1.1. Overview of Drug Metabolizing Enzymes (DMEs) and transporters
Drug metabolism can either lead to detoxification, bio-inactivation and/or elimination of the drug from the body. Metabolism can be broadly categorized into phases I and II. Phase I drug metabolizing enzymes (DMEs) primarily comprise of the Cytochrome (CYP) 450 family of enzymes. CYP3A4 is the most common isoform expressed in human liver and intestine accounting for ~30-60% of CYPs (Nebert & Russell, 2002) More than 50% of the currently marketed drugs are metabolized by CYP3A4 in humans (Guengerich, 1999). Phase II metabolism consists of conjugation reactions forming polar metabolites leading to enhanced excretion. Phase II reactions include glucuronidation (Uridine 5'-diphospho-glucuronosyltransferase, UGT) sulfation (Sulfotransferase, SULT), methylation (Methyltransferase), glutathione conjugation (Glutathione S-transferase, GST), etc. (Jancova et al., 2010, Meyer, 1996).
Drug transporters play a central role in the absorption, distribution, metabolism and elimination (ADME) processes of xenobiotics across the cellular barriers. They are broadly classified into uptake and efflux transporters which facilitate drug disposition in or out of the cells (Mizuno et al., 2003). Major transporters include, but are not limited to: multidrug resistant gene/P-glycoprotein (MDR/P-gp), multidrug resistance associated protein (MRP1-3), breast cancer resistance protein (BCRP), organic anion transporting peptides (OATPs) and organic cationic transporters (OCTs) (Mizuno et al., 2003, Mizuno & Sugiyama, 2002).
1.2. Altered drug metabolism and transport in pathophysiological conditions
Several studies have shown that drug metabolism and transport is disrupted during diseases and altered pathophysiological conditions primarily due to reductions in gene expression of these enzymes and transporters (Aitken et al., 2006, Kato, 1977). The transcription factors such as nuclear factor-κB (NF-κB), CAAT enhancer-binding protein (C/EBP) or nuclear transcription factor E2-related factor 2 (Nrf2) have been shown to regulate DME and transporter gene expression in vivo and in vitro (Gonzalez & Lee, 1996, Shen & Kong, 2009, Zordoky & El-Kadi, 2009). In addition to basal transcription factors, the xenobiotic nuclear receptors, pregnane X receptor (PXR), constitutive androstane receptor (CAR) heterodimerize with the central nuclear receptor, retinoid X receptor (RXR) α to regulate the expression of DME and transporter genes (Chen et al., 2004, Goodwin et al., 2002, Kast et al., 2002, Xie, 2008). Furthermore, nuclear receptors such as peroxisome proliferator activated receptor (PPAR), liver X receptor (LXR) or farsenoid X receptor (FXR) can also regulate DME and transporter gene expression (Xie, 2008). The orphan nuclear receptor, hepatocyte nuclear factor (HNF) 4α can regulate the gene expression of PXR and CAR mediated xenobiotic induction of CYP3A4 (Tirona et al., 2003).
Altered drug metabolism can lead to adverse drug reactions which account for ~10% of hospitalized cases (Deng et al., 2009, Maddox et al., 2010). However, due to underreporting, the actual incidences may be much higher (Lazarou et al., 1998., Pirmohamed et al., 2004). As early as 1960s, variations in drug metabolism were observed in patients or animals with diabetes (Dixon et al., 1961), cancer (Kato et al., 1963), hepatitis (Klotz et al., 1974, McHorse et al., 1974) or influenza (Kraemer et al., 1982). Changes in drug metabolism were also associated with a corresponding change in the pharmacodynamics (PD) of drugs (Dixon et al., 1961; Kato et al., 1968). These early studies prompted the researchers to study the alterations in DME and transporter gene expression and activity in pathophysiological conditions such as cancer, diabetes/obesity, rheumatoid arthritis (RA), non-alcoholic fatty liver disease (NAFLD) and cardiovascular diseases (CVDs) such as hypertension, heart failure, or stroke, etc. (Alkayed et al., 2002, Charles et al., 2006, Fisher et al., 2008, 2009a; 2009b, Thum & Borlak, 2002). Overall, the readers of this chapter will benefit from the discussions of the changes in expression of DMEs and transporters, and pharmacokinetics/pharmacodynamics (PK/PD) of clinically relevant medications in different pathophysiological conditions.
2. Infection and inflammation
2.1. Bacterial infections
2.1.1. Drug metabolizing enzymes
Most of the studies on regulation on DMEs have been documented with gram-negative bacteria. Of clinical relevance, sepsis induced by cecal ligation and puncture (CLP) is the most frequently used model owing to its close resemblance in the progression and characteristics of human sepsis (Wichterman et al., 1980). In a CLP rat model, total hepatic microsomal CYP content and activities were significantly reduced (Godellas et al., 1995). Infection of pigs with the gram-negative respiratory pathogen, Actinobacillus pleuropneumoniae, led to decreased clearances of antipyrine, caffeine, and acetaminophen 24 h after inoculation. This was further supported by decreased microsomal metabolism of several CYP-dependent substrates (Monshouwer et al., 1995). Another widely studied gram-negative pathogen, Citrobacter rodentium, is a natural murine pathogen which produces similar colonic pathology on the intestinal cells of the host as seen after enteropathogenic Escherichia coli infections in humans (Higgins et al., 1999). The mRNA and protein levels of CYP4F18 and 2D9 were induced in a live mouse model of inflammatory bowel disease induced by C. rodentium (Chaluvadi et al., 2009). The rapid down-regulation of CYP2Cs and CYP3As after intraperitoneal (i.p) injection and CYP4As after oral injection of C. rodentium were quantitatively and qualitatively different, suggesting that the effects of oral infection are not due to bacterial translocation to the liver (Chaluvadi et al., 2009).
