Qualitative and Quantitative Assessment of Cytochromes P450 mrna in Human

Transcription

1 Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 37 Qualitative and Quantitative Assessment of Cytochromes P450 mrna in Human Studies in the Liver, Blood and Gastrointestinal Mucosa MARI THÖRN ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2005 ISSN ISBN urn:nbn:se:uu:diva-5786

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3 To my daughters In memory of my dear parents and my niece Marie-Pierre Dammtussar kommer av sig själv. Allt annat måste man anstränga sig för. Anni Kjeldgaard

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5 List of Papers The thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I II III IV Finnström N, Thörn M, Lööf L and Rane A. Independent patterns of cytochromes P450 gene expression in liver and blood in patients with suspected liver disease. Eur J Clin Pharmacol (2001) 57: Thörn M, Lundgren S, Herlenius G, Ericzon B-G, Lööf L and Rane A. Gene expression of cytochromes P450 in liver transplants over time. Eur J Clin Pharmacol (2004) 60: Thörn M, Finnström N, Lundgren S, Rane A, and Lööf L. Cytochromes P450 and MDR-1 mrna expression along the human gastrointestinal tract. Published on the internet March, 30th 2005 by Brit J Clin Pharmacol Thörn M, Finnström N, Lundgren S, Rane A, and Lööf L. Expression of cytochrome P450 and MDR1 in patients with proctitis. Submitted to Eur J Clin. Pharmacol March 22 nd, 2005 Reprints were made with the permission of the publishers.

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7 Contents Introduction...11 Drug metabolism...11 Gastrointestinal tract and its role in drug metabolism...11 Why and how to study drug metabolism?...12 Cytochrome P450 and P-glycoprotein...12 CYP1A CYP1B CYP2E CYP3A Regulation of CYP3A CYP3A P-glycoprotein (P-gp)...19 The interplay between P-glycoprotein and CYP3A Liver transplantation and the metabolisation of calcineurin inhibitors...21 CYP, P-gp and inflammation...22 Aims of the thesis...24 Materials and methods...25 Blood and tissue sampling...25 mrna purification...26 Total RNA purification...26 cdna preparation...26 Real-time RT-PCR...26 Results...29 Paper I. Independent patterns of cytochrome P450 gene expression in human blood and liver...29 Paper II. Gene expression of cytochromes P450 in liver transplants over time...31 Paper III Cytochromes P450 and MDR-1 mrna expression along the human gastrointestinal tract...34 Paper IV Expression of cytochrome P450 and MDR-1 in patients with proctitis...36

8 Discussion...38 Expression pattern of different CYP enzymes in blood elements compared with the liver...38 Expression of CYP enzymes and P-gp in liver transplant grafts...39 Expression of CYP enzymes and P-gp in gastrointestinal mucosa...40 Conclusions...43 Svensk sammanfattning...44 Acknowledgements...46 References...49

9 Abbreviations Ah-receptor aromatic hydrocarbone receptor AUC area under curve CAR constitutive androstane receptor CD Crohn s disease cdna complementary DNA CYP Cytochrome P450 GI gastrointestinal IBD inflammatory bowel disease IBS irritable bowel syndrome LDLT living donor liver transplantation MDR-1 multi drug resistance 1 mrna messengerrna P-gp p-glycoprotein PXR pregnane x receptor RT-PCR reverse transcriptase polymerase chain reaction SNP single nucleotide polymorphism ssdna single stranded DNA UC ulcerative colitis

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11 Introduction Drug metabolism Many drugs and other xenobiotics entering the body have to be metabolised by enzymes before they can be eliminated from the body. Most compounds are lipophilic and biotransformation is therefore needed to make the compounds more hydrophilic so that they can be excreted in the bile or in the urine. This process usually requires two phases. In the phase I reaction, the foreign compound undergoes oxidation or demethylation mediated by the cytochrome P450 system. Most compounds require further metabolisation in the phase II reaction by the conjugation of a water-soluble polar group to a hydroxy group by glucuronidation, for example, or sulphation forming ether or ester linkages [1]. The liver is the main organ responsible for the biotransformation of drugs and other xenobiotics. Most drugs are absorbed intact from the small intestine after oral administration and are then transported via the portal system to the liver. In the liver, they undergo extensive metabolisation and this hepatic first-pass metabolism limits the oral availability of the drug. Some orally administered drugs, such as chlorpromazine, are extensively metabolised in the intestine. The intestinal metabolism may therefore contribute to the overall first-pass effect [2]. Other factors that limit oral bioavailability are hepatic blood flow and protein binding, as well as the chemical properties of the drug, such as its solubility, among other things [3]. Gastrointestinal tract and its role in drug metabolism Although the liver is the most important organ for drug metabolism, it has been shown that the metabolisation of certain orally administered drugs may be unaffected in patients with impaired liver function [4]. Earlier reports provided evidence that, even during the anhepatic phase of liver transplantation, metabolites of some drugs can be detected in the blood. This was shown by Kolars et al. in the case of cyclosporine [5] and by Paine et al. in the case of midazolam [6]. This was subsequently confirmed with cyclosporine [7], [8] and with digoxin and rifampin [9]. 11

