‘<Emphasis Type="BoldItalic">In Vitro</Emphasis>’, ‘<Emphasis Type="BoldItalic">In Vivo</Emphasis>’ and ‘<Emphasis Type="BoldItalic">In Silico</Emphasis>’ Investigation of the Anticancer Effectiveness of Oxygen-Loaded Chitosan-Shelled Nanodroplets as Potential Drug Vector

Purpose

Chitosan-shelled/decafluoropentane-cored oxygen-loaded nanodroplets (OLN) are a new class of nanodevices to effectively deliver anti-cancer drugs to tumoral cells. This study investigated their antitumoral effects ‘per se’, using a mathematical model validated on experimental data.

Methods

OLN were prepared and characterized either in vitro or in vivo. TUBO cells, established from a lobular carcinoma of a BALB-neuT mouse, were investigated following 48 h of incubation in the absence/presence of different concentrations of OLN. OLN internalization, cell viability, necrosis, apoptosis, cell cycle and reactive oxygen species (ROS) production were checked as described in the Method section.In vivo tumor growth was evaluated after subcutaneous transplant in BALB/c mice of TUBO cells either without treatment or after 24 h incubation with 10% v/v OLN.

Results

OLN showed sizes of about 350 nm and a positive surface charge (45 mV). Dose-dependent TUBO cell death through ROS-triggered apoptosis following OLN internalization was detected. A mathematical model predicting the effects of OLN uptake was validated on both in vitro and in vivo results.

Conclusions

Due to their intrinsic toxicity OLN might be considered an adjuvant tool suitable to deliver their therapeutic cargo intracellularly and may be proposed as promising combined delivery system.

     

Synthesis and pharmacological properties of alkylammonium salts of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>′-malonyl-<Emphasis Type="Italic">bis</Emphasis>-<Emphasis Type="Italic">p</Emphasis>-aminobenzoic acid

A series of ammonium salts of N,N′-malonyl-bis-p-aminobenzoic acid with some alkylamines have been obtained. It is established that these compounds possess hypotensive, antiarrhythmic, and anticoagulative activities depending on the structure of the alkylamine constituents.     

Components of convolvulin from <Emphasis Type="Italic">Quamoclit</Emphasis> × <Emphasis Type="Italic">multifida</Emphasis>

Alkaline hydrolysis of the ether-insoluble resin glycoside (convolvulin) fraction of the seeds of Quamoclit × multifida (syn. Q. sloteri House, Convolvulaceae), a hybrid between Q. pennat and Q. coccinea, gave three new glycosidic acids (maltifidinic acids C, D, and E) along with three known glycosidic acids (quamoclinic acids B, C, and D) and four organic acids (2S-methylbutyric, tiglic, 2R,3R-nilic, and 7S-hydroxydecanoic acids). The structures of the new glycosidic acids were characterized on the basis of spectroscopic data as well as chemical evidence.     

β-secretase (BACE1) inhibitors from <Emphasis Type="Italic">Perilla frutescens</Emphasis> var. <Emphasis Type="Italic">acuta</Emphasis>

In the course of screening for anti-dementia agents from natural products, two β-secretase (BACE1) inhibitors were isolated from the methanolic extract of Perilla frutescens var. acuta and identified as luteolin (1) and rosmarinic acid (2) with IC₅₀ values of 5.0×10⁻⁷ M and 2.1×10⁻⁵ M, respectively. They inhibited BACE1 in a non-competitive manner with a substrate in Dixon plots, suggesting that they might bind to either β-secretase subsite or to another regulatory site. Ki values of 1 and 2 were 6.2×10⁻⁵ M and 3.9×10⁻⁵ M, respectively. They were less inhibitory against other enzymes such as α-secretase (TACE), acetylcholine esterase (AchE), chymotrypsin, and elastase, indicating that they were relatively specific inhibitors of BACE1.     