Although, gram-positive infections account for more than 50% of the total community acquired infections (Martin et al., 2003), very few studies have linked the effects of gram-positive bacteria on regulation of DMEs. It was shown that in patients suffering from gram-positive bacteremia such as Pseudomonas or Staphylococcus infections, an increase in volume of distribution (Vd) and dilution of antimicrobial agents in plasma and extracellular fluids may occur, which needs careful monitoring of the dosage regimen (Pinder et al., 2002). Listeriosis, caused by Listeria monocytogenes, is one of the most critical food-borne diseases in humans. L. monocytogenes induced CNS infection in rodents significantly down-regulated mRNA, protein and activity of hepatic CYPs (Garcia Del Busto Cano & Renton, 2003).
The gram-negative bacterial component, lipopolysaccharide (LPS), and the gram-positive bacterial component, lipoteichoic acid (LTA), serve as sterile infection models by inducing inflammatory responses in animals (Ginsburg, 2002, Leemans et al., 2002). LPS can down-regulate the expression and activity of key hepatic, intestinal and renal DMEs in several animal species such as mice, rats or rabbits (Ghose et al., 2008, Sewer et al., 1996). Interestingly, studies have that changes in CYP expression and activity is dependent on the route of administrate at same dose of LPS (Shimamoto et al., 1998). We recently showed that LTA significantly down-regulated the gene expression of several phase I and phase II DMEs in mice (Ghose et al., 2009).
2.1.2. Drug transporters
Changes in expression of drug transporters can have significant impact on the safety and efficacy of the drugs. LPS treatment of mice significantly down-regulated P-gp and Mrp2, major transporters involved in disposition of clinically relevant drugs such as colchicine, verapamil, daunorubicin, cyclosporin A and the abundant food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (Dietrich et al., 2001, Petrovic et al., 2007). LPS-treated mice had significantly lower hepatic P-gp (30% of control) and increased P-gp expression in the kidney (140% controls) (Hartmann et al., 2001, 2005).
2.1.3. PK/PD studies
As early as 1980’s, it was shown that vaccination with the bacillus Calmette-Guerin (BCG) decreased clearance of theophylline in human volunteers (Gray et al., 1983). Altered PK was observed in other bacterial infections induced by Streptococcus pneumoniae (decreased antipyrine clearance) or Mycoplasma pulmonis (increased Tilmicosin plasma levels) (Modric et al., 1998, Sonne et al., 1985). LPS injections in animals and humans altered PK parameters such as maximum plasma concentration (Cmax, increase), area under the curve (AUC, increase), half-life (T1/2), Vd and clearance (CL, decrease) of several widely prescribed medications such as cisplatin, antipyrine, theophylline, hexobarbital, gentamicin and vancomycin (Gous et al., 1995, Hasegawa et al., 1994, Ishikawa et al., 1990, Shedlofsky et al., 1994). PK changes of drugs during bacterial infection or inflammation can profoundly affect the PD as well. E.g. turpentine oil-injected mice had very high anti-tumor activity of gimatecan compared to the controls (Frapolli et al., 2010). On the other hand, several studies have shown that inflammation does not affect the PD of drugs. E.g. despite of very high plasma concentrations of the calcium channel blocker, verapmil, or potassium channel antagonists, sotalol or propranolol; no change in the PD was seen in inflamed animals (Guirguis & Jamali, 2003, Kulmatycki et al., 2001, Mayo et al., 2000). The authors concluded this to be due to altered receptor-functioning or receptor-ligand binding exhibited by inflammation. Nevertheless, the above studies need further evaluations to delineate the disparities in altered drug metabolism caused by different bacterial infections or inflammation which has significant clinical implications for drug therapy in disease states.
2.2. Viral infections
2.2.1. Drug metabolizing enzymes
Viral infections can also stimulate the immune system releasing various inflammatory mediators from the immune cells (Mannering & Deloria, 1986). A survey of the literature reveals numerous studies on the effect of viral infections such as mouse-adapted influenza virus (Corbett & Nettesheim, 1973), Newcastle disease virus (Singh & Renton, 1981), encephalomyocarditis virus (Renton, 1981), chronic active hepatitis and cirrhosis (Schoene et al., 1972; Wilkinson, 1997) and HIV infection (Lee et al., 1993) on alteration of gene expression and activity of DMEs and oxidative pathways in animals and humans. Decreased levels of hepatic CYP1A2 were detected in children suffering from upper respiratory tract viral infections during an influenza outbreak (Chang et al., 1978; Kraemer et al., 1982). With exceptions of CYP2D6 mRNA and CYP1A2 activity, other major CYPs such as CYP2C9, 2C19, and 3A4 in HCV-infected PXB mice (chimeric mouse with human hepatocytes) were comparable to the non-infected controls (Kikuchi et al., 2010). Recombinant adenovirus injections in Sprague-Dawley rats led to significant down-regulation of renal CYP2E1 and hepatic CYP3A2 and CYP2C11 expression and activity, and induction of CYP4A protein expression (Callahan et al., 2005; Le et al., 2006).
