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Effect of cationized gelatins on the paracellular transport of drugs through caco-2 cell monolayers
Effect of Cationized Gelatins on the Paracellular Transport of Drugs Through Caco-2 Cell Monolayers TOSHINOBU SEKI,1 HIROSHI KANBAYASHI,1 TOMONOBU NAGAO,1 SUMIO CHONO,1 YASUHIKO TABATA,2 KAZUHIRO MORIMOTO1 1
Hokkaido Pharmaceutical University School of Pharmacy, 7-1 Katsuraoka-cho, Otaru, Hokkaido 047-0264, Japan
2
Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
Received 16 August 2005; revised 25 January 2006; accepted 13 February 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20616
ABSTRACT: Cationized gelatins, candidate absorption enhancers, were prepared by addition of ethylenediamine or spermine to gelatin and the effects of the resulting ethylenediaminated gelatin (EG) and sperminated gelatin (SG) on the paracellular transport of 5(6)-carboxyfluorescein (CF), FITC-dextran-4 (FD4), and insulin through caco-2 cell monolayers were examined. The Renkin function was used for characterization of the paracellular pathway and changes in the pore radius (R) and pore occupancy/ length ratio ("/L) calculated from the apparent permeability coefficients (Papp) of CF and FD4 are discussed. Ethylenediaminetetraacetic acid (EDTA) increased the R of the caco-2 cell monolayer and the Papp of all compounds examined was markedly increased by the addition of EDTA. On the other hand, EG and SG did not increase R and their enhancing effects were not as strong as those of EDTA. The increase in "/L could be the enhancing mechanism for the cationized gelatins. The number of pathways for water-soluble drugs, such as CF and FD4, in the caco-2 monolayers could be increased by the addition of the cationized gelatins. The ratios of the permeability coefficients of insulin (observed/ calculated based on the Renkin function) suggest that insulin undergoes enzymatic degradation during transport which is not inhibited by enhancers. ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:1393–1401, 2006
Keywords: paracellular transport; cationized gelatin; absorption enhancer; Renkin function; insulin; caco-2 cells; permeability; tight junction
INTRODUCTION Oral, nasal, or pulmonary drug delivery systems for peptide and protein drugs are needed to avoid the problems associated with parenteral formulations, such as tissue invasion, and also to improve patient compliance.1 The use of absorption enhancers2 and proteolytic enzyme inhibitors3 and suitably designed formulations4 are possible approaches to the development of novel delivery systems to increase the bioavailability of Correspondence to: Toshinobu Seki (Telephone: þ81-13462-1882; Fax: þ81-134-62-1848; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 95, 1393–1401 (2006) ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association
peptide and protein drugs. The absorption enhancers, which increase the permeability of drugs through the epithelial membranes without causing any tissue damage, will be especially useful for the delivery of drugs with a relatively high molecular weight (MW).5 Although many chemicals, including surfactants, bile salts, and fatty acids, have been evaluated as absorption enhancers, most of them produce some form of membrane damage.6,7 In order to choose a safe enhancer, a better understanding of the mode of action and mechanism of such compounds is required. Most of the candidate enhancers reported are able to open the tight junctions of epithelial cell layers and increase the paracellular permeability of drugs by a mechanism involving
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suppressed transepithelial resistance (TER) and increased permeability of mannitol and/or FITCdextrans.2 However, this evidence is not enough for a proper understanding of the change in the paracellular pathways.8 It has recently been reported that cationic polymers, including chitosan9,10 and poly-L-arginine11,12, are able to improve the absorption of peptide and protein drugs while causing negligible damage to the mucosal membrane. In previous reports, we showed that cationized gelatins with different numbers of amino groups enhanced the nasal absorption of insulin and the enhancing effects depended on the amino group content.13,14 The effect of the cationized gelatins on the LDH leaking from the nasal mucosa was relatively weak and similar to that of chitosan.13 The cationic polymers could interact with the luminal surface of mucus membranes directly by an ion–ion interaction and then induce signals that would open tight junctions resulting in intercellular permeation.9,11,15 However, the mechanism(s) of opening tight junctions is not yet fully understood. The aim of this study is to evaluate the mode of action of cationized gelatins, a new generation of permeation enhancers, on tight junctions. In recent years, our knowledge of tight junction structure has advanced dramatically and its molecular structure involving occludin, claudins, and ZO proteins has been described.16–19 Studies of the structure of claudins suggest the existence of aqueous pores in the tight junctions20 and at least two different sized pores are assumed to be present.21,22 A pore pathway a few Angstroms in radius could act as an absorption route for small water-soluble compounds.8 On the other hand, since relatively higher MW compounds, such as FITC-dextrans, are also able to pass through the epithelial cell layers of the intestines and nasal cavities, a pore pathway a few nanometers in radius could also be present in the tight junctions.23–25 Those smaller and larger pores might be physiologically regulated in different ways and the contribution of protein kinase A (PKA) and protein kinase C (PKC) to each process has been discussed.8,26 While activation of PKA may increase the ionic conductance of tight junctions without changing the barrier function for large molecules, activation of PKC may increase the permeability of large molecules.26 This PKC pathway may be one of the possible mechanisms governing the penetration enhancers for large molecules to allow them to pass through epithelial cell layers.27 These findings suggest that TER is not a good parameter to JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 6, JUNE 2006
evaluate the effects of candidate enhancers for large molecules8 and the barrier function of tight junctions for large molecules should be described using other parameters. Figure 1 shows models of permeation pathways (arrows) of water-soluble drugs having a relatively high MW through epithelial cell layers. Figure 1A assumes normal conditions; a few paracellular pathways are ‘‘open’’ to allow the passage of large molecules, such as FITC-dextrans.23–25 If the enhancers increase the size of the pathway, larger molecules will be able to pass via these routes (Fig. 1B). If the enhancers transform normal tight junctions into the pathways, the number will increase and the permeability coefficient of each drug should be proportionate to the number (Fig. 1C). If the candidate enhancers produce undesirable changes in the epithelial cell layers, such as emergence from the cells (I) and complete loss of barrier function by the membranes (II), the permeability of all penetrants will be higher but they will not be safe to use as enhancers (Fig. 1D). It is helpful to know which change (B–D) is induced by a candidate enhancer in order to identify the most effective enhancers.
