- Email: [email protected]
Modulation of Intestinal P-Glycoprotein Function by Cremophor EL and Other Surfactants by an In Vitro Diffusion Chamber Method Using the Isolated Rat Intestinal Membranes
Modulation of Intestinal P-Glycoprotein Function by Cremophor EL and Other Surfactants by an In Vitro Diffusion Chamber Method Using the Isolated Rat Intestinal Membranes YASUSHI SHONO, HISAYO NISHIHARA, YASUYUKI MATSUDA, SHIORI FURUKAWA, NAOKI OKADA, TAKUYA FUJITA, AKIRA YAMAMOTO Department of Biopharmaceutics, Kyoto Pharmaceutical University, Misasagi Yamashina-ku, Kyoto 607-8414 Japan
Received 29 July 2003; revised 24 October 2003; accepted 16 November 2003
ABSTRACT: Effects of various surfactants on the transport of rhodamine123, a P-glycoprotein (P-gp) substrate, across the isolated rat intestinal membranes were examined by an in vitro diffusion chamber system. The jejunal serosal-to-mucosal transport (Jsm) of rhodamine123 was more than threefold greater than its mucosal-toserosal transport (Jms), suggesting that the net movement of rhodamine123 across the rat jejunum was preferentially secretory direction. There exists a regional difference in the intestinal transport of rhodamine123 and the secretory directed transport was remarkably observed in the jejunum. The Jsm/Jms ratio of rhodamine123 decreased in the presence of 0.3 mM verapamil and 10 mM sodium azide (NaN3) þ 1 mM sodium fluoride (NaF), confirming that rhodamine123 might be secreted from the intestinal tissue into the lumen by a P-gp–mediated efflux system. Nonionic surfactants [0.1% Cremophor EL, Tween 80 and n-dodecyl-b-D-maltopyranoside (LM)] reduced the Jsm/ Jms ratio of rhodamine123, whereas its ratio was not influenced in the presence of 0.1% cationic surfactant (hexadecyltrimethylammonium bromide, C16TAB) and anionic surfactant (sodium dodecyl sulfate, SDS). Therefore, these findings suggested that charge of surfactants was possibly related to the action of these surfactants on the intestinal absorption of P-gp substrates. On the other hand, the transfer of rhodamine123 was not affected by the addition of Cremophor EL to the serosal side. Because the c.m.c. of Cremophor EL is 0.0095 w/v%, interactions between rhodamine123 and the micellar form of Cremophor EL may decrease the P-gp–mediated efflux of rhodamine123 at higher concentrations. In the kinetic analysis, the Vmax value (nmol/min/g wet tissue) of rhodamine123 decreased, although the Km value (mM) was constant in the presence of Cremophor EL. Therefore, Cremophor EL inhibited the efflux transport of rhodamine123 in a noncompetitive manner. Cremophor EL did not affect the transport of [14C]Gly-Sar and [3H]3-O-methyl-D-glucose, suggesting that the action of Cremophor EL might be P-gp specific. These findings indicated that nonionic surfactants including Cremophor EL and Tween 80 may be useful pharmaceutical excipients for inhibiting the function of P-gp, thereby increasing the intestinal absorption of various drugs, which are secreted by a P-gp–mediated efflux system in the intestine. ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:877–885, 2004
Keywords: intestinal absorption; intestinal secretion; P-glycoprotein; transporter; surfactant; pharmaceutical excipient; oral absorption Correspondence to: Akira Yamamoto (Telephone: þ81-75595-4661; Fax: þ81-75-595-4761; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 93, 877–885 (2004) ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association
INTRODUCTION P-glycoprotein (Pgp) is a plasma membrane glycoprotein of about 170 kDa that belongs to
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
877
878
SHONO ET AL.
the superfamily of ATP-binding cassette (ABC) transporters. P-gp can act as an energy-dependent drug efflux pump that lowers intracellular drug concentrations.1 Expressed in tumor cells, P-gp causes the MDR phenotype by the active extrusion of a wide range of cancer chemotherapeutic agents. In addition to being expressed in tumor cells, P-gp is also expressed in various normal tissues including liver, kidney, adrenal glands, brain, testis, and the intestinal brush border membranes.1 P-gp can transport a very broad range of substrates, including vinca alkaloids, anthracyclines, digoxin, and b-adrenergic agonists.2,3 It has been demonstrated that the intestinal P-gp, an ATP-dependent multidrug efflux pump, can be an active secretion system or an absorption barrier by transporting some drugs from the cells into the intestinal lumen. Therefore, intestinal absorption of drugs that are secreted by a P-gp– mediated efflux system can be improved by inhibiting the function of P-gp in the intestinal membrane. It is known that several excipients can reduce the function of P-gp in the intestine, thereby increasing the intestinal absorption of P-gp substrates.4–9 Nerurkar et al.5 reported that Cremophor EL and Tween 80 enhanced the absorptive transport of model peptide by inhibiting the secretory directed transport of this peptide in Caco-2 cells. More recently, Rege et al.7 demonstrated that various nonionic surfactants could inhibit the P-gp transporter, intestinal peptide transporter, and monocarboxy transporter in Caco-2 cells, although the mechanism was still unclear. In these previous studies, Caco-2 cell line, a human adenocarcinoma cell line, has been generally used to estimate drug permeability and substrate activity for efflux transport proteins such as P-gp. However, the expression levels of transporters in Caco-2 cells were usually variable and were dependent on the culture condition,10,11 which is one of the major disadvantages to estimate the function of P-gp in the presence or absence of some modulators and excipients using Caco-2 cells. Furthermore, the inhibitory mechanism of P-gp function by some surfactants has not been fully elucidated. In this study, therefore, we used an in vitro diffusion chamber system using the isolated rat intestinal membranes to estimate the effects of various surfactants with different charge on the intestinal transport of rhodamine123, a P-gp substrate. The inhibitory mode of P-gp function by Cremophor EL was also evaluated by a pharmaJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
cokinetic analysis. Furthermore, we examined the function of other transporters in the presence or absence of Cremophor EL and discussed the inhibitory mechanism of P-gp function and other transporters by these surfactants.
