Impact of Intestinal Transporters on Bioavailability

The importance of membrane transporter proteins has been well established with respect to the bioavailability of many therapeutic compounds. Notwithstanding their physiologic importance (e.g., fulfillment of nutrient requirements and waste trafficking), membrane transporter proteins can be particularly important due to their substrate specificity and kinetics and provide attractive therapeutic targets for pharmaceutical intervention. However, tissue and temporal expression patterns, as well as cellular localization, are all extremely important in determining the relevance of a particular transporter with respect to the contribution of transporter activity on the bioavailability of exogenous substrates. The cellular permeation of a compound is a function of a multitude of parameters and virtually unpredictable without adequate study. Moreover, given the obvious physiological relevance of many of the endogenous transporter protein substrates, it is also essential to explore the subtle interplay between different transporter proteins to elucidate the underlying mechanisms of transitional trafficking veiled by the net transcellular flux. In short, the interplay of various transporter protein mechanisms along with transport by parallel pathways can significantly impact on the overall bioavailability of a compound.

P-gp, possibly the most studied transporter protein, is expressed on the apical surface of intestinal epithelial mucosa where its primary function is to promote the removal of toxic xenobiotics, a side-effect of which is the active efflux of various pharmaceutical compounds. The role of P-gp in restricting intestinal absorption was clearly determined by observing two- to fivefold greater plasma concentrations of the HIV-1 protease inhibitors indinavir, nelfinavir, and saquinavir in mdr1a(-/—) mice, as compared to wild-type mice (Kim et al., 1998). Additionally, Chiou et al. (2000) have also reported the absolute bioavailability of tacrolimus increased from 22% in normal mice to 72% in P-gp knockout mice.

In lieu of functional knockout studies, a number of SNP have also been identified in the human MDR1 gene. A number of these SNPs have been associated with changes in P-gp expression, thereby affecting the pharmacokinetic profiles of a number of substrates (Hoffmeyer et al., 2000; Kim et al., 2001; Kurata et al., 2002). For example, Kurata et al. (2002) demonstrated the role of human MDR1 gene polymorphism on the bioavailability of digoxin, a known P-gp substrate. Previously, Hoffmeyer et al. (2000) showed that homozygosity for a polymorphism in exon 26 (C3435T) of the MDR1 gene was observed in 24% of a study sample population (n = 188) with these patients exhibiting significantly lower duodenal MDR1 expression as well as significantly increased plasma levels of digoxin. Furthermore, differences in the expression of P-gp, sP-gp, MDR3, MRP's (1-5), and lung resistance-associated protein (LRP) in the Caucasian and Chinese intestines have also been reported, suggesting variations in drug bioavailability due to ethnicity (Wang et al., 2004). Genetic polymorphism is not limited to the MDR1 gene, but has also been shown in both MRP1 and MRP2 in a Japanese population (Moriya et al., 2002). Clearly, there are multiple factors that influence inter-individual variability in drug absorption and disposition, and determining the precise contribution of genetic polymorphism to this variability in oral absorption will continue to be an interesting and challenging area in the future.

While genetic variations and/or environmental factors can obviously affect bioavailability, it is the interplay of various transporter proteins and other metabolic (e.g., cytochrome P-450s) and nonmetabolic (e.g., transduction factors) proteins that are more likely to influence individual therapy. One such example is the underlying interplay between P-gp and CYP3A4, a major drug-metabolizing enzyme (Watkins, 1997). As mentioned above, P-gp is expressed on the apical surface of intestinal epithelial mucosa where its primary function is to promote the removal of toxic xenobiotics, via active efflux into the intestinal lumen. CYP3A4, in contrast, is intracellularly localized and is a broad affinity, metabolizing enzyme. The coexpression of these two proteins results in an increase in drug metabolism through an increase in exposure to not only CYP3A4, but also any proteases, drug metabolizing enzymes, and the natural sink condition of the intestines. Therefore, the repeated exposure to both the harsh intestinal conditions, as well as the drug metabolizing enzyme, results in a net decrease in plasma concentrations, thus limiting overall bioavailability (Fig. 7.3) (Watkins, 1997; Benet et al., 2001).

