Mechanisms allowing protein delivery in nasal mucosa using NPL nanoparticles.

The intranasal administration of proteins using nanoparticles is a promising approach for several applications, especially for mucosal vaccines. Delivery of protein within the epithelial barrier is a key point to elicit an immune response and nano-carrier has to show no toxicity. The aim of this work was to elucidate the interactions of cationic porous nanoparticles loaded with protein delivery for antigen delivery in the nose. We investigated the loading, the cellular delivery and the epithelial transcytosis of proteins associated to these nanoparticles containing an anionic lipid in their core (NPL). NPL were highly endocytosed by airway epithelial cells and significantly improved the protein delivery into the cell. In vitro transcytosis studies showed that NPL did not modify the in vitro epithelial permeability suggesting no toxicity of these carriers. Moreover protein and NPL did not translocate the epithelial barrier. In vivo studies demonstrated that NPL prolonged the nasal residence time of the protein and no NPL were found beyond the epithelial barrier in vivo, precluding a negative side effect. All together these results establish the NPL as a bio-eliminable and optimal vaccine carrier.

Enteric-coated epichlorohydrin crosslinked dextran microspheres for site-specific delivery to colon.

Enteric-coated epichlorohydrin crosslinked dextran microspheres containing 5-Fluorouracil (5-FU) for colon drug delivery was prepared by emulsification-crosslinking method. The formulation variables studied includes different molecular weights of dextran, volume of crosslinking agent, stirring speed, time and temperature. Dextran microspheres showed mean entrapment efficiencies ranging between 77 and 87% and mean particle size ranging between 10 and 25 µm. About 90% of drug was released from uncoated dextran microspheres within 8 h, suggesting the fast release and indicated the drug loaded in uncoated microspheres, released before they reached colon. Enteric coating (Eudragit-S-100 and Eudragit-L-100) of dextran microspheres was performed by oil-in-oil solvent evaporation method. The release study of 5-FU from coated dextran microspheres was complete retardation in simulated gastric fluid (pH 1.2) and once the coating layer of enteric polymer was dissolved at higher pH (7.4 and 6.8), a controlled release of the drug from the microspheres was observed. Further, the release of drug was found to be higher in the presence of dextranase and rat caecal contents, indicating the susceptibility of dextran microspheres to colonic enzymes. Organ distribution and pharmacokinetic study in albino rats was performed to establish the targeting potential of optimized formulation in the colon.

Supramolecular gels of poly-α-cyclodextrin and PEO-based copolymers for controlled drug release.

The aim of this work was to prepare syringeable supramolecular gels of α-cyclodextrin-polymer (poly-αCD) with various poly(ethylene oxide) (PEO)-based copolymers, which can be suitable to form depots for controlled drug release. A series of water-soluble poly-αCDs was synthesized from αCD by crosslinking with epichlorohydrin in alkaline medium. The chemical composition of the polymers was characterized by NMR (αCD content>53%) and the molecular weight was evaluated using static light scattering (SLS). Supramolecular assemblies occurred by mixing poly-αCD (20-40% w/v) with a PEO-based polymer (i.e., PEG, Pluronic® F127 or Tetronic® 908) (10-15% w/v). Phase separation was observed and the αCD content in each phase was determined by means of the phenol-sulfuric acid colorimetric method. Formation of poly-αCD/PEO-based polymer 3D-supramolecular complexes was confirmed by diffusion-ordered NMR spectroscopy (DOSY) and X-ray diffractometry. The supramolecular assemblies showed good cytocompatibility against SAOS-2 cells and in the HET-CAM test. The supramolecular gels were able to sustain the release of vancomycin for at least 5 days at 37 °C, more efficiently than dispersions of each polymer component in separate. These results open new possibilities in the design of novel controlled delivery systems for the treatment of bone infections.

Two-photon fluorescence imaging and bimodal phototherapy of epidermal cancer cells with biocompatible self-assembled polymer nanoparticles.

