Introduction

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The intestinal absorption of water-soluble drugs is usually limited by their poor membrane permeability characteristics. Therefore, absorption enhancers have been often adopted to improve the absorption of these poorly absorbable drugs, including hydrophilic antibiotics and peptide and protein drugs. These absorption enhancers include surfactants, bile salts, chelating agents, and fatty acids (Lee and Yamamoto, 1990; Lee et al., 1991). As for surfactants and bile salts, our previous study indicated that rectal permeability of insulin was enhanced by the coadministration of various bile salts such as sodium glycocholate (NaGC), sodium taurocholate, and sodium deoxycholate (NaDC) (Yamamoto et al., 1992). Recently, Uchiyama et al. (1999) demonstrated that n-lauryl--D-maltopyranoside (LM), a nonionic surfactant, and bile salts such as NaGC and NaDC enhanced the permeability of insulin across the intestinal membrane. With regard to chelating agents, it was found that sodium salicylate and 5-methoxysalicylate remarkably enhanced the rectal absorption of insulin in rats (Nishihata et al., 1983; Aungst and Rogers, 1988). Furthermore, it was reported that linoleic acid (fatty acid)-surfactant-mixed micelles improved the intestinal absorption of streptomycin and gentamicin in rats (Muranishi, 1985, 1990). These different types of enhancers have been known to increase the intestinal absorption of poorly absorbable drugs by various mechanisms. These mechanisms involve increase in membrane fluidity, interaction with the ability of calcium ion to maintain the dimension of the intracellular space, solubilization of mucous membrane, change in nonprotein and protein sulfhydryl levels in mucosal tissues, increase in water flux, and reduction of the viscosity of mucus layer adhering to all mucosal surfaces (Lee and Yamamoto, 1990; Lee et al., 1991). However, the absorption enhancers with high effectiveness often cause damage and irritate the intestinal mucosal membrane (Swenson and Curatolo, 1992). Indeed, our previous studies indicated that there existed a almost linear relationship between the absorption-enhancing effects of various absorption enhancers in the small and large intestine and their membrane toxicity (Uchiyama et al., 1996; Yamamoto et al., 1996). Therefore, effective and less toxic absorption enhancers should be developed and used in clinical practice.

Recently, Salzman et al. (1995) reported that nitric oxide (NO) donors increased the permeability of water-soluble compounds across Caco-2 cell monolayers with neither loss of cell viability nor lactate dehydrogenase (LDH) release. In addition, Utoguchi et al. (1998) demonstrated that the rectal absorption of insulin was remarkably enhanced in the presence of NO donors. They also demonstrated the low cytotoxicity of NO donors as evaluated by the cell detachment and LDH release studies in Caco-2 cells. However, few studies have been carried out on the absorption-enhancing effect of NO donors in the intact intestinal membrane. In addition, the absorption-enhancing mechanisms of NO donors have not been examined in detail.

In this study, therefore, we examined the absorption-enhancing effect of NO donors on the intestinal permeability of water-soluble and poorly absorbable compounds using intact rat intestinal membrane. NOC5 [3-(2-hydroxy-1-(methylethyl)-2-nitrosohydrazino)-1-propanamine] and NOC12 [N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine] were chosen as models of NO donors. 5(6)-Carboxyfluorescein (CF) and fluorescein isothiocyanate-dextrans with average molecular weights of 4,000 (FD4) and 10,000 (FD10) were used as models of water-soluble compounds. We also investigated the absorption-enhancing mechanisms of these NO donors by the electrophysiological studies and their intestinal membrane toxicity by measuring LDH release from the intestinal membrane in rats.

	Experimental Procedures

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Materials. CF was kindly supplied from Eastman Kodak Co. (Rochester, NY). NOC5, NOC12, and 2-(4-carboxyphenyl) 4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide sodium salt (carboxy-PTIO) were purchased from Dojindo Laboratory (Kumamoto, Japan). FD4, FD10, NaGC, and sodium taurocholate were purchased from Sigma Chemical Co. (St. Louis, MO). NaDC and Testwako LDH kit were obtained from Wako Pure Chemical Industries Co. (Osaka, Japan). EDTA was obtained from Nacalai Tesque Inc. (Kyoto, Japan). LM was kindly supplied from Japan Fine Chemical Co. (Osaka, Japan). Sodium caprate (NaCap) was purchased from Tokyo Kasei Industries Co. (Tokyo, Japan). All other chemicals and solvents were of reagent grade.

