Murray S. Webb

Comparison of different hydrophobic anchors conjugated to poly(ethylene glycol): effects on the pharmacokinetics of liposomal vincristine

Poly(ethylene glycol) (PEG) conjugated lipids have been used to increase the circulation longevity of liposomal carriers encapsulating therapeutic compounds. PEG is typically conjugated to distearoylphosphatidylethanolamine (DSPE) via a carbamate linkage that results in a net negative charge on the phosphate moiety at physiological pH. It was anticipated that the presence of this negative charge could have deleterious effects on liposome pharmacokinetic characteristics. We describe here the synthesis of a new class of neutrally charged PEG-lipid conjugates in which the PEG moiety was linked to ceramide (CER). These PEG-CER conjugates were compared with PEG-DSPE conjugates for their effects on the pharmacokinetics of liposomal vincristine. PEG-CER (78% palmitic acid, C16) and PEG-DSPE achieved comparable increases in the circulation lifetimes of sphingomyelin/cholesterol (SM/chol) liposomes. However, PEG-DSPE significantly increased the in vitro and in vivo leakage rates of vincristine from SM/chol-based liposomes compared to vincristine leakage observed when PEG-CER was used. The increase in drug leakage observed in vitro that was due to the presence of PEG-DSPE was likely due to the presence of a negative surface charge. Analysis of the electrophoretic mobilities of these formulations suggested that the negative surface charges were shielded by approx. 80% by the PEG layer extending from the membrane surface. In contrast, formulations containing PEG-CER had no surface charge and no electrophoretic mobility. A comparison of the effects of the ceramide acyl chain length (C8 through C24) on the pharmacokinetics of SM/chol/PEG-CER formulations of vincristine demonstrated that longer acyl chains on the PEG-CER were associated with longer circulation lifetimes of the liposomal carriers and, consequently, higher plasma vincristine concentrations. These data suggest that the short chain PEG-ceramides underwent rapid partitioning from the vesicles after i.v. administration, whereas the longer chain PEG-ceramides had stronger anchoring properties in the liposome bilayers and partitioned slowly from the administered vesicles. These data demonstrate the utility of ceramide-based steric stabilizing lipids as well as the potential for developing controlled release formulations by manipulating the retention of the PEG-ceramide conjugate in liposome bilayers.

Interactions between soluble sugars and POPC (1-palmitoyl-2-oleoylphosphatidylcholine) during dehydration: vitrification of sugars alters the phase behavior of the phospholipid

The phase behavior of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) was characterized as a function of hydration in the presence of combinations of sugars representative of sugars found in seed embryos having differing degrees of desiccation tolerance. The tendency of the sugar mixes to vitrify was also monitored as a function of hydration. Using differential scanning calorimetry, it was found that all sugars diminished the increase in the gel-to-fluid phase transition temperature (Tm) of POPC that occurred upon dehydration of the pure lipid. These results are analyzed in terms of the osmotic and volumetric properties of sugars. Also, it was found that in those samples for which the glass transition temperature (Tg) was greater than the Tm of POPC, Tm was lowered by approx. 20 C degrees from the value for the fully hydrated lipid. X-ray diffraction data confirmed that acyl chain freezing was deferred to a lower temperature during cooling of vitrified samples. The significance of these results is discussed in terms of the ability of many organisms to tolerate desiccation.

A Comparison of Freezing Injury in Oat and Rye: Two Cereals at the Extremes of Freezing Tolerance

A detailed analysis of cold acclimation of a winter rye (Secale cereale L. cv Puma), a winter oat (Avena sativa L. cv Kanota), and a spring oat cultivar (Ogle) revealed that freezing injury of leaves of nonacclimated seedlings occurred at -2[deg]C in both the winter and spring cultivars of oat but did not occur in winter rye leaves until after freezing at -4[deg]C. The maximum freezing tolerance was attained in all cultivars after 4 weeks of cold acclimation, and the temperature at which 50% electrolyte leakage occurred decreased to -8[deg]C for spring oat, -10[deg]C for winter oat, and -21[deg]C for winter rye. In protoplasts isolated from leaves of nonacclimated spring oat, expansion-induced lysis was the predominant form of injury over the range of -2 to -4[deg]C. At temperatures lower than -4[deg]C, loss of osmotic responsiveness, which was associated with the formation of the hexagonal II phase in the plasma membrane and subtending lamellae, was the predominant form of injury. In protoplasts isolated from leaves of cold-acclimated oat, loss of osmotic responsiveness was the predominant form of injury at all injurious temperatures; however, the hexagonal II phase was not observed. Rather, injury was associated with the occurrence of localized deviations of the plasma membrane fracture plane to closely appressed lamellae, which we refer to as the “fracture-jump lesion.” Although the freeze-induced lesions in the plasma membrane of protoplasts of spring oat were identical with those reported previously for protoplasts of winter rye, they occurred at significantly higher temperatures that correspond to the lethal freezing temperature.

