New nystatin-related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces noursei.

Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.

Final report of the cosmetic ingredient review expert panel on the safety assessment of Polyisobutene and Hydrogenated Polyisobutene as used in cosmetics.

Polyisobutene and Hydrogenated Polyisobutene are homopolymers of isobutene. These ingredients are produced in a wide range of molecular weights. Polybutene is a chemically related cosmetic ingredient previously determined to be safe as used in cosmetic products. Polyisobutene is used in cosmetic products as a binder, film former, and nonaqueous viscosity-increasing agent. Hydrogenated Polyisobutene functions as a skin-conditioning agent-emollient and nonaqueous viscosity-increasing agent with a wide range of uses in cosmetic formulations. The estimated octanol water partition coefficient for Hydrogenated Polyisobutene and Polybutene is log K(ow) of 13.27 and the estimated water solubility was 5.6 x 10(-3) ng/L for Hydrogenated Polyisobutene and Polybutene. Acute oral toxicity testing demonstrated no effects other than lethargy in one rat study. The oral LD(50) was > 5.0 g/kg in rats. No short-term or subchronic animal toxicity data were available. A 2-year chronic oral toxicity study of Polybutene revealed no gross or microscopic pathological changes, and no changes in body weights or food consumption, hematological results, urology, or tumor formation that could be correlated with Polybutene ingestion, except that in the 20,000 ppm group, three out of six males that died between weeks 17 and 24 exhibited hematuria. In a 2-year chronic oral toxicity study of Polybutene in Beagle dogs, no abnormalities in body weight, food consumption, survival, behavioral patterns, hematology, blood chemistry, urinalysis, liver function, gross and histopathologic examinations, or organ weights and ratios were reported. In a three-generation reproductive study in Charles River albino rats that ingested Polybutene, none of the animals in successive generations differed from controls with regard to weight gain, litter size, the number of stillborn, and the number of viable pups during lactation. The survival, body weights, and reactions of test animals were comparable to those of controls. Neither Polyisobutene nor Hydrogenated Polyisobutene were ocular irritants, nor were they dermal irritants or sensitizers. Polyisobutene was not comedogenic in a rabbit ear study. Polyisobutene did not induce transformation in the Syrian hamster embryo (SHE) cell transformation assay, but did enhance 3-methylcholanthrene-induced transformation of C3H/10T1/2 cells. In a carcinogenicity study in mice, Polyisobutene was not carcinogenic, nor did it promote the carcinogenicity of 7,12-dimethylbenz(alpha)anthracene. Clinical patch tests uncovered no evidence of dermal irritation and repeat-insult patch tests with a product containing 4% Hydrogenated Polyisobutene or 1.44% Hydrogenated Polyisobutene found no reactions greater than slight erythema. These products also were not phototoxic or photoallergenic. The product containing 4% Hydrogenated Polyisobutene was not an ocular irritant in a clinical test. The Cosmetic Ingredient Review (CIR) Expert Panel recognized that there are data gaps regarding use and concentration of these ingredients. However, the overall information available on the types of products in which these ingredients are used and at what concentrations indicate a pattern of use, which was considered by the Expert Panel in assessing safety. Although there is an absence of dermal absorption data for Polyisobutene and Hydrogenated Polyisobutene, the available octanol water partition coefficient data and the low solubility in water suggest very slow absorption, so additional data are not needed. Gastrointestinal absorption is also not a major concern due to the low solubility of these chemicals. Although one in vitro study did report that Polyisobutene did promote cellular transformation, a mouse study did not find evidence of tumor promotion. Because lifetime exposure studies using rats and dogs exposed to Polybutene failed to demonstrate any carcinogenic or tumor promotion effect, and a three-generation reproductive/developmental toxicity study produced no adverse effects, the CIR Expert Panel does not believe these large, mostly insoluble polymers present any risks in the practices of use and concentration as described in this safety assessment.

Isolation and structure elucidation of cytotoxic polyacetylenes and polyenes from Echinacea pallida.

