In vitro drug delivery mediated by ecto-NAD+-glycohydrolase ligand-targeted liposomes.

We have studied the growth-inhibitory potency of methotrexate and methotrexate-gamma-aspartate encapsulated in liposomes conjugated to ligands of ecto-NAD+-glycohydrolase (Salord, J. et al., Biochim. Biophys. Acta 886 (1986) 64-75). The ability of targeted liposomes to enhance growth inhibition, which amounted to a 4-fold reduction of the drug concentration required to inhibit cell growth by 50% as compared to nontargeted liposomes, was observed only with cells expressing this ecto-enzyme activity, i.e., Swiss 3T3 fibroblasts and RAJI, a Burkitt-type lymphoma cell line. Delivery of the encapsulated drugs was inhibited by NH4Cl and varied with the endocytic capacity of the cells. Only small unilamellar vesicles affected the growth of the lymphoma cells, whereas the fibroblasts were more sensitive to large unilamellar vesicles. With vesicles of appropriate size, there was a good correlation between the specific binding of the targeted liposomes to cells and drug delivery. Our results suggest that ecto-NAD+-glycohydrolase can provide a recognition site on target cells and mediate the internalization of targeted liposomes by a mechanism most probably related to adsorptive endocytosis.

Preparation of neo-galactosylated liposomes and their interaction with mouse peritoneal macrophages.

In order to target liposomes to cells expressing at their surface a galactose-binding site we have prepared liposomes containing new synthetic galactolipids. Neo-galactosylated liposomes were prepared by covalently coupling beta-D-1-thiogalactopyranoside residues, substituted with a hydrophilic spacer-arm and functionalized with a sulfhydryl group, to preformed large unilamelar vesicles containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. The vesicles, having a galactose content above a threshold value of about 5 mol%, could be aggregated with Ricinus communis agglutinin. This aggregation was reversed by addition of excess free methyl beta-D-galactopyranoside, indicating that the surface glucidic moieties of these liposomes were accessible to the lectin. Compared to the control vesicles, the neo-galactosylated liposomes (containing 15 mol% galactose) presented in vitro an increased binding to cell possessing a beta-D-galactose specific receptor, i.e. resident mouse peritoneal macrophages. At 4 degrees C, the specific binding was about 2-fold, whereas at 37 degrees C it was increased to about 4-5-fold. This differential binding was largely unaffected by serum and, interestingly was much dependent on the degree of galactosylation of the liposomes, i.e. a threshold value of 5 mol% was needed to observe an increased binding of the targeted vesicles to the macrophages.

Studies on polyamine biosynthesis in Euglena gracilis.

Euglene gracilis (strain Z) was found to contain five polyamines which could be separated by high-pressure cation-exchange chromatography. 1,3-Diaminopropane, putrescine, norspermidine (N-(3-aminopropyl)-1,3-diaminopropane), spermidine and norspermine (N,N'-bis(aminopropyl)-1,3-diaminopropane) were identified. Biosynthesis of putrescine in E. gracilis proceeds through decarboxylation of L-ornithine, no arginine decarboxylase (EC 4.1.1.19) activity could be detected. The properties of the enzymes ornithine decarboxylase (EC 4.1.1.17) and S-adenosylmethionine decarboxylase (EC 4.1.1.50) in this alga were found to be similar to those of the enzymes isolated from animal tissues or yeast cells. A bioxynthetic scheme is proposed which relates the different polyamines occurring in E. gracilis.

Comparative role of polyamines in division and plastid differentiation of Euglena gracilis.

