Characterisation and implementation of the ERE-CALUX bioassay on indoor dust samples of kindergartens to assess estrogenic potencies.

Estrogen-like endocrine disrupting chemicals (EEDCs) can be found abundantly in the environment. Due to their low-dose effects and the large amount of unknown EEDCs, it is difficult to assess and manage possible human health risks. For young children, who are particularly vulnerable to endocrine disruption due to their development rate, indoor dust is one of the main routes of exposure. In this study, an estrogen responsive elements chemically activated luciferase gene expression (ERE-CALUX) bioassay was characterized and implemented for the analysis of 12 dust samples from kindergartens in Flanders and Brussels (Belgium). The human ovarian carcinoma BG 1CALUX cell line showed reproducible results and a low limit of detection (LOD). The effective concentration at 50% of the maximum response (EC50) yielded 497 fg/well, while the LOD was 16 fg/well. For all dust samples, full dose-response curves and their corresponding EC50 values could be calculated. All samples yielded bio-analytical equivalent concentrations (BEQs) that were significantly higher than the procedural blank level and ranged from 426 to 8710 pg E2 equivalents/g dust. A clear relationship was observed between a semi-quantitative interior score and the ERE-CALUX response of the samples. In addition, the concentration of phthalates, a major group of EEDCs used as plasticizers in plastics, was determined in the samples by GC-MS. Diisoheptyl phthalate (DiHP) and di(2-ethylhexyl) phthalate (DEHP) were present in every dust sample. A good correlation was found between ERE-CALUX activities and phthalate concentrations, when all phthalates except diisononyl phthalate (DiNP) and diisodecyl phthalate (DiDP), which do not bind to the estrogen receptor, were taken into account. This shows that the ERE-CALUX can provide relevant results concerning exposure to EEDCs from indoor dust. This article is part of a Special Issue entitled 'Endocrine disruptors & steroids'.

[Lotion with hydroquione - tretinoin]. / Lotion a l'hydroquinone et a la trétinoïne.

The observed precipitation in a lotion containing 4% of hydrochinon and 0,03% of tretinoine is evaluated. To dissolve both actives, 10% propyleneglycol is used and the alcohol concentration varied. From the results it is demonstrated that at least 54 ml of ethanol 96 degrees is necessary to dissolve both actives. It is also demonstrated that the addition of 0.2% of vitamin C, as antioxidant, is necessary to avoid coloration of the lotion, as a function of time, due to the presence of hydrochinon.

[Pharmacy compounding of medicinal shampoos: an update]. / Aspects galéniques des shampooings médicinaux: mise a jour.

Delivery of magistral (extemporaneous) preparations must comply with quality standards, as described in the "Guide to Good Officinal Pharmaceutical Practice" and the pharmacy's Quality Manual (Royal Decree of 21 January 2009). In this study, the shampoo Simplex Herdewijn, prepared with Texapon NSO, is compared with commercially available basic shampoos (Shampoo Fagron and Shampoo Simplex Conforma) with respect to its composition as well as its physicochemical properties. These basic shampoos were used to develop medicinal shampoos. The active compounds of these shampoos were: 0.5% hexachlorophene, 1% ichtammol, 1% cade oil or 2% liquor carbonis detergens. The necessary concentration of Cetomacrogol 1000, required to solubilise the active compounds was determined, as well as the amount of sodium chloride needed to adjust the viscosity of the shampoo. The viscosity of the basic and medicinal shampoos was determined by means of rheograms and by calculating their apparent viscosity. Additionally, a number of fundamental aspects of the formulation of shampoos are discussed in this paper. The results can be useful to pharmacists as guidelines for the preparation of medicinal shampoos. Beside patient instruction and pharmaceutical care, the intrinsic quality of magistral preparations is a prerequisite for therapeutic activity.

Assessment of the acrylamide intake of the Belgian population and the effect of mitigation strategies.

The acrylamide (AA) intake of the Belgian consumer was calculated based on AA monitoring data of the Belgian Federal Agency for the Safety of the Food Chain (FASFC) and consumption data of the Belgian food consumption survey coordinated by the Scientific Institute for Public Health (3214 participants of 15 years or older). The average AA exposure, calculated probabilistically, was 0.4 microg kg(-1) body weight (bw) day(-1) (P97.5 = 1.6 microg kg(-1) bw day(-1)), the main contributors to the average intake being chips (23%), coffee (19%), biscuits (13%), and bread (12%). Additionally, the impact of a number of AA mitigation scenarios was evaluated (German minimization concept, scenarios for mitigation from the literature, signal values), which is an important issue for public health as well as for policy-makers. Specific actions in cooperation with the food industry to reduce the AA content of foods seems to be a more efficient strategy than mere implementation of signal values. Considering that an important share of the AA intake is due to prepared meals, the catering industry as well as consumers need to be better informed on the various possibilities for keeping the AA content of meals as low as possible.

