Preclinical Testing of a Novel Niclosamide Stearate Prodrug Therapeutic (NSPT) Shows Efficacy Against Osteosarcoma.

Therapeutic advances for osteosarcoma have stagnated over the past several decades, leading to an unmet clinical need for patients. The purpose of this study was to develop a novel therapy for osteosarcoma by reformulating and validating niclosamide, an established anthelminthic agent, as a niclosamide stearate prodrug therapeutic (NSPT). We sought to improve the low and inefficient clinical bioavailability of oral dosing, especially for the relatively hydrophobic classes of anticancer drugs. Nanoparticles were fabricated by rapid solvent shifting and verified using dynamic light scattering and UV-vis spectrophotometry. NSPT efficacy was then studied in vitro for cell viability, cell proliferation, and intracellular signaling by Western blot analysis; ex vivo pulmonary metastatic assay model; and in vivo pharmacokinetic and lung mouse metastatic model of osteosarcoma. NSPT formulation stabilizes niclosamide stearate against hydrolysis and delays enzymolysis; increases circulation in vivo with t 1/2 approximately 5 hours; reduces cell viability and cell proliferation in human and canine osteosarcoma cells in vitro at 0.2-2 µmol/L IC50; inhibits recognized growth pathways and induces apoptosis at 20 µmol/L; eliminates metastatic lesions in the ex vivo lung metastatic model; and when injected intravenously at 50 mg/kg weekly, it prevents metastatic spread in the lungs in a mouse model of osteosarcoma over 30 days. In conclusion, niclosamide was optimized for preclinical drug delivery as a unique prodrug nanoparticle injected intravenously at 50 mg/kg (1.9 mmol/L). This increased bioavailability of niclosamide in the blood stream prevented metastatic disease in the mouse. This chemotherapeutic strategy is now ready for canine trials, and if successful, will be targeted for human trials in patients with osteosarcoma.

Micelle carriers based on macrogol 15 hydroxystearate for ocular delivery of terbinafine hydrochloride: In vitro characterization and in vivo permeation.

Non-ionic surfactant micelles are helpful for improving the diffusion of topically delivered drugs through the cornea. This study aimed to develop terbinafine hydrochloride (TH)-loaded micelles based on a soft non-ionic surfactant-macrogol 15 hydroxystearate (HS 15) and to investigate their in vivo cornea permeation. Briefly, 0.25% TH-loaded HS 15 micelles (TH-HNMs) were developed using a simple co-solvent method. Characterization of the TH-HNMs by Zetasizer and transmission electron microscopy (TEM) revealed that the spherical and discrete micellar droplets with a small size (13.22±0.73nm) and an electrically neutral surface (-2.15±0.39mV) were achieved. The drug entrapment efficiency of TH-HNMs was almost 100%. The release of TH from the micelles was pH dependent. 93.2±3.4% of encapsulated TH was released from the micelles in the PBS at pH5.0 within 6h, but only 0.122±0.020% of encapsulated TH was released in the PBS at pH7.4 within the same release time. TH-HNMs possessed good physical stability in the pH neutral medium (pH7.0).No obvious irritations were observed in rabbit eyes after ocular instillation of TH-HNMs. The in vivo corneal permeation study revealed that the TH-HNMs can penetrate into the corneal epithelium quickly and efficiently in mouse eyes. Good permeability was also noted in the stroma of mouse corneas with de-epithelialization. Compared with the TH oily solution, TH-HNMs delivered considerably increased levels of TH into rabbit corneas with or without de-epithelialization. In conclusion, the easily prepared, small, physically stable and biocompatible TH-HNMs with good ocular bioavailability hold great promise as an efficient carrier for topical ocular delivery of TH.

Kolliphor® HS 15 Micelles for the Delivery of Coenzyme Q10: Preparation, Characterization, and Stability.