MATERIALS AND METHODS Materials Rhodamine123, Cremophor EL, Tween 80, and n-dodecyl-b-D-malto-pyranoside (LM, purity; minimum 98%) were purchased from SigmaAldrich Chemical Co. Ltd. (St. Louis, MO). Sodium dodecyl sulfate (SDS; purity 95%), hexadecyltrimethylammonium bromide (C16TAB), verapamil, sodium azide (NaN3), and sodium fluoride (NaF) were obtained from Nacalai Tesque Inc., (Kyoto, Japan). 5(6)-Carboxyfluorescein (CF) was generously supplied by Eastman Kodak Co. (Rochester, NY). [14C]Gly-Sar (2.1 GBq/mmol) was purchased from Cambridge Research Biochemical (Cleveland, UK), and [3H]3-O-methyl-Dglucose (370 GBq/mmol) was obtained from NEMTM Life Science Products, Inc. All other reagents were of analytical grade. Preparation of Drug Solution Rhodamine123 and CF were dissolved in Krebs Ringer Bicarbonate Solution (KRBS) at pH 7.4 to yield a final concentration of 5, 10, 20, and 50 mM. In some experiments, 0.005 to 1% w/v of Cremophor EL, 0.1% w/v of various surfactants including Tween 80, LM, C16TAB, SDS, 10 mM of NaN3 þ 1 mM NaF or 0.3 mM verapamil were added to the drug solution. Transport of Rhodamine123 across the Intestinal Membrane by an In Vitro Diffusion Chamber System The transport of P-gp substrates across the rat intestinal membrane was studied with the diffusion chamber (Corning Coster Corp.).12 Male Wistar rats, weighing 280–350 g, were fasted overnight and were anesthesized with sodium pentobarbital (30 mg/kg). The studies examined in this article have been carried out in accordance with the guidelines of the animal ethics committee at Kyoto Pharmaceutical University. The intestine was exposed through a midline abdominal incision, removed, and washed in ice-cold saline. Intestinal segments, excluding Peyer’s patches,
MODULATION OF INTESTINAL P-GLYCOPROTEIN FUNCTION BY CREMOPHOR EL
were isolated and immersed in ice-cold KRBS. Segments were cut open and the intestinal sheets were mounted onto the pins of the cell, and the half-cells were clamped together. Drug solution (7 mL) was added to the donor site, whereas the same volume of drug-free buffer was added to the opposite site. The temperature of cells was maintained at 378C, and both fluids were circulated by gas lift with 95% O2/5% CO2. During the transport studies, aliquots were taken from the serosal side and the permeated drugs were assayed. The permeability flux of drugs at the steady state was calculated from the slope of linear portion of permeability-time profiles of drugs. The kinetic parameters, Michaelis-Menten constant (Km) and maximum velocity (Vmax) were calculated by nonlinear regression program using the software Sigma Plot version 4.0 (Jandel Scientific). The viability of intestinal membrane during the test period was monitored by measuring the transport of trypan blue dye and electrophysiological parameters. There was no transport of the 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 experiments. Equilibrium Dialysis Rhodamine123 was dissolved in KRBS at the concentration of 10 mM. The buffer solution (8 mL) was placed in a centrifuge tube as the outer fluid. Cellulose tube (Visking Co., #8/32) containing 1 mL of rhodamine123 solution with various concentrations of Cremophor EL was ligated at both ends, immersed in the buffer solution, and equilibrated for 12 h at 378C. After equilibration, the concentration of rhodamine123 in the outer layer was determined spectrofluorometrically as described below. In Vitro Initial Uptake Experiments Initial uptake of drugs was evaluated by mean of an in vitro everted sac experiment. Male Wistar rats, weighing 280–350 g, were fasted overnight and were anesthesized with sodium pentobarbital (30 mg/kg). Under pentobarbital anesthesia, the small intestine was washed with KRBS and quickly removed from the rat. The removed intestine was everted by a wire inserted through the lumen and 3 cm of jejunal everted sacs were prepared and ligated at both end. Sacs were preincubated in Krebs-Ringer buffer solution for 3 min. In the
879
inhibition studies, Cremophor EL was pretreated for 10 min. Then, initial uptake of drugs was studied for 1 min after the sacs were placed in test solution. After the uptake studies, sacs were washed with ice-cold saline and solubilized with soluene-350. The radioactivity in the serosal fluid was determined in a liquid scintillation system. The kinetic parameters for [14C]Gly-Sar and [3H]3-O-methyl-D-glucose uptake were calculated using the following equation. V0 ¼ Vmax S=ðKm þ SÞ þ Kd S; where V0 is the uptake rate of the drug (nmol/min/ g wet tissue), S is the drug concentration in the medium (mM), Km is the Michaelis constant (mM), Vmax is the maximum uptake rate by the saturable process (nmol/min/g wet tissue) and Kd is the coefficient of simple diffusion (nmol/min/g wet tissue/mM). The uptake data were fitted to the above equation by nonlinear least square regression analysis.
Determination (Assay) of Drugs The fluorescence intensity of rhodamine123 was measured with a fluorescence spectrophotometer (F-2000, Hitachi, Tokyo, Japan) at an excitation wavelength of 480 nm and an emission wavelength of 540 nm, respectively. Similarly, CF was determined spectrofluorometrically at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. [14C]Gly-Sar and [3H]3O-methyl-D-glucose were determined by a Liquid scintillation counter (BECKMAN LS6500).
Statistics Results are expressed as the mean of SE of at least three experiments. Statistical significance was assessed using the Student’s t-test or Dunnet’s test for multiple comparisons with p < 0.05 as the minimal level of significance.
RESULTS Permeability Characteristics of Rhodamine123 across the Intestinal Membranes Figure 1(a) shows the time course of absorptive (mucosal to serosal) and secretory (serosal to mucosal) transport of rhodamine123 across the rat jejunal membranes. As shown in this figure, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
880
SHONO ET AL.
Figure 1. Time course of absorptive (M to S), secretory (S to M) transport of rhodamine123 in the rat jejunal membranes and its regional different transport across the rat intestinal membranes. Results are expressed as the mean SE of at least three experiments. Keys: (*), absorptive (M to S) transport, (*), secretory (S to M) transport. (N.S.) not significant, *p < 0.05, **p < 0.01, compared with each absorptive (M to S) transport.
the jejunal serosal to mucosal transport (Jsm) of rhodamine123 was more than threefold greater than its mucosal to serosal transport (Jms), suggesting that the net movement of rhodamine123 across the rat jejunum was preferentially secretory direction. Figure 1(b) indicates a site difference of absorptive and secretory transport of rhodamine123 across the various intestinal membranes. In every region, the serosal to mucosal transport (Jsm) of rhodamine123 was greater than its mucosal to serosal transport (Jms). However, the secretory directed transport was more remarkably observed in the jejunum than the other intestinal segments, indicating that there exists a regional difference in the directional transport of rhodamine123 across the rat intestinal membrane. Based on the regional different absorption studies, we selected jejunum as a model region to estimate the function of P-gp in the presence or absence of various surfactants in the following studies. Effects of Various Surfactants on the Permeability of Rhodamine123 across the Intestinal Membranes Figure 2 shows the effects of various surfactants and inhibitors on the transport of rhodamine123 across the rat jejunal membranes. In this case, we evaluated the transport direction of rhodamine123 by calculating Jsm/Jms ratio of rhodamine123. As is evident from the figure, the Jsm/ JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
Figure 2. Effects of various surfactants on the transport of rhodamine123 across the rat jejunal membranes. Results are expressed as the mean SE of at least three experiments. (CEL); Cremophor EL, (LM); n-dodecylb-D-maltopyranoside, (SDS) sodium dodecyl sulfate, and (C16TAB) hexadecyltrimethylammonium bromide. (N.S.) not significant, *p < 0.05, **p < 0.01, compared with the control.
Jms ratio of rhodamine123 significantly decreased in the presence of 0.3 mM verapamil and 10 mM NaN3 þ 1 mM NaF, suggesting that the rhodamine123 might be secreted from the intestinal tissue into the lumen by a P-gp–mediated efflux system. Figure 2 also indicates Jsm/Jms ratio of rhodamine123 in the presence or absence of various surfactants. As shown in this figure, nonionic surfactants (0.1% Cremophor EL, Tween 80, and LM) reduced the Jsm/Jms ratio of rhodamine123, whereas its ratio was not influenced in the presence of 0.1% cationic surfactant (C16TAB) and anionic surfactant (SDS). The secretory transport flux of rhodamine123 in the control was 0.680 0.090 pmol/min/cm2, whereas its flux values in the presence of Cremophor EL, Tween 80 and LM were 0.456 0.060, 0.321 0.079, and 0.450 0.089 pmol/min/cm2, respectively. On the other hand, the transfer of rhodamine123 was not affected by the addition of Cremophor EL to the serosal side. Moreover, the transport of 5(6)-carboxyfluorescein (CF), a model drug transported by a passive diffusion was not affected in the presence of Cremophor EL to the mucosal side (data not shown). Therefore, these findings suggested that charge of surfactants is possibly related to the action of these surfactants on the intestinal absorption of P-gp substrates. In the subsequent studies, we selected Cremophor EL as a suitable P-gp modulator and examined its inhibitory action of P-gp in more detail.
MODULATION OF INTESTINAL P-GLYCOPROTEIN FUNCTION BY CREMOPHOR EL
881
Effect of Various Concentrations of Cremophor EL on the Transport of Rhodamine123 Figure 3 shows concentration-dependent effect of Cremophor EL on the absorptive and secretory transport of rhodamine123 in the rat jejunal membrane. There exists a bell-shaped profile in the relationship between the absorptive and secretory transport of rhodamine123 and concentration of Cremophor EL. We observed a greatest increase and decrease in the absorptive and secretory transport of rhodamine123 with 0.01– 0.1% Cremophor EL in rat jejunal membrane. This inhibitory effect of P-glycoprotein function almost disappeared when 1% of Cremophor EL was used. To elucidate the inhibitory action of P-gp function by various concentrations of Cremophor EL, we next examined the micellar interaction between rhodamine123 and Cremophor EL. Figure 4 shows % of rhodamine123 entrapped into the micelles of Cremophor EL in the presence of various concentrations of Cremophor EL. As shown in this figure, the amount of rhodamine123 entrapped into the micelles of Cremophor EL increased as the concentration of Cremophor EL increased. Therefore, at higher concentrations of Cremophor EL, rhodamine123 can be incorporated into the micelles of Cremophor EL and we observed a remarkable interaction between rhodamine123 and Cremophor EL. Because the c.m.c. of Cremophor EL is 0.0095% w/v, interactions between rhodamine123 and the micellar form of Cremophor EL may decrease the P-gp–mediated efflux of rhodamine123 at higher concentrations.
