Francis C. Szoka

Distribution in brain of liposomes after convection enhanced delivery; modulation by particle charge, particle diameter, and presence of steric coating

We have investigated the role of diameter, charge, and steric shielding on the brain distribution of liposomes infused by convection enhanced delivery (CED) using both radiolabeled and fluorescent-labeled particles. Liposomes of 40 and 80-nm diameter traveled the same distance but penetrated significantly less than a 10-kDa dextran; whereas 200-nm-diameter liposomes penetrated less than 80 nm liposomes. A neutral liposome shielded by polyethylene glycol (PEG; 2 kDa; 10% by mole) penetrated significantly farther than an unshielded liposome. Even when shielded with PEG, positive surface charge (10% by mole) significantly reduced the penetration radius compared to a neutral or negative charged liposome (10% by mole). A mathematical CED model including a term for liposome cell binding was applied to analyze the radius of particle penetration. Neutral liposomes had a binding constant of k=0.0010+/-0.0002 min-1, whereas for positive charged liposomes k increased 50-fold. The binding constant was independently verified using a degradable lipid radiolabel that eliminated from the brain with a 9.9+/-2.0 h half-life, equivalent to the calculated elimination constant k=0.0012+/-0.0002 min-1. During CED, liposomes accumulated in a subpopulation of perivascular cells within the brain. A non-degradable lipid radiolabel showed that lipid components remained within these perivascular brain cells for at least 2 days. To reduce this uptake, 100-fold molar excess of non-labeled liposomes were co-infused with labeled liposomes, which significantly increased liposome penetration. These studies suggest that optimization of therapeutic CED using particles such as drug-loaded liposomes, polymeric nanoparticles, non-viral DNA complexes, and viruses will require a strategy to overcome particle binding and clearance by cells within the CNS.

Barriers to carrier mediated drug and gene delivery to brain tumors.

Brain tumor patients face a poor prognosis despite significant advances in tumor imaging, neurosurgery and radiation therapy. Potent chemotherapeutic drugs fail when used to treat brain tumors because biochemical and physiological barriers limit drug delivery into the brain. In the past decade a number of strategies have been introduced to increase drug delivery into the brain parenchyma. In particular, direct drug administration into the brain tumor has shown promising results in both animal models and clinical trials. This technique is well suited for the delivery of liposome and polymer drug carriers, which have the potential to provide a sustained level of drug and to reach cellular targets with improved specificity. We will discuss the current approaches that have been used to increase drug delivery into the brain parenchyma in the context of fluid and solute transport into, through and from the brain, with a focus on liposome and polymer drug carriers.

Controlled drug release from a novel liposomal delivery system. II. Transdermal delivery characteristics

Abstract Recently, we reported the evaluation of a prototypal liposomal delivery system, which consisted of a thin agarose gel throughout which a model drug progesterone (PG), associated with multilamellar liposomes, was dispersed. The device was able to: (1) release PG into aqueous buffer solution with essentially zero-order kinetics for at least 24 hours, and (2) modulate PG delivery across hairless mouse skin in vitro. In this paper, we describe further research which examines the influence of lipid formulation on the transdermal delivery of PG from this system. Significant effects on the transdermal delivery of PG by the lipids employed were observed, despite similarities in the release behavior of different formulations into aqueous buffer. For example, transdermal delivery of PG from systems formulated with saturated acyi chain phospholipids (i.e. DMPC and DPPC) was an order of magnitude slower than that from cis-unsaturated phospholipid formulations (i.e. EPC and DOPC). The addition of a cis-unsaturated fatty acid to saturated acyl chain liposome devices increased the delivery rate of PG an order of magnitude. The results suggest the effective co-delivery of drug plus penetration enhancer under both serendipitous and deliberate circumstances.