Regulation of polyamine biosynthesis during growth and differentiation of Euglena gracilis was investigated. Increased activity of L-ornithine decarboxylase (EC 4.1.1.17), the enzyme which catalyzes the initial step in polyamine synthesis in Euglena, and accumulation of polyamines were observed prior to DNA replication in synchronous cultures of heterotrophically or photoautotrophically grown cells. In photoautotrophic cells three maxima of polyamine synthesis were observed during the light period of the cell cycle. The transition form quiescence to active growth was accompanied in heterotrophic Euglena by a very large stimulation of ornithine decarboxylase activity and polyamine synthesis; the decrease in growth potential of these cells was correlated with a decrease in polyamine levels. In contrast, differentiation of Euglena i.e. a shift from heterotrophic to photoautotrophic mode of living in the absence of division, led only to a minor stimulation of polyamine biosynthesis. Alpha-Methylornithine, an inhibitor of ornithine decarboxylase, blocked the growth of heterotrophic Euglena, and depletion of intracellular polyamines decreased the differentiation rate. Both events could be reversed by addition of putrescine to the growth medium. This study suggests that Euglena requires a minimal intracellular level of polyamines to grow and differentiate under optimal conditions. This requirement seems to be more stringent for cell division.

Neo-mannosylated liposomes: synthesis and interaction with mouse Kupffer cells and resident peritoneal macrophages.

In order to target liposomes to cells expressing at their surface mannose receptors, e.g. mouse Kupffer cells and peritoneal macrophages, we have developed a new synthetic strategy which allows a chemically well defined preparation of neo-mannosylated vesicles. alpha-D-Thiomannopyranoside residues, substituted with a hydrophilic spacer arm and functionalized with a sulfhydryl group, were covalently coupled to preformed large unilamellar vesicles containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. Liposomes, containing 15 mol% of mannosyl residues, were specifically aggregated with concanavalin A; this aggregation could be reversed by an excess of free methyl alpha-D-mannopyranoside indicating that the surface ligands were freely accessible to the lectin. The neo-mannosylated liposomes presented in vitro an increased binding to cells possessing alpha-D-mannose specific binding sites. At 37 degrees C a specific binding, up to 9-fold compared to control vesicles, was observed. These neo-mannosylated vesicles represent attractive tools for targeting bio-active molecules to macrophage-associated diseases.

Enhancement of gene delivery by an analogue of alpha-MSH in a receptor-independent fashion.

In order to transfect melanoma specifically by receptor-mediated endocytosis we prepared dioctadecyl aminoglycylspermine (lipospermine)--DNA complexes with [Nle(4),D-Phe(7)]-alpha-MSH(4--10), a pseudo-peptide analogue of alpha-melanocyte stimulating hormone (alpha-MSH) linked to a thiol-reactive phospholipid. With these complexes we obtained an up to 70-fold increase of transfection with B16-F1 melanoma cells. However when B16-G4F, an alpha-MSH receptor negative melanoma cell line was transfected, an up to 700-fold increased transfection efficiency was observed. The peptide hormone analogue was equally efficient when it was only mixed with lipospermine--DNA complexes without covalent coupling. In addition to melanoma cells we also obtained up to 30-fold increased transfection with BN cells (embryonic liver cells). Our data show that an alpha-MSH analogue increased transfection independently of the MSH receptor expression but reaches efficiencies approaching those obtained with peptides derived from viral fusion proteins. The absence of targeting of constructs containing [Nle(4),D-Phe(7)]-alpha-MSH(4-10) can probably be attributed due to the relatively modest number of MSH receptors at the surface of melanoma. We suggest, however, that the peptide hormone analogue used in this study has membrane-active properties and could be of interest as helper agent to enhance non-viral gene delivery presumably by endosomal-destabilizing properties.

Targeting of liposomes by covalent coupling with ecto-NAD+-glycohydrolase ligands.