12 It has been claimed that, in the case of cyclosporine, as much as 50% is metabolised in the intestine [3] [7]. This supports the hypothesis that some important metabolisation also takes place in the intestine. Why and how to study drug metabolism? Drug treatment is one of the most important ways of treating different medical conditions. It is well known that individuals respond differently to drug therapy. One individual may experience side-effects from a specific dose that has no effect on another individual. The classical way to study drug metabolism in vivo is to give a drug to an individual and measure the drug and/or its metabolites in blood and/or urine. However, this is a time-consuming method and there is a risk of adverse and toxic effects of the drug. Different in vitro methods have been developed in recent decades. Liver microsomes have been used to study cytochrome P450 enzymes, for example [10]. Liver slices can be used to study both phase I and phase II enzymes [11], which is an advantage, as it better reflects the conditions in vivo. In addition, primary cultures of hepatocytes can be used to study both phase I and phase II reactions. However, these two methods are expensive and require optimal conditions [12]. Recombinantly expressed drug metabolising enzymes can be used when the metabolic pathways of a drug are unknown [13]. With this method, however, the enzymes are expressed at high levels and in different proportions compared with the in vivo situation. The development of sensitive methods in molecular biology has facilitated the study of the gene expression of these enzymes in very limited tissue material. This method can also be used to study the expression of cytochrome P450 enzymes in tissues other than the liver. For ethical and practical reasons, liver tissue specimens are difficult to obtain. It would therefore be very convenient if studies of other tissues, i.e. blood as a surrogate organ, could be used to assess individual drug-metabolising capacity. Cytochrome P450 and P-glycoprotein Cytochrome P450 is a phylogenetically very old enzyme system and it is found throughout the animal and plant kingdoms. The oldest CYP gene evolved about 3 billion years ago and the most recent formed about 1 million years ago. When animals emerged onto land about million years ago, there was an increase in the number of CYP genes. It has been proposed that the reason for this was that plants contained compounds toxic 12

13 to animals. It was therefore necessary for the animals to have a system for detoxifying xenobiotics so that they could use the plants as food [14]. The human cytochrome P450 (CYP) superfamily consists of at least 55 enzymes. They are membrane-bound heme-containing proteins responsible for the metabolism of several endogenous compounds, such as steroids, bile acids and fatty acids, as well as drugs and other xenobiotics. The cytochrome P450 enzymes are arranged in families and subfamilies where enzymes sharing 40% of amino acid sequences are assigned to the same family and enzymes sharing 55% belong to the same subfamily. An Arabic number indicates the family, while a letter indicates the subfamily, and another Arabic number for the individual CYP, e.g. CYP3A4 [15]. Fig. 1 Bile acid synthesis CYP7 Hormone synthesis/ metabolism CYP8, CYP11, CYP17, CYP19, CYP21, CYP27, CYP46 CYP Drug metabolism CYP1, CYP2, CYP3 Fatty acid metabolism CYP4 Figure 1 The most important CYP families and their functions. Many CYP enzymes are polymorphically expressed. That means that different populations and individuals may have different drug-metabolising capacity. For example, people from Ethiopia and Saudi Arabia have very high CYP2D6 activity, because they have multiple genes for this enzyme. Drugs metabolised by this enzyme, such as antidepressants, therefore have a very high turnover and the effect of the medication is very limited with ordinary dosing regimens [16]. On the other hand, some people can have completely inactive alleles of this gene, resulting in an impaired ability to metabolise substrates of this enzyme [16]. Another important example is CYP2C9, which is the major CYP enzyme for the clearance of the anticoagulant warfarin. People with a low expression of this enzyme need very small doses of this drug to reach the therapeutic 13

14 interval, even though this polymorphism cannot explain all the variability in dose response to warfarin [17]. All CYP enzymes except CYP2D6 can also be induced or inhibited, which can significantly change the response to a drug [16]. Several methods for evaluating an individual s CYP activity have been described, including the intravenous erythromycin breath test [18] and the oral dapsone recovery ratio for CYP3A activity at least in a white population [19]. The intravenous 14 C-erythromycin breath test has been further evaluated with an oral formulation to aggregate liver and intestine CYP3A4 activity. However, this test also reflects intestinal P-gp. [20]. CYP1A2 activity has been evaluated by the caffeine breath test [21]. Multi-drug cocktails have also been developed; they include the Pittsburgh cocktail, which is a fiveprobe drug mix containing caffeine, chlorzoxazone, dapsone, debrisoquin and mephenytoin. Using this method, multiple CYP activity, CYP1A2, CYP2E1, CYP3A4, CYP2D6 and CYP2C19 respectively, can be evaluated simultaneously [22]. In newer versions of multi-drug cocktails, the probe drug for CYP3A4 has been changed to midazolam. For example, Blakey et al. [23] used caffeine, tolbutamide, debrisoquine, chlorzoxazone and midazolam to evaluate the activity of CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 respectively. However, these methods are often time consuming and expensive and there are risks of side-effects from the probe drugs. The number of known CYP alleles is constantly growing and there is an official CYP allele homepage, which is regularly updated ( The three major subfamilies involved in drug metabolism are the CYP1, CYP2 and CYP3 families. In these studies, we have primarily focused on CYP1A2, CYP1B1, CYP2E1, CYP3A4 and CYP3A5 but also the membrane-bound transporter P-glycoprotein (P-gp). An overview of a few of the most important substrates and inhibitors is given in Table 1. 14

