Chagas disease, or American trypanosomiasis, is a neglected illness that affects at least 8 million people in areas of disease endemicity in Latin America, and another 100 million people in the world are at risk of infection. The etiological agent of Chagas disease is the intracellular obligatory parasite Trypanosoma cruzi
, a hemoflagellate protozoan that is transmitted to humans by hematophagous insect vectors. However, other means of transmission, such as blood transfusion, organ transplantation, congenital transmission, and laboratory accidents, have been reported. Once the parasite enters the body, all types of nucleated cells in the human host are potential targets for infection (1
). The occurrence of this zoonosis in areas where the disease is not endemic, such as the United States and Europe, was reported to be mainly due to the migration of infected people (3
Benznidazole (BZL) is the only available drug for treatment of Chagas disease in most countries where it is endemic. It was reported that BZL is metabolized by a trypanosomal NADH-dependent type I nitroreductase that renders the cytotoxic and mutagenic agent glyoxal (4
). In mammalians, the nitro group is reduced to an amino group by a type II nitroreductase, with formation of free-radical intermediaries and reactive oxygen species (ROS) (4
Organs such as liver, intestine, and kidney play a key role in the metabolism and elimination of endogenous and exogenous compounds. Biotransformation involves phase I reactions catalyzed by isoforms of cytochrome P-450 (CYP) and phase II reactions catalyzed by glutathione S
-transferases (GST) and UDP-glucuronosyltransferases (UGT), among others. Metabolite excretion is mediated mainly by members of the ATP-binding cassette (ABC) family of transporters, such as P glycoprotein (P-gp/ABCB1/MDR1) and multidrug-resistance-associated protein 2 (Mrp2/ABCC2). P-gp transports a broad diversity of lipophilic and cationic compounds, including environmental pollutants and therapeutic agents such as cyclosporine, ritonavir, digoxin, and erythromycin. Mrp2 extrudes organic anions like bilirubin, bile salts, carcinogens, and therapeutic drugs (acetaminophen, ethynylestradiol, diclofenac, etc.) in the form of conjugates with glutathione (GSH), glucuronic acid, or sulfate (6
The relationship between biotransformation systems and transporters and its significance in drug disposition is well-recognized (7
). Changes in these proteins by endo- and xenobiotics may have a clinical impact on the normal functions of tissues with pharmacological and toxicological relevance, as mentioned above. Under these circumstances, the efficacy or toxicity of a very broad range of xenobiotics, including therapeutic agents, could be modified (6
Antiparasitic drugs have been shown to modulate biotransformation and transporter genes with an important impact on drug disposition. Bapiro et al. (9
) demonstrated that quinine and albendazole induced CYP isoforms in HepG2 cells at concentrations equivalent to those achieved in therapeutic protocols, warning about the risk of combining quinine or albendazole with other drugs that are metabolized by these systems. During antimalarial treatment with artemisinin, disease reactivation was associated with decreased artemisinin plasma levels. The authors of this work postulate that artemisinin induces its own elimination, probably by increasing first-pass metabolism (10
). Consistent with this postulate, Burk et al. (11
) have demonstrated that LS174T cells and primary human hepatocytes treated with artemisinin show selective induction of some isoforms of CYP and MDR1 mRNA expression.
Recently we observed that BZL induces the expression and activities of CYP3A4, GST-π, P-gp, and MRP2 in a concentration-dependent manner in HepG2 cells, used as model of human hepatocytes (12
). However, at present there is no information concerning the expression and activities of biotransformation systems and transporters in an in vivo
model and the potential consequences in pharmacokinetics of BZL or coadministered drugs. A study carried out with patients that received BZL for 30 days (7 mg/kg body weight [b.w.]day, twice a day) showed that the maximal plasma concentration after the first dose in the morning tends to decrease with treatment time (∼20% on average after 25 days of treatment) (13
). This finding suggests an increase in BZL metabolism and/or excretion that needs to be experimentally demonstrated.
Here we evaluate the expression and activities of biotransformation enzymes and transporter proteins in liver, intestine, and kidney from BZL-treated rats and how these changes modify the disposition of BZL.
The expression and activities of enzymatic systems and transporters can be altered by many factors, including diet, hormones, diseases, aging, and inducers. Thus, the effectiveness and/or toxicity of a broad range of xenobiotics, including therapeutic agents, could be modified.