We have developed herein an engineered polymer-based nanoplatform showing the convergence of two-photon fluorescence imaging and bimodal phototherapeutic activity in a single nanostructure. It was achieved through the appropriate choice of three different components: a ß-cyclodextrin-based polymer acting as a suitable carrier, a zinc phthalocyanine emitting red fluorescence simultaneously as being a singlet oxygen ((1)O2) photosensitizer, and a tailored nitroaniline derivative, functioning as a nitric oxide (NO) photodonor. The self-assembly of these components results in photoactivable nanoparticles, approximately 35 nm in diameter, coencapsulating a multifunctional cargo, which can be delivered to carcinoma cells. The combination of steady-state and time-resolved spectroscopic and photochemical techniques shows that the two photoresponsive guests do not interfere with each other while being enclosed in their supramolecular container and can thus be operated in parallel under control of light stimuli. Specifically, two-photon fluorescence microscopy allows mapping of the nanoassembly, here applied to epidermal cancer cells. By detecting the red emission from the phthalocyanine fluorophore it was also possible to investigate the tissue distribution after topical delivery onto human skin ex vivo. Irradiation of the nanoassembly with visible light triggers the simultaneous delivery of cytotoxic (1)O2 and NO, resulting in an amplified cell photomortality due to a combinatory effect of the two cytotoxic agents. The potential of dual therapeutic photodynamic action and two-photon fluorescence imaging capability in a single nanostructure make this system an appealing candidate for further studies in biomedical research.

Efficacy and safety of coadministration of rosuvastatin, ezetimibe, and colestimide in heterozygous familial hypercholesterolemia.

Aggressive low-density lipoprotein (LDL) cholesterol-lowering therapy is important for high-risk patients. However, sparse data exist on the impact of combined aggressive LDL cholesterol-lowering therapy in familial hypercholesterolemia (FH), particularly on side effects to changes in plasma coenzyme Q10 and proprotein convertase subtilisin/kexin type 9 levels. We enrolled 17 Japanese patients with heterozygous FH (12 men, 63.9 ± 7.4 years old) with single LDL receptor gene mutations in a prospective open randomized study. Permitted maximum doses of rosuvastatin (20 mg/day), ezetimibe (10 mg/day), and granulated colestimide (3.62 g/day) were introduced sequentially. Serum levels of LDL cholesterol decreased significantly by -66.4% (p <0.001) and 44% of participants achieved LDL cholesterol levels <100 mg/dl. There were no serious side effects or abnormal laboratory data that would have required the protocol to have been terminated except for 1 patient with myalgia. Coadministration of ezetimibe and granulated colestimide further lowered serum LDL cholesterol more than rosuvastatin alone without changing plasma coenzyme Q10 and proprotein convertase subtilisin/kexin type 9 levels. In conclusion, adequate introduction of this aggressive cholesterol-lowering regimen can improve the lipid profile of FH.

Development of a new delivery system consisting in "drug--in cyclodextrin--in nanostructured lipid carriers" for ketoprofen topical delivery.

A new delivery system based on drug cyclodextrin (Cd) complexation and loading into nanostructured lipid carriers (NLC) has been developed to improve ketoprofen therapeutic efficacy. The proposed strategy exploits both the solubilizing and stabilizing properties of Cds and the prolonged release, high tolerability and percutaneous absorption enhancer properties of NLC. Two different polymeric Cds, i.e. ß-Cd-epichlorohydrin polymer (EPI-ßCd) and carboxymethylathed-ß-Cd-epichlorohydrin polymer (EPI-CMßCd) were tested and two different techniques to obtain solid ketoprofen-polymeric Cd complexes (i.e. co-grinding and co-lyophilization) were compared, to investigate the influence of the preparation method on the physicochemical properties of the end product. EPI-ßCd was more effective than EPI-CMßCd in enhancing the solubility and dissolution properties of ketoprofen. Co-grinding in dry conditions was the best preparation technique of solid drug-Cd systems, allowing obtainment of homogeneous amorphous particles of nanometric range. NLC consisting in a mixture of Compritol® 888 ATO (glyceryl behenate) and Labrafac Lipophile were obtained by ultrasonication. Both empty and loaded NLC were suitably characterized for particle size, pH, entrapment efficiency and drug release behavior. The best (drug-Cd)-loaded NLC system, formulated into a xanthan hydrogel, exhibited drug permeation properties clearly better than those of the plain drug suspension or the plain drug-loaded NLC, in virtue of the simultaneous exploitation of the solubilizing effect of cyclodextrin and the penetration enhancer properties of NLC.

