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Release-Controlling Absorption Enhancement of Enterally Administered Ophiopogon Japonicus Polysaccharide by Sodium Caprate in Rats
Release-Controlling Absorption Enhancement of Enterally Administered Ophiopogon Japonicus Polysaccharide by Sodium Caprate in Rats XIAO LIN,1 DE-SHENG XU,2 YI FENG,1 SONG-MING LI,1 ZHI-LING LU,1 LAN SHEN1 1
Department of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2
Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200021, China
Received 16 May 2006; revised 6 June 2006; accepted 6 July 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20738
ABSTRACT: The aim of this study was to improve the intestinal absorption of Ophiopogon japonicus polysaccharide (OJP) by incorporating it together with sodium caprate (SC) into erodible matrices, designed to release OJP and SC at various rates over different periods of time. OJP, a graminan type fructosan with an average molecular weight in number of 3400 Da has been demonstrated to have anti-myocardial ischemic activity. The determination of OJP blood levels was carried out by the fluorescein isothiocyanate (FITC) prelabeling method. Matrix tablets, possessing different erosion rates, were prepared by changing the amounts of sodium alginate and using the two-layer tableting technique. Formulation effectiveness was evaluated by monitoring OJP plasma levels after intra-intestinal administration of each of the tablets to anesthetized rats. The findings indicate that all the SC containing formulations can significantly improve FITCOJP bioavailability. Compared with the formulations not containing SC, the increase varied from 5.6- to 20.8-fold for the worst and best SC containing formulations studied, respectively. Moreover, there were no statistically significant differences between the Cmax and AUC0–4 values obtained for three optimized formulations, which synchronously or nonsynchronously released both FITC-OJP and SC within 1–2 h. Their absorption enhancement effects were 2.1- to 3.6-fold higher than those of faster and slower release formulations studied. Fast delivery of the drug and its absorption adjuvant(s) contributes to their high concentrations at the absorption sites. However, at the same time, it leads to their short residence times and fast dilution by intestinal fluids. The better the balance between the two opposite effects for drug absorption, the more effective absorption enhancement would be obtained. ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:2534–2542, 2006
Keywords: polysaccharide; sodium caprate; sodium alginate; intestinal absorption enhancement; controlled release
INTRODUCTION Polysaccharides have generated considerable interest due to their various bioactivity and low toxicity. To date, several hundred polysaccharides Correspondence to: Yi Feng (Telephone: 0086-21-51322211; Fax: 0086-21-51322491; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 95, 2534–2542 (2006) ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association
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from a variety of sources such as animal, plant cell walls and fungal cells, have been discovered. The most common biological function of polysaccharides is their immunological character ranging from nonspecific stimulation of host immune system, resulting in anti-tumor, anti-viral, and anti-infective effects, to anti-oxidant, anti-mutagenic, or hematopoietic activity. Moreover, the clinical use of polysaccharides has expanded progressively as a result of the discovery of new
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bioactivities. For example, Ophiopogon japonicus polysaccharide (OJP), a highly water-soluble graminan-type fructosan with an average molecular weight in number of 3400 Da,1 has recently been demonstrated to have anti-myocardial ischemic activity.2 However, the injection-only nature of polysaccharide products poses a severe limitation to their longer-term use and/or important clinical uses for the treatment of nonacute life-threatening conditions. The low oral bioavailability of polysaccharides is due primarily to their large molecular size and hydrophilic character. To overcome this problem, one of the strategies used is to co-administer the effective and safe absorption enhancers, such as sodium caprate (SC), nitric oxide donors, chitosan derivatives, and thiolated polymers.3 SC is a wellrecognized absorption enhancer whose transient dilation effect on the paracellular pathway has been extensively documented using cell line4,5 and in situ rat models.6 In preliminary studies, we verified that the oral bioavailability of FITC-OJP was only 1.7% in the rat. Moreover, whereas FITC-OJP alone was poorly absorbed from all the intestinal regions of rats, it was rapidly absorbed when co-administered with SC, and the increase was significant and dose dependent in all regions. The best absorption enhancing site of SC for FITC-OJP was colon, followed by duodenum, jejunum and ileum in that order. In the human intestine, motility is intensive and the amounts of secreted fluid and mucus turnover are profound.7,8 Under such circumstances—‘‘open compartment’’ conditions with rapid dilution—the drug and its absorption enhancer(s) may separate right after their release from oral dosage forms. Therefore, it is reasonable to expect that the better absorption enhancing effect would be caused by relatively sustained release dosage forms rather than immediate release ones. However, the delivery rate should be carefully designed, since it contributes to both residence times and concentrations of the drug and its absorption enhancer(s) at the absorption sites, which are both important for the drug absorption. In the present study, OJP and SC were tested to ascertain the optimal oral co-administration way of the drug and its absorption enhancer.
MATERIALS AND METHODS Materials and Animals The preparation procedure of OJP involved extraction from the tube root of O. japonicus DOI 10.1002/jps
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(Cixi, Zhejiang province, China) with water followed by ethanol precipitation and chromatographic purification using DEAE Sepharose Fast Flow and Sephadex G-25 columns (Pharmacia, Uppsala, Sweden). Dextran from Leuconostoc ssp. with the standard molecular weight of 6000 Da (Dextran T-6) was purchased from Fluka (Buchs, Switzerland). Sodium caprate, fluorescein isothiocyanate (FITC) and 4-bromomethyl-6,7dimethoxycoumarin were purchased from Sigma (St. Louis, MO, USA). Sodium alginate (Keltone HVCR) was supplied by International Specialty Products, USA. All other chemicals used were of analytical grade. Male Sprague–Dawley rats weighing 250–300 g were supplied by Lab Animal Center of Shanghai University of Traditional Chinese Medicine. The rats were allowed to fast for 18–20 h with water freely available before the experiments. All animal studies were conducted in accordance with the Principles of Laboratory Animal Care (NIH publication #85–23, revised in 1985).
Labeling of OJP Using FITC Labeling of OJP using FITC was carried out according to the method used in a previous report with minor changes.9 Briefly, OJP (1 g) was dissolved in dimethyl sulfoxide (10 mL) containing a few drops of pyridine. FITC (0.1 g) was added, followed by dibutyltin dilaurate (20 mg), and the mixture was heated for 50 min at 958C. After the reaction, nine volumes of NaCl-saturated ethanol were then added to the reaction mixture. The resulting precipitates were recovered by centrifugation and washed with ethanol 10 times to fully remove free FITC and other excess reagents. The precipitates were dried using a vacuum pump, and the dry masses were measured. The yield was 0.89 g.