Figure 1. Models of permeation routes for watersoluble large molecules through epithelial cell layers and the effects of penetration enhancers on these pathways. A: Normal conditions; a few paracellular pathways are ‘‘open’’ to allow the passage of relatively large molecules, such as FD4. B: Increase in size of the pathway by enhancers; the pathways increase in size and larger molecules are able to pass via these routes. C: Increase in the number of pathways by enhancers; the pathway increase and the permeability coefficient of each drug should be proportional to the number. D: Damage to epithelial cell layers by enhancers; undesirable changes in epithelial cell layers, such as emergence of cells (I) and complete loss of barrier functions of membranes (II) increase the permeability of all penetrants. DOI 10.1002/jps
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EFFECT OF CATIONIZED GELATINS
In this study, cationized gelatins, candidate enhancers, were prepared by the addition of ethylenediamine or spermine to gelatin and the effects of the resulting ethylenediaminated gelatin (EG)13,14 and sperminated gelatin (SG) on the paracellular transport of 5(6)-carboxyfluorescein (CF), FITC-dextran-4 (FD4), and insulin through caco-2 cell monolayers28,29 were examined. Ethylenediaminetetraacetic acid (EDTA), which exhibits an absorption-enhancing effect by calcium chelation and causes marked damage to epithelia, was used as a comparison.30,31 The Renkin molecular sieving function was used for characterization of the paracellular pathway and changes in the equivalent cylindrical pore radius (R) and pore occupancy/length ratio ("/L) calculated from the apparent permeability coefficients (Papp) of CF and FD4 are discussed. In addition, the permeability coefficients of insulin were calculated using the Renkin function and the values obtained were compared with the observed values to evaluate the enzymatic degradation of insulin during the transport process.
MATERIALS AND METHODS Materials Gelatin (isoelectric point ¼ 9.0, MW 100 kDa) was kindly supplied by Nitta Gelatin Co. Ltd (Osaka, Japan). Spermine tetrahydrochloride, 1,2-ethylenediamine and EDTA (tetrasodium salt, EDTA 4Na) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Recombinant human insulin (28.7 IU/mg), FD4 (MW 4400), and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride were purchased from Sigma Chemical Co. (St Louis, MA). CF and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were purchased from Acros Organics (Morris Plains, NJ) and Nacalai Tesque (Kyoto, Japan), respectively. All other chemicals were of reagent grade and used as received. Diffusion coefficients of CF, FD4, and insulin were determined in our laboratory and the values have been reported previously (Table 1).32
Synthesis of Cationized Gelatins Gelatin was reacted with 1,2-ethylenediamine or spermine to obtain cationized gelatins in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride by a method previously reported.13,14 In brief, gelatin (10 g) DOI 10.1002/jps
Table 1. Diffusion Coefficient (Di) and Stokes– Einstein Radius (ri) of Drugs Used at 378C
CF FD4 Insulin a
MW
Dia (cm2/s) 106
ri (nm)
376.3 4400 5807.6
5.87 2.39 1.14
0.556 1.37 2.86
Reference 32.
was dissolved in 0.1 M phosphate buffer (pH 5.0, 250 mL). 1,2-Ethylenediamine (28.0 g, 0.466 mole) or spermine tetrahydrochloride (32.4 g, 0.093 mole) was added to the solution and then the pH of the solution was adjusted to 5.0 with hydrochloric acid. The resulting solution was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (5.35 g) and the total volume was adjusted to 500 mL by addition of phosphate buffer (pH 5.0). The reaction was allowed to take place at 378C for 18 h. The resulting aminated gelatins were purified by dialysis for 48 h then the aminated gelatin powder was obtained by lyophilization. Determination of Amino Group Content of Cationized Gelatins One milliliter of gelatin or cationized gelatin solution (0.50 mg/mL) in phosphate buffered saline (PBS, pH 7.4) was mixed with 1.0 mL sodium bicarbonate solution (4.0%) and 1.0 mL TNBS solution (0.10%). The mixture was kept at 408C for 2 h protected from light and then the absorbance of the solution at 415 nm was determined.13,14 A calibration curve was prepared for b-alanine. The primary amino group content was expressed as the amount of TNBS-reactive amino groups in 1 g gelatin or cationized gelatin. Since the addition of spermine introduced not only primary amino groups but also secondary groups to gelatin, the total amino group content (TA) of SG was calculated using the following Equation (1): TA ¼ ðPASG PAnative Þ 3 þ PAnative
ð1Þ
where PASG and PAnative are the primary amino group contents of SG and native-gelatin, respectively. Cell Culture Caco-2 cells were obtained from the Riken Gene Bank (Ibaragi, Japan). The cells were maintained in DMEM containing 10% heat-inactivated fetal JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 6, JUNE 2006
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Drug Transport Experiments
where (DMi/Dt) is the rate of the penetrant i appearing on the receiver side, A is area of the monolayers and C0,i is the initial concentration of i. The Renkin function (3) was used for characterization of the caco-2 cell monolayers33–35: r r 2 h r i i i F 1 2:104 ¼ 1 R R R r 3 r 5 i i i 0:95 ð3Þ þ 2:09 R R
Simultaneous transport of FD4, CF, and insulin through the caco-2 cell monolayers was observed at 378C. CF (30 mg/mL), FD4 (5.0%), insulin (100 IU/mL), and enhancers (0 or 0.20%) were dissolved in PBS (pH 7.4) and the solution (100 mL) was applied to the apical side. Hanks balanced salt solution (HBSS, pH 7.4, 0.60 mL) was used as the basolateral side solution. The HBSS was changed every 30 min for 2 h. TER was measured using Millicell1-ERS (Millipore, MA) before and after the transport experiments. HBSS was used as the apical and basolateral solutions.
where ri is the molecular radius of penetrant i which can be calculated from the diffusion coefficient (Di) as the Stokes–Einstein radius. Since CF and FD4 were not metabolized during the permeation process, the Papp of CF and FD4 (PappCF and PappFD4 ) were used for the calculation. R and "/L were obtained as the characteristic parameters for each monolayer using the following Equations (4) and (5) and diffusion parameters in Table 1.32,36 r e CF PappCF ¼ DCF F ð4Þ L R
calf serum, 40 mg/mL gentamicin and 1% nonessential amino acids, in a humidified atmosphere of 95% air and 5% CO2 at 378C. Cells from passage number 46 to 53 were seeded (4.5 105 cell /cm2) on polycarbonate filter inserts (pore size 0.4 mm, area 0.33 cm2, Transwell, Costar) and cultivated in the medium for 21–25 days before starting the drug transport experiments.
PappFD4 ¼
Analytical Methods FD4 and CF in the medium were isolated and determined by a gradient HPLC system (Shimadzu, Kyoto, Japan) consisting of a pump (LC-10AT), oven (CTO-6A), detector (RF-10A), and integrator (CR-5A).32 Separation was carried out using an analytical column (Shodex Asahipak NH2P-50 4E, 250 4.6 mm i.d.; Showa Denko, Kawasaki, Japan) and a gradient of 0.1 M triethanolamine þ 10% acetonitrile 0.1 M triethanolamine þ 10% acetonitrile þ 60 mM NaCl changing from 30:70 to 80:20 over 30 min. The flow-rate of the eluent was 1 mL/min, the oven was operated at 458C and the detector was operated at an excitation wavelength of 495 nm and an emission wavelength of 515 nm. The insulin concentration in the medium was determined using an enzyme immunoassay kit (YKO 60 Human Insulin EIA kit, Yanaihara Institute, Shizuoka, Japan) according to the manufacturer’s instructions.
