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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
G on the Oral Bioavailability of Low Bioavailable Therapeutic Agents
Pharmacokinetics-Biopharmaceutics Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore, Baltimore, Maryland (N.N.S., N.D.E.); and Division of Pediatric Gastroenterology and Nutrition and Gastrointestinal Section, Center for Vaccine Development, Department of Physiology, School of Medicine, University of Maryland at Baltimore, Baltimore, Maryland (A.F., M.T.)
Received June 24, 2004; accepted September 15, 2004.
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Abstract |
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G, the biologically active fragment of Zot, was isolated and shown to increase the in vitro transport and in vivo absorption of paracellular markers. The objective of this study was to examine the effect of G on the oral bioavailability of low bioavailable therapeutic agents. Jugular vein cannulated Sprague-Dawley rats were randomly assigned to receive the following treatments intraduodenally (ID): [3H]cyclosporin A, [3H]ritonavir, [3H]saquinavir, or [3H]acyclovir at (120 µCi/kg) alone, with protease inhibitors (PIs), or with G (720 µg/kg)/PI. Serial blood samples were collected, and plasma was analyzed for radioactivity. After ID administration with G/PI, Cmax significantly (p < 0.05) increased over a range of 197 to 5700%, whereas area under the plasma concentration time curve displayed significant increases extending over a range of 123.8 to 4990.3% for the investigated drugs. G significantly increased the in vivo oral absorption of some low bioavailable drugs in the presence of PI. This study suggests that G-mediated tight junction modulation, combined with metabolic protection, may be used to enhance the low oral bioavailability of certain drugs when administered concurrently.
). However, these absorption enhancers cause serious damage to the epithelial integrity, morphology, and function (e.g., exfoliation of the intestinal epithelium, diminished cell adhesion) (Thanou et al., 2000a).
Fasano et al. have identified zonula occludens toxin (Zot), a 45-kDa protein located in the cell envelope of Vibrio cholerae, which reversibly opens the tight junctions between cells and increases the paracellular transport of many drugs in a reversible and non toxic manner (Fasano et al., 1995, 1997; Fasano and Uzzau, 1997; Cox et al., 2001, 2002). In addition, Zot increased insulin oral bioavailability in diabetic rats (Fasano and Uzzau, 1997). Later, Di Pierro et al. and Uzzau et al. identified a receptor for Zot and Zonulin, an eukaryotic Zot analog that governs tight junctions permeability (Di Pierro et al., 2001) in the small intestinal epithelium, the nasal epithelium, the blood brain barrier endothelium, and the heart (Uzzau and Fasano, 2000). Studies in our laboratory have shown that Zot enhances the transport of drug candidates of varying mol. wt. (mannitol, polyethylene glycol 4000, inulin, and sucrose) or low bioavailability (paclitaxel, acyclovir, cyclosporin A, and doxorubicin) across Caco-2 cells and the bovine brain microvessel endothelial cells without modulating transcellular transport (Cox et al., 2001, 2002; Karyekar et al., 2003).
Recently, Di Pierro et al. (2001) introduced a smaller 12-kDa fragment of Zot, named G, as the biologically active fragment of Zot. Amino acid comparison between Zot active fragment and Zonulin, combined with site-directed mutagenesis experiments, confirmed the presence of an octapeptide receptor-binding domain toward the amino terminus of the processed Zot. Our earlier studies showed that G significantly increased the in vitro transport of paracellular markers in a nontoxic manner (Salama et al., 2003, 2004). Hence, this study is the first study to evaluate the effectiveness of G as an absorption enhancer after oral coadministration with low bioavailable therapeutic agents covering a wide range of mol. wt., lipophilicity/hydrophilicity, and efflux properties. Cyclosporin A is an immunosuppressant agent with high mol. wt., efflux properties and low oral bioavailability (F < 20%) (Ogino et al., 1999). A group of antiretroviral drugs was also investigated because of their enormous therapeutic value. Protease inhibitors, saquinavir and ritonavir, are used in HIV treatment. However, low or variable bioavailability is attributed to poor absorption or extensive first pass metabolism (Williams and Sinko, 1999). These drugs are subject to secretory transporters, mainly P-glycoprotein (P-gp). Acyclovir (mol. wt. 225.2) (200 mg 5 times/day for 7–10 days; http://www.niv.ac.za/virussa/virusa/vsa7_1.htm) is used to treat shingles and herpes virus infections with F = 15 to 20% (O'Brien and Campoli-Richards, 1989; Wagstaff et al., 1994).