To enhance the stability of coenzyme Q10 (CoQ10), Kolliphor® HS 15 (HS15) was employed as a carrier to build up a stable CoQ10-loaded micelle delivery system. The impact of micellar compositions, the preparation condition, and the preparation method on size characteristics, the solubilization efficiency, and micellar stability were investigated. The optimal preparation conditions were 1:6, 4, 0.2%, 118°C, and 25 min for CoQ10/HS15 mass ratio, pH value, the concentration of glucose, and the sterilization conditions. Upon these conditions, the particle size, polydispersity index (PDI), zeta potential, the entrapment efficiency, drug loading, and the critical micelle concentration (CMC) of CoQ10-loaded micelles were 19.76 nm, 0.112, -3.405 mV, 99.39%, 13.77%, and 5.623 × 10(-4) g/mL, respectively. Differential scanning calorimetry (DSC) analysis collectively corroborated that CoQ10 was entrapped into the micelles in amorphous form. The release pattern of drug was analyzed and proved to follow the first order. Additionally, the samples were exposed to the temperatures of 30°C for 6 months with more significant impact on their stabilities as compared to 4 and 25°C based on particle size and PDI. Under constant humidity with light protection long-term (25 ± 2°C, relative humidity (RH) 60 ± 10%, 18 months) conditions, there was no variation except minor changes of CoQ10 content of the samples. The shelf life of the micellar samples could be predicted as 24 months based on the stability results. Consequently, the CoQ10-loaded micelles showed excellent stabilities below 25°C as a potential drug candidate for further clinical applications.

4-Aminopyridyl-based CYP51 inhibitors as anti-Trypanosoma cruzi drug leads with improved pharmacokinetic profile and in vivo potency.

CYP51 is a P450 enzyme involved in the biosynthesis of the sterol components of eukaryotic cell membranes. CYP51 inhibitors have been developed to treat infections caused by fungi, and more recently the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. To specifically optimize drug candidates for T. cruzi CYP51 (TcCYP51), we explored the structure-activity relationship (SAR) of a N-indolyl-oxopyridinyl-4-aminopropanyl-based scaffold originally identified in a target-based screen. This scaffold evolved via medicinal chemistry to yield orally bioavailable leads with potent anti-T. cruzi activity in vivo. Using an animal model of infection with a transgenic T. cruzi Y luc strain expressing firefly luciferase, we prioritized the biaryl and N-arylpiperazine analogues by oral bioavailability and potency. The drug-target complexes for both scaffold variants were characterized by X-ray structure analysis. Optimization of both binding mode and pharmacokinetic properties of these compounds led to potent inhibitors against experimental T. cruzi infection.

Non-linear pharmacokinetics of octaarginine-modified lipid nanoparticles: barriers from in vitro to in vivo.

A rational development of an efficient siRNA delivery system is important for streamlining the RNAi-based drug development process. However, a huge gap frequently exists between in vitro and in vivo activity, which is the rate-limiting step for developing versatile nanoparticles. We report herein on a remarkable non-linearity in pharmacokinetics (PK), but not pharmacodynamics (PD) using octaarginine (R8) modified lipid nanoparticles in mice. A quantitative study of siRNA molecules between cultured cells and mouse liver revealed a high correlation between intracellular siRNA molecules and their RNAi activities, indicating that there was no significant difference in the efficiency in PD. In contrast, a remarkable difference was observed in the non-linearity in PK. Quantitative analysis of the time profile for siRNA showed that the percentage of siRNA accumulation in mice was severely decreased with decreasing input dose compared to in vitro data. These unexpected data reveal an important clue to bridging the gap between in vitro and in vivo activity.

Synthesis and application of 188Re-MN-16ET/Lipiodol in a hepatocellular carcinoma animal model.

