Improvement of the Solubility and Intestinal Absorption of Curcumin by N-Acyl Taurates and Elucidation of the Absorption-Enhancing Mechanisms

Abstract

In this study, the effects of N-acyl taurates (NATs) on the intestinal absorption of curcumin (CUR), a water-insoluble and poorly absorbed compound, were examined in rats. Sodium methyl lauroyl taurate (LMT) and sodium methyl cocoyl taurate (CMT) were the most effective in increasing the solubility and intestinal absorption of CUR. The intestinal membrane toxicity of the NATs was also evaluated by measuring the activity of lactate dehydrogenase (LDH), a toxicity marker. NATs did not increase the activity of LDH, suggesting that they may be safely administered orally. We further elucidated the absorption-enhancing mechanisms of NATs by using Caco-2 cells. In cellular transport studies, LMT and CMT reduced the transepithelial electrical resistance value of Caco-2 cells and increased the transport of 5(6)-carboxyfluorescein and CUR. Hence, the intestinal absorption enhancement by LMT and CMT was attributed to the synergistic effect of higher solubility and greater permeability of the cell layer towards CUR in the presence of the surfactants. In summary, co-administration of CUR with either LMT or CMT is a simple and effective method to enhance oral delivery of CUR.

Curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione; CUR) is a principal constituent of rhizomes of Curcuma longa (turmeric) and is well known for its versatile biological activities and pharmacological activities involving numerous molecular targets.^1–3) However, its medical benefits are restricted by low aqueous solubility, poor intestinal absorption, and chemical instability in the physiological environment, resulting in low bioavailability.⁴⁾ Many approaches have been developed to overcome these intrinsic barriers to bioavailability, including co-administration of CUR with inhibitors of glucuronidation, nano-formulations with lipids and polymers, amorphous solid dispersions, and prodrugs by conjugation.^5,6) These CUR formulations were administered orally and/or parenterally in both animal and human studies. Oral delivery is the preferred delivery route, given optimal patient compliance, for the therapy of chronic diseases like diabetes, arthritis, cancer, and human immunodeficiency virus.

Although the pharmacological effects of CUR have been demonstrated in many studies, there are still considerable concerns regarding the use of CUR e.g., organic solvent residues and the complex components of certain formulations would entail considerable risk with respect to quality control during commercial manufacture. In our opinion, therefore, a simple formulation with a multifunctional additive is a promising strategy for the pharmaceutical application of CUR.

It is known that the intestinal absorption of poorly absorbed drugs including insulin, calcitonin, and alendronate is significantly enhanced in the presence of various enhancers including surfactants, bile salts, chelating agents, fatty acids, nitric oxide donors, polyamines, chitosan oligomers, polyamidoamine (PAMAM) dendrimers, gemini surfactant, sucrose fatty acid esters, N-acyl amino acids, and N-acyl taurates.^7–16) Among these various chemical classes, acylated amino acids are surfactants with natural lipid-like structures and exhibit amphiphilic properties with high surface activity, as they are composed of a fatty acid and an amino acid, which are lipophilic and hydrophilic moieties respectively.¹⁷⁾ Because of their natural building blocks, these amino acid surfactants are regarded as “green” surfactants and are widely used in pharmaceutical, cosmetic, and food industries to replace petroleum-sourced surfactants. Moreover, acylated amino acids were found to exhibit biological activities that are potentially useful for the treatment of certain nervous, cardiovascular, and immune diseases.¹⁸⁾

N-Acyl taurines are a subset of acylated amino acids, some of which have been identified in the brain, liver, kidney, and skin.^19,20) They react with calcium channels, acting as signaling molecules in mammals. Derivatives of N-acyl taurines, N-acyl taurates (NATs), possess excellent properties of detergency and stability over the whole pH range in aqueous solution.^21–23) Owing to the variety of available fatty acids, the functionality of NATs may be modified to suit different purposes. Recently, we discovered a novel application of NATs as absorption enhancers, demonstrating improvement of the intestinal absorption of alendronate, a water-soluble and poorly absorbed drug.¹³⁾ Despite this former study, the absorption-enhancing effects of NATs for improving the intestinal absorption of other poorly absorbed drugs are not well understood.

