A novel thiocationic liposomal formulation of antisense oligonucleotides with activity against Mycobacterium tuberculosis.

This study describes the development of a novel thiocationic (OBEHYTOP) lipid-based formulation of phosphorothioate antisense oligonucleotides (PAOs) showing inhibitory activity against mycobacterium tuberculosis (mTB) as measured by an in vitro BACTEC 460TB assay. PAOs were designed based on sequences complementary to essential regions of the mycobacterial genome from published nucleic acid databases in GenBank. These included the superoxide dismutase sod A gene (TBS3), catalase-peroxidase katG gene (TBK1, TBK10), RNA polymerase beta-subunit rpo B gene (TBR5) and diaminopimelate decarboxylase lys A gene (TBL5). The effect of PAOs (TBS3, K1, K10, R5 and L5) alone on mTB was not significant compared with the no-drug control over a period of exposure of 150 h (ranges of -11.8 to +23.58% at 72 h; 15.26 to +25.82% at 96 h and -5.51 to +24.00% at 150 h). Liposomal formulations (10:5:2 OBEHYTOP:oleic acid:vitamin D3) of PAOs resulted in statistically significant (p < 0.05 in all cases) inhibition (ranges of -51.45 to -63.00% at 72 h; -56.75 to -67.96% at 96 h; -51.45 to -60.26% at 150 h) compared with PAOs alone, thiocationic liposomal control and liposomal components. Positive controls of streptomycin and isoniazid used at their minimum inhibitory concentrations of 2.00 and 0.10 microM, respectively, resulted in average % inhibition values of -94% and -97.36%, respectively, indicating that these thiocationic lipid-formulated PAOs showed inhibitory activity directed against mTB in vitro.

Structural requirements for cationic lipid mediated phosphorothioate oligonucleotides delivery to cells in culture.

A series of 2,3-dialkyloxypropyl quaternary ammonium lipids containing hydroxyalkyl chains on the quaternary amine were synthesized, formulated with dioleoylphosphatidylethanolamine (DOPE) and assayed for their ability to enhance the activity of an intercellular adhesion molecule 1 (ICAM-1) antisense oligonucleotide, ISIS 1570. Cationic liposomes prepared with hydroxyethyl, hydroxypropyl and hydroxybutyl substituted cationic lipid all enhanced the activity of the ICAM-1 antisense oligonucleotide. Cationic lipids containing hydroxypentyl quaternary amines only marginally enhanced the activity of ISIS 1570. Hydroxyethyl cationic lipids synthesized with dimyristyl (Cl4:0) and dioleyl (C18:1) alkyl chains were equally effective. Activity of cationic lipids containing saturated alkyl groups decreased as the chain length increased, i.e. the dimyristyl (C14:0) was more effective than dipalmityl (C16:0) lipid, which was more effective than distearyl (C18:0). The phase transition temperature of cationic lipids containing saturated aliphatic chains was 56 degrees C for the distearyl lipid, 42 degrees C for the dipalmityl lipid and 24 degrees C for the dimyristyl lipid. Cationic lipids with dioleyl alkyl chains required DOPE for activity, with optimal activity occurring at 50 mole%. In contrast, a dimyristyl containing cationic lipid did not require DOPE to enhance the activity of ISIS 1570. Formulation with different phosphatidylethanolamine derivatives, revealed that optimal activity was obtained with DOPE. These studies demonstrate that several cationic lipid species enhance the activity of phosphorothioate antisense oligonucleotides and provide further information on the mechanism by which cationic lipids enhance the activity of phosphorothioate oligodeoxynucleotides.

Lipid prodrugs of phosphonoacids: greatly enhanced antiviral activity of 1-O-octadecyl-sn-glycero-3-phosphonoformate in HIV-1, HSV-1 and HCMV-infected cells, in vitro.

