Silatecan 7-t-butyldimethylsilyl-10-hydroxycamptothecin (DB-67, Scheme 1) is a novel anti-cancer agent with superior blood stability and potent anti-cancer activity compared to other camptothecin analogues.1, 2 It has been recently approved by the FDA for phase I clinical studies.3 The biologically active lactone forms of camptothecins undergo pH dependent hydrolysis in solution to the inactive ring opened carboxylate forms.4–6 The carboxylate form is the favored species at equilibrium at physiological pH.6 In human blood this equilibrium may be further shifted towards the carboxylate due to its preferential binding to serum albumin.7–11 Efforts to synthesize camptothecins that remain in their active lactone form in blood resulted in the development of potent, highly lipophilic analogues with improved blood stability but poor water solubility.12 DB-67, along with other camptothecin analogues such as karenitecin and gimatecan, represent this new generation of blood stable but water insoluble camptothecins.13, 14 Novel formulation strategies are required to enable the delivery of these highly potent anti-cancer agents. Carriers that improve delivery of these agents to tumor tissue are also needed to diminish their toxic side effects.
Liposome technology has significant potential to improve formulation (e.g., solubility and stability) and tumor delivery-related issues that hinder the clinical advancement of these novel camptothecins. Lipid bilayer association has been previously found to improve solubility and stability of camptothecins.15–17 Liposomes are known to preferentially accumulate in tumor tissue after an i.v. injection and thus liposomal encapsulation offers the potential for improved antitumor specificity.18 Therefore, liposomal formulations are currently being investigated for various camptothecin analogues, several of which are in preclinical or clinical trials.19–23
One current strategy for liposomal encapsulation of camptothecins exploits the pH dependent lipid bilayer association of those compounds having an ionizable amine on the A or B-ring.21, 23–25 The ring-closed lactone has a greater membrane binding constant than the ring-opened carboxylate and therefore a low intraliposomal pH stabilizes the biologically active lactone form in lipid bilayers.15, 21, 23–25 A variety of loading techniques (e.g., pH, ammonium sulfate or ion gradient loading, metal ion complexation, etc.) developed to improve encapsulation efficiency of amphiphilic amines are applicable to amine-containing analogs or prodrugs of camptothecins.21, 23, 26–29 However, these techniques do not improve the loading efficiency of the lactone forms that lack an ionizable amine group such as DB-67 and camptothecin itself.
Another important consequence of the lack of an ionizable cationic functional group is the poor liposomal retention of the neutral analogs. For example, 28% of the amine-containing camptothecin, lurtotecan, was retained in liposomes 4 h after an i.v. injection20 while only 1% of the neutral camptothecin, SN-38, remained in the circulation 4 h after administration of a liposomal formulation.22 Premature leakage of the encapsulated drug fails to take advantage of the passive tumor targeting of liposomes and increases the potential for side-effects to healthy tissue.
To overcome such delivery-related issues with DB-67, efforts are underway in our laboratories to develop novel cationic and anionic prodrug strategies to improve liposomal encapsulation efficiency during formulation and drug retention in liposomes in vivo. A prerequisite to understanding the factors governing liposomal encapsulation, retention and release in vitro and in vivo is the knowledge of bilayer permeability of the biologically active and presumably bilayer permeating camptothecin lactone. To our knowledge, systematic studies of the lipid bilayer permeability of camptothecins including DB-67 have not been previously reported.
The objective of the current work was to understand the gel phase lipid bilayer permeability of the lactone form of DB-67 in vitro both in aqueous buffer and plasma since pegylated liposomes with a rigid gel phase bilayer have better retention of encapsulated drug in vivo.18 Previously, gel filtration and ultrafiltration methods have been used in these laboratories to study the bilayer permeability of various compounds.30–32 However the high lipophilicity (clog P = 5.4 ± 1.3) (calculated from Advanced Chemistry Development (ACD) Labs software) of DB-67 precluded the use of these methods due to ultrafilter membrane binding and the difficulty of maintaining good sink conditions in transport experiments. Therefore, a dynamic dialysis method to study membrane permeability of hydrophobic solutes has been developed and validated. A mathematical model has also been developed to calculate the bilayer permeability coefficients for hydrophobic solutes using dynamic dialysis and simulations were performed to identify situations where the method is suitable for quantifying liposome release kinetics of hydrophobic compounds. A theoretical estimate of the permeability coefficient for gel phase bilayer permeation of DB-67 was obtained using the bulk solubility-diffusion model and the recently developed “barrier-domain” solubility-diffusion model33 and compared to the experimental value. The kinetics of drug release from liposomes in vivo may be influenced by alteration of the bilayer barrier properties by serum proteins that adsorb to membranes34 and by the presence of trans-bilayer pH gradients. The hydrolysis kinetics of liposome entrapped versus free DB-67 lactone in rat plasma were monitored in the studies described herein to estimate the rate constant for drug release from liposomes in the presence of plasma proteins.