Chromatographic Analysis of FITC-OJP The liquid chromatographic apparatus consisted of a Shimadzu LC-20AB and a Shimadzu RF10AXL fluorescence detector, Kyoto, Japan set at Exl 495 nm and Eml 515 nm. Samples were separated by high-performance gel permeation chromatography using an 8 300-mm Shodex sugar KS-802 column (Tokyo, Japan). The mobile phase was 0.1 M phosphate buffer (pH 7.0) delivered at a flow rate of 0.5 mL/min. Under such chromatographic conditions, retention time
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stirring rate was selected according to the correlation of the erosion rate in vitro and the in vivo conditions. At predetermined time intervals, samples (4 mL) were withdrawn and replenished with the same volume of fresh buffer solution. Erosion kinetics of the tablets was measured gravimetrically at separate studies under the same conditions. At the same time intervals, the tablets were taken from the dissolution vessels and dried at 1058C until there was no further weight loss. Each study was performed in triplicate.
obtained for FITC-OJP was 13.2 min and no peaks corresponding to free FITC were determined.
Release-Controlled Formulations and In Vitro Release Studies Three types of synchronous release matrix tablets with increasing amounts of sodium alginate (S-1, S-2, and S-4) were prepared (Tab. 1). Their serial numbers reflect their overall erosion time (1, 2, and 4 h, respectively) as measured in vitro. Each tablet contained 9 mg of FITC-OJP and 34 mg of SC. The amount of SC incorporated was within the linear portion of the response (enhancement) curve of SC.6 Control tablets C-2 and C-4 without SC were prepared, which contained the same amounts of sodium alginate as formulations S-2 and S-4, respectively. CI-0, which did contained SC in the absence of sodium alginate, served as a control to emphasize the difference between the immediate release formulation and release-controlled formulations. Three types of nonsynchronous release matrix tablets (NS-1, NS-2, and NS4) composed of two layers were also prepared (Tab. 1). One layer contained FITC-OJP and the other contained SC; they were designed so that SC and FITC-OJP were released nonsynchronously. All tablets were 5 mm in diameter. Their hardness, excluding CI-0, was 3–4 kg. Drug release and erosion rates were studied in phosphate buffer (100 mL, pH 7.4), and performed in a Chinese Pharmacopoeia Type III dissolution apparatus, equipped with baskets at 25 rpm. This
FITC-OJP and SC Analysis in Buffer Solutions FITC-OJP concentrations in the withdrawn dissolution samples were measured spectrofluorimetrically (F-4500, Hitachi, Japan) at Exl 495 nm and Eml 515 nm. SC concentrations in the withdrawn samples were measured by HPLC, using sodium caprylate as an internal standard as described below. 0.25 mL taken from the withdrawn sample was mixed with sodium caprylate (0.25 mL, 0.1 mg/mL in phosphate buffer pH ¼ 7.4), 0.1 M HCl (1 mL), and dichloromethane (3 mL). The mixture was centrifuged at 4000 r/min for 5 min. Two milliliter of the dichloromethane phase was withdrawn and placed in a glass vial. After the dichloromethane phase was evaporated at 458C, 4-bromomethyl6,7-dimethoxycoumarin in acetonitrile (0.5 mL, 2 mg/mL) and triethylamine (10 mL) were added into the vial. The mixture was shaken for 40 min at 608C after which it was filtered through a
Table 1. Composition of the Immediate Release Matrix, CI-0, the Three FITC-OJP and SC Synchronous Release Matrices (S-1, S-2, and S-4), the FITC-OJP and SC Nonsynchronous Release Two-Layer Matrices NS-1, NS-2 and NS-4, and the Controls without SC (C-2 and C-4)
Formulation CI-0 S-1 S-2 S-4 NS-1 NS-1 NS-2 NS-2 NS-4 NS-4 C-2 C-4
1st layer 2nd layer 1st layer 2nd layer 1st layer 2nd layer
Sodium Alginate (mg)
FITC-OJP (mg)
SC (mg)
Dextran T-6 (mg)
Lactose (mg)
— 5 15 30 — 5 7 15 14 30 15 30
9 9 9 9 — 9 — 9 — 9 9 9
34 34 34 34 34 — 34 — 34 — — —
27 22 12 — — 36 — 26 — 11 12 —
— — — — — — — — — — 34 34
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membrane filter (pore size 0.45 mm) and injected into an analytical HPLC. The HPLC used was an Agilent 1100 series, equipped with a Shimadzu RF-530 fluorescence detector set at Exl 325 nm and Eml 398 nm. The column was a Kromasil KR100-5 C18 (250 4.6 mm). The mobile phase was acetonitrile/water (90:10, v/v) at a flow rate of 1.0 mL/min. Retention times obtained were 5.48 min for sodium caprylate and 8.77 min for SC. A linear relationship with a regression coefficient 0.9999 (n ¼ 6) was achieved between SC concentrations and the peak height ratios of SC and sodium caprylate.
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tion (Cmax) and the time to reach Cmax (Tmax) were determined directly from the plasma concentration-time profiles. The area under the plasma concentration-time curve (AUC) was calculated by the trapezoidal method from time zero to the final sampling time. Statistical Analysis Results were expressed as mean values standard deviation (SD). Statistical analyses were assessed using the Student’s t-test. Statistically significant differences were indicated by p values of <0.05.
In Vivo Evaluation Tablets were administered directly into the duodenum of the anesthetized rats as follows: 2 cm segment of the duodenum of rats was exposed through a midline incision. The tablets were administered through a 5 mm cut, 2 cm distal to the pylorus. After administration was completed, the cut was cannulated with plastic tubing to which a Shimadzu LC-5A HPLC pump was connected through an adapter. Then the abdominal wall was ligated. During the experiments, distilled water was pumped into the rat duodenum at a rate of 10 mL/min in order to imitate the supply of stomach fluid. Blood samples (400 mL) were withdrawn through a polyethylene catheter previously implanted in the femoral artery of each anesthetized rat at 15, 30, 60, 120, 180, and 240 min after the administration of tablets. Catheters were kept patent by flushing with 0.1 mL heparin (50 U/mL) between blood draws. Plasma was separated from blood cells by centrifugation using 0.2% ethylenediamine tetraacetic acid (EDTA) solution as an anticoagulant and stored at 208C until analysis.
