N-acyl-(alpha, gamma diaminobutyric acid)n hydrazide as an efficient gene transfer vector in mammalian cells in culture.

PURPOSE: 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 beta-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 beta-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 alpha, gamma-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.

Sebaceous-gland deposition of isotretinoin after topical application: an in vitro study using human facial skin.

As yet, topically applied isotretinoin fails to show convincing clinical efficacy in the treatment of severe recalcitrant acne. Although the reason for this is not known, it is possible that topical application results in low, pharmacologically inactive isotretinoin concentrations in the sebaceous glands, the most likely site of isotretinoin action. It has been suggested that topically applied liposomes enhance the delivery of drugs into the sebaceous glands. Accordingly, we compared the isotretinoin concentration in sebaceous glands and other skin compartments following topical application of small unilamellar vesicles, multilamellar vesicles, preformed vesicles (Natipide II) or mixed micelles of lecithin and bile salt. We found that the concentration of isotretinoin measured in the sebaceous glands varied between 0.17 and 1.57 ng/mg tissue. The comparison between ethanolic gel and liposomal or micellar gel did not reveal any significant difference. However, application of the Natipide formulation resulted in significantly lower isotretinoin concentrations in the sebaceous glands when compared to the ethanolic gel. Autoradiography and fluorescence microscopy indicated that isotretinoin penetrated the sebaceous glands along the follicular route. In conclusion, our in vitro study showed that, following topical administration, substantial amounts of isotretinoin were delivered to the sebaceous glands via the follicular route, whereby the ethanolic gel was as efficient as a liposomal or a mixed micellar gel.

Dioleoylmelittin as a novel serum-insensitive reagent for efficient transfection of mammalian cells.

Amphipathic peptides can be useful effectors to enhance gene delivery. However, peptide/DNA complexes usually require additional effectors, such as fusogenic lipids, to mediate efficient transfection. Due to weak and/or multiple interactions between the various components of the system, the transfecting complexes are often heterogeneous and unstable in biological fluids. Accordingly, a hybrid molecule resulting from the covalent coupling of an amphipathic, membrane-disturbing peptide to a lipid moiety might create a stable and efficient peptide-based gene transfer system. The present work describes such a novel hybrid molecule, dioleoylmelittin, resulting from the conjugation of dioleoylphosphatidylethanolamine-N-[3-(2-pyridyldithio)propionate] with [Cys1]melittin. Dioleoylmelittin had a lower hemolytic and membrane-disturbing activity than melittin. Size and zeta potential measurements, DNA gel electrophoresis, and electron microscopy showed that dioleoylmelittin, unlike melittin, was able to complex plasmid DNA to form spherical particles with a net positive charge and a diameter between 50 and 250 nm. These particles, prepared at an optimal 10/1 dioleoylmelittin/DNA ratio (w/w), mediated efficient transient transfection of reporter genes in cultured mammalian cells including primary cells. The luciferase activity induced by the dioleoylmelittin/DNA complex was 5-500-fold higher than that induced by a cationic lipid/DNA complex, depending on the cationic lipid and the cell-line. Surprisingly, the presence of 10-50% fetal calf serum during dioleoylmelittin-mediated transfection enhanced 1.5-3-fold gene expression. Dioleoylmelittin represents a new class of efficient peptide-based transfection reagents, especially suited for serum-sensitive cells.

Short-chain phospholipids enhance amphipathic peptide-mediated gene transfer.

Addition of short-chain phospholipids to the gramicidin S-DNA-dioleoyl phosphatidylethanolamine complex enhanced up to 6-fold beta-galactosidase expression in several cell-lines in vitro. Among the compounds tested, the most potent in enhancing transfection were the dicapryl- and the dicapryloyl phosphatidylcholine. In contrast, no significant enhancement of transfection was seen when short-chain phospholipids were mixed with cationic lipids. Short-chain phospholipid and gramicidin S may act simultaneously on the cell membrane to enhance gene transfer, yet resulting in no toxicity.

