Donna Cabral-Lilly

Combination of antitumor ether lipid with lipids of complementary molecular shape reduces its hemolytic activity.

Because the therapeutic use of the antitumor ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) is restricted by its hemolytic activity we explored the use of lipid packing parameters to reduce this toxicity by creating structurally optimized ET-18-OCH3 liposomes. We postulated that combination of ET-18-OCH3, which is similar in structure to lysophosphatidylcholine, with lipid molecules of complementary molecular shape (opposite headgroup/chain volume) would likely yield a stable lamellar phase from which ET-18-OCH3 exchange to red blood cell membranes would be curtailed. To quantitate the degree of shape complementarity, we used a Langmuir trough and measured the mean molecular area per molecule (MMAM) for monolayers comprised of ET-18-OCH3, the host lipids, and binary mixtures of varying mole percentage ET-18-OCH3. The degree of complementarity was taken as the reduction in MMAM from the value expected based on simple additivity of the individual components. The greatest degree of shape complementarity was observed with cholesterol: the order of complementarity for the ET-18-OCH3-lipid mixtures examined was cholesterol >> DOPE > POPC approximately DOPC. Phosphorus NMR and TLC analysis of aqueous suspensions of ET-18-OCH3 (40 mol%) with the host lipids revealed them to all be lamellar phase. For ET-18-OCH3 at 40 mol% in liposomes, the hemolytic activity followed the trend of the reduction in MMAM and was least for the ET-18-OCH3/cholesterol system (H50 = 661 microM ET-18-OCH3) followed by ET-18-OCH3/DOPE (H50 = 91 microM) and mixtures with POPC and DOPC which were comparable at H50 = 26 microM and 38 microM, respectively: the H50 concentration for free ET-18-OCH3 was 16 microM. This experimental strategy for designing optimized liposomes with a reduction in exchange, and hence toxicity, may be useful for other amphipathic/lipophilic drugs that are dimensionally compatible with lipid bilayers.

Intra and Inter-Molecular Interactions Dictate the Aggregation State of Irinotecan Co-Encapsulated with Floxuridine Inside Liposomes

PurposeThe inter/intramolecular interactions between drugs (floxuridine, irinotecan) and excipients (copper gluconate, triethanolamine) in the dual-drug liposomal formulation CPX-1 were elucidated in order to identify the physicochemical properties that allow coordinated release of irinotecan and floxuridine and maintenance of the two agents at a fixed, synergistic 1:1 molar ratio.MethodsRelease of irinotecan and floxuridine from the liposomes was assessed using an in vitro-release assay. Fluorescence, Nuclear Magnetic Resonance spectroscopy (NMR) and UV–Vis were used to characterize the aggregation state of the drugs within the liposomes.ResultsCoordinated release of the drugs from liposomes was disrupted by removing copper gluconate. Approximately 45% of the total irinotecan was detectable in the copper-containing CPX-1 formulation by NMR, which decreased to 19% without copper present in the liposomal interior. Formation of higher order, NMR-silent aggregates was associated with slower and uncoordinated irinotecan release relative to floxuridine and loss of the synergistic drug/drug ratio. Solution spectroscopy and calorimetry revealed that while all formulation components were required to achieve the highest solubility of irinotecan, direct drug-excipient binding interactions were absent.ConclusionsLong-range interactions between irinotecan, floxuridine and excipients modulate the aggregation state of irinotecan, allowing for simultaneous release of both drugs from the liposomes.

ASSOCIATION AND RELEASE OF PROSTAGLANDIN E1 FROM LIPOSOMES

PGE1-lipid interactions were studied in several liposome systems. Data from both circular dichroic (CD) measurements and differential scanning calorimetry (DSC) indicated that PGE1 in the protonated form seeks the less polar environment of the lipid bilayer. CD measurements made on PGE1 in solution showed that the wavelength of maximum absorbance red shifted approximately 8 nm with decreasing solvent polarity. The CD spectrum of liposomal PGE1 prepared in pH 4.5 but not pH 7.2 buffer was also red shifted. There was no red shift in the CD spectrum of PGE1 detected at pH 4.5 in the absence of phospholipid. DSC measurements on DSPC bilayers prepared with 5 mol% PGE1 at pH 4.5 but not pH 7.2 revealed an almost complete loss of the pre-transition as well as broadening of the main phase transition. The amount of 3H-PGE1 initially associated with EPC, POPC or DSPC liposomes was determined using size exclusion filters and centrifugation. This amount was found to be dependent on the pH of the buffer (pH 4.5 >> pH 7.2) and fluidity of the bilayer (EPC = POPC > DSPC), but independent of the lamellarity of the liposome. In all cases, addition of cholesterol reduced the amount of PGE1 associated with the liposome. The time-dependent release of PGE1 from the liposomes was determined by rapidly diluting the sample 100-fold into pH 7.2 buffer. Lipid saturation was a key factor influencing this release. Gel-phase liposomes of DSPC showed a rapid initial release (t(1/2) < 2 min) of PGE1, corresponding to the amount in the outer monolayer, followed by a very slow, almost negligible release of the remaining PGE1. A rapid initial release also occurred in fluid-phase membranes, followed by a more gradual release of the remaining PGE1 over several hours. This release rate could be slowed by increasing the lamellarity of these liposomes, or adding cholesterol to decrease the fluidity of the membrane.

Abstract 5464: Determination of total and encapsulated drug pharmacokinetics for CPX-351, a nanoscale liposomal fixed molar ratio of cytarabine-daunorubicin (Cyt:Daun)

Background: CPX-351 is a liposomal formulation that delivers elevated concentrations of Cyt and Daun after administration in vivo and maintains the two drugs at a 5:1 molar ratio shown to be synergistic in preclinical studies. Pharmacokinetic analysis of CPX-351 in advanced leukemia patients demonstrated prolonged plasma exposure of the drugs with quantifiable concentrations of both agents being observed as long as 7 days after the final CPX-351 dose. Here we developed a method to separate liposome-bound from unencapsulated drugs, thereby allowing the pharmacokinetics of encapsulated agents to be determined in comparison to total plasma drug concentrations. Methods: CPX-351 was administered to female Sprague-Dawley rats via IV bolus injection. Plasma samples were harvested at five timepoints from 1 – 24 hours. Unencapsulated drug analysis was conducted by addition of internal standards (gemcitabine (Cyt) and doxorubicin (Daun)) to the plasma, followed by a 5-fold dilution in buffer and ultrafiltration using Microcon YM-30. Unencapsulated drug levels were determined by reversed-phase HPLC-MS (SIM, 244.2 m/z (Cyt) and 528.3 m/z (Daun)). Total analysis was conducted by protein precipitation and analysis was determined by reversed-phase HPLC-UV (Cyt) and reversed-phase HPLC-Fluorescence (Daun). Results: A separation method was developed to separate 0.22 – 4.33% unencapsulated Cyt and 0.13 – 2.50% unencapsulated Daun from liposome in plasma matrix. Encapsulated drug was calculated by subtracting the amount of unencapsulated analyte from the corresponding amount of total analyte. The total drug curves and the calculated encapsulated drug curves from 1 – 24 hours were superimposable for Cyt and Daun. A maximum of 1.9% unencapsulated Cyt and 0.4% unencapsulated Daun was measured in the plasma at any time point. Conclusions: Unencapsulated drug levels in rat plasma are insignificant compared to the total drug levels such that levels of total and encapsulated are within the spread of the data and therefore indistinguishable. These results suggest that CPX-351 remains in circulation in rats as intact liposomes at least 24 hours post administration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5464. doi:10.1158/1538-7445.AM2011-5464