In this study, we, therefore, selected five different NATs with medium chain fatty acids and studied their effects on the solubility and intestinal absorption of CUR. Subsequently, the intestinal toxicity of the NATs was assessed, based on the activity of lactate dehydrogenase (LDH) released from the intestinal membrane. Finally, to elucidate the absorption-enhancing mechanism of NATs, the transport of 5(6)-carboxyfluorescein (CF) and CUR, and transepithelial electrical resistance (TEER) values in the presence of NATs were studied in vitro using Caco-2 cell monolayers.

MATERIALS AND METHODS

Materials

NATs were supplied by Nikko Chemical Co., Ltd. (Osaka, Japan). Curcumin (CUR), LDH-cytotoxic test wako, and albumin (from bovine serum, Cohn fraction V, pH 7.0) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). CF was supplied by the Eastman Kodak Company (Rochester, NY, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and modified Eagle’s medium (MEM) non-essential amino acid solution were purchased from Life Technologies Corporation (Carlsbad, CA, U.S.A.). Zero point twenty-five percent Trypsin–1 M ethylenediaminetetraacetic acid (EDTA) and antibiotic–antimycotic mixed stock solution (10000 U/mL penicillin, 10 mg/mL streptomycin, 25 mg/mL amphotericin B, 0.85% (w/v) saline) were prepared by Dojindo Laboratories (Kumamoto, Japan). Hank’s balanced salt (HBS; H6136-10X1L) was provided by Sigma-Aldrich Chemical Co., Ltd. (St. Louis, MO, U.S.A.). Caco-2 cells were bought from Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan). All other reagents used in the experiments were of analytical grade.

Preparation of CUR Suspensions

Sodium methyl lauroyl taurate (LMT, 1% (v/v)), sodium methyl cocoyl taurate (CMT, 1% (v/v)), sodium methyl myristoyl taurate (MMT, 1% (w/v)), sodium methyl palmitoyl taurate (PMT, 1% (w/v)), and sodium methyl stearoyl taurate (SMT, 1% (w/v)) solutions, were dissolved separately in phosphate-buffered saline solution (PBS; pH 6.5) containing 1% methyl cellulose (Nacalai Tesque Co., Ltd., Kyoto, Japan). The chemical structures of NATs used in the present study are shown in Fig. 1. According to the dose in the intestinal absorption, CUR was dispersed in each NAT solution at a concentration of 16.7 mg/mL, followed by agitation (Vortex-Genie 2; Scientific Industries, Inc., Bohemia, NY, U.S.A.) for 5 min at 25°C.

Fig. 1. Chemical Structure of the N-Acyl Taurates

Intestinal Absorption of CUR

Intestinal absorption in male Wistar rats was studied by using an in situ closed loop method.¹⁵⁾ The experiments were carried out in accordance with the guidelines of the Animal Ethics Committee at Kyoto Pharmaceutical University. Animals were fasted overnight for 16 h before the experiment but had free access to water. Following intraperitoneal injection of sodium pentobarbital at a dose of 32 mg/kg body weight, the anesthetized rats were placed under a heating lamp to maintain body temperature. The intestine was exposed through a midline abdominal incision and was rinsed with PBS (pH 6.5). After ligating the bile duct, the intestine was cannulated at both proximal and distal parts with polyethylene cannulas clipped by forceps. Three milliliters of CUR suspension, with or without NATs, warmed to 37°C, was injected into the intestinal loop at a dose of 200 mg/kg body weight,^24,25) through the cannula at the proximal part, which was then closed during the absorption study. The jugular vein was exposed, and 0.4 mL of blood sample was collected using heparinized syringes at predetermined time points up to 240 min. The blood samples were centrifuged immediately at 12000 rpm (Centrifuge 5417R; Eppendorf AG, Hamburg, Germany) for 5 min to obtain 150 µL of plasma, which was stored at −30°C until assay. CUR in the plasma was assayed by using HPLC.⁵⁾ HPLC was performed with a Shimadzu System (LC-20; Shimadzu Corporation, Kyoto, Japan) and a C18 reverse phase column (150 mm×4.6 mm, 5C18-AR-II, COSMOSIL, Nacalai Tesque Co., Ltd.) at 35°C. A mixture of 5% acetic acid and methanol (34/66, v/v) was used as the mobile phase. The flow rate was 1 mL/min and the detection wavelength was set at 420 nm.

