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Effects of pharmaceutical excipients on membrane permeability in rat small intestine
International Journal of Pharmaceutics 453 (2013) 363–370
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Effects of pharmaceutical excipients on membrane permeability in rat small intestine Yusuke Takizawa a,∗ , Hisanao Kishimoto a , Minami Nakagawa a , Nasa Sakamoto a , Yoshifusa Tobe a , Takahito Furuya a , Mikio Tomita b , Masahiro Hayashi c a Department of Drug Absorption and Pharmacokinetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan b Department of Drug Absorption and Pharmacokinetics, Tohoku Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai 981-8558, Japan c Laboratory of Molecular Pharmaceutics and Technology, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60, Nakaorui-machi, Takasaki, Gunma 370-0033, Japan
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Article history: Received 24 January 2013 Received in revised form 14 May 2013 Accepted 24 May 2013 Available online 3 June 2013 Keywords: Pharmaceutical excipient Membrane permeability Paracellular route Small intestine
a b s t r a c t Pharmaceutical excipients should not disturb the effects of drug therapy. In recent years, however, it has been reported that excipients induce some changes to the tight junction (TJ) and P-glycoprotein (P-gp), which can affect drug disposition. In this study, we examined the effects of 20 common pharmaceutical excipients from different classes on mucosal membrane and the differences of such effects among regions of the small intestine. We used the in vitro sac method in rat jejunum and ileum to study the effects of excipients on the membrane permeation of 5(6)-carboxyfluorescein (5-CF). 5-CF was used as a model of water-soluble compounds. In some dosage conditions of methyl--cyclodextrin, the membrane permeability of 5-CF was significantly increased in the jejunum, but such change was not observed in the ileum. Similarly, in the cases of sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose and croscarmellose sodium, the membrane permeability of 5-CF was significantly increased in the jejunum, but no change was observed in the ileum. On the other hand, in both the jejunum and the ileum, the membrane permeation of 5-CF was decreased with 0.02% (w/v) hydroxypropyl cellulose, but significantly increased with it at 0.20% (w/v). It was shown that excipients affected the membrane permeability of water-soluble compounds via the paracellular route, and these effects on absorption differed among regions of the small intestine. Moreover, in the case of 20 excipients, not only an increase in membrane permeability but also a decrease was observed. Therefore, it was suggested that a more effective formulation could be designed by changing the combination of excipients. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Pharmaceutical excipients ensure the safety of pharmaceutical products or their homogeneity, provide a pharmaceutical function, and increase the usefulness of pharmaceutical products. However, pharmaceutical excipients should not disturb the effects of drug therapy. In recent years, however, it has been reported that excipients induce some changes in the tight junction (TJ) and Pglycoprotein (P-gp), which can affect drug disposition (Uekama, 2004; Pathak et al., 2010; Boulmedarat et al., 2005; Fenyvesi et al., 2011; Cornaire et al., 2005).
∗ Corresponding author at: Department of Drug Absorption and Pharmacokinetics, School of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Tel.: +81 42 676 3168; fax: +81 42 676 3142. E-mail address: [email protected] (Y. Takizawa). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.05.055
Numerous studies on the interactions of drugs and pharmaceutical excipients have been reported, and led to the development of the concepts of pro-drug formulation and drug delivery system (DDS) (Sawada et al., 2003; Takahashi et al., 2005; Alvarez-Lorenzo and Concheiro, 2006). However, there are few reports that focused on the change of gastrointestinal mucosal membrane in association with pharmaceutical excipients (Tran et al., 2009; Merzlikine et al., 2009; Mudra and Borchardt, 2010). Because the influence of pharmaceutical excipients on the gastrointestinal mucosal membrane has not been discussed much (Takahashi et al., 2004; Mudra and Borchardt, 2010), there is very little information about the interaction between gastrointestinal mucosal membrane and different types of pharmaceutical excipient. Currently, the impact of pharmaceutical excipients on the gastrointestinal mucosal membrane is not considered in the selection criteria of pharmaceutical excipients in the pharmaceutical industry, and the pharmaceutical excipients are used on the basis of their maximum available amount and heuristic methods of different
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Table 1 Excipients used in current study. Diluents
Disintegrants