INTRODUCTION: Hepatocellular carcinoma is the most common form of primary hepatic carcinoma. A new N(2)S(2) tetradentate ligand, N-[2-(triphenylmethyl)thioethyl]-3-aza-19-ethyloxycarbonyl-3-[2-(triphenylmethyl)thioethyl]octadecanoate (H(3)MN-16ET), was introduced and labeled with (188)Re to create (188)Re-MN-16ET in the Lipiodol phase. The potential of (188)Re-MN-16ET/Lipiodol for hepatoma therapy was evaluated in a hepatocellular carcinoma animal model of Sprague-Dawley rats implanted with the N1S1 cell line. METHODS: Synthesis of H(3)MN-16ET was described, and characterization was identified by infrared, nuclear magnetic resonance and mass spectra. We compared the effects of transchelating agents (glucoheptonate or tartaric acid) and a reducing agent (stannous chloride) on the complexing of (188)Re-perrhenate and H(3)MN-16ET. Twenty-four rats implanted with hepatoma were injected with 3.7 MBq/0.1 ml of (188)Re-MN-16ET/Lipiodol or (188)Re-MN-16ET via transcatheter arterial embolization. Biodistribution experiments and single-photon emission computed tomography imaging were performed to investigate tumor accumulation. RESULTS: H(3)MN-16ET was proved to easily conjugate with the Re isotope and showed good solubility in Lipiodol. The radiochemical purity of (188)Re-MN-16ET/Lipiodol with 10 mg tartaric acid and stannous chloride was shown to be more than 90%. The major distribution sites of (188)Re-MN-16ET in Sprague-Dawley rats were hepatoma and the liver. However, the radioactivity at the tumor site postadministered with (188)Re-MN-16ET was quickly decreased from 9.15±0.23 (at 1 h) to 2.71%±0.18% of injected dose/g (at 48 h). The biodistribution and micro-single-photon emission computed tomography/computed tomography image data showed that (188)Re-MN-16ET/Lipiodol was selectively retained at the tumor site, with 11.55±1.44, 13.16±1.46 and 10.67%±0.95% of injected dose/g at 1, 24 and 48 h postinjection, respectively. The radioactivity in normal liver tissue was high but significantly lower than that of the tumors. CONCLUSION: H(3)MN-16ET is a suitable tetradentate ligand for (188)Re labeling. From the animal data, we suggest that (188)Re-MN-16ET/Lipiodol has the potential to be a therapeutic radiopharmaceutical for hepatoma treatment.

Final report on the safety assessment of sodium cetearyl sulfate and related alkyl sulfates as used in cosmetics.

Sodium cetearyl sulfate is the sodium salt of a mixture of cetyl and stearyl sulfate. The other ingredients in this safety assessment are also alkyl salts, including ammonium coco-sulfate, ammonium myristyl sulfate, magnesium coco-sulfate, sodium cetyl sulfate, sodium coco/hydrogenated tallow sulfate, sodium coco-sulfate, sodium decyl sulfate, sodium ethylhexyl sulfate, sodium myristyl sulfate, sodium oleyl sulfate, sodium stearyl sulfate, sodium tallow sulfate, sodium tridecyl sulfate, and zinc coco-sulfate. These ingredients are surfactants used at concentrations from 0.1% to 29%, primarily in soaps and shampoos. Many of these ingredients are not in current use. The Cosmetic Ingredient Review (CIR) Expert Panel previously completed a safety assessment of sodium and ammonium lauryl sulfate. The data available for sodium lauryl sulfate and ammonium lauryl sulfate provide sufficient basis for concluding that sodium cetearyl sulfate and related alkyl sulfates are safe in the practices of use and concentration described in the safety assessment.

Kinetic study of the prooxidant effect of alpha-tocopherol. Hydrogen abstraction from lipids by alpha-tocopheroxyl radical.

A kinetic study of the prooxidant effect of alpha-tocopherol was performed. The rates of allylic hydrogen abstraction from various unsaturated fatty acid esters (ethyl stearate 1, ethyl oleate 2, ethyl linoleate 3, ethyl linolenate 4, and ethyl arachidonate 5) by alpha-tocopheroxyl radical in toluene were determined, using a double-mixing stopped-flow spectrophotometer. The second-order rate constants (k (p)) obtained are <1 x 10(-2) M(-1 )s(-1) for 1, 1.90 x 10(-2) M(-1 )s(-1) for 2, 8.33 x 10(-2 )M(-1 )s(-1) for 3, 1.92 x 10(-1) M(-1 )s(-1) for 4, and 2.43 x 10(-1 )M(-1 )s(-1) for 5 at 25.0 degrees C. Fatty acid esters 3, 4, and 5 contain two, four, and six -CH(2)- hydrogen atoms activated by two pi-electron systems (-C=C-CH(2)-C=C-). On the other hand, fatty acid ester 2 has four -CH(2)- hydrogen atoms activated by a single pi-electron system (-CH(2)-C=C-CH(2)-). Thus, the rate constants, k (abstr)/H, given on an available hydrogen basis are k (p)/4 = 4.75 x 10(-3 )M(-1 )s(-1) for 2, k (p)/2 = 4.16 x 10(-2) M(-1 )s(-1) for 3, k (p)/4 = 4.79 x 10(-2 )M(-1 )s(-1) for 4, and k (p)/6 = 4.05 x 10(-2 )M(-1 )s(-1) for 5. The k (abstr)/H values obtained for 3, 4, and 5 are similar to each other, and are by about one order of magnitude higher than that for 2. From these results, it is suggested that the prooxidant effect of alpha-tocopherol in edible oils, fats, and low-density lipoproteins may be induced by the above hydrogen abstraction reaction.