15 Table 1. An overview of a few of the most important substrates and inhibitors CYP 1A2 Substrate endogenous exogenous Induced by caffeine theofyllamine smoking Inhibited by amiodarone 2E1 ketone bodies paracetamol halothane ethanol 3A4 3A5 endogenous steroids atorvastatin cyclosporine Ca ++ -antagonists erythromycin simvastatin terfenadine tacrol imus Ca ++ -antagonists claritromycin cyclosporine tacrolimus ethanol starving diabetes steroids St John s wort rifampin steroids propofol ketoconazole ciprofloxacin grapefruit juice ketoconazole ciprofloxacin grapefruit juice P-gp cyclosporine tacrolimus terfenadine rifampin St John s wort cyclosporine tacrolimus CYP1A2 Caffeine is metabolised by CYP1A2 and is often used as a probe substance for studies of CYP1A2 [24]. It also contributes to the metabolisation of at least different drugs, including theophylline [25], which is partly metabolised by CYP1A2. Cigarette smoke induces CYP1A2 and this leads to lower serum concentrations of theophylline in smokers [26]. CYP1A2 is also known to activate pro-carcinogens such as heterocyclic amines and arylamines [27]. CYP1A2 is generally considered to be a hepatic enzyme, even though this enzyme has been found in the prostate [28], oesophagus [29] and pancreas [30]. At least two mechanisms, one that regulates the constitutive expression and one that regulates the expression during induction, control the concentration of CYP1A2. It has been revealed that there are several regulatory elements in the 5 -flanking region of the CYP1A2 gene, including a xenobiotic responsive element sequence [27] and potential binding sites for transcription factors [31]. It is proposed that the induction of CYP1A2 is mediated at least in part by the Ah (aromatic hydrocarbon) receptor. The Ah receptor activates gene transcription by binding to an enhancer region in the CYP1A2 promoter [32]. It has also been claimed that the activation of pro-carcinogens by CYP1A2 is mediated by the Ah receptor [33]. It has also been shown that benzopyrene carcinogenicity is lost in mice lacking the Ah receptor [34]. 15

16 CYP1B1 This enzyme plays an important role in oestrogen metabolism [35] and it also plays a role in the activation of pro-carcinogens such as polycyclic aromatic hydrocarbons [36]. CYP1B1 does not act primarily in drug metabolism, but there is probably a vital endogenous substrate for this enzyme, as mutation on the human CYP1B1 gene causes congenital glaucoma [37] and it has also been shown that CYP1B1 expression is upregulated during pregnancy [38]. CYP1B1 mrna has been detected in different organs such as the liver, lymphocytes [39] and prostate [28]. However, at the protein level, it is mainly found in tumour cells [40]. There is also a report from Baron et al. [41] detecting CYP1B1 at both mrna and protein levels in human blood lymphocytes. When it comes to the activation of pro-carcinogens, it has been proposed that this is mediated by the Ah receptor, as in the case of CYP1A2 [32]. However, the basal expression regulation is distinct from that of CYP1A2, as it has been shown that it has a different promoter site [42]. CYP2E1 CYP2E1 is responsible for microsomal ethanol metabolism and it was described in the human liver in 1987 [43], [44]. It can also be induced by isoniazid and ethanol and is partly responsible for the metabolisation of acetaminophen [45]. Patients with alcoholic liver disease run an increased risk of developing acetaminophen hepatotoxicity, because ethanol induction increases the formation of reactive metabolites and, furthermore, the glutathione levels are depleted in these patients [46]. CYP2E1 is mainly expressed in the liver, but is also described in lymphocytes [47] and in the intestines [48]. The regulation of CYP2E1 is complex. It has been suggested that it has a post-transcriptional regulation [49]. The mechanism of ethanol induction has been a matter of controversy when it comes to whether it is a result of increased protein synthesis or is due to protein stabilisation. In rats given alcohol, acetone or other low molecular weight compounds, an increase in CYP2E1 protein but not in mrna levels has been observed and it was suggested that this protein stabilisation was mediated by the inhibition of the ubiquitin conjugation pathway [50]. It has also been shown that starvation and diabetes have an inducing effect on CYP2E1 gene expression, probably as a result of activation of the CYP2E1 gene transcription [51], and mrna stabilisation [52]. CYP2E1 is also involved in the activation of many chemical carcinogens, such as nitrosamines and benzene [33]. 16

17 CYP3A4 In overall terms, CYP3A4 is the most important CYP enzyme for drug metabolism and it is estimated that it accounts for the metabolisation of more than 50% of all the drugs in use today [53]. CYP3A4 is the most frequently expressed cytochrome in the liver [53], but it has also been found to be abundant in the gastrointestinal mucosa and especially in the small intestine [54], [55], [56], [57]. It has also been demonstrated in low levels in lymphocytes [58]. As it is the major drug-metabolising enzyme, interactions between different drugs that are metabolised, induced or inhibited by CYP3A4 are common. One well-known example relates to the electrocardiographic changes that take place with the concomitant administration of terfenadine and ketoconazole [59] and terfenadine and erythromycin [60]. Ketoconazole and erythromycin inhibit the metabolisation of terfenadine and cause higher plasma concentrations, which can lead to prolongation of the QT interval and the risk of a specific serious ventricular arrhythmia torsades de pointes. Another clinically important interaction is the increase in oral bioavailability that grapefruit juice produces on several CYP3A4 substrates, such as felodipine and nifedipine; leading to lower diastolic pressure and higher heart rate [61], terfenadine; which may lead to prolongation of the QT interval [62], and cyclosporine; where the absolute bioavailability of orally administered cyclosporine increases [63]. It has been suggested that this effect results from the selective downregulation of CYP3A4 in the small intestine [64]. CYP3A4 is also expressed polymorphically and this leads to insufficient drug concentrations in extensive metabolisers and high concentrations in the poor-metabolism phenotype [15]. The major endogenous substrates of CYP3A4 are steroids where the inactivation of testosterone, progesterone, androstendione and cortisol is catalysed by a CYP3A4-mediated 6 -hydroxylation [65] [66]. CYP3A4 also mediates the 6-hydroxylation of bile acids [67] [66]. Regulation of CYP3A4 It has been known that glucocorticoids induce CYP3A4 since this finding was described by Watkins et al. in 1985 [68], but there has been uncertainty about the role of the human glucocorticoid receptor (hgr) in CYP3A induction [69] [70]. However, the most important mechanism of induction of CYP3A is related to the pregnane X receptor (PXR), which was described by three different groups in 1998 [71], [72], [73]. This receptor, like CYP3A, is mainly expressed in the liver and intestines. PXR is a member of the nuclear receptor superfamily and activates the transcription of CYP3A genes [66]. Another receptor in this family is the constitutive androstane receptor (CAR). It has been shown that these two nuclear receptors share some xenobiotics and ster- 17

