Induction of Cytochromes P450 - Toxicology

Current Topics in Medicinal Chemistry 2004, 4, 1745-1766

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Induction of Cytochromes P450 Maurice Dickins* Pharmacokinetics, Dynamics and Metabolism, Pfizer Central Research, Sandwich, Kent, CT13 9NJ, UK Abstract: The induction of cytochromes P450 (CYPs) has been appreciated for some time but an understanding of the mechanisms involved has been poorly understood until recently. The discovery of the role of nuclear receptors such as the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) has provided a major trigger for research in this area. This work has provided an explanation for species differences in hepatic induction. The production of a PXR crystal structure in the presence and absence of known high affinity ligands has offered the possibility of predicting structures which may bind to the receptor and hence act as inducing agents in man. An improvement in the technology of hepatocyte culture, access to good quality human hepatocytes and the miniaturisation of cultured preparations has meant that the potential of this technique to predict induction in man has been realised. Molecular biological techniques have also proved essential in both the science and the quantitation of CYP induction. The use of transient transfection cell based systems coupled with reporter gene assays have meant that dose response curves can be generated for many chemicals. Assays have been developed to measure the increase of the corresponding CYP mRNAs in primary hepatocytes and some cell lines with a high degree of sensitivity and specificity (allowing the quantitation of closely related CYPs). Although CYP induction is not usually considered as a major drawback in drug development, the aim should be to eliminate or reduce the inducing effects of a new drug to a minimum. Thus, it is essential to increase our understanding of the complex mechanisms that regulate induction and to pay attention to both the dose and the physicochemical and structural properties of CYP inducing agents.

INTRODUCTION The concept of induction of drug metabolising enzymes has been known for several decades and a landmark review was published in the 1960s [1]. It was known that multiple administrations of some compounds such as phenobarbitone and carcinogens such as 3-methylcholanthrene could reduce the sleeping times of animals treated with short-acting hypnotic drugs such as pentobarbitone and zoxazolamine. These in vivo assays were used for some time in the pharmaceutical industry as assays for enzyme induction. More recently, hepatic enzyme induction has been assayed by the increased in vitro metabolism of CYP probe substrates, using hepatic microsomal fractions from the livers of animals treated with potential inducing agents as the enzyme source. However, it is now well recognised that animal data is inappropriate to predict induction in humans, because both the extent and pattern of CYP induction may differ markedly between animals and man. To this end, there have been significant advances in human in vitro methods to assess enzyme induction in man. The mechanisms of induction of CYPs have been the subject of intensive research. The earlier focus was on CYPs 1A, particularly when it was realised that these enzymes were involved in the generation of carcinogenic metabolites. *Address correspondence to this author at Pharmacokinetics, Dynamics and Metabolism, Pfizer Central Research, Sandwich, Kent, CT13 9NJ, UK; Tel: (44) – (0) 1304 – 644587; E mail: [email protected] 1568-0266/04 $45.00+.00

Induction of CYP1A1 was shown to involve increased transcriptional activation of the CYP1A1 gene, which resulted in an increase in the levels of the corresponding mRNA and increased production of the CYP1A1 enzyme. The relevance of the Ah (aryl or aromatic hydrocarbon) cytosolic receptor in the induction process was recognised and a number of polycyclic aromatic hydrocarbons (PAHs), including 3-methylcholanthrene, 3,4-benzo(a)pyrene and 2,3,7,8-tetrachloro-(p)-dioxin (TCDD) were shown to have high affinity for the Ah receptor and also be potent inducers of CYP1A1 [2]. The value of genetically different mice in the elucidation of mechanisms of induction was also shown in these studies investigating the induction of CYP1A1. The original work focused on two mouse strains which were either "responsive" or "non-responsive" to the inducing effects of chemical carcinogens. It was subsequently shown that the basis for this responsiveness was due to genetic differences in the Ah receptor in the two mouse strains which meant that CYP1A1 was inducible in the responsive strain by PAHs but was not induced in non-responsive mice [3]. The application of molecular biology techniques in recent years has greatly increased our understanding of the mechanisms of enzyme induction [4, 5]. Although induction of drug metabolising enzymes by phenobarbitone has been known for many years and the presence of a receptor (corresponding to the Ah receptor for CYP1A1 induction) was postulated [6], it is only within the last few years that major advances in this area have been made. The discovery that the nuclear receptor family of proteins was intimately © 2004 Bentham Science Publishers Ltd.

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Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16

involved in the gene regulation of CYP4A via the peroxisome proliferator-activated receptor (PPARα) receptor [7] led to a search which has revealed the identity of corresponding receptors which govern the regulation of CYPs 2B by phenobarbitone and of CYPs 3A by a wide range of chemicals. These receptors are the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) and their central roles in the regulation of not only CYPs but also other drug metabolising enzymes and transporters are becoming increasingly recognised [8]. In particular, animal and human forms of PXR have been widely studied and this has led to an increased knowledge of the reasons for species differences in patterns of enzyme induction by a range of chemicals [9]. The recent publications of crystal structures for human PXR (hPXR) and PPAR isoforms PPARα and PPARγ will further facilitate the identification of ligands for these receptors. Induction of CYPs, in particular the induction of human CYP3A4, is important because CYP3A4 is involved in the metabolism of about 50% of marketed drugs [10]. Recent advances in the field have culminated in the recognition that nuclear receptors such as those characterised for steroid hormones are involved in human CYP3A induction [11]. Although enzyme induction is not considered to be a major drawback in drug development, there may be some impact on the efficacy of coadministered drugs particularly those with a narrow therapeutic index. In these cases, the metabolism of the affected drug would be increased and its pharmacological effect would be reduced. In addition, some drugs may induce their own metabolism ( a phenomenon known as autoinduction) which would also lead to a

Maurice Dickins

reduction in therapeutic effect. Clinically relevant induction is encountered more frequently in certain therapeutic areas e.g. with drugs used to treat HIV, where the regime typically includes multiple drug therapy using high doses of the drugs. Thus, although drug interactions involving enzyme induction are significantly less common and less important than those due to enzyme inhibition, it is still advisable to avoid the use of enzyme inducing agents where possible. The major effects are on drug metabolising enzymes and transporter proteins, but the consequences of inductive effects on endogenous substrates of these proteins are not well understood. THE ROLE OF REGULATION

RECEPTORS

IN

ENZYME

The major mechanism for enzyme induction is via increased rates of transcription. Compounds which induce CYP gene expression through one of four receptordependent mechanisms (Fig. (1)) can transcriptionally activate many CYP genes which code for proteins in CYP families 1-4. These mechanisms can be thought of as being in two groups, each mediated by the nature of the receptors and their heterodimer partners: •

Ah receptor (with a basic “helix–loop-helix” DNA binding domain) which heterodimerises with the Ah receptor nuclear translocator, Arnt)

•

Nuclear receptors (proteins with “zinc finger” DNA binding domains) PXR, CAR and PPARα, all of which heterodimerise with RXR (retinoid X receptor)

Fig. (1). Transcription factors involved in CYP induction AhR = arylhydrocarbon receptor; ARNT = arylhydrocarbon receptor nuclear translocator protein; CAR = constitutive androstane receptor; PXR = pregnane X receptor; PPARα = peroxisome proliferator activated receptor α; RXR = retinoid X receptor.

Induction of Cytochromes P450

It should be noted that some enzymes are induced by other mechanisms which do not involve increased transcription. Enzymes which are primarily regulated by nontranscriptional means include CYP2E1, where increased metabolic function in response to inducing agents is mediated mainly through mRNA and protein stabilisation [12]. Ah Receptor The Ah receptor is a cytosolic helix-loop-helix protein which belongs to the Per - Arnt - Sim (PAS) family of transcription factors and stimulates the transcription of CYP1 genes in the presence of an appropriate inducing chemical. The Ah receptor is activated by binding to the inducing agent in the cytosol and the receptor ligand complex binds to the Ah receptor nuclear translocator (Arnt), forming a heterodimer complex. The complex then binds to response elements on the DNA upstream of the CYP1A1 gene in the nucleus and stimulates transcription of the target gene. It is worth mentioning here that a number of other genes have similar response elements upstream of their coding regions and hence are inducible by compounds which induce CYP1A1. These include CYP1A2, DT-diaphorase, certain isoforms of GSH-S-transferase and UGT and aldehyde dehydrogenase. Thus, it is clear that the Ah receptor not only regulates CYP1 genes but also a battery of other genes involved in drug metabolism. It should be noted that CYP1A1 is readily inducible in a range of extrahepatic cell types and cell lines, whereas CYP1A2 is liver specific and inducible in liver-derived tissues only.