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Materials and Methods |
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Methods
Animals. Jugular vein cannulated male Sprague-Dawley rats (250–275 g) were purchased from Harlan (Indianapolis, IN). Rats were housed individually in cages and allowed to acclimate at least 2 days after arrival before surgeries were performed. Rats were fed rat chow and water ad libitum and maintained on a 12-h light/dark cycle. The jugular vein cannula was kept patent by flushing with heparinized physiological saline and heparinized glycerol. The protocol for the animal studies was approved by the School of Pharmacy, University of Maryland Institutional Animal Care and Use Committee.
Intraduodenal Cannulation to Rats. Jugular vein cannulated Sprague-Dawley rats were anesthetized with i.p./i.m. injection of a ketamine/xylazine solution (80 mg/kg ketamine, 12 mg/kg xylazine). PE 50 was used for duodenal cannulation according to the method reported by Lukas and Moreton (1979). A pair of anchor blips were made in the PE 50 tubing (cannulas) via melting by hot air stream. A thin 25-gauge metal wire was inserted through the lumen of the cannula during blip formation to avoid its collapse by heating. Each cannula was tested for leakage by phosphate-buffered saline flushing. The abdomen and the back of the neck were shaved and skin was treated with antiseptic solution (BacDown parachlorometaxylenol). An incision was made in the epigastric region. The midline sector of the torso was identified and the abdominal muscles were separated by blunt dissection to expose the peritoneum. The duodenum was located and clamped gently. Purse stitches were made away from the blood capillaries and toward the stomach side (pylorus). The clamped duodenum was punctured using an 18-gauge needle. Cannulas were tailored to the same length and were inserted at approximately the same site in the punctured duodenum. The purse sutures were tightened around the cannula. The clamp was removed, and the cannula was flushed by physiological saline. The cannula was exteriorized from the muscle using a trochar and fixed by sutures. The trochar was used to guide the cannula beneath the skin to be exteriorized at the back of the neck. The muscle and skin were stitched separately. The wound was cleaned aseptically with betadine. Phosphate-buffered saline was infused very slowly to ensure the free flow of fluids through the intraduodenal cannula. Rats were allowed to recover from surgery for 2 days prior to the initiation of the study.
Oral Dosing with G. The animals were fasted for at least 12 h prior to the experiment, with free access to water. Rats (n = 3–6/group) were randomly assigned to receive intraduodenally (ID): [3H]drug (acyclovir, cyclosporin A, ritonavir, and saquinavir) (120 µCi/kg) with physiological saline, [3H]drug (120 µCi/kg)/protease inhibitors (PIs), or [3H]drug (120µci/kg)/PI with G (720 µg/kg). The PI mixture was composed of 30 mg/kg bestatin, 30 mg/kg captopril, and 67 mg/kg leupeptin. This mixture was previously shown to protect G against metabolic degradation in vivo (Salama et al., 2003, 2004). Bestatin, leupeptin, and captopril were selected because of their inhibitory effect on leucine aminopeptidase, aminopeptidase B, triaminopeptidase, angiotensin converting enzyme, serine and thiol proteases, calpain, cathepsin B, H, and L, and trypsin (Umezawa, 1976; Kuramochi et al., 1979; Knight, 1980; Aoyagi and Umezawa, 1981; Zimmerman and Schlaepfer, 1982; Zollner, 1993). The sequence of ID administration was PI or saline, followed by G or saline, and finally the drug over a period of 1 to 2 min for the test and control groups, respectively. The solution volume administered was adjusted to 2 ml with physiological saline. Serial blood samples (250 µl) were collected via the jugular vein cannula at: 0 (background), 5, 10, 20, 30, 45, 60, 90, 120, 240, 360, and 420 min and replaced by physiological saline (250 µl). Blood samples were centrifuged (13,000 rpm for 10 min), and plasma was obtained. Scintillation cocktail (5 ml) was added to the plasma samples, and radioactivity was measured by a Beckman Coulter LS 6500 multipurpose scintillation counter. At the end of the experiment, rats were sacrificed by CO2 asphyxiation, and the duodenum was inspected to ensure that the intraduodenal cannula was in place during dosing.