[Pharmacy compounding of an omeprazole suspension]. / Preparation magistrale d'une suspension d'omeprazole.

The problems of preparation of different pharmaceutically compounded formulations of prescribed omeprazole suspensions are discussed. Problems that can be cited are: inadequate preparation, chemical and physical stability problems, taste problems and low bioavailability. The formulation of the omeprazole suspension is optimized, taking into account the cited problems. At the same time, some formulations are presented, ensuring chemical stability of omeprazole and its bioavailability.

Influence of preparation method on itraconazole oral solutions using cyclodextrins as complexing agents.

In the literature, solubility values of itraconazole complexed with 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) were found which were still much too low to obtain the target concentration of 1 g itraconazole/100 ml, the concentration of the marketed itraconazole formulation Sporanox (Janssen Pharmaceutica). Therefore, we compared two preparation methods: the classical and the dissolving method to investigate if the method of preparation can have an influence on the solubility of itraconazole complexed with cyclodextrin (CD). With the classical method, the active compound and the CDs are jointly dissolved with a co-solvent, propylene glycol, in water. With the dissolving method, the active compound is first dissolved separately in a solvent in which it dissolves well, while the CDs are dissolved in water, before mixing. Three different CDs were used and compared for their complexing capacity with itraconazole. The complex formation of itraconazole with HP-beta-CD, sulfobutylether-7-beta-cyclodextrin (SBE-7-beta-CD) and maltosyl-beta-cyclodextrin (malt-beta-CD) was investigated at pH 2, in the presence of 10% propylene glycol for an oral solution. These three CDs were chosen as they can also serve in formulations for parenteral use. The method of preparation had an important influence on the complex formation. With the dissolving method, a much higher solubility of itraconazole was obtained using the same CD concentration than with the classical method. Inclusion capacity obtained with the dissolving method was comparable for HP-beta-CD and SBE-7-beta-CD: 1 g itraconazole/100 ml of 25% HP-beta-CD or of 30% SBE-7-beta-CD. In 100 ml of 40% malt-beta-CD only about 500 mg of itraconazole could be dissolved. With the classical method only around 160 mg itraconazole could be dissolved with 100 ml 40 % HP-beta-CD or SBE-7-beta-CD. Due to the fast preparation, once the CD amount is known by pretests, the dissolving method shows also an advantage for industrial production.

Selection of human immunodeficiency virus type 1 resistance against the pyranodipyrimidine V-165 points to a multimodal mechanism of action.

OBJECTIVES: We have previously identified the pyranodipyrimidines (PDPs) as a new class of integrase (IN) inhibitors. The most potent congener V-165 inhibits HIV-1 integration at low micromolar concentrations by inhibiting the binding of IN to the DNA. As part of pre-clinical studies with PDP, we wanted to investigate HIV resistance development against V-165 and to further characterize the physicochemical properties of the compound. METHODS: We selected PDP-resistant HIV-1 strains by growing the virus in the presence of increasing concentrations of V-165. The selected strains were analysed genotypically and phenotypically. Mutant IN enzymes were generated and evaluated in an enzymatic oligonucleotide-based assay for their activity and sensitivity to the different IN inhibitors. In addition, the antiviral effect of the compound on viral entry and integration was measured using quantitative PCR. RESULTS: Numerous mutations were detected in the RT, IN and env genes of the virus selected in the presence of V-165. Although V-165 inhibited integration in vivo as indicated by a decrease in the number of integrated proviruses, the compound also inhibited viral entry at a concentration of 19 microM. V-165 was poorly recovered from human hepatic microsomal matrix and 1% BSA. CONCLUSIONS: These data point to a multimodal mechanism of action. A quest for derivatives of V-165 that specifically target IN should be pursued.

Preparation and evaluation of paclitaxel-containing liposomes.