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are now thought to account for the marked species variation seen in the induction of CYPs 2B and 3A [9, 14, 15]. These nuclear receptors are ligand-activated transcription factors which each heterodimerise with another nuclear receptor 9-cis retinoic acid (retinoid X) receptor (RXR) following activation and bind to a corresponding response element upstream of the gene coding region of CYPs. The finding that the response elements for CYPs 2B, 3A and 4A contained motifs which were diagnostic for nuclear receptors strongly suggested that nuclear receptors were involved in the regulation of these genes. The hexameric consensus sequence motif AGGTCA or (A/G)(A/G)(G/T)TCA is found in the respective regulatory regions of genes of the inducible CYP families 2,3 and 4 in a range of species in various formats. The hexameric sequence is either separated by a single base pair as a direct repeat (DR-1 for PPAR/RXR, as a DR-3 (direct repeat with 3 base pair (bp) separation) for rat PXR/RXR, or as an everted repeat separated by 6 bp (ER-6) for human and rabbit PXR/RXR or as a direct repeat separated for 4/5 bp for CAR/RXR. Each receptor in the motif contacts one of the NR binding sites of the repeat Fig. (2).

Nuclear Receptors A number of nuclear receptors (NRs) have recently been implicated in mediating the effects of xenobiotics on CYP gene expression. These include the constitutive androstane receptor β (CAR-β), the pregnane X receptor (PXR) and the peroxisome proliferator- activated receptor α (PPARα) which are primarily involved in the regulation of CYPs 2B, 3A and 4A respectively. CAR and PXR are known as “orphan” nuclear receptors because the endogenous ligand for these receptors has not been identified to date. A nomenclature system for the nuclear receptors has been proposed following the guidelines of the CYP nomenclature. A NR prefix is used for nuclear receptor and the receptors are arranged in families and subfamilies. e.g. PXR and CAR are closely related and are in the same subfamily – they are designated NR1I2 and NR1I3 respectively[13]. Nuclear receptors have both a DNA binding domain (DBD) and a ligand binding domain (LBD). The DBD is highly conserved across species with typically 93-94 % amino acid sequence identity. LBD sequences in nuclear receptors typically also show a similar degree of homology (> 90%) between species. For example, human and mouse PPARα orthologues show 92% protein sequence identity in their LBDs. However, for certain receptors such as CAR and particularly PXR, the LBD sequence is very different across species (70-80% similarity) and it is these differences which

Fig. (2). Heterodimer of PXR-RXR binding to regulatory element upstream of target gene – varied arrangement of the AGGTCA consensus sequence.

It is important to recognise that these NRs are not only responsible for the regulation of CYP genes alone. A number of other drug metabolising enzymes (DMEs) have been shown to be regulated by NRs including UGTs [16] and the transporter P-glycoprotein (P-gp) [17]. PXR (Pregnane X Receptor) In 1998, a human orphan nuclear receptor, human pregnane X receptor (hPXR also known as SXR, [18], or PAR, [19] and its rodent orthologue mouse PXR (mPXR) were isolated as potential receptors which were involved in the regulation of human and rodent CYPs3A. The receptor is named due to the fact that it can be activated very efficiently by natural C21 steroids (pregnanes) [20,21]. However, a range of diverse chemicals and drugs can also activate PXR (Fig. (3) and Table (1). There are several lines of evidence which indicate that PXR is a key factor in the regulation of CYPs3A. •

CYPs3A are inducible by a wide range of structurally diverse compounds but there are major species differences in the response to these xenobiotics [9, 15]

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Maurice Dickins

O O

HO

O

O P

O

O

HO

P O

Hyperfori n (hP XR )

Cl

O

N

N

O

SR -12813 (hP XR )

Cl otri mazole (hP XR and m CAR)

HO AcO

OH OH

OH

O

N

MeO

OH

NH N O OH

O

H

N

H

NMe

O

O

Ri fampicin (hPXR)

RU-486 (M ifepristone) (hPXR )

Cl Cl

O Cl

N Cl

Cl

O

N

N O

N S

N

Cl

Cl CITCO (hCAR)

TCP OBOP (mCAR)

hCAR , hPXR = human C AR, PXR; mCAR = mouse CAR

Fig. (3). Structures of selected PXR and CAR activators.

•

PXR is expressed primarily in the liver and the intestine which are also the major sites of expression of CYPs3A [15, 20].

is a CYP3A inducer in human (CYP3A4) and rabbit (CYP3A6) in vivo and in vitro systems but is not an inducer of rat CYP3A23 [24].

•

CYP3A expression is dysregulated in PXR null (PXR “knockout”) mice [22, 23].

PXR receptors from a wide range of species have been cloned and characterised and the responses to a variety of known CYP3A inducers have been assessed in cell-based systems. Major differences have been seen in the sequences of the ligand binding domains of these receptors, although their DNA binding domains (DBDs) are about 95% identical [9]. The exception to this are rat and mouse PXR – these sequences share 95% amino acid identity throughout the receptor, and it is notable that there is also a close similarity in the nature of the compounds which activate PXR in these species [25]. However, the ligand binding domains (LBDs) of rabbit, rodent and human PXR share only about 75-80% sequence identity. This degree of divergence is unprecedented for

It has been known for some years that there are major species differences in xenobiotic induction of CYPs3A. Transfection studies using animal and human hepatocytes with reporter genes driven by CYP3A reporter sequences showed that species differences in CYP3A were a consequence of the host cell rather than differences in the CYP3A promoters. Thus the typical response to CYP3A inducers of a particular species is maintained in hepatocyte cultures of that species. e.g. pregnenolone carbonitrile (PCN) is a good inducer of rat CYP3A23 in both rat in vivo and in rat hepatocytes but does not induce rabbit CYP3A6 in rabbits in vivo or in rabbit hepatocytes. However, rifampicin


Table 1.


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Some PXR and CAR Activators

hPXR

mPXR

hCAR

mCAR

Rifampicin

PCN

CITCO

TCPOBOP

SR12813

RU-486

PB

PB

Hyperforin

Dexamethasone

Clotrimazole

Nifedipine Ritonavir 5-β-pregnane-3,20-dione RU-486 Lithocholic acid PB Lovastatin PCN = Pregnenolone 16α- carbonitrile ; PB = Phenobarbitone CITCO = 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime. TCPOBOP = 1,4-bis [2-(3,5-dichloropyridyloxy)]benzene hCAR, hPXR = human CAR, PXR ; mCAR, mPXR = mouse CAR, PXR

nuclear receptor orthologues. It is also notable that dog, pig and rhesus monkey show greater degrees of similarity to human in their LBDs [26] and this may well reflect a similar response to xenobiotics with regard to CYP3A induction – however, these species have not been as well studied as rodents. The expression of PXR has been investigated in human, rat, mouse and rabbit. For all four species, PXR mRNA was expressed at the greatest levels in the liver, with lower levels of expression in the gastrointestinal tract. PXR mRNA was also detectable in rabbit, but not rat or human kidney. PXR mRNA has also been detected in mouse kidney and stomach. Thus, the tissue expression of PXR reflects the known major sites of expression of CYPs 3A in the various species [9, 15, 20, 21, 25]. Experiments using PXR null mice have given further evidence for the involvement of PXR in CYP3A gene regulation. In experiments using these animals, no induction of the major inducible mouse CYP3A (Cyp3a11) was observed after treatment with PCN and dexamethasone [22, 23]. The increasing use of genetic techniques to investigate potentially functional polymorphisms has been extended to hPXR. One interesting finding was that of all the variants tested, none of them showed substitutions in the LBD region, suggesting that the binding pocket is highly conserved. Although a number of polymorphisms were identified, some in the coding region, there was little evidence of functional changes [27, 28]. CAR (Constitutive Androstane Receptor) A major recent advance in the understanding of the mechanism of enzyme induction by phenobarbitone (PB)