We have tested the feasibility of targeting liposomes via interaction with specific ecto-enzymes, i.e., enzymes which have their active site oriented to the external surface of the cell. 3,4-Dimethylpyridine adenine dinucleotide, a competitive inhibitor of ecto-NAD+-glycohydrolase, was substituted at N6 with a hydrophilic spacer arm, functionalized with a sulfhydryl group, and covalently linked to performed liposomes containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. We show that compared to control vesicles, the binding of the conjugated liposomes was greatly increased (up to 5-fold) to cells presenting ecto-NAD+-glycohydrolase activity (Swiss 3T3 fibroblasts, mouse peritoneal macrophages); in contrast, no specific binding was detected with hepatoma tissue culture cells, which lack this enzyme. Specific binding was found to depend on the ligand/lipid molar ratio of the vesicles and on the length of the arm. High concentrations of free 3,4-dimethylpyridine adenine dinucleotide virtually abolished the specific binding to cells of the targeted liposomes. Analysis of binding revealed that the ligand conjugated to the liposomes presented a functional affinity for 3T3 fibroblasts 15-fold superior to that of the free ligand.

Characterization of surface markers of continuously growing murine resident macrophages.

Conditions have been described which allow an in vitro indefinite multiplication of differentiated murine macrophages (Lombard et al: Biol Cell 53, 219, 1985). R. and MAY-1 cell lines, which were obtained, respectively, from mouse (Balb/c) spleen and resident peritoneal macrophages, have been further characterized. They present at their surface, besides the Mac-1 antigen and Fc-receptor, a mannose receptor which was characterized for its binding properties. This receptor is responsive for a specific phagocytosis of mannosylated particles, i.e., mannosylated latex beads or oil droplets containing mannosylated bovine serum albumin. Moreover, R and MAY-1 cells present an ectoenzyme profile (NAD+ glycohydrolase and nucleotide pyrophosphatase) similar to those of the corresponding resident macrophages.

Synthetic lipopeptides incorporated in liposomes: in vitro stimulation of the proliferation of murine splenocytes and in vivo induction of an immune response against a peptide antigen.

Amphiphilic lipopeptides, such as Pam3CysAlaGly and Pam3CysSerSer, were synthesized and incorporated into liposomes, and their ability to induce the proliferation of BALB/c mouse splenocyte was tested in vitro. When compared to monophosphoryl lipid A (MPL) the following potency order was found: liposomal lipopeptides > liposomal MPL > free (emulsified) lipopeptides. These results strongly depend on the size of the vesicles used: a mitogenic effect was observed only with lipopeptides incorporated within vesicles of diameter < or = 100 nm while lipopeptides in larger vesicles (diameter approximately 300 nm) gave no response. This may be related to the necessity for the liposome-associated lipopeptides to be endocytosed to reach putative intracellular targets. As immunoadjuvanticity seems to be linked to B-lymphocyte activation, the lipopeptides represent attractive alternatives to MPL for the realization of completely synthetic liposome-based peptide vaccine formulations. This was borne out by showing that Pam3CysAlaGly and Pam3CysSerSer, when incorporated in small unilamellar vesicles carrying a covalently conjugated synthetic peptide of sequence IRGERA, corresponding to an epitope of the C-terminal region of histone H3, were able to induce a potent and long-lasting immune response.

Induction of immune response against a short synthetic peptide antigen coupled to small neutral liposomes containing monophosphoryl lipid A.

We have investigated the parameters affecting the immunogenicity of a short synthetic hexapeptide associated with liposomes. The model peptide used had the sequence IRGERA which corresponds to the C-terminal hexapeptide region of histone H3. Immunogenicity was measured by the ability of anti-peptide antibodies to cross-react with the parent protein. By itself, the peptide was not able to induce significant antibody production. However, liposomes were shown to be able to render the peptide immunogenic, nevertheless a number of parameters were important: to be immunogenic the peptide had to be surface bound, rather than entrapped within the liposomes, and an adjuvant, monophosphoryl lipid A (MPLA), had to be present in the same population of liposomes. Additionally, the intensity and duration of the immune response were found to be dependent both on the charge of the liposomes; neutral liposomes yielding a longer lasting response than negatively charged liposomes, and on the immunisation schedule where a long time period between immunisation and boosting yielded a better result than a short time period. To account for these phenomena we propose a model in which surface-bound antigen targets liposomal MPLA to B lymphocytes specific for the antigen. These results demonstrate that liposomes containing the non-toxic adjuvant MPLA can act as carriers to induce a long-lasting IgG response against peptides, eliminating the need of protein carriers and conventional adjuvants. Such an approach may be useful for designing synthetic vaccines.