In vitro and in vivo studies on the complexes of glipizide with water-soluble ß-cyclodextrin-epichlorohydrin polymers.

The purpose of this study was to evaluate the potential of a newly modified cyclodextrin derivative, water-soluble ß-cyclodextrin-epichlorohydrin polymer (ß-CDP), as an effective drug carrier to enhance the dissolution rate and oral bioavailability of glipizide as a poorly water-soluble model drug. Inclusion complexes of glipizide with ß-CDP were prepared by the co-evaporation method and characterized by phase solubility, dissolution, and differential scanning calorimetry. The solubility curve was classified as type A(L), which indicated the formation of 1:1 complex between glipizide and ß-CDP. ß-CDP had better properties of increasing the aqueous solubility of glipizide compared with HP-ß-CD. The dissolution rate of drug from the ß-CDP complexes was significantly greater than that of the corresponding physical mixtures indicating that the formation of amorphous complex increased the solubility of glipizide. Moreover, the increment in drug dissolution rate from the glipizide/ß-CDP systems was higher than that from the corresponding ones with HP-ß-CD, which indicated that ß-CDP could provide greater capability of solubilization for poorly soluble drugs. Furthermore, in vivo study revealed that the bioavailability of glipizide was significantly improved by glipizide /ß-CDP inclusion complex after oral administration to beagle dogs.

One-generation reproductive toxicity study of epichlorohydrin in Sprague-Dawley rats.

This study was conducted to evaluate the potential reproductive toxicity of epichlorohydrin in a one-generation reproduction toxicity study in compliance with OECD Test Guideline 415. Twenty-four male and female rats per group were given epichlorohydrin by gavage at 0, 3.3, 10, and 30 mg/kg/day. Males were dosed for 10 weeks prior to and during mating. Females were dosed from 2 weeks before mating to day 21 of lactation. At 30 mg/kg, an increase in the incidence of clinical signs (i.e., nasal discharge, soft feces, depression, and piloerection), gross necropsy findings (i.e., cystic pustule of the epididymidis and enlargement of the kidney) and the weights of heart, liver, and epididymidis, a decrease in male fertility, and an increased incidence of histopathological changes of the testis, epididymidis, and kidney were observed. At 10 mg/kg, decreased male fertility and increased kidney weight and incidence of histopathological changes of the epididymidis were found. There was a slight, but nonsignificant, reduction in the male fertility index at the dose of 3.3 mg/ kg. Under these experimental conditions, the lowest-observed-adverse-effect level of epichlorohydrin was 3.3 mg/kg/day for parent animals and their offspring. The absolute toxic dose for parent animals and their offspring was estimated to be 10 mg/kg/day.

Colestimide lowers plasma glucose levels and increases plasma glucagon-like PEPTIDE-1 (7-36) levels in patients with type 2 diabetes mellitus complicated by hypercholesterolemia.

