prostate, breast and colon carcinogenesis (Surh and Chun, 2007). It has been reported in many studies that Cur suppresses growth of some tumoral cells lines (Shankar et al., 2007; Shankar and Srivastava, 2007; Deeb et al., 2007; Liu et al., 2007; Dhandapani et al., 2007; Mitra et al., 2006; Cao et al., 2007; Xia et al., 2007; Yoon and Liu, 2007). In addition, a recent completed phase II clinical trials suggests that Cur may prove to be useful for the chemoprevention of colon cancer in humans (Johnson and Mukhtar, 2007). Effects on drug-metabolizing enzymes, Pglycoprotein and pharmacokinetic interactions: Curcumin inhibited CYP1A2, CYP2C9, CYP2D6, CYP3A4 and CYP2B6 (Appiah-Opong et al., 2007a). Curcuma drugs might inhibit the catalytic activity of intestinal CYP3A4 (Hou et al., 2007) and recombinant human CYP1A2, CYP3A4, CYP2B6, CYP2C9 and CYP2D6 were inhibited in vitro (Appiah-Opong et al., 2007b). Results demonstrate that curcuminoids effectively inhibit MRP1-mediated transport (Chearwae et al., 2006) and also mediate down-regulation of intestinal P-gp (Zhang et al., 2007).
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Indole-3-carbinol (I3C) Preclinical and clinical evidences: I3C, a naturally occurring component of Brassica vegetables, such as cabbage, broccoli, and Brussels sprouts, induces a G1 cell cycle arrest of human breast cancer cells (Jump et al., 2007). I3C could inhibit bone metastasis of breast cancer by inhibiting CXCR4 and MMP-9 expression mediated via the inhibition of the NF-kappaB signaling pathway (Rahman et al., 2006). However, other studies have shown that I3C can induce apoptosis in G361 human melanoma cell (Kim et al., 2006) and in LNCaP prostate cancer cell (Hsu et al., 2006). Not only I3C but its condensation product 3,3'-diindolylmethane are acting by cytostatic mechanisms in human colon cancer cell lines (Pappa et al., 2007b). Furthermore, have been completed a phase I trial in women of the proposed chemopreventive activity that I3C could present (Reed et al., 2006) and also in women with histologically confirmed high-grade of vulvar intraepithelial neoplasia, which showed the potential therapeutic benefits of I3C. Meanwhile, more clinical and scientific investigations are required to support these findings (Naik et al., 2006). Effects on drug-metabolizing enzymes, Pglycoprotein and pharmacokinetic interactions: The potential of I3C to modulate P-gp expression was evaluated in vinblastine (VBL)-resistant K562 human leukemic cells (Arora et al., 2005). Other results indicate that I3C could be used as a novel modulator of P-gp-mediated multidrug resistance in vitro and may be effective as a dietary adjuvant in the treatment of cancers. Garlic Preclinical and clinical evidences: Garlic has long been used as a culinary spice and medicinal herb, cultivated in the Middle East and was first mentioned as medicine in China. Early trials suggested the potential of garlic to lower cholesterol and triglyceride levels in serum (Silgay and Neil, 1994; Warshafsky et al., 1993), but a recent trial has shown almost no benefit (Berthold, 1998). Garlic-derived organosulfur compounds induce apoptosis in many cancer cell lines (Lu et al., 2007) but not in Caco-2 and HT-29 cell lines (Jakubkov and Sedlk, 2006), human glioblastoma T98G and U87MG cells (Das et al., 2007) and some human prostate cancer cells (Kim et al., 2007; Xiao et al., 2006; Arunkumar et al., 2006). Garlic has also been shown to alter blood coagulation by increasing platelet aggregation and
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increasing fibrinolysis (Kiesewetter et al., 1990). Garlic has been used in a variety of conditions, including atherosclerosis, chronic candidiasis, hypertension, hyperlipidemia, hypertriglyceridemia, peptic-ulcer disease, peripheral vascular disease, and sicklecell anemia, and as a chemo-preventative agent for gastrointestinal tumors. Effects on drug-metabolizing enzymes, Pglycoprotein and pharmacokinetic interactions: In vitro studies have shown that garlic constituents modulate the activity of various CYP isozymes. Extracts of garlic inhibit CYP2C9, CYP2C19, CYP3A4, CYP3A5 and CYP3A7 activity, whereas no effect on CYP2D6 was found (Foster et al., 2001; Chen et al., 2003). Rats treated with diallyl sulfide, diallyl disulfide, allylmethyl sulfide and allyl mercaptan had a suppression of CYP2E1 activity as a result of competitive inhibition (Brady et al., 1991; 1988; Haber et al., 1994; Kwak et bal., 1994; Kwak et al., 1995; Reicks and Crankshaw, 1996; Guyonnet et al., 2000; Yang et al., 2001). In addition, diallyl sulfone is known to be a suicide inhibitor of CYP2E1 (Jin and Baillie, 1997). However, long-term administration led to enhanced activity and increased expression of CYP1A, CYP2B (Chen et al., 2003; Sheen et al., 1999; Dalvi, 1992) and CYP3A (Wu et al., 2002). Studies using in vitro and in vivo animal models have also indicated that various garlic