Ion conductors derived from cholic acid and spermine: importance of facial hydrophilicity on NA(+) transport and membrane selectivity.

A series of ion conductors have been synthesized in which the degree of facial hydrophilicity has been systematically varied. Specifically, conjugates have been prepared from cholic acid and spermine in which the hydrophilic face of each sterol bears methoxy (1), hydroxy (2), carbamate (3), or sulfate groups (4). The ability of these conjugates to promote the transport of Na(+) across phosphatidylcholine membranes of varying thickness has been investigated by (23)Na NMR spectroscopy. Examination of observed activities in three different phosphatidylcholine membranes has provided evidence for membrane-spanning dimers as the transport-active species. In the thinnest membranes investigated, made from 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (C14), Na(+)-transport activity was found to increase, substantially, with increasing facial hydrophilicity. In thicker membranes, made from 1,2-dioleoyl-sn-glycero-3-phosphocholine (C18), observed activities were found to decrease with increasing facial hydrophilicity; with a membrane of intermediate thickness, prepared from 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (C16), ion-conducting activity increased and then decreased, with continuous increases in facial hydrophilicity. The possible origins for these variations in activity are briefly discussed.

Molecular umbrella-assisted transport of glutathione across a phospholipid membrane.

A di-walled molecular umbrella (1a) has been synthesized by acylation of the terminal amino groups of spermidine with cholic acid, followed by condensation with bis(3-O-[N-1,2,3-benzotriazin-4(3H)-one]yl)-5,5'-dithiobis-2-nitrobenzoate (BDTNB), and displacement with glutathione (gamma-Glu-Cys-Gly, GSH). Replacement of the sterol hydroxyls with sulfate groups, prior to displacement with GSH, afforded a hexasulfate analogue 1b. Both conjugates have been found to enter large unilamellar vesicles (200 nm diameter, extrusion) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and to react with entrapped GSH to form oxidized glutathione (GSSG). Evidence for vesicular entry has come from the formation of oxidized glutathione (GSSG) within the interior of the vesicle, the appearance of the thiol form of the umbrella (USH), and the absence of release of GSH into the external aqueous phase. Results that have been obtained from monolayer experiments, together with the fact that the heavily sulfated conjugate is able to cross the phospholipid bilayer, have yielded strong inferential evidence for an "umbrella-like" action of these molecules as they cross the lipid bilayer.

Enhancing the "stickiness" of bile acids to cross-linked polymers: a bioconjugate approach to the design of bile acid sequestrants.

A series of cross-linked polymers have been synthesized by reaction of 2% cross-linked, chloromethylated polystyrene with a tertiary amine derivative (7) of cholic acid (prepared by reaction of cholic acid methyl ester with N,N-dimethyl-ethylenediamine), followed by exhaustive quaternization with trimethylamine. Increased loadings of 7 result in enhanced binding of taurocholate ion and a decrease in its rate of release upon exposure to 50 mM aqueous NaCl. Examination of particle-size effects indicates that film diffusion and particle diffusion both contribute to the observed kinetics. Interruption tests that were carried out confirmed that particle diffusion is kinetically important. The relevance of this bioconjugate approach to the design of bile acid sequestrants for the treatment of hypercholesterolemia is briefly discussed.

Efficacies of KY62 against Leishmania amazonensis and Leishmania donovani in experimental murine cutaneous leishmaniasis and visceral leishmaniasis.

Current therapy for leishmaniasis is unsatisfactory because parenteral antimonial salts and pentamidine are associated with significant toxicity and failure rates. We examined the efficacy of KY62, a new, water-soluble, polyene antifungal, against cutaneous infection with Leishmania amazonensis and against visceral infection with Leishmania donovani in susceptible BALB/c mice. Mice were infected with L. amazonensis promastigotes in the ear pinna and in the tail and were treated with KY62 or amphotericin B. The cutaneous lesions showed a remarkable response to therapy with KY62 at a dose of 30 mg per kg of body weight per day. At this dose, the efficacy of KY62 was equivalent to or better than that of amphotericin B at 1 to 5 mg/kg/day. Mice infected intravenously with 10(7) L. donovani promastigotes and treated with KY62 showed a 4-log reduction in the parasite burden in the liver and spleen compared to untreated mice. These studies indicate potent activity of KY62 against experimental cutaneous leishmaniasis caused by L. amazoniensis and against experimental visceral leishmaniasis caused by L. donovani.

