Relationships between the hydrophilic–lipophilic balance values of pharmaceutical excipients and their multidrug resistance modulating effect in Caco-2 cells and rat intestines

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Abstract

The effects of a series of pharmaceutical excipients, including Span 80, Brij 30, Tween 20, Tween 80, Myrj 52, and sodium lauryl sulfate (with increasing hydrophilic–lipophilic balance (HLB) values) on the intracellular accumulation, transport kinetics, and intestinal absorption of epirubicin were investigated in both the human colon adenocarcinoma (Caco-2) cell line and the everted gut sacs of rat jejunum and ileum. The possible use of these excipients as multidrug resistance (MDR) reversing agents also was examined. Epirubicin uptake experiments using a flow cytometer showed that these selected excipients markedly enhanced the intracellular accumulation of epirubicin in Caco-2 cells in a dose-dependent manner. The optimal effect on the epirubicin uptake was characteristic of excipients with intermediate HLB values ranging from 10 to 17. Moreover, the optimal net efficacy was observed for excipients with polyoxyethylene chains and intermediate chain length of fatty acid and fatty alcohol (monolaurate for Tween 20, monooleate for Tween 80, monostearate for Myrj 52, and lauryl alcohol for Brij 30). These excipients significantly increased apical to basolateral absorption and substantially reduced basolateral to apical efflux of epirubicin across Caco-2 monolayers. Furthermore, the addition of Tween 20, Tween 80, Myrj 52, and Brij 30 markedly enhanced mucosal to serosal absorption of epirubicin in the rat jejunum and ileum. This study suggests that inhibition of intestinal P-glycoprotein (P-gp), multidrug resistance associated protein family (MRPs), or other transporter proteins by pharmaceutical excipients may improve oral absorption of drugs in MDR spectrum. The optimal HLB values of surfactant systems with suitable hydrocarbon chains and polar groups are an important factor in designing promising epirubicin formulations for reversing MDR. In conclusion, therapeutic efficacy of epirubicin may be enhanced by the use of such low toxicity excipients as absorption enhancers and MDR modulators in formulations. This provides a potential strategy for improving bioavailability in the optimization of formulations for drugs performing intestinal absorption and secretion.

Introduction

The development of multiple drug resistance (MDR) appears to be a major impediment to the successful chemotherapy of human tumors. Drug efflux mediated by broad-specificity xenobiotic transporters present in the intestinal epithelium may be an important factor in the poor or variable absorption of orally administered drugs [1]. MDR has been usually associated with the overexpression of membrane transporter proteins, including the superfamily of the ATP-binding cassette (ABC) proteins. For example, P-glycoprotein (P-gp) and multidrug resistance associated protein family (MRPs) confer MDR in many normal or tumor tissues [2]. P-gp and MRPs are present in intestines and Caco-2 cells. The substrate specificity of MRPs is overlapping but distinct from that of P-gp, and includes both neutral and mildly cationic natural product cytotoxic drugs, such as epirubicin [3]. Both P-gp and MRPs exhibit substrate-stimulating or -inhibiting ATPase activity in crude membrane preparations and purified/reconstituted systems [4], [5].

Since the involvement of many secretory proteins in drug efflux in the gastrointestinal epithelium, their inhibition by drugs and excipients has become an important issue in improving oral drug delivery. Among the strategies for overcoming MDR, various P-gp or MRPs antogonizers have been evaluated for their effects in both the absorptive and secretory permeabilities of cytotoxic drugs [6]. In the search of less toxic MDR modulators, our previous studies have shown that bile salts, fatty acids, phospholipids, and surfactants were verified as potent absorption enhancers and efflux-reducing agents in Caco-2 cells and rat intestines [7], [8], [9]. Other researchers also tested the non-ionic detergents, such as Tween 80, Pluronic P85, and Cremophor EL in vitro and in vivo in animals and in humans for their potential to reverse MDR caused by Pgp and MRPs [10], [11], [12].

In this study, excipients with low toxicity were evaluated for their possible MDR-modulating activity. Excipients are usually added to the formulation to facilitate the preparation, patient acceptability and functioning of the dosage form. Excipients can be used as disintegrating agents, diluents, suspending agents, emulsifying agents, and solubilizing agents, etc. Among excipients, surfactants were found to have MDR modulator activity [13], [14], [15]. Some of the surfactants are pharmacologically inert excipients and are usually used as solubilizing agents, emulsifying agents and suspending agents in pharmaceutical dosage form preparation [16]. A number of these surfactants (e.g. Tween 80 and Cremophor EL) are commonly used vehicles for solubilizing lipophilic drugs (e.g. etoposide and Taxotere) or vitamins for oral or parenteral administration. Surfactants are also potent absorption enhancers to increase the solubility of hydrophobic macromolecules in the aqueous boundary layer [17]. Recently, Cremophor, Tween 80, and Solutol HS-15 have been proven to reverse MDR phenotype in cultured cells at concentrations likely to be achieved clinically [12], [14]. One of the possible mechanisms for their MDR reversing effects is to induce a change in the fluidity of the apical (or the basolateral) cell membrane, and thus inhibit activity of membrane spanning proteins, such as P-gp and MRP. In addition, Tween 80 was shown to modulate anthracycline and Vinca alkaloid resistance in MDR cells by inhibiting the binding of these drugs to P-gp [12], [18].