2.2.2. Drug transporters
A recent study showed that HIV-type 1 viral envelope glycoprotein gp120 decreased P-gp and Mrp expression levels in rat astrocytes (Ronaldson & Bendayan, 2006). However, due to the fact that HIV infected patients are on highly active antiretroviral therapy (HAART) consisting of numerous drugs, both, induction and suppression of drug transporters in HIV infection are reported (Giraud et al., 2010). Polyinosinic/polycytidylic acid [poly (I:C)] is widely used as a model of in vivo viral-induced inflammation. Poly (I:C) can induce interferons (IFNs) and pro-inflammatory cytokines such as interleukin (IL)-6, IL-10, IL-12, and tumor necrosis factor (TNF)-α. A significant down-regulation of key maternal hepatic and placental drug transporters and their endogenous substrates was observed upon i.p injection of poly (I:C) in pregnant rats (Petrovic & Piquette-Miller, 2010) However, Abcb1b (ATP-binding cassette sub-family B member 1) and Abcc3 (ATP-binding cassette sub-family C) were significantly induced. A recent study in PXB mice infected with hepatitis C virus (HCV) reported significantly higher expression of MRP4 and OATP2B1 and lower expression of OCT1 compared to non-infected mice (Kikuchi et al., 2010).
2.2.3. PK/PD studies
During the 1982 influenza B outbreak in King County, Washington, 11 children whose asthma had previously been controlled with a stable theophylline dose, developed theophylline toxicity on this same dose (Kraemer et al., 1982). These children had a significant decrease in CL and increase in T1/2 of theophylline. HIV infections could also lead to altered PK of levofloxacin and fluconazole (Goodwin et al., 1994, Tett et al., 1995). End-stage liver disease, which is largely the result of HCV infection, now accounts for up to 50% of deaths among persons with HIV-1 infection (Bica et al., 2001). A clinical study in HIV-HCV-coinfected patients showed significantly lower nelfinavir oral clearances in HIV+ and HCV+ patients with and without cirrhosis compared to HIV+ and HCV-negative patients (Regazzi et al., 2005). This presses the need for therapeutic drug monitoring in individualizing nelfinavir dosage in HIV-HCV-coinfected patients. In addition, an increase in AUC and Cmax of several anti-retrovirals are reported in HCV-infected patients with moderate liver impairment (Veronese et al., 2000, Wyles & Gerber, 2005). Other studies have also shown significantly higher AUC of docetaxel and reduced glomerular filtration rate, suggesting changes in renal CYP in rats injected with the recombinant adenovirus expressing β-galactosidase (Le et al., 2006, Wonganan et al., 2009). On the contrary, significantly reduced Cmax and AUC of ceftiofur hydrochloride were observed in pigs infected with porcine reproductive and respiratory syndrome virus compared to the uninfected pigs (Tantituvanont et al., 2009).
2.3. Mechanisms for altered drug metabolism in infections and inflammation
Bacterial or viral infections lead to activation of Toll-like receptor (TLR) signaling pathway, which leads to the induction of pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α in the immune cells. In the liver, TLRs are present on the cell surface of various immune cells (the resident macrophages or Kupffer cells) as well as the hepatocytes (Scott et al., 2009). Out of the 13 TLRs identified in mammals, TLR4 is activated by the gram-negative component, LPS, and TLR2 is activated by the gram-positive component, LTA (Aliprantis et al., 1999, Takeuchi et al., 1999). We and others have shown down-regulation of Cyp3a11 and P-gp in LPS sensitive TLR4 wild type (C3HeB/FeJ) mice could not be detected in TLR4-mutant (C3H/HeJ) mice (Ghose et al., 2008, Goralski et al., 2003, 2005). Recent data from our lab showed that down-regulation of gene expression of key hepatic phase I and phase II DMEs in TLR2+/+ mice by LTA was blocked in TLR2-/- mice (Ghose et al., 2009). We also observed that LTA down-regulated Mrp2, had no effect on Mrp3 and induced Mdr1b expression. Although, most of the studies have cited the role of Kupffer cell-derived TLRs in hepatic drug metabolism, we and others have also shown that LPS or LTA treatment of primary mouse hepatocytes can directly affect the DMEs via TLRs present on the hepatocytes, independent of cytokines (Ferrari et al., 2001, Ghose et al., 2011a). TLR-mediated signaling is initiated by the down-stream adaptor protein, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) (Kagan & Medzhitov, 2006, O'Neill & Bowie, 2007). We showed that TIRAP was involved only in TLR2-mediated regulation of DME and transporter genes (Ghose et al., 2011a), and not by TLR4 (Ghose et al., 2008).