Data Analysis. The amount of radiolabeled drug absorbed was converted to concentrations using the specific activity of the radio-labeled stock drug solution. The pharmacokinetic (PK) parameters were calculated using noncompartmental analysis based on the statistical moment theory using Winnonlin pharmacokinetic software package (Pharsight, Mountain View, CA). The area under the plasma concentration time curve was calculated using the linear trapezoidal rule. The highest observable concentration and the associated time point were defined as Cmax and Tmax, respectively. Data are presented as mean ± S.D. The percent enhancement ratio (%ER) for the PK parameters was calculated from the following equation:
 | (1) |
The SPSS statistical package was used for statistical comparisons of control and treatment groups using analysis of variance and Dunnett's post hoc test (p < 0.05).
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Results |
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G as an absorption enhancer in vivo. Male Sprague-Dawley rats cannulated in the jugular vein and the duodenum were randomly assigned to receive either radiolabeled drug with/without PI and G. The pharmacokinetic profile was characterized for each group, and the PK parameters were calculated using noncompartmental analysis. The effect of G on the oral bioavailability, as measured by the rate (Cmax and Tmax) and extent (AUC and Cmax) of drug absorption, was evaluated. The G dose selected for investigation was determined based on earlier studies with G and paracellular markers. Since 80% of protein enzymatic degradation comes from peptidases in the brush border or within the epithelial cell cytoplasm (Carino and Mathiowitz, 1999), PI were included in one treatment arm to protect G, a small peptide, against enzymatic digestion.
The HIV protease inhibitors, saquinavir and ritonavir, are used extensively for the treatment of HIV. When G was administered with the antiretroviral agents, G produced a significant improvement in the extent and/or rate of absorption of each anti-HIV drug (Tables 1 and 2). However, ritonavir showed a higher increase in AUC (188.1%) and Cmax (197%) (Fig. 1) compared with saquinavir/PI/G (%ER AUC 123.8% and Cmax 153.8%, respectively). In addition, ritonavir exhibited a large decrease in Tmax contrary to saquinavir, which displayed almost no change in Tmax for saquinavir/G/PI group versus saquinavir group. Saquinavir displayed more than 2-fold (p < 0.05) faster elimination with t1/2 of 195.7 min for saquinavir/G/PI group versus 473.6 min for saquinavir alone.