18 oid ligands [74] and that CAR mediates the transactivation of CYP3A4 by PXR [75]. St John s wort (Hypericum perforatum) induces hepatic drug metabolism through the activation of the PXR receptor, which leads to lower plasma concentrations of many drugs [76]. A dual effect of dexamethasone on CYP3A4 gene expression has been described by Pascussi et al. Lower concentrations of dexamethasone result in the glucocorticoid receptor-mediated expression of the PXR receptor, which activates CYP3A4. At higher concentrations, dexamethasone binds directly to the PXR receptor and induces CYP3A4. Glucocorticoids therefore play a dual role in CYP3A4 expression not only by controlling the expression of PXR and CAR under physiological conditions in the absence of xenobiotic inducers but also by being able to activate this receptor with a bolus dose equivalent to stress conditions [77]. The induction and inhibition of CYP3A4 can cause dramatic changes in the plasma concentration of drugs. It has been shown that the area under the plasma concentration time curve for orally administered midazolam is 400 times larger during treatment with itraconazole, which inhibits CYP3A4, than with treatment with rifampin, which is a strong inducer of CYP3A4 [78]. Rifampin has also been shown to induce CYP3A4 in the intestine [79]. In the case of other drugs, however, it seems that induction can be organ specific. For example, it has been shown that efavirenz, a non-nucleoside reverse transcriptase inhibitor, induces CYP3A4 in the liver but not in the intestine [80]. CYP3A5 This enzyme appears to share some of the substrates with CYP3A4, although it metabolises fewer substrates [81]. CYP3A5 is expressed in the adult liver of approximately 25% [82] to 74% [83] of the population. It is also expressed in the small bowel [82]. It has been shown to be the dominant CYP3A in the human stomach, colon, kidney and blood [57], [84], [56], [85], [86]. The expression of CYP3A5 is polymorphic and patients with at least one CYP3A5*1 allele express larger amounts of CYP3A5. In these individuals, CYP3A5 may account for 50% of the CYP3A pool and may contribute substantially to the metabolic clearance of CYP3A substrates [87]. There are few studies of the regulation of CYP3A5, but it has been suggested that it shares a common regulatory pathway with CYP3A4, possibly involving some elements in the 5 -flanking region [88]. The possession of single nucleotide polymorphisms (SNPs) is probably more important for the levels of CYP3A5, as described above [87], [89]. The true role of CYP3A5 in drug metabolism is under debate, because of partly conflicting data in the literature [90, 91]. However, in the case of tacrolimus, several reports have shown that CYP3A5 has a significant effect on the metabolism [92], [93], [94], [95]. In 18

19 contrast, in the case of nifedipine, the CYP3A5 genotype did not affect the plasma protein profile or the AUC [96]. P-glycoprotein (P-gp) The multidrug resistance gene (MDR-1), also called ABCB1 (ATP-binding cassette family B) [97], encodes for the membrane-bound, ATP-dependent transporter protein P-glycoprotein (P-gp). It first became renowned for the development of the multi-drug resistance phenomenon [98], especially in tumour cells. P-gp acts like a pump, which leads to a lower intracellular concentration of the substrate [99]. P-gp is found in epithelial cells in the bile canaliculi in the liver, the gut and in the kidneys, where it participates in the excretion of substances into the bile, the gut and the urine [100]. It is also an important part of the blood-brain barrier [101]. It is known that mdr-1a knockout mice develop a form of colitis, which can be prevented by antibiotics [102]. This indicates that P-gp plays a major role in the defence against intestinal bacteria and toxins. Moreover, it has raised the question of whether the reduced expression of MDR-1 may be a contributory factor in the pathogenesis of inflammatory bowel disease (IBD) [103]. It has been claimed that one of the polymorphisms in the MDR-1 gene, C3435T, which leads to a lower P-gp expression, is associated with increased susceptibility to ulcerative colitis (UC) [104]. However, this finding has been questioned [105], [106], [107]. In a study by Brant et al. [108], it was suggested that the MDR-1 Ala893 polymorphism is associated with IBD. These authors also questioned the finding by Schwab et al., as they failed to find evidence of an association between UC or Crohn s disease (CD) and the C3435T SNP. P-gp may also be induced, by rifampin, for example, and it has been shown that this induction can lower the plasma levels of digoxin [9]. It has also been shown that the induction of MDR-1 is mediated by PXR [109]. Apart from the role played by P-gp in the protection of vital tissues by the extrusion of xenotoxic agents, it has been proposed that it plays a part in normal tissue regeneration by protecting the stem cell population from cell death [110], [111], [112]. Recently, some authors have questioned the importance of P-gp in drug absorption, as many drugs have good oral bioavailability despite being P-gp substrates [113]. However, they also suggest that P-gp plays a very important role in the penetration of drugs into the central nervous system (CNS). For example, the development of non-sedating anti-histamines such as loratadine and cetirizine has resulted in the production of few CNS sideeffects, as these drugs are good p-gp substrates [114]. 19