Cytokines are involved in alteration of DMEs and transporters in vitro (Barker et al., 1992, Muntane-Relat et al., 1995). LPS-treatment of primary rat cocultures of hepatocytes and Kupffer cells significantly suppressed phenobarbital-mediated induction of CYP2B1 (Milosevic et al., 1999). This decrease was associated with a 5-fold induction in TNF-α released from the Kupffer cells in cocultures. In vitro studies with cytokine-treated rat or human hepatocytes led to decreased expression and activity of several drug transporters including efflux pumps such as P-gp, MRP2, 3 and 4, and BCRP, sodium-taurocholate cotransporting polypeptide (NTCP), a major sinusoidal transporter handling bile acids, uptake transporters such as OATP-B, OATP-C, and OATP-8 (Diao et al., 2010, Le Vee et al., 2008, Lee & Piquette-Miller, 2003, Sukhai et al., 2000, Vee et al., 2009).
However, recent evidence suggests cytokines may not be playing a major role in regulation of DMEs. Earlier studies in TNF-α-/- and IL-6-/- knockout mice revealed that DMEs were still down-regulated (Warren et al., 1999, 2001). A recent study by Kinloch et al in TNFR1−/−, IL1R1−/− and Kupffer cell depleted mice showed that only TNF-α, but not IL-1β and Kupffer cells, was involved in regulation of CYP3A11 and 3A25 oral C. rodentium infection (Kinloch et al., 2011). In addition, we showed that although down-regulation of DMEs was blocked in LTA-treated TIRAP-/- mice, hepatic cytokine gene expression remained unchanged (Ghose et al., 2011a).
Nitric oxide (NO), released from macrophages and hepatocytes during inflammation is also known to regulate DMEs (Morris & Billiar, 1994). However, contrasting results have been reported for the role of NO in regulation of DMEs in cytokine-treated primary rat hepatocytes (Carlson & Billings, 1996, Sewer & Morgan, 1997). IL-1β and TNF-α-mediated down-regulation of CYP protein was NO dependent, but not in IL-6 mediated down-regulation (Carlson & Billings, 1996). NO was also shown to regulate the suppression of UGT activities in cytokine-treated hepatocytes (Monshouwer et al., 1996).
Several studies have shown that inflammation-mediated activation of NF-κB plays a significant role in down-regulation of DMEs (Abdulla et al., 2005, Gilmore, 2006, Ke et al., 2001). NF-κB can either indirectly regulate CYP gene expression through mutual repression between NF-κB and nuclear receptors, or can directly regulate CYP gene expression through binding to NF-κB response element in the promoter region of CYP genes (Pascussi et al., 2003). Interaction of NF-κB with nuclear receptors during pathophysiological conditions can alter expression of DMEs (Gu et al., 2006). Inflammation-mediated activation of mitogen activated protein kinase (MAPK), c-Jun-N-terminal kinase (JNK), also regulates nuclear receptors and DMEs (Adam-Stitah et al., 1999, Yu et al., 1999). Recent experiments in human gastric carcinoma and pancreatic carcinoma cell lines suggested a prominent role of JNK activation in down-regulation of P-gp protein expression (Zhou et al., 2006). However, further detailed studies using in vitro models such as cell lines or primary hepatocytes, and specific inhibitors of these cell signaling components will significantly contribute in understanding the mechanistic regulation of DMEs and transporters during inflammation.
We speculate that down-regulation of nuclear receptors during inflammation might be involved in regulating the gene expression of DMEs and transporters in animal models (Ghose et al., 2004, 2008, 2009, Synold et al., 2001). We also showed that down-regulation of nuclear receptors by LPS in TLR4+/+ or by LTA in TLR2+/+ mice was blocked in TLR4 mutant or TLR2-/- mice (Ghose et al., 2008, 2009, 2011a). On the contrary, mRNA and protein expression of several CYPs did not differ in PXR-/- or PPAR-/- mice treated with LPS (Richardson & Morgan, 2005). Similarly, it was shown that PXR was least important in regulating several efflux and uptake drug transporters using PXR wild type or PXR null mice treated with LPS (Teng & Piquette-Miller, 2005). However, the down-regulation of Bsep and Mrp2 mRNA in IL6-treated wild type mice was attenuated in the PXR null mice. Thus, involvement of nuclear receptors in inflammation-mediated regulation of DMEs and transporters may depend on the nature of the inflammatory stimuli.