A new <Emphasis Type="BoldItalic">in vitro</Emphasis> lipid digestion – <Emphasis Type="BoldItalic">in vivo</Emphasis> absorption model to evaluate the mechanisms of drug absorption from lipid-based formulations

Purpose

In vitro lipid digestion models are commonly used to screen lipid-based formulations (LBF), but in vitro-in vivo correlations are in some cases unsuccessful. Here we enhance the scope of the lipid digestion test by incorporating an absorption ‘sink’ into the experimental model.

Methods

An in vitro model of lipid digestion was coupled directly to a single pass in situ intestinal perfusion experiment in an anaesthetised rat. The model allowed simultaneous real-time analysis of the digestion and absorption of LBFs of fenofibrate and was employed to evaluate the influence of formulation digestion, supersaturation and precipitation on drug absorption.

Results

Formulations containing higher quantities of co-solvent and surfactant resulted in higher supersaturation and more rapid drug precipitation in vitro when compared to those containing higher quantities of lipid. In contrast, when the same formulations were examined using the coupled in vitro lipid digestion – in vivo absorption model, drug flux into the mesenteric vein was similar regardless of in vitro formulation performance.

Conclusion

For some drugs, simple in vitro lipid digestion models may underestimate the potential for absorption from LBFs. Consistent with recent in vivo studies, drug absorption for rapidly absorbed drugs such as fenofibrate may occur even when drug precipitation is apparent during in vitro digestion.

     

<Emphasis Type="Italic">Buddlejol</Emphasis>, a new α-chymotrypsin inhibitor from <Emphasis Type="Italic">Buddleja asiatica</Emphasis>

Buddlejol (1), a new sterol, has been isolated from the ethyl acetate soluble fraction of the antispasmodic plant Buddleja asiatica along with stigmasterol (2), lignoceric acid (3), taraxerol (4) and α-amyrin (5), respectively. The structure of Buddlejol (1) was established as (24S)-stigmast-5,22-diene-7β-ethoxy-3β-ol by spectral analysis and comparison with closely related structures. Buddlejol revealed to be a competitive inhibitor of chymotrypsin with the Ki value of 10.60 µM as indicated by Lineweaver–Burk and Dixon plots and their re-plots against its chymotrypsin inhibition assay, while the other compounds showed less inhibitory potential. The bioassay-guided isolation was stimulated by the preliminary cytotoxic screening of various fractions of B. asiatica.     

Synthesis of novel <Emphasis Type="Italic">N</Emphasis>-acyl-<Emphasis Type="Italic">β</Emphasis>-<Emphasis Type="SmallCaps">d</Emphasis>-glucopyranosylamines and ureas as potential lead cytostatic agents

Novel classes of acetylated and fully deprotected N-acyl-β-d-glucopyranosylamines and ureas have been synthesized and biologically evaluated. Acylation of the per-O-acetylated β-d-glucopyranosylurea (5), easily prepared via its corresponding phosphinimine derivative, by zinc chloride catalyzed reaction of the corresponding acyl chlorides RCOCl (a–f) gave the protected N-acyl-β-d-glucopyranosylureas (6a–f), in acceptable-to-moderate yields. Subsequent deacetylation of analogues 6a–f under Zemplén conditions afforded the fully deprotected derivatives 7a,b,d,e,f, while the desired urea 7c was formed after treatment of 6c with dibutyltin oxide. All protected and unprotected compounds were examined for their cytotoxic activity in different L1210, CEM and HeLa tumor cell lines and were also evaluated against a broad panel of DΝΑ and RNA viruses. Derivative 7c exhibited cytostatic activity against the three evaluated tumor cell lines (IC₅₀ 9–24 μΜ) and might be the basis for the synthesis of structure-related derivatives with improved cytostatic potential. Only analogue 6f weakly but significantly inhibited the replication of parainfluenza-3 virus, Sindbis virus and Coxsackie virus B4 in cell cultures at concentrations of 45–58 μM.     