20 The interplay between P-glycoprotein and CYP3A4 P-gp shares many substrates and inhibitors with CYP3A4 [55], [115]. For example, it has been shown that cyclosporine and FK-506 (tacrolimus) are substrates of P-gp [116] and CYP3A4 [53], [117]. It has been shown that drug-drug interactions involving CYP3A4 can be modulated by P-gp [118]. In the enterocyte, P-gp is located on the apical plasma cell membrane and CYP3A4 is located in the endoplasmic reticulum [100], [54] Figure 2. This means that drugs absorbed in the intestinal epithelium are extruded back into the lumen by P-gp [99]. It was shown in an experimental model that this results in an increase in metabolism by CYP3A4 for substrates recognised by P-gp. This occurs because P-gp repeatedly circulates the substrate between the gut lumen and epithelial cells and prolongs the exposure to the metabolic enzyme [119]. It has also been shown that, if P-gp alone is inhibited, the extent of metabolisation of a substrate of CYP3A4 is reduced. For substrates not recognised by P-gp, there is no such decrease in CYP3A4-dependent metabolism [120]. It has also been shown that CYP3A4 and P-gp are regulated in the same way, at least in part, since the induction of both can be mediated by PXR [66], [109]. St John s wort increases the metabolism of drugs metabolised by CYP3A or transported by P-gp, mainly by inducing intestinal levels of this enzyme/transporter mediated by PXR [121]. P-gp P-gp CYP3A4 CYP3A4 Figure 2 Small bowel villi with a schematic sketch of the tip of the villi with P- glycoprotein (P-gp) in the apical membrane and CYP3A4 in the endoplasmic reticulum in the cytoplasm. 20

21 Liver transplantation and the metabolisation of calcineurin inhibitors Liver transplantation has dramatically improved the prognosis in patients with end-stage liver disease. The indication for liver transplantation is mainly chronic liver disease with decompensated cirrhosis or intractable symptomatic liver disease, such as primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), viral hepatitis and alcoholic liver disease. Other more unusual indications include acute liver failure resulting from paracetamol intoxication, for example, or selected cases of primary liver cancer. The one-year survival rates for non-malignant indications for liver transplantation is approaching 90% [122]. To prevent the rejection of the graft, immunosuppression, a combination of several drugs that include steroids and one of the calcineurin inhibitors, cyclosporine or tacrolimus, is used. The pharmacokinetics of cyclosporine [123], [124] and tacrolimus [124], [125], [126] vary considerably within and between liver-transplant patients, especially in the early post-transplant phase. There are several potential explanations for this variation. For example, poor graft function could result from ischaemia or rejection, which can influence the drug metabolism capacity unspecifically [123], [124], [127]. Furthermore, cyclosporine is partly bile dependent, so, when liver graft function is poor, the concentration falls, even though the microemulsion formula Neoral has improved absorption [128]. The absorption of tacrolimus, however, is not bile dependent and its concentration rises with poor graft function as the metabolic capacity falls. These drugs are metabolised predominantly by CYP3A4 [53, 117], but there are also data indicating that CYP3A5 plays a role [129, 130]. Cyclosporine and tacrolimus are also substrates for the transporter P-gp [116]. Recent studies have evaluated whether the genetic polymorphism of CYP3A4, CYP3A5, and MDR-1 has any effect on the blood concentrations of these drugs in kidney-transplant recipients. It has been shown that patients with the CYP3A5*1/*1 genotype require higher doses of tacrolimus, indicating that CYP3A5 plays a significant role in the metabolisation of tacrolimus [92], [93],[95], [94]. Hesselink et al. [92] also showed that carriers of the CYP3A4*1B allele required higher doses of tacrolimus. However, the MDR-1 C3435T polymorphism did not appear to play any role in the tacrolimus dose requirement. In the case of cyclosporine, none of these polymorphisms appeared to affect dose requirement in this study, which is in accordance with other reports [131], [132]. However, Haufroid et al. [94] found that individuals with the CYP3A5*1 allele also required higher doses of cyclosporine but not to the same extent as for tacrolimus. This has raised the question of whether genotyping for CYP3A5 polymorphism should be recommended before kidney transplantation, but this is still the subject of debate [133]. Oral midazolam has been evaluated as a probe for predicting oral cyclosporine metabolisation. However, Paine et 21

22 al. (2000) found no correlation and the authors concluded that there is no universal CYP3A probe, because every substrate will exhibit a unique mix of the different enteric processes that can affect oral drug bioavailability [128]. The expression of P-gp, but not CYP3A4, in the small intestine has been shown to correlate with tacrolimus concentration/dose ratio in a small bowel recipient [134] and in recipients of living-donor liver transplantation (LDLT) [135]. The expression of intestinal MDR-1 was also found to be a prognostic indicator of the outcome of LDLT, where high expression was correlated with poor survival [135]. It has also been shown in a study of rats that a reduction in the blood concentration of tacrolimus was associated with high-dose steroid therapy. This is caused by the induction of P-gp and CYP3A in the liver and the intestines. The authors had first seen this phenomenon in a patient after LDLT [136]. CYP, P-gp and inflammation It is known that inflammation reduces the level and activity of most CYP enzymes, at least in the liver. However, the level of reduction varies between the different CYP enzymes, as they appear to be regulated by different cytokines [137], [138]. Moreover, little is known about the effects of inflammation on CYP and MDR-1 expression in extrahepatic tissues, such as the gastrointestinal mucosa [137]. A reduction in the expression of these enzymes may influence the drug metabolisation capacity. This was shown in children with sepsis [139] and after surgical stress [140]. It has been shown that the mrna expression of CYP3A4 and MDR-1 is influenced by cytokines but in different directions. Whereas the expression of CYP3A4 decreases, the expression of MDR-1 increases in a colon carcinoma cell line (Caco-2 cells) [141]. Saclarides et al. showed that, in patients with ulcerative colitis, Pgp levels in inflamed mucosa increased, but the levels of P-gp also increased in long-standing inactive colitis [142]. However, it has also been shown that P- gp expression decreases in rat livers with induced inflammation [143]. Ulcerative colitis (UC) and Crohn s disease (CD) are inflammatory bowel diseases (IBD) of unknown pathogenesis. In UC, the inflammation is confined to the colon, but, in CD, every part of the gastrointestinal tract may be affected. The inflammation in CD also involves deeper parts of the mucosa compared with UC [144]. It has been suggested that IBD could be triggered by one or more metabolites of xenobiotics, from food or micro-organisms, for example, particularly in combination with polymorphism in the expression of CYP [145]. In a subgroup of patients with IBD, the inflammation is restricted to the rectal mucosa, proctitis. The most usual form of proctitis is ulcerative proctitis, even though Crohn s disease sometimes causes isolated proctitis. 22