The CUR peak plasma concentration (C_max) and the time to the peak concentration (T_max) were determined directly from the plasma concentration–time curves. The area under the curve (AUC) was calculated by the trapezoidal method from 0 to 240 min. The absorption enhancement ratio (ERa) of CUR, with or without NATs, was calculated as follows:   

The Intestinal Membrane Toxicity of NATs

The activity of LDH released from intestinal epithelial cells was examined in order to determine any intestinal toxicity of the NATs.^8,13) PBS (pH 6.5), 1% NATs, and 3% (v/v) Triton X-100 used as a positive control were administered into the intestinal loops of experimental animals in the same manner as that used for the in situ intestinal absorption experiments. At the end of 4 h, the intestine was washed with 30 mL of PBS (pH 7.4) and the washing solution was collected to determine the activity of LDH using the LDH-cytotoxicity test wako kit. The measurement was performed with a microplate multi-detection reader (Synergy HT with Gen5 Software; BioTek Instruments, Inc., Winooski, VT, U.S.A.).

Solubility of CUR with or without NATs

CUR was added to 1% NAT in PBS (pH 6.5) and agitated at 25°C for 5 min. The mixture was centrifuged at 12000 rpm for 5 min. The supernatant was assayed by HPLC. The solubility enhancement ratio (ERs) of CUR was calculated as follows:   

Cell Culture

Caco-2 cells were cultured in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) antibiotic–antimycotic mixed stock solution, and 100 mM MEM non-essential amino acid solution at 37°C in 5% CO₂ as reported-previously.^8,13,26) The passages used for cellular transport study were 57–65. Prior to the experiments, cells were seeded on polycarbonate inserts (Transwells, 12 mm in diameter, 0.4 µm pores, 1.12 cm²; Corning Incorporated, NY, U.S.A.) at an initial density of 1×10⁵ cells/well and were grown for 21 d. The medium was replaced every 2 d. The TEER values of cell layers were measured with a Millicell-ERS voltohmmeter (EMD Millipore Corporation, MA, U.S.A.). The cell monolayers with a TEER of >500 Ω·cm² were used for transport experiments in the present study.

Transport of Test Compounds by Caco-2 Cell Monolayers

Transport of CF and CUR across Caco-2 cells was studied from the apical to basolateral direction. Caco-2 cell monolayers were washed twice and incubated in HBS solution (HBSS) buffer at 37°C for 1 h. Transport experiments were initiated by replacing HBSS buffer with 0.5 mL of donor solution in the apical compartment. At predetermined time points up to 360 min, TEER was measured, followed by sampling 200 µL from 1.5 mL of the receiving solution in the basal compartment and adding an equivalent fresh medium.

CF (10 µM), with or without NAT, in HBSS (pH 6.5) was prepared as the donor solution. Transport from the apical compartment to the basolateral compartment (HBSS, pH 7.4) was determined to measure the fluorescence intensity with an excitation wavelength of 485 nm and an emission wavelength of 535 nm by a microplate multi-detection reader (Synergy HT with Gen5 Software; BioTek Instruments, Inc., Winooski, VT, U.S.A.).

CUR (2 mM), with or without NAT, in HBSS (pH 6.5) was prepared as the donor suspension, and HBSS (pH 7.4) containing 5% albumin was used as the basolateral medium. Transport from the apical compartment to the basolateral compartment was measured by HPLC (LC-20; Shimadzu Corporation, Kyoto, Japan) with a fluorescence detector (RF-10AXL; Shimadzu Corporation, Kyoto, Japan) at an excitation wavelength of 420 nm and an emission wavelength of 530 nm.²⁷⁾

The apparent permeability coefficient (P_app) in the Caco-2 cell model was calculated as follows:   

where P_app is the apparent parameter of permeability (cm/s), dQ/dt is the rate of the test compound appearance in the receiver side (pmol/min), A is the membrane surface area (1.12 cm²), and C₀ is the initial concentration or solubility of the test compound in donor solution or suspension (nM).