Figure 3. Concentration-dependent effects of Cremophor EL on absorptive and secretory transport of rhodamine123 in the rat jejunum. The concentration of Cremophor EL were 0.005–1% w/v. Results are expressed as the mean SE of at least three experiments.
Figure 4. Micellar interaction between rhodamine123 and Cremophor EL. The concentrations of Cremophor EL were 0.01–1% w/v. The percentage of rhodamine123 entrapped in the micelles of Cremophor EL (Micellar drug) (%) was calculated from the following equation: Micellar drug (%) ¼ (Fcontrol Fsample)/ Fcontrol 100.
Mode of Inhibitory action of P-gp Function by Cremophor EL Figure 5 indicates the inhibitory patterns of Cremophor EL on rhodamine123 transport across the rat jejunal membranes. Figure 5(a) shows the Michaelis Menten plot between net flux of rhodamine123 and rhodamine123 concentration. The net flux of rhodamine123 decreased by the addition of 0.1% Cremophor EL in every concentration of rhodamine123. Figure 5(b) shows Eadie-Hofstee plot between net flux of rhodamine123 and rhodamine123 concentration. In the
Figure 5. Inhibition pattern of Cremophor EL on rhodamine123 transport across the rat jejunal membrane. Results are expressed as the mean SE of at least three experiments. Keys: (*); rhodamine123 only, (*); rhodamine123 þ 0.1% Cremophor EL. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
882
SHONO ET AL.
kinetic analysis, the Vmax value of rhodamine123 decreased, although the Km value was constant in the presence of Cremophor EL. Therefore, Cremophor EL inhibited the Jsm of rhodamine123 in a noncompetitive manner. Effect of Cremophor EL on the Function of Other Transporters in Rat Intestinal Membrane Finally, to examine whether Cremophor EL may affect the function of other transporters in the rat intestinal membranes, we studied the effect of Cremophor EL on the function of glucose transporter and oligopeptide transporter in the intestine. [14C]Gly-Sar was used as a model substrate of oligopeptide transporter, while [3H]3-Omethyl-D-glucose was used as a model substrate of glucose transporter. As indicated in Table 1, Cremophor EL did not affect the kinetic parameters of transport of [14C]Gly-Sar and [3H]3-Omethyl-D-glucose, suggesting that Cremophor EL may specifically reduce the function of P-gp in the intestine.
DISCUSSION The present study demonstrated that the secretory directed transport of rhodamine123 was inhibited by various nonionic surfactants, although cationic and anionic surfactants did not inhibit the polarized transport of rhodamine123. In this study, rhodamine123 was used
as a typical model of P-gp substrate, because this compound was easily assayed and was widely used for evaluating the function of P-gp in the field of cancer chemotherapy as well as biopharmaceutics.13 As shown in Figure 1, the secretory transport of rhodamine123 was much greater than its absorptive transport. In addition, the Jsm/Jms ratio of rhodamine123 was clearly reduced in the presence of 0.3 mM verapamil and 1 mM NaF þ 10 mM NaN3. Therefore, we could confirm to estimate the function of P-gp using this in vitro Ussing chamber system. We observed a regional difference in the secretory directed transport of rhodamine123 in this study, and the polarized transport of rhodamine123 was the greatest in the jejunum. On the other hand, it was generally known that the expression of P-gp in the intestine was dominant in the lower part of the intestine.1 Furthermore, Yumoto et al. reported that the P-gp mediated transport of rhodamine123 was remarkably observed in the upper and lower ileum rather than duodenum and jejunum.14 Therefore, our present result was not in good agreement with the previous findings of site-dependent expression and function of P-gp, although the reason was not fully understood. However, Yumoto et al. evaluated the P-gp– mediated transport of rhodamine123 using verapamil (they regarded verapamil-induced inhibitory transport as the P-gp–mediated transport), while we evaluated the P-gp–mediated transport by calculating the absorptive and secretory transport ratio of rhodamine123 across the intestine.
Table 1. Effect of Cremophor EL on Kinetic Parameters of [14C]Gly-Sar and [3H]3-O-methyl-D-glucose (3-OMG) Uptake by the Rat Jejunal Everted Sacs Kinetic Parameters
Gly-Sar Control CEL 0.01% w/v 0.1% w/v 3-OMG Control CEL 0.01% w/v 0.1% w/v
Km (mM)
Vmax (nmol/min/g wet tissue)
Kd (nmol/min/g wet tissue/mM)
1.11 0.22
4.76 2.04
1.46 2.30
1.27 0.20 2.36 0.55
3.63 1.11 5.68 1.90
1.72 3.09 1.67 2.51
4.55 0.84
3.61 0.71
0.17 0.03
7.69 1.07 7.07 1.51
3.93 0.50 3.41 0.65
0.16 0.03 0.16 0.04
Results are expressed as the mean SE of at least three experiments. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
MODULATION OF INTESTINAL P-GLYCOPROTEIN FUNCTION BY CREMOPHOR EL
Therefore, such different evaluation method may be partly due to the discrepancy between the findings of Yumoto et al. and our present result. Of all the surfactants, we indicated that only nonionic surfactants including Cremophor EL, Tween 80, and LM could inhibit the function of Pgp in the intestine. This finding is well correlated with the previous finding of Nerurkar et al., who demonstrated that Cremophor EL and Tween 80 could inhibit the polarized transport of model peptide compound in Caco-2 cells.5 They also reported that Tween 80 up to 1% w/v and Cremophor EL up to 10% w/v did not cause any apparent damage to the Caco-2 cell monolayers within the experimental period of exposure.5 In addition, our present result indicated that the absorptive and secretory transport of CF, a paracellular marker compound, was not changed in the presence of Cremophor EL. Furthermore, Cremophor EL and Tween 80 are polyethoxylated excipients commonly added in pharmaceutical formulations to increase the aqueous solubility and drug candidates.15,16 These excipients, which are also added to food products, are considered to be nontoxic and inert.15,16 These findings suggested that Cremophor EL and Tween 80 increased the absorptive transport of rhodamine123 without any intestinal membrane damage, but they decreased the secretory transport of rhodamine123 by inhibiting the function of P-gp. On the other hand, it was known that LM had a strong absorption enhancing effect for various poorly absorbable drugs even at low concentrations. Murakami et al.17 demonstrated that various alkylsaccharides including LM enhanced the rectal absorption of 5(6)-carboxyfluorescein and fluorescein isothiocyanate labeleddextrans in rats. In addition, Uchiyama et al.18 reported that intestinal permeability of insulin was improved in the presence of LM. Therefore, the lack of the polarized transport of rhodamine123 in the presence of LM may be partly due to the increased absorptive transport of rhodamine123, although LM can inhibit the secretory transport of rhodamine123 by reducing the function of P-gp in the intestine. Unlike the nonionic surfactants, our present study indicated no influence in the transport of rhodamine123 with the ionic surfactants. The observed difference between nonionic and ionic surfactants in the inhibition of P-gp may be due to the differences in the binding affinities of these surfactants to the hydrophobic portion of P-gp molecule. In other words, the inhibitory action of P-gp function by Tween 80 and Cremophor EL may
883
be due to their specific binding to the hydrophobic domain of the P-gp, and this nonspecific binding may change its secondary and/or tertiary structure of the P-gp and reduce the function of P-gp. Regarding the effectiveness of Cremophor EL, there exists a bell-shaped profile in the relationship between rhodamine123 and the concentration of Cremophor EL. This result was also well correlated with the previous finding of Nerurkar et al.,5 who showed the increased fraction of P-gp substrate bound to micelle at higher concentrations of Cremophor EL. Furthermore, our present study using the eqilibrium dialysis method showed that the interaction of rhodamine123 with the micelle of Cremophor EL remarkably increased as the concentration of Cremophor EL increased and reached the above concentration of c.m.c. (0.0095%). Therefore, we suggest that the free fraction of Cremophor EL can contribute to inhibit the function of P-gp, thereby increasing the net transport of rhodamine123 across the intestinal membrane. In the kinetic analysis, the Vmax value of rhodamine123 decreased, although the Km value was constant in the presence of Cremophor EL. Therefore, Cremophor EL inhibited the efflux transport of rhodamine123 in a noncompetitive manner. Therefore, these nonionic surfactants can not act as substrates of P-gp, but can act as inhibitors. With respect to the inhibition mechanism of P-gp by these surfactant, nonionic surfactants such as Tween 80 and Cremophor EL are shown to integrate within the cell membranes and thus change their microviscosity.19 This may result in loosing the phospholipid bilayers of cells, resulting in the change of secondary and/or tertiary structure of membrane proteins, thus altering their biological activity. However, Rege et al. recently demonstrated that cholesterol and benzylalcohol, known as membrane fluidity modulators, did not change the transport of rhodamine123 in Caco-2 cells.7 Therefore, changes in membrane fluidity may not be a general mechanism underlying reduced P-gp activity in the intestine. Another important mechanism by which the function of P-gp reduced is the change in the level of protein kinase C (PKC). PKC, a phospholipid/ Ca2þ-dependent protein kinase, is known to phosphorylate several proteins for cellular functions, in response to extracellular stimuli. PKC activity has been linked to various transporters, such as P-gp and the peptide transporter.20 However, recent study demonstrated that staurosporine, a PKC inhibitor failed to inhibit the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
884
SHONO ET AL.