constituents used at high concentrations can induce CYP3A4 activity (Raucy, 2003) and conjugating enzymes (Kwak et al., 1994; Guyonnet et al., 1999; Wargovich et al., 1992; Munday and Mundsay, 1999). Reports suggest that CYP2E1 activity is also inhibited in humans receiving garlic for four weeks (Gurley et al., 2002). Clinical studies have shown that garlic no affect the activity of CYP1A2, CYP2D6 and CYP3A4 (Markowitz et al., 2003). Two trials indicated that it significantly decreases the systemic exposure to the HIV protease inhibitors saquinavir (Piscitelli et al., 2002) and ritonavir (Gallicano et al., 2003), which are metabolized by CYP3A4 (Fitzsimmons and Collins, 1997) and substrates for P-glycoprotein (Kim et al., 1998). Saw Palmetto Preclinical and clinical evidences: Saw palmetto (Serenoa repens) is a small low-growing palm tree native to southeastern North America. Saw palmetto is used in men with the hope of toning and strengthening the reproductive system, and
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specifically for symptoms of prostate enlargement (Shi et al., 2008). The main constituents of saw palmetto include carbohydrates, fixed oils, steroids, flavonoids, resin, tannin, and volatile oil (Sorenson and Sullivan, 2007). It has been reported that saw palmetto can inhibit prostate cancer proliferation. In women, the principal use of saw palmetto is to reduce ovarian enlargement and to increase the size of small, undeveloped mammary glands (Ernst, 2002). No drug interactions or medical contraindications with the use of saw palmetto have been reported, meanwhile, it would be prudent to avoid concomitant use with other hormonal therapies (eg, estrogen replacement therapy and oral contraceptives), which may provide an additive effect. Mangifera indica (Vimang) Preclinical and clinical evidences: Vimang is a nutritional supplement obtained from stem bark of Mangifera indica L. (mango). This aqueous extract is used in Cuba in presumably healthy people with environmental, nutritional risk factors, or population with chronic diseases to increment the quality of life through the increase of antioxidant mechanisms (Garrido-Garrido et al., 2007). Ethnomedical studies lead to a great improvement of quality of life in patients with cancer (Tamayo et al., 2001). Preclinical studies have demonstrated anti-angiogenic effects of Vimang in different cell lines (unpublished data). The active ingredient of Vimang formulations consists on a defined mixture of polyphenols, terpenoids, steroids, fatty acids and microelements that imparts unique properties to these formulations like antioxidant supplement (Nnez et al., 2002). Effects on drug-metabolizing enzymes, Pglycoprotein and pharmacokinetic interactions: Studies performed using rat hepatocytes culture showed Vimang did not modify CYP2C, 2D1, 2E1 and 3A1 activities, but it changed oxidations catalyzed by CYP1A2 and 2B1. It also reduced CYP1A2 activity, which may be caused either by a reduction of CYP1A2 levels induced by Vimang or by direct interaction of some component(s) of the extract with the catalytic function of the enzyme. The marked concentration-dependent decrease in CYP1A2 activity observed in hepatocytes shortly incubated with Vimang revealed a direct interference at the activity level (Rodeiro et al., 2007). Subsequent studies using human microsomes permitted to confirm previous findings on CYP1A activity observed on rat hepatocytes, but inhibition on
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human CYP3A activity was found. This apparent discrepancy could be due to different catalytic properties of rat CYP3A1 and human CYP3A4, and/or to differences in the metabolism of Vimang components in both species. No drug interactions with the use of Vimang have been reported, meanwhile, the in vitro results suggest that potential interactions with anticancer drugs metabolized for these isoforms must be considered. Others Scutellaria baicalensis (SB) is a Chinese herbal medicine historically used in anticancer therapy. Baicalin is the major anti-cancer component of SB (Kumagai et al., 2007). They inhibit the growth of H460 human lung nonsmall carcinoma cell line (Leung et al., 2007), LNCaP and PC-3 human prostate cancer cell lines (Ye et al., 2007) and malignant glioma cells (Scheck et al., 2006). Not only SB but other herbs such as, Artemisia annua (Chinese wormwood), Viscum album (European mistletoe), resveratrol and proanthocyanidin (grape seed extract), Magnolia officinalis (Chinese magnolia tree), Ginkgo biloba, quercetin, Poria cocos, Zingiber officinalis (ginger), Panax ginseng (ginseng), Rabdosia rubescens hora (Rabdosia), and Chinese destagnation herbs can enhance the efficacy of the conventional therapies or reduce toxicity (Sagar et al., 2006a), can inhibit angiogenesis (Yance and Sagar, 2006), tumour progression and reduce the risk of metastasis (Sagar et al., 2006b). The aqueous extract of Scutellariae