Characterization of Caco-2 Cell Monolayers The Papp of penetrant i (Pappi ) was calculated using the following Equation (2): Pappi ¼
DMi =Dt A C0;i
ð2Þ
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r " FD4 DFD4 F L R
ð5Þ
Statistical Analysis The mean values and their standard deviation (SD) were calculated in each experiment. The statistical significance of each treatment compared with the control group was evaluated by the Dunnett test. The Tukey–Kramer test was also used for the comparison of TER and StatView software (Ver.5.0, SAS Institute Inc., Raleigh, NC) was used for the calculations.
RESULTS Preparation of Cationized Gelatins Two different cationized gelatins, EG and SG, were prepared from gelatin and ethylenediamine or spermine. The amino group contents of the cationized gelatins were determined to monitor the increasing charge densities. Table 2 shows the primary amino group contents of the cationized gelatins and the calculated total amino group content of SG. The primary amino group content is the number of TNBS-reactive primary amino groups and is expressed as mmol/g gelatin. The values of EG and SG were higher than that of DOI 10.1002/jps
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Table 2. Amino Group Content of Cationized Gelatins Used
Native-gelatin EG SG
Primary Amino Group Contenta (NH2) (mmol/g gelatin) (SD)
Total Amino Group Contentb (NH2, ¼NH) (mmol/g gelatin)
0.255 (0.003) 0.823 (0.038) 0.660 (0.034)
— — 1.47
Mean values of three determinations are shown. a Amount of TNBS-reactive amino groups in 1 g gelatin or cationized gelatin. b Calculated amount of primary and secondary amino groups in 1 g SG (see text).
native gelatin, suggesting the addition of ethylenediamine or spermine to the gelatin. Since the addition of one spermine molecule transferred three amino groups to the gelatin, the calculated total amino group content of SG was higher than the amino group content of EG.
Effect of Cationized Gelatins and EDTA on the TER of Caco-2 Cell Monolayers The effects of cationized gelatins on the TER of the caco-2 cell monolayers were examined. The percentage TER of the caco-2 monolayers after the transport experiments compared with the initial values was reduced by the addition of cationized gelatins and EDTA (Fig. 2). This reduction represents an increase in the permeability of small ions through the monolayers. The tight junctions of the monolayer could be opened as a result. There was no difference in the effects of cationized gelatins and EDTA on the TER. Effect of Cationized Gelatins and EDTA on the Papp of CF, FD4, and Insulin Through Caco-2 Cell Monolayers CF, FD4, and insulin were simultaneously applied to the apical side of the caco-2 monolayers to determine their Papp in each monolayer. The penetration-enhancing effects of cationized gelatins and EDTA were evaluated in terms of the change in Papp for each penetrant. EG, SG, and EDTA showed significant enhancing effects for CF and FD4 (Fig. 3). The highest effect was obtained for EDTA: the enhancing ratios (ER; Papp with enhancer/Papp without enhancer) were 12.7 and 18.1 for CF and FD4, respectively. On DOI 10.1002/jps
Figure 2. Effects of penetration enhancers on the change in transepithelial resistance (TER) of caco-2 cell monolayers. The values of TER after transport experiments for 2 h are shown as a percentage of the initial values. Each enhancer was applied as a 0.2% solution. Each data set is the mean SD (n ¼ 3–5). **Significant difference compared with the control (p < 0.01) in the Dunnett test. There was no significant difference between EG, SG, and EDTA in the Tukey–Kramer test.
the other hand, the enhancing effects of EG and SG were not as potent although the effects on the TER were similar to that of EDTA. Similar enhancing effects were observed for insulin (observed P in Table 3). The enhancing effects of EDTA were more marked for higher MW penetrants (ER ¼ 12.7, 18.2, and 41.1 for CF, FD4, and insulin, respectively). On the other hand, the ER values of EG and SG were similar for CF, FD4, and insulin (ER of EG ¼ 2.6, 2.8, and 2.3 for CF, FD4, and insulin, ER of SG ¼ 4.2, 4.4, and 5.6 for CF, FD4, and insulin, respectively). Effect of Cationized Gelatins and EDTA on the Membrane Parameters of Caco-2 Cell Monolayers The membrane parameters of each caco-2 cell monolayer were calculated from PappCF , PappFD4 and previously reported diffusion parameters of CF and FD4 (Table 1) based on the Renkin function. The effects of cationized gelatins and EDTA on R and "/L of the monolayers are shown in Figure 4. In the case of EDTA, both R and "/L increased significantly. The increase in R could be due to removal of calcium ions from the intercellular space by chelation. On the other hand, cationized gelatins did not increase R of the monolayer. Since the increased ratios of "/L are 2.5 and 4.1 for EG and SG, respectively, the enhancing effects of cationized gelatins could be mainly due to the increased "/L. This result suggests that the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 6, JUNE 2006
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cationized gelatins transform normal tight junctions into the penetration pathways for CF and FD4 to increase the number in the monolayers as shown in Figure 1C. Although the effects of EDTA could be as shown in Figure 1B, since R is a mean value, it might include undesirable effects like those shown in Figure 1D. Comparison of Calculated Permeability Coefficients of Insulin With Observed Values
Figure 3. Effects of penetration enhancers on the permeability coefficients of CF (A) and FD4 (B) through caco-2 cell monolayers. Each enhancer was applied as a 0.2% solution. Each data set is the mean SD (n ¼ 3–5). *Significant difference compared with the control (p < 0.05) in the Dunnett test. **Significant difference compared with the control (p < 0.01) in the Dunnett test.
The permeability of insulin through the caco-2 cell monolayers could be calculated from the membrane parameters and the diffusion parameters of insulin based on the Renkin function. In our previous report, the observed permeability coefficient of insulin through a cellulose membrane was in agreement with that calculated from the membrane parameters and the diffusion parameters of insulin using the Renkin function.32 In the case of biological membranes, such as the caco-2 monolayers, we did not expect this agreement, because insulin would undergo enzymatic degradation during the transport process. Table 3 shows the observed and calculated permeability coefficients of insulin through the caco-2 cell monolayers. The lower observed values could be related to the degradation of insulin and the interaction of insulin with biological components. The permeability ratios (observed/calculated) were about 0.2 and the values did not change following the addition of the cationic gelatins and EDTA. This result suggests that the enhancers did not inhibit the degradation of insulin and/or the interactions reducing the permeability of insulin.