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Since the investigated therapeutic molecules are susceptible to first pass metabolism, the effect of PI on drug absorption was tested after ID administration to rats. The administration of PI with ritonavir led to 145.3% increase in the AUC0–7 h and 127% increase in Cmax compared with ritonavir alone. In comparison, the coadministration of G/PI with ritonavir resulted in 188.1% increase in AUC0–7 h and 197% increase in Cmax. The increase in Cmax (39.4 ng/ml) was statistically significant after inclusion of G in the treatment in comparison with the Cmax observed with ritonavir/PI (25.4 ng/ml) and ritonavir (20.0 ng/ml) groups. In addition, inclusion of G in the treatment resulted in a significant increase in AUC0–7 h (12598.8 ng/ml/min) compared with ritonavir (6696.4 ng/ml/min). Ritonavir plasma levels were significantly higher for the ritonavir/G/PI group compared with those of ritonavir group. These observations suggest that coadministration of PI with ritonavir increased the amount of drug absorbed and raised plasma drug levels due to metabolic protection, yet inclusion of G with PI led to significantly larger increases in the oral absorption of ritonavir. For saquinavir, the increase in AUC0–2 h was statistically significant for the saquinavir group (225.2 ng/ml/min) compared with the saquinavir/PI group (270.3 ng/ml/min) and saquinavir/PI/G with PI (278.8 ng/ml) (Fig. 2). In addition, the Tmax was altered by PI and by G/PI compared with the saquinavir group. Furthermore, Cmax for saquinavir was increased by G administration (4.0 ng/ml) compared with the administration of saquinavir (2.6 ng/ml) and with saquinavir/PI (3.0 ng/ml). These observations show that the administration of G with PI increased the amount of saquinavir absorbed to a very limited extent (%ER = 123.8% versus 120% for saquinavir/PI/G and saquinavir/PI, respectively); however, this increase was not statistically significant. This coincides with the high metabolic susceptibility of saquinavir, which implies that metabolic protection against first pass metabolism will contribute significantly to an increase in its absorption. Collectively, the increased oral absorption in case of G with PI for the HIV protease inhibitors is a combined effect of metabolic protection of the drug and increase in the fraction of drug absorbed paracellularly via G-mediated tight junction modulation, yet the contribution of G appears to be marginal.
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Similarly, PI with/without G significantly enhanced both the rate and extent of acyclovir absorption. The Cmax and AUC0–2 h of acyclovir were significantly (p < 0.05) increased by 216.7 and 145.9%, respectively, for the acyclovir/G/PI group versus acyclovir group, yet no statistical difference was observed in Cmax for acyclovir/PI versus acyclovir group. The AUC0–2 h and Cmax for acyclovir/G/PI (8.9 ng/ml/min and 0.13 ng/ml, respectively) as well as for acyclovir/PI (8.1 ng/ml/min and 0.08 ng/ml, respectively) were higher than those of acyclovir group (6.1 ng/ml/min and 0.06 ng/ml, respectively) (Table 1). A decline in Tmax from 52 min for the control group to 20 min for G-treated group was observed; however, G had a minimal effect on t1/2 (254.4 versus 227.6 min for acyclovir/G/PI and acyclovir, respectively) (Fig. 3). In addition, the coadministration of G with acyclovir/PI resulted in significantly higher acyclovir plasma levels up to 20 min, which were not observed after coadministration of PI alone with acyclovir. The 13.1% higher AUC0–2 h increase and the 83.4% higher Cmax increase obtained due to G coadministration relative to acyclovir/PI suggest that the absorption enhancement achieved could be confounded with the effect of PI-metabolic protection and that, similar to saquinavir, the absorption of acyclovir will benefit significantly by metabolic protection, whereas the contribution of paracellular modulation by G appears minimal.
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On the other hand, the PK parameters for cyclosporin A, an immunosuppressant drug highly susceptible to efflux, displayed significant alteration with G administration (Table 1). Being a peptide, it would be expected that cyclosporin A would be extensively metabolized in the GI tract by enzymes and intestinal flora and observations similar to those of G with the antivirals were expected. However, seven of the amino acids of cyclosporin A are N-methylated and may, therefore, minimize but do not completely prevent degradation of the drug in the GI tract (Fahr, 1993). The main site of metabolism for cyclosporin A is CYP3A in the liver and intestinal membranes (Kronbach et al., 1988). In addition, cyclosporin A is a CYP3A4 and P-gp inhibitor. When cyclosporin A was coadministered with PI alone, Cmax, AUC0–7 h, and Tmax were not significantly changed. However, the inclusion of G with PI led to dramatic increase in the absorption of cyclosporin A significantly higher than either cyclosporin A alone or cyclosporin A/PI administration (Fig. 4). The increase in AUC0–7 h (102 versus 87.2 versus 5090.1 ng/ml/min for cyclosporin A, cyclosporin A/PI, and cyclosporin A/G/PI, respectively) and Cmax (0.4 versus 0.3 versus 22.8 ng/ml for cyclosporin A, cyclosporin A/PI, and cyclosporin A/G/PI, respectively) following paracellular absorption modification reached 4990 and 5700%, respectively.