Bioassay-guided fractionation of n-hexane extracts of Echinacea pallida (Asteraceae) roots led to the isolation and structure elucidation of two polyacetylenes (1, 3) and three polyenes (2, 4, 5). Two are known hydroxylated compounds, namely 8-hydroxy-pentadeca-(9E)-ene-11,13-diyn-2-one (1) and 8-hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one (2). Two dicarbonylic constituents, namely pentadeca-(9E)-ene-11,13-diyne-2,8-dione (3) and pentadeca-(9E,13Z)-dien-11-yne-2,8-dione (4), were isolated and characterized for the first time. Furthermore, the structure elucidation of pentadeca-(8Z,13Z)-dien-11-yn-2-one (5) is described. The structure of the compounds isolated was determined on the basis of UV, IR, NMR (including 1D and 2D NMR experiments, such as 1H-1H gCOSY, gHSQC-DEPT, gHMBC, gNOESY) and MS spectroscopic data. The cytotoxic activity of the isolated constituents against MIA PaCa-2 human pancreatic adenocarcinoma cells was evaluated in the concentration range 1-100 microg/ml. Results show that the hydroxylated compounds (1, 2) have low cytotoxicity, while the more hydrophobic polyacetylenes (3) and polyenes (4, 5) displayed moderate activity.

Biocompatibility and characterization of polylactic acid/styrene-ethylene-butylene-styrene composites.

Polylactic acid (PLA)/styrene-ethylene-butylene-styrene (SEBS) composites were prepared by melt blending. Differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WXRD) were used to characterize PLA and PLA/SEBS composites in terms of their melting behavior and crystallization. Curves from thermal gravimetric analysis (TGA) illustrated that thermostability increased with SEBS content. Further morphological analysis of PLA/SEBS composites revealed that SEBS molecules were not miscible with PLA molecules in PLA/SEBS composites. The tensile testing for PLA and PLA/SEBS composites showed that the elongation at the break was enhanced, but tensile strength decreased with increasing SEBS content. L929 fibroblast cells were chosen to assess the cytocompatibility; the cell growth of PLA was found to decrease with increasing SEBS content. This study proposes possible reasons for these properties of PLA/SEBS composites.

Polyene--sterol interaction and selective toxicity.

From permeability experiments carried out with series of amphotericin B derivatives in both biological and model membranes, it was concluded that derivatives, whose carboxyl group at the C18 position is blocked by substitution, are much more efficient at inducing permeability in ergosterol-containing than in cholesterol-containing membranes, whereas derivatives whose carboxyl group is free and ionizable are equally efficient in both membranes types. Binding measurements on erythrocyte membranes showed that all amphotericin B derivatives simply partition between membrane lipids and aqueous medium, according to their lipid solubility. There is no relationship between binding and efficiency in inducing permeability. Permeability studies carried out on lipidic vesicles containing various sterols showed that: 1) derivatives having their carboxyl free induced permeability of the 'channel' type, regardless of the sterol present, and no detectable permeability in sterol-free membranes; 2) derivatives whose carboxyl group is blocked induce channels only in membranes containing ergosterol or sterols having an alkyl side chain identical to that of ergosterol. In the presence of other sterols or in sterol-free membranes, their ionophoric activity is poor and always of the 'mobile-carrier' type. A model of polyene-sterol interaction is proposed, accounting for the data obtained with both biological and model membranes.

Polyene antibiotics: relative degrees of in vitro cytotoxicity and potential effects on tubule phospholipid and ceramide content.

Polyene antibiotic administration is limited by dose-dependent nephrotoxicity. The latter is believed to be mediated by polyene anchoring to plasma membrane cholesterol, resulting in pore formation, abnormal ion/solute flux, adenosine triphosphate (ATP) declines, and, ultimately, a loss of tubule viability. The relative nephrotoxicity of these agents and their liposomal preparations has remained poorly defined. Thus, freshly isolated mouse proximal tubules or cultured human proximal tubule (HK-2) cells were exposed to either nystatin, amphotericin B, or three different polyene liposomal preparations (Nyotran, AmBisome, or Abelcet; 4 to 64 microg/mL). The impact of these agents on (1) plasma membrane injury (sodium-driven ATP consumption, assessed by ATP-adenosine diphosphate [ADP] ratios); (2) cellular susceptibility to superimposed injury (chemical hypoxia or ferrous ammonium sulfate-mediated oxidative stress; assessed by lactate dehydrogenase release); and (3) membrane cholesterol, phospholipid, and ceramide expression was assessed. Amphotericin B was more cytotoxic than nystatin (approximately 25% to 50% greater ATP-ADP ratio declines). Most of this toxicity could be eliminated by polyene liposomal formulation. Nevertheless, the liposomal polyenes still fully sensitized tubule cells to superimposed chemical hypoxic (antimycin/deoxyglucose), but not oxidant, attack. Nystatin and amphotericin B caused acute increments in tubule sphingomyelin-phosphatidylcholine ratios and ceramide content (indicating an impact on the plasma membrane extending beyond the classic view of pore formation with ion flux). In conclusion, (1) nystatin is seemingly less cytotoxic than amphotericin B (in contrast to the prevailing clinical view); (2) liposomal formulation markedly decreases this cytotoxicity; (3) despite this reduced toxicity, liposomal polyenes are still able to render tubule cells more vulnerable to selected forms of superimposed injury; and (4) acute alterations in plasma membrane phospholipid and ceramide expression are previously unrecognized consequences and potential mediators of polyene-mediated tubular cell attack.