Estudo descritivo sobre a antibioticoprofilaxia na rupturaprematura pré-termo de membranas / Perinatal results of antibioticprophyxalis with erythromycin in preterm premature rupture of the membranes

Objetivo: avaliar os resultados perinatais do uso profilático de estearato de eritromicina nas pacientesinternadas na unidade de gestação alto risco da Maternidade Carmela Dutra (MCD), Florianópolis–SC, com diagnóstico de ruptura prematura pré-termo de membranas (RPM). Métodos: estudo descritivo com análise de todas as pacientes internadas com o diagnóstico de RPM e com idade gestacional entre 20 semanas e 33 semanas e cinco dias. Foram excluídas da pesquisa gestantes com históriade hipersensibilidade à eritromicina, com sinais clínicos e/ou laboratoriais de corioamnionite, que estavam emtrabalho de parto ou que faziam uso de antibióticos no momento da internação. A amostra obtida entre 1º de abril de 2007 e 15 de maio de 2008 foi de 22 pacientes. Resultados e conclusões: o tempo médio de latência foi de 12 dias. Não houve casos confirmados decorioamnionite. Uma (4,54%) gestante desenvolveu quadro de endometrite puerperal. Não houve óbitos maternos. Dois (9,09%) recém-nascidos desenvolveram sepse. A taxa de óbito neonatal foi de 13,63%. Apesarda nossa pequena casuística, o uso de eritromicina nas pacientes com RPM parece estar associado a umadiminuição na taxa de corioamnionite.

Objective: The purposes of this study were to evaluate perinatal results of the prophylactic use of erythromycin to patients admitted in the high-riskgestation unit at Carmela Dutra Maternity Hospital, Florianópolis – SC with preterm premature rupture ofmembranes (PROM). Methods: We performed a descriptive analysis ofall patients with PROM and gestational age between 20 weeks and 33 weeks plus 5 days. Patients with erythromycin allergy, with chorioamnionitis signs orwomen who already being prescribed antibiotics were excluded from this study. Enrolment was from April 1,2007, until May 15, 2008. Twenty-two women had been followed up in this study. Results and Conclusions: The medium latency period was 12 days. There was not confirmed chorioamnionitis case. The occurrence of endometritis was 4,54%. There was not maternal death. The occurrence of neonatal sepsis was 9,09% and theoccurrence of neonatal deaths was 13,63%. Despite our small casuistry, the prophylactic use of erythromycinseems to reduce the chorioamnionitis rate.

Antitumor activity and pharmacokinetics of liposomes containing lipophilic gemcitabine prodrugs.

BACKGROUND: Gemcitabine is a deoxycytidine analogue that exhibits antitumoral activity against adenocarcinomas of the colon, lung and pancreas. After intravenous injection, gemcitabine is rapidly converted to the inactive metabolite 2'-deoxy-2',2'-difluorouridine by cytidine deaminase. MATERIALS AND METHODS: To improve the pharmacokinetic behavior and the antitumor activity of the drug, the gemcitabine prodrug, 4-(N)-stearoylgemcitabine (C18gem) was incorporated in liposomes and both the pharmacokinetic and the in vivo activity of this formulation intravenously or peritumorally administered in nude female CR1:Nu/Nu(CD-1)BR mice grafted with HT-29 and KB 396p cells were studied. RESULTS: The C18gem-liposomes showed both higher plasma half life and tumor regression than control and gemcitabine. CONCLUSION: The incorporation of C18gem-prodrug in liposomes increased the plasma half life of the drug resulting in increased accumulation in the tumor cells and a higher level of antitumoral efficacy. The results obtained with different tumors sensitive to gemcitabine support the efficacy of this proposed drug delivery system.

Dynamics and partitioning of spin-labeled stearates into the lipid domain of stratum corneum.