Statistical Analyses

Results are expressed as the mean±standard error (S.E.) of at least three experiments. Statistical significance determinations between experimental groups were performed using Dunnett’s test; p<0.05 was considered significant. Significance levels are denoted as (n.s.) not significantly different, * p<0.05, ** p<0.01, and *** p<0.001.

RESULTS

Effect of NATs on the Intestinal Absorption of CUR

The effect of NATs on the intestinal absorption of CUR was assessed by using an in situ closed loop method. As shown in Fig. 2 and Table 1, the intestinal absorption of CUR was markedly enhanced in the presence of some of the different NATs tested at an initial concentration in each case of 1% (v/v or w/v). The rank order of the enhancing effects was LMT≧MMT≧CMT>SMT>PMT. LMT, MMT, and CMT produced enhancement ratios of 47, 43, and 42, respectively. In the presence of SMT, the absorption of CUR was improved 21-fold, while there was no discernible change in intestinal absorption in PMT formulation.

Fig. 2. Absorption of Curcumin (200 mg/kg) from the Small Intestines of Rats in the Presence of 1% N-Acyl Taurates

CUR, curcumin; LMT, sodium methyl lauroyl taurate; CMT, sodium methyl cocoyl taurate; MMT, sodium methyl myristoyl taurate; PMT, sodium methyl palmitoyl taurate; SMT, sodium methyl stearoyl taurate. Results are expressed as the mean±S.E. of 3–4 experiments. Keys: (○) CUR, (▲) +1% (v/v) LMT, (■) +1% (v/v) CMT, (◆) +1% (w/v) MMT, (□) +1% (w/v) PMT, (△) +1% (w/v) SMT.

Table 1. Pharmacokinetic Parameters of Curcumin in the Presence of 1% N-Acyl Taurates after Intestinal Administration to Rats

Group	Cmax (µg/mL)	Tmax (min)	AUC0→240 min (µg/mL·min)	ERa
CUR	0.004±0.004	30±0	0.090±0.090	1
+1% (v/v) CMT	0.027±0.005	30±0	3.8±0.68*	42
+1% (v/v) LMT	0.016±0.007	27±12	4.2±0.34**	47
+1% (w/v) MMT	0.037±0.007	60±17	3.9±1.1*	43
+1% (w/v) PMT	0.003±0.001	60±0	0.10±0.020	1
+1% (w/v) SMT	0.020±0.001	150±24	1.9±1.6	21

CUR, curcumin; LMT, sodium methyl lauroyl taurate; CMT, sodium methyl cocoyl taurate; MMT, sodium methyl myristoyl taurate; PMT, sodium methyl palmitoyl taurate; SMT, sodium methyl stearoyl taurate. Results are expressed as the mean±S.E. of 3–4 experiments. ** p<0.01, * p<0.05, compared with CUR (control).

Evaluation of Intestinal Membrane Damage Caused by NATs

Intestinal membrane damage was evaluated by measuring the activity of LDH released from the intestinal tissue. Figure 3 shows that none of the tested NATs increased the observed activity of LDH. However, the activity of LDH in the presence of 3% Triton X-100 was significantly higher than that of the control. NATs did not, therefore, cause serious membrane damage to the intestinal mucosa.

Fig. 3. Assay of Lactate Dehydrogenase Activity in the Intestinal Wash Solution after 240 min Administration of N-Acyl Taurates

LMT, sodium methyl lauroyl taurate; CMT, sodium methyl cocoyl taurate; MMT, sodium methyl myristoyl taurate; PMT, sodium methyl palmitoyl taurate; SMT, sodium methyl stearoyl taurate. Results are expressed as the mean±S.E. of 3–4 experiments. N.S. means no significant difference compared with PBS (control). * p<0.05, compared to PBS.

Solubility of CUR in the Presence of NATs

The solubility of CUR in NAT formulations was determined to compare their relative solubilizing potencies. As listed in Table 2, all NATs (1% v/v or w/v) increased the solubility of CUR and the rank order of solubilizing ability was CMT>LMT>MMT>SMT>PMT. Following dispersal in 1% (v/v) LMT and CMT, the solubility of CUR was measured as 29 and 35 µg/mL, respectively, more than 4000-fold greater than the control.