3.1. Drug metabolizing enzymes
Owing to the fact that, most anticancer drugs have a very low or narrow therapeutic index, alteration of DMEs can lead to life-threatening adverse drug reactions or increased risk of treatment failure in patients undergoing chemotherapy. Decreased hepatic microsomal DME activity was detected in tumor bearing rats with Walker carcinosarcoma 256, where impaired metabolism of hexobarbital, strychnine and meprobamate was observed (Kato et al., 1963). Due to difficulties in obtaining human liver tissue from cancer patients, an Engelbreth-Holm-Swarm (EHS) sarcoma mouse model bearing transgenic CYP3A4/lacZ gene was developed (Charles et al., 2006). Reduced hepatic levels of the transgene-derived β-galactosidase, as quantified by o-nitrophenyl-β-D-galactopyranoside assay, and Cyp3a11 mRNA and protein was observed in these mice (Charles et al., 2006). Tumors derived from the surface of the ovary account for the vast majority of ovarian tumors (approximately 80%). Altered gene expression ratio of CYP3A4/ABCB1 (P-gp) in cancer cells grown from epithelial ovarian tumors had significant contribution in altering docetaxel disposition (DeLoia et al., 2008). On the other hand, there was no significant correlation in CYP2C8/ABCB1 ratio suggesting that paclitaxel disposition may require additional critical gene products. The expression of several phase II DMEs was also characterized in EHS tumor-bearing mice (Charles et al., 2006). Out of 8 GSTs studied, six were reduced and two unchanged; SULT1A1 was increased while SULT2A1 and UGT2B5 were reduced, and no change was observed in UGT1A7. Tamoxifen remains the first-line targeted treatment for the estrogen receptor α-positive breast cancer patients and undergoes metabolism in the breast tissue which also consists of several DMEs (Williams & Phillips, 2000). In a study examining the role of methylation patterns of genes responsible for tamoxifen metabolism, higher methylation rate of N-acetyl transferase-1 (NAT1), a phase II DME gene, was observed in human breast cancer tissues compared to control breast tissues (Kim et al., 2010).
3.2. Drug transporters
Changes in the genetic variability in clinical specimens as well as over expression of ABC transporter family in tumors have been shown to play a critical role in multidrug resistance to several anticancer drugs (Hoffmeyer et al., 2000, Robinson et al., 1997, Yoh et al., 2004, Young et al., 1999). A recent study showed significant reductions in the mRNA levels of Mdr2, Mrp2, Mrp3, Ntcp, Oatp 2, bile salt export pump (Bsep), Bcrp, whereas Mdr1a and Oatp1 remained unchanged (Sharma et al., 2008).
3.3. PK/PD studies
Cancer-induced changes in the PK and PD profiles of several drugs have been documented since the late 1960s (Kato et al., 1968, Rosso et al., 1968, 1971). In a clinical study, the absorption rate constant, apparent Vd and serum CL of penbutolol (antihypertensive drug) were significantly reduced in the cancer group (Aguirre et al., 1996). PD effect (reduction in heart rate) of penbutolol did not vary statistically in respect to baseline values in cancer patients (Aguirre et al., 1996). Reduction in the metabolism of omeprazole (CYP2C19 substrate) has also been observed in patients with advanced cancer (Williams et al., 2000). Reduced CYP3A expression resulted in >2 fold increase in the sleep time in tumor bearing mice receiving the widely used sedative-hypnotic, midazolam, (CYP3A specific substrate) (Charles et al., 2006).
3.4. Mechanisms of cancer-mediated altered drug metabolism
Since the 1800s, it was observed that chronic inflammation is frequently associated with the onset and progression of various cancers (Balkwill & Mantovani, 2001). A strong association between cancer progression and induction of cytokines or acute phase reactive proteins in tumors is documented (Burke & Balkwill, 1996, Burke et al., 1996, Naylor et al., 1993). E.g. EHS tumor-bearing mice had significantly higher circulating plasma levels of IL-6 (25 pg/ml) compared to the control mice (below detection limit). IL-6 mediated activation of JNK was also evident in EHS tumor-bearing mice, which again prompts the important role of JNK in regulation of DMEs. Studies have shown that TLR expression is enhanced in tumor cells lines (Yu & Chen, 2008). However, the role of TLRs in alteration of DMEs and transporters in cancer has never been investigated.
The role of NF-κB activation in acute inflammation has been suggested in carcinogenesis (Karin et al., 2002, Lind et al., 2001). Cancer-mediated alteration of DMEs and transporters may possibly be regulated by over-expression of NF-κB. A recent study highlighted the role of extra hepatic malignancies in down-regulation of PXR and CAR in tumor-bearing mice (Kacevska et al., 2011). This study prompts to link the reduction in nuclear receptors with altered drug metabolism in cancer. However, additional studies with nuclear receptor knockout animal models with tumors will help identify their direct role in regulation of DMEs and transporters. Overall, all these studies imply that tumor-mediated inflammation may play an integral role in drug response and toxicity of various anticancer agents.