Phosphonoformate (PFA) effectively inhibits viral polymerases but is relatively ineffective in virus-infected cells in tissue culture. A lipid prodrug of phosphonoformate was synthesized by coupling the phosphonate residue of phosphonoformate to the sn-3 hydroxyl of 1-O-octadecyl-sn-glycerol. This prodrug, 1-O-octadecyl-sn-glycero-3-phosphonoformate (ODG-PFA), was 93-fold more active than phosphonoformate in cells infected with the AD169 strain of cytomegalovirus (CMV), and 111-147-fold more active in cells infected with three human clinical isolates of CMV. The compound was also 44-fold more active in human immunodeficiency virus-1 (HIV-1) infected cells and 43-fold more active in cells infected with herpes simplex virus (HSV). Studies of the mechanisms of increased antiviral activity indicate that 1-O-octadecyl-sn-glycero-3-[14C]phosphonoformate is taken up more extensively than the free drug by the host MRC-5 human lung fibroblasts. Intracellular enzymes convert 1-O-octadecyl-sn-glycero-3-phosphonoformate to phosphonoformate. This conversion does not occur in the tissue culture medium containing fetal bovine serum (FBS) or in MRC-5-conditioned medium. In view of its greatly increased in vitro potency and selectivity, 1-O-octadecyl-sn-glycero-3-phosphonoformate may be useful in treating viral diseases.

Phosphatidylazidothymidine and phosphatidyl-ddC: assessment of uptake in mouse lymphoid tissues and antiviral activities in human immunodeficiency virus-infected cells and in Rauscher leukemia virus-infected mice.

During the early stages of human immunodeficiency virus (HIV) infection, although symptoms are absent and viral replication in peripheral blood mononuclear cells is low, substantial levels of HIV replication can be documented in lymphoid tissue [G. Pantaleo, C. Graziosi, J.F. Demarest, L. Butini, M. Montroni, C.H. Fox, J.M. Orenstein, D.P. Kotler, and A.S. Fauci, Nature (London) 362:355-358, 1993, and J. Embretsen, M. Zupancic, J.L. Ribas, A. Burke, P. Racz, K. Tenner-Tacz, and A.T. Haase, Nature (London) 362:359-362, 1993]. This observation suggests that earlier treatment of HIV infection may be indicated and that strategies for enhancing drug targeting to the lymphoid tissue reservoris of HIV infection may be beneficial. To address this issue, we synthesized dioleoylphosphatidyl-ddC (DOP-ddC) and dipalmitoylphosphatidyl-3'-azido-3'-deoxythymidine (DPP-AZT), phospholipid prodrugs which form lipid bilayers and which are readily incorporated into liposomes. The anti-HIV activity of DOP-ddC was similar to that of ddC in HIV type 1-infected HT4-6C cells, but DPP-AZT was considerably less active than AZT in HT4-6C cells. Liposomes containing DOP-[3H]ddC or DPP-[3H]AZT administered intraperitoneally to mice produced greater levels of total radioactivity over time in plasma, spleen, and lymphoid tissue relative to the results with [3H]ddC and [3H]AZT, respectively. DPP-AZT administered intraperitoneally in liposomes as a single daily dose to mice infected with Rauscher leukemia virus prevented increased spleen weight and reverse transcriptase levels in serum with a dose-response roughly comparable to that of AZT given continuously in the drinking water. DOP-ddC, DPP-AZT, and lipid conjugates of other antiretroviral nucleosides may provide higher levels of drug over time in plasma and in lymph nodes and spleen, important reservoirs of HIV infection, and may represent an interesting alternative approach to antiviral nucleoside treatment of AIDS.

Antiviral activity of phosphatidyl-dideoxycytidine in hepatitis B-infected cells and enhanced hepatic uptake in mice.