Table 2. Solubility of Curcumin in the Presence of 1% N-Acyl Taurates

Group	Solubility (µg/mL)	ERs
CUR	0.007±0.001	1
+1% (v/v) CMT	35±0.34***	5000
+1% (v/v) LMT	29±0.69***	4143
+1% (w/v) MMT	5.7±0.88***	814
+1% (w/v) PMT	0.63±0.14	90
+1% (w/v) SMT	0.73±0.10	104

CUR, curcumin; LMT, sodium methyl lauroyl taurate; CMT, sodium methyl cocoyl taurate; MMT, sodium methyl myristoyl taurate; PMT, sodium methyl palmitoyl taurate; SMT, sodium methyl stearoyl taurate. Results are expressed as the mean±S.E. of at least three experiments. *** p<0.001, compared with CUR (control).

Effect of LMT and CMT on the TEER of Caco-2 Cell Monolayers

TEER values of Caco-2 cell monolayers, an index of the integrity of the tight junctions, were monitored during transport studies. As demonstrated in Fig. 4, the reduction of TEER was dependent on the concentration of either LMT or CMT in the range of 0.003–0.1% (v/v), and TEER values decreased substantially when Caco-2 were exposed to high concentration of NATs. In the comparison of TEER between two NAT groups at 0.01%, it is clear to observe that CMT had a stronger efficacy on the cell layers than LMT.

Fig. 4. Effect of Sodium Methyl Lauroyl Taurate and Sodium Methyl Cocoyl Taurate on TEER in Caco-2 Cell Monolayers

CF, 5(6)-carboxyfluorescein; LMT, sodium methyl lauroyl taurate; CMT, sodium methyl cocoyl taurate. Results are expressed as the mean±S.E. of three experiments. Some error bars of S.E. are within the size of symbols. Keys: (○) CF, (▲) +0.003% (v/v) LMT, (■) +0.01% (v/v) LMT, (◆) +0.1% (v/v) LMT, (△) +0.003% (v/v) CMT, (□) +0.01% (v/v) CMT, (◇) +0.1% (v/v) CMT.

Effect of LMT and CMT on the Cellular Transport of CF across Caco-2 Cell Monolayers

In order to elucidate the absorption-enhancing mechanisms of LMT and CMT in the intestine, CF was used as a marker compound to investigate the potential transport of drugs. In light of the permeation profiles in Fig. 5A, both LMT and CMT at 0.1% increased the permeation of CF considerably. The P_app values of CF were significantly increased to 11.6±0.2 (×10⁻⁶) and 12.6±0.7 (×10⁻⁶) cm/s by the addition of LMT and CMT at high concentration, respectively (Fig. 5B). In addition, P_app of CF in 0.01% CMT was 2.9-fold higher than that in the control, while this index in other groups was similar with the control.

Fig. 5. Effect of Sodium Methyl Lauroyl Taurate and Sodium Methyl Cocoyl Taurate on Cellular Transport of 5(6)-Carboxyfluorescein in Caco-2 Cell Monolayers

CF, 5(6)-carboxyfluorescein; LMT, sodium methyl lauroyl taurate; CMT, sodium methyl cocoyl taurate. Results are expressed as the mean±S.E. of three experiments. Some error bars of S.E. are within the size of symbols. *** p<0.01, compared with CF (control). Keys: (○) CF, (▲) +0.003% (v/v) LMT, (■) +0.01% (v/v) LMT, (◆) +0.1% (v/v) LMT, (△) +0.003% (v/v) CMT, (□) +0.01% (v/v) CMT, (◇) +0.1% (v/v) CMT.

Effect of LMT and CMT on the Cellular Transport of CUR across Caco-2 Cell Monolayers

The permeation profile and apparent permeability coefficient in Fig. 6 present a significant influence on the permeation of CUR produced by LMT or CMT. Similar to the transport of CF, the permeation of CUR through Caco-2 cell monolayers was significantly improved by either LMT or CMT at 0.1%. Compared to the control value of 7.05±0.45 (×10⁻⁷) cm/s, P_app was 6.74±0.38 (×10⁻⁶) cm/s and 5.36±0.60 (×10⁻⁶) when CUR was co-administered with LMT or CMT, respectively.