Table 3. Observed and Calculated Permeability Coefficient of Insulin Through Caco-2 Cell Monolayers
Control þEGb þSGb þEDTA4Nab
Observed P (Papp) 109 cm/s (SD)
Calculated Pa 109 cm/s (SD)
0.70 (0.36) 1.64 (0.59) 3.91c (0.97) 28.8d (3.2)
3.89 (1.59) 15.5 (10.7) 20.6 (7.2) 179d (91)
Ratio of P (observed/calculated) (SD) 0.19 0.16 0.20 0.19
(0.09) (0.15) (0.05) (0.11)
Mean values are shown (n ¼ 3–5). a R and e/L obtained from permeability coefficients of simultaneously applied CF and FD4 in each permeation experiment were used for the calculation. b 0.2% solution was used. c Significant difference compared with the control (p < 0.05) in the Dunnett test. d Significant difference compared with the control (p < 0.01) in the Dunnett test. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 6, JUNE 2006
DOI 10.1002/jps
EFFECT OF CATIONIZED GELATINS
Figure 4. Effects of enhancers on the values of R (A) and e/L (B) of caco-2 cell monolayers. Each enhancer was applied as a 0.2% solution. Each data set is the mean SD (n ¼ 3–5). *Significant difference compared with the control (p < 0.05) in the Dunnett test. **Significant difference compared with the control (p < 0.01) in the Dunnett test.
DISCUSSION In this study, the mode of action of cationized gelatins, which should be related to their efficiency and toxicity, was examined in caco-2 cell monolayers and was compared with that of EDTA which is known to cause marked epithelial damage. Two different cationized gelatins, EG and SG, were used as cationic polymers to evaluate the penetration-enhancing effects on the transport of CF, FD4, and insulin through caco-2 cell monolayers. The Renkin function was used to characterize the monolayers and the effects of the cationized gelatins. Simultaneous application of multicomponents allowed the characteristics of each monolayer to be determined. DOI 10.1002/jps
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The absorption-enhancing effects of cationic polymers depend on their chemical composition, MW and charge density. Natsume et al.11,12 examined the effect of MW and charge density on the enhancing effect of cationic polymers and found that the charge density was the most important parameter for the enhancement of the nasal absorption of FD4. The cationic polymers could interact with the luminal surface of mucus membranes directly by an ion–ion interaction and then induce signals that would open tight junctions resulting in intercellular permeation.9,11,15 In a previous report, we examined the enhancing effects of EGs with different amino group contents on the nasal absorption of insulin.14 The effects of EGs on insulin absorption were correlated with those on CF absorption and depended on the amino group content, suggesting a mechanism similar to that for other cationic polymers.27 In this study, SG with spermine as a moiety was compared with an EG. Since SG showed increased effects on the permeation of all penetrants compared with EG, which had a higher primary amino group content but a lower total amino group content than SG, the secondary amino group in SG may also contribute to the enhancing effect. The effects of EDTA were compared with those of EG and SG. The rank order of the penetration enhancement was EDTA > SG > EG for all penetrants, but there was no significant difference in the change in TER, suggesting that TER is not a useful parameter for such investigations. The R and "/L of each caco-2 cell monolayer were calculated from the corresponding PappCF and PappFD4 , and the diffusion parameters of CF and FD4 (Table 1) based on the Renkin function. The R values were increased by addition of EDTA. A similar increase in the R of caco-2 cell monolayers has been reported following the addition of EGTA.8,24 Calcium chelation could widen the cell junctions30 and such widening might be an advantage for the delivery of larger molecules.12 On the other hand, the cationized gelatins could increase the number of pathways rather than their size. The action of the cationized gelatins might have an advantage in terms of reversibility. Since absorption of pancreatic proteolytic enzymes, for example, trypsin and chymotrypsin having an MW of 23000–26000, could cause injury to the underlying tissue, excessive widening of the junctions is not desirable. Although the action of cationized gelatins is not effective for large molecules, it is good as far as increased safety is concerned. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 6, JUNE 2006
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In the case of insulin absorption, inhibition of proteolysis could be one of the mechanisms of absorption enhancement.37 In order to evaluate the contribution of inhibition to the enhancing effects of the cationized gelatins, the calculated permeability of insulin through the caco-2 cell monolayers was compared with the Papp of insulin.32 The ratios of the values (observed/calculated) were not affected by the addition of the cationized gelatins, suggesting that the cationized gelatins do not inhibit the degradation of insulin. The use of protease inhibitors together with cationized gelatins could be a way of obtaining a higher bioavailability of insulin after mucosal application.31 In summary, the cationized gelatins exhibited enhancing effects on the permeation of CF, FD4, and insulin through caco-2 cell monolayers. They were able to increase the number rather than the size of the pathways for water-soluble large molecules in the caco-2 cell monolayers. The cationized gelatins are good candidates as safe absorption enhancers which will only mildly modify the permeability of the paracellular pathway, retaining the sieving property of the epithelial membranes.
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DOI 10.1002/jps
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22. Fihn BM, Sjo¨qvist A, Jodal M. 2000. Permeability of the rat small intestinal epithelium along the villuscrypt axis: Effects of glucose transport. Gastroenterology 119:1029–1036. 23. Shimazaki T, Tomita M, Sadahiro S, Hayashi M, Awazu S. 1998. Absorption-enhancing effects of sodium caprate and palmitoyl carnitine in rat and human colons. Dig Dis Sci 43:641–645. 24. Mineo H, Amano M, Chiji H, Shigematsu N, Tomita F, Hara H. 2004. Indigestible disaccharides open tight junctions and enhance net calcium, magnesium, and zinc absorption in isolated rat small and large intestinal epithelium. Dig Dis Sci 49:122– 132. 25. Miyamoto M, Natsume H, Satoh I, Ohtake K, Yamaguchi M, Konayashi D, Sugibayashi K, Morimoto Y. 2001. Effect of poly-L-arginine on the nasal absorption of FITC-dextran of different molecular weights and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats. Int J Pharm 226:127–138. 26. Karczewski J, Groot J. 2000. Molecular physiology and pathophysiology of tight junctions III. Tight junction regulation by intracellular messengers: Differences in response within and between epithelia. Am J Physiol Gastrointest Physiol 279:G660– G665. 27. Ohtake K, Maeno T, Ueda H, Natsume H, Morimoto Y. 2003. Poly-L-arginine predominantly increases the paracellular permeability of hydrophilic macromolecules across rabbit nasal epithelium in vitro. Pharm Res 20:153–160. 28. Dorkoosh FA, Broekhuizen CAN, Borchard G, Rafiee-Tehrani M, Verhoff JC, Junginger HE. 2004. Transport of octreotide and evaluation of mechanism of opening the paracellular tight junctions using superporous hydrogel polymers in caco2 cell monolayers. J Pharm Sci 93:743–752.