Paclitaxel, an antitumoral drug, is poorly soluble in aqueous media. Therefore, in a commercialised formulation (Taxol), paclitaxel (30 mg active compound) is dissolved in polyethoxylated castor oil (Cremophor EL) and ethanol. After dilution of Taxol in aqueous media paclitaxel tends to precipitate. Several side effects, attributed to the surfactant Cremophor EL, occur, e.g. bronchospasm, hypotension, neuro- and nephrotoxicity, and anaphylactic reactions. To eliminate these side effects, the solubility of paclitaxel was enhanced using liposomes instead of Cremophor EL. The amount of entrapped paclitaxel in crystal-free liposomes was 0.5 mg/ml liposome suspension, i.e. almost 85 times the native solubility. Thus, 30 mg paclitaxel had to be dissolved in 60 ml liposome suspension, of either multi-lamellar vesicles (MLV's) or of small unilamellar vesicles (SUV's) with 5% sucrose as cryoprotector. No precipitation was observed after dilution of the MLV-formulation with (physiological) water or with 5% aqueous dextrose solution, which proves their suitability for administration with perfusions. The chemical stability of paclitaxel in the prepared MLV's stored at 4 degrees C was demonstrated during a period of 5 months. The chemical degradation to conjugated dienes and hydroperoxides, two oxidative degradation products of EPC, was negligible (less than 1%).

Quality evaluation of chloroquine, quinine, sulfadoxine-pyrimethamine and proguanil formulations sold on the market in East Congo DR.

OBJECTIVE: Drug quality may be poor in many regions of the world. Our first aim was to verify whether the dose of the active compounds in various antimalarial medicines on the market in East Congo conforms to the quality requirements of the European Pharmacopoeia (Ph. Eur.). The second aim was to check the extent to which simple methods of analysis could be used to evaluate drug quality. METHODS: The formulations analysed included tablets, injections and syrups of chloroquine (CQ), quinine, sulfadoxine-pyrimethamine (SP) and proguanil. Ultraviolet (UV) spectrophotometry was used to quantify CQ and quinine in tablets and injections. Thin layer chromatography was used to identify the preservative(s) in the syrups. As the drug form (base or salt) in the tablets, is rarely declared, the estimated dose was calculated using both forms. High-performance liquid chromatography (HPLC) was used to check for assay interference and for measuring SP combinations. RESULTS AND DISCUSSION: When the dose declaration on the label was assumed to be of the salt form, 33% of CQ batches were underdosed and two of eight batches of quinine were underdosed by about 25% and 15% respectively. When the base form was assumed, only one batch of CQ tablets conformed. The underdosed batches contained about 50-66% of the claimed amount for CQ. The dose of quinine in the different batches of tablets was in the range 62-86%. For the CQ syrup, interference by the preservative Nipagin, confirmed by HPLC-UV, was observed with UV-spectrophotometry at 257 nm but not at 342 nm. The results for CQ syrup using UV-spectrophotometry at 342 nm and HPLC-UV at 257 nm were comparable and showed compliance with the European Pharmacopoeia limits of 95-105%. One of two batches of CQ injections and one of four batches of quinine injections were overdosed by about 14% and 8% respectively. The SP tablets were analysed by using HPLC-UV only. All five batches were underdosed in sulfadoxine (91-94%) but still met the United States Pharmacopeial (USP) limit of 90-110%. Two batches were slightly overdosed in pyrimethamine (106% and 108% respectively) while one batch contained neither active ingredient. The one batch of proguanil analysed, met the Ph. Eur. quality requirement (98.7%). CONCLUSION: Simple methods of analysis like UV-spectrophotometry can be used to check drug quality routinely. A substantial proportion of the antimalarial drugs sold on the Congo DR market is of poor quality. Some batches contain little or no drug. This is a serious threat to public health in the region of Congo DR.

Investigation on the photostability of tretinoin in creams.

In this investigation, the photodegradation of some tretinoin cream formulations was evaluated. Several oils were selected to prepare the cream formulations: olive oil, maize oil, castor oil, isopropyl myristate and Miglyol 812. A solubility study showed that tretinoin is best soluble in castor oil (0.60g/100ml), followed by isopropyl myristate, maize oil, Miglyol 812 and olive oil, respectively, 0.35, 0.30, 0.29 and 0.22g/100ml. The photostability of tretinoin in oils is comparable with the photostability of a tretinoin lotion (ethanol/propylene glycol 50/50), castor oil and olive oil giving slightly better results than the other oils. Investigation of the photodegradation of tretinoin in o/w creams, prepared with the same oils as mentioned above, revealed that tretinoin is far more stable in the cream formulations than in the respective oils, however it is not clear whether this is due to the formulation or due to a different irradiation technique. Tretinoin seemed to be most stable in the olive oil cream, followed by the castor oil cream. However microscopic investigation revealed the presence of tretinoin crystals in the olive oil cream, while the other creams were free of it. As a conclusion, one can say that the cream prepared with castor oil seems to be the most suitable one, in terms of solubility of tretinoin and in terms of photostability.