Binders
Lubricants
Sustained release substrate
Methyl--cyclodextrin Lactose hydrate Corn starch Microcrystalline cellulose Sodium carboxymethyl starch Low substituted Hydroxypropyl cellulose Croscarmelose sodium Hydroxypropyl cellulose Hydroxypropylmethyl cellulose Povidone (K29/32) Povidone (K90) Pullulan Talc stearic acid Calcium stearate Magnesium stearate Glyceryl monostearate Soft anhydrous silicic acid Ethyl cellulose Methyl cellulose
M--CD LH CS MCC SCMS L-HPC CCS HPC HPMC PVP (K29/32) PVP (K90)
SA CS MS GMS SAAS EC MC
entities within this industry. Moreover, it has been reported that the pharmacological effect differs between brand-name drugs and generic equivalents (Wang et al., 2010; Fujimura et al., 2011), which may be due to a difference of pharmaceutical excipients used. In recent years, the market for generic drugs has continued to expand; therefore, information about the equivalence of brand-name drugs and their generic counterparts is becoming increasingly important for patients and the pharmaceutical industry. Against this background, this paper focuses on the effect of pharmaceutical excipients on gastrointestinal mucosal membrane. As shown in Table 1, we examined the effects of 20 kinds of pharmaceutical excipient, which are major pharmaceutical excipients, on the membrane permeation of paracellular markers. In addition, we examined the effect of each pharmaceutical excipient at two different concentrations. 2. Materials and methods 2.1. Materials 5(6)-Carboxyfluorescein (5-CF) was purchased from Sigma Aldrich Co. Ltd. (Tokyo, Japan). All other reagents were of analytical grade or better. 2.2. Animals Male Wistar rats (8 weeks old) were purchased from Tokyo Laboratory Animals Science Co., Ltd. (Tokyo, Japan). All animal experiments were performed in accordance with the guidelines of Tokyo University of Pharmacy and Life Sciences. The animals were fasted for 18 h before starting the experiment. Water was freely available during fasting. 2.3. Membrane permeation experiments using the in vitro sac method Anesthesia was performed by the intraperitoneal administration of Somnopentyl® (pentobarbital sodium, 50 mg/kg) and rats were placed on a heating pad to maintain their body temperature at 37 ◦ C. The abdomen was opened by a midline longitudinal incision and 6 cm jejunal (5 cm below the Treitz ligament) and ileal (5 cm above the cecum) segments were removed and cannulated at both ends with plastic tubing. The experimental system was assembled with a silicone stopper and beaker (Fig. 1) before the intestinal tract
Fig. 1. Diagram of the experimental apparatus of the in vitro sac method.
dried, Krebs–Henselite bicarbonate buffer (KHBB, NaCl, 126 mM, KCl, 5 mM, NaHCO3 1.4 mM, Na2 HPO4 0.95 mM, NaH2 PO4 ·2H2 O 4.85 mM, CaCl2 2 g/L, and glucose 3.5 mM; pH 6.5) was added to the receiver side (serosal side, 40 mL) and the donor side (mucosal side, 5 mL), and pre-incubation was carried out for 10 min. After pre-incubation, 5 mL of KHBB solution containing 5-CF (10 M) with or without pharmaceutical excipient was added to the donor side. Sampling from the receiver side was performed at the following time points: 0, 20, 40, 60, 80, 100 and 120 min after the administration of 5-CF. After the membrane permeation experiment, the intestinal tract was removed from the experimental system, and its effective surface area was measured. In addition, drug solution and buffer warmed to 37 ◦ C were used in the experiment, and the experiment was performed at 37 ◦ C. All samples were processed immediately after collection. The concentrations of 5-CF on the receiver side and the donor side were measured using a fluorescent spectrophotometer (FP-6500; HITACHI, Tokyo, Japan). The area under the concentration curve of receiver (AUC) of 5-CF was calculated by the trapezoidal method. 2.4. Statistical analysis All results are expressed as the mean ± SE. Statistically significant differences between groups were analyzed using Tukey’s test; P < 0.05 was considered significant. 3. Results and discussion 3.1. Effect of diluents on membrane permeation in rat small intestine Before examination of pharmaceutical excipient, the administration of methyl--cyclodextrin (M--CD) at a high concentration (3.25 and 13.0% [w/v]), which was reported to have a significant absorption-enhancing effect, was confirmed to be a positive control of the increase of permeation via the paracellular route. By the administration of 13.0% (w/v) M--CD, the AUC of 5-CF was significantly increased approximately 2-fold compared with that in the no additive condition in both jejunum and ileum (data not shown). Therefore, if the AUC of 5-CF is increased approximately 2-fold by the administration of each pharmaceutical excipient, it is
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Fig. 2. Changes in AUC of 5-CF from jejunum (a) and ileum (b) by diluent administration. Data represent means and S.E. (n = 4–10 for each condition). *P < 0.05 vs. no additive condition.