DOI 10.1002/jps
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29. Salama NN, Fasano A, Thakar M, Eddington ND. 2004. The effect of DG on the transport and oral absorption of macromolecules. J Pharm Sci 93: 1310–1319. 30. Tomita M, Hayashi M, Awazu S. 1994. Comparison of absorption-enhancing effect between sodium caprate and disodium ethylenediaminetetraacetate in caco-2 cells. Biol Pharm Bull 17:753– 755. 31. Quan YS, Fujita T, Tohara D, Tsuji M, Kohyama M, Yamamoto A. 1999. Transport kinetics of leucine enkephalin across caco-2 monolayers: Quantitative analysis for contribution of enzymatic and transport barrier. Life Sci 64:1243–1252. 32. Hosoya O, Chono S, Saso Y, Juni K, Morimoto K, Seki T. 2004. Determination of diffusion coefficients of peptides and prediction of permeability through a porous membrane. J Pharm Pharmacol 56:1501–1507. 33. Renkin EM. 1954. Filtration, diffusion, and molecular sieving through porous cellulose membranes. J Gen Physiol 20:225–243. 34. Knipp GT, Ho NFH, Barsuhn CL, Borchardt RT. 1997. Paracellular diffusion in caco-2 cell monolayers: Effect of perturbation on the transport of hydrophilic compounds that vary in charge and size. J Pharm Sci 86:1105–1110. 35. Nakahara N, Tavelin S, Artursson P. 2004. Contribution of the paracellular route to pH-dependent epithelial permeability to cationic drugs. J Pharm Sci 93:2972–2984. 36. Adson A, Raub TJ, Burton PS, Barsuhn CL, Hilgers AR, Audus KL, Ho NFH. 1994. Quantitative approaches to delineate paracellular diffusion in cultured epithelial cell monolayers. J Pharm Sci 83:1529–1536. 37. Hirai S, Ikenaga T, Matsuzawa T. 1978. Nasal absorption of insulin in dogs. Diabetes 27:296–299.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 6, JUNE 2006
Hokkaido Pharmaceutical University School of Pharmacy, 7-1 Katsuraoka-cho, Otaru, Hokkaido 047-0264, Japan
2
Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
Received 16 August 2005; revised 25 January 2006; accepted 13 February 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20616
ABSTRACT: Cationized gelatins, candidate absorption enhancers, were prepared by addition of ethylenediamine or spermine to gelatin and the effects of the resulting ethylenediaminated gelatin (EG) and sperminated gelatin (SG) on the paracellular transport of 5(6)-carboxyfluorescein (CF), FITC-dextran-4 (FD4), and insulin through caco-2 cell monolayers were examined. The Renkin function was used for characterization of the paracellular pathway and changes in the pore radius (R) and pore occupancy/ length ratio ("/L) calculated from the apparent permeability coefficients (Papp) of CF and FD4 are discussed. Ethylenediaminetetraacetic acid (EDTA) increased the R of the caco-2 cell monolayer and the Papp of all compounds examined was markedly increased by the addition of EDTA. On the other hand, EG and SG did not increase R and their enhancing effects were not as strong as those of EDTA. The increase in "/L could be the enhancing mechanism for the cationized gelatins. The number of pathways for water-soluble drugs, such as CF and FD4, in the caco-2 monolayers could be increased by the addition of the cationized gelatins. The ratios of the permeability coefficients of insulin (observed/ calculated based on the Renkin function) suggest that insulin undergoes enzymatic degradation during transport which is not inhibited by enhancers. ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:1393–1401, 2006
Keywords: paracellular transport; cationized gelatin; absorption enhancer; Renkin function; insulin; caco-2 cells; permeability; tight junction
INTRODUCTION Oral, nasal, or pulmonary drug delivery systems for peptide and protein drugs are needed to avoid the problems associated with parenteral formulations, such as tissue invasion, and also to improve patient compliance.1 The use of absorption enhancers2 and proteolytic enzyme inhibitors3 and suitably designed formulations4 are possible approaches to the development of novel delivery systems to increase the bioavailability of Correspondence to: Toshinobu Seki (Telephone: þ81-13462-1882; Fax: þ81-134-62-1848; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 95, 1393–1401 (2006) ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association
peptide and protein drugs. The absorption enhancers, which increase the permeability of drugs through the epithelial membranes without causing any tissue damage, will be especially useful for the delivery of drugs with a relatively high molecular weight (MW).5 Although many chemicals, including surfactants, bile salts, and fatty acids, have been evaluated as absorption enhancers, most of them produce some form of membrane damage.6,7 In order to choose a safe enhancer, a better understanding of the mode of action and mechanism of such compounds is required. Most of the candidate enhancers reported are able to open the tight junctions of epithelial cell layers and increase the paracellular permeability of drugs by a mechanism involving
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suppressed transepithelial resistance (TER) and increased permeability of mannitol and/or FITCdextrans.2 However, this evidence is not enough for a proper understanding of the change in the paracellular pathways.8 It has recently been reported that cationic polymers, including chitosan9,10 and poly-L-arginine11,12, are able to improve the absorption of peptide and protein drugs while causing negligible damage to the mucosal membrane. In previous reports, we showed that cationized gelatins with different numbers of amino groups enhanced the nasal absorption of insulin and the enhancing effects depended on the amino group content.13,14 The effect of the cationized gelatins on the LDH leaking from the nasal mucosa was relatively weak and similar to that of chitosan.13 The cationic polymers could interact with the luminal surface of mucus membranes directly by an ion–ion interaction and then induce signals that would open tight junctions resulting in intercellular permeation.9,11,15 However, the mechanism(s) of opening tight junctions is not yet fully understood. The aim of this study is to evaluate the mode of action of cationized gelatins, a new generation of permeation enhancers, on tight junctions. In recent years, our knowledge of tight junction structure has advanced dramatically and its molecular structure involving occludin, claudins, and ZO proteins has been described.16–19 Studies of the structure of claudins suggest the existence of aqueous pores in the tight junctions20 and at least two different sized pores are assumed to be present.21,22 A pore pathway a few Angstroms in radius could act as an absorption route for small water-soluble compounds.8 On the other hand, since relatively higher MW compounds, such as FITC-dextrans, are also able to pass through the epithelial cell layers of the intestines and nasal cavities, a pore pathway a few nanometers in radius could also be present in the tight junctions.23–25 Those smaller and larger pores might be physiologically regulated in different ways and the contribution of protein kinase A (PKA) and protein kinase C (PKC) to each process has been discussed.8,26 While activation of PKA may increase the ionic conductance of tight junctions without changing the barrier function for large molecules, activation of PKC may increase the permeability of large molecules.26 This PKC pathway may be one of the possible mechanisms governing the penetration enhancers for large molecules to allow them to pass through epithelial cell layers.27 These findings suggest that TER is not a good parameter to JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 6, JUNE 2006
evaluate the effects of candidate enhancers for large molecules8 and the barrier function of tight junctions for large molecules should be described using other parameters. Figure 1 shows models of permeation pathways (arrows) of water-soluble drugs having a relatively high MW through epithelial cell layers. Figure 1A assumes normal conditions; a few paracellular pathways are ‘‘open’’ to allow the passage of large molecules, such as FITC-dextrans.23–25 If the enhancers increase the size of the pathway, larger molecules will be able to pass via these routes (Fig. 1B). If the enhancers transform normal tight junctions into the pathways, the number will increase and the permeability coefficient of each drug should be proportionate to the number (Fig. 1C). If the candidate enhancers produce undesirable changes in the epithelial cell layers, such as emergence from the cells (I) and complete loss of barrier function by the membranes (II), the permeability of all penetrants will be higher but they will not be safe to use as enhancers (Fig. 1D). It is helpful to know which change (B–D) is induced by a candidate enhancer in order to identify the most effective enhancers.