Controlled drug release from a novel liposomal delivery system. I: Investigation of transdermal potential

Abstract The in vitro release behavior of a novel liposome-based drug delivery device has been characterized. The system consists of a molded agarose matrix in which the model drug (progesterone) was dispersed either free or associated with one of four lipid formulations: egg-phosphatidylcholine (EPC) liposomes, EPC/cholesterol (2:1) liposomes, Intralipid® emulsion, and dipalmitoylphosphatidylcholine (DPPC) liposomes. Drug release rates from the devices into aqueous buffer were measured at 37° C. The free progesterone release rate decreased rapidly over 24 h with over 90% delivered. The liposomal patches, on the other hand, imposed apparent zero-order kinetics: for example, both the EPC and DPPC systems delivered their progesterone payloads at about 1%/h over 24 h. Further, the EPC and DPPC patches significantly slowed transdermal drug delivery across excised hairless mouse skin. The EPC device retarded throughput to one-half the control value, the DPPC system reduced the transport kinetics by an order of magnitude. The results support two hypotheses: (a) the liposomal-based reservoir system can modulate drug input via the skin, (b) the zero-order release of progesterone from liposomes is determined by slow interfacial transport out of the bilayer into the surrounding aqueous medium.

Antitumor Effect of Folate-Targeted Liposomal Doxorubicin in KB Tumor-Bearing Mice after Intravenous Administration

The effect of folate-targeted liposomal doxorubicin (FTL-Dox) has been well characterized in folate receptor (FR) overexpressing tumors in vitro, particularly in KB human carcinoma cells. However, there are few studies evaluating the in vivo efficacy of FTL-Dox in KB murine xenograft models. In this study, we investigated the antitumor activity of FTL-Dox injected intravenously in mice bearing KB tumors. Folate ligands comprising of folate-polyethyleneglycol-distearoylphosphatidylethanolamine (FA-PEG-DSPE) were synthesized with different MW PEG. To design an optimum FTL-Dox formulation for therapeutic studies, we prepared various FTLs and characterized their in vitro targeting and in vivo tissue biodistribution. Mice were administered a single intravenous injection of free Dox, nontargeted PEGylated liposomal Dox (PL-Dox), or FTL-Dox. FTLs and PLs accumulated similarly in tumor tissue, despite FTLs’ faster clearance from circulation. Mice treated with FTL-Dox 20 mg/kg had a slightly greater tumor growth inhibition and almost a 50% increase in life span than mice receiving PL-Dox 20 mg/kg (P = 0.0121; log-rank test). We conclude that FTLs administered systemically have the potential to enhance the delivery of anticancer drugs in vivo; however, their removal by FR expressing normal tissues may have to be blocked if the benefits of tumor targeting are to be realized.

MOLECULAR MODELING OF THE INTESTINAL BILE ACID CARRIER : A COMPARATIVE MOLECULAR FIELD ANALYSIS STUDY

A structure–binding activity relationship for the intestinal bile acidtransporter has been developed using data from a series of bile acid analogsin a comparative molecular field analysis (CoMFA). The studied compoundsconsisted of a series of bile acid–peptide conjugates, withmodifications at the 24 position of the cholic acid sterol nucleus, andcompounds with slight modifications at the 3, 7, and 12 positions. For theCoMFA study, these compounds were divided into a training set and a test set,comprising 25 and 5 molecules, respectively. The best three-dimensionalquantitative structure–activity relationship model found rationalizesthe steric and electrostatic factors which modulate affinity to the bile acidcarrier with a cross-validated, conventional and predictive r2of 0.63, 0.96, and 0.69, respectively, indicating a good predictive model forcarrier affinity. Binding is facilitated by positioning an electronegativemoiety at the 24–27 position, and also by steric bulk at the end of theside chain. The model suggests substitutions at positions 3, 7, 12, and 24that could lead to new substrates with reasonable affinity for the carrier.

Antigen presentation by B cells and macrophages of cytochrome c and its antigenic fragment when conjugated to the surface of liposomes

An in vitro antigen presentation system was used to study how antigens coupled to the surface of phospholipid vesicles (liposomes) are presented to antigen specific T cells. Liposome-bound pigeon cytochrome c (PCC) was 30-40-fold more potent than free PCC when peritoneal macrophages were the presenting cell. B cells presented surface-bound PCC, albeit less efficiently than unmodified PCC. Surface-bound peptide epitope was presented by both cell types, but not as efficiently as unmodified peptide. With the T cell epitope, antigen processing was not required since glutaraldehyde fixed cells could present surface-bound peptide.