BACKGROUND: Colestimide has been reported to lower blood glucose levels in patients with type 2 diabetes complicated by hypercholesterolemia. AIM: To examine the mechanism by which colestimide decreases plasma glucose levels in the above patients. METHODS: A total of 16 inpatients with type 2 diabetes complicated by hypercholesterolemia received colestimide for 1 week after their plasma glucose levels stabilized. We measured plasma glucose, serum immunoreactive insulin (IRI), serum lipid, plasma glucagon, and plasma glucagon-like peptide-1 (GLP-1) levels. These variables at baseline and 1 week of colestimide administration were compared. RESULTS: Preprandial plasma glucose levels (baseline: 132 +/- 33 mg/dL vs. completion: 118 +/- 43 mg/dL, P=0.073) tended to decrease after colestimide administration, while 1-hr postprandial plasma glucose levels (baseline: 208 +/- 49 mg/dL vs. completion: 166 +/- 30 mg/dL, P<0.001) and 2-hr postprandial plasma glucose levels (baseline: 209 +/- 56 mg/dL vs. completion: 178 +/- 39 mg/dL, P=0.015) decreased significantly at 1 week of colestimide administration. The 2-hr postprandial plasma GLP-1 level was significantly (P=0.015) higher at 1 week of colestimide administration as compared with the baseline level, while there were no significant changes in preprandial and 1-hr postprandial plasma GLP-1 levels. CONCLUSIONS: The GLP-1-increasing activity of colestimide may explain, at least in part, the mechanism of its blood glucose-lowering activity in patients with type 2 diabetes complicated by hypercholesterolemia.

Effect of oral colestimide on the elimination of high-dose methotrexate in patients with primary central nervous system lymphoma--case report.

Delayed methotrexate (MTX) elimination occurred in two patients with primary central nervous system lymphoma undergoing high-dose MTX treatment. Oral administration of the anion exchange resin colestimide, which binds MTX effectively in vitro, effectively accelerated MTX elimination. Colestimide probably interrupts the enterohepatic circulation, and is a potential oral antidote to MTX toxicity.

Polymorphonuclear leukocytes may impair endothelial function: results of crossover randomized study of lipid-lowering therapies.

OBJECTIVES: To examine whether polymorphonuclear leukocytes (PMNs) in hypercholesterolemia (HC) are activated to generate large amount of superoxide in vivo and hence impair endothelial function and, if so, whether statins, which possess anti-inflammatory properties, may restore PMN-mediated endothelial dysfunction. METHODS AND RESULTS: At baseline, subjects with HC showed impaired endothelial function (P<0.001), estimated by flow-mediated vasodilation of the brachial artery, and increased susceptibility of low-density lipoprotein (LDL) to oxidation (P<0.0001) compared with control subjects. PMNs obtained from HC produced greater amount of superoxide (P<0.0001), showed higher adhesiveness to cultured endothelial cells (HUVECs) (P<0.0001), and impaired endothelial nitric oxide synthase (eNOS) Ser1177 phosphorylation of HUVECs compared with controls (P<0.001). Crossover administration of fluvastatin or colestimide for 3 months lowered LDL to the same levels (P<0.001 for both). Endothelial function was restored (P<0.0001). LDL oxidation (P<0.0001) and superoxide release from PMNs (P<0.0001) were diminished only in fluvastatin but not in colestimide arm. Fluvastatin attenuated PMN adhesion to HUVECs (P<0.0001) and restored eNOS Ser1177 phosphorylation of HUVECs (P<0.001). CONCLUSIONS: Statins may improve endothelial function at least in part by inactivating neutrophils independently of LDL reduction. Our results raise a novel concept that polymorphonuclear leukocytes may attack endothelia and play a pivotal role in the pathogenesis of atherosclerosis.

Identification and quantitative determination of 3-chloro-2-hydroxypropylmercapturic acid and alpha-chlorohydrin in urine of rats treated with epichlorohydrin.