Figure 1. Models of permeation routes for watersoluble large molecules through epithelial cell layers and the effects of penetration enhancers on these pathways. A: Normal conditions; a few paracellular pathways are ‘‘open’’ to allow the passage of relatively large molecules, such as FD4. B: Increase in size of the pathway by enhancers; the pathways increase in size and larger molecules are able to pass via these routes. C: Increase in the number of pathways by enhancers; the pathway increase and the permeability coefficient of each drug should be proportional to the number. D: Damage to epithelial cell layers by enhancers; undesirable changes in epithelial cell layers, such as emergence of cells (I) and complete loss of barrier functions of membranes (II) increase the permeability of all penetrants. DOI 10.1002/jps
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In this study, cationized gelatins, candidate enhancers, were prepared by the addition of ethylenediamine or spermine to gelatin and the effects of the resulting ethylenediaminated gelatin (EG)13,14 and sperminated gelatin (SG) on the paracellular transport of 5(6)-carboxyfluorescein (CF), FITC-dextran-4 (FD4), and insulin through caco-2 cell monolayers28,29 were examined. Ethylenediaminetetraacetic acid (EDTA), which exhibits an absorption-enhancing effect by calcium chelation and causes marked damage to epithelia, was used as a comparison.30,31 The Renkin molecular sieving function was used for characterization of the paracellular pathway and changes in the equivalent cylindrical pore radius (R) and pore occupancy/length ratio ("/L) calculated from the apparent permeability coefficients (Papp) of CF and FD4 are discussed. In addition, the permeability coefficients of insulin were calculated using the Renkin function and the values obtained were compared with the observed values to evaluate the enzymatic degradation of insulin during the transport process.
MATERIALS AND METHODS Materials Gelatin (isoelectric point ¼ 9.0, MW 100 kDa) was kindly supplied by Nitta Gelatin Co. Ltd (Osaka, Japan). Spermine tetrahydrochloride, 1,2-ethylenediamine and EDTA (tetrasodium salt, EDTA 4Na) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Recombinant human insulin (28.7 IU/mg), FD4 (MW 4400), and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride were purchased from Sigma Chemical Co. (St Louis, MA). CF and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were purchased from Acros Organics (Morris Plains, NJ) and Nacalai Tesque (Kyoto, Japan), respectively. All other chemicals were of reagent grade and used as received. Diffusion coefficients of CF, FD4, and insulin were determined in our laboratory and the values have been reported previously (Table 1).32
Synthesis of Cationized Gelatins Gelatin was reacted with 1,2-ethylenediamine or spermine to obtain cationized gelatins in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride by a method previously reported.13,14 In brief, gelatin (10 g) DOI 10.1002/jps
Table 1. Diffusion Coefficient (Di) and Stokes– Einstein Radius (ri) of Drugs Used at 378C
CF FD4 Insulin a
MW
Dia (cm2/s) 106
ri (nm)
376.3 4400 5807.6
5.87 2.39 1.14
0.556 1.37 2.86
Reference 32.
was dissolved in 0.1 M phosphate buffer (pH 5.0, 250 mL). 1,2-Ethylenediamine (28.0 g, 0.466 mole) or spermine tetrahydrochloride (32.4 g, 0.093 mole) was added to the solution and then the pH of the solution was adjusted to 5.0 with hydrochloric acid. The resulting solution was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (5.35 g) and the total volume was adjusted to 500 mL by addition of phosphate buffer (pH 5.0). The reaction was allowed to take place at 378C for 18 h. The resulting aminated gelatins were purified by dialysis for 48 h then the aminated gelatin powder was obtained by lyophilization. Determination of Amino Group Content of Cationized Gelatins One milliliter of gelatin or cationized gelatin solution (0.50 mg/mL) in phosphate buffered saline (PBS, pH 7.4) was mixed with 1.0 mL sodium bicarbonate solution (4.0%) and 1.0 mL TNBS solution (0.10%). The mixture was kept at 408C for 2 h protected from light and then the absorbance of the solution at 415 nm was determined.13,14 A calibration curve was prepared for b-alanine. The primary amino group content was expressed as the amount of TNBS-reactive amino groups in 1 g gelatin or cationized gelatin. Since the addition of spermine introduced not only primary amino groups but also secondary groups to gelatin, the total amino group content (TA) of SG was calculated using the following Equation (1): TA ¼ ðPASG PAnative Þ 3 þ PAnative
ð1Þ
where PASG and PAnative are the primary amino group contents of SG and native-gelatin, respectively. Cell Culture Caco-2 cells were obtained from the Riken Gene Bank (Ibaragi, Japan). The cells were maintained in DMEM containing 10% heat-inactivated fetal JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 6, JUNE 2006
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Drug Transport Experiments
where (DMi/Dt) is the rate of the penetrant i appearing on the receiver side, A is area of the monolayers and C0,i is the initial concentration of i. The Renkin function (3) was used for characterization of the caco-2 cell monolayers33–35: r r 2 h r i i i F 1 2:104 ¼ 1 R R R r 3 r 5 i i i 0:95 ð3Þ þ 2:09 R R
Simultaneous transport of FD4, CF, and insulin through the caco-2 cell monolayers was observed at 378C. CF (30 mg/mL), FD4 (5.0%), insulin (100 IU/mL), and enhancers (0 or 0.20%) were dissolved in PBS (pH 7.4) and the solution (100 mL) was applied to the apical side. Hanks balanced salt solution (HBSS, pH 7.4, 0.60 mL) was used as the basolateral side solution. The HBSS was changed every 30 min for 2 h. TER was measured using Millicell1-ERS (Millipore, MA) before and after the transport experiments. HBSS was used as the apical and basolateral solutions.