function of P-gp, suggesting that inhibition of PKC was not the mechanism of surfactant-induced transporter inhibition of P-gp function.7 Our present results indicated that Cremophor EL inhibit the function of P-gp, but did not reduce the other transporters including glucose and peptide transporters. This finding concurs with the previous findings of Rege et al.,7 who showed the negative effect of Cremophor EL on the function of peptide transporter in Caco-2 cells, although Cremophor EL decreased the function of monocarboxy transporter. Therefore, these findings suggested that the inhibitory effect of Cremophor EL was not general phenomenon in all the transporters. As for the clinical application of Cremophor EL, Cremophor EL has been used clinically as a vehicle for the hydrophobic drugs such as taxol and cyclosporin A. In particular, taxol is formulated as 50% Cremophor EL and 50% ethanol. Therefore, the concentration of Cremophor EL, which we used in this study, is not as high compared with that in clinical application. Presumably, these surfactants might be useful for not only improving the solubility of anticancer drugs but also increasing the uptake of anticancer drugs by inhibiting the function of P-glycoprotein on the surface of the tumor cells. In conclusion, nonionic surfactants including Cremophor EL and Tween 80 were effective to inhibit the P-gp–mediated efflux system, thereby improving the intestinal absorption of poorly absorbable drugs that are secreted by P-gp from the cells into the lumen. Furthermore, this in vitro diffusion chamber system is suitable to evaluate the function of P-gp in the presence or absence of various surfactants and the other candidates of P-gp modulators.
REFERENCES 1. Hunter J, Hirst BH. 1997. Intestinal secretion of drugs: The role of P-glycoprotein and related drug efflux systems in limiting oral drug absorption. Adv Drug Del Rev 25:129–157. 2. Terao T, Hisanaga E, Sai Y, Tamai I, Tsuji A. 1996. Active secretion of drugs from the small intestinal epithelium in rats by P-glycoprotein functioning as an absorption barrier. J Pharm Pharmacol 48: 1083–1089. 3. Emi Y, Tsunashima D, Ogawara K, Higaki K, Kimura T. 1998. Role of P-glycoprotein as a secretory mechanism in quinidine absorption from rat small intestine. J Pharm Sci 87:295–299. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
4. Wagner D, Spahn-Langguth H, Hanafy A, Koggel A, Langguth P. 2001. Intestinal drug efflux: formulation and food effect. Adv Drug Del Rev 50: S13–S31. 5. Nururkar MM, Burton PS, Borchardt RT. 1996. The use of surfactants to enhance the permeability of peptides through Caco-2 cells by inhibition of an apically polarized efflux system. Pharm Res 13: 528–534. 6. Nerurkar MM, Ho NFH, Burton PS, Vidmar TJ, Borchardt RT. 1997. Mechanistic roles of neutral surfactants on concurrent polarized and passive membrane transport of a model peptide in Caco-2 cell. J Pharm Sci 86:813–821. 7. Rege BD, Kao JPY, Polli JE. 2002. Effects of nonionic surfactants on membrane transporters in Caco2 cell monolayers. Eur J Pharm Sci 16:237–246. 8. Hugger ED, Audus KL, Borchardt RT. 2002. Effects of poly(ethylene glycol) on efflux transporter activity in Caco-2 cell monolayers. J Pharm Sci 91: 1980–1990. 9. Hugger ED, Novak BL, Burton PS, Audus KL, Borchardt RT. 2002. A comparison of commonly used polyethoxylated pharmaceutical excipients on their ability to inhibit P-glycoprotein activity in vitro. J Pharm Sci 91:1991–2000. 10. Anderle P, Niederer E, Rubas W, Hilgendorf C, Spahn-Langguth H, Wunderli-Allenspach H, Merkle HP, Langguth P. 1998. P-glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: The influence of culturing condition and drug exposure on P-gp expression levels. J Pharm Sci 87: 757–762. 11. Hosoya K, Kim KJ, Lee VHL. 1996. Age-dependent expression of P-glycoprotein, gp170 in Caco-2 cell monolayers. Pharm Res 13:885–890. 12. Grass GM, Sweetana SA. 1988. In vitro measurement of gastrointestinal tissue permeability using a new diffusion cell. Pharm Res 5:372–376. 13. Fontaine M, Elmquist WF, Miller DW. 1996. Use of rhodamine123 to examine the functional activity of P-glycoprotein in primary cultured brain microvessel endothelial cell monolayers. Life Sci 59: 1521–1531. 14. Yumoto R, Murakami T, Nakamoto Y, Hasegawa R, Nagai J, Takano M. 1999. Transport of rhodamine123, a P-glycoprotein substrate, across rat intestine and Caco-2 cell monolayers in the presence of cytochrome P-450 3A-related compounds. J Pharmacol Exp Ther 289:149–155. 15. Woodcock DM, Jefferson S, Linsenmeyer ME, Crowther PJ, Chojnowski GM, Williams B, Bertoncello I. 1990. Reversal of the multidrug resistance phenotype with Cremophor EL, a common vehicle for water-insoluble vitamins and drugs. Cancer Res 50:4199–4203. 16. Zordan-Nudo T, Ling V, Liu Z, George E. 1993. Effects of nonionic detergents on P-glycoportein
MODULATION OF INTESTINAL P-GLYCOPROTEIN FUNCTION BY CREMOPHOR EL
drug binding and reversal of multidrug resistance. Cancer Res 53:5994–6000. 17. Murakami M, Kusanoi Y, Takada K, Muranishi S. 1992. Assessment of enhancing ability of mediumchain alkylsaccharides as new absorption enhancers in rat rectum. Int J Pharm 79:159–169. 18. Uchiyama T, Sugiyama T, Quan YS, Kotani A, Okada N, Fujita T, Muranishi S, Yamamoto A. 1999. Enhanced permeability of insulin across the rat intestinal membrane by various absorption enhancers: Their intestinal mucosal toxicity and
885
absorption-enhancing mechanism of n-lauryl-b-Dmaltopyranoside. J Pharm Pharmacol 51:1241– 1250. 19. Dudeja PK, Anderson KM, Harris JS, Buckingham L, Coon JS. 1995. Reversal of multidrug resistance to membrane lipid fluidity. Arch Biochem Biophys 319:309–315. 20. Castro AF, Horton JK, Vanoye CG, Altenberg CA. 1999. Mechanism of inhibition of P-glycoproteinmediated drug transport by protein kinase C blockers. Biochem Pharmacol 58:1723–1733.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