Two new <Emphasis Type="Italic">β</Emphasis>-carboline alkaloids from the stems of <Emphasis Type="Italic">Picrasma quassioides</Emphasis>

Two new β-carboline alkaloids, 1-acetyl-4-methoxy-8-hydroxy-β-carboline (1) and 1-acetyl-4,8-dimethoxy-β-carboline (2), together with 10 known compounds; seven β-carboline alkaloids (3–9), two canthin-6-one alkaloids (10 and 11), and one quassinoid (12) were isolated from the stems of Picrasma quassioides. The structure of the new compounds 1 and 2 were determined by spectroscopic analyses including 1D- and 2D-NMR and HRMS interpretation. All the isolates (1–12) were evaluated for their cytotoxicity against human ovarian carcinoma A2780 and SKOV3 cell lines using MTT assays. Of the isolates, compounds 5–7 exhibited the most potent cytotoxicity on both A2780 and SKOV3 cell lines in vitro.     

The interaction of host genetic factors and <Emphasis Type="Italic">Helicobacter</Emphasis> <Emphasis Type="Italic">pylori</Emphasis> infection

Helicobacter pylori plays an important role in the development of atrophic gastritis that represents the most recognized pathway in multistep gastric carcinogenesis. Recent studies suggest that a combination of host genetic factors, bacterial virulence factors, and environmental and lifestyle factors determine the severity of gastric damage and the eventual clinical outcome of Helicobacter pylori infection. As to bacterial virulence factors, a high proportion of Japanese strains are cagA⁺vacAs1. The CagA protein is injected from attached Helicobacter pylori into gastric epithelial cells and the CagA-SHP-2 interactions elicit cellular changes that increase the risk of carcinogenesis. Host cytokine gene polymorphisms and a frequent single nucleotide polymorphism in the PTPN11 gene that encodes SHP-2 may associate with gastric atrophy among Helicobacter pylori-infected subjects. Prevention of gastric cancer requires the development of better screening strategies for determining eradication candidates and further improvement of treatments of Helicobacter pylori infection. Received 6 August 2006; accepted 21 August 2006     

Toxicity of abamectin to the terrestrial isopod <Emphasis Type="Italic">Porcellio</Emphasis> <Emphasis Type="Italic">scaber</Emphasis> (Isopoda,Crustacea)

To determine effects of the antiparasitic veterinary drug abamectin on the isopod Porcellio scaber, animals were exposed for 21 days to Lufa 2.2 soil spiked at concentrations of 3–300 mg/kg dry soil. After exposure, abamectin residues in the isopods were analysed using a novel analytical method. Toxicity was evaluated on different levels of biological organisation: biochemical, cellular and the individual organism. Measurements included glutathione S-transferase (GST) activity and stability of cell membranes in the digestive gland, animal mass gain or loss, food consumption, behaviour and mortality. LC50 for the effect of abamectin on survival of P. scaber was 71 mg/kg dry soil. The most obvious sublethal effects were reduced food consumption and decreased body mass (NOEC 3 mg/kg dry soil). Additionally, loss of digging activity and reduced GST activity (NOEC 30 mg/kg dry soil) and cell membrane destabilization (NOEC 10 mg/kg dry soil) were recorded. Abamectin only slightly accumulated in the isopods, with bioaccumulation factors always being <0.1. Based on these results and current information on environmental levels of abamectin, it is not likely that isopods will be affected by abamectin, but further studies with exposure through faeces are recommended.     

Shape-Memory Terpolymer Rods with 17-β-estradiol for the Treatment of Neurodegenerative Diseases: an <Emphasis Type="BoldItalic">In Vitro</Emphasis> and <Emphasis Type="BoldItalic">In Vivo</Emphasis> Study

Purpose

Estradiol (E2)-loaded poly(L-lactide-co-glycolide-trimethylenecarbonate) (P(L-LA:GA:TMC)) rods with shape-memory were developed for the treatment of neurodegenerative diseases. Usefulness of the extrusion method in the obtaining process was also considered. The influence of structural and surface properties during hydrolytic degradation was developed. The possible therapeutic aspect of rods with E2 was determined.