RESULTS AND DISCUSSION Characterization of FITC-OJP FITC-OJP obtained with different reaction times (5, 10, 50, or 120 min) was analyzed chromatographically. No remarkable difference in the elution position and shape of FITC-OJP was observed with prolongation of the reaction time, indicating no alteration in the length of the FITC-OJP chain during the reaction. FITC was randomly conjugated to hydroxyl groups of OJP at a frequency of 0.004 mol of FITC per mole of fructose. The FITC label assumes negative charge at physiologic pH. However, because the degree of substitution is very low, the kinetics of FITC-OJP is expected to be closer to the kinetics of neutral OJP. From the chromatograms of plasma samples for the in vivo studies of tablets, no other peaks corresponding to the metabolic intermediate oligosaccharide of FITC-OJP and free FITC were observed, which indicated that FITC-OJP, just like OJP,10 was stable during its in vivo course and could be determined spectrofluorimetrically.
Blood FITC-OJP Level Analysis To a 100-mL portion of plasma sample, 40 mL of 1 M perchloric acid was added to facilitate the precipitation of plasma proteins. After centrifugation at 10000 r/min for 2 min, the supernatant of the sample was transferred to another clean tube into which 1 M NaOH (30 mL) and phosphate buffer (0.9 mL, pH 7.4) were added. The mixture was then determined spectrofluorimetrically at Exl 495 nm and Eml 515 nm. The limit of detection for FITC-OJP was 0.05 mg/mL. The calibration curve for FITC-OJP was linear up to 6 mg/mL (R ¼ 0.9998, n ¼ 8). The peak concentraDOI 10.1002/jps
In Vitro Evaluation By changing the amounts of sodium alginate and using the two-layer tableting technique, we were able to accomplish synchronous or nonsychronous releases of FITC-OJP and SC over different durations (Fig. 1). In the rat intestine the formulations S-1, S-2, and S-4 eroded within 1, 2, and 4 h, respectively and the immediate release formulation, CI-0, eroded within 20 min. Figure 2 displays the linear relationship between the cumulative releases of FITC-OJP from the formulations (S-1, S-2, and S-4) and the cumulative
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Figure 1. The in vitro release kinetics of FITC-OJP (open triangles) and SC (open circles) from the synchronous release formulations S-1 (A), S-2 (B) and S-4 (C) and the nonsynchronous release formulations NS-1 (D), NS-2 (E) and NS-4 (F). Each point represents the mean SD of three experiments.
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molecules, that have enabled it to be used as a matrix for the entrapment and/or delivery of a variety of proteins and cells.12 In Vivo Evaluation Results are shown in Figure 3 and Table 2, which indicate that not only SC was necessary for the enhanced absorption of FITC-OJP, but the release
Figure 2. Relationship between the cumulative release of FITC-OJP from the formulations S-1 (open circles), S-2 (open triangles) and S-4 (open square) and the cumulative erosion of the corresponding matrix. Shown are the mean values of three studies.
erosion of the corresponding matrix. The slopes of the regression lines were 1.0, indicating that the FITC-OJP release mainly depended on the matrix erosion. Although FITC-OJP and SC are both highly water-soluble, there is a marked difference between their molecular weights. The average molecular weight in peak of OJP is 5000 Da; however, the molecular weight of SC is only 194.2 Da. The difference brings into question how to attain the synchronous releases of them from one matrix. The findings of this study suggested that erodible matrices could serve that purpose over a predetermined time slot. The matrices used were made of physical mixtures of sodium alginate, SC and FITC-OJP. Owing to the proper natures of sodium alginate, including fast hydration, swelling and erosion, the rates of advancement of the swelling front into the glassy polymer and the attrition of the rubbery-state polymer were made equal so that the diffusional path length for the drug and hence the zero-order release remained nearly constant. The zero-order release of highly water-soluble compounds from hydrophilic gels can also be obtained by different polymer combinations such as hydroxypropylmethylcellulose and carboxymethylcellulose sodium, as has been shown previously.11 In addition, alginate also has several unique properties, such as a relatively inert aqueous environment within the matrix and a high gel porosity which allows for high diffusion rates of macroDOI 10.1002/jps
Figure 3. Plasma levels of FITC-OJP after intraintestinal administration of (A) CI-0 (open circles), S-1 (closed triangles) and NS-1 (open triangles) and (B) S-2 (open circles), NS-2 (closed circles), S-4 (open triangles), NS-4 (closed triangles), C-2 (open square) and C-4 (closed square). Each point represents the mean SD of four experiments.
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Table 2. Pharmacokinetics Parameters after Intra-Intestinal Administration of Synchronous and Nonsynchronous Formulations to Rats (Means SD, n ¼ 4) Formulation CI-0 S-1 S-2 S-4 NS-1 NS-2 NS-4 C-2 C-4
Tmax (h)
Cmax (mg/mL)
AUC0–4 (h mg/mL)
Fa (%)
0.25 0.50 1.00 0.50 0.25 0.50 0.50 1.00 3.00
15.0 2.6 29.1 5.9 21.3 4.6 11.2 2.4 12.8 2.4 23.4 4.8 9.6 2.5 0.9 0.4 0.7 0.4
19.7 5.6 46.4 11.2 52.0 7.3 20.5 3.7 19.8 2.5 42.0 6.6 14.2 5.6 2.5 1.2 2.3 1.0
11.8 3.4* 27.8 6.7** 31.2 4.4*** 12.3 2.2* 11.8 1.5* 25.2 3.9** 8.5 3.4{ 1.5 0.7 1.3 0.6
a
The derived absolute bioavailability values (F) were calculated after intravenous administration of 4 mg of FITC-OJP per rat. At this dose the AUC0–4 was found to be 74.1 9.7 h mg/mL. *p < 0.01 compared with C-2. **p < 0.05 compared with CI-0. ***p < 0.01 compared with CI-0. { p < 0.05 compared with C-2.