Received 29 July 2003; revised 24 October 2003; accepted 16 November 2003
ABSTRACT: Effects of various surfactants on the transport of rhodamine123, a P-glycoprotein (P-gp) substrate, across the isolated rat intestinal membranes were examined by an in vitro diffusion chamber system. The jejunal serosal-to-mucosal transport (Jsm) of rhodamine123 was more than threefold greater than its mucosal-toserosal transport (Jms), suggesting that the net movement of rhodamine123 across the rat jejunum was preferentially secretory direction. There exists a regional difference in the intestinal transport of rhodamine123 and the secretory directed transport was remarkably observed in the jejunum. The Jsm/Jms ratio of rhodamine123 decreased in the presence of 0.3 mM verapamil and 10 mM sodium azide (NaN3) þ 1 mM sodium fluoride (NaF), confirming that rhodamine123 might be secreted from the intestinal tissue into the lumen by a P-gp–mediated efflux system. Nonionic surfactants [0.1% Cremophor EL, Tween 80 and n-dodecyl-b-D-maltopyranoside (LM)] reduced the Jsm/ Jms ratio of rhodamine123, whereas its ratio was not influenced in the presence of 0.1% cationic surfactant (hexadecyltrimethylammonium bromide, C16TAB) and anionic surfactant (sodium dodecyl sulfate, SDS). Therefore, these findings suggested that charge of surfactants was possibly related to the action of these surfactants on the intestinal absorption of P-gp substrates. On the other hand, the transfer of rhodamine123 was not affected by the addition of Cremophor EL to the serosal side. Because the c.m.c. of Cremophor EL is 0.0095 w/v%, interactions between rhodamine123 and the micellar form of Cremophor EL may decrease the P-gp–mediated efflux of rhodamine123 at higher concentrations. In the kinetic analysis, the Vmax value (nmol/min/g wet tissue) of rhodamine123 decreased, although the Km value (mM) was constant in the presence of Cremophor EL. Therefore, Cremophor EL inhibited the efflux transport of rhodamine123 in a noncompetitive manner. Cremophor EL did not affect the transport of [14C]Gly-Sar and [3H]3-O-methyl-D-glucose, suggesting that the action of Cremophor EL might be P-gp specific. These findings indicated that nonionic surfactants including Cremophor EL and Tween 80 may be useful pharmaceutical excipients for inhibiting the function of P-gp, thereby increasing the intestinal absorption of various drugs, which are secreted by a P-gp–mediated efflux system in the intestine. ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:877–885, 2004
Keywords: intestinal absorption; intestinal secretion; P-glycoprotein; transporter; surfactant; pharmaceutical excipient; oral absorption Correspondence to: Akira Yamamoto (Telephone: þ81-75595-4661; Fax: þ81-75-595-4761; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 93, 877–885 (2004) ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association
INTRODUCTION P-glycoprotein (Pgp) is a plasma membrane glycoprotein of about 170 kDa that belongs to
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
877
878
SHONO ET AL.
the superfamily of ATP-binding cassette (ABC) transporters. P-gp can act as an energy-dependent drug efflux pump that lowers intracellular drug concentrations.1 Expressed in tumor cells, P-gp causes the MDR phenotype by the active extrusion of a wide range of cancer chemotherapeutic agents. In addition to being expressed in tumor cells, P-gp is also expressed in various normal tissues including liver, kidney, adrenal glands, brain, testis, and the intestinal brush border membranes.1 P-gp can transport a very broad range of substrates, including vinca alkaloids, anthracyclines, digoxin, and b-adrenergic agonists.2,3 It has been demonstrated that the intestinal P-gp, an ATP-dependent multidrug efflux pump, can be an active secretion system or an absorption barrier by transporting some drugs from the cells into the intestinal lumen. Therefore, intestinal absorption of drugs that are secreted by a P-gp– mediated efflux system can be improved by inhibiting the function of P-gp in the intestinal membrane. It is known that several excipients can reduce the function of P-gp in the intestine, thereby increasing the intestinal absorption of P-gp substrates.4–9 Nerurkar et al.5 reported that Cremophor EL and Tween 80 enhanced the absorptive transport of model peptide by inhibiting the secretory directed transport of this peptide in Caco-2 cells. More recently, Rege et al.7 demonstrated that various nonionic surfactants could inhibit the P-gp transporter, intestinal peptide transporter, and monocarboxy transporter in Caco-2 cells, although the mechanism was still unclear. In these previous studies, Caco-2 cell line, a human adenocarcinoma cell line, has been generally used to estimate drug permeability and substrate activity for efflux transport proteins such as P-gp. However, the expression levels of transporters in Caco-2 cells were usually variable and were dependent on the culture condition,10,11 which is one of the major disadvantages to estimate the function of P-gp in the presence or absence of some modulators and excipients using Caco-2 cells. Furthermore, the inhibitory mechanism of P-gp function by some surfactants has not been fully elucidated. In this study, therefore, we used an in vitro diffusion chamber system using the isolated rat intestinal membranes to estimate the effects of various surfactants with different charge on the intestinal transport of rhodamine123, a P-gp substrate. The inhibitory mode of P-gp function by Cremophor EL was also evaluated by a pharmaJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
cokinetic analysis. Furthermore, we examined the function of other transporters in the presence or absence of Cremophor EL and discussed the inhibitory mechanism of P-gp function and other transporters by these surfactants.