Methods

The extruded rods were incubated in a PBS solution (pH 7.4, 37°C, 240 rpm). The amount of released E2 in vitro conditions was estimated by UV-VIS method. The following methods in the degradation of rods were applied: NMR, DSC, FTIR, GPC, SEM, and optical microscopy. Changes in water uptake and weight loss were also determined. In vivo study was performed on rats. Measurements of E2 level were performed before and after ovariectomy of rats using ELISA method. A sample of tissue adjacent to the site of the rod implantation was analysed under an optical microscope.

Results

A stable and steady degradation process ensured zero-order release of E2. The in vivo study indicated a significant increase in the E2 level in serum after ovariectomy. Moreover, structural and surface features indicated that the extrusion method was appropriate for obtaining E2-loaded rods.

Conclusions

Shape-memory P(L-LA:GA:TMC) rods with E2 are an adequate proposal for further research in the field of neurological disorders.

     

A glycerol α-<Emphasis Type="SmallCaps">d</Emphasis>-glucuronide and a megastigmane glycoside from the leaves of <Emphasis Type="Italic">Guettarda</Emphasis><Emphasis Type="Italic">speciosa</Emphasis> L.

From the 1-BuOH-soluble fraction of a MeOH extract of the leaves of Guettarda speciosa L., two new compounds (1, 2) were isolated together with six known compounds. Spectroscopic analysis of 1 and 2 established their structures to be derivatives of a glycerol α-glucuronide and a megastigmane glycoside, respectively. HPLC analysis of the hydrolyzate of 1 confirmed the presence of d-glucuronic acid in the structure, and the modified Mosher’s method established the absolute structure of 2.     

Poly(ε-caprolactone)-<Emphasis Type="Italic">Block</Emphasis>-poly(ethyl Ethylene Phosphate) Micelles for Brain-Targeting Drug Delivery: <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Valuation

Purpose  

The purpose of this work was to investigate the potential of poly(ε-caprolactone)-block-poly(ethyl ethylene phosphate) (PCL-PEEP) micelles for brain-targeting drug delivery.     

Structure–activity relationship study of secondary metabolites from <Emphasis Type="Italic">Mesua beccariana</Emphasis>, <Emphasis Type="Italic">Mesua ferrea</Emphasis> and <Emphasis Type="Italic">Mesua congestiflora</Emphasis> for anti-cholinesterase activity

Our search for potential anti-acetylcholinesterase (AChE) inhibitors for treatment of Alzheimer’s disease has led to the discovery of two bioactive compounds, α-mangostin (11) and congestiflorone acetate (13). This discovery was achieved from a preliminary screening of the anti-AChE activity on the extracts of three Mesua species namely M. ferrea, M. beccariana and M. congestiflora using Ellman’s method. The pure metabolites, 1–12 which were isolated from the Mesua species, along with a synthetic derivative, compound 13 were then evaluated for their activities in order to identify the compounds that correspond to the enzyme inhibitory activities. Compounds 11 and 13 were found to give significant anti-AChE activities with IC₅₀ values of 17.51 and 20.25 µM.     

Dosage Form Development, <Emphasis Type="BoldItalic">in Vitro</Emphasis> Release Kinetics,and <Emphasis Type="BoldItalic">in Vitro–in Vivo</Emphasis> Correlation for Leuprolide Released from an Implantable Multi-reservoir Array