4. Diabetes and obesity
4.1. Drug metabolizing enzymes
Another prevalent pathophysiological condition affecting millions of people in the world is the occurrence of diabetes and obesity. As per the latest statistical report, 366 million people in the world will have diabetes by 2030 (Wild et al., 2004). Dixon et al demonstrated that alloxan-induced diabetes decreased hexobarbital, chlorpromazine, and codeine metabolism in male rats (Dixon et al., 1961, 1963). Although, streptozotocin-induced diabetes in rats and hamsters significantly induced hepatic and renal CYP2E1 and 4A2 protein levels (Chen et al., 1996, Shimojo et al., 1993), suggesting altered metabolism of ketones and fatty acids in diabetes, hepatic CYP2E1 protein levels remained unchanged in streptozotocin-induced diabetic mice livers (Chen et al., 1996, Sakuma et al., 2001). A recent study showed differential effects of alloxan-induced diabetes on protein expression and activity of CYP2E1 (increased) and CYP2B4 (decreased) in rabbits (Arinc et al., 2005). Altered gene expression of DMEs in genetically obese zucker fatty rats (reduction in CYP2B1/2 and Mrp3) and db/db mice (increase in CYP2B10) are also reported (Xiong et al., 2002, Yoshinari et al., 2006a). Studies have reported interesting results on DME gene and protein expression for different diet-induced obese (DIO) animal models. E.g. Although Cyp3a11 gene and protein expression were significantly reduced in both long term (12 weeks) and short term treatment (1 week) of high fat diet (HFD), Cyp2c9 gene expression was significantly reduced only in the short term HFD treatment (Yoshinari et al., 2006b). We recently showed that mRNA levels of the phase II DMEs (Ugt1a1, Sult1a1, Sultn) were reduced ~30-60% in mice fed high-fat diet (HFD, 60% kcal fat for 14 weeks) compared to low fat diet (LFD, 10% kcal fat) mice (Ghose et al., 2011b). RNA levels of Cyp2e1 and Cyp1a2 were unaltered in HFD mice. These findings indicate that regulation of CYPs is dependent on the model of diabetes and obesity, and is tissue, isoform and species-specific.
4.2. Drug transporters
Streptozotocin treatment in rats increased hepatic levels of Mdr2, leading to increased phospholipid secretion into bile (van Waarde et al., 2002). Another study also showed that the hepatic expression of uptake transporters (Oatp1a1, 1a4, 1b2, 1a6, 2b1, and Ntcp) in diabetic mice decreased significantly compared to the wild type controls (Cheng et al., 2008). Our recent study showed no effect of high fat in DIO mice on gene expression of hepatic transporters (Mrp2 and 3, and Mdr1b) (Ghose et al., 2011b).
4.3. PK/PD studies
Obesity-associated alterations in phase II metabolism were reported in 1980’s. E.g. clearances of oxazepam and lorazepam, widely used benzodiazepines and excreted as glucuronide conjugates, were significantly increased in obese patients (Abernethy et al., 1983). Similarly, increased metabolism of chlorzoxazone (CYP2E1 substrate) to 6-hydroxychlorzoxazone was observed in obese individuals. This was attributed to increased CYP2E1 activity associated with obesity (O'Shea et al., 1994). Animal studies performed using a diabetes mellitus rat model (induced by alloxan or streptozotocin treatment) have reported altered PK of drugs such as acetaminophen, chlorzoxazone, theophylline, clarithromycin, furosemide, and methotrexate (Baek et al., 2006, Kim et al., 2005a, 2005b, Park et al., 1996, 1998, Watkins & Sherman, 1992). Although, no changes in PD of atracurium were reported in obese animals compared to lean control (Varin et al., 1990), triazolam-induced sedation in obese humans increased significantly compared to normal weight men (Derry et al., 1995). We also observed similar disparities in the PD of midazolam, CYP3A substrate, (increased sleep time) and zoxazolamine, CYP2E1 substrate (no change) in DIO mice (Ghose et al., 2011b). This can be attributed to decrease in CYP3A and no change in CYP2E1 expression. Thus, the differential effects of obesity on PD of drugs may depend on the DME, or the drug or the target organ itself.
4.4. Mechanisms of altered drug metabolism in diabetes/obesity
The major pathophysiological manifestation in diabetes/obesity is characterized by low-level chronic and local inflammation, such as release or over expression of TNF-α and C-reactive protein in adipose tissue (Hotamisligil et al., 1993, Wellen & Hotamisligil, 2005). However, the role of inflammation in regulation of DMEs and transporters in diabetes/obesity remains unclear. Hormonal regulation of DMEs in diabetes/obesity has also been addressed before (Thummel & Schenkman, 1990). Although an increase in mRNA or protein levels of CYP2E1 have been observed in obese patients (Lucas et al., 1998), db/db mice showed no such effects (Yoshinari et al., 2006a). This can possibly be due to hyperinsulinemia leading to a faster turnover (shorter CYP2E1 mRNA half-life) by insulin (De Waziers et al., 1995). Various studies have shown that phosphatidylinositol-3-kinase (PI3K) signaling, using PI3K inhibitors, wortmannin and LY294002, ameliorated insulin-mediated decrease in CYP2E1 and phase II enzymes (α-GST) mRNA (Kim et al., 2006, Kim & Novak, 2007, Woodcroft et al., 2002).
Interestingly, lower expression of CAR and CYP2B in obese Zucker rats and ~2 fold induction in obese and genetically diabetic mice (db/db) on HFD (Xiong et al., 2002, Yoshinari et al., 2006b) were reported. This discrepancy in obese Zucker rats and db/db mice in regulating expression profiles of CYPs and nuclear receptors can be explained by the difference in the position of mutation of leptin receptor gene (Chua et al., 1996, Lee et al., 1996). We recently showed that expression of PXR and CAR; and protein levels of RXRα were significantly reduced in HFD mice (Ghose et al., 2011b). Thus, a complex set of processes including but not limited to cytokines, nuclear receptors, insulin sensitization or downstream signaling molecules, may regulate DMEs and transporters in diabetes/obesity.