Dideoxycytidine (ddC) inhibits the replication of hepatitis B virus (HBV) but its clinical use is limited by peripheral neuropathy. We synthesized dioleoylphosphatidyl-ddC (DOP-ddC), a phospholipid prodrug of ddC which forms lipid bilayers and is readily incorporated into liposomes. The 90% effective dose (ED90) of DOP-ddC was 18 microM vs. 7 microM for ddC. However, in HBV-infected human hepatoma cells (2.2.15 cells), DOP-ddC was less toxic in vitro. When liposomal DOP-[5,6-3H]ddC was administered intraperitoneally to mice, drug levels in liver were 40 times greater than [5,6-3H]ddC when expressed as area under curve. Liposomal DOP-ddC also provided higher levels of drug in lymph nodes and spleen, important accessory sites of HBV replication. Plasma levels of drug remained above the ED90 six times longer with DOP-ddC than with ddC. DOP-ddC levels in sciatic nerve, the major site of toxicity, were not significantly different from levels observed with free ddC. The phospholipid prodrug approach is a general one which may readily be applied to other antiviral nucleosides for HBV.

Synthesis and antiviral activity of 3'-azido-3'-deoxythymidine triphosphate distearoylglycerol: a novel phospholipid conjugate of the anti-HIV agent AZT.

Phospholipid conjugates of 3'-azido-3'-deoxythymidine (AZT) show activity against the human immunodeficiency virus (HIV) in vitro. In a previous report (K.Y. Hostetler, L.M. Stuhmiller, B.H.M. Lenting, H. van den Bosch and D.D. Richman (1991), J. Biol. Chem. 265, 6112-6117) the syntheses and anti-HIV activities of AZT mono- and diphosphate diglyceride have been described. We now report on the synthesis, characterization and biological activity of 3'-azido-3'-deoxythymidine triphosphate distearoylglycerol (AZTTP-DSG). The compound was prepared by the condensation of AZT diphosphate with distearoylphosphatidic acid morpholidate in anhydrous pyridine at room temperature and purified by means of high-performance liquid chromatography using a silica column. Characterization was performed with 31P-NMR and IR analyses and determination of the fatty acid, phosphorus and nucleoside content of the product. AZTTP-DSG inhibited HIV-1 replication in both CEM and HT4-6C cells at a level intermediate in potency between its mono- and diphosphate analogs. The IC50 values of AZTTP-DSG were 0.33 and 0.79 microM in these two cell lines, respectively. In addition, AZTTP-DSG was less toxic to CEM cells in vitro than the other AZT liponucleotides and reduced viable cell numbers in this cell type by 50% at 1000 microM. Initial studies on the metabolism of AZTTP-DSG revealed that both AZT and AZT monophosphate were liberated from the lipid pro-drug by a rat liver mitochondrial enzyme preparation. These phospholipid derivatives of AZT nucleotides represent pro-drugs for the intracellular delivery of phosphorylated antiviral nucleoside analogs.

Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations.

The application of cationic liposome reagents has advanced DNA and mRNA transfection research in vitro, and data are accumulating which show their utility for in vivo gene transfer. However, chemical structure-activity data leading to a better mechanistic understanding of their biological activity is still limited. Most of the cationic lipid reagents in use today for this application are formulated as liposomes containing two lipid species, a cationic amphiphile and a neutral phospholipid, typically dioleoylphosphatidylethanolamine (DOPE). The studies reported here examine the effects of some systematic chemical structural changes in both of these lipid components. Cationic and neutral phospholipids were formulated together as large multilamellar vesicles (MLV) or small sonicated unilamellar vesicles (SUV) in water, and each formulation was assayed quantitatively in 96-well microtiter plates under 64 different assay conditions using COS.7 cells and an RSV-beta-galactosidase expression plasmid. The cationic lipid molecules used for these studies were derived from a novel series of 2,3-dialkyloxypropyl quaternary ammonium compounds containing a hydroxyalkyl moiety on the quaternary amine. A homologous series of dioleylalkyl (C18:1) compounds containing increasing hydroxyalkyl chain lengths on the quaternary amine were synthesized, formulated with 50 mol % DOPE, and assayed for transfection activity. The order of efficacy was ethyl > propyl > butyl > pentyl > 2,3-dioleyloxypropyl-1-trimethyl ammonium bromide (DOTMA). DOTMA, which is commercially available under the trademark Lipofectin Reagent, lacks a hydroxyalkyl moiety on the quaternary amine. A homologous series of hydroxyethyl quaternary ammonium derivatives with different alkyl chain substitutions were synthesized, formulated with 50 mol % DOPE, and assayed in the transfection assay. The order of transfection efficacy was dimyristyl (di-C14:0) > dioleyl (di-C18:1) > dipalmityl (di-C16:0) > disteryl (di-C18:0). The addition of 100 microM chloroquine in the transfection experiment enhanced the activity of the dioleyl compound by 4-fold and decreased the activity of the dimyristyl compound by 70%. For each of the compounds and formulations examined in this report, large multilamellar vesicles (MLV; diameter 300-700 nm) were more active than small unilamellar vesicles (SUV; diameter 50-100 nm). The neutral phospholipid requirements for transfection activity in COS.7 cells with these cationic lipid molecules were examined.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyclovir diphosphate dimyristoylglycerol: a phospholipid prodrug with activity against acyclovir-resistant herpes simplex virus.