In contrast to the efflux transporter proteins where substrate activity is a negative attribute of potential drug candidates, influx transporters (i.e., peptide transporters) provide a unique opportunity to specifically design and develop therapeutic agents with significantly higher molecular recognition for potentially increasing intestinal drug absorption and bioavailability. This strategy has been utilized in the development of prodrugs exhibiting high affinity for PepT1, as observed in the synthesis of L-Val-acyclovir (valacyclovir) prodrug of acyclovir (Friedrichsen et al., 2002; Bhardwaj et al., 2005b), although affinity for other transporters in addition to PepT1 cannot be ruled out (Phan et al., 2003; Landowski et al., 2003). Acyclovir has an oral bioavailability between 15 and 30% (Fletcher and Bean, 1985), while


Small Intestine

S + Met

Intestinal Elimination

Figure 7.3. Interplay of P-gp and CYP3A4 affecting the absorption of substrates (S) and potential metabolites (Met) from a representative intestinal epithelium cell. Substrate enters the enterocyte through any number of mechanisms and can either enter the circulation, be metabolized by CYP3A4, or effluxed into the intestinal lumen via P-gp. The effluxed substrate may then be either reabsorbed into the enterocyte where the process may then be repeated, or excreted through intestinal elimination. The reabsorbed substrate may then undergo additional metabolism, be absorbed into the circulation, or be effluxed again. (Modified from Benet and Cummins (2001))

Soul-Lawton et al. (1995) estimated a 54.2% mean bioavailability of acyclovir in healthy volunteers after a single dose of valacyclovir. Other studies have demonstrated three to fivefold increases in bioavailability for valacyclovir as compared to acyclovir (Weller et al. 1993; Steingrimsdottir et al., 2000). A more detailed review of the clinical advantages and pharmacokinetics of valacyclovir were elaborated by MacDougall and Guglielmo (2004). It is also important to mention one study by Kimberlin et al. (1998) where preliminary pharmacokinetic data for acyclovir were contrasted with valacyclovir therapy in pregnant women receiving herpes simplex virus suppressive therapy. Valacyclovir therapy resulted in an increased acyclovir bioavailability and increased acyclovir concentrations in the amniotic fluid with no evidence of a preferential accumulation in the fetus (1.7 mean maternal/umbilical vein plasma ratios for valacyclovir to 1.3 for acyclovir therapy). Other prodrugs, particularly angiotensin-converting enzyme inhibitors (e.g., captopril), have also shown to be PepT substrates, with important consequences in their pharmacokinetics (Sugawara et al., 2000; Shu et al., 2001).

Another strategy to increase drug bioavailability via peptide transporters is by stimulating PepT-like transport by increasing the proton driving force. Nozawa et al. (2003) used a proton-releasing polymer, Eudragit L100-55, to increase the luminal/intracellular proton gradient to stimulate PepT1 transport. Incremental increases in intestinal transport activity were seen. Nielsen et al. (2002), Kunta and Sinko (2004), and Steffansen et al. (2004, 2005) have reviewed additional examples of therapeutic applications of the PepT1 transport pathway.

Organic anion transporter proteins have also been shown to influence drug absorption of therapeutic compounds. For instance, the intestinal absorption of the

H1-histamine receptor antagonist fexofenadine is primarily mediated by OATP-B not P-gp (Dresser et al., 2002). Inhibition of OATP-B, by ingestion of grape fruit juice, decreased the fexofenadine area under the plasma concentration-time curve (AUC), the peak plasma drug concentration (Cmax), and the urinary excretion values to 30-40% of those compared with the compound ingested with water in human. Since grape fruit juice is a more potent inhibitor of OATPs compared to P-gp, the reduced oral bioavailability of fexofenadine is suggested to result from limited influx process via OATP-B (Dresser et al., 2002). Furthermore, the drug may be cycled between the enterocyte and the intestinal lumen, which may also result in increased metabolism and/or hydrolysis, as mentioned above. However, this model has not been studied specifically for this compound.

Further evidence suggests that OATP-B may be involved in the intestinal uptake of pravastatin, as mentioned in Sect. 7.3.2. These data suggest OATP-B not only mediates absorption of anionic compounds, but that its activity may be optimum at the acidic surface microclimate of the small intestine. In addition, infusion of pravastatin directly into the stomach resulted in maximal absorption in the duodenum and enhanced the bioavailability of pravastatin (Triscari et al., 1995). This observation correlates well with the expression patterns of OATP-B in human enterocytes, where OATP-B was immunohistochemically localized at the apical membrane of intestinal epithelial cells (Kobayashi et al., 2003). Therefore, determinations of the intestinal expression patterns of other important OATP/Oatp family members could have critical clinical relevance. Unfortunately, limited in vivo data exist to ascertain the full functional significance of this transporter family with respect to oral drug absorption.