Squalene epoxide cyclase and microemulsion.

Squalene epoxide cyclase was extracted from microsomal preparations of rat liver using anionic, cationic and non-ionic microemulsions. The anionic microemulsion was the best with respect to protein solubilisation, extracted cyclase activity and stability of this activity over time. The activity assay showed cyclase activity to be higher in anionic microemulsion than in buffer in the presence of surfactant. Calcium chloride in the anionic microemulsion had a stabilising effect and less total protein seemed to be extracted.

Immunogenicity of new heterobifunctional cross-linking reagents used in the conjugation of synthetic peptides to liposomes.

We have investigated the immunogenicity of six thiol-reactive heterobifunctional cross-linking reagents that permit the conjugation of cysteine carrying peptides to the surface of liposome containing monophosphoryl lipid A. Such constructs elicit an immune response against short synthetic peptides and our aim was to find the least immunogenic linkers to limit potential carrier-induced epitopic suppression. For that purpose the properties of three new polyoxyethylene linkers of different lengths and thiol-reactive moieties (maleimide, bromoacetyl, dithiopyridine) were compared to known derivatives obtained by reacting the classical reagents SMPB and SPDP or N-succinimidyl bromoacetate with phosphatidylethanolamine. The least immunogenic linkers were the bromoacetate derivatives whereas those containing a maleimide group evoked a significant anti-linker immune response. In addition, using IRGERA as a model peptide, we found that all six liposomal constructs strongly elicited the production of anti-peptide IgG antibodies. This immune response was therefore independent of the length of the linkers (ranging between 0.3 and 1.6 nm) and of the nature of the linkage. between the peptide and the thiol-reactive moieties of the cross-linkers, i.e. stable thioether or bio-reducible disulfide bonds.

Structural and stereoelectronic requirements for the inhibition of mammalian 2,3-oxidosqualene cyclase by substituted isoquinoline derivatives.

2,3-Oxidosqualene lanosterol-cyclase (OSC; EC 5.4.99.7) is an attractive target for the design of compounds that block hepatic cholesterol biosynthesis. (4a alpha, 5 alpha, 6 beta, 8a beta)-Decahydro-5,8a-dimethyl-2-(1,5,9-trimethyldecyl)-6- isoquinolinol (1) and simplified analogs have been devised to inhibit this enzyme by mimicking the postulated pro-C-8 high-energy intermediary carbocation occurring during the cyclization-rearrangement pathway. In order to gain an understanding into the mechanism by which these types of molecules inhibit OSC, we have synthesized a series of substituted isoquinoline derivatives 3 and investigated the structural and stereoelectronic requirements, and their stringency, that make 3 potential high-energy intermediate analogs of OSC. Determination of the IC50 values of the different compounds with rat liver microsomal cyclase, allowed the study of the relative importance of (i) the nature and the stereochemistry of the nitrogen side chain, (ii) the presence of methyl groups at C-5 and C-8a (ring junction), (iii) the presence and stereochemistry of the C-6 hydroxyl group, (iv) the nature of the ring junction, and (v) the absolute configuration of the bicyclic system. The resulting structure-activity relationships seem to validate the mechanism of action of these inhibitors as analogs of a pro-C-8 high-energy intermediate and delineate the minimal requirements for the design of efficient isoquinoline-based, or simplified, OSC inhibitors.

Effects of a 2,3-oxidosqualene-lanosterol cyclase inhibitor 2,3:22,23-dioxidosqualene and 24,25-epoxycholesterol on the regulation of cholesterol biosynthesis in human hepatoma cell line HepG2.