KY-62, a polyene analog of amphotericin B, for treatment of murine candidiasis.

KY-62 is a water-soluble analog of amphotericin B. In vitro testing of five clinical isolates of Candida albicans showed KY-62 to have potency similar to that of amphotericin B. KY-62 was administered to mice infected intravenously with C. albicans. In vivo, KY-62 was effective in immunocompetent mice, with potency similar to that of amphotericin B. KY-62 was well tolerated up to 30 mg/kg of body weight per dose, an amount that would be lethal with amphotericin B. KY-62 was less effective in mice rendered neutropenic with 5-fluorouracil. The addition of flucytosine had little effect. KY-62 may have potential for clinical development.

Evidence for highly cooperative binding between molecular umbrella-spermine conjugates and DNA.

Double- and tetrawalled molecular umbrella-spermine conjugates (I and II) have been synthesized, and their binding to calf thymus DNA (CT-DNA), poly[d(AT)], and poly[d(GC)] compared with that of a single-walled analogue (III). At moderate salt concentrations (8 mM NaCl), I and II show significantly greater affinity toward each DNA, relative to III; at high salt concentrations (150 mM NaCl), strong binding of I and II (but not III) was maintained toward poly[d(GC)]. Examination of the influence of I-III on the melting behavior of poly[d(AT)] has provided strong evidence that the binding of I and II reflects highly cooperative interactions among DNA-bound conjugates and that the DNA duplex serves as a nucleation site for umbrella aggregation. The implications of these findings for the rational design of novel drug conjugates that operate at the nuclear level, and also novel transfection agents, are briefly discussed.

Antimicrobial activities of squalamine mimics.

We investigated the antimicrobial properties of compounds with structural features that were designed to mimic those of squalamine, an antibiotic isolated from the stomach of the dogfish shark. The mimics, like squalamine, are sterol-polyamine conjugates. Unlike squalamine, the mimics were simple to prepare, at high yield, from readily available starting materials. Several squalamine mimics showed activity against gram-negative rods, gram-positive cocci including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and fungi. Some had little or no hemolytic activity. The hydrophobicity of the sterol backbone and the length and the cationic charge of the side chains appeared to be critical determinants of activity. One of the squalamine mimics, SM-7, was bactericidal against Escherichia coli, Pseudomonas aeruginosa, and S. aureus; its activity was decreased by divalent or monovalent cations and by bovine serum albumin. Subinhibitory concentrations of SM-7 markedly enhanced the antimicrobial activity of rifampin against gram-negative rods. These results suggest that the compounds may disrupt an outer membrane of gram-negative rods. Squalamine mimics are a new class of broad-spectrum antimicrobial agents. The antagonism of their activity by serum and albumin and their hemolytic properties may limit their use as systemic agents. The squalamine mimics, because of their potencies, broad spectra of antimicrobial activity, and potential for systemic toxicity, appear to be good candidates for development as topical antimicrobial agents.

Supramolecular surfactants: amphiphilic polymers designed to disrupt lipid membranes.

Simple polyesters derived from poly(ethylene glycol)s and alpha, omega-dicarboxylic acids exhibit a broad range of activity in disrupting phospholipid membranes. This activity has been analyzed by measuring the release of liposome-encapsulated 5(6)-carboxy-fluorescein (CF). Comparison with an analogous monomeric surfactant, and with Triton X-100, demonstrates that macromolecular activity is a sensitive function of the size of the hydrophobic and hydrophilic segments within each repeat unit, and that high disrupting power is possible. In vitro studies with the human immunodeficiency virus type-1 have revealed that those polyesters which exhibit the highest membrane disrupting power also provide significant protection for human CD4+ lymphocytes against HIV-1. The potential for adjusting and utilizing these "supramolecular surfactants" in medicine is briefly discussed.

Interactions of polymerizable phosphatidylcholine vesicles with blood components: relevance to biocompatibility.