5. Non-alcoholic fatty liver disease
5.1. Drug metabolizing enzymes
Non-alcoholic fatty liver disease (NAFLD) is highly prevalent with an estimated world population between 14% and 24% being affected. NAFLD comprises of symptoms ranging from simple steatosis (fatty liver) to the more severe non-alcoholic steatohepatitis (NASH, fatty liver with infiltration of inflammatory cells) to progressive hepatic fibrosis and to cirrhosis (Reynaert et al., 2005). Alteration of hepatic CYP2E1 was first noted in humans with NASH (Weltman et al., 1998). Later studies have shown significant contribution of NAFLD (comprising of both, simple stage fatty liver as well as NASH) on expression and activity of DMEs in animals (Fisher et al., 2008, 2009a, 2009b). Similarly, in vitro studies in primary human or animal hepatocyte cell cultures from steatotic or non-steatotic livers showed a profound impact of steatosis on the metabolic functionality of hepatocytes (Donato et al., 2007, Fisher et al., 2004, Gomez-Lechon et al., 2004). Significant reductions in CYP1A2, 2C9, 2E1 and 3A4 activities in fat-overloaded hepatocytes were observed compared with control hepatocytes obtained from the same liver sample (Fisher et al., 2008).
5.2. Drug transporters
Decreased mRNA and protein expression of uptake transporters such as NTCP, OATP1a1, 1a4, 1b2 and 2b1; and OAT 2 and 3 were observed in NAFLD (Fisher et al., 2009a).
5.3. PK/PD studies
Studies have shown interesting results with acetaminophen (APAP) PK in rats and humans with NAFLD. Children with NAFLD had significantly higher concentrations of APAP-glucuronide (APAP-G) in serum and urine compared with controls, with no significant differences in PK of APAP among the 2 groups (Barshop et al., 2011). Another study showed that biliary concentrations of APAP-sulfate (APAP-S), APAP-G, and APAP-glutathione were reduced in MCD (methionine- and choline-deficient) rats (Lickteig et al., 2007a). However, plasma levels of APAP-G were also elevated in MCD rats, similar to that observed in children (Barshop et al., 2011). A clinical study evaluated the effect of NAFLD on PK of silymarin (Schrieber et al., 2008). The AUC0-24h for the sum of total silymarin flavonolignans was ~3-4 fold higher in patients with NAFLD (p<0.03), compared with healthy volunteers.
5.4. Mechanisms of altered drug metabolism in NAFLD
Several mechanisms have been proposed for the effect of NAFLD on altered drug metabolism. Deposition of fat in human hepatocytes can lead to a marked impairment in CYP mRNA and activity (Donato et al., 2006). Fisher et al observed intense staining for IL-1β in steatotic livers, indicating that experimental steatosis and NASH results in increased hepatocellular inflammation (Fisher et al., 2009a). Studies have shown ambiguous results on expression of nuclear receptors and transcription factors in NAFLD (Fisher et al., 2009b, Hardwick et al., 2010, Lickteig et al., 2007b). Except for PXR, which was significantly increased by 1.4 fold, the other nuclear receptors (AhR, CAR, PPARα and Nrf2) were not altered (Fisher et al., 2008). Therefore, various factors need to be taken into account for improved pharmacotherapy in patients with NAFLD.
6. Cardiovascular disorders
6.1. Drug metabolizing enzymes
CYPs in humans are responsible for metabolizing a large number of cardiovascular medications, including β-blockers, calcium channel blockers and angiotensin receptor antagonists (Abernethy & Flockhart, 2000). Alteration in DMEs could be of particular clinical relevance in patients with heart failure because these patients take more than 10 medications on average. Although, not detected in the normal human heart, failing hearts expressed CYP11B1 and 11B2 (Young et al., 2001). Surprisingly, an up-regulation in CYP2J2, 1B1, 2E1, 4A10 and 2F2 gene expression was reported in the failing heart (Tan et al., 2002). Increased cardiac CYP11B2 mRNA was associated with increased myocardial fibrosis and the severity of left ventricular dysfunction in patients with heart failure (Satoh et al., 2002). It was shown that the production of testosterone metabolites, including dihydrotestosterone and androstenedione, was significantly increased in hypertrophic human hearts (Thum & Borlak, 2002). Transient ischemic attacks (TIA) are risk factors for strokes. A recent study showed that cerebral infarct size was reduced in TIA-preconditioned animals and CYP2C11 mRNA and protein were coincidentally increased in the brain after experimentally induced TIA (Johnston, 2004). Genetic polymorphisms of DMEs are commonly associated with heart failure and hypertension (Kivisto et al., 2005). E.g. a study in Japanese subjects reported that CYP2C9 wild type carriers had lower systolic blood pressure after losartan (metabolizes to the active metabolite EXP3174) therapy than poor metabolizers (Sekino et al., 2003).
