Enhanced intestinal absorption of salmon calcitonin (sCT) from proliposomes containing bile salts

doi:10.1016/j.jconrel.2005.05.016

Journal of Controlled Release

Volume 106, Issue 3, 2 September 2005, Pages 298-308

https://doi.org/10.1016/j.jconrel.2005.05.016 Get rights and content

Abstract

Purpose

The feasibility of using proliposomes containing salmon calcitonin (sCT) and absorption enhancing agents, as an oral delivery system, to improve the intestinal absorption of sCT was explored using rats and Caco-2 cell systems.

Methods

Seventeen surfactants were examined for their effects with reference to accelerating the permeability of sCT (300 μg/ml) across Caco-2 cell monolayers, and damage to the intestinal epithelial cells, as measured by the change in transepithelial electrical resistance (TEER) across the cell monolayer. Proliposomes containing sCT (0.75%, w/w) and sodium taurodeoxycholate (NaTDC, 2.5%, w/w) (TDC proliposomes) were prepared according to the standard method using sorbitol and phosphatidylcholine as core and wall-forming materials, respectively, administered intra-duodenally to rats, and plasma concentrations of sCT were subsequently determined by LC–MS.

Results

Among the surfactants examined, some bile salts including NaTDC appeared to be the most advantageous when estimated based on the balance between the permeation enhancement (e.g., a 10.8-fold increase in the permeability of sCT for 0.1% NaTDC) and damage to the cells (e.g., a 3.55-fold decrease in the TEER value for 0.1% NaTDC). The administration of TDC proliposomes resulted in a 7.1-fold increase in the bioavailability (i.e., 0.49%) of sCT, when administered duodenally to rats. The size of the reconstituted liposomes in water was significantly smaller (e.g., 23.2 nm, number weighted diameter), and the entrapment efficiency (EE) of sCT in the reconstituted liposomes was 2.8-fold larger (54.9%), for TDC proliposomes, compared to proliposomes prepared without NaTDC (sCT proliposomes).

Conclusion

A 7.1-fold increase in the bioavailability of sCT could be achieved from the TDC proliposomes. In addition to the intrinsic activity of the bile salt to fluidize the membrane, the simultaneous delivery of sCT and NaTDC to the site of absorption in the intestine via proliposomes and the subsequent formation of lipophilic ion-pair complexes between sCT and NaTDC at the site might have been contributing factors in this outstanding absorption enhancement.

Introduction

Non-parenteral delivery is by far the most convenient route to drug delivery, especially when repeated or routine administration is necessary [1]. Salmon calcitonin (sCT), which plays a crucial role in both calcium homeostasis and the treatment of bone disease such as osteoporosis [2], [3], is such a drug for which appropriate oral dosage forms need to be developed. Like many other proteins or peptides, the oral bioavailability of sCT is very low due to enzymatic degradation in the gastrointestinal (GI) tract [4], [5] and poor permeation across intestinal epithelial cells [6]. As a result, less than 0.1% of the bioavailability of sCT was obtained following intra-duodenal, -colonic and -ileac administration in rats and dogs [7], [8].

In a previous study [9], we reported a 3.6-fold increase in the apparent permeability (P_app) of sCT across Caco-2 cell monolayers (i.e., 6.14 × 10^− 7 cm/s) when sCT was applied in the form of proliposomes, a free-flowing particulate dosage form that immediately forms a liposomal dispersion in water [9], [10]. However, the P_app for the proliposomes is still quite low compared to the value that is generally known to be necessary for favorable intestinal absorption (i.e., 1 × 10^− 6 cm/s) [11]. In cases, the addition of appropriate absorption enhancers has frequently been attempted. However, in most cases, absorption enhancers also damage intestinal epithelial cells, limiting their use at high levels. Thus, the preferential delivery of absorption enhancers to the site of absorption rather than to the intestinal bulk fluid would be highly desirable [12]. Liposomes have often been considered to be potential candidates for achieving this objective [13]. In the present study, sCT and an appropriate absorption enhancer were formulated in the form of proliposomes, a precursor dosage form of liposomes, and examined for the feasibility as a dosage form that can increase intestinal absorption of sCT. Caco-2 cell monolayers were employed in the assessment of the permeability of sCT and the toxicity of absorption enhancers or dosage forms to the intestinal membrane, and the in vivo duodenal absorption of sCT in rats was measured, in an attempt to estimate bioavailability.

Section snippets

Materials

Synthetic sCT (3431.9 Da, purity > 99%) was purchased from A&PEP Inc. (Yeongi, Korea) and stored in a deep freezer (− 70 °C) before use. l-α-Phosphatidylcholine (PC, powder type from frozen egg yolk, > 99%), sorbitol (> 98%), chloroform (anhydrous, > 99%), trifluoroacetic acid (TFA), Dulbecco's modified Eagle's medium, non-essential amino acid solution, penicillin–streptomycin, Hank's balanced salts solution (HBSS) and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), sodium glycocholate

Selection of sodium taurodeoxycholate as an efficient absorption enhancer

The amount of sCT transported from the apical to the basolateral side across Caco-2 cell monolayers was less than 0.1% of the dose for a 45 min period (data not shown) under the given experimental conditions. The apparent permeability (P_app) calculated for 300 μg/ml sCT was 1.35 ± 0.21 × 10^− 7 cm/s (mean ± SD, n = 3, Table 1), consistent with our previous data (i.e., 1.71 ± 0.32 × 10^− 7 cm/s, [9]). The value is 10.4-fold greater compared to the transport of [¹⁴C] mannitol (i.e., 1.30 ± 0.28 × 10^− 8 cm/s),

Discussion

sCT, like many other peptides and proteins, is a hydrophilic and cationic (isoelectric point of 10.4) molecule with a relatively high molecular weight (3431.9 Da) that is soluble in water but only sparingly soluble in organic solvents. The polarity of sCT, which appears to be associated with basic amino acid residues (such as arginine, histidine and cysteine) in the molecule, might be reduced in the presence of appropriate counter anions if sCT interacts with the anions to form lipophilic

Acknowledgement

This research was supported, in part, by a grant M10115000003-01A130000311 from the Critical National Technology Program, Ministry of Science and Technology, Republic of Korea.

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