considered a substantial change of membrane permeation through the paracellular route. We examined the influence on the membrane permeation of pharmaceutical excipients that are widely used in the pharmaceutical industry. The concentrations of pharmaceutical excipients were decided from the clinical dosage. Each category of pharmaceutical excipient is included in a tablet (200 mg) as follows (diluents: 20–30%, disintegrants: 5–10%, binders: 5–10%, lubricants: 1%, sustained release substrate: 20–30%). Because the tablet is taken with 100–200 mL of water, we set the dose (concentration) of each pharmaceutical excipient from the volume of water and the amount of pharmaceutical excipient included in a tablet. To do this, we performed a membrane permeation experiment on 5-CF, which is a paracellular marker (Dorkoosh et al., 2004; Salama et al., 2004), using the in vitro sac method in rat small intestine. 5-CF was not reported to be recognized by P-gp and CYP, so we examined the effect of verapamil as a P-gp inhibitor and ketoconazole as a CYP inhibitor on the membrane permeation of 5-CF using the in vitro sac method. As a result, no significant changes of membrane permeation of 5-CF were observed by the administration of verapamil and ketoconazole (data not shown). It was clarified that P-gp and CYP had no influence on the membrane permeation of 5-CF in the in vitro sac method. Therefore, it was considered that the changes in
membrane permeation of 5-CF were caused by a change of paracellular permeation. In addition, we examined the regional difference between the jejunum and the ileum in terms of the influence of pharmaceutical excipients. First, we present the results of diluents. Diluents were used at concentrations of 0.08% and 0.8% (w/v). Numerous studies of M--CD have been reported, and there are reports about the membrane permeation of paracellular markers (Maruyama et al., 2009; Sugibayashi et al., 2009; Doi et al., 2011). In this study, the experiments were performed at lower concentrations than in these previous reported studies. In the dosage condition of M--CD, membrane permeation of 5-CF was significantly increased in a concentration-dependent manner of M--CD compared with the condition with no additive in the jejunum. However, no significant change was observed in the ileum (Fig. 2). As described above, the increase of membrane permeation of paracellular marker by the administration of M--CD has already been reported (Maruyama et al., 2009; Sugibayashi et al., 2009; Doi et al., 2011), but our results showed that the effect of M--CD was observed at a concentration lower than that reported previously. In addition, the presence of a regional difference in this effect in rat small intestine was observed. Because the effects of lactose hydrate (LH), corn starch (CS) and microcrystalline cellulose (MCC) on the membrane
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Fig. 3. Changes in AUC of 5-CF from jejunum (a) and ileum (b) by disintegrant administration. Data represent means and S.E. (n = 4–10 for each condition). *P < 0.05 vs. no additive condition.
permeation of paracellular marker have not been reported, it is important to clarify the effects of these diluents on gastrointestinal mucosal membrane. No significant changes of paracellular permeability in both the jejunum and the ileum by the administration of the above 3 kinds of diluents were observed (Fig. 2). From these results, it was considered that diluents might have an influence on paracellular permeability in rat small intestine.
3.2. Effect of disintegrants on membrane permeation in rat small intestine Disintegrants were used at concentrations of 0.02% and 0.2% (w/v). For sodium carboxymethyl starch (SCMC), low-substituted hydroxypropyl cellulose (L-HPC) and croscarmellose sodium (CCS), in relation to the membrane permeation of paracellular marker, the membrane permeability of 5-CF in the jejunum was significantly increased by the administration of all 3 kinds of disintegrant at concentrations of both 0.02% and 0.2% (w/v). On the other hand, no significant increase of membrane permeation of 5-CF was observed in the ileum (Fig. 3). Regional differences in the effects on the
membrane permeation of a paracellular marker by disintegrants were thus observed in rat small intestine. 3.3. Effect of binders on membrane permeation in rat small intestine Binders were used at concentrations of 0.02% and 0.2% (w/v). We selected hydroxypropyl cellulose (HPC), hydroxypropylmethyl cellulose (HPMC), povidone (K29/32), povidone (K90) and pullulan as binders, and examined their effects on the gastrointestinal mucosal membrane. The membrane permeation of 5-CF in both the jejunum and the ileum was significantly decreased by the addition of 0.02% (w/v) HPC, but it was significantly increased by the addition of 0.2% (w/v) HPC. On the other hand, no significant change of paracellular permeability was observed in both the jejunum and the ileum by the addition of HPMC (Fig. 4). From these results, it was observed that the effect of HPMC on gastrointestinal mucosal membrane differed from that of HPC. The difference between HPC and HPMC is the presence or absence of a methyl group. It is not clear whether this difference participates in the influence on membrane permeation, so further detailed examination is necessary.
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Fig. 4. Changes in AUC of 5-CF from jejunum (a) and ileum (b) by binder administration. Data represent means and S.E. (n = 4–10 for each condition). *P < 0.05 vs. no additive condition.
The membrane permeation of 5-CF tended to increase in both the jejunum and the ileum by the addition of 0.02% and 0.2% (w/v) povidone (K29/32), and it was significantly increased by the addition of 0.02% (w/v) povidone (K29/32) in the jejunum. Significant change in the membrane permeation of 5-CF by the administration of povidone (K90) was not observed in the jejunum, but was observed in the ileum. However, the membrane permeation of 5-CF was significantly decreased at 0.02% (w/v) and was significantly increased at 0.2% (w/v) (Fig. 4). There is a difference in viscosity between povidone (K29/32) and povidone (K90). Therefore, it is necessary to examine whether povidone acts directly on the mucous membrane. No significant change of paracellular permeability between the jejunum and the ileum by the addition of pullulan was observed (Fig. 4). Further examination on binders is necessary while considering the composition of the solution.
change of membrane permeation of 5-CF in both the jejunum and the ileum by the addition of talc, SA and CS was observed. Although no effect on the membrane permeation of 5-CF by the addition of SA was observed, its membrane permeation was significantly increased in a concentration-dependent manner of MS compared with that in the condition with no additive in the jejunum and the ileum. In addition, paracellular permeation was significantly increased by the addition of 0.004% (w/v) GM in the jejunum (Fig. 5). From these results, the influence on the gastrointestinal mucous membrane factor of the compounds including SA was not clarified. A significant increase of paracellular permeation in the jejunum was observed by the addition of 0.04% (w/v) SAAS (Fig. 5). Lubricants can affect the paracellular permeability in rat small intestine at a considerably low concentration.
