Partitioning of homologous nicotinic acid ester prodrugs (nicotinates) into dipalmitoylphosphatidylcholine (DPPC) membrane bilayers

Abstract

The partitioning behavior of a series of perhydrocarbon nicotinic acid esters (nicotinates) between aqueous solution and dipalmitoylphosphatidylcholine (DPPC) membrane bilayers is investigated as a function of increasing alkyl chain length. The hydrocarbon nicotinates represent putative prodrugs, derivatives of the polar drug nicotinic acid, whose functionalization provides the hydrophobic character necessary for pulmonary delivery in a hydrophobic, fluorocarbon solvent, such as perfluorooctyl bromide. Independent techniques of differential scanning calorimetry and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy measurements are used to analyze the thermotropic phase behavior and lipid bilayer fluidity as a function of nicotinate concentration. At increasing concentrations of nicotinates over the DPPC mole fraction range examined (X(DPPC)=0.6-1.0), all the nicotinates (ethyl (C2H5); butyl (C4H9); hexyl (C6H13); and octyl (C8H17)) partition into the lipid bilayer at sufficient levels to eliminate the pretransition, and decrease and broaden the gel to fluid phase transition temperature. The concentration at which these effects occur is chain length-dependent; the shortest chain nicotinate, C2H5, elicits the least dramatic response. Similarly, the DPH anisotropy results demonstrate an alteration of the bilayer organization in the liposomes as a consequence of the chain length-dependent partitioning of the nicotinates into DPPC bilayers. The membrane partition coefficients (logarithm values), determined from the depressed bilayer phase transition temperatures, increase from 2.18 for C2H5 to 5.25 for C8H17. The DPPC membrane/water partitioning of the perhydrocarbon nicotinate series correlates with trends in the octanol/water partitioning of these solutes, suggesting that their incorporation into the bilayer is driven by increasing hydrophobicity.

Figure 1

Chemical structure of the perhydrocarbon nicotinates.

Figure 2

Typical calorimetric scans for mixtures of DPPC with (A) Ethyl nicotinate - C2H5, (B) Butyl nicotinate – C4H9, (C) Hexyl nicotinate – C6H13 and (D) Octyl nicotinate – C8H17 in excess water. The mole fraction of DPPC is indicated beside each scan.

Figure 3

Partial phase diagrams of mixtures of DPPC with (A) C2H5, (B) C4H9, (C) C6H13 and (D) C8H17 in excess water. All data points are average of three experiments ± SD. (■) Onset temperature of main transition; (◆) DPPC melting temperature, T_m; (×) offset temperature of main transition; (▲) onset temperature of pretransition; (●) DPPC pretransition temperature, T_p; and (Δ) offset temperature of pretransition.

Figure 4

Half width of the (A) pretransition and (B) main phase transition of DPPC with (▲) C2H5, (■) C4H9, (◆) C6H13 and (×) C8H17. All data points represent average of three experiments ± SD.

Figure 5

Normalized anisotropy of DPH fluorescent probe in DPPC bilayers as a function of temperature and varying nicotinate concentrations for (A) C2H5 (B) C4H9 (C) C6H13 and (D) C8H17.

Figure 6

Melting temperature, T_m(◆) and phase transition width, ΔT_r (●) of the DPPC phase transition as function of concentrations of (A) C2H5, (B) C4H9, (C) C6H13 and (D) C8H17 measured by DPH fluorescence anisotropy.

Figure 7

Correlation of experimental DPPC bilayer-aqueous partition coefficients with predicted K_o/w partitioning values [11] (Δ) and published octanol-water values [31] (◆). (●)Trend with cytotoxicity data [11].