MATERIALS AND METHODS Materials Rhodamine123, Cremophor EL, Tween 80, and n-dodecyl-b-D-malto-pyranoside (LM, purity; minimum 98%) were purchased from SigmaAldrich Chemical Co. Ltd. (St. Louis, MO). Sodium dodecyl sulfate (SDS; purity 95%), hexadecyltrimethylammonium bromide (C16TAB), verapamil, sodium azide (NaN3), and sodium fluoride (NaF) were obtained from Nacalai Tesque Inc., (Kyoto, Japan). 5(6)-Carboxyfluorescein (CF) was generously supplied by Eastman Kodak Co. (Rochester, NY). [14C]Gly-Sar (2.1 GBq/mmol) was purchased from Cambridge Research Biochemical (Cleveland, UK), and [3H]3-O-methyl-Dglucose (370 GBq/mmol) was obtained from NEMTM Life Science Products, Inc. All other reagents were of analytical grade. Preparation of Drug Solution Rhodamine123 and CF were dissolved in Krebs Ringer Bicarbonate Solution (KRBS) at pH 7.4 to yield a final concentration of 5, 10, 20, and 50 mM. In some experiments, 0.005 to 1% w/v of Cremophor EL, 0.1% w/v of various surfactants including Tween 80, LM, C16TAB, SDS, 10 mM of NaN3 þ 1 mM NaF or 0.3 mM verapamil were added to the drug solution. Transport of Rhodamine123 across the Intestinal Membrane by an In Vitro Diffusion Chamber System The transport of P-gp substrates across the rat intestinal membrane was studied with the diffusion chamber (Corning Coster Corp.).12 Male Wistar rats, weighing 280–350 g, were fasted overnight and were anesthesized with sodium pentobarbital (30 mg/kg). The studies examined in this article have been carried out in accordance with the guidelines of the animal ethics committee at Kyoto Pharmaceutical University. The intestine was exposed through a midline abdominal incision, removed, and washed in ice-cold saline. Intestinal segments, excluding Peyer’s patches,
MODULATION OF INTESTINAL P-GLYCOPROTEIN FUNCTION BY CREMOPHOR EL
were isolated and immersed in ice-cold KRBS. Segments were cut open and the intestinal sheets were mounted onto the pins of the cell, and the half-cells were clamped together. Drug solution (7 mL) was added to the donor site, whereas the same volume of drug-free buffer was added to the opposite site. The temperature of cells was maintained at 378C, and both fluids were circulated by gas lift with 95% O2/5% CO2. During the transport studies, aliquots were taken from the serosal side and the permeated drugs were assayed. The permeability flux of drugs at the steady state was calculated from the slope of linear portion of permeability-time profiles of drugs. The kinetic parameters, Michaelis-Menten constant (Km) and maximum velocity (Vmax) were calculated by nonlinear regression program using the software Sigma Plot version 4.0 (Jandel Scientific). The viability of intestinal membrane during the test period was monitored by measuring the transport of trypan blue dye and electrophysiological parameters. There was no transport of the 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 experiments. Equilibrium Dialysis Rhodamine123 was dissolved in KRBS at the concentration of 10 mM. The buffer solution (8 mL) was placed in a centrifuge tube as the outer fluid. Cellulose tube (Visking Co., #8/32) containing 1 mL of rhodamine123 solution with various concentrations of Cremophor EL was ligated at both ends, immersed in the buffer solution, and equilibrated for 12 h at 378C. After equilibration, the concentration of rhodamine123 in the outer layer was determined spectrofluorometrically as described below. In Vitro Initial Uptake Experiments Initial uptake of drugs was evaluated by mean of an in vitro everted sac experiment. Male Wistar rats, weighing 280–350 g, were fasted overnight and were anesthesized with sodium pentobarbital (30 mg/kg). Under pentobarbital anesthesia, the small intestine was washed with KRBS and quickly removed from the rat. The removed intestine was everted by a wire inserted through the lumen and 3 cm of jejunal everted sacs were prepared and ligated at both end. Sacs were preincubated in Krebs-Ringer buffer solution for 3 min. In the
879
inhibition studies, Cremophor EL was pretreated for 10 min. Then, initial uptake of drugs was studied for 1 min after the sacs were placed in test solution. After the uptake studies, sacs were washed with ice-cold saline and solubilized with soluene-350. The radioactivity in the serosal fluid was determined in a liquid scintillation system. The kinetic parameters for [14C]Gly-Sar and [3H]3-O-methyl-D-glucose uptake were calculated using the following equation. V0 ¼ Vmax S=ðKm þ SÞ þ Kd S; where V0 is the uptake rate of the drug (nmol/min/ g wet tissue), S is the drug concentration in the medium (mM), Km is the Michaelis constant (mM), Vmax is the maximum uptake rate by the saturable process (nmol/min/g wet tissue) and Kd is the coefficient of simple diffusion (nmol/min/g wet tissue/mM). The uptake data were fitted to the above equation by nonlinear least square regression analysis.
Determination (Assay) of Drugs The fluorescence intensity of rhodamine123 was measured with a fluorescence spectrophotometer (F-2000, Hitachi, Tokyo, Japan) at an excitation wavelength of 480 nm and an emission wavelength of 540 nm, respectively. Similarly, CF was determined spectrofluorometrically at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. [14C]Gly-Sar and [3H]3O-methyl-D-glucose were determined by a Liquid scintillation counter (BECKMAN LS6500).
Statistics Results are expressed as the mean of SE of at least three experiments. Statistical significance was assessed using the Student’s t-test or Dunnet’s test for multiple comparisons with p < 0.05 as the minimal level of significance.
RESULTS Permeability Characteristics of Rhodamine123 across the Intestinal Membranes Figure 1(a) shows the time course of absorptive (mucosal to serosal) and secretory (serosal to mucosal) transport of rhodamine123 across the rat jejunal membranes. As shown in this figure, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
880
SHONO ET AL.
Figure 1. Time course of absorptive (M to S), secretory (S to M) transport of rhodamine123 in the rat jejunal membranes and its regional different transport across the rat intestinal membranes. Results are expressed as the mean SE of at least three experiments. Keys: (*), absorptive (M to S) transport, (*), secretory (S to M) transport. (N.S.) not significant, *p < 0.05, **p < 0.01, compared with each absorptive (M to S) transport.
the jejunal serosal to mucosal transport (Jsm) of rhodamine123 was more than threefold greater than its mucosal to serosal transport (Jms), suggesting that the net movement of rhodamine123 across the rat jejunum was preferentially secretory direction. Figure 1(b) indicates a site difference of absorptive and secretory transport of rhodamine123 across the various intestinal membranes. In every region, the serosal to mucosal transport (Jsm) of rhodamine123 was greater than its mucosal to serosal transport (Jms). However, the secretory directed transport was more remarkably observed in the jejunum than the other intestinal segments, indicating that there exists a regional difference in the directional transport of rhodamine123 across the rat intestinal membrane. Based on the regional different absorption studies, we selected jejunum as a model region to estimate the function of P-gp in the presence or absence of various surfactants in the following studies. Effects of Various Surfactants on the Permeability of Rhodamine123 across the Intestinal Membranes Figure 2 shows the effects of various surfactants and inhibitors on the transport of rhodamine123 across the rat jejunal membranes. In this case, we evaluated the transport direction of rhodamine123 by calculating Jsm/Jms ratio of rhodamine123. As is evident from the figure, the Jsm/ JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
Figure 2. Effects of various surfactants on the transport of rhodamine123 across the rat jejunal membranes. Results are expressed as the mean SE of at least three experiments. (CEL); Cremophor EL, (LM); n-dodecylb-D-maltopyranoside, (SDS) sodium dodecyl sulfate, and (C16TAB) hexadecyltrimethylammonium bromide. (N.S.) not significant, *p < 0.05, **p < 0.01, compared with the control.
Jms ratio of rhodamine123 significantly decreased in the presence of 0.3 mM verapamil and 10 mM NaN3 þ 1 mM NaF, suggesting that the rhodamine123 might be secreted from the intestinal tissue into the lumen by a P-gp–mediated efflux system. Figure 2 also indicates Jsm/Jms ratio of rhodamine123 in the presence or absence of various surfactants. As shown in this figure, nonionic surfactants (0.1% Cremophor EL, Tween 80, and LM) reduced the Jsm/Jms ratio of rhodamine123, whereas its ratio was not influenced in the presence of 0.1% cationic surfactant (C16TAB) and anionic surfactant (SDS). The secretory transport flux of rhodamine123 in the control was 0.680 0.090 pmol/min/cm2, whereas its flux values in the presence of Cremophor EL, Tween 80 and LM were 0.456 0.060, 0.321 0.079, and 0.450 0.089 pmol/min/cm2, respectively. On the other hand, the transfer of rhodamine123 was not affected by the addition of Cremophor EL to the serosal side. Moreover, the transport of 5(6)-carboxyfluorescein (CF), a model drug transported by a passive diffusion was not affected in the presence of Cremophor EL to the mucosal side (data not shown). Therefore, these findings suggested that charge of surfactants is possibly related to the action of these surfactants on the intestinal absorption of P-gp substrates. In the subsequent studies, we selected Cremophor EL as a suitable P-gp modulator and examined its inhibitory action of P-gp in more detail.
MODULATION OF INTESTINAL P-GLYCOPROTEIN FUNCTION BY CREMOPHOR EL
881
Effect of Various Concentrations of Cremophor EL on the Transport of Rhodamine123 Figure 3 shows concentration-dependent effect of Cremophor EL on the absorptive and secretory transport of rhodamine123 in the rat jejunal membrane. There exists a bell-shaped profile in the relationship between the absorptive and secretory transport of rhodamine123 and concentration of Cremophor EL. We observed a greatest increase and decrease in the absorptive and secretory transport of rhodamine123 with 0.01– 0.1% Cremophor EL in rat jejunal membrane. This inhibitory effect of P-glycoprotein function almost disappeared when 1% of Cremophor EL was used. To elucidate the inhibitory action of P-gp function by various concentrations of Cremophor EL, we next examined the micellar interaction between rhodamine123 and Cremophor EL. Figure 4 shows % of rhodamine123 entrapped into the micelles of Cremophor EL in the presence of various concentrations of Cremophor EL. As shown in this figure, the amount of rhodamine123 entrapped into the micelles of Cremophor EL increased as the concentration of Cremophor EL increased. Therefore, at higher concentrations of Cremophor EL, rhodamine123 can be incorporated into the micelles of Cremophor EL and we observed a remarkable interaction between rhodamine123 and Cremophor EL. Because the c.m.c. of Cremophor EL is 0.0095% w/v, interactions between rhodamine123 and the micellar form of Cremophor EL may decrease the P-gp–mediated efflux of rhodamine123 at higher concentrations.