Preparation of the Drug Solution. CF, FD4, and FD10 were dissolved in a modified Ringer's solution adjusted to pH 7.4 to yield a final concentration of 0.1 mM. In certain experiments, the dosing solutions were added to NO donors at concentrations of 0.01 to 1 mM. In the inhibition studies, carboxy-PTIO (0.2 or 0.4 mM), an NO scavenger, was applied to drug and NO donor solution.

Intestinal Permeability Studies. Intestinal permeability of drugs was examined in a modified Ussing chamber (surface area, 0.3 cm²) using stripped rat intestine for 3 h (Yodoya et al., 1994; Asada et al., 1995; Tanaka et al., 1996; Uchiyama et al., 1998). Male Wistar rats, weighing 200 to 250 g, were used. The studies examined in this article have been carried out in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. The intestine was excised and rinsed in phosphate-buffered solution. The experimental segments were obtained and the underlying muscularis was removed before mounting in a modified Ussing chamber. The first 10-cm portion of the small intestine from the stomach (duodenum) was removed and the second 10-cm portion of the small intestine was used as the jejunum. Similarly, the first 10-cm portion of the large intestine from the ileo-cecal junction was removed and the second 10-cm portion of the large intestine was used as the colon. Modified Ringer's solution (2.5 ml) was added to the serosal side. An equal volume of drug solution was added to the mucosal side. In certain experiments, NO donor was applied to the drug solution in the mucosal side or buffer solution in the serosal side. Mixing was performed by bubbling with 95% O₂, 5% CO₂ gas. At predetermined times up until 150 min, solution was sampled from the serosal side and the concentrations of drugs were determined spectrofluorometrically. The apparent permeability coefficients (Papp) were calculated by the relationship Papp = dX_R/dT·1/A·C₀, where Papp is the apparent permeability coefficient in centimeters per second, X_R is the amount of the drugs in moles in the receptor side, A is the diffusion area (i.e., in square centimeters), and C₀ is the initial concentration of drugs in the donor side in moles per milliliter. The viability of intestinal membrane during the test period was monitored by measuring the transport of trypan blue dye and electrophysiological parameters, including potential difference, short-circuit current, and membrane resistance. There was no transport of dye during the incubation and no remarkable change of the electrophysiological parameters, confirming that the viability of the intestinal membrane was maintained during the transport experiment.

Pretreatment with NOC12. The intestine was removed by the same method as described above. The intestinal membrane in the mucosal side was pretreated with 0.1 or 1 mM NOC12 for 10 min. After the exposure of NOC12, the intestinal membrane was washed with modified Ringer's solution and was mounted in the Ussing chamber. The intestinal permeability experiment was studied in the same manner as described under Intestinal Permeability Studies.

Assessment of Membrane Damage. To evaluate membrane damage, the release of LDH from the colonic membrane was measured. LDH is a cytosolic enzyme, and its presence in the apical compartment is generally regarded as evidence of cell membrane damage (Schasteen et al., 1992). For LDH studies, 50-µl aliquots were withdrawn from the donor site at the end of the experiments. The amount of LDH released from the intestinal membrane in the presence or absence of absorption enhancers was determined with a Testwako LDH kit (Wako Pure Chemical Industries Co.). Absorption enhancers used in this experiment did not interfere with the LDH assay.

Electrical Measurements. The transmucosal electrical potential difference and the short-circuit current were measured at 10-min intervals. The transepithelial electrical resistance (TEER) was calculated by Ohm's law. TEER reached steady state about 20 min after mounting the membrane, at which point the experiment was started. TEER was monitored for 60 min in the presence or absence of NOC12. For subsequent experiments, we used the membranes whose TEERs were in the range of 70 to 120 ohm · cm².

Analytical Methods. The florescence intensity of CF was measured with a fluorescence spectrofluorometer (F-2000; Hitachi, Tokyo, Japan) at an excitation wavelength of 490 nm and an emission wavelength of 520 nm, respectively. Similarly, FD4 and FD10 were determined spectrofluorometrically at an excitation wavelength of 490 nm and an emission wavelength of 520 nm.