Infection with herpes simplex viruses (HSVs) resistant to treatment with acyclovir (9-[(2-hydroxyethoxy)-methyl]guanine, Zovirax) is a growing clinical problem in patients with AIDS and other immunosuppressed states. Most virus isolates resistant to acyclovir are deficient or defective in virally coded thymidine kinase (TK), which converts acyclovir to acyclovir monophosphate in virus-infected cells. To restore acyclovir efficacy, we synthesized acyclovir diphosphate dimyristoylglycerol, an analog of a naturally occurring phospholipid, CDP-diacylglycerol. Its biological activity was tested in WI38 human lung fibroblasts infected with the acyclovir-resistant DM21 strain of HSV, which is TK negative due to an 816-base-pair deletion in the TK coding region. Acyclovir diphosphate dimyristoylglycerol has substantial activity in DM21-infected cells (IC50 = 0.25 microM), whereas acyclovir and acyclovir monophosphate were ineffective (IC50 > 100 microM). Similar results were obtained in TK-altered and TK-deficient strains of HSV-1 and in acyclovir-resistant isolates of HSV-2 obtained from two AIDS patients. The phospholipid prodrug is active by means of TK-independent metabolic pathways that liberate acyclovir monophosphate inside the host cell. Acyclovir phosphates were 56 times greater in WI38 human lung fibroblasts incubated for 24 hr with [8-3H]acyclovir diphosphate dimyristoylglycerol relative to acyclovir. Acyclovir monophosphate added to the culture medium (outside the cell) did not circumvent the acyclovir resistance of the TK-negative DM21 mutant, presumably due to its conversion to acyclovir by phosphatases. Acyclovir diphosphate diacylglycerol prodrugs may be useful in treating TK-deficient mutant and wild-type strains of HSV.

The interaction of protein-bound heparin and antithrombin III.

When commercially prepared porcine mucosal heparin is added to human plasma, some of the heparin fractions form a complex with antithrombin III activating it to an immediate inhibitor of thrombin as well as of other serine proteases. Certain fractions of heparin may complex with other proteins such as alpha 2-macroglobulin, another progressive inhibitor of thrombin. Without complexing with antithrombin III, this protein-bound heparin fraction(s) still retains the capacity to activate it to an immediate inhibitor of thrombin. Protamine sulfate inactivates those heparin fractions that bind to antithrombin III but not those bound to alpha 2-macroglobulin. Activated antithrombin III may undergo a molecular change in the presence of protamine which not only changes it back to a progressive inhibitor but makes it resistant to activation by the protein-bound heparin fraction(s). However, it can still be reactivated by other heparin fractions in fresh whole heparin. The observations presented may help explain heparin "rebound" in patients believed adequately neutralized with protamine.