6.2. Drug transporters
A recent study demonstrated a selective disease-dependent regulation of the high-affinity carnitine transporter, OCTN2, in patients with dilated cardiomyopathy, whereas the other OCT(N)s were unaffected (Grube et al., 2011).
6.3. PK/PD studies
It was shown that lidocaine plasma clearance was significantly decreased in patients with cardiac failure and this was associated with decreased liver blood flow (Thomson et al., 1971). Another group also observed reduced plasma clearance of lignocaine in patients suffering from myocardial infarction without cardiac failure (Prescott et al., 1976). Thus, the mounting evidence for the effect of CVDs on DMEs and transporters needs to be extended for further PK/PD studies.
6.4. Mechanisms of altered drug metabolism in CVDs
Failing or hypertensive hearts are susceptible to infiltration by pro-inflammatory cytokines and reactive oxygen species induced by stress (Fliser et al., 2004). Studies have shown that increased circulating levels of TNF-α and IL-6 in patients with congestive heart failure were inversely proportional to CYP2C19 and CYP1A2 activity (Frye et al., 2002). Similarly, down-regulation of OCTN2 expression in patients with dilated cardiomyopathy inversely correlated with cardiac CD3+ T-cell count (Grube et al., 2011). In addition, cardiac cytokine release may affect OCTN2 expression during cardiomyopathy associated with inflammation.
7. Rheumatoid Arthritis (RA)
7.1. Drug metabolizing enzymes
Rheumatic diseases are estimated to affect up to 1.1% of the world’s population (Harris, 1980). Various studies have shown that gene expressions of DMEs are altered in adjuvant arthritis (AA) rats (Achira et al., 2002b, 2002c, Projean et al., 2005). Similarly, activities of CYP3A were significantly decreased in AA rats compared to control rats (Uno et al., 2007).
7.2. Drug transporters
Decreased activity of hepatic P-gp in the isolated perfused liver of AA rats was reported (Achira et al., 2002c, Uno et al., 2007). Decrease in P-gp activity corresponded with the decreased levels of Mdr1a mRNA and P-gp protein in AA rats.
7.3. PK/PD studies
PK/PD changes such as elevated plasma levels of acebutolol, cyclosporin A, propranolol and prolongation of sleep time with pentobarbital were observed in AA rats compared to normal rats (Dipasquale et al., 1974, Piquette-Miller & Jamali, 1992, 1993, Shibata et al., 1993). Based on these early observations, recent studies have also shown altered PK of methotrexate, T-5557 (novel anti-inflammatory agent) and doxorubicin in AA animals (Achira et al., 2002a, 2002b, 2002d). Although, a significant increase in the plasma concentrations of verapamil in rats and humans with underlying arthritis were reported, there were no changes in the PD of verapamil (prolongation of PR interval) (Mayo et al., 2000, Sattari et al., 2003). This discrepancy was then attributed to a decrease in the receptor-ligand affinity in inflammation (Laporte et al., 1998, Shore et al., 1997).
7.4. Mechanisms of altered drug metabolism in RA
AA animal models represent a systemic inflammatory disease with bone and cartilage changes similar to those observed in RA (Williams et al., 1992). Down-regulation of hepatic P-gp in AA rats was attributed to elevated levels of cytokines such as TNF-α and IL-6 but not IL-1β (Philippe et al., 1997). Similarly, increased plasma concentrations of drugs in AA rats correlated with increased serum TNF-α level (Sattari et al., 2003). Several in vitro and in vivo studies have shown up-regulation of NF-κB in RA and osteoarthritis (Handel et al., 1995, Mor et al., 2005). It was recently demonstrated that PXR and CAR expression in small intestine was decreased in arthritis (Kawase et al., 2007b). In another study, bilirubin elimination was significantly decreased in collagen-induced arthritis (CIA) rats compared to normal rats (Kawase et al., 2007a), which was attributed to decreased expression of CAR in CIA rats. Thus, overall these studies imply that inflammatory pathways may be involved in the regulation of DMEs and transporters in arthritis.
A common theme of this chapter is that a multiplex of mechanisms are responsible for alterations of DMEs, transporters and PK/PD of drugs in different pathophysiological conditions. It is well-established that changes in gene expression of enzymes and transporters can lead to disruption in drug disposition in altered pathophysiological conditions including infection/inflammation, cancer, obesity, CVD, rheumatoid arthritis, etc. Studies show that induction of inflammatory mediators is an underlying factor common to all these pathophysiological conditions and may contribute to altered drug disposition in disease states. In addition, the generally accepted role of cytokines in alterations of DMEs and transporters needs further evaluation. We have established the involvement of Toll-like receptor signaling pathway in the regulation of DMEs and transporters, and our studies point to the role of cytokine-independent pathways in the liver. The role of transcription factors and nuclear receptors in the regulation of DMEs and transporters in disease states need further investigation. There is an urgent need to develop models for delineating the roles of individual inflammatory mediators or nuclear receptors in altered drug disposition in disease states. Understanding alterations of drug disposition in disease states is critical in predicting and preventing undesirable effects of clinically-relevant medications.