Andreas Supersaxo

Effect of Molecular Weight on the Lymphatic Absorption of Water-Soluble Compounds Following Subcutaneous Administration

The lymphatic absorption of four water-soluble compounds with different molecular weights (MW) was determined by measuring their cumulative recovery in lymph draining from the site of s.c. administration in sheep. The cumulative recoveries (% of dose, mean ± SD; N = 3) were 4.0 ± 1.5 (5-fluoro-2′-deoxyuridine, MW 246.2), 21.0 ± 7.1 (inulin, MW 5200), 38.6 ± 6.7 (cytochrome c, MW 12,300), and 59.5 ± 7.7 [human recombinant interferon (rIFN) alpha-2a, MW 19,000], respectively. Our data show that in the investigated MW range, there is a linear relationship between the molecular weight and the proportion of the dose absorbed lymphatically. An increase in molecular weight results in an increased lymphatic absorption. Molecules with MW > 16,000 are absorbed mainly by the lymphatics which drain the application site. The knowledge gained in this investigation may help to improve the mode of administration and therapeutic efficacy of endogenous proteins whose targets are lymphoid cells (e.g., interferons, interleukins). Practical implications for the clinical use of such proteins are discussed.

Recombinant Human Interferon Alpha-2a: Delivery to Lymphoid Tissue by Selected Modes of Application

The effect of different parenteral administration routes (i.d., s.c., i.v.), infusion rates, and albumin contents of the drug vehicle on the cumulative recovery of recombinant human interferon alpha-2a (rIFN alpha-2a) in lymph and on its concentration in blood and lymph was determined in sheep. Blood samples were withdrawn from a jugular vein catheter and lymph was collected from a cannulated efferent popliteal lymphatic duct. The concentration of rIFN alpha-2a in lymph and blood plasma samples was measured by an enzyme immunoassay. Following i.v. infusion of 2 × 107 U of rIFN alpha-2a, the peak concentrations measured in blood plasma and lymph, respectively, were 8250 and 14 U/ml. The concentration measured in lymph after i.d. or s.c. administration of the same dose was about 105 times higher (peak concentration, 3.1 × 106 U/ml), while blood plasma levels remained low (peak concentration, 315 U/ml). The cumulative recovery of rIFN alpha-2a in lymph following i.d. or s.c. administration was 59.2 ± 7% (mean ± SD; N = 8) and was affected neither by the infusion rate nor by the coadministration of albumin. Our data indicate that following i.d. or s.c. administration, rIFN alpha-2a (MW 19,000) is absorbed mainly by the lymphatics. This results in high levels of rIFN alpha-2a in the lymph which drains from the application site to the regional lymph nodes. The knowledge gained in this investigation may help to improve the mode of administration and therapeutic efficacy of protein drugs whose targets are lymphoid cells.

Mixed Micelles as a Proliposomal, Lymphotropic Drug Carrier

Four lipophilic, low molecular weight drugs solubilized in phosphatidylcholine–bile salt mixed micelles were injected s.c. into the hind legs of sheep and their cumulative recoveries in lymph draining from the site of application were determined. Surprisingly, the cumulative recoveries (percentage of dose) varied between less than 1 and 60%. We found that there is a correlation between the lipophilicity of the drug (log P octanol/water ∼ Rm° value) and the proportion of the dose absorbed by the lymphatic route. Drugs with Rm° values >10 are absorbed preferentially by the lymphatics (>50% of dose), whereas compounds with Rm° values <4 are hardly absorbed at all by the lymphatics (<10% of dose). By applying the prodrug principle we demonstrated that it is also possible to target drugs with Rm° values <4 to the lymphatics. Furthermore, the analysis of the collected lymph samples by gel filtration, quasi-elastic light scattering, and electron microscopy revealed that, following s.c. administration, mixed micelles are converted into homogeneous, unilamellar vesicles. In conclusion, these results suggest that mixed micelles may represent a suitable delivery system for low molecular weight drugs whose targets are lymphoid cells. In addition, for drugs where liposomal application leads to a therapeutic advantage, the thermo-dynamically stable mixed micelle could be a good alternative to the liposome. However, for both applications a high drug lipophilicity is a prerequisite.

N-Acyl-(α,γ Diaminobutyric Acid)n Hydrazide as an Efficient Gene Transfer Vector in Mammalian Cells in Culture

AbstractPurpose. This study investigates the structure/activity relationship of a series of N-acyl-peptides (lipopeptides) for the transfection of mammalian cells. Methods. Lipopeptides comprising 1 to 3 basic amino-acids and a single fatty acid chain were synthesized. Transfecting complexes between lipopeptide, plasmid DNA and dioleoyl phosphatidylethanolamine were prepared and applied on cells in culture. Transfection efficiency was evaluated by measuring β-galactosidase activity 48 h post-transfection. Lipopeptide-DNA binding was also investigated by physical means and molecular modelling. Results. Besides the length of the fatty acid chain, the nature of the basic amino-acid and the C-terminal group were crucial parameters for high transfection efficiency. The N-acyl-(diaminobutyric acid)n derivatives were the most potent transfecting agents among those tested and induced a β-galactosidase activity 2 to 20 times higher than the N-acyl-lysine, -ornithine or -diaminopropionic acid derivatives. Furthermore, a hydrazide C-terminal modification greatly enhanced transfection efficiency for all compounds tested. The reason why α, γ-diaminobutyric acid hydrazide-based lipopeptides were the most potent in transfection is not fully understood but could be related to their high DNA binding. Conclusions. Poly- or oligo-diaminobutyric acid containing or not a hydrazide C-terminus could advantageously be used in peptide-based gene delivery systems.

Transient Transfection in Mammalian Cells

The aim of the presented work was to develop a cost-effective and easily scaleable transient transfection system with mammalian cells grown in serum-free suspension culture. For this purpose the cationic polyethylenimine (PEI) and the novel hybrid molecule dioleoyl-melittin (DOM) were used. Both substances are highly efficient transfection reagents for mammalian cells and have been described recently. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were performed in fresh medium (1/10 culture volume) and then added to the cells (indirect method).

Synergistic Enhancement of Transient Expression by Dioleoyl-Melittin (DOM) and Polyethylenimine (PEI) in Mammalian Cells in Suspensionculture

Dioleoyl-melittin, which is a conjugate of dioleoyl-phosphatidyl-ethanolamine-N-3-(2-pyridyldithio) propionate with the amphipatic peptide melittin Gly-Cys 1 represents a new class of peptide-based reagent for efficient transfection of mammalian cells. In this work we investigated the transfection efficiency of dioleoyl-melittin (DOM) combined with polyethylenimine (PEI) using HEK293(EBNA) cells grown in serum-free suspension cultures. Gene expression was monitored using the luciferase reporter gene and the human soluble TNF receptor p55 gene (TNFRp55) inserted into different vectors. Our data clearly show that DOM together with PEI exhibited synergistic enhancement for gene expression in EBNA cells. At the optimal DNA-DOM-PEI weight ratio the efficiency of transfection increases significantly compared to corresponding PEI and DOM transfection at low DNA concentration. In summary, our data show that dioleoyl-melittin and polyethylenimine act synergistically in transfecting 293(EBNA) cells grown in serum-free suspension culture.