rates of SC and FITC-OJP were very important for the enhancement of FITC-OJP absorption, caused by SC. FITC-OJP plasma levels after the administration of the formulations without SC (C-2 and C-4) were as low as those obtained after oral administration of 0.9 mL of FITC-OJP in water (10 mg/ mL). The absolute bioavailability values (F) of FITC-OJP obtained for C-2 and C-4 were 1.5% and 1.3%, respectively. On the other hand, all the SC containing formulations can significantly improve FITC-OJP bioavailability. Compared with the formulations not containing SC, the increase varied from 5.6- to 20.8-fold for the worst and best SC containing formulations NS-4 and S-2, respectively. SC induces tight junctions opening in the intestinal epithelium by activating phospholipase C which, in turn, causes calcium release from intracellular stores.5,13 However, its most important property in the context of this study was its rapid onset and short residual effect.4,6 This property contributes to the safety of SC. In the in vivo studies, the distance of the tablets from the site of administration was also measured. It was found that the distance of the tablets passed was proportional to their erosion properties. The faster erosion progressed the longer distance of the tablets passed while being eroded. Moreover, the smaller the matrix became, the faster the matrix moved. That is, the dilution effect in the intestine became more and more significant with the erosion of the matrix with time. This together with the rapid on (2 min)-off (15 min) effect of SC,4,6 the better absorption enhancement effect of
SC at the upper intestine and the rather fast elimination of OJP from blood10 can explain the phenomenon that the Tmax values for all the SC containing formulations were more or less shorter than the in vitro release times of SC and OJP (Fig. 1 and Tab. 2). Absorption enhancing effects of the formulations CI-0 and NS-1, both of which released SC within 25 min, were 2–3 times less than those of the formulations S-1, NS-2, and S-2, all of which released SC within 1–2 h (Fig. 1 and Tab. 2). It is obvious that the relatively lower F values obtained for CI-0 and NS-1 was caused by the too fast release of SC, which led to its rapid dilution and separation from FITC-OJP due to fast spreading over large surface area of the intestine and the different diffusion behaviors of the small molecular absorption enhancer and the large molecular polysaccharide. Although the largest Cmax value and AUC0–4 value were obtained for the synchronous release formulations S-1 and S-2, respectively, there were no statistically significant differences between the Cmax and AUC0–4 values obtained for S-1, S-2 and the nonsynchronous release formulation NS-2 (Tab. 2), indicating that the exactly synchronized release of drug and its absorption enhancer(s) from the same matrix was not necessary for obtaining optimized absorption enhancing effect of absorption enhancer(s). This somewhat complements and differs with the results reported by Baluom et al.14 who emphasized the importance of synchronized release and found that the oral sulpiride (a poorly water-soluble antipsychotic drug) bioavailability obtained for the matrix synchronously releasing
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SC and sulpiride within 1 h was 2.3- to 8.0fold higher than those of faster and slower synchronous or nonsynchronous release formulations. In addition, they failed to investigate the nonsynchronous release matrix that released SC within 1 h but drug within 2 h, which was verified in our study to have no significant difference in absorption enhancing effect with two synchronous release matrices that synchronously released SC and drug within 1 and 2 h, respectively. As for the less erodible formulations, FITC-OJP plasma levels after the administration of the formulations S-4 and NS-4 were both low and close to those obtained after the administration of CI-0 (Fig. 3). In addition, the AUC0–4 value was 3.6-fold lower after NS-4 administration compared with S-2 administration, although the release rates of SC from the two formulations were similar. The result showed that the release rate of FITC-OJP, which contributed to the drug concentration at the absorption site, could also have a remarkable influence on the absorption enhancement when the effects of SC obtained for different formulations were similar.
CONCLUSIONS Fast delivery of the drug and its absorption adjuvant(s) contributes to their high concentrations at the absorption sites. However, at the same time, it leads to their short residence times and fast dilution by intestinal fluids. The better the balance between the two opposite effects for drug absorption, the more effective absorption enhancement would be obtained. In this study we have demonstrated that absorption enhancement effects of the formulations synchronously or nonsynchronously releasing both FITC-OJP and SC within 1–2 h were similar and 2.1- to 3.6-fold higher than the faster and slower FITC-OJP and SC release formulations studied. Moreover, all the SC containing formulations can significantly improve FITC-OJP bioavailability. Compared with the formulations not containing SC, the increase varied from 5.6- to 20.8-fold for the worst and best SC containing formulations NS-4 and S-2, respectively. As the release of the system suggested in our study mainly depends on the matrix erosion and the erosion rate can be regulated easily by changing the amount and/or type of matrix polymer(s), this simple formulative DOI 10.1002/jps
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approach should be useful for other macromolecules and absorption adjuvants.
ACKNOWLEDGMENTS Financial support from Shanghai Municipal Education Commission (Grant No. 04CC23) National Natural Science Foundation of China (Grant No. 30371685) and Shanghai Leading Academic Discipline Project (Project No. T0301) is gratefully acknowledged.
REFERENCES 1. Xu DS, Feng Y, Lin X, Deng HL, Fang JN, Dong Q. 2005. Isolation, purification and structural analysis of a polysaccharide MDG-1 from Ophiopogon Japonicus. Acta Pharmaceutica Sinica 40:636– 639. 2. Xu DS, Feng Y, Zhou YH, Zhang XC, Lin X, Deng HL. 2004 . Active components of polysaccharide of Ophiopogon japonicus on acute myocardial ischemia. Chin Tradit Patent Med 26:832–837. 3. Lin X, Xu DS, Feng Y. 2003. Research progress on intestinal absorption enhancers for macromolecular drugs. Chin Pharm J 38:741–744. 4. Hayashi M, Sakai T, Hasegawa Y, Nishikawahara T, Tomioka H, Iida A, Shimizu N, Tomita M, Awazu S. 1999. Physiological mechanism for enhancement of paracellular drug transport. J Control Rel 62: 141–148. 5. Anderberg EK, Lindmark T, Artursson P. 1993. Sodium caprate elicits dilatations in human intestinal tight junctions and enhances drug absorption by the paracellular route. Pharm Res 10:857–864. 6. Baluom M, Friedman M, Rubinstein A. 1998. The importance of intestinal residence time of absorption enhancer on drug absorption and implication on formulative considerations. Int J Pharm 176: 21–30. 7. Shameem M, Katori N, Aoyagi N, Kojima S. 1995. Oral solid controlled release dosage forms: Role of GI-mechanical destructive forces and colonic release in drug absorption under fasted and fed conditions in humans. Pharm Res 12:1049–1054. 8. Phillips SF. 1973. Integration of secretory and absorptive function. Mayo Clin Proc 48:630– 633. 9. de Belder AN, Granath K. 1973. Preparation and properties of fluorescein-labelled dextrans. Carbohydr Res 30:375–378. 10. Lin X, Xu DS, Feng Y, Shen L. 2005. Determination of Ophiopogon japonicus polysaccharide in plasma by HPLC with modified postcolumn fluor-
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escence derivatization. Anal Biochem 342:179– 185. 11. Lin X, Chen JM. 2002. Several approaches to prepare matrices containing soluble drug with zero-order release kinetics. Chin Pharm J 37: 192–195. 12. Gombotz WR, Wee SF. 1998. Protein release from alginate matrices. Adv Drug Deliv Rev 31:267–285.