23 Proctitis is usually treated with topical enemas or suppositories containing steroids or 5-ASA. This is sometimes combined with oral preparations of these drugs. In spite of this, some patients with proctitis do not respond to treatment [146]. In this context, a contributory factor may be high expression of P-gp, as it has been shown that patients with CD who express higher levels of P-gp more often fail to respond to glucocorticoid therapy [147]. 23

24 Aims of the thesis To study the gene expression of some CYP enzymes in the blood and relate them to the same enzymes in the liver to evaluate whether a blood sample could be used as a surrogate tissue for studying drug metabolism. To study the gene expression of CYP450 and P-gp during the preservation phase and initial phase of liver transplantation and in relation to plasma levels of cyclosporine and tacrolimus. To study the gene expression and distribution of CYP450 and P-gp in the gut in normal mucosa and in patients with inflammatory bowel disease in inflamed gastrointestinal mucosa and in normal mucosa from the same patient. 24

25 Materials and methods Blood and tissue sampling The project and the collection of liver, blood and gastrointestinal samples was approved by the ethics committees at Uppsala University (all papers) and the Karolinska Institutet at Karolinska University Hospital (Paper II). All the patients were only included after informed consent for each study. The liver biopsies (5-10 mg) were obtained as surplus material from routine percutaneous needle biopses performed for histopathological analysis in patients with suspected liver disease and in relation to liver transplantation. The gastrointestinal samples were obtained as extra biopsies in connection with clinical routine endoscopic examination. The biopsies were snap frozen in 70 C liquid nitrogen and stored until further processing. The blood samples for Paper I were collected in EDTA tubes in connection with the liver biopsies. RNA isolation from the blood samples was performed within one hour. The different studies are outlined in Fig 3. Paper I: Independent patterns of cytochrome P450 gene expression in liver and blood in patients with supected liver disease. Paper II: Gene expression of cytochromes P450 in liver transplants over time. Paper III:Cytochromes P450 and MDR1 mrna expression along the human gastrointestinal tract Paper IV: Expression of cytochrome P450 and MDR1 in patients with proctitis. Aim:To compare CYP mrnaexpression in liver and blood. Aim: To evaluate CYP and P-gp mrnaexpression in líver transplants over time. : Aim:To evaluate CYP and P-gp mrnaexpression in the gastrointestinal tract. Aim:To investigate CYP and P-gp mrnaexpression in normal vs inflamed gastrointestinal mucosa. Pat. No : 13 Pat. No : 20 Pat. No : 27 Pat. No : 11 Tissue:Liver, blood CYP:1A2,1B1,2E1, and 3A4 Method:Real-time PCR Tissue:Liver CYP:3A4, 3A5, 2E1, 1A2 and P-gp Method:Real-time PCR Tissue:Gastrointestinal mucosa CYP:3A4, 3A5, 2E1, and P-gp Method: Real-time PCR Tissue:Gastrointestinal mucosa CYP:3A4, 3A5, 2E1, and P-gp Method: Real-time PCR Figure 3 Schematic overview of the studies in this thesis. 25

26 mrna purification The purification of mrna from the liver and gastrointestinal mucosa biopsies was performed using the Quickprep mrna purification kit (Amersham Biosciences, Sweden). Briefly, the liver samples were homogenised mechanically and mixed with oligo(dt) cellulose followed by several washing procedures. After washing, the mrna was eluted with pre-warmed buffer from the kit. After ethanol (95%) precipitation and re-suspension in 50 ul RNAse free water, the amount of mrna was measured at a wavelength of 260 nm, with correction for background at 320 nm. Total RNA purification Total RNA was purified from percutaneous liver biopsies using the SV Total RNA isolation system (Promega) and from blood samples using the RNeasy Blood Mini kit (Qiagen) for the analyses in Paper I. Briefly, a silica column based system where RNA is bound to the column, washed and subsequently eluted with pre-warmed buffer. In Paper II, the purification of total RNA, for comparative analyses vs. mrna, from three human liver donor samples was performed using the phenol-chloroform precipitation technique, as described by Chomczynski & Sacchi [148]. cdna preparation cdna was synthesised using the First Strand cdna synthesis kit (Amersham Biosciences, Sweden). For each synthesis, 80 ng of the collected mrna was used. The sample was diluted with RNAse free water to a volume of 8 µl. For total RNA, 500 ng was used. From gastrointestinal biopsies for Papers III and IV, 50 ng of the collected mrna was used. Real-time RT-PCR The principle of real-time RT-PCR is outlined in Fig 4. This method makes use of probes with fluorescent dyes to measure the PCR products. Specific primers and probes for each analysed CYP enzyme and P-gp were used. The probes were labelled with 6-carboxy-4, 7,2, 7 -tetrachloro-fluorescein (TET ) or 6-carboxyfluorescein (FAM ) dyes at the 5 end and a dark quencher (Dabcyl) at the 3 -end of the sequence (SGS, Sweden, or Cybergene AB, Sweden). The TATA box binding protein (TBP) was used for the normalisation of the mrna values in the liver vs. blood work. It was labelled with the fluorescent VIC. Since TBP was variably expressed in this material, the housekeeping -actin gene was used as an internal control gene 26

