Eric Mayhew

Activity of paclitaxel liposome formulations against human ovarian tumor xenografts.

Although the current clinical formulation of paclitaxel (Taxol®) is an important new anti‐cancer agent, it has significant side effects, some of which are related to its formulation in Cremophor/ethanol, Paclitaxel is difficult to formulate for i.v. administration because of its poor aqueous solubility. Here, we report the therapeutic effects of 2 liposome formulations of paclitaxel against human ovarian A121 tumor growing as an s.c. xenograft in athymic nude mice. The liposome formulations used were ETL and TTL, which have 1 or 3 lipid components, respectively. TTL was used as a reconstituted lyophilate or as a stable aqueous suspension. ETL was used as a reconstituted lyophilate only. Both paclitaxel‐liposome formulations were much better tolerated than Taxol® after i.v. or i.p. administration. The acute reactions seen after Taxol® administration did not occur when paclitaxel‐liposome formulations were administered. All ETL and TTL preparations significantly delayed A121 tumor growth similarly to Taxol at equivalent doses and schedules. Based on pharmacokinetic data, it is possible that paclitaxel rapidly dissociates from ETL or TTL after i.v. administration and distributes in a manner similarly to Taxol. ETL and TTL formulations may be useful clinically not only for eliminating toxic effects of the Cremophor/ethanol vehicle but also for allowing alterations in route and schedule of drug administration. Int. J. Cancer, 71:103–107, 1997.

A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes.

High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC >> DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component.

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.

Hydrolyzable hydrophobic taxanes: synthesis and anti-cancer activities.

A series of taxane prodrugs with 2-bromoacyl chains attached at the 2′-position of the paclitaxel side chain, varying from six, eight, 12, 14 to 16 carbons in length, were synthesized, characterized and evaluated against human breast MCF-7 cancer cell line for their growth inhibitory (GI50) activities. The GI50 is the drug concentration required to inhibit cell growth by 50%. For comparison, hydrophobic taxanes varying in acyl chain lengths from six to 16 carbons were also synthesized and compared for their G050s with taxanes having equivalent bromoacyl chain lengths. The bromoacyl taxanes bearing six, eight and 12 carbon acyl chain lengths had GI50 values very similar to parent paclitaxel. The GI50 was 3 nM for three taxanes versus 1 nM for paclitaxel on the MCF-7 cell line. Increasing the acyl chain length to 14 or 16 carbons resulted in a significant decrease in cytotoxicity and an increase in the GI50 to 20 or 70 nM, respectively. In general, the GI50 values were directly related to the bromoacyl chain lengths in cultured tumor cells. Unlike bromoacyl taxanes, the taxanes lacking bromine in their acyl chain composition were 50- to 250-fold less active, suggesting that the heteroatom facilitated the hydrolysis of acyl chains to yield free paclitaxel. These differences in growth inhibitory activities may indirectly reflect differences in the susceptibility of the acyl chain to bromine-induced hydrolysis after association of the derivative with cell membranes. Liposome formulations of 2-bromoacyl taxanes bearing six, eight, 12 and 16 carbons were prepared and tested in SCID mice against a xenografted human ovcar-3 ovarian tumor. In vivo results showed that bromoacyl taxanes with a longer chain were therapeutically more efficacious than those with a short chain, presumably due to slow hydrolysis of the prodrug followed by sustained delivery of paclitaxel to the tumor.

Growth inhibitory effects of liposome-associated 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine.

The growth inhibitory effects of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) and various liposome compositions of ET-18-OCH3 were compared in a standardized growth inhibition assay utilizing a diverse tumor cell line panel including cell lines expressing multidrug resistance. ET-18-OCH3 and ELL-12 (4∶3∶1∶2, dioleoylphosphatidylcholine/cholesterol/dioleoylphosphatidylethanolamine-glutaric acid/ET-18-OCH3), an optimal liposomal ET-18-OCH3 formulation, inhibited growth in the micromolar range in drug-sensitive and-resistant cells. In general, ET-18-OCH3-liposomes were about twofold less growth inhibitory than ET-18-OCH3. However, the known hemolytic effects of ET-18-OCH3 were greatly reduced, up to 20 or more times, by liposome association. The effects of ET-18-OCH3 and ELL-12 were compared in intracellular [Ca2+] modulation and DNA fragmentation assays. ET-18-OCH3 elicited both concentration- and serum-dependent transient and permanent increases in intracellular [Ca2+]. In contrast, ELL-12 did not modulate intracellular [Ca2+]. ET-18-OCH3 and ELL-12 similarly affected DNA fragmentation, which may be indicative of apoptosis. The results suggest that, although the specific growth inhibitory effects of ET-18-OCH3 and ELL-12 are similar, associating ET-18-OCH3 with stable well-characterized liposomes eliminates nonspecific cell membrane-associated lytic effects.