We have studied the biocompatibility properties of polymerizable phosphatidylcholine bilayer membranes, in the form of liposomes, with a view toward the eventual utilization of such polymerized lipid assemblies in drug carrier systems or as surface coatings for biomaterials. The SH-based polymerizable lipid 1,2-bis[1,2-(lipoyl)dodecanoyl]-sn-glycero-3-phosphocholine (dilipoyl lipid, DLL) and the methacryl-based lipid 1,2-bis[(methacryloyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (dipolymerizable lipid, DPL) were studied in comparison to 'conventional' zwitterionic or charged phospholipids. We examined binding of serum proteins to liposomes and effects of liposomes on fibrin clot formation and on platelet aggregation. All types of liposomes tested bound complex mixtures of serum proteins with IgG being the most abundant bound component. DPL vesicles and anionic vesicles bound substantially more protein than other vesicle types. Polymerized DPL vesicles uniquely bound a protein of about 53 kDa which was not bound to other types of phosphatidylcholine liposomes. Likewise polymerized DPL vesicles, but not other types of phosphatidylcholine vesicles, caused a marked alteration in coagulation as measured by activated partial thromboplastin time (APTT) and prothrombin time (PT) tests; this effect was shown to be due to binding and depletion of clothing factor V by the DPL polymerized vesicles. Polymerized DPL liposomes and DLL liposomes in polymerized or nonpolymerized form, were without substantial effect on platelet aggregation. However, DPL nonpolymerized vesicles, while not causing aggregation, did impair ADP-induced aggregation of platelets. These studies suggest that SH based polymerizable lipids of the DLL type may be very suitable for in vivo use in the contexts of drug delivery systems or biomaterials development. Methacryloyl-based lipids of the DPL type seem to display interactions with the hemostatic process which militate against their in vivo utilization.

Polymerized phospholipid vesicles containing amphotericin B: evaluation of toxic and antifungal activities in vitro.

We have prepared lipid vesicles (liposomes) composed of polymerized bis[12-(methacryloyloxy)dodecanoyl]-L-alpha-phosphatidylcholine (DPL) which contain the antifungal polyene antibiotic amphotericin B (AMB). It was necessary to devise a novel method for incorporating AMB into the liposomes subsequent to polymerization. The polymer liposome AMB was as effective as AMB in "conventional" liposomes in terms of inhibiting fungal growth in vitro. However, in contrast to "conventional" liposomes, the polymerized DPL vesicles did not protect mammalian cells against AMB induced toxicity.

Polymerized phosphatidylcholine vesicles as drug carriers.

Polymerized ammonium and phosphatidylcholine vesicles have been synthesized via poly(methacrylate) and poly(disulfide) formation. A functionalized polymeric liposome has also been synthesized and effectively conjugated with alpha chymotrypsin. Vesicles derived from 2 have been studied most extensively as potential carriers of drugs. The are extremely stable toward physical perturbation and exposure to organic solvents or detergents, and should be able to maintain their integrity and homogeneity during pharmaceutical manufacturing procedures, including lyophilization. The fact that they retain substantial quantities of lipophilic substances in the presence of detergent concentrations in excess of those found in the upper gastrointestinal tract suggests that they should be able to carry and selectively release lipophilic drugs in the lower reaches of the GI tract. Moreover, the apparent lack of acute toxicity of these vesicles, as indicated by intravenous administration to outbred mice, further suggests that such photopolymerized liposomes may represent a unique and valuable carrier for the controlled delivery of certain types of therapeutic agents, especially by the oral route.

Interactions of polymerized phospholipid vesicles with cells. Uptake, processing and toxicity in macrophages.

We have studied the uptake of photopolymerized multilamellar vesicles composed of bis(1,2(methacryloyloxy)dodecanoyl)-L-alpha-phosphatidylchol ine (DPL) by mouse peritoneal macrophages in vitro. Vesicles composed of polymerized DPL are taken up more rapidly and extensively than vesicles composed of conventional phosphatidylcholine. The uptake of radioactive DPL vesicles was not blocked by incubation with unlabelled phosphatidylcholine vesicles in either the fluid or gel state. Likewise, fluid-phase negatively charged vesicles failed to block uptake of DPL vesicles, whereas solid-phase negatively charged vesicles did have a blocking effect. A radioactive lipophilic marker (dipalmitoylphosphatidyl[N-methyl-3H]choline) incorporated into DPL vesicles was metabolized at essentially the same rate whether the vesicles were polymerized or not. Nonpolymerized DPL vesicles were quite toxic to macrophages, whereas polymerized DPL vesicles or vesicles composed of conventional phosphatidylcholines were not toxic.