The EPR spectra of the positional isomers n-doxyl stearic acids (n-DSA), with n=5, 12 and 16, and 5-doxyl methyl stearate (5-DMS) structured in the lipid domain of intact stratum corneum (SC), are characterized by the thermodynamic equilibrium of two distinct spectral components provided by two different motional states of the spin-labeled chains. A two-component model used in the EPR spectra simulations provided the relative populations of the components, allowing for the calculation of the thermodynamic profile. Based on a detailed investigation, the more motionally restricted population of spin labels (component 1) is found to arise when the spin label is hydrogen-bonded to the polar surfaces of the membranes, while the less motionally restricted population (component 2) is generated by spin labels nonhydrogen-bonded and more deeply inserted in the hydrophobic core. The 5-DSA is bound tightly to the polar surfaces (DeltaG(o)2 --> 1=-1.75 kcal/mol and DeltaH(o)2 --> 1=-13.8 kcal/mol), whereas the more lipophilic 5-DMS has a major spin population stabilized in the hydrophobic core (DeltaG(o)2 --> 10.57 kcal/mol and DeltaH(o)2 --> 1=-9.1 kcal/mol). Upon lipid-depleting SC increases the interactions of the probe with the polar surfaces, thereby decreasing its rotational diffusion. In contrast, the treatment of SC with oleic acid, a permeation enhancer, drastically increases the mobility of the spin labels, particularly that of component 1, and the thermodynamic equilibrium shifts towards the formation of component 2. A mechanism for water permeation in SC is also proposed.

The microencapsulated ascorbic acid release in vitro and its effect on iron bioavailability.

The present study was carried out to examine the stability of microencapsulated ascorbic acid in simulated-gastric and intestinal situation in vitro and the effect of microencapsulated ascorbic acid on iron bioavailability. Coating materials used were polyglycerol monostearate (PGMS) and medium-chain triacylglycerol (MCT), and core materials were L-ascorbic acid and ferric ammonium sulfate. When ascorbic acid was microencapsulated by MCT, the release of ascorbic acid was 6.3% at pH 5 and 1.32% at pH 2 in simulated-gastric fluids during 60 min. When ascorbic acid was microencapsulated by PGMS, the more ascorbic acid was released in the range of 9.5 to 16.0%. Comparatively, ascorbic acid release increased significantly as 94.7% and 83.8% coated by MCT and PGMS, respectively, for 60 min incubation in simulated-intestinal fluid. In the subsequent study, we tested whether ascorbic acid enhanced the iron bioavailability or not. In results, serum iron content and transferring saturation increased dramatically when subjects consumed milks containing both encapsulated iron and encapsulated ascorbic acid, compared with those when consumed uncapsulated iron or encapsulated iron without ascorbic acid. Therefore, the present data indicated that microencapsulated ascorbic acid with both PGMS and MCT were effective means for fortifying ascorbic acid into milk and for enhancing the iron bioavailability.

Effect of Terplex/VEGF-165 gene therapy on left ventricular function and structure following myocardial infarction. VEGF gene therapy for myocardial infarction.

BACKGROUND: We used a novel lipopolymeric gene delivery system, TeplexDNA, to transfect myocardium with plasmid vascular endothelial growth factor-165 (pVEGF) and evaluated the ability of pVEGF to preserve left ventricular function and structure after coronary ligation in a rabbit model. METHODS: New Zealand white rabbits underwent circumflex coronary ligation after direct intramyocardial injection of either Terplex alone or Terplex + 50 microg pVEGF-165. Serial echocardiography and histologic studies were performed (n = 12/group). Mortality did not differ between groups. The data is reported as the mean +/- standard deviation. RESULTS: Over the 21 days following coronary ligation, pVEGF-165-treated animals demonstrated significant improvement in fractional shortening (20-25%, p = 0.02), long axis two-dimensional ejection fraction (42-51%, p=0.02) and short axis m-mode ejection fraction (46-54%, p = 0.02). No significant improvements were noted in the control group. VEGF-treated animals had a 50% increase in peri-infarct vessel density and a trend towards a smaller infarct size (20% vs. 29%, p = 0.10). In animals receiving pVEGF-165, the diastolic ventricular area increased from 1.87 +/- 0.24 cm2 prior to ligation to 2.19 +/- 0.23 cm2 at 21 days following ligation, compared to an increase from 1.84 +/- 0.38 to 2.54 +/- 0.55 cm2 over the same period in control animals (p = 0.03). Similarly, the systolic ventricular area in VEGF-165 animals increased from 1.06 +/- 0.26 cm2 prior to ligation to 1.50 +/- 0.29 cm2 at 21 days following ligation, compared to an increase from 1.16 +/- 0.30 to 1.86 +/- 0.43 cm2 over the same period in the control animals (p = 0.04). CONCLUSION: TerplexDNA mediated delivery of plasmid VEGF administered at the time of coronary occlusion improves left ventricular function and reduces left ventricular dilation following myocardial infarction.