N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol (8-azadecalin 1), a high-energy intermediate analogue for the 2,3-oxidosqualene-lanosterol cyclase, was found to be a powerful (IC50 approximately 0.1 microM) inhibitor of cholesterol biosynthesis in human hepatoma HepG2 cells. In analogy with other mammalian cells grown in the presence of cyclase inhibitors, the decrease in C27-sterol formation was accompanied by an accumulation of 2,3-oxidosqualene, 2,3:22, 23-dioxidosqualene, and by the formation of a compound characterized as 24,25-epoxycholesterol, a repressor of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase activity. In order to assess the cyclase as a potential pharmacological target for the design of hypocholesterolemic drugs, it is important to test whether inhibitors of this enzyme are able to act synergistically on the biosynthesis of cholesterol, i.e. by decreasing the amount of lanosterol formed and by repressing the regulatory HMG-CoA reductase via the formation of regulatory oxysterols. The accumulation of 24,25-epoxycholesterol in relationship to the decrease of C27-sterol biosynthesis and of HMG-CoA reductase activity showed only a partial correlation: e.g. at [1] = 100 x IC50 only a 50% reduction in enzyme activity could be attained. In contrast, when HepG2 cells were treated with 2,3:22,23-dioxidosqualene or 24,25-epoxycholesterol, excellent correlations were found between the inhibition of C27-sterol biosynthesis and the repression of HMG-CoA reductase activity, which was almost complete at the highest concentrations of these epoxides (10(-5) M). Altogether, our results suggest that treatment of HepG2 cells with a cyclase inhibitor such as 8-azadecalin (1) does not lead to an intracellular accumulation of repressor molecules high enough to fully trigger a regulatory pathway resulting in a complete down-regulation of HMG-CoA reductase. At intermediary concentrations of cyclase inhibitors (IC50), however, a synergistic mode of action of these inhibitors seems plausible.

Inhibition of cholesterol biosynthesis in 3T3 fibroblasts by 2-aza-2,3-dihydrosqualene, a rationally designed 2,3-oxidosqualene cyclase inhibitor.

The effect of 2-aza-2,3-dihydrosqualene, a new compound designed to inhibit the 2,3-oxidosqualene-lanosterol cyclase [A. Duriatti et al., Biochem. Pharmac. 34, 2765 (1985)] was studied as inhibitor of cholesterol biosynthesis in Swiss 3T3 fibroblasts. Treatment with the drug of cells which were grown for 2 days in a delipidated medium resulted in a marked decrease of [14C]acetate incorporation into the C27-sterol fraction. An IC50 = 0.3 microM was calculated when the cells were preincubated for a period of 4 hr with 2-aza-2,3-dihydrosqualene. This inhibition was correlated with an intracellular accumulation of 2,3-[14C]oxidosqualene and of 2,3:22,23-[14C]dioxidosqualene, indicating that the cyclase was indeed an intracellular target of the drug. A precursor-product relationship of the accumulated [14C]squalene oxide(s) and the [14C]sterols was demonstrated in chase experiments in the absence of drug. Sterols more polar than cholesterol were also detected in treated fibroblasts and in the cells which underwent chase experiments; they were mainly composed of 24,25-epoxycholesterol. The C27-[14C]sterols of [14C]acetate pulse labeled cells consisted in a mixture of desmosterol and cholesterol; treatment of the cells with 2-aza-2,3-dihydrosqualene resulted in a decreased conversion of desmosterol into cholesterol indicating that the delta 24-sterol reductase might be another target of the drug. 2-Aza-2,3-dihydrosqualene at 1 microM affected normal growth of 3T3 fibroblasts, this effect could be prevented by addition of exogeneous cholesterol (50 microM). Growth arrest of the treated cells was correlated with a decrease in cellular sterol content to less than 40% of controls. About 30% of the C27-sterol fraction, of the treated cells, was desmosterol. Our work demonstrates that 2-aza-2,3-dihydrosqualene is a valuable new inhibitor of cholesterol biosynthesis in mammalian cells.