13. Hochman J, Artursson P. 1994. Mechanism of absorption enhancement and tight junction regulation. J Control Rel 29:253–267. 14. Baluom M, Friedman M, Assaf P, Haj-Yehia AI, Rubinstein A. 2000. Synchronized release of sulpiride and sodium decanoate from HPMC matrices: A rational approach to enhance sulpiride absorption in the rat intestine. Pharm Res 17:1071–1076.
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Department of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2
Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200021, China
Received 16 May 2006; revised 6 June 2006; accepted 6 July 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20738
ABSTRACT: The aim of this study was to improve the intestinal absorption of Ophiopogon japonicus polysaccharide (OJP) by incorporating it together with sodium caprate (SC) into erodible matrices, designed to release OJP and SC at various rates over different periods of time. OJP, a graminan type fructosan with an average molecular weight in number of 3400 Da has been demonstrated to have anti-myocardial ischemic activity. The determination of OJP blood levels was carried out by the fluorescein isothiocyanate (FITC) prelabeling method. Matrix tablets, possessing different erosion rates, were prepared by changing the amounts of sodium alginate and using the two-layer tableting technique. Formulation effectiveness was evaluated by monitoring OJP plasma levels after intra-intestinal administration of each of the tablets to anesthetized rats. The findings indicate that all the SC containing formulations can significantly improve FITCOJP bioavailability. Compared with the formulations not containing SC, the increase varied from 5.6- to 20.8-fold for the worst and best SC containing formulations studied, respectively. Moreover, there were no statistically significant differences between the Cmax and AUC0–4 values obtained for three optimized formulations, which synchronously or nonsynchronously released both FITC-OJP and SC within 1–2 h. Their absorption enhancement effects were 2.1- to 3.6-fold higher than those of faster and slower release formulations studied. Fast delivery of the drug and its absorption adjuvant(s) contributes to their high concentrations at the absorption sites. However, at the same time, it leads to their short residence times and fast dilution by intestinal fluids. The better the balance between the two opposite effects for drug absorption, the more effective absorption enhancement would be obtained. ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:2534–2542, 2006
Keywords: polysaccharide; sodium caprate; sodium alginate; intestinal absorption enhancement; controlled release
INTRODUCTION Polysaccharides have generated considerable interest due to their various bioactivity and low toxicity. To date, several hundred polysaccharides Correspondence to: Yi Feng (Telephone: 0086-21-51322211; Fax: 0086-21-51322491; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 95, 2534–2542 (2006) ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association
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from a variety of sources such as animal, plant cell walls and fungal cells, have been discovered. The most common biological function of polysaccharides is their immunological character ranging from nonspecific stimulation of host immune system, resulting in anti-tumor, anti-viral, and anti-infective effects, to anti-oxidant, anti-mutagenic, or hematopoietic activity. Moreover, the clinical use of polysaccharides has expanded progressively as a result of the discovery of new
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bioactivities. For example, Ophiopogon japonicus polysaccharide (OJP), a highly water-soluble graminan-type fructosan with an average molecular weight in number of 3400 Da,1 has recently been demonstrated to have anti-myocardial ischemic activity.2 However, the injection-only nature of polysaccharide products poses a severe limitation to their longer-term use and/or important clinical uses for the treatment of nonacute life-threatening conditions. The low oral bioavailability of polysaccharides is due primarily to their large molecular size and hydrophilic character. To overcome this problem, one of the strategies used is to co-administer the effective and safe absorption enhancers, such as sodium caprate (SC), nitric oxide donors, chitosan derivatives, and thiolated polymers.3 SC is a wellrecognized absorption enhancer whose transient dilation effect on the paracellular pathway has been extensively documented using cell line4,5 and in situ rat models.6 In preliminary studies, we verified that the oral bioavailability of FITC-OJP was only 1.7% in the rat. Moreover, whereas FITC-OJP alone was poorly absorbed from all the intestinal regions of rats, it was rapidly absorbed when co-administered with SC, and the increase was significant and dose dependent in all regions. The best absorption enhancing site of SC for FITC-OJP was colon, followed by duodenum, jejunum and ileum in that order. In the human intestine, motility is intensive and the amounts of secreted fluid and mucus turnover are profound.7,8 Under such circumstances—‘‘open compartment’’ conditions with rapid dilution—the drug and its absorption enhancer(s) may separate right after their release from oral dosage forms. Therefore, it is reasonable to expect that the better absorption enhancing effect would be caused by relatively sustained release dosage forms rather than immediate release ones. However, the delivery rate should be carefully designed, since it contributes to both residence times and concentrations of the drug and its absorption enhancer(s) at the absorption sites, which are both important for the drug absorption. In the present study, OJP and SC were tested to ascertain the optimal oral co-administration way of the drug and its absorption enhancer.
MATERIALS AND METHODS Materials and Animals The preparation procedure of OJP involved extraction from the tube root of O. japonicus DOI 10.1002/jps
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(Cixi, Zhejiang province, China) with water followed by ethanol precipitation and chromatographic purification using DEAE Sepharose Fast Flow and Sephadex G-25 columns (Pharmacia, Uppsala, Sweden). Dextran from Leuconostoc ssp. with the standard molecular weight of 6000 Da (Dextran T-6) was purchased from Fluka (Buchs, Switzerland). Sodium caprate, fluorescein isothiocyanate (FITC) and 4-bromomethyl-6,7dimethoxycoumarin were purchased from Sigma (St. Louis, MO, USA). Sodium alginate (Keltone HVCR) was supplied by International Specialty Products, USA. All other chemicals used were of analytical grade. Male Sprague–Dawley rats weighing 250–300 g were supplied by Lab Animal Center of Shanghai University of Traditional Chinese Medicine. The rats were allowed to fast for 18–20 h with water freely available before the experiments. All animal studies were conducted in accordance with the Principles of Laboratory Animal Care (NIH publication #85–23, revised in 1985).