3.4. Effect of lubricants on membrane permeation in rat small intestine
3.5. Effect of sustained release substrates on membrane permeation in rat small intestine
Lubricants were used at concentrations of 0.004% and 0.04% (w/v). We selected talc, stearic acid (SA), calcium stearate (CS), magnesium stearate (MS), glyceryl monostearate (GMS) and soft anhydrous silicic acid (SAAS) as lubricants, and examined their effects on the gastrointestinal mucosal membrane. No significant
Sustained release substrates were used at concentrations of 0.08% and 0.8% (w/v). We selected ethyl cellulose (EC) and methyl cellulose (MC) as sustained release substrates. Although no significant change of paracellular permeability in the ileum was observed by the addition of EC and MC at both concentrations, a significant
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Fig. 5. Changes in AUC of 5-CF from jejunum (a) and ileum (b) by lubricant administration. Data represent means and S.E. (n = 4–10 for each condition). *P < 0.05 vs. no additive condition.
increase was observed by the addition of 0.08% (w/v) EC and 0.8% (w/v) MC in the jejunum (Fig. 6). From these results, it was shown that there are many pharmaceutical excipients that influence drug permeation via the paracellular route, but the mechanisms of increasing effects and decreasing effects were not clarified. Moreover, it is very important to determine the properties of each pharmaceutical excipient. 3.6. Regional difference in the influence of pharmaceutical excipients To evaluate the regional difference in the influence of pharmaceutical excipients on gastrointestinal mucosal membrane definitively, the rates of change of AUC in the jejunum and the ileum were plotted, as shown in Figs. 2–6. As shown in Fig. 7, in the jejunum, the effect of increasing paracellular permeation was observed for numerous pharmaceutical excipients. On the other hand, in the ileum, an increase and a decrease in paracellular permeation by the addition of pharmaceutical excipients were observed at the same level. Accordingly, the jejunum was shown to be more susceptible to the administration of pharmaceutical excipients than the ileum. From this result, it was shown that the influence on the gastrointestinal mucosal membrane of pharmaceutical excipients differs between the jejunum and the ileum. Regarding the difference between jejunum and ileum, a difference in the expression of the claudin family, structural components of
tight junctions, was reported (Markov et al., 2010), but a clear difference of function of tight junction between jejunum and ileum has not been clarified. Therefore, the cause of this regional difference was not clarified in this study, so future studies are needed. In this study, pharmaceutical excipients were used at concentrations typical of practical formulations. Thus, the concentrations of pharmaceutical excipients in this study are lower than those in previous reported studies, in association with their effects on the gastrointestinal mucosa. However, changes in membrane permeation were observed by the administration of numerous pharmaceutical excipients in this study. On the other hand, we showed only the results of changes in paracellular permeation by the addition of pharmaceutical excipients. Although various absorption-enhancing mechanisms have been reported (Ishizawa et al., 1987; Lee and Yamamoto, 1990; Lee et al., 1991; Tomita et al., 1995; Hayashi et al., 1999), further studies are necessary to clarify the mechanism of the membrane permeation-enhancing effect by these pharmaceutical excipients. Pharmaceutical excipients not only affect drug transport via paracellular permeation, but may also affect transcellular permeation and transporters. It is also important to study further the effect of pharmaceutical excipients on drug permeation via the transcellular route and certain transporters.
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4. Conclusion It was shown that pharmaceutical excipients affected the membrane permeability of water-soluble compounds via the paracellular route, and a regional difference in these effects on absorption was observed within the small intestine. Moreover, in the case of 20 pharmaceutical excipients, not only an increase of the membrane permeability but also a decrease was observed. In order to use drugs more effectively, it was suggested that more effective formulations should be designed by changing the combination of pharmaceutical excipients. Acknowledgements The authors thank Mr. Yasuhiko Ito, Miss Haruka Ishizaka, Miss Yukako Ohno, Mr. Yuki Aizawa and Miss Nozomi Goto for technical assistance. References
Fig. 6. Changes in AUC of 5-CF from jejunum (a) and ileum (b) by sustained release substrate administration. Data represent means and S.E. (n = 4–10 for each condition). *P < 0.05 vs. no additive condition.
Fig. 7. Correlation between ratio of changes of AUC in jejunum and that of ileum. Open circle: lower concentration of each pharmaceutical excipient. Closed circle: higher concentration of each pharmaceutical excipient. Data represent means and S.E. (n = 4–10 for each condition).