Drugs with antiparasitic properties regulate biotransformation and transporter genes with an important impact on drug disposition (9–11
). BZL is the only drug available for the treatment of Chagas disease in most countries where it is endemic. Although BZL can be coadministered with other drugs (diuretics, antibiotics, antiretrovirals, immunosuppressants, etc,), no study has been conducted to evaluate its effects on the systems involved in drug disposition. Recently we observed an increase in P-gp, MRP2, CYP3A4, and GST-π protein levels with a concomitant enhancement in their activities in cells from a hepatoma cell line, HepG2, treated with BZL (200 μM, 48 h) (12
). Here we studied the effects of BZL treatment on phase I and phase II biotransformation enzymes and ABC efflux transporters in rat liver, small intestine, and kidney and the consequences in BZL disposition.
At present little is known about the effects of BZL on biotransformation systems. In phase I, the CYP3A subfamily as a whole significantly influences drug bioavailability in humans, since 40 to 50% of all drug metabolism involves some degree of CYP3A-mediated oxidation (33
). It was reported that pentobarbital-induced sleep time is extended after acute administration of BZL to rats (30 mg/kg b.w., i.p.). This effect was associated with inhibition of the hepatic microsomal biotransformation systems aminopyrine and ethylmorphine N
-demethylase by covalent interactions with BZL electrophilic metabolites (noncompetitive inhibition) (34
). In addition, Workman et al. (35
) observed that pharmacological concentrations of BZL were able to inhibit lomustine hydroxylation by CYP in mouse liver. They postulate that this BZL effect could explain modification of lomustine pharmacokinetics and enhanced response of mouse tumor to this drug. Our present study demonstrates a modest increase in the overall expression of CYP3A members only in liver of BZL-treated rats after 3 days of treatment (100 mg/kg b.w./day). Thus, despite the increase in CYP3A protein expression, the predominant effect of BZL seems to be inhibition of enzyme activities that can lead to drug-drug interactions.
In phase II metabolism, the expression of UGT1A was not modified by BZL in any tested tissues, indicating that glucuronidation is not essentially affected, at least for the isoforms recognized by our antibody, which represent the most relevant isoenzymes involved in the glucuronidation of phenol derivatives such as acetaminophen and endogenous compounds such as bilirubin (23
It is known that reactive electrophilic metabolites are metabolized and excreted by a GSH-dependent process that reduces their toxicity. The conjugation reaction is generally catalyzed by GSTs, a family of detoxification enzymes that protects cellular macromolecules from attack by reactive electrophiles (36
). The 2-amino reduction of BZL is catalyzed by isoforms of cytochrome P-450 nitroreductases present in host cells, rendering electrophilic metabolites (5
). In the therapeutic protocol, BZL is orally administered. It is possible that increased global GST activity and expression of GST-μ in liver and GST-α in jejunum and ileum by BZL represents a presystemic compensatory mechanism to cope with overproduction of electrophilic metabolites.
Apart from overexpression of biotransformation systems, increased clearance of endo- and xenobiotics is also frequently associated with higher levels of efflux transporter proteins, such as P-gp and/or MRPs (8
). In this study, we observed upregulation of P-gp and Mrp2 proteins by BZL mainly in liver and jejunum. A global approach was used to further evaluate whether induction of these transporters had functional consequences. With an in vivo
model, we demonstrated increased transport activity for P-gp in liver using a typical substrate, Rh123, in agreement with transporter upregulation. It is known that Rh123 is also a substrate for breast cancer resistance protein (Bcrp) (38
). However, its influence in increased Rh123 transport is unlikely, since we did not observe changes in Bcrp levels (unpublished results). The induction of hepatic Mrp2 is usually followed by an increase in the excretion rate of its substrates. The slight increment in the biliary excretion of DNP-SG and DNP-CG observed in BZL-treated rats did not correlate with the significant increase in Mrp2 protein expression. This could be due to synthesis of a nonfunctional protein (e.g., Mrp2 not localized to canalicular membrane), the presence of other compounds competing with Mrp2-mediated excretion, or a nonsaturating concentration of CDNB. To further clarify this issue, we estimated the transport activity of Mrp2 in isolated hepatocytes. In the isolation process, hepatocytes are washed out of the intracellular compounds, including derivatives from BZL metabolism that could compete with DNP-SG for secretion via Mrp2. In this model, we found a direct correlation between activity and increased protein levels, suggesting that the in vivo
results could be, at least in part, a consequence of competition among DNP-SG and other potential Mrp2 substrates, such as BZL-thiol or glucuronic conjugates. Thus, overexpression of these transporters in liver may result in faster biliary excretion of drugs that are their substrates, depending on their relative affinities diminishing their effectiveness and/or toxicity.