Minoxidil-containing dosage forms: skin retention and after-rinsing hair-growth promotion.

Three kinds of topical dosage forms of minoxidil (MXD), namely vesicles, double emulsions, and an inclusion complex with hydoxypropyl-beta-cyclodextrin (HP-beta-CD), were prepared. The skin retention of MXD in the preparations was evaluated in vitro using hairless mouse skins. After applying the preparations onto the skin and rinsing it, the amount of the drug left on the skin was determined using HPLC. Retention was the highest when the drug was encapsulated in cationic vesicles. Nonionic vehicle, the double emulsion, and HP-beta-CD left no significant amount of the drug after rinsing the skin. Thus, an ionic interaction between the cationic vehicle and negatively charged skin is likely responsible for the relatively high skin retention. In vivo hair growth-promotion effect of each dosage form was investigated, in which the sample application onto the clipped backs of female mice (C57BL6) and the subsequent rinsing of the backs were done once a day for 30 days. Only MXD in the cationic vesicles had hair growth promotion effect, possibly due to significant skin retention.

Preparation of albumin preferential surfaces on poly(vinyl chloride) membranes via surface self-segregation.

Poly(vinyl chlorides)-graft-[omega-stearyl-poly(ethylene oxide)] (PVC-g-SPEO), which has a poly(vinyl chloride) (PVC) backbone, poly(ethylene oxide) (PEO) side chain, and stearyl end groups, has been synthesized. Self-organizing blends of the amphiphilic comb polymer in poly(vinyl chlorides) have been examined as a means to create albumin preferential surfaces on polymer films. X-ray photoelectron spectroscopy (XPS) analysis indicates substantial surface segregation of the PVC-g-SPEO. A surface concentration of 59.9 EO wt % is achieved by the solution casting and heat treatment of a film with a bulk concentration of only 3.78 EO wt %. In the aqueous environment, the surface rearrangement of PVC-g-SPEO/PVC blend film is limited and presents a high interfacial energy and high depolar component of interfacial energy due to the "tail-like" SPEO side chain. Protein adsorption tests confirm that PVC-g-SPEO/PVC blend films absorb high levels of albumin and dramatically resist fibrinogen adsorption. Surfaces to attract and reversibly bind albumin, which might diminish the occurrence of thrombosis, inflammation, and infection, are developed by self-organizing blends of the amphiphilic comb polymer in poly(vinyl chlorides).

The effect of fatty acid substitution on the in vitro release of amphotericin B from micelles composed of poly(ethylene oxide)-block-poly(N-hexyl stearate-L-aspartamide).

The effect of fatty acid substitution on the in vitro release of amphotericin B (AmB) from micelles composed of poly(ethylene oxide)-block-poly[N-(6-hexyl stearate)-L-aspartamide] (PEO-b-PHSA) was investigated. PEO-b-PHSA at 11, 50 and 70% of stearic acid substitution self assembled into micelles that effectively encapsulate AmB by solvent evaporation and dialysis methods. The sustained release of AmB from PEO-b-PHSA micelles was evidenced, by measuring the transfer of the drug to lipid vesicles [dipalmitoyl phosphatidylcholine:cholesterol:dimyristoyl phosphatidyglycerol (3:1:0.25)]. The release of AmB for PEO-b-PHSA micelles was markedly influenced by the degree of fatty acid substitution--as it increased, the release of AmB slowed. Accordingly, drug release was found to correlate with haemolysis induced by AmB encapsulated in PEO-b-PHSA micelles. At 11% stearic acid substitution, encapsulation of AmB had little effect on the drug's ability to induce untoward haemolysis. In contrast, AmB stably encapsulated in PEO-b-PHSA micelles at 50 and 70% caused no hemolysis up to 20 microg/ml. Lastly, PEO-b-PHSA micelles at 50 and 70% were able to elute entirely as micelles during size-exclusion chromatography, indicating their stability toward dissociation after dilution. The results point to a nanoscopic drug depot that may release AmB at controlled rates.

Influence of neutron activation factors on matrix tablets for site specific delivery to the colon.