STABILITY OF ASSOCIATION OF 1-O-OCTADECYL-2-O-METHYL-SN-GLYCERO-3-PHOSPHOCHOLINE WITH LIPOSOMES IS COMPOSITION DEPENDENT

The ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), has anticancer activity, but it has serious side-effects, including hemolysis, which prevent its optimal use. We surmised if ET-18-OCH3 could be stably associated with liposomes, less free ET-18-OCH3 would be available for lytic interaction with red cells. Liposome composition variables investigated included acyl chain saturation, phospholipid head group and mole ratio of Chol and ET-18-OCH3. It was found that attenuation of hemolysis was strongly liposome composition dependent. Some ET-18-OCH3 liposome compositions were minimally hemolytic. For example, whereas the HI5 (drug concentration required to cause 5% human red cell lysis) was 5-6 microM for free ET-18-OCH3, it was approximately 250 microM for DOPC (dioleoylphosphatidylcholine):Chol (cholesterol):DOPE-GA (glutaric acid derivatized DOPE):ET-18-OCH3, (4:3:1:2) and 640 microM for DOPE (dioleyolphosphatidylethanolamine):Chol:DOPE-GA:ET-18-OCH3 (4:3:1:2) liposomes. Efflux of carboxyfluorescein (CF) from liposomes and Langmuir trough determinations of mean molecular area of lipids in monolayers (MMAM) were used as indicators of membrane packing and stability. Incorporation of ET-18-OCH3 in liposomes reduced the MMAM. Reduction in CF permeation was correlated with reduction in hemolysis. The most stable liposomes included components, such as cholesterol, DOPC and DOPE, which have complementary shapes to ET-18-OCH3.

Increased cell-surface receptor expression on U-937 cells induced by 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine

Abstract Association of the ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) with liposomes (ELL-12) reduces acute toxicity while maintaining or enhancing anticancer activity in experimental tumor models. ELL-12 has been shown to induce apoptosis by a cytochrome-c-dependent caspase-mediated pathway, which results in proteolytic cleavage of poly(ADP-ribose) polymerase and lamins, but the antitumor effects of ET-18-OCH3 or ELL-12 could result from tumor cell differentiation or activation. Here we compared the effects of ET-18-OCH3 and ELL-12 on the expression of cell-surface proteins associated with cell differentiation and/or activation in U-937 cells. Phorbol 12-myristate 13-acetate and all-trans-retinoic acid, which induce differentiation in U-937 cells, up-regulated CD11b (MAC1 α-integrin) and CD82 and down-regulated CD71 (transferrin receptor) in a time- and dose-dependent manner. In contrast, ET-18-OCH3 and ELL-12 up-regulated both CD71 and CD11b and did not have any effect on expression of CD82 in U-937 cells, suggesting that the ELL-12 may activate these cells rather than induce differentiation. Further evidence of activation was that ET-18-OCH3 and ELL-12 strongly induced tumor necrosis factor α production by U-937 cells.

INHIBITION OF CELL DIVISION BUT NOT NUCLEAR DIVISION BY 1- O -OCTADECYL-2- O -METHYL- Sn -GLYCERO-3-PHOSPHOCHOLINE

1‐ O ‐Octadecyl‐2‐ O ‐methyl‐glycero‐3‐phosphocholine (ET‐18‐OCH3) selectively inhibits the growth of cancer cells. Here we show that in some cell types ET‐18‐OCH3and liposome‐associated ET‐18‐OCH3inhibit cell division without concurrent inhibition of nuclear division, leading to multinucleate cell formation, and cell death through apoptosis. Cell cycle analysis revealed that ET‐18‐OCH3‐treated U‐937 cells continued to move through the cell cycle, but many cells were not able to divide and instead accumulated as tetraploid cells or octaploid cells in the G0/G1 phase of the cell cycle. Inhibition of cytokinesis has been shown to be paralleled by activation of U‐937 cells, including upregulation of some cell‐surface markers, acquisition of phagocytic activity, and secretion of tumor necrosis factor (TNF)‐α (Pushkareva et al., 2000). Furthermore, treatment of cells with ET‐18‐OCH3results in the accumulation of apoptotic cells in time‐ and dose‐dependent manner. It is possible that inhibition of cytokinesis may be related to cytoskeletal effects.