Mannosylated liposomes as carriers for hamycin in the treatment of experimental aspergillosis in Balb/C mice.

Hamycin incorporated into mannosylated liposomes produced less toxicity and enhanced antifungal activity in experimental aspergillosis in balb/c mice in vivo. Incorporation of cholesterol into mannosylated liposomes led to a decrease in hamycin toxicity. The LD50 (mg/kg) values of hamycin contained in SPC/Chol/DPPE-Man (molar ratio 4:5:1) lipsomes was 2.8 whereas that in SPC/DPPE-Man liposomes (molar ratio 9:1) was 1.4. Incorporation of cholesterol into mannosylated liposomes increased the survival rates of infected animals: 70% survival was recorded after 7 days therapy as well as reduced fungal load in lung, liver, spleen and kidney. HPLC studies of distribution of hamycin in various tissues showed a reduction in the concentration of the liposomal drug in circulation compared to that observed for free drug and neutral liposomes after 1 hour.

Characterization of Hexsyn, a polyolefin rubber.

Hexsyn is the Goodyear Tire and Rubber Company tradename for a polyolefin rubber synthesized from 1-hexene with 3-5% methylhexadiene as the source of residual double bonds for vulcanization. Under license from Goodyear, this same polymer has been manufactured by Lord Corporation for the hinge portion of finger joint prostheses using the tradename Bion. This rubber is currently licensed to the University of Akron and to the Cleveland Clinic Foundation for use in biomedical applications, and is being used primarily for biocompatible and highly fatigue resistant rubber components in ventricular assist and artificial heart systems. Results are presented from the physical, mechanical, and biological characterization of Hexsyn. Procedures are described for the synthesis, compounding, and post-molding extraction for Hexsyn. The physical testing of Hexsyn reported includes determinations of its density at 23 and 37 degrees C, initial hardness and hardness after aging in oxygen, blood, pseudoextracellular fluid and polyethylene glycol 600, typical molecular weights determined by gel permeation chromatography/low angle laser light scattering and intrinsic viscosity, thermal analyses by differential scanning calorimetry of Hexsyn gum, and vulcanized Hexsyn after exposure to blood and blood/fatigue conditions. Also reported are results of differential thermal analyses, thermomechanical analyses of virgin and annealed samples, and thermogravimetric analyses conducted in helium and in air. Dynamic mechanical analyses of Hexsyn include Clash-Berg and Rheovibron tests. Swelling was conducted to determine lot-to-lot and sheet-to-sheet variation for quality control and also a number of solvents were used so that the polymer-solvent interaction parameters could be determined. The permeability of Hexsyn to water, water vapor, and a variety of gases is reported. The permeability by contact angle measurements, refractive index, residual solvent analyses, migration of blood components into Hexsyn, melt rheology by Monsanto Rheometer, resistance to acids, and typical mold shrinkage for Hexsyn are reported. Mechanical testing of Hexsyn includes tensile strength, elongation, and tensile stress (modulus) at 23 and 37 degrees C and after conditions including exposure to blood, pseudoextracellular fluid, polyethylene glycol, oxygen, 100% relative humidity, and fatigue testing. Stress/strain calibration curves, flexural rigidity after aging in blood, tension set, compression set, stress relaxation, and the effect of repeated cycling on the elastic modulus are presented along with the results of Pico abrasion, skid resistance tests on wet concrete.(ABSTRACT TRUNCATED AT 400 WORDS)

Respiratory toxicity in mice exposed to mattress covers.