Fig. 6. Effect of Sodium Methyl Lauroyl Taurate and Sodium Methyl Cocoyl Taurate on Cellular Transport of Curcumin in Caco-2 Cell Monolayers

CUR, curcumin; LMT, sodium methyl lauroyl taurate; CMT, sodium methyl cocoyl taurate. Results are expressed as the mean±S.E. of three experiments. Some error bars of S.E. are within the size of symbols. *** p<0.001, ** p<0.01, compared with CUR (control). Keys: (○) CUR, (◆) +0.1% (v/v) LMT, (◇) +0.1% (v/v) CMT.

DISCUSSION

The present study demonstrates that NATs can enhance the solubility and intestinal absorption of CUR in rats. Previously, we found that they could increase the intestinal absorption of alendronate, one of the typical bisphosphonates.¹³⁾ Therefore, the present findings are well correlated with our previous results, although the physicochemical characteristics of CUR, a poorly water-soluble compound, are quite different from those of alendronate, a water soluble, hydrophilic drug.

In the present study, of the various NATs that were assessed, CMT and LMT were the most effective for improving the intestinal absorption of CUR. Therefore, the absorption-enhancing effects were dependent on the type of fatty acid chain component of the molecule. Lauric acid is the single acyl component of LMT, while CMT is derived from coconut oil, in which lauric acid is also the main fatty acid.²⁸⁾ Therefore, lauric acid, the common component of both treatments might be of particular importance for improving the intestinal absorption of CUR. In contrast, the solubilizing and absorption-enhancing effects of PMT and SMT were restricted by their intrinsic low solubility at physiological temperature owing to their longer fatty acid chains. PMT exhibited a moderate absorption-enhancing effect but SMT had no effect. Furthermore, we calculated the bioavailability and absorption rate of CUR by a deconvolution method.²⁹⁾ In the presence of CMT, LMT, MMT, and SMT, the bioavailability % of CUR at 240 min after administration were 17.7, 18.9, 17.7, and 9.2%, respectively. Therefore, the bioavailability % of CUR is similar among CMT, LMT, and MMT groups, which is 2-fold higher than that in SMT group. On the other hand, the absorption rates of CUR at 60 min post-administration were also calculated to be 1.11, 1.23, 1.20 (ng/min) in CMT, LMT, and MMT groups, separately, while SMT showed lower absorption rate of CUR at 0.28 (ng/min). These findings suggested that CMT, LMT, and MMT have a fast onset of enhancement action on the absorption of CUR. In contrast, SMT shows relatively slow absorption-enhancing effect for improving the intestinal absorption of CUR. However, we could not calculate these two parameters in the control and PMT groups due to the low absorption of CUR. Hence, differences in enhancement may be explained by variation in the properties of NATs originating from the different fatty acid chains.

The present study has indicated that NATs can act as absorption enhancers in a similar manner to fatty acids. It has been established that the intestinal absorption of poorly absorbed drugs was improved by medium-chain fatty acids and their derivatives.³⁰⁾ A possible mechanism is that fatty acids cleaved and released from NAT structures may have increased the intestinal absorption of CUR in the present study. However, preliminary studies indicated that the absorption-enhancing actions of fatty acids were much smaller than those of NATs. Therefore, it seems implausible that fatty acid release increased the intestinal absorption of CUR in rats.

As shown in Fig. 7, a good sigmoidal relationship (R=0.8725) between the solubility and the intestinal absorption of CUR with and without NATs, indicates that the solubility is an important factor that contributed to the absorption enhancement of CUR by NATs. It was reported that critical micelle concentration (CMC) of LMT was 0.3% (w/v),³¹⁾ and CMT was expected to have a similar CMC due to its the main composition of LMT. As a result, CUR was entrapped in the micelles formed by LMT or CMT in this study, which acted as a drug reservoir for the intestinal absorption of CUR. In this case, it also should be noted that the increase in the dissolution rate of CUR by solubilization is the other factor increasing the intestinal absorption of CUR by N-acyl taurates. However, the intestinal absorption of CUR was not changed when the drug solubility increased more than 5 µg/mL. This finding suggests that the rate-limiting step was changed from the apparent solubility of drugs in micelles to the cellular permeation.