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 approximately Rm degrees value) and the proportion of the dose absorbed by the lymphatic route. Drugs with Rm degrees values greater than 10 are absorbed preferentially by the lymphatics (greater than 50% of dose), whereas compounds with Rm degrees values less than 4 are hardly absorbed at all by the lymphatics (less than 10% of dose). By applying the prodrug principle we demonstrated that it is also possible to target drugs with Rm degrees values less than 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 thermodynamically stable mixed micelle could be a good alternative to the liposome. However, for both applications a high drug lipophilicity is a prerequisite.

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 greater than 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 x 10(7) 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 10(5) times higher (peak concentration, 3.1 x 10(6) 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.

Effect of interferon-alpha-2a on the output of recirculating lymphocytes from single lymph nodes.

The output of recirculating lymphocytes from cannulated popliteal lymph nodes in sheep was measured after administration of human recombinant interferon (rIFN)-alpha-2a. Interferon (IFN) injection caused a dramatic decrease in lymphocyte output from lymph nodes. Following a single s.c. or i.d. injection of 2 x 10(7) U IFN into the drainage area of the popliteal lymph node, lymphocyte output fell to below 1% of the pre-treatment level and remained depressed for up to 35 hr. A substantial decrease in lymphocyte output from cannulated nodes also occurred after IFN was injected either i.v., into the skin of the opposite non-cannulated hind leg or into an afferent lymphatic vessel leading to the popliteal lymph node. After the period of depressed lymphocyte output, a seemingly compensatory surge of cell traffic occurred that lasted 2-3 days. During this phase there was a relative increase in the proportion of CD4+ T cells in lymph. Similar changes occurred after each treatment in animals given multiple doses of IFN. These effects are unlikely to be antigen-induced since there was no blast cell response in any treated animal. The analysis of blood and lymph plasma samples showed that the most severe depression of lymphocyte output was associated with high levels of IFN, while there was no apparent correlation between the reduction in lymphocyte traffic and concentrations of cortisol in plasma. These results suggest that IFN-alpha-2a is involved directly in the regulation of lymphocyte output from lymph nodes.

The antitumour effect of lipophilic derivatives of 5-fluoro-2'-deoxyuridine incorporated into liposomes.

Lipophilic prodrugs of 5-fluoro-2'-deoxyuridine (FUdR), namely 5'-O-palmitoyl-5-fluoro-2'-deoxyuridine (5'-O-palm-FUdR) and 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine (3',5'-O-dipalm-FUdR), were incorporated into bilayer liposomes. Prodrug incorporation into positively charged liposomes was quantitative and stable, homogeneous bilayer vesicles were obtained. The maximal amounts of prodrug incorporation are 200 micrograms for 5'-O-palm-FUdR and 90 micrograms for 3',5'-O-dipalm-FUdR per mg egg phosphatidylcholine as matrix lipid. The prodrug-liposome preparations were tested in vivo against mammary carcinoma 13/C, Lewis lung carcinoma and L1210 leukaemia and compared to the cytostatic activity of free FUdR and of the prodrugs dissolved in peanut oil. Intraperitoneally administered prodrugs either incorporated into liposomes or dissolved in peanut oil inhibited tumour growth in all animals. The comparison of the doses required for tumour growth inhibition showed that both prodrugs were active at concentrations 20-75 times lower as compared to unmodified FUdR. However, due to the increased toxicity of the prodrug-liposome preparations, the therapeutic index of the parent drug FUdR could not be improved. The cytostatic effect of the prodrug preparations may be explained by altered pharmacokinetic properties of the FUdR derivatives and the additional sustained release action the liposomes are providing. A further increase of the antitumour activity may be obtained by the attachment of tumour-specific antibodies to the surface of such prodrug-containing liposomes.