Epichlorohydrin (ECH) is used in many industrial processes. Different toxic effects of ECH were found in rodents. The metabolism of ECH was investigated before in rats using [14C]ECH. The aim of this investigation was the development of non-radioactive quantitative analytical methods for measuring two urinary metabolites of ECH, namely 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) and alpha-chlorohydrin (alpha-CH). The identity of CHPMA and alpha-CH excreted in urine of rats treated with 5 to 35 mg/kg ECH was confirmed by GC-MS. The quantitative analysis of CHPMA, involving ethyl acetate extraction from acidified urine and subsequent methylation and analysis by gas chromatography-flame photometric detection (GC-FPD), showed a method limit of detection of 2 micrograms/ml. The analysis of alpha-CH based on ethyl acetate extraction and subsequent analysis by GC-ECD, showed a method limit of detection of 2 micrograms/ml. CHPMA and alpha-CH derivatives could be determined quantitatively down to concentrations of 0.5 and 0.4 micrograms/ml urine, respectively, by selected-ion monitoring GC-MS under EI conditions. Cumulative urinary excretion of CHPMA and alpha-CH by rats treated with ECH were found to be 31 +/- 10 and 1.4 +/- 0.6% (n = 13) of the ECH dose, respectively. For CHPMA, the dose-excretion relationship suggested partially saturated ECH metabolism. For alpha-CH, the doe-excretion relationship was linear. With fractionated urine collection it was found that approximately 74 and 84% of the total cumulative excretion of CHPMA and alpha-CH, respectively, took place within the first 6 h after administration of ECH. From these investigations it is concluded that the GC-FPD and GC-ECD based methods developed are sufficiently sensitive to measure urinary excretion of CHPMA and alpha-CH in urine from rats administered 5 to 35 mg/kg ECH. It is anticipated that the analysis of CHPMA and alpha-CH based on GC-MS may be sufficiently sensitive to investigate urinary excretion from humans occupationally exposed to ECH.

Comparison of contact site cancer potency across dose routes: case study with epichlorohydrin.

Risk assessment for airborne carcinogens is often limited by a lack of inhalation bioassay data. While extrapolation from oral-based cancer potency factors may be possible for some agents, this is not considered feasible for contact site carcinogens. The change in contact sites (oral: g.i. tract; inhalation: respiratory tract) when switching dose routes leads to possible differences in tissue sensitivity as well as chemical delivery. This research evaluates the feasibility to extrapolate across dose routes for a contact site carcinogen through a case study with epichlorohydrin (EPI). EPI cancer potency at contact sites is compared across three bioassays involving different dose routes (gavage, drinking water, inhalation) through the use of dosimetry models to adjust for EPI delivery to contact sites. Results indicate a large disparity (two orders of magnitude) in potency across the three routes of administration when expressed as the externally applied dose. However, when expressed as peak delivered dose, inhalation and oral potency estimates are similar and overall, the three potency estimates are within a factor of seven. The results suggest that contact site response to EPI is more dependent upon the rate than the route of delivery, with peak concentration the best way to extrapolate across dose routes. These results cannot be projected to other carcinogens without further study.

Toxicity studies of epichlorohydrin in Sprague-Dawley rats.

Adult male and female Sprague-Dawley rats received epichlorohydrin via gavage in distilled water for 10 consecutive days at dose levels of 3, 7, 19, and 46 mg/kg-day, and for 90 days at dose levels of 1, 5, and 25 mg/kg-day. Epichlorohydrin did not adversely effect mortality, but toxicity, at the higher doses, was evident by: 1) losses in body weight gain and organ weights, 2) reductions in food and water consumption, and 3) in the hematological and microscopic examinations in both study periods. Significant decreases in erythrocyte count, hemoglobin, and hematocrit levels were found in the high dose level in males after 10 and 90 days. Dose-related increases in kidney and liver weights were observed in both sexes at 25 mg/kg-day in the 90-day study and in various organs for both 19 and 46 mg/kg-day in the 10-day study. Histopathological examination identified the forestomach as the primary target organ for both sexes and in both studies with significant dose-related increases in mucosal hyperplasia (acanthosis) and hyperkeratosis. Based on the data presented, a lowest observable adverse effect level (LOAEL) for oral exposure of Sprague-Dawley rats to epichlorohydrin is 3 mg/kg-day for 10 days and 1 mg/kg-day is suggested as the no observed adverse effect level (NOAEL) for a 90 day oral exposure. These conclusions were the same whether the lesions were analyzed for each sex individually or whether the data in each study was pooled.

[Ontogenetic characteristics of the body response to chronic exposure to chemical substances]. / Ontogeneticheskie osobennosti otvetnykh reaktsii organizma na khronicheskoe vozdeistvie khimicheskikh veshchestv.