was made following the discovery of CAR. PB induction cannot be reproduced in continuous cell lines and culture conditions often require optimisation for maximal induction using primary hepatocyte cultures. The groups of Anderson and Negishi identified a 51 base pair (bp) PB-responsive enhancer module (PBREM) in the upstream regions of rat, mouse and human 2B genes [29-31]. The PBREM typically consists of two nuclear receptor (NR) sites which sandwich a nuclear factor 1 (NF1) binding site but only the NR sites are crucial for the response to phenobarbitone-type inducers. Furthermore, the NR sites, but not the central NF1 site are highly conserved between rodent and human genes. CAR binds to each of the PBREM NR sites as a heterodimer with the retinoid X receptor RXR [32]. Negishi and co-workers have shown that the hepatic nuclear receptor CAR is the key regulatory factor which interacts with the PBREM and results in the induction primarily of CYP2B genes (PB-type induction). However, a PBREM has also been found in the UGT1A1 ( the major bilirubin glucuronosyltransferase) regulatory region and is activated by a mechanism involving CAR [16]. This indicates that several of the responses due to PB may be mediated through CAR – a study using differential gene expression to investigate this has indicated that about half of the mouse genes affected by PB were under regulation by CAR [33]. However the mechanism by which CAR exerts its effects appear to be different from both PXR and PPAR, which both act following an initial direct binding of the inducing agent to the ligand binding domain of the receptor. In contrast to PXR, CAR is sequestered in the cytosol of hepatocytes and translocates into the nucleus upon activation (in response to an inducing agent such as phenobarbital)[34]. This translocation phenomenon is common among steroid receptors such as GR (glucocorticoid receptor). However, in

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cell lines such as HepG2, CAR is located in the nucleus [35], even in the absence of CAR ligands or CYP2B inducers. This is in contrast to the situation in primary cells and in vivo, where CAR is predominantly localized in the cell cytoplasm in the absence of CAR ligands or CYP2B inducers. Thus, nuclear translocation of CAR may be a first step in its activation. However, the effects of phenobarbitone on CYP2B expression are blocked by the phosphatase inhibitor okadaic acid, which suggests that dephosphorylation of CAR, rather than direct ligand binding, is involved in its translocation into the nucleus [34]. It seems that activation of CAR thus consists of a number of steps, with nuclear translocation being an early event. It can be independent of ligand binding as illustrated by the fact that phenobarbitone, a well characterized mCAR and hCAR activator, does not bind to hCAR at concentrations up to 1mM [14]. CAR is an unusual receptor in that it shows a high level of constitutive transcriptional activity and can activate expression of reporter gene constructs in the absence of added ligand. Metabolites of the endogenous steroid androstane (androstanol and androstenol) bind to mouse CAR (mCAR) and appear to inhibit the interaction of CAR with the steroid receptor co-activator 1 (SRC-1)[36]. This suggests that “deactivation” is mediated by direct binding to the receptor and that the androstane metabolites are acting as inverse agonists. Thus CAR may be thought of as a “repressed” nuclear receptor in the presence of these steroid metabolites. However, all known mCAR repressors including androstenol have no effect on hCAR [14] and illustrate the problems of extrapolating data from mouse to human [37]. CAR is also activated by some ligands in a manner more characteristic of the other nuclear receptors. Thus the compound 1,4-bis [2-(3,5-dichloropyridyl-oxy)]benzene (TCPOBOP) is a potent activator of mCAR but not of human CAR [14] (Table 1). This is reflected by the fact that TCPOBOP is a potent inducer of mouse CYP2B (Cyp2b10) but not of the human orthologue CYP2B6 which substantiates the hypothesis that CYPs2B are regulated by species-specific forms of CAR. TCPOBOP thus differs from PB in that it is a genuine ligand for mCAR and binds directly to the receptor whereas PB causes translocation of CAR from the cytoplasm to the nucleus [14]. However, the first hCAR agonist , 6-(4-chlorophenyl)imidazo[2,1b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) (Fig. (3) and Table (1) has been identified [38] and this should further increase our understanding of the hCAR pharmacophore and mechanism of activation. Experiments using CAR null mice (CAR "knockout") have shown that the response to phenobarbitone and TCPOBOP is lost when these compounds were administered to mice which lacked the receptor. CAR null mice showed no increase in liver weight when treated with the CYP2B inducers and showed no increased transcription of the Cyp2b10 gene. In addition, PB protected wild type, but not CAR deficient mice from paralysis caused by the administration of the anesthetic agent zoxazolamine. These results demonstrate that CAR is essential for responses to PB type inducers [39].

Maurice Dickins

Recent data have also implicated CAR in the induction of Cyp3a in mice, since the inducing effect of PB or TCPOBOP on Cyp3a was lost in CAR null mice [40]. GR (Glucocorticoid Receptor) The role of GR in the regulation of CYP expression has long been a controversial issue. Glucocorticoids such as dexamethasone are known to be effective inducing agents of rodent CYP3A but dexamethasone is a weak inducer of human CYPs. There is little experimental evidence of the direct involvement of GR in the control of CYPs. However, a number of groups using primary cultured hepatocytes have shown that the inclusion of glucocorticoids (GR ligands) such as dexamethasone or hydrocortisone at submicromolar concentrations ( i.e. concentrations which would only activate GR and not PXR or CAR) enhance the induction of CYPs 2B and 3A. Maurel’s group have suggested a likely explanation for this effect using primary cultured human hepatocytes [41-43]. The expression of the relevant nuclear receptors themselves i.e. CAR, PXR and their heterodimer partner RXR is positively regulated by dexamethasone and endogenous glucocorticoids [41-45]. Thus GR could contribute indirectly to CYP induction, by upregulation of the nuclear receptors which themselves control expression of CYPs. The concentration of the glucocorticoid present in the medium is critical – submicromolar concentrations of GR ligands will control the expression of PXR and CAR. In human serum, the major circulating glucocorticoid is hydrocortisone which is present at concentrations ranging from 0.1 to 0.45 µM [42]. However, dexamethasone is a weak activator of hPXR [14, 46] and concentrations of at least 50µM are required for marked activation of hPXR in reporter gene assays [46]. Using HepG2 cells, the response to glucocorticoids was further dissected by cotransfection experiments with hGR (human glucocorticoid receptor) and hPXR. HepG2 cells were transfected with a 1 kB portion of the CYP3A4 upstream regulatory region which includes response elements for both hGR and hPXR [47]. Hydrocortisone was used as the inducing agent and plasmids containing either hGR, hPXR or both were added to the cultures. The data indicated that hPXR had a predominant role in regulation of CYP3A4, but hGR had an additive effect, a finding that was also seen for rifampicin. The data also suggest that at physiological concentrations, the dominant role of GR is as a regulator of the nuclear receptors rather than as ligands or direct activators of the receptors themselves. Experiments using GR null mice have shown that GR appears to be essential for the induction of CYP2B by steroids such as dexamethasone or PCN in this species [48]. However, induction of CYP3A by glucocorticoids was fully functional in GR null mice The constitutive level of CYP3A prior to induction also remained unaltered in GR null mice compared to the wild type animals. These data indicate that induction of CYP3A is not affected by GR whereas CYP2B induction by steroids is markedly impaired. However, induction of CYPs 3A and 2B by PB in the GR null mouse was diminished by a third compared to wild type mice. Taken together, these data suggest that GR is important in the steroid-mediated up regulation of CYP2B but


not CYP3A in the mouse. However, induction by PB shows a different effect of inducing agents and response elements [48]. In human hepatocytes, Maurel and others have shown that the constitutive expression of CYP2B6 is not changed in the presence of glucocorticoids, whereas CYP3A4 basal expression is increased. Dexamethasone alone did not increase CYP2B6 expression at low concentrations (which would activate GR but not PXR) but this did increase CYP2B6 expression induced by typical hPXR ligands in this system [49]. The interplay of the GR, PXR and CAR receptors together with their differing affinities for various types of inducing agent, all suggest that the overall induction process is due to a combination of nuclear receptor mediated effects.