Labeling of OJP Using FITC Labeling of OJP using FITC was carried out according to the method used in a previous report with minor changes.9 Briefly, OJP (1 g) was dissolved in dimethyl sulfoxide (10 mL) containing a few drops of pyridine. FITC (0.1 g) was added, followed by dibutyltin dilaurate (20 mg), and the mixture was heated for 50 min at 958C. After the reaction, nine volumes of NaCl-saturated ethanol were then added to the reaction mixture. The resulting precipitates were recovered by centrifugation and washed with ethanol 10 times to fully remove free FITC and other excess reagents. The precipitates were dried using a vacuum pump, and the dry masses were measured. The yield was 0.89 g.
Chromatographic Analysis of FITC-OJP The liquid chromatographic apparatus consisted of a Shimadzu LC-20AB and a Shimadzu RF10AXL fluorescence detector, Kyoto, Japan set at Exl 495 nm and Eml 515 nm. Samples were separated by high-performance gel permeation chromatography using an 8 300-mm Shodex sugar KS-802 column (Tokyo, Japan). The mobile phase was 0.1 M phosphate buffer (pH 7.0) delivered at a flow rate of 0.5 mL/min. Under such chromatographic conditions, retention time
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stirring rate was selected according to the correlation of the erosion rate in vitro and the in vivo conditions. At predetermined time intervals, samples (4 mL) were withdrawn and replenished with the same volume of fresh buffer solution. Erosion kinetics of the tablets was measured gravimetrically at separate studies under the same conditions. At the same time intervals, the tablets were taken from the dissolution vessels and dried at 1058C until there was no further weight loss. Each study was performed in triplicate.
obtained for FITC-OJP was 13.2 min and no peaks corresponding to free FITC were determined.
Release-Controlled Formulations and In Vitro Release Studies Three types of synchronous release matrix tablets with increasing amounts of sodium alginate (S-1, S-2, and S-4) were prepared (Tab. 1). Their serial numbers reflect their overall erosion time (1, 2, and 4 h, respectively) as measured in vitro. Each tablet contained 9 mg of FITC-OJP and 34 mg of SC. The amount of SC incorporated was within the linear portion of the response (enhancement) curve of SC.6 Control tablets C-2 and C-4 without SC were prepared, which contained the same amounts of sodium alginate as formulations S-2 and S-4, respectively. CI-0, which did contained SC in the absence of sodium alginate, served as a control to emphasize the difference between the immediate release formulation and release-controlled formulations. Three types of nonsynchronous release matrix tablets (NS-1, NS-2, and NS4) composed of two layers were also prepared (Tab. 1). One layer contained FITC-OJP and the other contained SC; they were designed so that SC and FITC-OJP were released nonsynchronously. All tablets were 5 mm in diameter. Their hardness, excluding CI-0, was 3–4 kg. Drug release and erosion rates were studied in phosphate buffer (100 mL, pH 7.4), and performed in a Chinese Pharmacopoeia Type III dissolution apparatus, equipped with baskets at 25 rpm. This
FITC-OJP and SC Analysis in Buffer Solutions FITC-OJP concentrations in the withdrawn dissolution samples were measured spectrofluorimetrically (F-4500, Hitachi, Japan) at Exl 495 nm and Eml 515 nm. SC concentrations in the withdrawn samples were measured by HPLC, using sodium caprylate as an internal standard as described below. 0.25 mL taken from the withdrawn sample was mixed with sodium caprylate (0.25 mL, 0.1 mg/mL in phosphate buffer pH ¼ 7.4), 0.1 M HCl (1 mL), and dichloromethane (3 mL). The mixture was centrifuged at 4000 r/min for 5 min. Two milliliter of the dichloromethane phase was withdrawn and placed in a glass vial. After the dichloromethane phase was evaporated at 458C, 4-bromomethyl6,7-dimethoxycoumarin in acetonitrile (0.5 mL, 2 mg/mL) and triethylamine (10 mL) were added into the vial. The mixture was shaken for 40 min at 608C after which it was filtered through a
Table 1. Composition of the Immediate Release Matrix, CI-0, the Three FITC-OJP and SC Synchronous Release Matrices (S-1, S-2, and S-4), the FITC-OJP and SC Nonsynchronous Release Two-Layer Matrices NS-1, NS-2 and NS-4, and the Controls without SC (C-2 and C-4)
Formulation CI-0 S-1 S-2 S-4 NS-1 NS-1 NS-2 NS-2 NS-4 NS-4 C-2 C-4
1st layer 2nd layer 1st layer 2nd layer 1st layer 2nd layer
Sodium Alginate (mg)
FITC-OJP (mg)
SC (mg)
Dextran T-6 (mg)
Lactose (mg)
— 5 15 30 — 5 7 15 14 30 15 30
9 9 9 9 — 9 — 9 — 9 9 9
34 34 34 34 34 — 34 — 34 — — —
27 22 12 — — 36 — 26 — 11 12 —
— — — — — — — — — — 34 34
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membrane filter (pore size 0.45 mm) and injected into an analytical HPLC. The HPLC used was an Agilent 1100 series, equipped with a Shimadzu RF-530 fluorescence detector set at Exl 325 nm and Eml 398 nm. The column was a Kromasil KR100-5 C18 (250 4.6 mm). The mobile phase was acetonitrile/water (90:10, v/v) at a flow rate of 1.0 mL/min. Retention times obtained were 5.48 min for sodium caprylate and 8.77 min for SC. A linear relationship with a regression coefficient 0.9999 (n ¼ 6) was achieved between SC concentrations and the peak height ratios of SC and sodium caprylate.
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tion (Cmax) and the time to reach Cmax (Tmax) were determined directly from the plasma concentration-time profiles. The area under the plasma concentration-time curve (AUC) was calculated by the trapezoidal method from time zero to the final sampling time. Statistical Analysis Results were expressed as mean values standard deviation (SD). Statistical analyses were assessed using the Student’s t-test. Statistically significant differences were indicated by p values of <0.05.