Statistical Analyses. Results were expressed as the mean ± S.E. and statistical significance was assessed by the Student's t test

	Results

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Effects of NO Donors on the Permeability of CF across the Intestinal Membrane. Figure 1 shows the effects of various concentrations of NOC5 and NOC12 on the permeability of CF across the colonic membrane. The NO donors significantly increased the permeability of CF across the colonic membrane and the absorption-enhancing effects of these NO donors were concentration-dependent over the range of 0.01 to 0.1 mM. However, 0.1 mM NO donors had almost the same absorption-enhancing effects as those at 1 mM, suggesting that the absorption-enhancing effect of NOC12 was saturable at a higher concentration.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Effects of various concentrations of NOC5 and NOC12 on the permeability of CF across the colonic membrane. Each value represents the mean ± S.E. of three to six experiments. *P < 0.05, **P < 0.01, significantly different compared with the control.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Regional differences in the effects of NOC5 and NOC12 on the permeability of CF across the jejunal and colonic membranes. The concentration of NO donors was 0.1 mM. Each value represents the mean ± S.E. of three to six experiments. *P < 0.05, **P < 0.01, significantly different compared with the control. n.s., not significantly different compared with the control.

Effect of NOC12 on the Colonic Permeability of Various Compounds with Different Molecular Weights. We also examined the effect of NOC12 (0.1 mM) on the colonic transport of various compounds with different molecular weights. Figure 3 indicates the relationship between the effect of NOC12 on the permeability of various compounds across the colonic membrane and their molecular weights. NOC12 also increased the permeability of FD4 across the colonic membrane, although we found no significant increase in the colonic permeability of FD10 in the presence of NOC12. Overall, the absorption-enhancing effect of NOC12 reduced as the molecular weights of compounds increased. Therefore, the degree of absorption-enhancing effect of NOC12 was dependent on the molecular weights of compounds.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Relationship between effect of NOC12 (0.1 mM) on Papp value of water-soluble compounds across the colonic membrane and their molecular weights. Each value represents the mean ± S.E. of four to six experiments. *P < 0.05, **P < 0.01, significantly different compared with the control. n.s., not significantly different compared with the control.

Effect of NOC12 Applied in the Serosal Side on the Permeability of CF across the Colonic Membrane. The effect of NOC12 (0.1 mM) applied in the serosal side on the permeability of CF across the colonic membrane was investigated. As shown in Fig. 4, no significant absorption-enhancing effect of NOC12 was observed when applied in the serosal side, although we found a remarkable absorption-enhancing effect of NOC12 applied in the mucosal side. Therefore, the absorption-enhancing effect of NOC12 was dependent on its administration side.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of administration side of NOC12 (0.1 mM) on the permeability of CF across the colonic membrane. Control (), NOC12 applied in serosal side (), NOC12 applied in mucosal side (). Each value represents the mean ± S.E. of four to six experiments. **P < 0.01, significantly different compared with the control. n.s., not significantly different compared with the control.

Effect of Pretreatment with NOC12 on the Permeability of CF across the Colonic Membrane. The effect of pretreatment with NOC 12 on the permeability of CF across the colonic membrane was examined. In this case, the colonic membrane was pretreated with NOC12 (0.1 mM) for 10 min and the permeability of CF across the colonic membrane was studied (Fig. 5). The absorption-enhancing effect of NOC12 pretreated to the colonic membrane was much less than that by the coadministration with NOC12, suggesting that the absorption-enhancing effect of NOC12 (0.1 mM) was reversible and less toxic to the colonic membrane. However, there was no significant difference on the absorption-enhancing effect of NOC12 (1 mM) between coadministration and pretreatment studies.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effect of pretreatment with NOC12 (0.1 or 1 mM) on the permeability of CF across the colonic membrane. Coadministration of NOC12 (), pretreatment with NOC12 (). Each value represents the mean ± S.E. of four to six experiments. *P < 0.05, **P < 0.01, significantly different compared with the control.

Assessment of Colonic Membrane Toxicity by NO Donors. Figure 6 shows the release of LDH, a biological membrane damage marker, from the colonic membrane in the presence or absence of various absorption enhancers, including NOC12. NaDC and EDTA caused an approximate 4-fold increase in LDH release over the control, indicating high intestinal membrane toxicity of these enhancers. In contrast, LM, NaGC, and NaCap caused a minor release of LDH, similar to control levels. In addition, the amount of LDH in the presence of NOC12 was much less than that with NaDC and EDTA. Therefore, NOC12 as well as NaGC, NaCap, and LM were less toxic to the colonic membrane.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Release of LDH from the colonic membrane in the presence of NOC12 (0.1 mM) and other various absorption enhancers (20 mM). Each value represents the mean ± S.E. of three to four experiments. *P < 0.05, **P < 0.01, significantly different compared with the control.