where ri is the molecular radius of penetrant i which can be calculated from the diffusion coefficient (Di) as the Stokes–Einstein radius. Since CF and FD4 were not metabolized during the permeation process, the Papp of CF and FD4 (PappCF and PappFD4 ) were used for the calculation. R and "/L were obtained as the characteristic parameters for each monolayer using the following Equations (4) and (5) and diffusion parameters in Table 1.32,36 r e CF PappCF ¼ DCF F ð4Þ L R
calf serum, 40 mg/mL gentamicin and 1% nonessential amino acids, in a humidified atmosphere of 95% air and 5% CO2 at 378C. Cells from passage number 46 to 53 were seeded (4.5 105 cell /cm2) on polycarbonate filter inserts (pore size 0.4 mm, area 0.33 cm2, Transwell, Costar) and cultivated in the medium for 21–25 days before starting the drug transport experiments.
PappFD4 ¼
Analytical Methods FD4 and CF in the medium were isolated and determined by a gradient HPLC system (Shimadzu, Kyoto, Japan) consisting of a pump (LC-10AT), oven (CTO-6A), detector (RF-10A), and integrator (CR-5A).32 Separation was carried out using an analytical column (Shodex Asahipak NH2P-50 4E, 250 4.6 mm i.d.; Showa Denko, Kawasaki, Japan) and a gradient of 0.1 M triethanolamine þ 10% acetonitrile 0.1 M triethanolamine þ 10% acetonitrile þ 60 mM NaCl changing from 30:70 to 80:20 over 30 min. The flow-rate of the eluent was 1 mL/min, the oven was operated at 458C and the detector was operated at an excitation wavelength of 495 nm and an emission wavelength of 515 nm. The insulin concentration in the medium was determined using an enzyme immunoassay kit (YKO 60 Human Insulin EIA kit, Yanaihara Institute, Shizuoka, Japan) according to the manufacturer’s instructions.
Characterization of Caco-2 Cell Monolayers The Papp of penetrant i (Pappi ) was calculated using the following Equation (2): Pappi ¼
DMi =Dt A C0;i
ð2Þ
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r " FD4 DFD4 F L R
ð5Þ
Statistical Analysis The mean values and their standard deviation (SD) were calculated in each experiment. The statistical significance of each treatment compared with the control group was evaluated by the Dunnett test. The Tukey–Kramer test was also used for the comparison of TER and StatView software (Ver.5.0, SAS Institute Inc., Raleigh, NC) was used for the calculations.
RESULTS Preparation of Cationized Gelatins Two different cationized gelatins, EG and SG, were prepared from gelatin and ethylenediamine or spermine. The amino group contents of the cationized gelatins were determined to monitor the increasing charge densities. Table 2 shows the primary amino group contents of the cationized gelatins and the calculated total amino group content of SG. The primary amino group content is the number of TNBS-reactive primary amino groups and is expressed as mmol/g gelatin. The values of EG and SG were higher than that of DOI 10.1002/jps
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Table 2. Amino Group Content of Cationized Gelatins Used
Native-gelatin EG SG
Primary Amino Group Contenta (NH2) (mmol/g gelatin) (SD)
Total Amino Group Contentb (NH2, ¼NH) (mmol/g gelatin)
0.255 (0.003) 0.823 (0.038) 0.660 (0.034)
— — 1.47
Mean values of three determinations are shown. a Amount of TNBS-reactive amino groups in 1 g gelatin or cationized gelatin. b Calculated amount of primary and secondary amino groups in 1 g SG (see text).
native gelatin, suggesting the addition of ethylenediamine or spermine to the gelatin. Since the addition of one spermine molecule transferred three amino groups to the gelatin, the calculated total amino group content of SG was higher than the amino group content of EG.
Effect of Cationized Gelatins and EDTA on the TER of Caco-2 Cell Monolayers The effects of cationized gelatins on the TER of the caco-2 cell monolayers were examined. The percentage TER of the caco-2 monolayers after the transport experiments compared with the initial values was reduced by the addition of cationized gelatins and EDTA (Fig. 2). This reduction represents an increase in the permeability of small ions through the monolayers. The tight junctions of the monolayer could be opened as a result. There was no difference in the effects of cationized gelatins and EDTA on the TER. Effect of Cationized Gelatins and EDTA on the Papp of CF, FD4, and Insulin Through Caco-2 Cell Monolayers CF, FD4, and insulin were simultaneously applied to the apical side of the caco-2 monolayers to determine their Papp in each monolayer. The penetration-enhancing effects of cationized gelatins and EDTA were evaluated in terms of the change in Papp for each penetrant. EG, SG, and EDTA showed significant enhancing effects for CF and FD4 (Fig. 3). The highest effect was obtained for EDTA: the enhancing ratios (ER; Papp with enhancer/Papp without enhancer) were 12.7 and 18.1 for CF and FD4, respectively. On DOI 10.1002/jps
Figure 2. Effects of penetration enhancers on the change in transepithelial resistance (TER) of caco-2 cell monolayers. The values of TER after transport experiments for 2 h are shown as a percentage of the initial values. Each enhancer was applied as a 0.2% solution. Each data set is the mean SD (n ¼ 3–5). **Significant difference compared with the control (p < 0.01) in the Dunnett test. There was no significant difference between EG, SG, and EDTA in the Tukey–Kramer test.
the other hand, the enhancing effects of EG and SG were not as potent although the effects on the TER were similar to that of EDTA. Similar enhancing effects were observed for insulin (observed P in Table 3). The enhancing effects of EDTA were more marked for higher MW penetrants (ER ¼ 12.7, 18.2, and 41.1 for CF, FD4, and insulin, respectively). On the other hand, the ER values of EG and SG were similar for CF, FD4, and insulin (ER of EG ¼ 2.6, 2.8, and 2.3 for CF, FD4, and insulin, ER of SG ¼ 4.2, 4.4, and 5.6 for CF, FD4, and insulin, respectively). Effect of Cationized Gelatins and EDTA on the Membrane Parameters of Caco-2 Cell Monolayers The membrane parameters of each caco-2 cell monolayer were calculated from PappCF , PappFD4 and previously reported diffusion parameters of CF and FD4 (Table 1) based on the Renkin function. The effects of cationized gelatins and EDTA on R and "/L of the monolayers are shown in Figure 4. In the case of EDTA, both R and "/L increased significantly. The increase in R could be due to removal of calcium ions from the intercellular space by chelation. On the other hand, cationized gelatins did not increase R of the monolayer. Since the increased ratios of "/L are 2.5 and 4.1 for EG and SG, respectively, the enhancing effects of cationized gelatins could be mainly due to the increased "/L. This result suggests that the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 6, JUNE 2006
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cationized gelatins transform normal tight junctions into the penetration pathways for CF and FD4 to increase the number in the monolayers as shown in Figure 1C. Although the effects of EDTA could be as shown in Figure 1B, since R is a mean value, it might include undesirable effects like those shown in Figure 1D. Comparison of Calculated Permeability Coefficients of Insulin With Observed Values
Figure 3. Effects of penetration enhancers on the permeability coefficients of CF (A) and FD4 (B) through caco-2 cell monolayers. Each enhancer was applied as a 0.2% solution. Each data set is the mean SD (n ¼ 3–5). *Significant difference compared with the control (p < 0.05) in the Dunnett test. **Significant difference compared with the control (p < 0.01) in the Dunnett test.