Purpose Implanted multi-reservoir arrays improve dosing control relative to osmotic pumps or polymer depots. The limited reservoir volume requires concentrated formulations. This report describes the development of a stable solid phase formulation of leuprolide acetate for chronic in vivo delivery from a multi-reservoir microchip and examines the correlation between in vitro release kinetics and serum pharmacokinetics. Materials and Methods Concentrated formulations (>10% w/v) were prepared using small volume processing methods. Drug yield, release kinetics, and formulation stability were evaluated in vitro by HPLC. The correlation between in vitro and in vivo kinetic data was determined for a solid formulation by direct comparison of data sets and using absorption kinetics calculated from the Wagner–Nelson equation. Results High yield and the control of release kinetics by altering peptide formulation or reservoir geometry were demonstrated. Lyophilized leuprolide in a soluble solid matrix exhibited reproducible release kinetics and was stable (>95% leuprolide monomer) after 6 months at 37°C. A strong correlation was found between in vitro release kinetics and in vivo absorption by direct comparison of data sets and using the Wagner–Nelson absorption (slopes of 1.01 and 0.91; R² 0.99). Conclusions Reproducible releases of a stable solid leuprolide formulation from a multi-reservoir microchip were achieved in vitro. Chronic pulsatile release was subsequently performed in vivo. Comparison of in vitro and in vivo data reveals that pharmacokinetics were controlled by the rate of release from the device.     

<Emphasis Type="BoldItalic">In Vitro</Emphasis> and <Emphasis Type="BoldItalic">In Vivo</Emphasis> Characterization of Drug Nanoparticles Prepared Using PureNano™ Continuous Crystallizer to Improve the Bioavailability of Poorly Water Soluble Drugs

Purpose

The aim of this study was to enhance the dissolution and oral absorption of poorly water-soluble active pharmaceutical ingredients (APIs) using nanoparticle suspensions prepared with a PureNano? continuous crystallizer (PCC).

Method

Nanoparticle suspensions were prepared with a PCC, which is based on microfluidics reaction technology and solvent–antisolvent crystallization. Phenytoin, bezafibrate, flurbiprofen, and miconazole were used as model APIs. These APIs were dissolved in ethanol and precipitated by the addition of water and polyvinyl alcohol. Batch crystallization (BC) using a beaker was also performed to prepare the suspensions. Both PCC and BC formulations were freeze-dried before being characterized in vitro and in vivo.

Results

The particle sizes of the nanoparticle suspensions prepared with the PCC were smaller than those prepared by BC. The dissolution rate of each API in vitro significantly increased after crystallization. Reducing the particle size of either the BC or PCC formulation led to increased API flux across Caco-2 cell monolayers. PCC preparations showed higher plasma concentrations after oral administration, demonstrating the advantages of a fast dissolution rate and increased interaction with the gastrointestinal tract owing to the smaller particle size.

Conclusions

PCC can continuously produce nanoparticle APIs and is an efficient approach for improving their oral bioavailability.

     

Thyroid Function in Nestling Tree Swallows and Eastern Bluebirds Exposed to Non-Persistent Pesticides and <Emphasis Type="BoldItalic">p</Emphasis>, <Emphasis Type="BoldItalic">p</Emphasis>′-DDE in Apple Orchards of Southern Ontario,Canada

This study examined the associated effects of pesticides and persistent residues of p,p-DDE on thyroid function in 16-day-old tree swallows (Tachycineta bicolor) and 12-day-old eastern bluebirds (Sialia sialis). Apple orchards sprayed with pesticides in current use and reference sites were chosen for study in southern Ontario, Canada, during 2000–2001. We assessed thyroid hormone concentrations (plasma and muscle thyroxine [T₄], and triiodothyronine [T₃]) and the response to a challenge with thyroid stimulating hormone (TSH) as well as parameters of thyroid histology. Individual nests were exposed to as many as seven individual pesticide applications and up to five pesticide mixtures. Concentrations of p,p-DDE in eggs from both species were highest in orchard sites, and ranged from 0.05 to 5.44 mg/kg wet weight for tree swallows, and from 0.17 to 95.4 mg/kg for eastern bluebirds. In 2000, tree swallows from sprayed orchards had elevated plasma T₄ concentrations, and thyroid glands with collapsed follicles, hypertrophic epithelia, and higher follicular epithelial cells relative to chicks from reference sites. Plasma T₄ concentration was positively correlated with the total number of pesticide mixtures applied during egg incubation through chick rearing. In 2001, basal plasma T₃ concentration in tree swallow chicks did not differ between orchard and reference sites, though there were differences among orchards; T₃ was positively correlated with total number of individual sprays applied during egg incubation through chick rearing. Tree swallows challenged with TSH showed no significant difference in thyroid hormone concentration or follicular epithelial cell height between exposure groups; however, a significantly greater proportion of chicks from reference sites were found to have thyroids with focal or diffuse hypertrophic epithelia relative to orchard chicks. In 2001, bluebirds from sprayed orchards had significantly higher T₃ concentrations in response to TSH challenge; this response was positively correlated with the total number of pesticides applied during egg incubation through chick rearing.     