Fig. 7. The Relationship between Solubility and Intestinal Absorption in the Presence of N-Acyl Taurates

We examined the stability of CUR at near neutral pH, mimicking the physiological environment in the intestine. Our preliminary study demonstrated that CUR was stable for 4 h (data not shown), suggesting that degradation of CUR within the intestinal fluid may be ruled out. We, therefore, conclude that, in this work, NATs acted as solubilizing agents and absorption enhancers to promote the permeability of CUR across the intestinal membrane.

As shown in Fig. 3, we also evaluated whether these NATs might cause significant damage to the intestinal membrane. LDH is a cytosolic enzyme probe to measure the toxicity of absorption enhancers.^8,13,26,32) The LDH activity in the presence of NATs did not increase significantly compared with the negative control, but Triton X-100 (3%, v/v) increased the activity of LDH, indicating that NATs did not cause any serious membrane damage to intestinal tissue. Therefore, NATs are regarded as very safe surfactants for use as absorption enhancers for CUR,³³⁾ and the intestinal absorption of CUR was not promoted due to the damage to the intestinal membrane.

Caco-2 cells are isolated from the human colon carcinoma cells and retain many characteristics of human enterocytes. Caco-2 cells form a polarized monolayer with tight junctions and microvilli at the apical side and express transport proteins of the ATP binding cassette (ABC) transporter super family.³⁴⁾ In order to further elucidate the absorption-enhancing mechanism of NATs, CMT and LMT were tested using a Caco-2 permeability model system. P_app values of CF in the presence of both NATs were more than 90 times higher than that of the control. This permeation enhancement was accompanied by a substantial reduction in TEER. Furthermore, these findings correlate well with our previous work where CMT was shown to be effective at improving the intestinal absorption of water-soluble alendronate.¹³⁾ The P_app of CUR was improved 7.6- and 9.2-fold in CMT and LMT formulations, respectively, irrespective of the effect of enhanced solubility. These results clearly highlighted that both CMT and LMT accelerated the permeation of CUR via a paracellular pathway, most probably by opening the tight junctions between constituent cells of the monolayer.

Recently, it was demonstrated that the loosening of tight junctions might be regulated by tight junction-related proteins including occludin, claudins, and ZO-1. Previously, we found that the expression level of claudin-4 was reduced in the presence of palmitoyl sarcosinate, an N-acyl amino acid.¹³⁾ It may, therefore, be possible that LMT and CMT might reduce the expression levels of claudin proteins, loosen the tight junctions of the intestinal epithelium, and hence increase intestinal permeation and absorption of CUR. Furthermore, since the paracellular permeation is dependent on the molecular weight of compounds, it is assumed that LMT and CMT would enhance the intestinal absorption of poorly absorbable drugs with similar molecular size to CUR (MW=368) through the paracellular pathway. Indeed, our previous study indicated that N-acyl taurates including LMT and CMT could improve the intestinal absorption of poorly absorbable drugs including alendronate.¹³⁾

In addition, our previous study indicated that N-acyl amino acids, such as palmitoyl sarcosinate, increased the fluidity of the plasma membrane of epithelial cells, thereby increasing the permeation of drugs via a transcellular pathway.¹³⁾ Therefore, CMT and LMT may increase intestinal membrane fluidity, thereby increasing intestinal permeability and absorption of CUR. It is, therefore, necessary to consider the further absorption enhancing mechanisms of LMT and CMT in future studies.

CONCLUSION

In conclusion, LMT and CMT were the most effective of the N-acyl taurates tested at increasing the solubility and intestinal absorption of CUR. These NATs did not increase the activity of LDH released from intestinal cells, suggesting the safety of their application for oral administration. In cellular transport studies, LMT and CMT reduced the TEER values of Caco-2 cells and increased the transport of CF and CUR. Therefore, the intestinal absorption enhancement by LMT and CMT might be attributed to the synergistic effect of higher solubility and increased permeation of CUR through cellular layers in their presence. Therefore, co-administration with either LMT or CMT is a simple and effective method to enhance the oral delivery of CUR.

Acknowledgments

This work is in part supported by Kyoto Pharmaceutical University and Zhang Fen Jun Scholarship Fund (Kyoto City International Foundation).

Conflict of Interest

The authors declare no conflict of interest.

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