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Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Effects of pharmaceutical excipients on membrane permeability in rat small intestine Yusuke Takizawa a,∗ , Hisanao Kishimoto a , Minami Nakagawa a , Nasa Sakamoto a , Yoshifusa Tobe a , Takahito Furuya a , Mikio Tomita b , Masahiro Hayashi c a Department of Drug Absorption and Pharmacokinetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan b Department of Drug Absorption and Pharmacokinetics, Tohoku Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai 981-8558, Japan c Laboratory of Molecular Pharmaceutics and Technology, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60, Nakaorui-machi, Takasaki, Gunma 370-0033, Japan
a r t i c l e
i n f o
Article history: Received 24 January 2013 Received in revised form 14 May 2013 Accepted 24 May 2013 Available online 3 June 2013 Keywords: Pharmaceutical excipient Membrane permeability Paracellular route Small intestine
a b s t r a c t Pharmaceutical excipients should not disturb the effects of drug therapy. In recent years, however, it has been reported that excipients induce some changes to the tight junction (TJ) and P-glycoprotein (P-gp), which can affect drug disposition. In this study, we examined the effects of 20 common pharmaceutical excipients from different classes on mucosal membrane and the differences of such effects among regions of the small intestine. We used the in vitro sac method in rat jejunum and ileum to study the effects of excipients on the membrane permeation of 5(6)-carboxyfluorescein (5-CF). 5-CF was used as a model of water-soluble compounds. In some dosage conditions of methyl--cyclodextrin, the membrane permeability of 5-CF was significantly increased in the jejunum, but such change was not observed in the ileum. Similarly, in the cases of sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose and croscarmellose sodium, the membrane permeability of 5-CF was significantly increased in the jejunum, but no change was observed in the ileum. On the other hand, in both the jejunum and the ileum, the membrane permeation of 5-CF was decreased with 0.02% (w/v) hydroxypropyl cellulose, but significantly increased with it at 0.20% (w/v). It was shown that excipients affected the membrane permeability of water-soluble compounds via the paracellular route, and these effects on absorption differed among regions of the small intestine. Moreover, in the case of 20 excipients, not only an increase in membrane permeability but also a decrease was observed. Therefore, it was suggested that a more effective formulation could be designed by changing the combination of excipients. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Pharmaceutical excipients ensure the safety of pharmaceutical products or their homogeneity, provide a pharmaceutical function, and increase the usefulness of pharmaceutical products. However, pharmaceutical excipients should not disturb the effects of drug therapy. In recent years, however, it has been reported that excipients induce some changes in the tight junction (TJ) and Pglycoprotein (P-gp), which can affect drug disposition (Uekama, 2004; Pathak et al., 2010; Boulmedarat et al., 2005; Fenyvesi et al., 2011; Cornaire et al., 2005).
∗ Corresponding author at: Department of Drug Absorption and Pharmacokinetics, School of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Tel.: +81 42 676 3168; fax: +81 42 676 3142. E-mail address: [email protected] (Y. Takizawa). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.05.055
Numerous studies on the interactions of drugs and pharmaceutical excipients have been reported, and led to the development of the concepts of pro-drug formulation and drug delivery system (DDS) (Sawada et al., 2003; Takahashi et al., 2005; Alvarez-Lorenzo and Concheiro, 2006). However, there are few reports that focused on the change of gastrointestinal mucosal membrane in association with pharmaceutical excipients (Tran et al., 2009; Merzlikine et al., 2009; Mudra and Borchardt, 2010). Because the influence of pharmaceutical excipients on the gastrointestinal mucosal membrane has not been discussed much (Takahashi et al., 2004; Mudra and Borchardt, 2010), there is very little information about the interaction between gastrointestinal mucosal membrane and different types of pharmaceutical excipient. Currently, the impact of pharmaceutical excipients on the gastrointestinal mucosal membrane is not considered in the selection criteria of pharmaceutical excipients in the pharmaceutical industry, and the pharmaceutical excipients are used on the basis of their maximum available amount and heuristic methods of different
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Table 1 Excipients used in current study. Diluents
Disintegrants
Binders
Lubricants
Sustained release substrate