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required as a heterodimer partner for a number of additional nuclear receptors to bind DNA. In the adult liver, RXRα is the most abundant of the three RXRs (RXRα, RXRβ and RXRγ), suggesting that it may a have a major role as a heterodimer partner with CAR, PXR and PPARs [55]. Experiments using RXRα null mice showed that RXRα functions as a master regulator for basal CYP gene expression. Decreased basal expression of the genes regulated by CAR, PXR and PPARα was observed in the RXRα null mice indicating that RXRα is essential for maintenance of constitutive levels of expression of the genes for CYPs 2B, 3A and 4A. The levels of CAR, PXR and PPARα were unchanged or slightly increased in RXRα null mice. In contrast, expression of the Cyp7a gene in RXRα null mice was markedly increased compared to the wild type animals [55].

PPARs (Peroxisome Proliferator-activated Receptors) PPARα is expressed in metabolically active tissues including the liver, kidney and heart. A number of drugs designed to decrease lipid levels such as the fibrates (e.g. clofibrate, ciprofibrate and bezafibrate) are PPARα ligands. They cause peroxisome proliferation in rats and mice and upregulate the corresponding CYP4A genes in these species leading ultimately to liver carcinogenesis in these species. PPARα null mice fail to show the characteristic biochemical and morphological response to peroxisome proliferators, thus implicating PPARα as a key mediator of this response [44, 45]. PPARα ligands do not cause these effects in nonresponsive species such as guinea pig and man. This is despite the findings that guinea pig PPARα is functional [50] and that humans retain the coding potential for a functional PPARα [51]. The lack of response in these species may in part be due to the much lower amount of the PPARα in the liver of these species compared with the mouse[5, 50, 51]. Richert and others [52] have shown that there is an upregulation of other PPARα target genes (CYP2B and UGT) in human hepatocytes despite minimal effects on CYP4A and peroxisomal enzymes, the major enzymes which are induced in rodents. These data further support the fact that human hepatocytes do not respond to a PPARα ligand, clofibric acid, in the same way as rodents, but some other target genes are enhanced [52]. A number of substituted thiazolidinediones which are ligands for PPARγ are used therapeutically as treatment for Type II diabetes. Several of these PPARγ -specific ligands such as troglitazone, rosiglitazone and pioglitazone have also been shown to upregulate CYPs in human hepatocytes, particularly CYP3A4 [53, 54] and troglitazone is a hPXR activator [9]. Only troglitazone is also a clinically relevant CYP3A4 inducer, and this has been withdrawn from the market due to hepatotoxicity and idiosyncratic liver failure. These effects may or may not be related to chronic exposure to this enzyme inducing agent. RXR (Retinoid X Receptor) The retinoid X receptors are unique among the nuclear receptors in that they bind to DNA as a homodimer and are

Cross–talk of Receptors There is a high degree of similarity between the receptors CAR and PXR. Both are members of the same nuclear receptor subfamily (NR1) and share about 40% similarity in their LBDs. In addition, these receptors share a number of the same ligands or activators such as phenobarbitone and clotrimazole [14]. However, it appears that PXR is not only more promiscuous than CAR with respect to the greater range of compounds which activate this receptor, it may also dominate CAR with regard to expression of CYP3A. In a cell-based assay in which both hCAR and hPXR were coexpressed , hPXR was shown to be the dominant regulator [14]. Nonetheless, in experiments with PXR null mice, which showed the predicted lack of response towards typical CYP3A inducing agents PCN and dexamethasone, induction of Cyp3a11 by PB was not affected. Induction of mouse Cyp2b10 was similar in both wild type and PXR null mice, indicating that mPXR does not regulate mouse Cyp2b induction [22, 23]. This is supported by the experimental evidence that indicates mouse Cyp2b induction is primarily mediated via mCAR [8]. Functional GR was maintained in the PXR null mice – this was confirmed by the finding that induction of tyrosine aminotransferase (TAT) by dexamethasone was similar in the wild type and PXR null mice [56]. These data suggest that induction of mouse Cyp3a is not solely mediated by PXR and must be under some other regulatory control. The most likely candidate for PBmediated increase of mouse Cyp3a is mCAR and recent work has supported this idea. Induction of Cyp3a in mice by PB or TCPOBOP is dependent on CAR, as the effect was lost in CAR knockout mice [40]. It has been shown that CAR can bind to response elements in both the CYP2B6 and CYP3A4 promoter region and that PB activation can drive increased transcription of both genes [14, 31]. In addition, whereas PXR is known to regulate CYP3A expression, it has also been shown that hPXR can upregulate the human CYP2B6 gene by binding PXR-RXR heterodimers in the presence of relevant inducing agents [57].

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The weight of evidence thus demonstrates that CAR and PXR are capable of regulating common genes through the same elements, suggesting that cross talk between the two signalling pathways is an important means of generating a protective response to a xenobiotic challenge [8, 11, 58-60]. The overlap in the response element recognition for CAR and PXR establishes the presence of multiple mechanisms for the regulation of CYPs. Nuclear Receptors Regulate Additional CYPs as well as other Important Proteins Involved in the Disposition of Xenobiotics Treatment with compounds such as rifampicin and PB are known to be implicated in the regulation of a number of other drug metabolising enzymes besides the CYPs which they primarily induce, namely CYPs 3A and 2B respectively. Other CYPs, conjugating enzymes and as shown more recently transporters are also inducible following treatment with certain enzyme inducing agents [17, 22, 23, 61]. Human hepatocytes have been identified as the system which represents the best in vitro cell based assay for the assessment of human induction [56]. Treatment of human hepatocyte cultures with rifampicin or PB results in increased expression of CYPs3A4 and 2B6 and also a number of members of the CYP2C subfamily, notably CYPs 2C8 and 2C9 and, to a lesser extent, CYP2C19 [43, 62-64]. Increased transcription of the CYP2C subfamily members are typically much smaller (2-6 fold) than that of CYPs 3A4 (70-100 fold) and 2B6 (10-15 fold) measured by RNAse protection assays together with functional catalytic activity increases using probe substrates. mRNA levels in human hepatocytes for additional CYPs such as CYP2A6 together with certain UGTs, GSTs and FMOs have also shown to be increased by rifampicin treatment using the technique of differential gene expression together with RNAse protection assay [65]. Identification of nuclear receptor response elements in the regulatory regions of the CYPs2C and UGT1A1 genes strongly implicate the involvement of PXR and/or CAR in the upregulation of these genes [16, 43, 66, 67]. PXR is now known to activate the transcription of various CYP genes but also genes for transporters such as MDR-1, the multi drug resistance protein (which codes for the efflux protein P-glycoprotein, P-gp) and OATP-2, organic anion transporter 2, an influx protein involved with drug uptake [22, 23]. Rifampicin and the anticancer drug taxol (a substrate for CYPs 2C8 and 3A4 as well as P-gp) upregulated both CYPs2C8 and 3A4 together with P-gp in human hepatocytes [17]. Experiments with LS180 (human carcinoma cells) transfected with a PXR construct confirmed the requirement for PXR in the upregulation of P-gp in the intestine. A DR-4 motif which binds PXR has also been identified in the upstream enhancer region of the MDR-1 gene [61]. PXR has also been shown to regulate the expression of the gene which codes for the multidrug resistance related protein (the MRP-2 gene) [68, 69] involved in the excretion of acidic drugs and drug conjugates such as glucuronides.

Maurice Dickins

Protective Effects of PXR Activators Lithocholic acid (LCA) is a toxic bile acid that causes cholestasis when administered to rats. This toxic effect was shown to be prevented by administration of the antiglucocorticoid PCN, which is a known ligand for PXR. Using PXR null mice, it was shown that PXR mediated the hepatoprotective effects of PCN against LCA induced toxicity by a combination of effects [22, 23]. •

Treatment with LCA results in marked induction of Cyp3a11 and Oatp2 in wild type mice – this induction was not observed in PXR null mice, indicating that LCA is a PXR ligand which mediates regulation of both Cyp3a11 and Oatp2

•

PXR null mice were not protected by PCN against the toxic effects of LCA, and hence PXR is required for the protective effect

•

PCN induces Cyp3a11 in PXR wild type mice but not in PXR null mice

•

PCN downregulates CYP7a1, cholesterol 7α hydroxylase, which leads to the decreased formation of bile acids such as LCA - this mechanism is lost in PXR null mice

•

PCN induced Oatp2 in the wild type mouse but not in PXR null mice – Oatp2 is therefore controlled by PXR and its upregulation increases the rate of uptake of LCA into the liver where it can be metabolised by Cyp3a to less toxic metabolites.