To evaluate factors that might contribute to the rise in prevalence of childhood asthma, we allowed groups of male Swiss-Webster mice to breath the emissions of six brands of waterproof crib mattress covers for 1 h. We used a computerized version of ASTM-E-981 test method to monitor respiratory frequency, pattern, and airflow velocity. Single exposure to the emissions of these mattress covers caused various combinations of sensory irritation, pulmonary irritation, and decreases in mid-expiratory airflow velocity. At the peak effects of these emissions, sensory irritation ranged from 9% to 51% of the breaths, pulmonary irritation ranged from 4% to 16% of the breaths, and airflow limitation ranged from 9% to 38% of the breaths. Three brands caused airflow limitation that persisted for at least 24 h after a single 1-h exposure of naive mice to these emissions. Repeat exposures to the emissions of four brands caused more marked effects (i.e., up to 96% of the breaths showing sensory irritation, up to 44% of the breaths showing pulmonary irritation, and up to 75% of the breaths showing airflow limitation). Histological evaluation of the lungs revealed a mild inflammatory response, with focal collections of polymorphonuclear leukocytes and edema, but there were no eosinophils and no bronchial mucosa changes. We used gas chromatography/mass spectrometry to evaluate one of the test atmospheres, and there was evidence of chemicals for which toxic properties have been documented previously. The results of our study demonstrated that some mattress covers emit mixtures of chemicals that can cause a variety of acute toxic effects in mice, including asthma-like reactions.

Liposomal hamycin in the control of experimental aspergillosis in mice: relative toxicity, therapeutic efficacy and tissue distribution of free and liposomal hamycin.

Therapeutic efficacy of liposomal Hamycin has been evaluated in an animal model system for aspergillosis in Balb/c mice. Hamycin was intercalated into soya phosphatidyl choline (SPC), SPC: choline (1:1, vol./vol.) and DMPC liposomes. A single dose of either 0.1 mg/kg, 0.25 mg/kg or 0.5 mg/kg of liposomal Hamycin and 0.1 mg/kg of free Hamycin was injected (i.v.) into animals infected with Aspergillus fumigatus. An increase in the survival rate of animals along with decrease in fungal count in various organs was observed with liposomal administration. Incorporation of cholesterol into liposomes decreased the in vivo toxicity of Hamycin in a dose dependent manner. However, antifungal activity both in the presence and absence of cholesterol showed marked variation as compared to that of non-aromatic polyenes, e.g. amphotericin B. Analysis of Hamycin distribution by HPLC in various tissues revealed higher blood concentration of this drug, when given in free form, compared to its liposomised form. These studies suggest that liposomal Hamycin is more effective than free Hamycin in controlling the experimental Aspergillosis.

Chemical hazards analysis of resilient flooring for healthcare.

This article addresses resilient flooring, evaluating the potential health effects of vinyl flooring and the leading alternatives-synthetic rubber, polyolefin, and linoleum-currently used in the healthcare marketplace. The study inventories chemicals incorporated as components of each of the four material types or involved in their life cycle as feedstocks, intermediary chemicals, or emissions. It then characterizes those chemicals using a chemical hazard-based framework that addresses persistence and bioaccumulation, human toxicity, and human exposures.

Chemotherapeutic activity of liposomal SJA-95: a new polyene macrolide antibiotic in experimental aspergillosis and cryptococcosis.

The incidence of systemic fungal infections that has risen dramatically over the past three decades has propelled a continuous need for more potent antifungal drugs. The purpose of this research was to evaluate the chemotherapeutic activity of a new heptaene polyene macrolide antibiotic (SJA-95) and liposomal incorporated SJA-95 (lip. SJA-95) in a mouse model of aspergillosis and cryptococcosis respectively. Lip. SJA-95 was prepared in our laboratory by the proliposome method involving incorporation of the antifungal into the proliposome mixture and its subsequent conversion into a liposomal dispersion by a simple dilution step. Treatment with free SJA-95 and lip. SJA-95, both in aspergillosis and cryptococcosis, progressively prolonged the survival time and decreased the fungal loads in vital organs respectively. A higher LD(50) value of lip. SJA as compared to that of free SJA-95 was indicative of reduced toxicity of lip. SJA-95. Our findings suggest lip. SJA-95 treatment results in prolonged survival time, effective microbiological clearance and reduced toxicity that might help to establish its usefulness as a chemotherapeutic agent in systemic fungal infections with fewer adverse reactions.

[Evaluation of biological safety of continuous carbon-fiber reinforced polyolefin as hard tissue repair].

OBJECTIVE: To evaluate the biological safety of continuous carbon-fiber reinforced polyolefin as hard tissue repair material. METHODS: Biocompatibility of the material was evaluated through hemolysis test, pyrogen test, skin irritation test, cytotoxicity test, ames test, in vitro chromosome aberration test, and bone marrow cells Micronuclei test. RESULTS: No obvious hemolysis, pyrogenic characteristics, sensitivity, cytotoxicity, and mutagenicity were observed. CONCLUSION: The continuous carbon-fiber reinforced polyolefin composite material is of good biological safety. It meets all the demand made by biological safety as hard tissue repair material.

