<Emphasis Type="Italic">In Vitro</Emphasis> and<Emphasis Type="Italic">in vivo</Emphasis> studies on the complexes of vinpocetine with hydroxypropyl-β-cyclodextrin

The purpose of this study was to evaluate complexes of vinpocetine (VIN), a poorly water-soluble base type drug, with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) in aqueous environment and in solid state, with or without citric acid (CA) as an acidifier of the complexation medium. The apparent stability constant (Kc) calculated by phase solubility was 282 M(-1) and the complexation in solution was structurally characterized by 1H-NMR which showed VIN was likely to fit into the cyclodextrin cavity with its phenyl ring and ethyl ester bond. Solid complexes of VIN and HP-beta-CD were prepared by kneading (KE), co-evaporating (CE) and freeze-drying (FD) methods. Physical mixtures were prepared for comparison. The study in the solid state included the differential scanning calorimetry (DSC), X-ray diffractometry (XRD) and infrared absorption spectroscopy (IR). From these analyses, CE and FD products were found in amorphous state, allowing to the conclusion of strong evidences of inclusion complex formation. However, the dissolution test showed that only VIN/HP-beta-CD+CA complexes by CE and FD method could provide satisfying dissolution behavior (rapid, complete and lasting) when compared to that of VIN/HP-beta-CD complexes. Interestingly, the addition of CA in inclusion complexes could significantly decrease the amount of HP-beta-CD needed to solubilize the same amount of VIN and thereby reducing the formulation bulk. Furthermore, in-vivo study revealed that the bioavailability of VIN after oral administration to rabbits (n=6) was significantly improved by VIN/HP-beta-CD+CA inclusion complex.     

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.     

<Emphasis Type="Italic">In Vivo</Emphasis> and<Emphasis Type="Italic">In Vitro</Emphasis> anti-inflammatory activities of α-linolenic acid isolated from actinidia polygama fruits

The fruit of Actinidia polygama (AP) has long been used as a folk medicine in Korea for the treatment of pain, rheumatoid arthritis and inflammation. In the present study, bioassay-guided fractionation of AP led to the separation and identification of a polyunsaturated fatty acid, alpha-linolenic acid (ALA), which was found to show anti-inflammatory activity. The anti-inflammatory effects of ALA, using acetic acid or carrageenan-induced inflammation models, were investi gated in mice or rats, respectively. ALA significantly inhibited the acetic acid-induced vascular permeability in a dose dependent manner (34.2 and 37.7% inhibition at doses of 5 and 10 mg/ kg, respectively). ALA also significantly reduced a rat paw edema induced by a single treatment of carrageenan. To investigate the mechanism of the anti-inflammatory action of ALA, the effects of ALA on lipopolysaccharide (LPS)-induced responses in the murine mac rophages cell line, RAW 264.7, were examined. Exposure of LPS-stimulated cells to ALA inhibited the accumulation of nitrite and prostaglandin E2 (PGE2) in the culture medium. Consistent with these observations, the protein and mRNA expression levels of iNOS and COX-2 enzyme were markedly inhibited by ALA in a dose dependent manner. These results suggest that the anti-inflammatory activity of ALA might be due to the suppression of the expressions of iNOS and COX-2 mRNA.     

Imiquimod Induces Apoptosis in Human Endometrial Cancer Cells <Emphasis Type="Italic">In vitro</Emphasis> and Prevents Tumor Progression <Emphasis Type="Italic">In vivo</Emphasis>

Purpose

The increasing incidence of endometrial cancer (EC), in younger age at diagnosis, calls for new tissue-sparing treatment options. This work aims to evaluate the potential of imiquimod (IQ) in the treatment of low-grade EC.

Methods

Effects of IQ on the viabilities of Ishikawa and HEC-1A cells were evaluated using MTT assay. The ability of IQ to induce apoptosis was evaluated by testing changes in caspase 3/7 levels and expression of cleaved caspase-3, using luminescence assay and western blot. Apoptosis was confirmed by flow cytometry and the expression of cleaved PARP. Western blot was used to evaluate the effect of IQ on expression levels of Bcl-2, Bcl-xL, and BAX. Finally, the in vivo efficacy of IQ was tested in an EC mouse model.

Results

There was a decrease in EC cell viability following IQ treatment as well as increased caspase 3/7 activities, cleaved caspase-3 expression, and Annexin-V/ 7AAD positive cell population. Western blot results showed the ability of IQ in cleaving PARP, decreasing Bcl-2 and Bcl-xL expressions, but not affecting BAX expression. In vivo study demonstrated IQ’s ability to inhibit EC tumor growth and progression without significant toxicity.

Conclusions

IQ induces apoptosis in low-grade EC cells in vitro, probably through its direct effect on Bcl-2 family protein expression. In, vivo, IQ attenuates EC tumor growth and progression, without an obvious toxicity. Our study provides the first building block for the potential role of IQ in the non-surgical management of low-grades EC and encouraging further investigations.