These data also suggest a mechanism by which rifampicin, used in the treatment of cholestasis, could exert its effects. Nuclear Receptor Crystal Structures A number of the nuclear receptors have been crystallised in a ligand bound form. The calculated volume of the binding cavity for the nuclear receptors varies considerably. For example, the binding sites of hPXR and hPPARα are considerably larger than those of many other nuclear receptors including the oestrogen receptor (hER), and hRXR (Table 2) [70, 71]. This reflects the variety of ligands which can bind to PXR and PPAR – both receptors can bind high molecular weight ligands (such as rifampicin for PXR (MW = 823). In contrast, nuclear receptors such as thyroid hormone receptor, ER (oestrogen receptor), VDR (vitamin D receptor) and RXR are highly selective for their specific ligands. PXR has evolved to detect a wide range of structurally diverse chemicals. PXR is able to bind both small and large ligands due to the presence of a 12 amino acid flexible loop which may expand the ligand-binding cavity of hPXR. The binding of a large compound such as rifampicin may alter the conformation of this loop, allowing the binding pocket to be enlarged thus facilitating the access of larger ligands [70]. The resulting new binding pocket for hPXR would have an increased volume of 1600 Å3 [72]. hPXR has been crystallized in the absence and in the presence of two high affinity ligands, SR12813 [70] and hyperforin [73]. A


Table 2.


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The Volume of the Binding Cavity for Nuclear Receptors

Receptor

Volume ( Å3 )

Ligand

hPXR

1294

None (apo hPXR)

hPXR

1280

SR12813

hPXR

1544

Hyperforin

hCAR

1170

hPPARα

1400

hER

450

hRXR

687

hVDR

910

comparison of the three crystal structures, apo-hPXR (no ligand), SR12813 and hyperforin structures indicates that hPXR can change its shape and size to accommodate particular ligands. In the SR12813-PXR structure, the binding pocket shrinks slightly whereas for the hyperforinPXR complex, it expands by 250 Å3 [73]. In addition, site directed mutagenesis of putative contact residues have shown that forms of PXR can be generated which show higher affinity for SR12813 and correspondingly lower affinity for hyperforin (compared to “wild-type” hPXR) and other mutants which show the opposite behaviour [73].

modeling studies suggest that the ligand binding pocket of CAR is unlikely to accommodate the variety of ligands which can fit in the PXR binding site [26]. In contrast to hPXR, hCAR lacks structural flexibility in the ligand binding site due to the absence of a corresponding surface loop described for hPXR [72]. Thus hCAR is unlikely to have the diversity of ligands known for hPXR and hence is a less promiscuous receptor.

The ligand binding domain (LBD) of hPXR is structurally based on a three-layered helix which is common to all nuclear receptors [74]. Ligand binding induces significant conformational changes in the folding of the LBD and leads to the recruitment of various coactivator proteins including the steroid receptor co-activator, SRC-1. Previous studies had shown that SR12813 could bind in three different orientations to hPXR [70] and it was not known which was the favoured conformation. However, more recent crystallography studies by Watkins and others have shown that binding of SRC-1 to the surface of hPXR limits the binding of SR12813 to a single conformation, generating a single, active complex [75]. It is not yet known how applicable these results are to other hPXR ligands.

There are a number of systems used to study enzyme induction. In most cases, they could be applied to studies in animal species and humans. Since there are well characterised species differences in patterns of enzyme induction, it should be recognised that data from animals may well be relevant to that species alone. There are known to be large differences in both the nature of the enzymes induced and the extent of induction when comparing animals and man. As a result, there is an increasing emphasis on the use of human in vitro systems, which should give a better prediction of the induction potential in man [77].

The ligand binding domain of hPXR is predominantly lipophilic, but it contains a small number of polar residues (5/28 amino acid residues) [70, 76]. These polar residues appear to have an important role in dictating the ligand specificity of hPXR. Using site directed mutagenesis, four of the polar residues in mouse PXR (mPXR) were mutated to the corresponding amino acids present at the corresponding positions in hPXR. The result was a mouse-human hybrid PXR (“humanised mouse” PXR) which had the ligand specificity and transient transactivation properties of authentic hPXR. This finding indicates that only a small number of amino acids in the LBD of PXR may confer species specificity of PXR. The crystal structure of CAR is not yet available but a homology model has been constructed using hPXR and human Vitamin D receptor (hVDR) as templates [72]. The

CYP INDUCTION IN ANIMALS AND MAN – METHODOLOGY

However, induction in animal species, especially those used as Toxicology species such as the rat, is also of importance since it may explain changes in drug kinetics or other findings seen following multiple administration of a test chemical [78]. Well known examples include •

induction of CYPs4A and subsequent liver carcinogenesis in rodents following the administration of compounds which cause hepatic peroxisome proliferation [79].

•

induction of thyroxine glucuronidation in the rat (but not the mouse) by a number of drugs and xenobiotics leading to increased thyroid weight and subsequent tumour promotion in this organ [80].

Systems and Methodologies to Investigate Induction The major systems and methods used to study enzyme induction are: -

1754


Systems •

Ex vivo samples from animals treated with test chemicals

•

Primary hepatocyte cultures and immortalised cell lines

Methodologies •

Immunoblotting for CYPs, Phase II enzymes and transporters

•

mRNA assays including RT-PCR, Northern blotting and gene arrays

•

Reporter gene assays and nuclear receptor binding assays

•

Catalytic / marker substrate assays

•

In silico methods – prediction of PXR ligands from chemical structure

•

Clinical methodology

Systems Ex- vivo Samples from Animals Since there are well characterised species differences in patterns of enzyme induction, it should be recognised that data from animals may well be relevant to that species alone. There are known to be large differences in both the nature of the enzymes induced and the extent of induction when comparing animals and man [24] as shown in Table (3). Humans and rabbits but not rodents are responsive to rifampicin whereas rat and mouse 3A is readily inducible by PCN, in contrast to humans and rabbits. All these species respond to dexamethasone but the effect is weakest in man. Phenobarbitone induces predominantly CYP2B in rodents, but 3A is preferentially induced in man and rabbit. Finally, peroxisome proliferators induce CYP4A in rodents but not in other species. The most established technique is to take liver samples from animals (usually rats) treated with multiple doses of the test chemical and assay total CYP content and the catalytic properties of the samples (compared to control animals). A Table 3.

Maurice Dickins

number of probe substrates which are selective for individual CYP isozymes are frequently used to assess induction using liver microsomal preparations [81]. This method is advantageous as the liver samples are generally taken from animals being treated in Toxicology studies, therefore any effects seen can be directly related to the Toxicology study. It may be possible to implicate changes in drug kinetics e.g. increased clearance with increasing dose to a change in the levels of CYPs measured by increased probe substrate turnover. For these studies, it is useful to have data from animals of the same strain which have been dosed with prototype inducing agents to selectively increase CYP levels to compare the magnitude of the effect seen with the test chemical [82]. Hepatocytes Primary cultures of human hepatocytes are recognised as the most appropriate in vitro system [56, 83]for investigating potential human CYP inducing agents [62, 64, 84-86]. These cultures can be used not only to generate samples for mRNA analysis of induction using techniques described below but also for analysis of CYP protein and functional analysis using probe substrates. Data comparing results from cell based reporter gene assays and human hepatocytes to investigate clinically relevant human CYP inducers have shown good correlation [46]. However, the availability of fresh human hepatocytes will always be limited and there is a need to maximise this resource [87]. Although cryopreserved rat [81] and human [88] hepatocytes have been shown to respond to CYP inducing agents, it is only recently that this technique has become more widely used for studies of enzyme induction in cultured, cryopreserved human cells [89-91]. Liver Cell Lines Liver cell lines are generally thought to be poor models for assaying CYP induction [92, 93]. This is mainly due to their dedifferentiated state relative to that of intact liver and freshly isolated hepatocytes and the fact that CYPs are constitutively expressed at very low levels [94]. HepG2 (human hepatoma) cells still retain some of the hepatic phenotype, and some induction response has been shown

Species Differences in Drug Induced CYPs -induction of CYPs in Animals does not Necessarily Reflect Induction in Man

Species and major CYPs induced Compound Man Rifampicin

3A>2B

Dexamethasone

3A

PCN Phenobarbitone

Rat/Mouse

Rabbit 3A

3A

3A

3A 3A>2B

PPs PCN = pregnenolone-16α-carbonitrile ; PPs = peroxisome proliferators

2B>3A 4A>2B

3A


[95], but are markedly inferior to primary cultured human hepatocytes as a system to assess human enzyme inducing potential. However, work has been published describing a new human hepatoma line BC2 which is able to respond to prototype human CYP inducing agents [96]. A recent innovation in the use of human hepatocytes for screening of compounds as potential inducers is the immortalised human hepatocyte. Cells have been treated with the Simian virus SV40 T antigen as the immortalising gene and selective culture conditions have maintained the hepatocyte phenotype. A hepatocyte clonal line has been selected and shown to respond to known human inducing agents [97, 98]. Methodologies Immunoblotting The profile of induction can also be assessed by using immuno- or Western blotting to estimate the respective levels of the individual CYPs using antibodies which are selective for CYPs 1A, 2B, 3A and 4A. The CYP apoprotein is detected as the haem moiety is denatured under the conditions used in the separation of the proteins by electrophoresis. This method is relatively time consuming and is at best semi-quantitative, but gives a useful visual estimate of the level of CYP isoform induction [46].