baicalensis Georgi has inhibitory activity against P-gp 170, a multiple drug resistant gene product. Baicalein, one of the major flavones, was found to be responsible for this activity (Lee et al., 2004). In contrast, grapefruit juice inhibits intestinal CYP3A4 (Fattinger and Meier, 2002). Caffeic acid phenethyl ester (CAPE), an active component of propolis, has been implicated in the regulation of cell growth and apoptosis in SW480 (He et al., 2006) and HCT116 human colon cancer cells in vitro (Xiang e al., 2006) and C6 glioma cells in vivo (Tseng and Lee, 2006). Recently, two new propolis-derived prenylflavanones (propolin A and propolin B) showed also induce apoptosis (Chen et al., 2007). CAPE and other plant-derived polyphenols such as, resveratrol, silymarin, flavopiridol, emodin, piperine, oleandrin, ursolic acid, and betulinic acid can sensitize tumor cells and inhibit pathways that lead to cancer treatment resistance (Garg et al., 2005).
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Figure 2. ATP-binding cassette drug transporters involved in the transport of chemotherapeutic drugs.
9-aminocamptothecin, daunorubicin, epirubicin, etoposide, lurtotecan, mitoxantrone, SN-38, topotecan.
BCRP
methotrexate, SN-38G, sulfinpyrazone, vinblastine.
M R P 2
Drug transporters
M R P 1
P-glycoprotein
chlorambucil, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin, irinotecan, melphalan, methotrexate, mitoxantrone, teniposide, topotecan, vinblastine, vincristine.
actinomycin D, bisantrene, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, irinotecan, mitomycin C, mitoxantrone, paclitaxel, tamoxifen, teniposide, tipifarnib, topotecan, vinblastine, vincristine.
Figure 3. Metabolizing enzymes involved in the metabolism of chemotherapeutic drugs.
CYP1A1, CYP1A2
CYP2A6
CYP2B6, CYP2C9
Dacarbazine.
Cyclophosphamide, ifosfamide, tegafur.
Cyclophosphamide, ifosfamide.
CYP2C8
CYP2C19
CYP2D6
Cyclophosphamide, ifosfamide, paclitaxel.
Teniposide.
Tamoxifen, doxorubicin, vinblastine.
CYP2E1 CYP3A4 Dacarbazin Teniposide, etoposide, epipodophyllotoxin, cyclophosphamide, ifosfamide, vindesine, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, irinotecan, tamoxifen, tipifarnib, gefitinib, imatinib.
CYP3A5
Etoposide, tipifarnib.
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Tomatoes (Lycopersicon esculentum Mill.) are the principal dietary source of lycopene (Lic), which is one of carotenoid and is highly beneficial in preventing some diseases such as the cancer (Wan et al., 2007). Lic has been suggested the most promising agents for decreased prostate cancer risk (Thompson, 2007; Peters et al., 2007; Von Lw et al., 2007; Jian et al., 2007) and it can affect growth of different human tumour cell lines (Palozza et al., 2007; Salman et al., 2007). Although, in 2004, the U.S. Food and Drug Administration (FDA) found no credible evidence to support an association between Lic intake and a reduced risk of prostate, lung, colorectal, gastric, breast, ovarian, endometrial, or pancreatic cancer and also found no credible evidence for an association between tomato consumption and a reduced risk of lung, colorectal, breast, cervical, or endometrial cancer (Kavanaugh et al., 2007). Herbal remedies with the potential to modulate cytochrome P-450 activity and thus participate in interactions with conventional drugs include Milk thistle, Angelica dahurica, ginseng, Danshen and liquorice (Ioannides, 2002). Clinical relevance of natural productsanticancer drug interactions An increasing number of cancer patients are using CAM in combination with their conventional chemotherapeutic treatment. Therefore patients who are subjected to combination treatments face considerable risk of drug-drug interaction. Importantly, although there are plenty of experimental data available, large epidemiological trials to underline the antitumoral effects of natural products-anticancer drug combination are scarce. Hence, if potential drug interactions are to be predicted, it is essential that the ability of herbal products to interfere with drug-metabolizing enzyme systems is fully established. Most of the herbal formulations or pure compounds extracted from herbals are known to enhance the cytotoxic effect of drugs through different mechanisms. For example, the polyphenol mangiferin, a C-glucosylxanthone, potentiates cisplatin-, vincristin-, doxorubicin-, etoposide-, adriamycin- and araC-mediated cell death through suppression of NF-B activation (Sarkar et al., 2004). An additive antiproliferative effect was note with the combination of genistein, a soy derived