27 to enable comparisons between samples in liver transplants and in the gastrointestinal studies. The -actin probe was coupled to the fluorescent FAM. As long as the reporter (R) and quencher (Q) are close to each other, no fluorescence is detected (Fig 4; strand displacement). After the probe has bound to the ssdna, the primer elongates during the PCR and the enzyme will use its exonuclease activity to degrade the probe, thereby separating the reporter from the quencher (Fig 4; cleavage). When the reporter dye is released from the quencher, fluorescence becomes detectable. With an increasing number of cycles, there is an exponential increase in reporter dye fluorescence (Fig 4; cleavage, polymerisation completed). The result is shown in graphs as depicted in Figure 5. Standard curves were generated from three or four standards in different concentrations run at the same time as the unknown samples. All the samples were run in duplicate and at least two no template controls (NTC) were included in every run. Real time PCR using Tacman chemistry Polymerisation Forward primer 5 3 R Probe Q 3 5 Strand Displacement (elongation) Cleavage - R R Q Q (exonuclease activity) R Q Polymerisation complete Figure 4 Real-time PCR: The specific primer and the intact probe, which contains the reporter (R) and the quencher (Q), bind to DNA. AmpliTaque Gold elongates the primer and exhibits 5 -exonuclease activity. When the distance between the reporter and the quencher is long enough, i.e. after cleavage, the fluorescent light from the reporter is detected. 27

28 Rn Threshold Ct 1 Ct 2 Number of cycles Figure 5 This graph describes the formation of the PCR product. The number of cycles is on the x-axis. On the y-axis, the Rn value is shown; this is the intensity of fluorescence from the reporter normalised to the internal fluorescent probe ROX. The threshold (horizontal line) is defined at a level where all the amplification curves are in the early logarithmic phase. The Ct value is defined as the number of cycles where the amplification curve crosses the threshold. The higher starting amount of the template gives a lower Ct value. The amount of material at Ct 1 is consistently higher than the amount at Ct 2. 28

29 Results Paper I. Independent patterns of cytochrome P450 gene expression in human blood and liver (Published in Eur J Clin. Pharmacol (2001) 57: ) Patients with suspected liver disease, mainly mild to severe steatosis, for whom a percutaneous liver biopsy was planned, were asked to participate in the study. A blood sample was collected from 13 patients at the same time as the liver biopsy. A small part (5-10 mg) of the biopsy was used in the study and the rest was sent for histopathological diagnosis. The expression of CYP1A2, CYP2E1, CYP3A4 and CYP1B1 was investigated. The first three enzymes were included because of their importance as catalysts of drug and xenobiotic metabolism, while CYP1B1 was included for its prevalence at high expression levels in the blood. The expression of all CYP enzymes except CYP1B1 was higher in the liver than in the blood. CYP2E1 was the enzyme with the highest mrna expression in the liver, whereas CYP1B1 had the lowest expression in the liver. In contrast, the CYP1B1 mrna levels were the highest of the investigated enzymes in the blood (p< ) (Fig 6 and Fig 7). They were consistently high in all the blood samples, whereas, in most blood samples, the other enzymes, and CYP1A2 and CYP2E1 in particular, were expressed at very low levels and were just 1/1000 of the levels found in the liver. The gene expression of CYP3A4 in the blood was just 3% of the expression level in the liver. The inter-individual variation in CYP mrna was considerable. In the liver, the variation was 28-, 18-, 18- and 26-fold for CYP1A2, CYP1B1, CYP2E1 and CYP3A4 respectively. In the blood, the variation was higher for CYP1A2, CYP2E1 and CYP3A4; 45-, 27- and 60-fold respectively. However, for CYP1B1, the variation in blood expression was less, only 11- fold. No correlation was found between the blood and liver expression of any of the investigated enzymes (ANOVA multivariate analyses). 29

30 CYP 1A2 CYP 1B1 CYP 2E1 CYP3A4 Figure 6 CYP gene expression in liver and blood as displayed by real-time PCR. The black curves indicate the liver-specific expression and the grey curves the expression in blood. LIVER BLOOD CYP1A2 CYP1B1 CYP2E1 CYP3A4-10 CYP1A2 CYP1B1 CYP2E1 CYP3A4 Figure 7 CYP gene expression pattern in liver and blood. CYP1A2, CYP1B1, CYP2E1 and CYP3A4 mrna levels were determined in paired liver and blood samples using real-time PCR. The results are presented as box plots with the horizontal limits of the boxes representing the 25 th and the 75 th percentiles. The vertical lines represent the 95% confidence intervals. 30

31 Paper II. Gene expression of cytochromes P450 in liver transplants over time (Published in Eur J Clin Pharmacol (2004) 60(6): ) Real-time RT-PCR was used for quantitative analyses of mrna specific to CYP3A4, CYP3A5, CYP2E1 and CYP1A2 cytochromes, as well as P-gp. The gene expression of -actin was used as a standard to enable comparisons between samples. CYP3A4 was chosen because it is the principal enzyme for the metabolism of the immunosuppressive drugs cyclosporine [123] and tacrolimus [117]. CYP3A5 and P-gp were analysed because of their close relationship with CYP3A4 and their common array of substrates and inhibitors [81], [118]. CYP1A2 and CYP2E1 were included because of their importance in drug and xenobiotic metabolisation [15]. Biopsies were obtained, after informed consent, before the transplantation (A), after the reperfusion of the liver graft (B). Biopsies were also obtained in the event of suspected rejection (C) and at the one-year follow-up (D) The gene expression of the enzymes is shown in Table 2. CYP2E1 mrna expression was about 1000 times higher than that of CYP3A4. Moreover, CYP3A5 and CYP1A2 were expressed at higher levels than CYP3A4 in the A (p<0.0001), B (p<0.0001) and D biopsies (p<0.05) (Kruskal-Wallis). P-gp was expressed at a lower and relatively constant level (see Table 2 and Fig 8). The median mrna expression increased significantly from biopsy B to D for CYP3A4 (p<0.01), CYP3A5 (p<0.1), CYP1A2 (p<0.01) and CYP2E1 (p<0.05) (Wilcoxon matched pairs test). There was a large inter-individual variation in the expression of the enzymes. For example, the CYP3A4 mrna expression varied 60-fold. To validate the finding of higher CYP3A5 mrna levels compared with the levels of CYP3A4, which was an unexpected result, we tested our method in different ways. We have evaluated whether the use of mrna instead of total RNA would influence the results by purifying both mrna and total RNA from three human donor livers. The analysis revealed no difference in the quantification. We have also analysed the gene expression of these CYP enzymes in four different liver donor samples and one surgical surplus sample from the intestine. The same liver and intestine samples were analysed using a similar method by another laboratory to exclude systematic errors in our laboratory. The results were found to be comparable. We found no correlation between the serum bilirubin levels or the prothrombin index and the investigated mrnas. Although we found no correlation between the plasma level of cyclosporine (r=0.24) or tacrolimus (r=0.26) and the level of gene expression for CYP3A4, we found a relationship between 31