The impact of the neutron activation procedure, i.e. incorporation of samarium oxide (Sm(2)O(3)) and neutron irradiation, on the compression properties (including the crushing strength) and in vitro dissolution of potential colonic delivery systems based on matrix tablets of amidated pectin (Am.P) or two types of hydroxypropyl methylcellulose (HPMC) was investigated. The neutron activation factors did not influence the compression properties of the tablets. Replacement of magnesium stearate with samarium stearate in directly compressed Am.P tablets to achieve both radiolabelling and lubrication resulted in a greater extent of concentration-dependent reduction of the crushing strength. Dissolution tests demonstrated that irradiation increased the release of the model drug ropivacaine from the tablets. The extent of this increase was unexpectedly low considering the previously observed degradation of the polymer expressed as an irradiation-induced viscosity reduction in solutions prepared from the polymers. Delayed-release coating with Eudragit L 100 protected the HPMC tablets against the release-increasing effect of irradiation until the late phases of release. Sm(2)O(3) retarded the release to a varying extent depending on particle characteristics. Incorporation of Sm(2)O(3) in the coating layer did not influence the release. However, one-third of the radioactivity leached from the coating within 60 min in 0.1 M HCl.

Electrogenicity of hepatocellular fatty acid uptake.

Sensitivity of cellular fatty acids uptake to the membrane potential difference is still a matter of controversy. For direct evaluation of potential sensitivity the effect of changing membrane potential on uptake of a fluorescent long chain fatty acid derivative, 12-NBD-stearate, in isolated rat hepatocytes, was examined. Changes in membrane potential were achieved by patch clamp procedures. Fatty acid influx was simultaneously determined by recording of cell fluorescence. Hyperpolarization from -30 to -70 mV accelerated fatty acid influx whereas depolarization to +50 mV reduced uptake. After obtaining equilibrium hyperpolarization increased cell fluorescence, whereas depolarization pushed NBD-stearate out of cells. Potential sensitivity of uptake was dependent on the fatty acid concentrations in the medium with most prominent effects at low unbound concentrations. These data show that, at low fatty acid concentrations, uptake is, in part, driven by an intracellular negative electric membrane potential.

L-FABP and I-FABP expression increase NBD-stearate uptake and cytoplasmic diffusion in L cells.

The effects of intestinal and liver fatty acid binding protein (I- and L-FABP, respectively) expression on single-cell fatty acid uptake, internalization, and cytoplasmic diffusion were determined in transfected L cell fibroblasts. These parameters were measured using the nonesterifiable fluorescent fatty acid probe 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate) and fluorescence digital imaging. In single-cell fluorescence imaging experiments, L-FABP-expressing cells, but not I-FABP-expressing cells, increased NBD-stearate uptake 1.7-fold compared with control cells. Both I- and L-FABP increased the cytoplasmic diffusion rate of the internalized NBD-stearate 2.6- and 1.9-fold, respectively, compared with control cells. However, increased NBD-stearate lateral membrane mobility was observed only in L-FABP-expressing cells. After incubation of the cells with 4 microM NBD-stearate at 37 degrees C for 30 min, fluorescence deconvolution imaging indicated that NBD-stearate was localized primarily into lipid droplets in all cell lines. The differential effect of these proteins on fatty acid uptake and intracellular trafficking in single cells illustrates a possible difference in the physiological function of I- and L-FABP in intact cells.

Sterol carrier protein-2 expression increases NBD-stearate uptake and cytoplasmic diffusion in L cells.

The effects of sterol carrier protein-2 (SCP-2) expression on fatty acid uptake and cytoplasmic diffusion were determined using L cell fibroblasts transfected with cDNA encoding either the 15- or 13. 2-kDa form of SCP-2. Cis-parinarate and 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate) were used as nonesterifiable fluorescent fatty acid probes. NBD-stearate and cis-parinarate uptake was rapid and saturable. In 15-kDa SCP-2-expressing cells, the extent of NBD-stearate and cis-parinarate uptake was increased 1.4- and 1. 2-fold, respectively, compared with control. In the 13.2-kDa SCP-2-expressing cells, the extent of NBD-stearate and cis-parinarate uptake was increased 1.3- and 1.1-fold, respectively, compared with control cells. NBD-stearate cytoplasmic diffusion was increased 1.5-fold in 15-kDa SCP-2-expressing cells, but not in 13. 2-kDa SCP-2-expressing cells, compared with control cells. After incubation with NBD-stearate for 30 min at 37 degrees C, fluorescence imaging indicated that NBD-stearate was localized primarily in lipid droplets in all cell lines. These results suggest that SCP-2 may be involved not only in fatty acid uptake but also in intracellular fatty acid trafficking.