Methyl--cyclodextrin Lactose hydrate Corn starch Microcrystalline cellulose Sodium carboxymethyl starch Low substituted Hydroxypropyl cellulose Croscarmelose sodium Hydroxypropyl cellulose Hydroxypropylmethyl cellulose Povidone (K29/32) Povidone (K90) Pullulan Talc stearic acid Calcium stearate Magnesium stearate Glyceryl monostearate Soft anhydrous silicic acid Ethyl cellulose Methyl cellulose
M--CD LH CS MCC SCMS L-HPC CCS HPC HPMC PVP (K29/32) PVP (K90)
SA CS MS GMS SAAS EC MC
entities within this industry. Moreover, it has been reported that the pharmacological effect differs between brand-name drugs and generic equivalents (Wang et al., 2010; Fujimura et al., 2011), which may be due to a difference of pharmaceutical excipients used. In recent years, the market for generic drugs has continued to expand; therefore, information about the equivalence of brand-name drugs and their generic counterparts is becoming increasingly important for patients and the pharmaceutical industry. Against this background, this paper focuses on the effect of pharmaceutical excipients on gastrointestinal mucosal membrane. As shown in Table 1, we examined the effects of 20 kinds of pharmaceutical excipient, which are major pharmaceutical excipients, on the membrane permeation of paracellular markers. In addition, we examined the effect of each pharmaceutical excipient at two different concentrations. 2. Materials and methods 2.1. Materials 5(6)-Carboxyfluorescein (5-CF) was purchased from Sigma Aldrich Co. Ltd. (Tokyo, Japan). All other reagents were of analytical grade or better. 2.2. Animals Male Wistar rats (8 weeks old) were purchased from Tokyo Laboratory Animals Science Co., Ltd. (Tokyo, Japan). All animal experiments were performed in accordance with the guidelines of Tokyo University of Pharmacy and Life Sciences. The animals were fasted for 18 h before starting the experiment. Water was freely available during fasting. 2.3. Membrane permeation experiments using the in vitro sac method Anesthesia was performed by the intraperitoneal administration of Somnopentyl® (pentobarbital sodium, 50 mg/kg) and rats were placed on a heating pad to maintain their body temperature at 37 ◦ C. The abdomen was opened by a midline longitudinal incision and 6 cm jejunal (5 cm below the Treitz ligament) and ileal (5 cm above the cecum) segments were removed and cannulated at both ends with plastic tubing. The experimental system was assembled with a silicone stopper and beaker (Fig. 1) before the intestinal tract
Fig. 1. Diagram of the experimental apparatus of the in vitro sac method.
dried, Krebs–Henselite bicarbonate buffer (KHBB, NaCl, 126 mM, KCl, 5 mM, NaHCO3 1.4 mM, Na2 HPO4 0.95 mM, NaH2 PO4 ·2H2 O 4.85 mM, CaCl2 2 g/L, and glucose 3.5 mM; pH 6.5) was added to the receiver side (serosal side, 40 mL) and the donor side (mucosal side, 5 mL), and pre-incubation was carried out for 10 min. After pre-incubation, 5 mL of KHBB solution containing 5-CF (10 M) with or without pharmaceutical excipient was added to the donor side. Sampling from the receiver side was performed at the following time points: 0, 20, 40, 60, 80, 100 and 120 min after the administration of 5-CF. After the membrane permeation experiment, the intestinal tract was removed from the experimental system, and its effective surface area was measured. In addition, drug solution and buffer warmed to 37 ◦ C were used in the experiment, and the experiment was performed at 37 ◦ C. All samples were processed immediately after collection. The concentrations of 5-CF on the receiver side and the donor side were measured using a fluorescent spectrophotometer (FP-6500; HITACHI, Tokyo, Japan). The area under the concentration curve of receiver (AUC) of 5-CF was calculated by the trapezoidal method. 2.4. Statistical analysis All results are expressed as the mean ± SE. Statistically significant differences between groups were analyzed using Tukey’s test; P < 0.05 was considered significant. 3. Results and discussion 3.1. Effect of diluents on membrane permeation in rat small intestine Before examination of pharmaceutical excipient, the administration of methyl--cyclodextrin (M--CD) at a high concentration (3.25 and 13.0% [w/v]), which was reported to have a significant absorption-enhancing effect, was confirmed to be a positive control of the increase of permeation via the paracellular route. By the administration of 13.0% (w/v) M--CD, the AUC of 5-CF was significantly increased approximately 2-fold compared with that in the no additive condition in both jejunum and ileum (data not shown). Therefore, if the AUC of 5-CF is increased approximately 2-fold by the administration of each pharmaceutical excipient, it is
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Fig. 2. Changes in AUC of 5-CF from jejunum (a) and ileum (b) by diluent administration. Data represent means and S.E. (n = 4–10 for each condition). *P < 0.05 vs. no additive condition.