Anti-tumor activity of liposome encapsulated fluoroorotic acid as a single agent and in combination with liposome irinotecan.

To test the hypothesis that co-delivery of synergistic drug combinations in the same liposome provides a better anti-tumor effect than the drugs administered in separate liposomes, fluoroorotic acid (FOA) alone and in combination with irinotecan (IRN) were encapsulated in liposomes and evaluated for their anti-tumor activity in the C26 colon carcinoma mouse model. A new chaotropic loading strategy was devised wherein FOA was dissolved in 7 M urea to increase its solubility. This enabled the passive loading of FOA into liposomes at a high concentration. IRN was remote loaded into liposomes that contained the ammonium salt of the multi-valent 1,2,3,4-butanetetracarboxylic acid with a greater than 90% efficiency and at a drug to lipid ratio of 0.2:1. When the two molecules were loaded into the same liposome, FOA was used to remote load IRN. Modulation of the drug/lipid ratio, temperature, and loading time allowed for consistent co-encapsulation of FOA+IRN at various molar ratios. The anti-tumor activity of L-FOA, L-IRN, L-FOA-IRN (5:1), and the L-FOA+L-IRN mixture (5:1) were examined in the C26 mouse model. The maximum tolerated dose of L-FOA was 10 mg/kg given weekly as compared to 100 mg/kg of the non-encapsulated FOA. Delivering two drugs in the same liposome provided a statistically better anti-tumor effect than delivering the drugs in separate liposomes at the same drug ratio. However, the synergistic activity of the 5:1 ratio of free drugs measured on C26 cells in vitro was not observed in the C26 tumor mouse model. These findings point out the challenges to the design of synergistic treatment protocols based upon results from in vitro cytotoxicity studies. L-FOA at 10 mg/kg as a single agent provided the best anti-tumor efficacy which supports previous suggestions that L-FOA has useful properties as a liposome dependent drug.

Use of the intestinal and hepatic bile acid transporters for drug delivery

Abstract There are several possible approaches that can be used to increase the oral absorption of drugs. Recently, utilization of carrier-mediated transport mechanisms in the intestinal tract to increase the bioavailability of drugs has drawn a great deal of attention. This review describes the potential use of this approach, using the bile acid carrier system in both the intestinal tract and the liver. The physiology, ontogeny and molecular biology of the ileal and hepatic transporters are described and an overview is presented of the compounds that are transported by these carriers. Furthermore, the structural requirements for recognition by these carrier systems is presented and the current utilization of this mechanism is reviewed. The importance of carrier-mediated bile acid transport in drug delivery is addressed.

Transdermal iontophoresis of amino acids and peptides in vitro

Abstract The effects of penetrant properties (lipophilicity and charge) and of vehicle pH on the iontophoretically enhanced delivery of amino acids, their N-acetylated derivatives, and eight tripeptides, of the general structure alanine-X-alanine, have been examined in vitro. The penetrants were (a) 9 amino acids (five were zwitterionic, two positively charged and two negatively charged), (b) four N-acetylated amino acids, which carry a net negative charge at pH 7.4, and (c) peptides which were blocked both at the carboxyl terminus using the mixed anhydride reaction with t-butylamine, and at the amino terminus by acetylation with 14C-acetic anhydride; the central residue (X) was varied widely by selecting one of five neutral amino acids, two negatively chargeable moieties (aspartic and glutamic acids), and a positively chargeable species (histidine). Iontophoresis at constant current (0.36 mA/cm2), using Ag/AgCl electrodes, was conducted across freshly excised hairless mouse skin. The diffusion cells used were designed so that both anode and cathode were situated on the same (epidermal) side of a single piece of skin. Overall, it was found that the results of this research support the principle of enhanced peptide delivery across the skin by iontophoresis.