It is clear that OCT family members function primarily in the elimination of cationic drugs and other xenobiotics in the kidney and the liver; however, their role with respect to intestinal transport is poorly understood. Studies suggest that OCTs, especially OCT1, mediate the basolateral uptake of organic cations from the blood to the intestine and may play an important role in the secretion of compounds to the small intestinal lumen. Jonker et al. (2001) demonstrated that the intestinal secretion of TEA was reduced about twofold after i.v. administration in Oct1(-/—) knockout mice. Wang et al. (2002) further reported decreased met-formin distribution to the mouse small intestine due to OCT1 knockout. Additionally, the role of OCTs in the active intestinal elimination of drugs is supported in part by the finding that in the presence of cephalexin, a known OCT substrate (Karlsson et al., 1993), plasma AUC of ciprofloxacin increased, while intestinal clearances decreased in rats (Dautrey et al., 1999). The role of other efflux membrane transporters may also contribute to this mechanism and cannot be ruled out.

Unfortunately, targeting OCTs to increase oral bioavailability of drug substrates does not seem to be an attractive approach since these substrates are excreted across the sinusoidal membrane into the hepatocyte and further across the canalic-ular membrane to the bile by an as yet unidentified H+ -organic cation exchanger and/or by P-gp. In contrast, targeting OCTNs could be an efficient method to increase drug bioavailability, by taking advantage of both the transporter's tissue and cellular localizations. OCTNs are localized to the apical membranes of intestinal and renal cells. As such, the transporter can effectively increase drug bioavailability via the absorptive intestinal OCTNs, while simultaneously maintaining blood concentrations by renal resorption through OCTNs. In one such example, the peak concentration and AUC of sulpiride were decreased by the concomitant administration of substrates or inhibitors of OCTN1 and OCTN2 in rats (Watanabe etal., 2004).

Similar to the synthesis of valacyclovir, additional strategies have been developed to exploit the activity and selectivity of specific transporters. One such example is the development of anticancer agent prodrugs, which has been developed to improve not only their physicochemical properties, but also to promote their selectivity and thus reduce undesirable toxicity effects. Floxuridine and gemc-itabine are anticancer agents where studies have shown the feasibility of achieving enhanced transport and selective antiproliferative action of amino acid ester pro-drugs in cell systems overexpressing PepTl (Vig et al., 2003; Landowski et al., 2005; Song et al., 2005). However, one should question the relevance of PepTl transport for these particular compounds in normal physiologic systems in light of the fact that they are also potential substrates for other transporter families, such as the nucleoside transporters. However, the broad substrate specificity, tissue expression, and physiologic relevance of their substrates limit the utility of both nucleoside and monocarboxylate transporters as effective drug targets (see Sects. 7.3.4 and 7.3.5). This example illustrates one of the many problems inherent to the study of drug transport in that overlapping specificity of many of these transporter systems confounds the applicability of transport data to physiologic systems. Technology hurdles also limit the feasibility of studying multiple transporter systems simultaneously, thereby further limiting our understanding of the relevance of a particular transporter to overall transcellular flux.

Drug transporters play an important role in intestinal drug absorption and secretion, and can be major determinants of oral bioavailability. Transporters exhibit affinity for an extraordinary range of compounds and provide great insight for advancing the field of rational drug design. However, understanding the limitations associated with transporter research will aid future scientists in understanding the subtle interactions of each of the distinct transporter families, and may help elucidate their overlapping specificities as well as provide additional therapeutic targets. By understanding the substrate specificity, transport mechanism, and expression profile of transporters, efficient intestinal absorption may be made feasible by strategies including appropriately modifying either the structural recognition elements of NCEs or through rational formulation design to tailor delivery to windows of optimized drug delivery. In addition, complete understanding of the mechanisms of intestinal absorption of various drugs and the underlying regulation of drug transporters could help pharmaceutical scientists to predict the intra-and interindividual variability inherent to the study of oral bioavailability.

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