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particularly useful because it can be used to study the relative expression of multiple genes simultaneously before and after treatment with a potential inducing agent. Using this technique, rifampicin has been shown to induce a number of CYP genes in primary human hepatocytes as well as other proteins involved in drug disposition [65]. Reporter Gene Systems and Nuclear Receptor Binding Assay The in vitro transactivation cell based assay is widely used to investigate the effects of chemical-induced changes in gene expression [14, 15, 36, 58, 104, 105]. The most common design of this experiment is to transiently transfect a cell line with an appropriate reporter gene construct and challenge the system with the test chemical at a suitable range of concentrations. The reporter gene data is then recorded and plotted to produce a dose response curve. The activation of a variety of receptors [8] can be assessed using this technique, but the method is illustrated with reference to a PXR receptor reporter gene assay described below [106]. Two plasmid constructs are typically used in studies to investigate CYP enzyme induction (Fig. 4).

Transcription – mRNA Assay These techniques rely on the fact that the majority of induction is under transcriptional control and thus measurement of the corresponding mRNAs can be used to assess the extent of induction. The methods are advantageous in that very small samples of liver or hepatocytes are required from which the RNA can be processed. The inherent sensitivities of the methods means that very small changes in the levels of mRNA can be detected and they can distinguish between very closely related CYPs e.g. 3A4, 3A5, 3A7. However, it is currently uncertain how the fold changes in mRNA expression relate to the fold changes seen with other methods (such as catalytic activity). The changes in mRNA levels may be many fold greater than the corresponding change in the metabolism of a probe substrate [56]. The use of appropriate selective primers means that measurements of the induction of individual CYPs can be made - catalytic assays and Western blotting typically only assess the levels of CYP subfamilies (i.e. CYPs1A, 2B etc) rather than CYP1A1, 1A2, 2B1, 2B2 etc. Various applications of this technology are being used to assess induction of human CYPs including Northern blot analysis [9, 85], quantitative real time PCR (polymerase chain reaction technology) [99, 100], the branched chain DNA assay [46, 101] and the Invader® assay which measures mRNA directly [97, 102]. Another use of this technique is to study the potential induction of non-CYP drug metabolising enzymes and other proteins such as nuclear receptors and transporters. Microarray technology is another approach which is useful for studying mRNA levels within cells [103]. It is

Fig (4). Schematic view of a reporter gene sytem for hPXR.

The reporter plasmid contains the reporter gene e.g. chloramphenicol acetyltransferase (CAT), secretory placental alkaline phosphatase (SPAP) or luciferase and also contains a control element for the gene of interest e.g. CYP3A4, typically part of the upstream regulatory region. For this assay, the construct consists of the PXR response element within the CYP3A1 regulatory region linked to minimal thymidine kinase (tk) promoter and the SPAP reporter gene. The part of this region used in the construct can vary in length from a short (about 20 bp) ER-6 sequence which recognises the hPXR-RXR heterodimer, to a 1kb fragment of the upstream CYP3A4 regulatory region and includes response elements for hPXR, hGR, hER and the hepatic nuclear factors HNF-4 and HNF-5. The 1 kb sequence has been used to investigate the effects of receptors additional to PXR (such as GR) on the induction process by including a plasmid which codes for GR [47].

1756


An expression plasmid containing the sequence necessary to transiently express the nuclear receptor of interest e.g PXR, CAR, PPAR is constructed. Species differences in ligand mediated activation can readily be studied by including the species specific receptor sequence in this plasmid. For example, the hPXR expression plasmid contains nucleotides 1-1608 from the hPXR clone and is necessary to generate full length PXR in the host cells. Typically , PXR will not be expressed in the cell types used in the assay e.g. CV-1 cells (monkey) or HepG2 and HuH7 (human hepatoma cell lines). The two plasmids are transfected into the host cells using a liposome or calcium phosphate based system such as the cationic agent Lipofectamine or Cellphect, respectively and the cells are tested for reporter gene activity. There are many effective reagents available for transfecting cells and the optimal reagent may be plasmid and cell dependent. The cells are then treated with the test compounds and the reporter gene activity is assayed. An increase in the reporter gene activity will be detected if the compound is a nuclear receptor ligand which is directly proportional to increased nuclear receptor activation, and a dose response curve can be generated. El-Sankary et al [105] have described an assay using PXR to generate EC50 (potency) and maximal induction (efficacy) values to rank CYP3A4 inducing agents. For hPXR ligands, rifampicin is included as a positive control inducing agent and its activity at 10 µM used as a 100% value. However, a systematic approach to compare the degree of activation of PXR with enhanced CYP3A4 mediated catalytic activity in human hepatocytes has shown that a PXR reporter gene assay generates complementary data to a functional assay in a target cell population [46]. More recently, hepatoma cells stably transfected with a CYP3A4-luciferase gene construct and hPXR have been described which can determine the potential of compounds to activate PXR. [107]. This system would offer advantages over a transient transfection system. An alternative assay to the reporter gene format is the nuclear receptor binding assay. Using this latter approach, only ligand affinity is assayed and hence it would not be known if the ligand was an activator or a repressor of a particular receptor. Jones and others have described an assay for hPXR using a radiolabelled high affinity ligand, SR12813 [9, 14, 106].

Maurice Dickins

substrates and inhibitors of these enzymes [108]. One such approach involves the construction of models based on crystal structures of bacterial CYPs, mammalian CYP2C5 and most recently human CYP2C9 [109]. The recent publication of crystal structures of hPXR [70, 73, 75] and a homology model of hCAR based on the hPXR crystal structure [72] have meant that the structures of ligands of these receptors may become better defined. However, the number of known, clinically relevant hPXR ligands is very small. Attempts have been made to generate a classification method of PXR ligands based on both affinity and results from hPXR / reporter gene assays using transient transfection systems [105, 110]. The majority of the data relies on the percentage activation of the reporter gene (relative to the response obtained with rifampicin) rather than on EC50 (a measure of affinity) because a dose response curve is infrequently obtained. A pharmacophore model of hPXR has been published to describe the chemical space in the hPXR LBD, using EC50 values generated from both binding assays and transient transfection reporter gene assays to generate the model [111]. Ligands with a range of affinities for PXR were included such as hyperforin (the putative active component of St John’s Wort), EC50 = 25 nM; SR12813 , EC50 = 120 nM, and rifampicin, EC50 = 700nM. This approach may prove to be complementary to other in silico methods of hPXR classification. However, since the PXR binding cavity can change depending on the bound ligand, the ability to generate a set of rules to describe potential PXR ligands will prove difficult using only conventional rigid docking in the hPXR crystal structure. A potential way forward may be to use flexible docking of the respective ligands and proteins together with more dynamic pharmacophore models [112]. At present, prediction of induction using in silico models based on hPXR alone is fraught with difficulties. There are few well characterized clinically relevant CYP inducing agents and in a few cases, e.g. phenobarbitone and phenytoin, drugs may induce in part by mechanisms other than via hPXR, potentially via hCAR. A model may be constructed using induction data generated in human hepatocytes – an intact human liver cell system may be expected to mimic the in vivo situation more realistically than a reporter gene system. However, it is likely that a combination of approaches will be needed to generate a successful in silico model. Clinical Methodology for CYP3A4 Induction in Man

Catalytic Activities

In Silico Screening of PXR Ligands

A wide variety of compounds have been shown to induce human CYPs, although CYP induction does not necessarily increase the metabolism of the inducing agent. For example, omeprazole is an inducer of CYP1A2 [113] whereas the major enzyme involved in omeprazole metabolism is CYP2C19 [114, 115]. Phenytoin and troglitazone are CYP2C substrates but induce CYP3A4. (see Tables 4 and 5). Other compounds, which are themselves CYP3A4 substrates, induce their own metabolism by up-regulation of CYP3A4.