Antibacterial Efficacy of Gentamicin Encapsulated in pH-Sensitive Liposomes against an In Vivo Salmonella enterica Serovar Typhimurium Intracellular Infection Model

ABSTRACT Encapsulation of gentamicin in liposomes can be used to achieve intracellular delivery and broaden the clinical utility of this drug. We have previously described a novel, rationally designed, pH-sensitive liposomal carrier for gentamicin that has superior in vitro efficacy against intracellular infections compared to the efficacies of both free gentamicin and non-pH-sensitive liposomal controls. This liposomal carrier demonstrated pH-sensitive fusion that was dependent on the presence of unsaturated phosphatidylethanolamine (PE) and the pH-sensitive lipid N-succinyldioleoyl-PE. The pharmacokinetics and biodistribution of the free and liposomal gentamicin were examined in mice bearing a systemic Salmonella enterica serovar Typhimurium infection. Encapsulation of gentamicin in pH-sensitive liposomes significantly increased the concentrations of the drug in plasma compared to those of free gentamicin. Furthermore, the levels of accumulation of drug in the infected liver and spleen were increased by 153- and 437-fold, respectively, as a result of liposomal encapsulation. The increased accumulation of gentamicin in the liver and spleen effected by liposomal delivery was associated with 104-fold greater antibacterial activity than that associated with free gentamicin in a murine salmonellosis model. These pH-sensitive liposomal antibiotic carriers with enhanced in vitro activity could be used to improve both in vivo intracellular drug delivery and biological activity.

Dehydration-induced lamellar-to-hexagonal-II phase transitions in DOPE/DOPC mixtures

Plasma membranes of protoplasts isolated from non-acclimated rye plants undergo a transition from the bilayer to the inverted hexagonal (HII) phase during freeze-induced dehydration at -10 degrees C. It has been suggested (Bryant, G. and Wolfe, J. (1989) Eur. Biophys. J. 16, 369-372) that the differential hydration of various membrane components may induce fluid-fluid demixing of highly hydrated (e.g., PC) from poorly hydrated (PE) components during dehydration. This could yield a PE-enriched domain more prone to form the HII phase. We have examined the lyotropic phase behavior of mixtures of DOPE and DOPC at 20 degrees C by freeze-fracture electron microscopy, differential scanning calorimetry, and X-ray diffraction. HII phase formation was favored by higher proportions of DOPE and lower water contents. Mixtures of 1:1 and 1:3 DOPE/DOPC had a hydration-dependent appearance of two L alpha phases at water contents just above those at which the HII phase occurred. The hydration-dependence of the lamellar repeat spacings suggested that the DOPE-enriched domains preferentially underwent the L alpha-to-HII phase transition. Mixtures of 3:1 DOPE/DOPC did not separate into two L alpha phases during dehydration. These data suggest that the differential hydration characteristics of various membrane components may induce their lateral fluid-fluid demixing during dehydration.

Preclinical pharmacology, toxicology and efficacy of sphingomyelin/cholesterol liposomal vincristine for therapeutic treatment of cancer

Purpose: To establish the pharmacodynamic relationships between drug biodistribution and drug toxicity/efficacy, a comprehensive preclinical evaluation of sphingomyelin/cholesterol (SM/chol) liposomal vincristine and unencapsulated vincristine in mice was undertaken. Methods: Pharmaceutically acceptable formulations of unencapsulated vincristine and liposomal vincristine at drug/lipid ratios of 0.05 or 0.10 (wt/wt) were evaluated for toxicity, antitumor activity and pharmacokinetics following intravenous administration. Results: Mice given liposomal vincristine at 2 mg/kg vincristine had concentrations of vincristine in blood and plasma at least two orders of magnitude greater then those achieved after an identical dose of unencapsulated drug. One day after administration of the liposomal vincristine, there were at least tenfold greater drug quantities, relative to unencapsulated vincristine, in the axillary lymph nodes, heart, inguinal lymph nodes, kidney, liver, skin, small intestines and spleen. Increased plasma and tissue exposure to vincristine as a result of encapsulation in SM/chol liposomes was not associated with increased drug toxicities. Treatment of the murine P388 ascitic tumor with a single intravenous dose of unencapsulated drug at 2, 3 and 4 mg/kg, initiated 1 day after tumor cell inoculation, resulted in a 33 to 38% increase in lifespan. In contrast, long-term survival rates of 50% or more were achieved in all groups treated with the SM/chol liposomal vincristine formulations at doses of 2, 3 and 4 mg/kg. At the 4 mg/kg dose, eight of ten and nine of ten animals survived past day 60 when treated with SM/chol liposomal vincristine prepared at the 0.05 and 0.1 drug/lipid ratios, respectively. Conclusions: Overall, increased and prolonged plasma concentrations of vincristine achieved by liposomal encapsulation were correlated with dramatically increased antitumor activity in comparison with the unencapsulated drug, but no correlations could be established between pharmacokinetic parameters and toxicity.