Mannose-coated liposomal hamycin in the treatment of experimental leishmaniasis in hamsters.

Liposomal hamycin was found to elicit enhanced microbicidal activity and reduced toxicity in experimental leishmaniasis in a hamster model under in vivo conditions. Mannose-coated liposomal hamycin was seen to produce increased therapeutic efficacy as judged from the lowering of spleen parasite load. At an equivalent dose of 0.5 mg/kg, every 3 days for a total of three doses in 7 days, the mannose-coated liposomal hamycin was found to be most effective compared to either of the liposomal hamycin or the free hamycin. Because of the reduced toxicity as judged from the blood pathology, tissue histology, and specific enzyme level related to normal liver function, mannose-coated liposomal hamycin resulted in 80 to 100% survival for a period of 15-18 days. Hamycin intercalated in sterol-rich liposomes showed reduced hemolytic activity but comparable therapeutic efficacy as was found with ordinary liposomes.

Liposomal hamycin in the control of experimental aspergillosis in mice: effect of phosphatidic acid with and without cholesterol.

Hamycin incorporated into liposomes containing phosphatidylcholine (SPC) and phosphatidic acid (PA) had reduced toxicity and an enhanced antifungal activity in experimental aspergillosis in balb/c mice. Incorporation of cholesterol into liposomes led to a dose dependent decrease in the toxicity of hamycin. The LD50 (mg/kg) of hamycin contained in SPC/cholesterol/PA (molar ratio 4:5:1) liposomes was 2.8 whereas that in SPC/PA liposomes (molar ratio 9:1) was 0.35. Although the free drug had little or no protective effect on the animals, those administered liposomal hamycin at an equivalent dose (0.1 mg/kg) in the absence of cholesterol (SPC/PA; molar ratio 9:1) showed 90% survival after seven days of therapy. On the other hand the presence of cholesterol in the carrier phosphatidic acid liposomes (SPC/cholesterol/PA; molar ratio 4:5:1) at a similar dose (0.1 mg/kg) led to a 60% survival over the same time period. Hamycin incorporation in phosphatidic acid liposomes both in the presence or absence of cholesterol was found to be effective in reducing the fungal load in lung, liver, spleen and kidney. Studies with distribution of hamycin in various tissues by HPLC showed a significant reduction in the concentration of the liposomal drug in circulation as compared to those seem after administration of free drug.

A comparison of in vitro toxicity and antifungal efficacy of membrane-active drugs after liposome encapsulation.

The membrane-active ionophores were observed to possess antifungal activity against Candida albicans 336 and were toxic to human erythrocytes. Liposome encapsulation of these drugs significantly reduced their toxicity to erythrocytes but resulted in the loss of their antifungal potency. These results are compared with membrane-active polyenes which maintained their antifungal activity after encapsulation into liposomes. Liposomal-ionophores, however, showed antifungal activity along with low concentrations of Amphotericin B indicating the presence of synergism between these drugs.

Neoglycoprotein conjugated liposomes as macrophage specific drug carrier in the therapy of leishmaniasis.

The potential utility of neoglycoprotein conjugated multilamellar liposomes as macrophage specific drug delivery system was studied using hamycin as the model drug and visceral leishmaniasis as the model macrophage disease. Hamycin, a polyene antibiotic, was found to have a growth inhibitory effect on cultured Leishmania donovani promastigotes at a concentration of 0.05 microgram/ml. Hamycin entrapped in neoglycoprotein conjugated liposome (neohamysome) eliminated intracellular amastigotes of L. donovani in peritoneal macrophages 10 and 1.5 times more efficiently than did the free and liposome entrapped drug (hamysome), respectively. Moreover, neohamysome possibly could completely eliminate splenic intracellular parasites in a 45 day BALB/c mouse model of visceral leishmaniasis at a dose of 1.5 mg/Kg/day given for 4 consecutive days. Hamysome at a similar dose had 80% parasite suppressive effect whereas free drug could not be administered more than the dosage of 0.5 mg/Kg/day due to mortality problem. Neohamysome and hamysome were generally less toxic than the free drug as judged by erythrocyte lysis and several clinical parameters of liver toxicity. These results suggest a possible use of neoglycoprotein conjugated liposomes in macrophage-associated diseases.