Computational approaches have been widely used for the investigation of the ligand binding sites of CYPs and have given important insights into the structural requirements for

In man, induction is typically measured by two methods – administration of a probe substrate for CYP3A4 or the assessment of endogenous steroid metabolism following

Assessment of enzyme induction by using measurement of catalytic activities of induced CYPs using appropriate model substrates is recommended by the regulatory authorities as the gold standard. Reporter systems were felt only to be appropriate for initial screens since their predictive value is yet to be established [83].


treatment with the potential inducing agent. Midazolam has frequently been used as a probe substrate for the assessment of CYP3A4 induction [116-118]. Midazolam is a useful probe because it is a good substrate for CYP3A4 and is also metabolised by both hepatic and intestinal CYP3A4 [119]. Erythromycin is another CYP3A4 substrate used to assay for CYP3A induction in man. Unlike midazolam, erythromycin is a substrate for both CYP3A4 and P-glycoprotein. The erythromycin test was originally developed a number of years ago with intravenously administered radiolabelled (14C) substrate to assess CYP3A4 enzyme activity in vivo [120, 121]. The method uses the fact that radiolabel is excreted in the breath as radiolabelled CO2, a result of the major route of CYP3A4 mediated metabolism of erythromycin (N-demethylation). CYP3A4 activity could thus be assayed without the need for blood sampling and a specific assay for the metabolite. However, administration by the intravenous route will measure hepatic activity only and an oral stable-labelled (13C) formulation of erythromycin has been developed. An interaction study with rifampicin and erythromycin has been carried out, using both the IV (14C) and oral (13C) erythromycin formulations. The results suggested that both CYP3A4 induction (hepatic and intestinal) and P-glycoprotein induction (intestinal) were involved in the rifampicin-erythromycin interaction, rather than an effect on CYP3A4 alone [122]. The most widely used non-invasive marker of CYP3A induction is by measurement of 6β-OH cortisol/cortisol ratios following treatment with potential inducing agents. A number of drugs that are known, clinically relevant inducers of CYP3A4 have been shown to significantly increase this ratio such as rifampicin [123], phenobarbitone [124], phenytoin [125], troglitazone [126] and St John’s wort [127]. A new non-invasive assay for CYP3A4 induction has been described involving measurement of 4β-OH cholesterol [128]. Phenytoin, a CYP3A4 inducer, was shown to increase the 4β-OH cholesterol/cholesterol ratio in man and the formation of 4β-OH cholesterol was catalysed by CYP3A4 in vitro. Valproate, which does not induce CYP3A4 in man, had no effect on the 4β-OH cholesterol/cholesterol ratio. Table 4.

Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 16 Troglitazone

[138, 139]

Ritonavir [140] St John’s Wort [141] Dexamethasone [142] Modafinil [143] Bosentan [144] Avasimibe [145]

Table 5.

Examples of Compounds which Activate PXR and /or Induce CYPs In Vitro

5β-Pregnane-3,20 dione

Rifampicin

Hydrocortisone

Pregnenolone -16αcarbonitrile

RU-486

Troglitazone

Dexamethasone

SR-12813

Nicardipine

Clotrimazole

Hyperforin

Trans-nonachlor

Simvastatin

Lovastatin

Lithocholic acid

Taxol

Phenobarbitone

Carbamazepine

Phenytoin

Ritonavir

Avasimibe

References [9, 14, 20, 23, 46, 64, 68, 85, 86, 105, 145]

INDUCTION STUDIES WITH SERIES OF COMPOUNDS – LIMITED STRUCTURE ACTIVITY RELATIONSHIPS Taxol and Taxotere O O O

O

O N

OH

O OH

Compounds which Induce CYP 3A in the Clinic

HO O

O

H O

O

Macrolides

O

Taxol

Rifampicin [117, 118, 129] Rifabutin [129, 130] Antiepileptics

OH

Phenobarbitone [131, 132] O

Carbamazepine [116] Phenytoin [116] Topiramate [133, 134]

O

N

OH O

OH HO O

Nevirapine [135] Taxotere Others

O

O

NNRTIs

Efavirenz [136, 137]

1757

O

H O

O O

1758


Maurice Dickins

O O NH N

NH

S

N

O

S N

O

O O

Piogl itazone

Rosigli tazone O NH

HO

O

S

O

O Trogli tazone

shown to be inducers of CYP3A4 in vitro [54, 147] although only troglitazone has been shown to be a clinically relevant CYP3A4 inducing agent [138, 139] and troglitazone also induces the 6β-OH cortisol/cortisol ratio at clinical doses[126]. Troglitazone was a more potent inducer of both CYP3A4 and CYP2B6 although the capacity to induce maximum CYP3A4 expression (Emax) was greater for rosiglitazone and pioglitazone. These data suggest that all three compounds have the potential to induce CYP3A4, if sufficient concentrations could be achieved at the target sites of the liver and the gut. However, only troglitazone induces CYP3A4 in the clinic whereas neither rosiglitazone [148, 149]nor pioglitazone [150] elicit clinically relevant changes in pharmacokinetics of co-administered CYP3A substrates. It is notable that troglitazone was given at a much greater dose (200-400 mg/day) than either rosiglitazone (4-8 mg/day) or pioglitazone 45 mg/day [54].

Taxol (paclitaxel) was shown to be a potent PXR activator and induced not only CYP3A4 but also CYP2C8 in primary human hepatocytes. In contrast, Taxotere (docetaxel), a closely related structure did not activate PXR and neither CYPs3A4 nor 2C8 were induced. In addition, neither of the two major metabolites of taxol (6α-OH and 3’p-OH formed by CYPs 2C8 and 3A4 respectively) were activators of PXR [17]. However, the apparent difference in inducing properties between paclitaxel and docetaxol may be, in part, due to differential uptake in human hepatocytes, where total exposure to paclitaxel was three-fold higher than docetaxol [146]. Thiazolidinediones Three thiazolidinedione compounds have been investigated with respect to induction of CYP3A4. Troglitazone, rosiglitazone and pioglitazone have been

HO HO OH AcO

OH OH

OH

MeO

O

OH

O

OH

O

O NH

NH N N O

O N

O

OH

O O

OH

N

O

Rifampi cin

Rifapentine

O O

O O

O

O O

O N

N

O N O

O Ri fabutin

N

N N



Macrolide Antibiotics

of induction mainly driven by its poor lipophilicity. However, induction is also not indicated in clinical studies for the other statins, despite showing marked induction potential in in vitro systems. The statins are all lipophilic, given at relatively low doses of 10-80 mg /day, are typically rapidly metabolised by CYP3A4 (with the exception of pravastatin) and hence would be unlikely to act as inducers in vivo.

Rifampicin and rifapentine are potent inducers of CYP3A4 whereas rifabutin is a much weaker inducer of CYP3A4, as shown using two catalytic markers for CYP3A4 induction [151, 152]. These data are in good agreement with in vivo data that measured increased urinary excretion of 6βOH cortisol after treatment with rifampicin (274% increase) and rifabutin (26%) [129], and a study using rifapentine showed a 189% increase in this parameter [153]. Although all three compounds have similar structures, both of the potent inducers rifampicin and rifapentine have a piperazinyl iminomethyl group which is lacking in rifabutin.