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phytoestrogen, and tamoxifen on cancerous breast cells (Tanos et al., 2002). Another example of the effect of CAM on the pharmacokinetic of anticancer drugs is St Johns Wort (SJW, Hypericum perforatum). In cancer patients using SJW, a widely used herbal product, in combination with irinotecan, the plasma levels of SN-38, the active metabolite of irinotecan, were 42% lower (Mathijssen et al., 2002). Chemoprevention by edible phytochemicals is now considered to be an inexpensive, readily applicable, acceptable and accessible approach to cancer control and management but more study is warranted. The clinical importance of herb-drug interactions depends on many factors associated with the particular herb, drug, drugs transporters (Fig. 2) and drugs metabolizing enzyme (Fig. 3). Herbs should be appropriately labelled to alert consumers to potential interactions when concomitantly used with drugs and to recommend a consultation with their general practitioners and other medical carers (Hu et al., 2005). CONCLUSIONS We here shown the rich variety of natural products related with cancer prevention and treatment. As the intake of CAM is increasing, extra care must be taken with the consumption of these products when using coventional cancer treatments as the wide molecular mechanisms they can target may interact with those of the standard therapies. It is a fact how many cancer patients use CAM and standard therapies together without the knowledge/advice of their healthcare providers. Knowledge about how these products modulate the many metabolizing enzymes and/or drug transporters, which may lead tolower therapeutic efficacy and/or adverse effects of conventional drugs is limited. It is therefore important that physicians are aware of the possibility of CAMdrug interactions; more when the resultant action of it could are not known or not enough studied. Physicians must be prepared to discuss CAM use with their patients and to be aware of potential CAManticancer drug interactions without prejudices.
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Dalvi RR. 1992. Alterations in hepatic phase I and phase II biotransformation enzymes by garlic oil in rats. Toxicol Lett 60:299-305. Das A, Banik NL and Ray SK. 2007. Garlic compounds generate reactive oxygen species leading to activation of stress kinases and cysteine proteases for apoptosis in human glioblastoma T98G and U87MG cells. Cancer 110:10831095. Deeb D, Jiang H, Gao X, Al-Holou S, Danyluk AL, Dulchavsky SA and Gautam SC. 2007. Curcumin [1,7-bis(4-hydroxy-3methoxyphenyl)-1-6-heptadine-3,5-dione; C21H20O6] sensitizes human prostate cancer cells to tumor necrosis factor-related apoptosis-inducing ligand/Apo2L-induced apoptosis by suppressing nuclear factor-kappaB via inhibition of the prosurvival Akt signaling pathway. J Pharmacol Exp Ther 321:616-625. Dhandapani KM, Mahesh VB and Brann DW. 2007. Curcumin suppresses growth and chemoresistance of human glioblastoma cells via AP-1 and NFkappaB transcription factors. J Neurochem 102:522-538. El Touny LH and Banerjee PP. 2007. Akt GSK-3 pathway as a target in genistein-induced inhibition of TRAMP prostate cancer progression toward a poorly differentiated phenotype. Carcinogenesis 28:1710-1717. Ernst E. 2002. The risk-benefit profile of commonly used herbal therapies: Ginkgo, St. Johns wort, ginseng, echinacea, saw palmetto, and kava. Ann Intern Med 136:42-53. Evans AM. 2000. Influence of dietary components on the gastrointestinal metabolism and transport of drugs. Ther Drug Monit 22:131-136. Fattinger K and Meier-Abt A. 2002. Interactions of phytotherapeutic drugs, foods and drinks with medicines. Ther Umsch 59:292-300. Ferguson LR and Philpott M. 2007. Cancer prevention by dietary bioactive components that target the immune response. Curr Cancer Drug Targets 7:459-464. Fimognari C and Hrelia P. 2007. Sulforaphane as a promising molecule for fighting cancer. Mutat Res 635:90-104. Fimognari C, Lenzi M, Sciuscio D, Cantelli-Forti G and Hrelia P. 2007. Cell-cycle specificity of sulforaphane-mediated apoptosis in Jurkat T-leukemia cells. In Vivo 21:377-380. Fitzsimmons ME and Collins JM. 1997. Selective biotransformation of the human immunodeficiency virus protease inhibitor saquinavir by human small-intestinal cytochrome P450 3A4: Potential contribution to high-risk first-pass metabolism. Drug Metab Dispos 25:256-266. Foster BC, Foster MS and Vandenhoek S. 2001. An in vitro evaluation of human cytochrome P450 3A4 and Pglycoprotein inhibition by garlic. J Pharm Pharm Sci 4:176184. Gallicano K, Foster B and Choudhri S. 2003. Effect of short-term administration of garlic supplements on single-dose ritonavir pharmacokinetics in healthy volunteers. Br J Clin Pharmacol 55:199-202. Garg AK, Buchholz TA and Aggarwal BB. 2005. Chemosensitization and radiosensitization of tumors by plant polyphenols. Antioxid Redox Signal 7:1630- 1647. Garrido-Garrido G, Martnez-Snchez G, Pardo-Andreu G, Garca-Rivera D, Hernndez-Casaa P, Rodeiro-Guerra I, Guevara-Garca M, Delgado-Hernndez R and Nez-Sells AJ. 2007. Recent advances in the research & development of an aqueous stem bark extract obtained from Mangifera
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Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas

Volumen 7, Nmero 6, 2008

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Intracellular fluorescence ng Rho-123/mg protein

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Figure 5 Influence of some phenolics on P-gp expression

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Chieli and Romiti, in preparation

Romiti et al., 2008

EGCG-epigallocatechin gallate; n.d.- non determined; WB- Western blot

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Hypericum extract concentration (g/ml)

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

300 Intracellular fluorescence (% of control) 250 200 150 100 50 0

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

2008 The authors, licensee BLACPMA ISSN 0717 7917

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Intracellular fluorescence ng Rho-123/mg protein

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Figure 5 Influence of some phenolics on P-gp expression

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Chieli and Romiti, in preparation

Romiti et al., 2008

EGCG-epigallocatechin gallate; n.d.- non determined; WB- Western blot

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

Hypericum extract concentration (g/ml)

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

300 Intracellular fluorescence (% of control) 250 200 150 100 50 0

Chieli and Romiti

P-glycoprotein modulation by natural compounds in HK-2 cells

2008 The authors, licensee BLACPMA ISSN 0717 7917

Transportadores de tipo ABC: consecuencias de su interaccin con flavonoides

lvarez de Felipe y Pulido Duarte

Transportadores ABC y flavonoides

lvarez de Felipe y Pulido Duarte

PATIENT'S DETAILS SURNAME _ Other names _

Date started _// _/_/___

Date stopped _/_/_ /_/__

Therapeutic indication

Date stopped _/_/_ _/_/_ _/_/_ _/_/_ _/_/___

Therapeutic indication

Yes No _____ _ _ _____

Date reaction started _// _// _// _//

Date reaction ended _/_/_ _/_/_ _/_/_ _/_/_

Outcome eg. fatal recovered, continuing