It is known that P-gp and Mrp2 expression levels vary inversely along the small intestine (29
). Mrp2 is the most highly expressed in the proximal intestine, whereas P-gp expression is higher distally (40
). In our study, the major induction of both transporters occurred at the proximal segment of the small intestine. Interestingly, the increase in P-gp levels was extended beyond the ileum, its normal site of expression. When the intestinal excretion rate of Rh123 was tested in the in vivo
model, we observed a significant increase in BZL-treated rats in comparison with controls, in agreement with P-gp upregulation. Again, Bcrp levels did not vary between groups (unpublished results). For intestinal Mrp2, when in vivo
activity was measured, a higher DNP-SG/CG elimination rate was detected in BZL-treated rats than in controls, consistent with the upregulation of this protein by the drug. Consequently, the substantial increases in P-gp and Mrp2 expression rates and activities in proximal intestine can lead to an increased secretion of substances that are present in blood or a reduced absorption of drugs that are orally administered, including BZL itself.
Whether the current findings on the induction of biotransformation and transport systems also occur in patients receiving BZL is not known. The usual doses used for the treatment of Chagas disease vary between 5 and 10 mg/kg b.w. administered for 30 to 60 days, or even up to 5 months in the case of disease reactivation (41
). In our study, 100 mg/kg b.w./day for 3 consecutive days was the dose of BZL that showed inductive effect. The BZL plasma concentration measured 24 h after the last injection in rats was 15 μM on average, similar to that found in patients (13 to 26 μM) 24 h after the last dose of a 30-day treatment (13
). In general, rodents need a higher dose of a given compound to reproduce the same effects as in humans. Thus, an inducer effect of BZL in patients cannot be ruled out because the time of treatment is more extended than in our experimental approach. Drug-drug interactions could be particularly important in chagasic patients under immunosuppressant treatment with cyclosporine, corticosteroids, and azathioprine for heart or kidney transplantation (42
) or in HIV patients infected with T. cruzi
and receiving antiretrovirals (44
In addition, changes in BZL pharmacokinetics would also be expected. In support of this hypothesis, Raaflaub (13
) observed that the maximal plasma concentration in male patients that received BZL (7 mg/kg b.w./day for 30 days, twice a day) tends to decrease with the course of treatment (−20% on average after 25 days), suggesting an increase in BZL metabolism (autoinduction) and/or excretion and/or limited absorption. Here, the calculated BZL pharmacokinetic parameters show a higher elimination rate from plasma in the treated group than in controls. The lower AUC observed in plasma from BZL pretreated rats in comparison with controls can result from a higher amount of BZL excreted in bile 90 min post-BZL administration. In addition, as a lower maximum concentration in plasma (Cmax
) was achieved in the BZL group, reduced BZL absorption is suggested. The decreased mucosa to serosa transport of BZL in intestinal sacs confirms this assumption. The participation of P-gp in BZL efflux was observed in P-gp knockdown HepG2 cells (12
). Further experiments are needed to corroborate the contribution of this transporter or any other in BZL transport in an in vivo
model. The data from the current study suggest the possibility of a progressive decrease in BZL absorption and/or increase in BZL metabolism/elimination after therapeutic administration. Unfortunately, we found no studies in the literature evidencing this possibility or a link with decreased therapeutic efficacy.
Although the reason CYP3A, GST, P-gp, and Mrp2 in kidney are not induced by BZL is unknown, it seems to be organ specific. The pregnane X-receptor (PXR) is a nuclear receptor that controls the expression of phase I and phase II biotransformation enzymes as well as xenobiotics transporters (6
). PXR is highly expressed in liver and to a lesser extent in small intestine in humans, rats, mice, and rabbits. Interestingly, these are the same tissues where biotransformation and transporter systems are most highly expressed and induced by BZL. Lower levels of PXR have also been detected in kidney (46
). The knockdown of PXR in HepG2 cells was able to abolish the induction of CYP3A4, GST-π, P-gp, and MRP2 by BZL, suggesting that this nuclear receptor is involved in BZL effects (12
). We postulate that the differential induction of studied systems in liver and intestine versus those in kidney could be related to tissue-specific differences in PXR expression and/or other transcription factors. Further studies are required to elucidate the mechanisms underlying the effects of BZL on these systems (i.e., transcriptional versus nontranscriptional, nuclear receptor participation, etc.).
In conclusion, BZL increases the expression and activities of P-gp and Mrp2 mainly in liver and proximal intestine along with upregulation of hepatic CYP3A and hepatic and intestinal GST. These findings suggest that under BZL treatment, drug-drug interactions could appear, especially at the excretion level, the limiting pathway in the depuration of endogenous and exogenous compounds.