Potent inhibition of cholesterol biosynthesis in 3T3 fibroblasts by N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol, a new 2,3-oxidosqualene cyclase inhibitor.

N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol, a new compound rationally designed to inhibit the 2,3-oxidosqualene cyclase (M. Taton et al., Biochem. biophys. Res. Commun. 138, 764, 1986) was studied as an inhibitor of cholesterol biosynthesis in Swiss 3T3 fibroblasts. Treatment of cells, which were grown for 2 days in a delipidated medium, resulted in a dramatic decrease of [14C]acetate incorporation into the C27-sterol fraction. An IC50 of 20 nM was calculated, which classes this drug amongst the most powerful cholesterol biosynthesis inhibitors acting at the 2,3-oxidosqualene-lanosterol cyclase tested so far on mammalian cells. The inhibition of the C27-sterols synthesis was correlated with the accumulation of 2,3-[14C]oxidosqualene and of 2,3:22,23-[14C]dioxidosqualene indicating that the cyclase was indeed an intracellular target of the drug. A minor secondary target was identified as the sterol-8-ene isomerase. Cells treated with the inhibitor also accumulated sterols more polar than cholesterol which could originate, for example, from the metabolization of 2,3:22,23-dioxidosqualene. Treatment of the cells with increasing concentrations of the drug resulted in a progressive reduction of the HMG-CoA reductase activity (up to 50% of control). The drug affected normal growth of the fibroblasts and growth arrest was correlated with a decrease in cellular cholesterol content to less than 50% of control. This work indicates that N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol is a potent and promising new tool in the inhibition of cholesterol biosynthesis in mammalian cells.

Inhibition of 2,3-oxidosqualene cyclase and sterol biosynthesis by 10- and 19-azasqualene derivatives.

The inhibition of 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7) (OSC) by new azasqualene derivatives, mimicking the proC-8 and proC-20 carbocationic high-energy intermediates of the cyclization of 2,3-oxidosqualene to lanosterol, was studied using pig liver microsomes, partially purified preparations of OSC, and yeast microsomes. The azasqualene derivatives tested were: 6E- and 6Z-10aza-10,11-dihydrosqualene-2,3-epoxide 17 and 18, 19-aza-18,19,22,23-tetrahydrosqualene-2,3-epoxide 19 and its corresponding N-oxide 20, and 19-aza-18,19,22,23-tetrahydrosqualene 21. The compounds 17 and 19 (i.e. the derivatives bearing the 2,3-epoxide ring and the same geometrical configuration as the OSC substrate) were effective inhibitors, as shown by the Ki obtained using partially purified OSC: 2.67 microM and 2.14 microM, respectively. Compound 18, having an incorrect configuration and the 19-aza derivative 21, lacking the 2,3-epoxide ring, were poor inhibitors, with IC50 of 44 microM and 70 microM, respectively. Compound 21 was a competitive inhibitor of OSC, whereas 17 and 19 were noncompetitive inhibitors, and showed a biphasic time-dependent inactivation of OSC, their apparent binding constants being 250 microM and 213 microM, respectively. The inhibition of sterol biosynthesis was studied using human hepatoma HepG2 cells. The incorporation of [14C] acetate in the C27 sterols was reduced by 50% by 0.55 microM 17, 0.22 microM 19, and 0.45 microM 21, whereas 2 microM 18 did not affect sterol biosynthesis. In the presence of 17, 19 and 21, only the intermediate metabolites 2,3-oxidosqualene and 2,3,22,23-dioxidosqualene accumulated, demonstrating a very specific inhibition of OSC.

In vitro inhibition of animal and higher plants 2,3-oxidosqualene-sterol cyclases by 2-aza-2,3-dihydrosqualene and derivatives, and by other ammonium-containing molecules.

