Partition behaviour of acids and bases in a phosphatidylcholine liposome–buffer equilibrium dialysis system

doi:10.1016/S0928-0987(97)00278-9

European Journal of Pharmaceutical Sciences

Volume 5, Issue 4, 1 July 1997, Pages 223-231

https://doi.org/10.1016/S0928-0987(97)00278-9 Get rights and content

Abstract

Apparent partition coefficients (D) of (RS)-[³H]propranolol, ¹⁴C-lidocaine, ¹⁴C-diazepam, ¹⁴C-warfarin, ¹⁴C-salicylic acid and ¹⁴C-cyclosporine A were determined in a phosphatidylcholine (PhC) liposome–buffer system with equilibrium dialysis (37°C, pH 2 to 11). The resulting pH-dependent partition diagrams represent a superposition of Henderson–Hasselbalch equations for the pK_a of drug and PhC. True partition coefficients were calculated for the neutral (P_n) as well as for the ionized (P_i) molecules. For all drugs P_n was higher than P_i. A decrease in D was found at pH values where the phosphate group of PhC was protonated (pH≤2). With extruded liposomes D values were lower than with detergent (cholate) dialysis liposomes, which indicates that the residual cholate (1 molecule per 700 PhC molecules) has an influence on the partition behaviour.

Introduction

Drugs penetrate a number of cell barriers before they reach the site of action. An essential physico-chemical parameter to understand the behaviour of a compound towards membranes is the partition coefficient which is a measure of its lipophilicity. The partition coeffient is expressed as the distribution ratio of a solute between a lipophilic and a hydrophilic phase. In vivo biological membranes represent the lipophilic and aqueous compartments the hydrophilic phase. To determine true partition coefficients (P) several conditions have to be fulfilled (Nernst, 1891): constant temperature, non-miscible phases, stability of the compounds and low solute concentrations resulting in an activity coefficient of 1. Several methods have been used to determine the partition coefficient of a molecule. The classical partition system makes use of an organic solvent, e.g., octanol, as lipophilic phase and a buffer solution as hydrophilic phase (Leo et al., 1971). New approaches comprise artificial membranes, i.e., liposomes (Betageri and Rogers, 1987, Betageri et al., 1989) as lipophilic phase. Early experiments were hampered by the fact that inhomogeneous populations of multilamellar liposomes resulted in poor reproducibility and also Nernst conditions were not always fulfilled. As a consequence a standardized partition system with unilamellar egg phosphatidylcholine (PhC) liposomes was developed (Pauletti and Wunderli-Allenspach, 1994). It makes use of equilibrium dialysis which permits measurements of concentrations without disturbing the equilibrium. Temperature is kept constant and the stability of drug and liposomes is controlled. With radiolabelled drugs the detection limit is kept low so that the activity coefficient equals 1. With this combined approach the Nernst conditions for the determination of P are fulfilled. It is the first system which permits quantitative comparison of various compounds over a wide pH range (2–11) under strictly controlled conditions. The first model drug in the equilibrium dialysis system was the base (RS)-[³H]propranolol (Pauletti and Wunderli-Allenspach, 1994). The highest apparent partition coefficients (D) were found where the drug and the lipid molecules were electrostatically neutral. To describe the D–pH-diagram quantitatively, data were fit with Henderson–Hasselbalch equations (Krämer and Wunderli-Allenspach, 1996). The pK_a of both the drug (pK_a 9.24) and the PhC (pK_a∼2) appeared as inflection points in the diagram. It is obvious that not only the neutral but also the ionized drug molecules can be associated with the lipid bilayer. Therefore, the assumption of Brodie and Hogben (1957)that only the neutral compound is found in the lipophilic phase has to be modified for lipid membranes. It can thus be postulated that both P, the one for neutral (P_n) as well the one for ionized (P_i) molecules, are relevant for the in vivo partition behaviour of a drug.

In the present work, additional drugs have been tested. The aim was to examine the partition behaviour of acids (¹⁴C-warfarin, ¹⁴C-salicylic acid), bases (¹⁴C-lidocaine, ¹⁴C-diazepam) and a neutral compound (¹⁴C-cyclosporine A) over a wide pH range in the equilibrium dialysis system with PhC–liposomes. With all tested compounds D–pH partition diagrams followed exactly the Henderson–Hasselbalch equations. The liposome–buffer partition system was also used to study the influence of residual cholate, i.e., liposomes prepared by extrusion were compared with those prepared by detergent dialysis. With (RS)-[³H]propranolol an increase in the partition coefficient was found in the presence of residual cholate.

Section snippets

Chemicals

PhC, grade 1, was purchased from Lipid Products (Nutfield, Surrey, UK). (RS)-propranolol·HCl, lidocaine·HCl, diazepam, salicylic acid and warfarin were from Sigma, (St. Louis, MO, USA). The radiolabelled (RS)-l-[4-³H]propranolol (533 GBq/mmol), 2-¹⁴C-diazepam (2.04 GBq/mmol) and ¹⁴C-warfarin (3α-acetonyl[α-¹⁴C]benzyl-4-hydroxycoumarin, 2.11 GBq/mmol) were from Amersham (Little Chalfont, Buckinghamshire, UK). From DuPont (Stevenage, Hertfordshire, UK) were carbonyl-¹⁴C-lidocaine (2.1 GBq/mmol)

pH-Dependent stability of liposomes

Liposomes were prepared by either the extrusion method or by the detergent dialysis procedure with cholate as detergent (see Section 2). Their diameter as well as the polydispersity factor were measured by DLS. The cumulants method for extruded liposomes yielded a z average mean value of 94.4±7.7 nm and a polydispersity factor of 0.087±0.030 (n=7). For detergent dialysis liposomes the corresponding values were: z average mean 68.6±1.3 nm, and the polydispersity factor 0.083±0.025 (n=3).

The

Discussion

The pH-dependent partition diagram of the model compound (RS)-[³H]propranolol in a standardized PhC liposome–buffer system can be fit with superposed Henderson–Hasselbalch equations of the solute and the PhC (Pauletti and Wunderli-Allenspach, 1994). In the present work the same partition system, which comprises unilamellar PhC liposomes as lipophilic phase, was tested with various drugs.

In the pH-dependent partition diagrams for the bases ¹⁴C-lidocaine and (RS)-[³H]propranolol a plateau was

Acknowledgements

We would like to thank Sandoz Pharma for the generous gift of cyclosporine A. The excellent technical assistance of Christina Jakits for HPLC analysis is gratefully acknowledged.

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