Figure 3. Concentration-dependent effects of Cremophor EL on absorptive and secretory transport of rhodamine123 in the rat jejunum. The concentration of Cremophor EL were 0.005–1% w/v. Results are expressed as the mean SE of at least three experiments.
Figure 4. Micellar interaction between rhodamine123 and Cremophor EL. The concentrations of Cremophor EL were 0.01–1% w/v. The percentage of rhodamine123 entrapped in the micelles of Cremophor EL (Micellar drug) (%) was calculated from the following equation: Micellar drug (%) ¼ (Fcontrol Fsample)/ Fcontrol 100.
Mode of Inhibitory action of P-gp Function by Cremophor EL Figure 5 indicates the inhibitory patterns of Cremophor EL on rhodamine123 transport across the rat jejunal membranes. Figure 5(a) shows the Michaelis Menten plot between net flux of rhodamine123 and rhodamine123 concentration. The net flux of rhodamine123 decreased by the addition of 0.1% Cremophor EL in every concentration of rhodamine123. Figure 5(b) shows Eadie-Hofstee plot between net flux of rhodamine123 and rhodamine123 concentration. In the
Figure 5. Inhibition pattern of Cremophor EL on rhodamine123 transport across the rat jejunal membrane. Results are expressed as the mean SE of at least three experiments. Keys: (*); rhodamine123 only, (*); rhodamine123 þ 0.1% Cremophor EL. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
882
SHONO ET AL.
kinetic analysis, the Vmax value of rhodamine123 decreased, although the Km value was constant in the presence of Cremophor EL. Therefore, Cremophor EL inhibited the Jsm of rhodamine123 in a noncompetitive manner. Effect of Cremophor EL on the Function of Other Transporters in Rat Intestinal Membrane Finally, to examine whether Cremophor EL may affect the function of other transporters in the rat intestinal membranes, we studied the effect of Cremophor EL on the function of glucose transporter and oligopeptide transporter in the intestine. [14C]Gly-Sar was used as a model substrate of oligopeptide transporter, while [3H]3-Omethyl-D-glucose was used as a model substrate of glucose transporter. As indicated in Table 1, Cremophor EL did not affect the kinetic parameters of transport of [14C]Gly-Sar and [3H]3-Omethyl-D-glucose, suggesting that Cremophor EL may specifically reduce the function of P-gp in the intestine.
DISCUSSION The present study demonstrated that the secretory directed transport of rhodamine123 was inhibited by various nonionic surfactants, although cationic and anionic surfactants did not inhibit the polarized transport of rhodamine123. In this study, rhodamine123 was used
as a typical model of P-gp substrate, because this compound was easily assayed and was widely used for evaluating the function of P-gp in the field of cancer chemotherapy as well as biopharmaceutics.13 As shown in Figure 1, the secretory transport of rhodamine123 was much greater than its absorptive transport. In addition, the Jsm/Jms ratio of rhodamine123 was clearly reduced in the presence of 0.3 mM verapamil and 1 mM NaF þ 10 mM NaN3. Therefore, we could confirm to estimate the function of P-gp using this in vitro Ussing chamber system. We observed a regional difference in the secretory directed transport of rhodamine123 in this study, and the polarized transport of rhodamine123 was the greatest in the jejunum. On the other hand, it was generally known that the expression of P-gp in the intestine was dominant in the lower part of the intestine.1 Furthermore, Yumoto et al. reported that the P-gp mediated transport of rhodamine123 was remarkably observed in the upper and lower ileum rather than duodenum and jejunum.14 Therefore, our present result was not in good agreement with the previous findings of site-dependent expression and function of P-gp, although the reason was not fully understood. However, Yumoto et al. evaluated the P-gp– mediated transport of rhodamine123 using verapamil (they regarded verapamil-induced inhibitory transport as the P-gp–mediated transport), while we evaluated the P-gp–mediated transport by calculating the absorptive and secretory transport ratio of rhodamine123 across the intestine.
Table 1. Effect of Cremophor EL on Kinetic Parameters of [14C]Gly-Sar and [3H]3-O-methyl-D-glucose (3-OMG) Uptake by the Rat Jejunal Everted Sacs Kinetic Parameters
Gly-Sar Control CEL 0.01% w/v 0.1% w/v 3-OMG Control CEL 0.01% w/v 0.1% w/v
Km (mM)
Vmax (nmol/min/g wet tissue)
Kd (nmol/min/g wet tissue/mM)
1.11 0.22
4.76 2.04
1.46 2.30
1.27 0.20 2.36 0.55
3.63 1.11 5.68 1.90
1.72 3.09 1.67 2.51
4.55 0.84
3.61 0.71
0.17 0.03
7.69 1.07 7.07 1.51
3.93 0.50 3.41 0.65
0.16 0.03 0.16 0.04
Results are expressed as the mean SE of at least three experiments. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
MODULATION OF INTESTINAL P-GLYCOPROTEIN FUNCTION BY CREMOPHOR EL
Therefore, such different evaluation method may be partly due to the discrepancy between the findings of Yumoto et al. and our present result. Of all the surfactants, we indicated that only nonionic surfactants including Cremophor EL, Tween 80, and LM could inhibit the function of Pgp in the intestine. This finding is well correlated with the previous finding of Nerurkar et al., who demonstrated that Cremophor EL and Tween 80 could inhibit the polarized transport of model peptide compound in Caco-2 cells.5 They also reported that Tween 80 up to 1% w/v and Cremophor EL up to 10% w/v did not cause any apparent damage to the Caco-2 cell monolayers within the experimental period of exposure.5 In addition, our present result indicated that the absorptive and secretory transport of CF, a paracellular marker compound, was not changed in the presence of Cremophor EL. Furthermore, Cremophor EL and Tween 80 are polyethoxylated excipients commonly added in pharmaceutical formulations to increase the aqueous solubility and drug candidates.15,16 These excipients, which are also added to food products, are considered to be nontoxic and inert.15,16 These findings suggested that Cremophor EL and Tween 80 increased the absorptive transport of rhodamine123 without any intestinal membrane damage, but they decreased the secretory transport of rhodamine123 by inhibiting the function of P-gp. On the other hand, it was known that LM had a strong absorption enhancing effect for various poorly absorbable drugs even at low concentrations. Murakami et al.17 demonstrated that various alkylsaccharides including LM enhanced the rectal absorption of 5(6)-carboxyfluorescein and fluorescein isothiocyanate labeleddextrans in rats. In addition, Uchiyama et al.18 reported that intestinal permeability of insulin was improved in the presence of LM. Therefore, the lack of the polarized transport of rhodamine123 in the presence of LM may be partly due to the increased absorptive transport of rhodamine123, although LM can inhibit the secretory transport of rhodamine123 by reducing the function of P-gp in the intestine. Unlike the nonionic surfactants, our present study indicated no influence in the transport of rhodamine123 with the ionic surfactants. The observed difference between nonionic and ionic surfactants in the inhibition of P-gp may be due to the differences in the binding affinities of these surfactants to the hydrophobic portion of P-gp molecule. In other words, the inhibitory action of P-gp function by Tween 80 and Cremophor EL may
883
be due to their specific binding to the hydrophobic domain of the P-gp, and this nonspecific binding may change its secondary and/or tertiary structure of the P-gp and reduce the function of P-gp. Regarding the effectiveness of Cremophor EL, there exists a bell-shaped profile in the relationship between rhodamine123 and the concentration of Cremophor EL. This result was also well correlated with the previous finding of Nerurkar et al.,5 who showed the increased fraction of P-gp substrate bound to micelle at higher concentrations of Cremophor EL. Furthermore, our present study using the eqilibrium dialysis method showed that the interaction of rhodamine123 with the micelle of Cremophor EL remarkably increased as the concentration of Cremophor EL increased and reached the above concentration of c.m.c. (0.0095%). Therefore, we suggest that the free fraction of Cremophor EL can contribute to inhibit the function of P-gp, thereby increasing the net transport of rhodamine123 across the intestinal membrane. In the kinetic analysis, the Vmax value of rhodamine123 decreased, although the Km value was constant in the presence of Cremophor EL. Therefore, Cremophor EL inhibited the efflux transport of rhodamine123 in a noncompetitive manner. Therefore, these nonionic surfactants can not act as substrates of P-gp, but can act as inhibitors. With respect to the inhibition mechanism of P-gp by these surfactant, nonionic surfactants such as Tween 80 and Cremophor EL are shown to integrate within the cell membranes and thus change their microviscosity.19 This may result in loosing the phospholipid bilayers of cells, resulting in the change of secondary and/or tertiary structure of membrane proteins, thus altering their biological activity. However, Rege et al. recently demonstrated that cholesterol and benzylalcohol, known as membrane fluidity modulators, did not change the transport of rhodamine123 in Caco-2 cells.7 Therefore, changes in membrane fluidity may not be a general mechanism underlying reduced P-gp activity in the intestine. Another important mechanism by which the function of P-gp reduced is the change in the level of protein kinase C (PKC). PKC, a phospholipid/ Ca2þ-dependent protein kinase, is known to phosphorylate several proteins for cellular functions, in response to extracellular stimuli. PKC activity has been linked to various transporters, such as P-gp and the peptide transporter.20 However, recent study demonstrated that staurosporine, a PKC inhibitor failed to inhibit the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
884
SHONO ET AL.