considered a substantial change of membrane permeation through the paracellular route. We examined the influence on the membrane permeation of pharmaceutical excipients that are widely used in the pharmaceutical industry. The concentrations of pharmaceutical excipients were decided from the clinical dosage. Each category of pharmaceutical excipient is included in a tablet (200 mg) as follows (diluents: 20–30%, disintegrants: 5–10%, binders: 5–10%, lubricants: 1%, sustained release substrate: 20–30%). Because the tablet is taken with 100–200 mL of water, we set the dose (concentration) of each pharmaceutical excipient from the volume of water and the amount of pharmaceutical excipient included in a tablet. To do this, we performed a membrane permeation experiment on 5-CF, which is a paracellular marker (Dorkoosh et al., 2004; Salama et al., 2004), using the in vitro sac method in rat small intestine. 5-CF was not reported to be recognized by P-gp and CYP, so we examined the effect of verapamil as a P-gp inhibitor and ketoconazole as a CYP inhibitor on the membrane permeation of 5-CF using the in vitro sac method. As a result, no significant changes of membrane permeation of 5-CF were observed by the administration of verapamil and ketoconazole (data not shown). It was clarified that P-gp and CYP had no influence on the membrane permeation of 5-CF in the in vitro sac method. Therefore, it was considered that the changes in
membrane permeation of 5-CF were caused by a change of paracellular permeation. In addition, we examined the regional difference between the jejunum and the ileum in terms of the influence of pharmaceutical excipients. First, we present the results of diluents. Diluents were used at concentrations of 0.08% and 0.8% (w/v). Numerous studies of M--CD have been reported, and there are reports about the membrane permeation of paracellular markers (Maruyama et al., 2009; Sugibayashi et al., 2009; Doi et al., 2011). In this study, the experiments were performed at lower concentrations than in these previous reported studies. In the dosage condition of M--CD, membrane permeation of 5-CF was significantly increased in a concentration-dependent manner of M--CD compared with the condition with no additive in the jejunum. However, no significant change was observed in the ileum (Fig. 2). As described above, the increase of membrane permeation of paracellular marker by the administration of M--CD has already been reported (Maruyama et al., 2009; Sugibayashi et al., 2009; Doi et al., 2011), but our results showed that the effect of M--CD was observed at a concentration lower than that reported previously. In addition, the presence of a regional difference in this effect in rat small intestine was observed. Because the effects of lactose hydrate (LH), corn starch (CS) and microcrystalline cellulose (MCC) on the membrane
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Fig. 3. Changes in AUC of 5-CF from jejunum (a) and ileum (b) by disintegrant administration. Data represent means and S.E. (n = 4–10 for each condition). *P < 0.05 vs. no additive condition.
permeation of paracellular marker have not been reported, it is important to clarify the effects of these diluents on gastrointestinal mucosal membrane. No significant changes of paracellular permeability in both the jejunum and the ileum by the administration of the above 3 kinds of diluents were observed (Fig. 2). From these results, it was considered that diluents might have an influence on paracellular permeability in rat small intestine.
3.2. Effect of disintegrants on membrane permeation in rat small intestine Disintegrants were used at concentrations of 0.02% and 0.2% (w/v). For sodium carboxymethyl starch (SCMC), low-substituted hydroxypropyl cellulose (L-HPC) and croscarmellose sodium (CCS), in relation to the membrane permeation of paracellular marker, the membrane permeability of 5-CF in the jejunum was significantly increased by the administration of all 3 kinds of disintegrant at concentrations of both 0.02% and 0.2% (w/v). On the other hand, no significant increase of membrane permeation of 5-CF was observed in the ileum (Fig. 3). Regional differences in the effects on the
membrane permeation of a paracellular marker by disintegrants were thus observed in rat small intestine. 3.3. Effect of binders on membrane permeation in rat small intestine Binders were used at concentrations of 0.02% and 0.2% (w/v). We selected hydroxypropyl cellulose (HPC), hydroxypropylmethyl cellulose (HPMC), povidone (K29/32), povidone (K90) and pullulan as binders, and examined their effects on the gastrointestinal mucosal membrane. The membrane permeation of 5-CF in both the jejunum and the ileum was significantly decreased by the addition of 0.02% (w/v) HPC, but it was significantly increased by the addition of 0.2% (w/v) HPC. On the other hand, no significant change of paracellular permeability was observed in both the jejunum and the ileum by the addition of HPMC (Fig. 4). From these results, it was observed that the effect of HPMC on gastrointestinal mucosal membrane differed from that of HPC. The difference between HPC and HPMC is the presence or absence of a methyl group. It is not clear whether this difference participates in the influence on membrane permeation, so further detailed examination is necessary.
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Fig. 4. Changes in AUC of 5-CF from jejunum (a) and ileum (b) by binder administration. Data represent means and S.E. (n = 4–10 for each condition). *P < 0.05 vs. no additive condition.
The membrane permeation of 5-CF tended to increase in both the jejunum and the ileum by the addition of 0.02% and 0.2% (w/v) povidone (K29/32), and it was significantly increased by the addition of 0.02% (w/v) povidone (K29/32) in the jejunum. Significant change in the membrane permeation of 5-CF by the administration of povidone (K90) was not observed in the jejunum, but was observed in the ileum. However, the membrane permeation of 5-CF was significantly decreased at 0.02% (w/v) and was significantly increased at 0.2% (w/v) (Fig. 4). There is a difference in viscosity between povidone (K29/32) and povidone (K90). Therefore, it is necessary to examine whether povidone acts directly on the mucous membrane. No significant change of paracellular permeability between the jejunum and the ileum by the addition of pullulan was observed (Fig. 4). Further examination on binders is necessary while considering the composition of the solution.
change of membrane permeation of 5-CF in both the jejunum and the ileum by the addition of talc, SA and CS was observed. Although no effect on the membrane permeation of 5-CF by the addition of SA was observed, its membrane permeation was significantly increased in a concentration-dependent manner of MS compared with that in the condition with no additive in the jejunum and the ileum. In addition, paracellular permeation was significantly increased by the addition of 0.004% (w/v) GM in the jejunum (Fig. 5). From these results, the influence on the gastrointestinal mucous membrane factor of the compounds including SA was not clarified. A significant increase of paracellular permeation in the jejunum was observed by the addition of 0.04% (w/v) SAAS (Fig. 5). Lubricants can affect the paracellular permeability in rat small intestine at a considerably low concentration.