The permeability of insulin through the caco-2 cell monolayers could be calculated from the membrane parameters and the diffusion parameters of insulin based on the Renkin function. In our previous report, the observed permeability coefficient of insulin through a cellulose membrane was in agreement with that calculated from the membrane parameters and the diffusion parameters of insulin using the Renkin function.32 In the case of biological membranes, such as the caco-2 monolayers, we did not expect this agreement, because insulin would undergo enzymatic degradation during the transport process. Table 3 shows the observed and calculated permeability coefficients of insulin through the caco-2 cell monolayers. The lower observed values could be related to the degradation of insulin and the interaction of insulin with biological components. The permeability ratios (observed/calculated) were about 0.2 and the values did not change following the addition of the cationic gelatins and EDTA. This result suggests that the enhancers did not inhibit the degradation of insulin and/or the interactions reducing the permeability of insulin.
Table 3. Observed and Calculated Permeability Coefficient of Insulin Through Caco-2 Cell Monolayers
Control þEGb þSGb þEDTA4Nab
Observed P (Papp) 109 cm/s (SD)
Calculated Pa 109 cm/s (SD)
0.70 (0.36) 1.64 (0.59) 3.91c (0.97) 28.8d (3.2)
3.89 (1.59) 15.5 (10.7) 20.6 (7.2) 179d (91)
Ratio of P (observed/calculated) (SD) 0.19 0.16 0.20 0.19
(0.09) (0.15) (0.05) (0.11)
Mean values are shown (n ¼ 3–5). a R and e/L obtained from permeability coefficients of simultaneously applied CF and FD4 in each permeation experiment were used for the calculation. b 0.2% solution was used. c Significant difference compared with the control (p < 0.05) in the Dunnett test. d Significant difference compared with the control (p < 0.01) in the Dunnett test. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 6, JUNE 2006
DOI 10.1002/jps
EFFECT OF CATIONIZED GELATINS
Figure 4. Effects of enhancers on the values of R (A) and e/L (B) of caco-2 cell monolayers. Each enhancer was applied as a 0.2% solution. Each data set is the mean SD (n ¼ 3–5). *Significant difference compared with the control (p < 0.05) in the Dunnett test. **Significant difference compared with the control (p < 0.01) in the Dunnett test.
DISCUSSION In this study, the mode of action of cationized gelatins, which should be related to their efficiency and toxicity, was examined in caco-2 cell monolayers and was compared with that of EDTA which is known to cause marked epithelial damage. Two different cationized gelatins, EG and SG, were used as cationic polymers to evaluate the penetration-enhancing effects on the transport of CF, FD4, and insulin through caco-2 cell monolayers. The Renkin function was used to characterize the monolayers and the effects of the cationized gelatins. Simultaneous application of multicomponents allowed the characteristics of each monolayer to be determined. DOI 10.1002/jps
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The absorption-enhancing effects of cationic polymers depend on their chemical composition, MW and charge density. Natsume et al.11,12 examined the effect of MW and charge density on the enhancing effect of cationic polymers and found that the charge density was the most important parameter for the enhancement of the nasal absorption of FD4. The cationic polymers could interact with the luminal surface of mucus membranes directly by an ion–ion interaction and then induce signals that would open tight junctions resulting in intercellular permeation.9,11,15 In a previous report, we examined the enhancing effects of EGs with different amino group contents on the nasal absorption of insulin.14 The effects of EGs on insulin absorption were correlated with those on CF absorption and depended on the amino group content, suggesting a mechanism similar to that for other cationic polymers.27 In this study, SG with spermine as a moiety was compared with an EG. Since SG showed increased effects on the permeation of all penetrants compared with EG, which had a higher primary amino group content but a lower total amino group content than SG, the secondary amino group in SG may also contribute to the enhancing effect. The effects of EDTA were compared with those of EG and SG. The rank order of the penetration enhancement was EDTA > SG > EG for all penetrants, but there was no significant difference in the change in TER, suggesting that TER is not a useful parameter for such investigations. The R and "/L of each caco-2 cell monolayer were calculated from the corresponding PappCF and PappFD4 , and the diffusion parameters of CF and FD4 (Table 1) based on the Renkin function. The R values were increased by addition of EDTA. A similar increase in the R of caco-2 cell monolayers has been reported following the addition of EGTA.8,24 Calcium chelation could widen the cell junctions30 and such widening might be an advantage for the delivery of larger molecules.12 On the other hand, the cationized gelatins could increase the number of pathways rather than their size. The action of the cationized gelatins might have an advantage in terms of reversibility. Since absorption of pancreatic proteolytic enzymes, for example, trypsin and chymotrypsin having an MW of 23000–26000, could cause injury to the underlying tissue, excessive widening of the junctions is not desirable. Although the action of cationized gelatins is not effective for large molecules, it is good as far as increased safety is concerned. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 6, JUNE 2006
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In the case of insulin absorption, inhibition of proteolysis could be one of the mechanisms of absorption enhancement.37 In order to evaluate the contribution of inhibition to the enhancing effects of the cationized gelatins, the calculated permeability of insulin through the caco-2 cell monolayers was compared with the Papp of insulin.32 The ratios of the values (observed/calculated) were not affected by the addition of the cationized gelatins, suggesting that the cationized gelatins do not inhibit the degradation of insulin. The use of protease inhibitors together with cationized gelatins could be a way of obtaining a higher bioavailability of insulin after mucosal application.31 In summary, the cationized gelatins exhibited enhancing effects on the permeation of CF, FD4, and insulin through caco-2 cell monolayers. They were able to increase the number rather than the size of the pathways for water-soluble large molecules in the caco-2 cell monolayers. The cationized gelatins are good candidates as safe absorption enhancers which will only mildly modify the permeability of the paracellular pathway, retaining the sieving property of the epithelial membranes.
9.
10.
11.
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14.
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