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Discussion |
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G, the biologically active fragment of Zot, displays the intrinsic activity of modulating tight junctions and thereby increasing paracellular drug transport (Di Pierro et al., 2001). Zot/G reversibly opens tight junctions through the activation of a cascade of intracellular events with protein kinase C 
-related polymerization of actin filaments and resulting in modification of the cytoskeleton (Fasano et al., 1995). In earlier in vitro studies, G increased the transport of paracellular markers (
3-fold) without toxicity manifestations (Salama et al., 2003, 2004), contrary to other absorption enhancers (Duizer et al., 1998; Sakai et al., 1998; Dodane et al., 1999). In vivo studies showed that protease inhibitors are necessary to minimize G-enzymatic degradation secondary to proteases/peptidases (Salama et al., 2003). After oral administration, G/PI displayed a high intrinsic biological activity with 3-fold higher Cmax and AUC0–6h for mannitol, whereas no significant differences were observed with G alone. Furthermore, G was also capable of improving the AUC and Cmax for macromolecules up to 6.6- and 13-fold, respectively (Salama et al., 2004). Studies of the effect of PI alone on the absorption of paracellular markers at BBB (Karyekar, 2002) and the gastrointestinal tract (Salama et al., 2004) versus paracellular marker alone, marker/G, and marker/G/PI showed that PI alone caused no significant difference in the absorption or tissue concentration of the tested paracellular marker. Therefore, collectively, administration of PI alone or G alone did not result in significant absorption improvement, and G/PI absorption enhancement is due to metabolic protection of G.
The low oral bioavailability (F = 3–20%) of the investigated drugs has been largely attributed to P-gp-mediated efflux and/or first pass metabolism. Modification of drug transport route by G to overcome the effect of efflux and increase their oral absorption was investigated. Since the investigated drugs are susceptible to first pass metabolism, the administration of PI, originally added to protect G, with most drugs resulted in increased drug absorption, and the addition of G to PI/drug treatment led to further increases in PK parameters and drug plasma levels, which were statistically significant (p < 0.05) compared with drug administration alone (Figs. 1, 2, 3, 4). However, the absorption enhancement observed with drug/G/PI coadministration was comparable with that observed for drug/PI administration with some of the investigated drugs (saquinavir and acyclovir).
The effect of G/PI was most prolonged for ritonavir and cyclosporin A, with plasma drug levels significantly higher than control levels for 7 h versus an elevation for 2 h with other drugs. This observation might be because cyclosporin A and ritonavir are CYP3A inhibitors, a characteristic that distinguishes these two compounds from the other investigated agents (Clarke and Rivory, 1999; Jover et al., 2002). The in vitro enhancement effect of G in Caco-2 cells (lacks CYP3A4; Schmiedlin-Ren et al., 2001), lack of in vivo effect of G alone, along with the large increases in rate and extent of absorption of cyclosporin A and ritonavir (CYP3A4 inhibitors) after G/PI administration, suggest that "exogenous" G may be susceptible to metabolism by cytochrome P450 (47 isoforms in rat), the major enzyme family responsible for the biotransformation of a variety of endogenous substrates and xenobiotics. Three CYP gene families (CYP1, CYP2, and CYP3) have been postulated to be responsible for most drug metabolism in humans and rats (Wrighton et al., 1996). Consequently, it is possible that CYP3A inhibition contributes to metabolic protection of G and, therefore, extending its effect for a prolonged period. The metabolic profile for G has to be closely studied to validate this assumption. In addition, G possible interaction with efflux transporters has to be addressed to account for the differential increase in effects observed with cyclosporin A (P-gp inhibitor) and ritonavir (not a P-gp inhibitor in vivo). The difference in G in vivo effect is the resultant effect of differences in drug mol. wt., lipophilicity/hydrophilicity, and relative extent of susceptibility to efflux transporters.