Chronic intoxication by carbon oxide and long-term exposure to hypoxic hypoxia produced more favourable reactions in animals, exposed to these in the early terms of ontogenesis. The observed regularity was not found under chronic exposure to styrene and epichlorohydrin ++. A supposition is made on the evolutionary predetermination of this effect pertaining to unfavourable factors of hypoxic nature.

Acute inhalation exposure to epichlorohydrin transiently decreases rat sperm velocity.

The effect of inhaled epichlorohydrin on rat sperm motility characteristics was evaluated. Male F-344 rats were exposed to 100 ppm epichlorhydrin via inhalation for 4 hr in the morning of Day 0 and killed immediately and on Days 1, 2, 6, and 14 postexposure. Videotapes of cauda epididymal sperm were analyzed (300-350 sperm/sample) with a Hamilton Thorn motility analyzer (HTM-2000, Hamilton Thorn Research, Danvers, MA). Epichlorohydrin did not affect the percentage of motile sperm at any time. However, transient changes in sperm velocity were found. On Day 1 postexposure mean progressive (straight line) and mean path (smoothed curvilinear) velocity were significantly decreased to 80 and 85% of control, respectively. The progressive velocities of sperm from both control and treated rats were normally distributed, indicating a general effect of epichlorohydrin on all sperm as opposed to a more severe effect on a specific sperm subpopulation. Sperm velocity was not significantly affected at later times. Other endpoints (testis and epididymis weights, testicular spermatid counts, and cauda epididymal sperm reserves) were unaltered by epichlorohydrin. Thus, inhaled epichlorohydrin at 100 ppm produced specific, transient decreases in rat sperm velocity. Furthermore, computer-assisted sperm analysis was able to detect these relatively subtle, toxicant-induced changes in rat sperm velocity, demonstrating the utility of this technology in reproductive toxicology studies.

[Use of Acudex Polfa in the treatment of chronic crural ulcers]. / Zastosowanie Acudexu Polfa do leczenia przewleklych owrzodzen podudzi.

Eighty-four patients with chronic crural ulcers were treated with Acudex powder (Dextranomer) Polfa. The mean duration of the treatment was 8 weeks (3-15 weeks). Complete healing of ulcers was achieved in 44 cases (52%), improvement in 10 (12%). The preparation caused a relatively quick clearing of ulcers and marinal epithelialization. In 7 cases the treatment had to be stopped due of allergic reactions around the ulcers or major pains. The remaining patients tolerated the drug very well. Outpatient treatment was also possible.

Genotoxic effects of a sub-acute low-level inhalation exposure to a mixture of carcinogenic chemicals.

A study was conducted using a combined testing protocol (CTP), to determine whether short-term biological end-points, singly or in combination, are sufficiently sensitive to identify damage induced by exposure to ambient levels of industrial chemicals. A small-scale inhalation set-up which is both economical and easy to assemble was designed. Mice were exposed to 4 concentrations of a custom-blend mixture of benzene, chloroprene, epichlorohydrin and xylene in a ratio of 2:2:1:2, respectively. The concentrations for benzene, chloroprene and xylene were 0, 0.1, 1.0 and 10 ppm each. Concentrations for epichlorohydrin were half those for the other components. Groups of 22 males and 22 female mice were exposed to each concentration of the mixture for 3 and 6 weeks. Selected biological end-points including urine mutagenesis, bone marrow cell aberrations and micronuclei, spleen lymphocyte aberrations and liver enzyme induction were monitored. The spleen lymphocyte aberrations and liver enzyme induction were the most sensitive end-points. The lymphocytes showed a significant induction of chromosome aberrations from exposure for 3 weeks to all 3 concentrations of the mixtures. After 6 weeks of exposure, significant induction of aberrations was observed after exposure to low and medium concentrations but not to the high concentration. This lack of response at the high concentration after 6 weeks exposure, appeared to correlate with a significant induction of glutathione S-transferase in the liver. Since this enzyme is known to detoxify 3 of the 4 chemicals in our mixture, it may indicate a detoxification mechanism after enzyme induction. The present study indicates that the CTP is sufficiently sensitive to identify toxicological effects after exposure to ambient levels of a gas mixture.