In Vivo Evaluation Tablets were administered directly into the duodenum of the anesthetized rats as follows: 2 cm segment of the duodenum of rats was exposed through a midline incision. The tablets were administered through a 5 mm cut, 2 cm distal to the pylorus. After administration was completed, the cut was cannulated with plastic tubing to which a Shimadzu LC-5A HPLC pump was connected through an adapter. Then the abdominal wall was ligated. During the experiments, distilled water was pumped into the rat duodenum at a rate of 10 mL/min in order to imitate the supply of stomach fluid. Blood samples (400 mL) were withdrawn through a polyethylene catheter previously implanted in the femoral artery of each anesthetized rat at 15, 30, 60, 120, 180, and 240 min after the administration of tablets. Catheters were kept patent by flushing with 0.1 mL heparin (50 U/mL) between blood draws. Plasma was separated from blood cells by centrifugation using 0.2% ethylenediamine tetraacetic acid (EDTA) solution as an anticoagulant and stored at 208C until analysis.
RESULTS AND DISCUSSION Characterization of FITC-OJP FITC-OJP obtained with different reaction times (5, 10, 50, or 120 min) was analyzed chromatographically. No remarkable difference in the elution position and shape of FITC-OJP was observed with prolongation of the reaction time, indicating no alteration in the length of the FITC-OJP chain during the reaction. FITC was randomly conjugated to hydroxyl groups of OJP at a frequency of 0.004 mol of FITC per mole of fructose. The FITC label assumes negative charge at physiologic pH. However, because the degree of substitution is very low, the kinetics of FITC-OJP is expected to be closer to the kinetics of neutral OJP. From the chromatograms of plasma samples for the in vivo studies of tablets, no other peaks corresponding to the metabolic intermediate oligosaccharide of FITC-OJP and free FITC were observed, which indicated that FITC-OJP, just like OJP,10 was stable during its in vivo course and could be determined spectrofluorimetrically.
Blood FITC-OJP Level Analysis To a 100-mL portion of plasma sample, 40 mL of 1 M perchloric acid was added to facilitate the precipitation of plasma proteins. After centrifugation at 10000 r/min for 2 min, the supernatant of the sample was transferred to another clean tube into which 1 M NaOH (30 mL) and phosphate buffer (0.9 mL, pH 7.4) were added. The mixture was then determined spectrofluorimetrically at Exl 495 nm and Eml 515 nm. The limit of detection for FITC-OJP was 0.05 mg/mL. The calibration curve for FITC-OJP was linear up to 6 mg/mL (R ¼ 0.9998, n ¼ 8). The peak concentraDOI 10.1002/jps
In Vitro Evaluation By changing the amounts of sodium alginate and using the two-layer tableting technique, we were able to accomplish synchronous or nonsychronous releases of FITC-OJP and SC over different durations (Fig. 1). In the rat intestine the formulations S-1, S-2, and S-4 eroded within 1, 2, and 4 h, respectively and the immediate release formulation, CI-0, eroded within 20 min. Figure 2 displays the linear relationship between the cumulative releases of FITC-OJP from the formulations (S-1, S-2, and S-4) and the cumulative
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Figure 1. The in vitro release kinetics of FITC-OJP (open triangles) and SC (open circles) from the synchronous release formulations S-1 (A), S-2 (B) and S-4 (C) and the nonsynchronous release formulations NS-1 (D), NS-2 (E) and NS-4 (F). Each point represents the mean SD of three experiments.
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molecules, that have enabled it to be used as a matrix for the entrapment and/or delivery of a variety of proteins and cells.12 In Vivo Evaluation Results are shown in Figure 3 and Table 2, which indicate that not only SC was necessary for the enhanced absorption of FITC-OJP, but the release
Figure 2. Relationship between the cumulative release of FITC-OJP from the formulations S-1 (open circles), S-2 (open triangles) and S-4 (open square) and the cumulative erosion of the corresponding matrix. Shown are the mean values of three studies.
erosion of the corresponding matrix. The slopes of the regression lines were 1.0, indicating that the FITC-OJP release mainly depended on the matrix erosion. Although FITC-OJP and SC are both highly water-soluble, there is a marked difference between their molecular weights. The average molecular weight in peak of OJP is 5000 Da; however, the molecular weight of SC is only 194.2 Da. The difference brings into question how to attain the synchronous releases of them from one matrix. The findings of this study suggested that erodible matrices could serve that purpose over a predetermined time slot. The matrices used were made of physical mixtures of sodium alginate, SC and FITC-OJP. Owing to the proper natures of sodium alginate, including fast hydration, swelling and erosion, the rates of advancement of the swelling front into the glassy polymer and the attrition of the rubbery-state polymer were made equal so that the diffusional path length for the drug and hence the zero-order release remained nearly constant. The zero-order release of highly water-soluble compounds from hydrophilic gels can also be obtained by different polymer combinations such as hydroxypropylmethylcellulose and carboxymethylcellulose sodium, as has been shown previously.11 In addition, alginate also has several unique properties, such as a relatively inert aqueous environment within the matrix and a high gel porosity which allows for high diffusion rates of macroDOI 10.1002/jps
Figure 3. Plasma levels of FITC-OJP after intraintestinal administration of (A) CI-0 (open circles), S-1 (closed triangles) and NS-1 (open triangles) and (B) S-2 (open circles), NS-2 (closed circles), S-4 (open triangles), NS-4 (closed triangles), C-2 (open square) and C-4 (closed square). Each point represents the mean SD of four experiments.