Side Effects of Interferon-<Emphasis Type="Italic">α</Emphasis> Therapy

Aim

Interferon-α (IFN-α) has been extensively explored for its efficacy in various disease conditions and is currently used as a standard treatment in several of these. Its use is accompanied by a wide variety of possible side effects. These side-effects may hamper reaching and maintaining the dose needed for maximal therapeutic effect while their occurrence can outweigh clinical benefit of IFN-α treatment. This review addresses the toxicity profile of IFN-α, the presumed pathophysiology of the different side effects and the strategies to handle these.

Methods

Computerized searches were used and cross-references of articles and books were checked.

Results

Adverse effects due to IFN-α have been described in almost every organ system. Many side-effects are clearly dose-dependent. Taken together, occurrence of flu-like symptoms, hematological toxicity, elevated transaminases, nausea, fatigue, and psychiatric sequelae are the most frequently encountered. Although insight in the mechanisms accounting for IFN-α-related toxicities has improved in recent years, much remains to be elucidated. Guidelines on the management of these untoward sequelae are mostly based on clinical experience, while many side-effects can only be adequately handled by dose adjustment or cessation of treatment.

Conclusion

Further research on the mechanisms underlying both therapeutic effects and adverse events is warranted. Hopefully, this will lead to better identification of those patients who are likely to benefit from treatment without experiencing severe toxicities.

     

Synthesis and <Emphasis Type="BoldItalic">in Vitro</Emphasis> Evaluation of a Novel Chitosan–Glutathione Conjugate

Purpose It was the aim of this study to synthesize and characterize a novel chitosan–glutathione (GSH) conjugate providing improved mucoadhesive and permeation-enhancing properties.Methods Mediated by carbodiimide and N-hydroxysuccinimide, glutathione was covalently attached to chitosan via the formation of an amide bond. The adhesive properties of chitosan–GSH conjugate were evaluated in vitro on freshly excised porcine mucosa via tensile studies and the rotating cylinder method. The cohesive properties and stability of the resulting conjugate were evaluated by disintegration test and by oxidation experiments, respectively. The permeation-enhancing effect of the chitosan–GSH/GSH system was evaluated in Ussing chambers by using rhodamine 123 as model compound.Results The obtained conjugate displayed 265.5 μmol immobilized free thiol groups and 397.9 μmol disulfide bonds per gram polymer. Because of the formation of disulfide bonds within the polymer, the stability of matrix tablets could be strongly improved. In tensile studies, the total work of adhesion of the conjugate was determined to be 9.9-fold increased in comparison to unmodified chitosan. Results from the rotating cylinder method showed more than 55-fold increase in the adhesion time of thiolated chitosan vs. unmodified chitosan. In addition, the conjugate in combination with GSH displayed a 4.9-fold higher permeation-enhancing effect compared with unmodified chitosan.Conclusions Because of the improved mucoadhesive and cohesive properties, and the strong permeation-enhancing effect of the chitosan–GSH conjugate/GSH system, the novel thiolated chitosan seems to represent a promising multifunctional excipient for various drug delivery systems.