32 low CYP3A4 expression and high, normalised serum levels of cyclosporine/tacrolimus and vice versa (Table 3). These last calculations were made solely on the C and D biopsies at steady-state plasma levels. Log Ratio CYP mrna/ -actin mrna ,1 0,01 0,001 0,0001 A B C D CYP2E1 CYP1A2 CYP3A5 CYP3A4 P-gp Figure 8 The median values of CYP mrna in all investigated samples. The values are normalised on the basis of -actin (BA) gene expression (logarithmic scale). The time points for biopsies are shown on the x-axis: A before transplantation, B after reperfusion, C suspected rejection, D one-year follow-up. The vertical lines from the symbols indicate the 25 th and 75 th percentiles. Logarithmic y-scale. 32

33 Table 2. Cytochrome P450 and P-gp mrna in relation to -actin mrna CYPmRNA/ -actin mrna ratio x 10-3 Median, 75% percentile, 25% percentile BIOPSY A B C D CYP3A CYP3A CYP1A CYP2E P-gp Biopsy A: Before surgery B: After reperfusion C: Suspected rejection D: One-year follow-up Table 3. Relation between CYP3A4 expression and normalized drug plasma concentration (Plasmalevel/dose*weight) of cyclosporine/tacrolimus. The level was defined as low if below the median value and high if higher or equal to the median value. The figures indicate the distribution (%) of low and high levels among 21 observations with simultaneous sampling from blood and liver tissue. CYP3A4 expression ratio (%) Low High Normalised drug Low 14 33* concentration (%) High 39* 14 *= Fischer s exact two-tailed test (p<0.0001) 33

34 Paper III Cytochromes P450 and MDR-1 mrna expression along the human gastrointestinal tract (Published online by Brit J Clin Pharmacol, March 30th 2005 In this study, we evaluated the expression of CYP2E1, CYP3A4, CYP3A5 and P-gp throughout the gastrointestinal tract in 27 individuals. Biopsies were obtained after informed consent in conjunction with routine combined endoscopy of the upper and lower gastrointestinal tract. The patients were referred to evaluate different bowel symptoms and the most frequent diagnosis was irritable bowel syndrome (IBS). Five biopsies were taken from each individual from the following locations: stomach, duodenum, right colon, left colon and rectum. The gene expression was analysed with real-time PCR, as described above. We also evaluated the expression of CYP1A2 in a few patients, but, as we found low or no expression of this enzyme, we did not carry on with these analyses. The gene expression of the investigated enzymes is shown in Table 4 and Figure 9. Among the investigated enzymes, the gene expression of CYP2E1 was highest in all locations (p<0.05-p<0.0001) except in the right colon, where CYP3A5 was equally expressed (Wilcoxon matched pairs test). The peak CYP2E1 gene expression was observed in the stomach and the duodenum (p<0.01, Friedman ANOVA) but with large inter-individual variability (Kendall's coefficient of concordance = 0.16). The levels of CYP2E1- specific mrna in the duodenum exceeded the levels of CYP3A4, CYP3A5 and P-gp by approximately 7, 6 and 300 times respectively. CYP3A4 expression was highest in the duodenum (p<0.001, Friedman ANOVA), with no or low expression in the stomach and colon. The expression of CYP3A5 specific mrna was highest in the duodenum and stomach (p< , Friedman ANOVA). The level of expression was higher than that of CYP3A4 in the other parts of the GI tract, apart from the duodenum. P-gp expression increased from the stomach and duodenum and reached its highest levels in the left colon (p< , Friedman ANOVA). When we evaluated the effect of smoking on mrna expression, we did not find any difference between smokers (n=8) and non-smokers (n=19) for any of the investigated enzymes. Nor did we find a difference related to gender (f=15, m=12), alcohol consumption (more than 50g, n=7) or daily medication (n=16) (Mann-Whitney U-test). 34

35 Table 4. CYP specific mrna/ -actin specific mrna ratio: median (Q25-Q75) CYP2E1 CYP3A4 CYP3A5 P-gp Stomach ( ) ( ) ( ) ( ) Duodenum ( ) ( ) ( ) Right colon ( ) ( ) Left colon ( ) ( ) Rectum ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) CYPmRNA ratio (Log) 100 1e-9 2E1 3A4 3A5 PGP 2E1 3A4 3A5 PGP 2E1 3A4 3A5 PGP 2E1 3A4 3A5 PGP 2E1 3A4 3A5 PGP Stomach Duodenum Rightcolon Left colon Rectum Figure 9 The gene expression of CYP enzymes and P-gp in the gastrointestinal tract. CYP2E1, CYP3A4, CYP3A5 and P-gp gene expression were determined at five locations in the gastrointestinal tract. The results are presented as box plots for all the investigated enzymes/transporter at each location. The horizontal line in the box represents the median. The results are presented as box plots with the horizontal limits of the boxes representing the 25 th and the 75 th percentiles. The vertical lines represent the extreme values. Logarithmic y-scale. 35