Encapsulation of Vincristine in Liposomes Reduces its Toxicity and Improves its Anti-Tumor Efficacy

AbstractVincristine is a potent therapeutic agent with activity against a variety of tumor types. It is a cell-cycle specific agent which has exhibited enhanced anti-tumor activity when delivered in liposomal form. Vincristine can be encapsulated into large unilamellar vesicles in response to a transmembrane pH gradient with trapping efficiencies approaching 100%. The extent of vincristine encapsulation, and the subsequent retention of the drug within the liposomes, both in vitro and in vivo, are strongly dependent on the lipid composition of the liposome and on the magnitude of the transmembrane pH gradient. Liposomal formulations of vincristine have been optimized for both liposome circulation longevity, drug retention characteristics and in vivo antitumor activity. When compared to free vincristine, these formulations significantly increase the levels of vincristine remaining in the plasma after i.v. administration. These formulations also significantly increase the delivery of vincristine to tumor sit...

Freeze-Induced Membrane Ultrastructural Alterations in Rye (Secale cereale) Leaves

Freezing injury in protoplasts isolated from leaves of nonaccli-mated rye (Secale cereale cv Puma) is associated with the formation of the inverted hexagonal (HII) phase. However, in protoplasts from cold-acclimated rye, injury is associated with the occurrence of localized deviations in the fracture plane, a lesion referred to as the “fracture-jump lesion.” To establish that these ultrastructural consequences of freezing are not unique to protoplasts, we have examined the manifestations of freezing injury in leaves of non-acclimated and cold-acclimated rye by freeze-fracture electron microscopy. At -10[deg]C, injury in nonacclimated leaves was manifested by the appearance of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and by the frequent occurrence of the HII phase. The HII phase was not observed in leaves of cold-acclimated rye frozen to -35[deg]C. Rather, injury was associated with the occurrence of the fracture-jump lesion between the plasma membrane and closely appressed cytoplasmic membranes. Studies of the time dependence of HII phase formation in nonacclimated leaves indicated that freeze-induced dehydration requires longer times in leaves than in isolated protoplasts. These results demonstrate that the freeze-induced formation of the HII phase in nonacclimated rye and the fracture-jump lesion in cold-acclimated rye are not unique to protoplasts but also occur in the leaves from which the protoplasts are isolated.

Preparation, characterization, and biological analysis of liposomal formulations of vincristine.

Vincristine is a dimeric Catharanthus alkaloid derived from the Madagascan periwinkle that acts by binding to tubulin and blocking metaphase in actively dividing cells. While vincristine is widely used in the treatment of a number of human carcinomas, its use is associated with dose-limiting neurotoxicity, manifested mainly as peripheral neuropathy. It is known that the therapeutic activity of vincristine can be significantly enhanced after its encapsulation in appropriately designed liposomal systems. Enhanced efficacy is also associated with a slight decrease in drug toxicity. Thus, the therapeutic index of vincristine can be enhanced significantly through the use of a liposomal delivery system. Vincristine may be encapsulated into liposomes of varying lipid composition by several techniques, including passive loading, pH gradient loading, and ionophore-assisted loading. However, most research has focused on the encapsulation of vincristine in response to a transbilayer pH gradient, which actively concentrates the drug within the aqueous interior of the liposome. This chapter details the preparation and evaluation of liposomal vincristine. Specifically, we elaborate on the components (choice of lipids, molar proportions, etc.), methods (preparation of liposomes, drug loading methods, etc.), critical design features (size, surface charge, etc.), and key biological endpoints (circulation lifetime, bioavailability, efficacy measurements) important to the development of a formulation of vincristine with enhanced therapeutic properties.

Effects of COR6.6 and COR15am Polypeptides Encoded by COR (Cold-Regulated) Genes of Arabidopsis thaliana on Dehydration-Induced Phase Transitions of Phospholipid Membranes

Cold acclimation of Arabidopsis thaliana includes the expression of cold-regulated (COR) genes and the accumulation of COR polypeptides. The hydration characteristics of two COR polypeptides, COR6.6 and COR15am, have been determined and their effects on the dehydration-induced liquid crystalline-to-gel and lamellar-to-hexagonal II phase transitions in phospholipid mixtures have been examined. After dehydration at osmotic pressures between 8 and 150 MPa, the water content of the COR polypeptides was less than that of bovine serum albumin, with COR15am the least hydrated: bovine serum albumin > COR6.6 > COR15am. Neither COR6.6 nor COR15am altered the dehydration-induced gel lamellar -> fluid lamellar phase transition temperature of either dipalmitoylphosphatidylcholine or dioleoylphosphatidylcholine (DOPC). In multilamellar vesicles of dioleoylphosphatidylethanolamine:DOPC (1:1, mol:mol) prepared by either freeze-thaw or reverse-phase evaporation methods, neither COR6.6, COR15am, nor bovine serum albumin altered the incidence of the dehydration-induced formation of the inverted hexagonal phase as a function of osmotic pressure. However, a specific ultrastructural alteration[mdash] the formation of a striated surface morphology in the lamellar domains[mdash]was observed in mixtures of dioleoylphosphatidylethanolamine:DOPC that were dehydrated in the presence of COR15am. Nevertheless, neither COR6.6 nor COR15am appears to participate in a specific protein-phospholipid interaction that alters the dehydration-induced phase behavior of phospholipid vesicles.