Comparison of free iodine as a function of the dilution of two commercial povidone-iodine formulations.

Binding studies by means of equilibrium dialysis on two different Povidone-lodine-solutions reveal that the amount of available iodine and free iodine is very different as such and after dilution. The free iodine concentration in the Braunol concentrate was found to be ca. 22 mg/L and in the iso-Betadine concentrate only ca. 2.1 mg/L, despite the total amount of available iodine in iso-Betadine being higher than that of Braunol. As the bactericidal level of free iodine is characterised by concentrations >5 ppm, Braunol can be employed as a disinfectant as such, iso-Betadine has to be diluted before use. In both concentrates more than 99% of available iodine is present as reservoir for free iodine. Concerning the results as a function of dilution, it was demonstrated that, for both solutions, free iodine reaches a maximum after a 50-fold dilution (ca. 31 mg/L and ca. 51 mg/L for iso-Betadine and Braunol respectively). After dilution, a more constant level of free iodine was observed in the Braunol than in the iso-Betadine solution, and this is attributed to the present molar ratio of I(2)/I- and the addition of iodate in the former. The pH for both solutions approximates that of the skin, as such and after dilution. In summary, it can be stated that Braunol is superior to iso-Betadine as to the release of free iodine in both the undiluted as well as in the diluted form.

In vitro study of the pulmonary translocation of nanoparticles: a preliminary study.

Recent studies indicate that inhaled ultrafine particles can pass into the circulation. To study this translocation in an in vitro model three types of pulmonary epithelial cells were examined. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. TEER was too low in A549 cells. In these preliminary experiments, TEER values of 1007+/-300 and 348+/-62 Omega cm2 were reached for the Calu-3 cell line, using permeable membranes of 0.4 and 3 microm pore size, respectively. Growing primary rat type II pneumocytes on 0.4 microm pores, a TEER value of 241+/-90 Omega cm2 was reached on day 5; on 3 microm pores, no acceptable high TEER value was obtained. Translocation studies were done using 46 nm fluorescent polystyrene particles. When incubating polystyrene particles on membranes without a cellular monolayer, significant translocation was only observed using 3 microm pores: 67.5% and 52.7% for carboxyl- and amine-modified particles, respectively. Only the Calu-3 cell line was used in an initial experiment to investigate the translocation: on 0.4 microm pores no translocation was observed, on 3 microm pores approximately 6% translocation was observed both for carboxyl- and amine-modified particles.

Inclusion complexation of diazepam with different cyclodextrins in formulations for parenteral use.

A parenteral formulation for the water-insoluble benzodiazepine diazepam was developed. Different cyclodextrins (CDs) suitable for parenteral injection: hydroxypropyl-beta-cyclodextrin (HP-beta-CD), hydroxy-propyl-gamma-cyclodextrin (HP-gamma-CD), sulfobutylether-7-beta-cyclodextrin (SBE-7-beta-CD) and maltosyl-beta-cyclodextrin (malt-beta-CD) were used as alternatives to cosolvents to increase solubility. The increase in solubility displayed a concentration dependency for the four CDs used. Diazepam's solubility is enhanced linearly as a function of each CD concentration. The highest improvements in solubility (dissolved concentration circa 3.5 mg/ml in 40% CD) were found by adding HP-beta-CD or SBE-7-beta-CD. The additional use of polyvinylpyrrolidone (PVP) did not further increase the solubility of diazepam with HP-beta-CD. A parenteral aqueous diazepam solution was prepared containing 10 mg diazepam/5 ml 30% HP-beta-CD or SBE-7-beta-CD solution. The preparations are in agreement with the requirements for parenteralia. Sterilisation by filtration is required since autoclaving degrades the active compound. The stability of the preparations, with and without pH adjustment to pH 5, was investigated during 18 months and during this period no noticeable degradation was observed.