     

Development of Stealth Liposome Formulation of 2′-Deoxyinosine as 5-Fluorouracil Modulator: <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Study

Purpose The aims of this study were to develop a stealth, pegylated liposomal formulation of 2′-deoxyinosine (d-Ino), a 5-fluorouracil (5-FU) modulator, to evaluate its efficacy in vitro and in tumor-bearing mice, and to study its pharmacokinetics in rats. Method After designing a pegylated liposome encapsulating d-Ino (L-d-Ino), we evaluated its efficacy as 5-FU modulator in vitro. Antiproliferative assays, thymidylate synthase (TS) inhibition, and apoptosis studies were carried out to check whether an optimization of 5-FU action was achieved on the 5-FU-resistant SW620 cell line. Animal pharmacokinetic and ex vivo studies were next performed to confirm that L-d-Ino displayed a slower plasma elimination pattern than free d-Ino. Finally, effects on tumor growth of L-d-Ino + 5-FU combination was evaluated in xenografted mice. Results We developed a stable, sterile, and homogenous 100-nm population of pegylated liposomes encapsulating 30% of d-Ino. Liposomal d-Ino exhibited a strong potential as 5-FU modulator in vitro by enhancing TS inhibition and subsequent apoptosis induction, while displaying a better pharmacokinetic profile in animals, with a near seven times clearance reduction as compared with the free form. When used in tumor-bearing mice in combination with 5-FU, our results showed next that the association led to 70% of tumor reduction with a doubling median survival time as compared with untreated animals, whereas 5-FU alone was ineffective. Conclusion Our data show that liposomal d-Ino, through an optimized pharmacokinetic profile, displays apotenteffect as fluoropyrimidines modulator, both in vitro and in xenografted mice. Besides, we showed here that itispossible to reverse a resistant phenotype to 5-FU, a major drug extensively described in clinical oncology.     

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.     

<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.     

How Close Are We to Profiling Immunogenicity Risk Using <Emphasis Type="Italic">In Silico</Emphasis> Algorithms and <Emphasis Type="Italic">In Vitro</Emphasis> Methods?: an Industry Perspective

In silico HLA-binding algorithms and in vitro T cell-based assays as predictive tools for human immunogenicity risk have made inroads in the biotherapeutic drug discovery and development process. Currently, these tools are being used only for candidate selection or characterization and not for making a go/no-go decision for further development. A clear limitation for a broader implementation is the lack of correlation between the predicted T cell epitope content/immune reactivity potential of a biotherapeutic and the subsequent ADA-related clinical immunogenicity outcome. The current state of technologies and their pros and cons were discussed as a part of the 2016 AAPS National Biotechnology Conference in a themed session. A review of the advances in the area and the session talks along with the ensuing discussions are summarized in this commentary.     

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.     

‘<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.

     

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     

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.     

Effect of a β-glucan from <Emphasis Type="Italic">Aureobasidium</Emphasis> on TGF-β<Subscript>1</Subscript>-modulated <Emphasis Type="Italic">in vitro</Emphasis> dermal wound repair

The objective of the present study was to determine if Aureobasidium-originated beta-glucan (beta-glucan) modulated transforming growth factor (TGF)-β₁-mediated wound healing. Proliferation of and collagen production by human dermal fibroblast cells were measured during in vitro dermal wound repopulation after treatment with 100, 10, 1, 0.1 and 0.01 mg/mL β-glucan and 1 ng/mL TGF-β₁. Control group was treated without β-glucan or 1 ng/mL TGF-β₁. TGF-β₁ significantly decreased the optical density at A₅₇₀ (a measure of fibroblast cell number) and increased procollagen production compared with the control. Also, fibroblast migration into wound defects decreased. The reductions in fibroblast proliferation and migration were significantly and dose-dependently inhibited by β-glucan (at 0.1 mg/mL or higher). However, glucan did not affect procollagen production. Thus, β-glucan may aid wound healing mediated by TGF-β₁, an important cytokine in this context.     

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.     

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.     

Virtual Thorough QT (TQT) Trial—Extrapolation of <Emphasis Type="Italic">In Vitro</Emphasis> Cardiac Safety Data to <Emphasis Type="Italic">In Vivo</Emphasis> Situation Using Multi-Scale Physiologically Based Ventricular Cell-wall Model Exemplified with Tolterodine and Fesoterodine

QT interval prolongation typically assessed with dedicated clinical trials called thorough QT/QTc (TQT) studies is used as surrogate to identify the proarrhythmic risk of drugs albeit with criticism in terms of cost-effectiveness in establishing the actual risk of torsade de pointes (TdP). Quantitative systems toxicology and safety (QSTS) models have potential to quantitatively translate the in vitro cardiac safety data to clinical level including simulation of TQT trials. Virtual TQT simulations have been exemplified with use of two related drugs tolterodine and fesoterodine. The impact of bio-relevant concentration in plasma versus estimated heart tissue exposure on predictions was also assessed. Tolterodine and its therapeutically equipotent metabolite formed via CYP2D6 pathway, 5-HMT, inhibit multiple cardiac ion currents (I_Kr, I_Na, I_CaL). The QSTS model was able to accurately simulate the QT prolongation at therapeutic and supra-therapeutic dose levels of tolterodine well within 95% confidence interval limits of observed data. The model was able to predict the QT prolongation difference between CYP2D6 extensive and poor metaboliser subject groups at both dose levels thus confirming the ability of the model to account for electrophysiologically active metabolite. The QSTS model was able to simulate the negligible QT prolongation observed with fesoterodine establishing that the 5-HMT does not prolong QT interval even though it is a blocker of hERG channel. With examples of TOL and FESO, we demonstrated the utility of the QSTS approaches to simulate virtual TQT trials, which in turn could complement and reduce the clinical studies or help optimise clinical trial designs.     

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.     

A guanidine derivative from seeds of <Emphasis Type="Italic">Plantago asiatica</Emphasis>

A new guanidine derivative named plantagoguanidinic acid was isolated from the seeds of Plantago asiatica. The structure was elucidated by two-dimensional (2D) nuclear magnetic resonance (NMR) spectral and other spectral methods.