Effect of Carboxy-PTIO on the Absorption-Enhancing Effect of NOC12. To clarify whether the absorption-enhancing effect of NOC12 was mediated by NO, we examined the absorption-enhancing effect of NOC12 in the presence of carboxy-PITO, a NO scavenger. As shown in Fig. 7, the absorption-enhancing effect of NOC12 was significantly inhibited by the coadministration of carboxy-PTIO (0.4 mM), suggesting that NO can regulate the absorption-enhancing effect of water-soluble drugs in the gut.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Effect of carboxy-PTIO (c-PTIO) on the permeability of CF across the colonic membrane in the presence of NOC12. Each value represents the mean ± S.E. of four to six experiments. **P < 0.01, significantly different compared with the control.

Effect of NOC12 on the TEER of the Colonic Membrane. The TEER of the colonic membrane was measured in the presence of NOC12. As shown in Fig. 8, NOC12 at 0.1 and 1 mM significantly decreased the TEER value of the colonic membrane, although we found no significant effect on the TEER of the colonic membrane in the presence of 0.01 mM NOC12. Furthermore, the decreased TEER value by NOC12 (0.1 mM) recovered to the control level in the presence of 0.4 mM carboxy-PTIO. Therefore, these findings suggest that the absorption-enhancing mechanism of NOC12 partly includes the dilation of the tight junction in the epithelium via a paracellular route.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Effect of NOC12 on the transepithelial electrical resistance of rat colonic membrane. Control (), 0.01 mM NOC12 (), 0.1 mM NOC12 (), 1 mM NOC12 (), 0.1 mM NOC12 + 0.4 mM carboxy-PTIO (c-PTIO) (). Each value represents the mean ± S.E. of three to four experiments. **P < 0.01, significantly different compared with the control.

	Discussion

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The present study demonstrated that the absorption-enhancing effects of NO donors were dose-dependent over the concentration range of 0.01 to 1 mM (Fig. 1). Utoguchi et al. (1998) reported that the absorption-enhancing effect of S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, for rectal insulin absorption was dose-dependent over the range of 0.25 to 4.0 mg in rats. Similarly, Salzman et al. (1995) reported that incubation with sodium nitroprusside (SNP), an NO donor, resulted in a concentration-dependent increase in the transepithelial transport of fluorescein sulfonic acid in Caco-2 cells. Thus, our present findings concur with the previous results of absorption-enhancing effects of other NO donors, SNAP, and SNP.

In this study, we found regional differences in the absorption-enhancing effect of NOC12 on the permeability of CF across the jejunal and colonic intestinal membranes (Fig. 2). We previously reported that the absorption-enhancing effects of NaCap, LM, and EDTA for ebiratide and insulin absorption were greater in the colon than those in the jejunum (Yamamoto et al., 1997; Uchiyama et al., 1999). Especially, the permeability of insulin coadministered with LM in the colonic membrane was about 6 times higher than in the control, although its jejunal permeability was almost the same. Therefore, the present findings were consistent with our previous results of absorption enhancers applied in the different intestinal regions.

As indicated in Fig. 3, the degree of absorption-enhancing effect of NOC12 was dependent on the molecular weights of compounds. The absorption-enhancing effect of NOC12 decreased as the molecular weight of drugs increased in this study. This finding suggests that NOC12 may loosen the tight junctions of the epithelium to some extent, but the enlargement of the pore radius existing in the paracellular pathway may not be enough to improve the intestinal absorption of macromolecular compounds. On the other hand, we previously reported that NaGC, NaCap, and EDTA showed the highest promoting effects of drug with an approximate molecular weight of 4,000 for their nasal absorption (Yamamoto et al., 1993). Furthermore, Morita et al. (1993) reported that the maximal enhancing effect of NaGC and NaCap was noted in the pulmonary absorption of drug with an approximate molecular weight of 10,000, although EDTA showed the same results as in the nasal absorption. Consequently, these results indicated that the molecular weight of drugs is one of the most essential factors regulating the absorption-enhancing actions of absorption enhancers, including NOC12, although there was no optimal molecular weight of drugs for improving their intestinal absorption by NO donors in the present study.

We found no significant absorption-enhancing effect of NOC12 when applied in the serosal side, although a remarkable absorption-enhancing effect of NOC12 was seen when applied in the mucosal side (Fig. 4). Furthermore, our present study suggests that NOC12 may enhance the paracellular permeability of drugs in the gut because CF, a water-soluble compound, is mainly transported via a paracellular route. In contrast to our present finding, Noach et al. (1993) reported that the absorption-enhancing effect of EDTA applied to the basal side of the Caco-2 cells was much greater than that applied in the apical side. EDTA is known to be a chelating agent that forms a chelate compound with calcium ion existing at the tight junction in the membrane, resulting in increased permeability of the paracellular route (Cassidy and Tidball, 1967). The reason for the different effects between NOC12 and EDTA is not clearly understood. Presumably, the absorption-enhancing mechanism of NOC12 was different from the enhancing action of EDTA, although NOC12 also increased the permeability of drugs via a paracellular route, like EDTA.