3.4. Effect of lubricants on membrane permeation in rat small intestine
3.5. Effect of sustained release substrates on membrane permeation in rat small intestine
Lubricants were used at concentrations of 0.004% and 0.04% (w/v). We selected talc, stearic acid (SA), calcium stearate (CS), magnesium stearate (MS), glyceryl monostearate (GMS) and soft anhydrous silicic acid (SAAS) as lubricants, and examined their effects on the gastrointestinal mucosal membrane. No significant
Sustained release substrates were used at concentrations of 0.08% and 0.8% (w/v). We selected ethyl cellulose (EC) and methyl cellulose (MC) as sustained release substrates. Although no significant change of paracellular permeability in the ileum was observed by the addition of EC and MC at both concentrations, a significant
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Fig. 5. Changes in AUC of 5-CF from jejunum (a) and ileum (b) by lubricant administration. Data represent means and S.E. (n = 4–10 for each condition). *P < 0.05 vs. no additive condition.
increase was observed by the addition of 0.08% (w/v) EC and 0.8% (w/v) MC in the jejunum (Fig. 6). From these results, it was shown that there are many pharmaceutical excipients that influence drug permeation via the paracellular route, but the mechanisms of increasing effects and decreasing effects were not clarified. Moreover, it is very important to determine the properties of each pharmaceutical excipient. 3.6. Regional difference in the influence of pharmaceutical excipients To evaluate the regional difference in the influence of pharmaceutical excipients on gastrointestinal mucosal membrane definitively, the rates of change of AUC in the jejunum and the ileum were plotted, as shown in Figs. 2–6. As shown in Fig. 7, in the jejunum, the effect of increasing paracellular permeation was observed for numerous pharmaceutical excipients. On the other hand, in the ileum, an increase and a decrease in paracellular permeation by the addition of pharmaceutical excipients were observed at the same level. Accordingly, the jejunum was shown to be more susceptible to the administration of pharmaceutical excipients than the ileum. From this result, it was shown that the influence on the gastrointestinal mucosal membrane of pharmaceutical excipients differs between the jejunum and the ileum. Regarding the difference between jejunum and ileum, a difference in the expression of the claudin family, structural components of
tight junctions, was reported (Markov et al., 2010), but a clear difference of function of tight junction between jejunum and ileum has not been clarified. Therefore, the cause of this regional difference was not clarified in this study, so future studies are needed. In this study, pharmaceutical excipients were used at concentrations typical of practical formulations. Thus, the concentrations of pharmaceutical excipients in this study are lower than those in previous reported studies, in association with their effects on the gastrointestinal mucosa. However, changes in membrane permeation were observed by the administration of numerous pharmaceutical excipients in this study. On the other hand, we showed only the results of changes in paracellular permeation by the addition of pharmaceutical excipients. Although various absorption-enhancing mechanisms have been reported (Ishizawa et al., 1987; Lee and Yamamoto, 1990; Lee et al., 1991; Tomita et al., 1995; Hayashi et al., 1999), further studies are necessary to clarify the mechanism of the membrane permeation-enhancing effect by these pharmaceutical excipients. Pharmaceutical excipients not only affect drug transport via paracellular permeation, but may also affect transcellular permeation and transporters. It is also important to study further the effect of pharmaceutical excipients on drug permeation via the transcellular route and certain transporters.
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4. Conclusion It was shown that pharmaceutical excipients affected the membrane permeability of water-soluble compounds via the paracellular route, and a regional difference in these effects on absorption was observed within the small intestine. Moreover, in the case of 20 pharmaceutical excipients, not only an increase of the membrane permeability but also a decrease was observed. In order to use drugs more effectively, it was suggested that more effective formulations should be designed by changing the combination of pharmaceutical excipients. Acknowledgements The authors thank Mr. Yasuhiko Ito, Miss Haruka Ishizaka, Miss Yukako Ohno, Mr. Yuki Aizawa and Miss Nozomi Goto for technical assistance. References
Fig. 6. Changes in AUC of 5-CF from jejunum (a) and ileum (b) by sustained release substrate administration. Data represent means and S.E. (n = 4–10 for each condition). *P < 0.05 vs. no additive condition.
Fig. 7. Correlation between ratio of changes of AUC in jejunum and that of ileum. Open circle: lower concentration of each pharmaceutical excipient. Closed circle: higher concentration of each pharmaceutical excipient. Data represent means and S.E. (n = 4–10 for each condition).
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