Grace Y. Kao

A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells

The development of long-circulating formulations of liposomes (S-liposomes), sterically stabilized with lipid derivatives of poly(ethylene glycol) (PEG), has increased the likelihood that these liposomes, coupled to targeting ligands such as antibodies, could be used as drug carriers to deliver therapeutic drugs to specific target cell populations in vivo. We have developed a new methodology for attaching monoclonal antibodies to the terminus of PEG on S-liposomes. A new end-group functionalized PEG-lipid derivative pyridylthiopropionoylamino-PEG- distearoylphosphatidylethanolamine (PDP-PEG-DSPE) was synthesized for this purpose. Incorporation of PDP-PEG-DSPE into S-liposomes followed by mild thiolysis of the PDP groups resulted in formation of reactive thiol groups at the periphery of the lipid vesicles. Efficient attachment of maleimide-derivatized antibodies took place under mild conditions even when the content of the functionalized PEG-lipid in S-liposomes was below 1% of total lipid. The resulting S-immunoliposomes showed efficient drug remote loading, slow drug release rates and increased survival times in circulation compared to liposomes lacking PEG. When antibodies recognizing several different tumor-associated antigens were coupled to the PEG terminus of S-liposomes a significant increase in the in vitro binding of liposomes to the target cells was observed. The binding of S-immunoliposomes containing entrapped doxorubicin to their target cell population resulted in increased cytotoxicity compared to liposomes lacking the targeting antibody.

Evaluation of the semicarbazones, thiosemicarbazones and bis-carbohydrazones of some aryl alicycylic ketones for anticonvulsant and other biological propertie

Summary A number of aryl alicyclic ketones were converted to their corresponding semicarbazones, thiosemicarbazones and bis-carbohydrazones. Anticonvulsant activity was displayed by most of the compounds in the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) screens when given intraperitoneally to mice. However, on oral administration to rats, a marked selective activity in the MES screen only was noted. X-ray crystallography on five semicarbazones was undertaken in order to find correlations between the shapes of these molecules and anticonvulsant properties. The thiosemicarbazones displayed greater cytotoxicity to P388D1 and L1210 cells than the semicarbazones while a number of human tumors and different viruses were, in general, insensitive to representative compounds.

A novel, long-circulating, and functional liposomal formulation of antisense oligodeoxynucleotides targeted against MDR1.

The goal of this study was to develop a small, stable liposomal carrier for antisense oligodeoxynucleotides (asODN) that would have high trapping efficiencies and long circulation times in vivo. Traditional cationic liposomes aggregate to large complexes and, when injected intravenously, rapidly accumulate in the liver and lung. We produced charge-neutralized liposome-asODN particles by optimizing the charge interaction between a cationic lipid and negatively charged asODN, followed by a procedure in which a layer of neutral lipids coated the exterior of the cationic lipid-asODN particle. The coated cationic liposomes had an average diameter of 188 nm and entrapped 85–95% of the asODN. The biodistribution and pharmacokinetics of an 18-mer 125I-labeled phosphorothioate ODN formulated by this method were determined after tail vein injection in mice. The majority of the asODN was cleared from blood, with a half-life of >10 hours compared with <1 hour for free asODN. When coupled with an anti-CD19 targeted antibody, this formulation was also effective at delivering an MDR1 asODN to a multidrug-resistant human B-lymphoma cell line in vitro, decreasing the activity of P-glycoprotein. No inhibition was found for nontargeted formulations or for free asODN. A number of therapeutic opportunities exist for the use of small, stable, long-circulating, and targetable liposomal carriers such as this, with high trapping efficiencies for asODN.

Evaluation of some Mannich bases of cycloalkanones and related compounds for cytotoxic activity

Abstract A number of Mannich bases of cycloalkanones and related quaternary ammonium compounds were prepared for cytotoxic evaluation in order to examine the theory that sequential release of alkylating agents produces increased bioactivity compared to related compounds containing only 1 potential alkylating site. Many of the compounds had significant activity against murine L1210 cells and various human tumours. Some correlations between structure and activity were noted but the biological data did not support the view that potential sequential liberation of cytotoxic species produced compounds with increased potency. The formation of various oximes and oxime benzoates as candidate prodrugs was achieved but in general these compounds were not cytotoxic at the concentrations utilized. This observation may be due to the fact that the oximes were much more stable in deuterated phosphate buffered saline over a period of 48 h at 37°C than the Mannich bases, as revealed by 1 H-NMR spectroscopy.

Synthesis and cytotoxic evaluation of some styryl ketones and related compounds

Summary A number of 1-aryl-4-methyl-1-penten-3-ones 1 were converted to the corresponding Mannich bases 2 and analogues 3 . Attempts to form the azines 4 from several members in series 1 led to the isolation of the corresponding pyrazolines 5 or aryl aldehyde azines 6 . Replacement of the isopropyl group of a compound in series 1 by methyl and ethyl functions led to ketones that reacted with hydrazine producing the corresponding azines. The Mannich bases displayed greater activity than the precursor ketones towards murine P388 and L1210 leukemia cells as well as to a panel of human tumour cell lines. Certain of the Mannich bases had selective toxicity towards some human tumour cell lines and others to L1210 cells (in contrast to human T lymphocytes). Several drug-resistant cell lines were shown to be free from cross resistance to a number of the Mannich bases.

Transendothelial Movement of Liposomes In Vitro Mediated by Cancer Cells, Neutrophils or Histamine

A two‐chamber culture system has been used to examine the ability of small liposomes to cross an endothelial cell barrier in response to various stimuli. Transendothelial transit of liposomes was almost negligible in the presence of intact, healthy endothelial cells (EC). Addition of histamine induced a concentration‐dependent increase in the movement of liposomes across the EC monolayer. In the presence of polymorphonuclear neutrophils (PMNs), migrating in response to a chemotactic gradient of N‐Formil‐Met‐Leu‐Phe (fMLP), both liposomes and IgG crossed EC monolayer by a paracellular pathway, largely independent of an association with the PMNs. The presence of cancer cell, growing in the lower chamber or the presence of cancer cell‐conditioned media, also resulted in the passage of liposome across the EC. We conclude that EC monolayers are sufficiently disrupted by several physiologically relevant stimuli to allow for the transendothelial passage of liposomes. These results have important implications for the therapeutic use of liposome in the treatment of cancer or other inflammatory processes.

Use of a Two Chamber Culture Apparatus to Examine Transendothelial movement of liposomes in vitro

AbstractA two chamber culture apparatus has been used to examine the ability of small liposomes to cross an endothelial cell barrier in response to various stimuli. Transendothelial transit of liposomes was almost negligible in the presence of intact, healthy endothelial cell (EC3) monolayers. Inflammatory mediators such as histamine or polymorphonuclear neutrophils (PMISIs), resulted in liposomes or IgG crossing EC monolayers by a paracellular pathway, but only at high concentrations of the mediators. We conclude that EC monolayers can be disrupted sufficiently by inflammatory mediators to allow for the transendothelial passage of liposomes but, because of the high concentrations involved, this is likely to have relevance for the extravascular delivery of liposome-entrapped drugs only in the treatment of inflammation and solid tumors, and not be of general physiological relevance. These results have implications for the type of diseases which would be most susceptible to therapy with liposomal drugs.