function of P-gp, suggesting that inhibition of PKC was not the mechanism of surfactant-induced transporter inhibition of P-gp function.7 Our present results indicated that Cremophor EL inhibit the function of P-gp, but did not reduce the other transporters including glucose and peptide transporters. This finding concurs with the previous findings of Rege et al.,7 who showed the negative effect of Cremophor EL on the function of peptide transporter in Caco-2 cells, although Cremophor EL decreased the function of monocarboxy transporter. Therefore, these findings suggested that the inhibitory effect of Cremophor EL was not general phenomenon in all the transporters. As for the clinical application of Cremophor EL, Cremophor EL has been used clinically as a vehicle for the hydrophobic drugs such as taxol and cyclosporin A. In particular, taxol is formulated as 50% Cremophor EL and 50% ethanol. Therefore, the concentration of Cremophor EL, which we used in this study, is not as high compared with that in clinical application. Presumably, these surfactants might be useful for not only improving the solubility of anticancer drugs but also increasing the uptake of anticancer drugs by inhibiting the function of P-glycoprotein on the surface of the tumor cells. In conclusion, nonionic surfactants including Cremophor EL and Tween 80 were effective to inhibit the P-gp–mediated efflux system, thereby improving the intestinal absorption of poorly absorbable drugs that are secreted by P-gp from the cells into the lumen. Furthermore, this in vitro diffusion chamber system is suitable to evaluate the function of P-gp in the presence or absence of various surfactants and the other candidates of P-gp modulators.
REFERENCES 1. Hunter J, Hirst BH. 1997. Intestinal secretion of drugs: The role of P-glycoprotein and related drug efflux systems in limiting oral drug absorption. Adv Drug Del Rev 25:129–157. 2. Terao T, Hisanaga E, Sai Y, Tamai I, Tsuji A. 1996. Active secretion of drugs from the small intestinal epithelium in rats by P-glycoprotein functioning as an absorption barrier. J Pharm Pharmacol 48: 1083–1089. 3. Emi Y, Tsunashima D, Ogawara K, Higaki K, Kimura T. 1998. Role of P-glycoprotein as a secretory mechanism in quinidine absorption from rat small intestine. J Pharm Sci 87:295–299. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
4. Wagner D, Spahn-Langguth H, Hanafy A, Koggel A, Langguth P. 2001. Intestinal drug efflux: formulation and food effect. Adv Drug Del Rev 50: S13–S31. 5. Nururkar MM, Burton PS, Borchardt RT. 1996. The use of surfactants to enhance the permeability of peptides through Caco-2 cells by inhibition of an apically polarized efflux system. Pharm Res 13: 528–534. 6. Nerurkar MM, Ho NFH, Burton PS, Vidmar TJ, Borchardt RT. 1997. Mechanistic roles of neutral surfactants on concurrent polarized and passive membrane transport of a model peptide in Caco-2 cell. J Pharm Sci 86:813–821. 7. Rege BD, Kao JPY, Polli JE. 2002. Effects of nonionic surfactants on membrane transporters in Caco2 cell monolayers. Eur J Pharm Sci 16:237–246. 8. Hugger ED, Audus KL, Borchardt RT. 2002. Effects of poly(ethylene glycol) on efflux transporter activity in Caco-2 cell monolayers. J Pharm Sci 91: 1980–1990. 9. Hugger ED, Novak BL, Burton PS, Audus KL, Borchardt RT. 2002. A comparison of commonly used polyethoxylated pharmaceutical excipients on their ability to inhibit P-glycoprotein activity in vitro. J Pharm Sci 91:1991–2000. 10. Anderle P, Niederer E, Rubas W, Hilgendorf C, Spahn-Langguth H, Wunderli-Allenspach H, Merkle HP, Langguth P. 1998. P-glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: The influence of culturing condition and drug exposure on P-gp expression levels. J Pharm Sci 87: 757–762. 11. Hosoya K, Kim KJ, Lee VHL. 1996. Age-dependent expression of P-glycoprotein, gp170 in Caco-2 cell monolayers. Pharm Res 13:885–890. 12. Grass GM, Sweetana SA. 1988. In vitro measurement of gastrointestinal tissue permeability using a new diffusion cell. Pharm Res 5:372–376. 13. Fontaine M, Elmquist WF, Miller DW. 1996. Use of rhodamine123 to examine the functional activity of P-glycoprotein in primary cultured brain microvessel endothelial cell monolayers. Life Sci 59: 1521–1531. 14. Yumoto R, Murakami T, Nakamoto Y, Hasegawa R, Nagai J, Takano M. 1999. Transport of rhodamine123, a P-glycoprotein substrate, across rat intestine and Caco-2 cell monolayers in the presence of cytochrome P-450 3A-related compounds. J Pharmacol Exp Ther 289:149–155. 15. Woodcock DM, Jefferson S, Linsenmeyer ME, Crowther PJ, Chojnowski GM, Williams B, Bertoncello I. 1990. Reversal of the multidrug resistance phenotype with Cremophor EL, a common vehicle for water-insoluble vitamins and drugs. Cancer Res 50:4199–4203. 16. Zordan-Nudo T, Ling V, Liu Z, George E. 1993. Effects of nonionic detergents on P-glycoportein
MODULATION OF INTESTINAL P-GLYCOPROTEIN FUNCTION BY CREMOPHOR EL
drug binding and reversal of multidrug resistance. Cancer Res 53:5994–6000. 17. Murakami M, Kusanoi Y, Takada K, Muranishi S. 1992. Assessment of enhancing ability of mediumchain alkylsaccharides as new absorption enhancers in rat rectum. Int J Pharm 79:159–169. 18. Uchiyama T, Sugiyama T, Quan YS, Kotani A, Okada N, Fujita T, Muranishi S, Yamamoto A. 1999. Enhanced permeability of insulin across the rat intestinal membrane by various absorption enhancers: Their intestinal mucosal toxicity and
885
absorption-enhancing mechanism of n-lauryl-b-Dmaltopyranoside. J Pharm Pharmacol 51:1241– 1250. 19. Dudeja PK, Anderson KM, Harris JS, Buckingham L, Coon JS. 1995. Reversal of multidrug resistance to membrane lipid fluidity. Arch Biochem Biophys 319:309–315. 20. Castro AF, Horton JK, Vanoye CG, Altenberg CA. 1999. Mechanism of inhibition of P-glycoproteinmediated drug transport by protein kinase C blockers. Biochem Pharmacol 58:1723–1733.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004