2-Aza-2,3-dihydrosqualene and related molecules, a series of new compounds designed as analogues of the transient carbocationic high energy intermediate, occurring in the oxirane ring opening during the cyclization of 2,3-oxidosqualene, were tested in vitro as inhibitors of the microsomal 2,3-oxidosqualene cyclase of animals (rat liver) and of higher plants (maize, pea). These molecules proved to be good and specific inhibitors for the cyclases of both phyla. The inhibition is due to positively charged species and is sensitive to the steric hindrance around the nitrogen-atom. 4,4,10 beta-Trimethyl-trans-decal-3 beta-ol and 4,10 beta-dimethyl-trans-decal-3 beta-ol, which have previously been described (J.A. Nelson et al., J. Am. chem. Soc. 100, 4900 (1978] as inhibitors of the 2,3-oxidosqualene cyclase of chinese hamster ovary cells, were found to be non-competitive inhibitors of the rat liver microsomal enzyme and presented no activity towards the higher plants cyclases. Aza derivatives of these decalines (A. Rahier et al., Phytochemistry, in press), which were aimed to mimic the C-8 carbocationic intermediate occurring during later steps of the 2,3-oxidosqualene cyclization did not inhibit the cyclases. This result underlines the theoretical limitations of the high energy analogues concept in designing enzyme inhibitors. Amongst other molecules tested, 2,3-epiminosqualene was found to be a reversible, non-competitive inhibitor of the cyclases; similarly U18666A was a very potent inhibitor of the microsomal cyclases. In contrast AMO 1618, a known anticholesterolemic agent reported previously to act at the level of the 2,3-oxidosqualene cyclization step, was not found per se to act on the cyclases.

Latex beads phagocytosis capacity and ecto-NAD+ glycohydrolase activity of rat brain microglia cells in vitro.

Conditions are described which allow the preparation in vitro of pure (greater than 95%) microglial cell cultures isolated from newborn rat brain. Such ameboid cells cultivated in vitro can efficiently phagocytize opsonized latex beads and are capable of ingesting more (100-200 beads of 1.1 micron diameter per cell) and larger (6.4 microns) particles than other nerve cells, such as oligodendrocytes and astroglia. The microglial cells also show an important ecto-NAD+ glycohydrolase activity which is characteristic of phagocytic cells. We noted that the phagocytic capacity and ecto-NAD+ glycohydrolase of these cells were correlated and increased notably during the in vitro culture. Microglia cultivated in vitro appear to be a good model to study the activation of phagocytic properties in the central nervous system and corresponding modulation by natural or pharmacological immunomodulators.

Drug design based on biosynthetic studies: synthesis, biological activity, and kinetics of new inhibitors of 2,3-oxidosqualene cyclase and squalene epoxidase.

Various classes of inhibitor of 2,3-oxido squalene cyclase have been synthesized and tested on rat liver and Saccharomyces cerevisiae microsomes, 3T3 fibroblast cultures, and various bacteria, fungi, and yeasts. The compounds include azasqualenes, azasqualanes, bis-azasqualenes, bis-azasqualanes, and N-oxide and ammonium derivatives of squalene. In order to better mimic the transition state involved in the SN2-like opening of 2,3-oxidosqualene, we synthesized squalene N-methyloxaziridine. Other derivatives tested were N-methylimine, aminalic hydroperoxide, and N-methylamide. We also attempted to produce new "suicide" inhibitors of SO cyclase, such as a squalenoid epoxide vinyl ether. Many of the products described inhibited the various cyclases, the best having an IC50 of 0.3 microM on plants and 1.5 microM on rat liver microsomes, and good antibacterial and antifungal activity. In a search for inhibitors of squalene epoxidase, a series of mono- and bifunctional squalenoid acetylenes and allenes were synthesized. Some of them proved to be inhibitors of squalene epoxidase.