[Liposomes as carriers of lipophilic cytosine arabinoside and fluorodeoxyuridine derivatives. Their cytostatic effect and possibilities of tumor cell specific therapy]. / Liposomen als Träger von lipophilen Cytosinarabinosid- und Fluorodeoxyuridin-Derivaten. Ihre zytostatische Wirkung und Möglichkeiten zur tumorzellspezifischen Therapie.

A method of preparation for large volumes of sterile, homogenous bilayer liposomes as carriers of lipophilic cytostatic prodrugs is described. Liposomes in the size range of 60 to 120 nanometers are produced through the dialysis of lipid/prodrug/detergent micelles by means of a capillary dialysis system. The cytostatic effect of cytosine arabinoside (ara-C) prodrug liposomes against L1210 leukemia showed to be superior to free ara-C treatment in respect to drug dosage and treatment schedule. Fluorodeoxyuridine (FUdR) prodrug-liposomes were active against various solid tumors, although with less pronounced effects, but at 20-60 times lower drug concentrations as compared to free FUdR. To further improve the cytostatic effect of such prodrug-containing liposomes, methods for the linkage of tumor-cell-specific antibodies to the liposomes are discussed. The targeted delivery of cytostatic drugs by such means might improve their antitumor effects and reduce the untoward toxic side-effects.

Treatment of murine L1210 lymphoid leukemia and melanoma B16 with lipophilic cytosine arabinoside prodrugs incorporated into unilamellar liposomes.

Lipophilic prodrugs of I-beta-D-arabinofuranosyl cytosine (Ara-C), namely N4- and 5'-oleyl-I-beta-D-arabinofuranosyl cytosine (N4-oleyl-ara-C, 5'-oleyl-ara-C) and N4-palmitoyl-I-beta-D-arabinofuranosyl cytosine (N4-palm-ara-C) were incorporated into liposomes of various lipid compositions. The phospholipid vesicles were prepared by controlled dialysis of lipid/prodrug/detergent micelles yielding homogeneous and stable unilamellar liposomes. The liposome size ranged from 70 to 120 nm depending on the lipid composition and the amounts of prodrug incorporated. Depending on the total amount of micellized Ara-C prodrugs, a maximal incorporation rate of 250 micrograms prodrug per mg egg phosphatidylcholine was achieved. The incorporation efficiency was in the range of 85 to 97%. The in vivo antitumor activity of the liposome preparations against L1210 lymphoid leukemia was clearly superior by factors of 2-8 depending on the therapy schedule and route of drug application. The treatment of melanoma B16 with free Ara-C as well as with the prodrug-liposomes exhibit rather weak antitumor activity. Drug application by means of prodrug-liposomes yields equal or higher tumor-inhibitory effects at drug concentrations which were 2-4 times lower than those of free Ara-C. Although drug tolerance and myelosuppression studies with Swiss albino mice revealed that the prodrug-liposomes were 5-10 times more toxic than free Ara-C, a substantial improvement of efficiency could be observed. The incorporation of the ara-C prodrugs into liposomes provides protection against fast degradation and systemic elimination which might be an explanation for the improved antitumor effect of these preparations.

5'-O-Palmitoyl- and 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine--novel lipophilic analogues of 5'-fluoro-2'-deoxyuridine: synthesis, incorporation into liposomes and preliminary biological results.

5'-O-palmitoyl- and 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine were prepared by the reaction of 5-fluoro-2'-deoxyuridine in dimethylacetamide with palmitic acid chloride. The incorporation of the synthesized prodrugs into liposomes composed of egg phosphatidylcholine/stearylamine/cholesterol/alpha-tocopherol at a molar ratio of 10:1:2:0.05 was nearly quantitative; homogeneous bilayer vesicles (75 nm diameter) were obtained. Preliminary tolerance studies revealed that the prodrug-liposome preparations are about 20-60 times more toxic than the parent drug. The prodrugs incorporated into liposomes were 10 to 30 times more active against murine colon 38 carcinoma compared to the free drug. In comparison to the administration of the prodrugs in peanut oil the liposomal preparations seem to exert improved effects and represent a valuable drug delivery system for parenteral applications.