Experimental designed optimisation and stability evaluation of dry suspensions with artemisinin derivatives for paediatric use.

There is a great need for oral anti-malaria preparations especially for small children, which are easy to administer and keep their stability under tropical conditions. The purpose of this work was therefore to develop a dry suspension, containing one of the artemisinin derivatives, namely artesunate, artemether and dihydroartemisinin using fast wetting suspending agents, i.e. xanthan gum and Avicel CL611. For the optimisation of these two variables, namely the suspending agent's content, a Doehlert design was applied. Via preliminary tests on sedimentation behaviour, the limits of both products were determined, respectively 0.1-0.4% (w/v) and 1.0-2.5% (w/v). As responses, sedimentation as a function of time, viscosity and price of the suspension, were evaluated. The stability tests of the reconstituted suspensions showed bad results for artesunate, even when the pH was adapted. In contrast, dihydroartemisinin showed only 10% degradation within 10 days and artemether was stable at least 21 days. Practically the last one was able to foresee a chemically and physically stable suspension at least during the administration period (5 to 7 days) and was therefore selected for further optimisation concerning taste and appearance. Based on the results of selection tests for the colourant, sweetener and taste masking agent, the following composition was proposed for a suitable dry powder with artemether (AM) as active compound to prepare 100 ml reconstituted suspension: AM 300 mg, Avicel CL611 2 g, xanthan gum 200 mg, crystalline saccharose 35 g, citric acid monohydrate 150 mg, Nipagine 80 mg, Nipasol 20 mg, sodium saccharinate 250 mg, tutti-frutti 250 mg and Sunset yellow 10 mg.

Development of a reversed-phase thin-layer chromatographic method for artemisinin and its derivatives.

In this study a clear separation between seven analogues of artemisinin on thin-layer chromatography (TLC) is presented. The developed TLC method is carried out on a RP-C18 thin-layer plate using acetonitrile-water (50:25 v/v) as the mobile phase. Spots are visualized by derivatization with an acidified 4-methoxybenzaldehyde reagent in methanol-water. This method allows the separation of a diverse group of compounds that have versatile hydrophilic/lipophilic characteristics; namely artemisinin, artesunate (AS), artelinic acid (AL), arteether (AE), both isomers of artemether (AM) (alpha and beta), dihydroartemisinin, and desoxyartemisinin. Separation of some degradation products and impurities, down to 2%, allows quality control and stability investigation of all actives in raw material and pharmaceutical formulations. The method is further developed via densitometric measurement for quantitative determination purposes for AL and AS. The derivatization technique is evaluated, showing good stability and reproducibility of the coloring process. Percent relative standard deviation values are less than 5% for replicates, and linearity is obtained in the range of 0.5 to 8 microg. A comparative study with high-performance liquid chromatography (HPLC) on a C18 column, applying the same mobile phase, proves the suitability of the TLC method, in which almost all presented analytes are separated from each other. In contrast, HPLC requires at least a 20-min analysis to chromatograph all of the compounds and only betaAM and AE are clearly separated from each other and from the other compounds.

Novel inhibitors of HIV-1 integration.

Human immunodeficiency virus (HIV) is the etiological agent of the acquired immune deficiency syndrome (AIDS). The current strategy for the treatment of HIV infection is called Highly Active Antiretroviral Therapy (HAART) and is based on cocktails of drugs that are currently approved by the Food and Drug Administration. These drugs include compounds that target the viral entry step and the enzymes reverse transcriptase or protease. The introduction of HAART has dramatically changed the landscape of HIV disease. Death from AIDS-related diseases has been reduced significantly since HAART came into use. Nevertheless it is not clear how long clinical benefit will last taking into account the emergence of multiple drug-resistant viral strains. Addition of new anti-HIV drugs targeting other steps of the viral replication cycle may increase the potency of inhibition and delay resistance development. HIV integrase is an essential enzyme in the HIV life cycle and is an attractive target for new drug development. Despite years of intensive research, only two classes of compounds that inhibit integration have been identified until now, namely the diketo acids and the pyranodipyrimidines. In this review we will point to new potential antiviral targets related to retroviral integration that are amenable to drug development. We will describe the pitfalls of currently used integrase assays and propose new strategies and technologies for the discovery of HIV integration inhibitors. Furthermore, we will describe the two classes of integrase inhibitors and discuss their antiviral activity, molecular mechanism of anti-HIV action and the selection of HIV resistance against these drugs.