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Table 2. Pharmacokinetics Parameters after Intra-Intestinal Administration of Synchronous and Nonsynchronous Formulations to Rats (Means SD, n ¼ 4) Formulation CI-0 S-1 S-2 S-4 NS-1 NS-2 NS-4 C-2 C-4
Tmax (h)
Cmax (mg/mL)
AUC0–4 (h mg/mL)
Fa (%)
0.25 0.50 1.00 0.50 0.25 0.50 0.50 1.00 3.00
15.0 2.6 29.1 5.9 21.3 4.6 11.2 2.4 12.8 2.4 23.4 4.8 9.6 2.5 0.9 0.4 0.7 0.4
19.7 5.6 46.4 11.2 52.0 7.3 20.5 3.7 19.8 2.5 42.0 6.6 14.2 5.6 2.5 1.2 2.3 1.0
11.8 3.4* 27.8 6.7** 31.2 4.4*** 12.3 2.2* 11.8 1.5* 25.2 3.9** 8.5 3.4{ 1.5 0.7 1.3 0.6
a
The derived absolute bioavailability values (F) were calculated after intravenous administration of 4 mg of FITC-OJP per rat. At this dose the AUC0–4 was found to be 74.1 9.7 h mg/mL. *p < 0.01 compared with C-2. **p < 0.05 compared with CI-0. ***p < 0.01 compared with CI-0. { p < 0.05 compared with C-2.
rates of SC and FITC-OJP were very important for the enhancement of FITC-OJP absorption, caused by SC. FITC-OJP plasma levels after the administration of the formulations without SC (C-2 and C-4) were as low as those obtained after oral administration of 0.9 mL of FITC-OJP in water (10 mg/ mL). The absolute bioavailability values (F) of FITC-OJP obtained for C-2 and C-4 were 1.5% and 1.3%, respectively. On the other hand, all the SC containing formulations can significantly improve FITC-OJP bioavailability. Compared with the formulations not containing SC, the increase varied from 5.6- to 20.8-fold for the worst and best SC containing formulations NS-4 and S-2, respectively. SC induces tight junctions opening in the intestinal epithelium by activating phospholipase C which, in turn, causes calcium release from intracellular stores.5,13 However, its most important property in the context of this study was its rapid onset and short residual effect.4,6 This property contributes to the safety of SC. In the in vivo studies, the distance of the tablets from the site of administration was also measured. It was found that the distance of the tablets passed was proportional to their erosion properties. The faster erosion progressed the longer distance of the tablets passed while being eroded. Moreover, the smaller the matrix became, the faster the matrix moved. That is, the dilution effect in the intestine became more and more significant with the erosion of the matrix with time. This together with the rapid on (2 min)-off (15 min) effect of SC,4,6 the better absorption enhancement effect of
SC at the upper intestine and the rather fast elimination of OJP from blood10 can explain the phenomenon that the Tmax values for all the SC containing formulations were more or less shorter than the in vitro release times of SC and OJP (Fig. 1 and Tab. 2). Absorption enhancing effects of the formulations CI-0 and NS-1, both of which released SC within 25 min, were 2–3 times less than those of the formulations S-1, NS-2, and S-2, all of which released SC within 1–2 h (Fig. 1 and Tab. 2). It is obvious that the relatively lower F values obtained for CI-0 and NS-1 was caused by the too fast release of SC, which led to its rapid dilution and separation from FITC-OJP due to fast spreading over large surface area of the intestine and the different diffusion behaviors of the small molecular absorption enhancer and the large molecular polysaccharide. Although the largest Cmax value and AUC0–4 value were obtained for the synchronous release formulations S-1 and S-2, respectively, there were no statistically significant differences between the Cmax and AUC0–4 values obtained for S-1, S-2 and the nonsynchronous release formulation NS-2 (Tab. 2), indicating that the exactly synchronized release of drug and its absorption enhancer(s) from the same matrix was not necessary for obtaining optimized absorption enhancing effect of absorption enhancer(s). This somewhat complements and differs with the results reported by Baluom et al.14 who emphasized the importance of synchronized release and found that the oral sulpiride (a poorly water-soluble antipsychotic drug) bioavailability obtained for the matrix synchronously releasing
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SC and sulpiride within 1 h was 2.3- to 8.0fold higher than those of faster and slower synchronous or nonsynchronous release formulations. In addition, they failed to investigate the nonsynchronous release matrix that released SC within 1 h but drug within 2 h, which was verified in our study to have no significant difference in absorption enhancing effect with two synchronous release matrices that synchronously released SC and drug within 1 and 2 h, respectively. As for the less erodible formulations, FITC-OJP plasma levels after the administration of the formulations S-4 and NS-4 were both low and close to those obtained after the administration of CI-0 (Fig. 3). In addition, the AUC0–4 value was 3.6-fold lower after NS-4 administration compared with S-2 administration, although the release rates of SC from the two formulations were similar. The result showed that the release rate of FITC-OJP, which contributed to the drug concentration at the absorption site, could also have a remarkable influence on the absorption enhancement when the effects of SC obtained for different formulations were similar.
CONCLUSIONS Fast delivery of the drug and its absorption adjuvant(s) contributes to their high concentrations at the absorption sites. However, at the same time, it leads to their short residence times and fast dilution by intestinal fluids. The better the balance between the two opposite effects for drug absorption, the more effective absorption enhancement would be obtained. In this study we have demonstrated that absorption enhancement effects of the formulations synchronously or nonsynchronously releasing both FITC-OJP and SC within 1–2 h were similar and 2.1- to 3.6-fold higher than the faster and slower FITC-OJP and SC release formulations studied. Moreover, all the SC containing formulations can significantly improve FITC-OJP bioavailability. Compared with the formulations not containing SC, the increase varied from 5.6- to 20.8-fold for the worst and best SC containing formulations NS-4 and S-2, respectively. As the release of the system suggested in our study mainly depends on the matrix erosion and the erosion rate can be regulated easily by changing the amount and/or type of matrix polymer(s), this simple formulative DOI 10.1002/jps
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approach should be useful for other macromolecules and absorption adjuvants.
ACKNOWLEDGMENTS Financial support from Shanghai Municipal Education Commission (Grant No. 04CC23) National Natural Science Foundation of China (Grant No. 30371685) and Shanghai Leading Academic Discipline Project (Project No. T0301) is gratefully acknowledged.
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escence derivatization. Anal Biochem 342:179– 185. 11. Lin X, Chen JM. 2002. Several approaches to prepare matrices containing soluble drug with zero-order release kinetics. Chin Pharm J 37: 192–195. 12. Gombotz WR, Wee SF. 1998. Protein release from alginate matrices. Adv Drug Deliv Rev 31:267–285.
13. Hochman J, Artursson P. 1994. Mechanism of absorption enhancement and tight junction regulation. J Control Rel 29:253–267. 14. Baluom M, Friedman M, Assaf P, Haj-Yehia AI, Rubinstein A. 2000. Synchronized release of sulpiride and sodium decanoate from HPMC matrices: A rational approach to enhance sulpiride absorption in the rat intestine. Pharm Res 17:1071–1076.
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