Polymer-drug compatibility: a guide to the development of delivery systems for the anticancer agent, ellipticine.

To establish a method for predicting polymer-drug compatibility as a means to guide formulation development, we carried out physicochemical analyses of polymer-drug pairs and compared the difference in total and partial solubility parameters of polymer and drug. For these studies, we employed a range of biodegradable polymers and the anticancer agent Ellipticine as the model drug. The partial and total solubility parameters for the polymer and drug were calculated using the group contribution method. Drug-polymer pairs with different enthalpy of mixing values were analyzed by physicochemical techniques including X-ray diffraction and Fourier transform infrared. Polymers identified to be compatible [i.e., polycaprolactone (PCL) and poly-beta-benzyl-L-aspartate (PBLA)] and incompatible [i.e., poly (d,l-lactide (PLA)], by the above mentioned methods, were used to formulate Ellipticine. Specifically, Ellipticine was loaded into PBLA, PCL, and PLA films using a solvent casting method to produce a local drug formulation; while, polyethylene oxide (PEO)-b-polycaprolactone (PCL) and PEO-b-poly (d,l-lactide) (PLA) copolymer micelles were prepared by both dialysis and dry down methods resulting in a formulation for systemic administration. The drug release profiles for all formulations and the drug loading efficiency for the micelle formulations were also measured. In this way, we compared formulation characteristics with predictions from physicochemical analyses and comparison of total and partial solubility parameters. Overall, a good correlation was obtained between drug formulation characteristics and findings from our polymer-drug compatibility studies. Further optimization of the PEO-b-PCL micelle formulation for Ellipticine was also performed.

Use of micellar media for the fluorimetric determination of ellipticine in aqueous solutions.

Ellipticine is a pyridocarbazole alkaloid with interesting antitumour activity. Use of neutral ellipticine is hampered by its very low water solubility and therefore this compound has been administered as a salt; however, nitrogen quaternization modifies the antitumour properties of ellipticine. Potential alternatives to quaternization include the use of cyclodextrins, and also the use of micellar media. The latter possibility is explored in this work as an analytical tool. The results obtained with model anionic (SDS), cationic (CTAB) and neutral (Brij-35) surfactants are described. Fluorimetric analysis shows that ellipticine solubilizes completely in the presence of all these compounds, as a result of its aromatic, planar structure. The use of micellar media considerably increases the slopes of the calibration curves with improved correlation coefficients (e.g. 0.8904 in water and 0.9982 with SDS). Micellar media also modify proton transfer processes, as a consequence of the apolar environment of the micellar phase. Deprotonation of ellipticine is hampered in SDS because of the relationship between this process and the surface charge of the micelles. Finally, fluorescence quenching in micellar media has been studied, it being found that surfactants provide protection towards this phenomenon.

Determination of 9-hydroxyellipticine by redox colorimetry.

The chemical reactions involved in the decomposition of 9-hydroxyellipticine (9-OH-E), an anticancer agent, in polar solvents is explained. The reactions, which involve the formation of 9-oxo-ellipticine and the addition of a nucleophilic acid on the C10 site of the heterocyclic system, have been used to measure 9-OH-E quantitatively by colorimetry in solution and by reflection on paper surfaces. A method for the stabilization of 9-OH-E in polar solvents is proposed.

Acid-base properties of ellipticine bound to DNA, micelles and liposomes.

We have determined the acid-base properties of the alkaloid ellipticine, bound to DNA and to micelles and liposomes, taken as models for membranes, in the prospect of characterizing the actual structure of the bound ligand, this being relevant to the mode of action of the drug. The acid-base properties of ellipticine bound to sonicated calf thymus DNA and SDS micelles are similar as regards their pK values and their dependence on NaCl concentrations. This observation is satisfactorily understood in terms of sodium ion condensation around the negative phosphate and sulphate groups. The slope of pK vs log(Na+) is -1, a value predicted by Friedman theory. The pK of ellipticine bound to cationic (CTAB, DDTAB) or to neutral (Triton X100) micelles and to neutral liposomes (PC) is significantly lower than water (7.4), and, in contrast to the situation in DNA and SDS micelles, does not vary with addition of NaCl. Interestingly, this result is good evidence for ellipticine having a specific pK when bound to a hydrophobic structure. This view is likely to hold for ellipticine bound to DNA.

Kinetic and thermodynamic studies on drug-DNA interactions in the ellipticine series.

The temperature-jump (T-jump) method has been used to investigate the binding mechanism to calf-thymus DNA of ellipticine and some of its derivatives. The results show that the plant alkaloid, ellipticine, interacts with DNA at a unique intercalation site whereas most of its synthetic derivatives, such as ellipticinium, 9-hydroxy-ellipticinium and related alkyl-oxazolopyridocarbazoles recognize two distinct DNA sites. Parallel analysis of kinetic data and DNA lengthening abilities of these derivatives suggests that only one of these two DNA sites is an intercalation site. Owing to the determination of the genuine number of drug-DNA complexes (inferred from T-jump experiments) and with the results of thermodynamic investigations (Van't Hoff plots), further characterization of the molecular interactions involved in the binding process was proposed. Thus, the formation of the unique intercalation complex of ellipticine was found to be entropy driven whereas binding of drugs which recognize the second class of binding sites was essentially enthalpy driven. These different thermodynamic behaviors suggest that intercalation essentially results from hydrophobic solvent structure effects in contrast to the second binding mode which principally arises from hydrogen bonding interactions through DNA grooves.

Antibody recognition of substituted ammonium ions. Modulation by the counterion.

Antibodies directed against elliptinium acetate, a quaternary ammonium compound with antineoplastic activity in man, were obtained in rabbits, after conjugation of the drug with haemocyanin. These antibodies are specific for the quaternary ammonium structure. However, the recognition of the drug can be markedly decreased (ten-fold) by changing the associated counterion. These observations were extended to other ellipticine derivatives that exist in two forms: acetate and chloride. In each case, the recognition of the acetate form was 7-11-fold higher than that of the chloride form. These results could be explained by high-energy strengths existing between the cation and the anion, resulting in a paired-ion antigen. This represents the first identification of antibodies directed to a paired-ion structure, with specificity for both the cation and anion used for immunization. Such results are relevant in the construction of immunoassays for quaternary ammonium compounds.

Determination of ellipticine in biological samples by high-performance liquid chromatography.

Ellipticine, a plant alkaloid effective against murine leukemias and solid tumors, is presently undergoing toxicological assessment prior to clinical trial. A rapid, sensitive, reversed-phase high-performance liquid chromatographic method employing an internal standard was developed for the detection of ellipticine and its principal metabolite 9-hydroxyellipticine after extraction from biological samples. The method was successfully applied to the quantitation of ellipticine in mouse blood and tissues after intravenous administration of ellipticine and to mouse blood levels of drug after oral administration. Similar success was achieved in determinations of ellipticine and 9-hydroxyellipticine in samples of spiked human blood and plasma. Mouse blood ellipticine levels monitored over 3 h after the intravenous administration of drug demonstrated a biphasic decline with a terminal half-life of 52 min.