As shown in Fig. 5, the absorption-enhancing effect of 0.1 mM NOC12 pretreated to the colonic membrane was much less than that by the coadministration with 0.1 mM NOC12, whereas NOC12 at a higher concentration irreversibly increased in the present study. On the contrary, our previous study demonstrated that the absorption-enhancing effect of sodium deoxycholate, one of the most toxic enhancers, pretreated to the colonic membrane was almost the same as its effect coadministered with drug, suggesting the irreversible effect of NaDC to the intestinal membrane (Uchiyama et al., 1999). Thus, we confirmed that the absorption-enhancing effect of NOC12 at a lower concentration was reversible and less toxic to the colonic membrane. However, the half-life for release rate of NO from NOC12 is relatively slow (about 120 min) and it might be possible that this slow release of NO from NOC12 appeared to be a irreversible action of NOC12 at 1 mM.

In the membrane toxicity experiments, release of LDH was significantly increased in the presence of NaDC and EDTA in the colonic membrane (Fig. 6). It was reported that NaDC, a hydrophobic bile salt, caused irreversible damage to the intestinal membrane by solubilizing and disrupting the membrane component, although its absorption-enhancing effect was considerable (Dawson et al., 1960). Similarly, Yamashita et al. (1987) reported that a high concentration of EDTA causes functional damage to the intestinal membrane, whereas it loosens the tight junctions of the epithelium due to its chelating activity with calcium at a lower concentration. Therefore, the high LDH values in the present study may be related to the toxicity of these absorption enhancers. Our present findings indicated that little or no release of LDH was observed in the presence of NOC12, like LM, NaGC, and NaCap (Fig. 6). With regard to LM, Murakami et al. (1992) reported that no apparent histological change was observed in the rectal mucosa by exposure of LM, which agrees with our present finding. In addition, Hirai et al. (1981) reported that the absorption-enhancing action of NaGC on the nasal absorption of insulin was reversible, and there was no marked histological change of the nasal epithelium by exposure of NaGC. Furthermore, NaCap is now clinically used in a commercial rectal suppository as an absorption enhancer for sodium ampicillin in Japan. Taking these findings of LDH release and pretreatment studies together, we suggest that NOC12 as well as LM, NaGC, and NaCap are the more effective and relatively less toxic absorption enhancers used in this study. This finding was also supported by the previous finding that SNP-induced hyperpermeability was not due to loss of cell viability, as confirmed by ultrastructure, unaltered LDH release, and ability to recover baseline permeability (Salzman et al., 1995).

The absorption-enhancing effect of NOC12 was inhibited by the coadministration of carboxy-PTIO (Fig. 7). Utoguchi et al. (1998) reported that the absorption-enhancing effect of SNAP was inhibited by carboxy-PTIO and the rectal absorption of insulin was decreased to the control level in the presence of carboxy-PTIO. Unno et al. (1997) also reported that coincubation of Caco-2 monolayers with several free-radical scavengers and peroxynitrous acid scavengers ameliorated the hyperpermeability induced by 3-morpholinosydnominine, an NO donor. Consequently, our present finding concurs with the previous results. These findings indicated that the absorption-enhancing effect of NOC12 was mediated by NO and NO can regulate the intestinal absorption of water-soluble drugs.

By electrophysiological studies, NOC12 at 0.1 and 1 mM significantly decreased the TEER value of the colonic membrane, although we found no significant effect on the TEER of the colonic membrane in the presence of 0.01 mM NOC12. Furthermore, the decreased TEER value by NOC12 recovered to the control level in the presence of carboxy-PTIO. Anderberg et al. (1993) reported that NaCap elicited dilatations in the tight junctions of the Caco-2 cell epithelium, thereby decreasing the membrane resistance and increasing drug absorption by the paracellular route. Therefore, these findings suggest that the absorption-enhancing mechanism of NOC12 partly includes the dilation of the tight junction in the epithelium via a paracellular route.

In conclusion, our present studies suggest that the intestinal absorption of water-soluble drugs was enhanced by NO donors and the NO donors may be useful to enhance the intestinal absorption of poorly absorbable drugs.