The biological activity of another series of absorption enhancers, the chitosans, has been extensively evaluated to modify the absorption of peptides (bioavailability < 1%) after coadministration in vivo. Trimethyl chitosan 60 (TMC60, 1%, 60% degree of trimethylation) increased the absolute bioavailability of octreotide, a peptide drug model, by 16%, with a 5-fold increase in Cmax after intrajejunal administration to rats (Thanou et al., 2000b), yet 5 and 10% (w/v) TMC60 increased octreotide oral bioavailability by 14.5- and 7.7-fold after intrajejunal administration to pigs (Thanou et al., 2001). In addition, TMC40 (1%) and TMC60 (1%) produced 8- and 16-fold increases in buserelin bioavailability after ID administration to rats (Thanou et al., 2000a). Other proposed absorption enhancers, such as the synthetic bile acid derivative cholylsarcosine (20 mM), increased the absorption efficiency of octreotide and desmopressin by 13.5- and 14.5-fold, respectively (Michael et al., 2000). Thus, most reported absorption enhancers produced in vivo absorption enhancements up to 15-fold for proteins/peptide drugs which are restricted to paracellular transport. On the contrary, our experiments show that G increased the absorption of drugs, which permeate the cell and are susceptible to first pass metabolism and efflux transporters, up to 50-fold, besides paracellularly transported macromolecules (Salama et al., 2004).
Strategies to increase absorption (by absorption enhancers), decrease degradation (by peptidase inhibitors), or both could overcome GI absorption barriers. The addition of PI to one of the treatment arms was aimed at protecting G from metabolic degradation. However, as such, some tested therapeutic agents exhibited a combined absorption enhancement effects via metabolic protection and paracellular modulation. The combined effect of absorption enhancement and metabolic protection was previously addressed for Bowman-Birk inhibitor with chitosan-EDTA conjugate (Bernkop-Schnurch and Pasta, 1998) and antipain with chitosan-insulin (Bernkop-Schnurch et al., 1997).
In conclusion, G significantly increased the Cmax and AUC for some investigated drugs up to 57- and 50-fold, respectively, in the presence of PI. However, for drugs highly susceptible to metabolic degradation, G-mediated absorption enhancement appeared to be minimal compared with PI metabolic protection. G mechanism of action suggests significant benefit to macromolecules (mainly absorbed paracellularly). A mixture of G/PI might benefit drugs whose low oral bioavailability is due to comparable contributions of efflux susceptibility and first pass metabolism. Following metabolic protection by structural modification or formulation approaches, G has the potential (as evidenced by cyclosporin A enhanced absorption) to act as a novel absorption enhancer for drugs susceptible to efflux transporters by modification of their transport route.
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Footnotes |
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ABBREVIATIONS: Zot, zonula occludens toxin; HIV, human immunodeficiency virus; P-gp, P-glycoprotein; ID, intraduodenal(ly); PI, protease inhibitor; PK, pharmacokinetic; ER, enhancement ratio; AUC, area under the curve; GI, gastrointestinal; TMC, trimethyl chitosan.
Address correspondence to: Dr. Natalie D. Eddington, Pharmacokinetics-Biopharmaceutics Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore, Health Sciences Facility II, 20 Penn Street, Room 543, Baltimore, MD 21201. E-mail: neddingt{at}rx.umaryland.edu
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