In the current study, the effects of a series of pharmaceutical excipients, including Span 80, Brij 30, Tween 20, Tween 80, Myrj 52, and sodium lauryl sulfate (with increasing hydrophilic–lipophilic balance (HLB) values) on the intracellular accumulation, transport kinetics, and intestinal absorption of epirubicin were evaluated in both the human colon adenocarcinoma (Caco-2) cell line and the everted gut sacs of rat jejunum and ileum. Furthermore, the functional involvement of P-gp in this process was verified by the addition of verapamil, one typical P-gp modulator. Flow cytometry was used to determine if the resistance could be modulated by these pharmaceutical excipients by measuring the change in the intracellular fluorescent epirubicin concentrations [19], [20]. The human colon adenocarcinoma (Caco-2) cell line was used as a human intestinal epithelial cell model, because these cells can differentiate spontaneously to enterocytelike cells under conventional cell culture conditions [21], [22].

In addition, the relationship between HLB values of these excipients and the degree of their MDR reversing effects was also investigated. For this purpose, acacia and tragacanth were also included in this study. However, due to the complex structure and polydisperse nature of these two excipients, further evaluation of the relationship between their structural characteristics and MDR reversing activity is not in the scope of this study. The structural characteristics, molecular weight, critical micelle concentrations (CMC), and HLB values of all the excipients used in this study are listed in Table 1.

Section snippets

Materials

Epirubicin was purchased from Pharmacia and Upjohn (Milano, Italy). Span 80, Brij 30, Tween 20, Tween 80, Myrj 52, acacia, tragacanth, and sodium dodecyl sulfate (SDS) were purchased from Sigma (St. Louis, MO, USA). All cell culture medium and reagents were purchased from Gibco BRL (Grand Island, NY, USA). Most of the other chemical reagents were purchased from either Merck (Darmstadt, Germany) or Sigma (St. Louis, MO, USA). Tyrode’s solution was prepared by dissolving 24 g of NaCl (137 mM), 3

Effect of pharmaceutical excipients on the uptake of epirubicin in Caco-2 cells

As illustrated in Fig. 1, the results show that all the selected excipients substantially enhanced the uptake of epirubicin into Caco-2 cells in a dose-dependent manner, as measured by flow cytometer. The enhancement factor was ranged from 1.18±0.03 for Span 80 and SLS at the concentration of 20 μM, to 2.01±0.05 for Tween 80 at the concentration of 200 μM. Verapamil, a conventional P-gp substrate, exhibited more pronounced enhancement on the intracellular epirubicin accumulation compared with

Discussion

Our results depict the enhancing effects of excipients, such as Span 80, Tween 20, Tween 80, Myij 52, Brij 30, and SLS, on the intracellular accumulation and transepithelial flux of epirubicin in Caco-2 cells, as well as intestinal absorption of epirubicin in rats. The enhancement could not be solely explained by the solubilization of epirubicin by these pharmaceutical surfactant excipients. This improvement occurred, most notably, in the surfactant concentrations below their CMC. The similar

Acknowledgements

This work was supported by grant NSC 91-2320-B-041-011 from the National Science Council, Taiwan.

References (35)

  • M Sakai et al.

    Effects of absorption enhancers on the transport of model compounds in Caco-2 cell monolayers: assessment by confocal laser scanning microscopy

    J. Pharm. Sci.

    (1997)
  • E.S Park et al.

    Enhancing effect of polyoxyethylene alkyl ethers on the skin permeation of ibuprofen

    Int. J. Pharm.

    (2000)
  • T.F Ferguson et al.

    The effect of surfactants upon mammalian cells in vitro

    Food Cosmet. Toxicol.

    (1976)
  • A.V Kabanov et al.

    Pluronic block copolymers for overcoming drug resistance in cancer

    Adv. Drug Deliv. Rev.

    (2002)
  • H Schuldes et al.

    Reversal of multidrug resistance and increase in plasma membrane fluidity in CHO cells with R-verapamil and bile salts

    Eur. J. Cancer

    (2001)
  • V.D Makhey et al.

    Characterization of the regional intestinal kinetics of drug efflux in rat and human intestine and in Caco-2 cells

    Pharm. Res.

    (1998)
  • J Kvackajova-Kisucka et al.

    Drug transporters and their role in multidrug resistance of neoplastic cells

    Gen. Physiol. Biophys.

    (2001)
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