Ca channel Antagonists H N

Certain HMG (Hydroxymethylglutaryl)-CoA reductase inhibitors, also known as statins, have been shown to increase the CYP4A4 reporter gene activity in PXR cotransfection systems. Lovastatin was a potent activator of human PXR in CV-1 cells [20], and both lovastatin and simvastatin (but not pravastatin) activated transcription in a separate study in HepG2 human hepatoma cells [105]. The induction of CYPs in primary human hepatocyte culture by statins was investigated and induction of CYP3A and CYP2B6 was found for lovastatin, simvastatin, fluvastatin and atorvastatin whereas pravastatin was inactive [85] and supported the data of El-Sankary et al.[105]. Although pravastatin is chemically dissimilar to the other statins in that it is much more hydrophilic [154] and requires active transport to enter the hepatocyte [155], it effectively blocked cholesterol synthesis in the hepatocytes as did the other statins. Thus, access to the target receptors in the liver is not restricted for pravastatin and it appears that the compound has limited interaction with CYPs and associated mechanisms

H

H

O O

O N

O

+

N+

O-

O

ONi fedipine

Nicardi pine

Induction of human CYPs by the calcium channel antagonists nifedipine and verapamil was first shown by Strom et al. [156] using primary human hepatocytes. Drocourt et al. (2001) [64] have extended these studies to additional compounds in the dihydropyridine antagonist series and have shown induction of CYP3A by nifedipine, nicardipine and isradipine and a calcium channel agonist BK8644. As for the statins, the dihydropyridine calcium channel antagonists (with the exception of amlodipine) are rapidly and extensively metabolised [157] and hence do not act as inducing agents in clinical use.

O

O

O

O

H

O O

O

H O

N

O

O

O

H O

O

HO

OH

HO

O

H N

O

HMG CoA Reductase Inhibitors

HOOC

O

H

H

HO Lovastatin

Pravastatin

S imvastatin

O

N O

N

O N

O Na F

F Fluvastatin

O

O O

O

O

1759

Atorvastatin

Ca

1760


H N

O

O

Cl N

Other NNRTI compounds such as HBY-097 have been reported to induce the clearance of indinavir by induction of CYP3A4 in man [161] and the related analogue GW420867 induces CYP3A4 in primary human hepatocytes [162].

O

N

N

Maurice Dickins

HIV Protease Inhibitors

F 3C

N

Nevirapine

Ritonavir is a widely used HIV protease inhibitor, better known as a potent inhibitor of CYP3A4 than as an inducer. However, ritonavir has been shown to act as an inducing agent of methadone metabolism on chronic dosing [140]. This is thought to be due to the net effect of ritonavir switching from marked inhibition to weak induction with continuous dosing of ritonavir. It should be noted that human hepatocytes treated with ritonavir showed a marked inhibition of testosterone 6β -hydroxylase but induction of CYP3A4 was also observed by immunoblotting for CYP3A4 [46, 56]. Similar data has also been reported for another HIV protease inhibitor DPC 681 [163]. Although the compound was shown to induce its own metabolism in man, testosterone 6β -hydroxylase in human hepatocytes was inhibited by DPC 681. Further support for the inducing properties of both ritonavir and DPC 681 is that both have been shown to be activators of human PXR [46, 163]. Ritonavir binds to hPXR (ligand binding assay) and activates hPXR with an EC50 of ~ 2 uM, corresponding to the circulating concentration of the drug and hence is a pharmacologically relevant concentration. Other protease inhibitors are either much weaker ligands (saquinavir) or do not activate hPXR [68].

Efavirenz H N

O

S S

N O

O

HBY-097

Agents Used in the Treatment of HIV Infection NNRTI Compounds Nevirapine and efavirenz are the two most widely studied non–nucleoside reverse transcriptase inhibitors which induce CYP3A4, and induce the metabolism of CYP3A4 substrates such as HIV protease inhibitors (reviewed in Barry et al. [158]. The drug used in the treatment of opiate addiction, methadone, is also a CYP3A4 substrate. Recent studies have shown that drug addicts with HIV infections are susceptible to opiate withdrawal symptoms, due to increased metabolism of methadone by nevirapine [135] and efavirenz [136]. Efavirenz was also shown to induce hepatic CYP3A4 but not intestinal CYP3A4 [137] in contrast to rifampicin which induces CYP3A4 in liver and intestine [159]. Efavirenz has also been shown to activate hPXR [160].

THE CLINICAL RELEVANCE OF HUMAN IN VITRO DATA ON INDUCTION A typical enzyme inducing agent is lipophilic and is unlikely to be cleared rapidly from the body. The body’s response to administration of an inducer is to increase the levels of proteins such as CYP3A4, other CYPs, conjugating enzymes such as UGT (UDP-glucuronosyltransferase) and transporter efflux proteins which will increase the rate of

S O

N O

O N

N

O

H

N

N

N

N

S

OH

O

O

H

O

Ri tonavi r

s aquinavir

O H N

N H

O

O S

N H

O

N OH

DP C-681

N O NH 2

NH O

F

N

N

NH 2


elimination of a co-administered compound. For example, phenobarbitone can increase the clearance of compounds which are metabolized by CYP3A4 (cyclosporin) CYP2C9 (warfarin) and UGT (lamotrigine). In addition, some compounds will induce their own metabolism (autoinduction); carbamazepine is an extensively studied autoinducer [164, 165] and other compounds which show this characteristic include troglitazone, ritonavir, efavirenz and cyclophosphamide [166]. Although a large number of compounds have now been shown to activate PXR, the major receptor involved in the up-regulation of CYP3A4, it is noteworthy that not all the compounds bring about clinically significant induction. The concentrations of test compounds used in vitro should be representative of the concentrations achievable during clinical use. The relationship between EC50 or “potency” and the %max or “efficacy” in a PXR reporter gene assay may be difficult to interpret in terms of clinical relevance. Compounds which are potent hPXR activators such as hyperforin, a constituent of St John’s wort (hPXR EC50 = 25nM) or rifampicin (hPXR EC50 about 1µM and is given at high doses) are clinically relevant inducing agents in man. However, other compounds which elicit a strong response in this assay may in practice be non inducers in man e.g. lovastatin and simvastatin which are rapidly metabolised by CYP3A4. It should be noted that clinically relevant induction of ADME targets occurs far less frequently than inhibition. Induction studies are also more difficult to carry out experimentally than inhibition studies, but the advent of newer technologies such as mRNA analysis has greatly increased the throughput of compounds. Moreover, increased transcription is the predominant mechanism for human enzyme induction, although protein or mRNA stabilisation may contribute in some cases. The magnitude of the increased levels of message to functional de novo synthesised protein remains to be established, although estimates have been made. For example, a ratio of about 5:1 has been reported between the fold induction observed in CYP3A4 mRNA levels and that of a corresponding enzyme activity in human hepatocytes in response to rifampicin [56]. Functional assays using probe substrates for individual CYPs will continue to be needed for induction assays. However, difficulty in the interpretation of the induction response may arise if the test compound is also an inhibitor of CYP3A4, particularly a potent inhibitor such as ritonavir [46]. In such cases, any increase in the catalytic assay used may be nullified by the inhibitory action of the test compound. An increase over control in other in vitro assays such as mRNA, PXR or immunoblot would indicate that the test compound had the potential to be an inducer of CYP3A4 in man. Uptake transporters may play an important role in enzyme induction. It has recently been shown that rifampicin is a substrate for the human liver specific uptake transporter OATP-C and that OATP-C expression potentiates PXR activation [167]. Furthermore, whereas functionally relevant polymorphisms of hPXR are rarely evident in man, human OATP-C has a number of naturally occurring variant forms which have markedly impaired function [167, 168]. This may in part explain the large inter-individual differences in


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the degree of hepatic CYP3A4 induction seen in man following treatment with an inducer such as rifampicin [123]. Additional variation may also be due to the sites of induction – for example, rifampicin induces CYP3A4 in both the liver and the intestine, whereas efavirenz appears to induce hepatic CYP3A4 alone [137]. A human inducing agent is typically characterised by a chronic dosing regime using large doses of the chemical compound e.g. rifampicin and drugs used in the treatment of HIV. A reduction of the level of dosing, which may be achieved by improvement of the PK properties of the compounds together with increased potency at the target receptor should reduce the likelihood of induction in the clinic [169, 170]. The introduction of in vitro screens such as human hepatocyte assays and higher throughput assays for CYP mRNA and PXR activation means that issues of potential human CYP induction can now be addressed. Nevertheless, the regulatory authorities recognise the need to investigate enzyme induction during drug development and this will result in the further development and validation of in vitro assays to test for induction using human systems [83, 171]. REFERENCES [1] [2]

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