Thiazolyl-isatin derivatives: Synthesis, in silico studies, in vitro biological profile against breast cancer cells, mRNA expression, P-gp modulation, and interactions of Akt2 and VIM proteins.

The literature reports that thiazole and isatin nuclei present a range of biological activities, with an emphasis on anticancer activity. Therefore, our proposal was to make a series of compounds using the molecular hybridization strategy, which has been used by our research group, producing hybrid molecules containing the thiazole and isatin nuclei. After structural planning and synthesis, the compounds were characterized and evaluated in vitro against breast cancer cell lines (T-47D, MCF-7 and MDA-MB-231) and against normal cells (PBMC). The activity profile on membrane proteins involved in chemoresistance and tumorigenic signaling proteins was also evaluated. Among the compounds tested, the compounds 4c and 4a stood out with IC50 values of 1.23 and 1.39 µM, respectively, against the MDA-MB-231 cell line. Both compounds exhibited IC50 values of 0.45 µM for the MCF-7 cell line. Compounds 4a and 4c significantly decreased P-gp mRNA expression levels in MCF-7, 4 and 2 folds respectively. Regarding the impact on tumorigenic signaling proteins, compound 4a inhibited Akt2 in MDA-MB-231 and compound 4c inhibited the mRNA expression of VIM in MCF-7.

An Integrated Approach to the Anti-Inflammatory, Antioxidant, and Genotoxic Potential of Portuguese Traditional Preparations from the Bark of Anacardium occidentale L.

Anacardium occidentale L. stem bark Traditional Herbal Preparations (AoBTHPs) are widely used in traditional medicine to treat inflammatory conditions, such as diabetes. The present study aims to evaluate the anti-inflammatory, antioxidant, and genotoxic potential of red and white Portuguese AoBTHPs. Using a carrageenan-induced rat paw edema model, a significant anti-edema effect was observed for all tested doses of white AoBTHP (40.2, 71.5, and 127.0 mg/kg) and the two highest doses of red AoB THP (71.5 and 127.0 mg/kg). The anti-edema effect of red AoBTHP's highest dose was much more effective than indomethacin 10 mg/kg, Trolox 30 mg/kg, and Tempol 30 mg/kg. In DPPH, FRAP, and TAC using the phosphomolybdenum method, both types of AoBTHPs showed similar antioxidant activity and no genotoxicity up to 5000 µg/plate in the Ames test. The LC-UV/DAD-ESI/MS fingerprint allowed the identification of gallic and protocatechuic acids as the two main marker compounds and the presence of catechin, epicatechin, epigallocatechin gallate, and ellagic acid in both AoBTHPs. The obtained results support the validation of red and white AoB and their THPs as anti-inflammatory agents and contribute to the possible development of promising new therapeutic options to treat inflammatory conditions.

Bioguided Identification of Active Antimicrobial Compounds from Asphodelus bento-rainhae and Asphodelus macrocarpus Root Tubers.

Root tubers of Asphodelus bento-rainhae subsp. bento-rainhae (AbR), a vulnerable endemic species, and Asphodelus macrocarpus subsp. macrocarpus (AmR) have traditionally been used in Portugal to treat inflammatory and infectious skin disorders. The present study aims to evaluate the in vitro antimicrobial activity of crude 70% and 96% hydroethanolic extracts of both medicinal plants, specifically against multidrug-resistant skin-related pathogens, to identify the involved marker secondary metabolites and also to assess the pre-clinical toxicity of these medicinal plant extracts. Bioguided fractionation of the 70% hydroethanolic extracts of both species using solvents of increasing polarity, namely diethyl ether (DEE: AbR-1, AmR-1), ethyl acetate (AbR-2, AmR-2) and aqueous (AbR-3, AmR-3) fractions, enabled the identification of the DEE fractions as the most active against all the tested Gram-positive microorganisms (MIC: 16 to 1000 µg/mL). Furthermore, phytochemical analyses using TLC and LC-UV/DAD-ESI/MS techniques revealed the presence of anthracene derivatives as the main constituents of DEE fractions, and five known compounds, namely 7'-(chrysophanol-4-yl)-chrysophanol-10'-C-beta-D-xylopyranosyl-anthrone (p), 10,7'-bichrysophanol (q), chrysophanol (r), 10-(chrysophanol-7'-yl)-10-hydroxychrysophanol-9-anthrone (s) and asphodelin (t), were identified as the main marker compounds. All these compounds showed high antimicrobial activity, particularly against Staphylococcus epidermidis (MIC: 3.2 to 100 µg/mL). Importantly, no cytotoxicity against HepG2 and HaCaT cells (up to 125 µg/mL) for crude extracts of both species and genotoxicity (up to 5000 µg/mL, with and without metabolic activation) for AbR 96% hydroethanolic extract was detected using the MTT and Ames tests, respectively. Overall, the obtained results contribute to the concrete validation of the use of these medicinal plants as potential sources of antimicrobial agents in the treatment of skin diseases.

Anacardium occidentale Bark as an Antidiabetic Agent.

Anacardium occidentale L. is used throughout the world to treat type 2 diabetes. In Portugal, a traditional herbal preparation made with stem bark of this species (AoBTHP) has been used for more than 30 years to treat this pathology. The AoBTHP was standardized on total phenolic content, and its hypoglycemic activity was assessed using db/db mice (n = 26) for 92 days. Three doses (40.2, 71.5, and 127.0 mg/kg/day, per os) were tested, and glibenclamide (5 mg/kg/day) was used as positive control. During the study, glycemia was measured under non-fasting or fasting states. In sequence, thin-layer chromatography bioautographic assays were used for the detection of possible alpha- and beta-glucosidase inhibitors. A significant hypoglycemic effect in fasting glycemia in days 31 and 57 was observed with the three tested doses. The 71.5 mg/kg and 127.0 mg/kg AoBTHPs significantly reduced non-fasting glycemia on day 24. The highest dose showed the most significant hypoglycemic effect. Gallic acid was identified as the major alpha- and beta-glucosidase inhibitor. The 127 mg/kg/day AoBTHP dose showed a greater glucose-lowering effect than glibenclamide. For the first time, a standardized AoBTHP was tested using an in vivo diabetes model, and its usage was preclinically validated for type 2 diabetes treatment. The hypoglycemic activity of an AoBTHP can be related to the presence of alpha- and beta-glucosidase inhibitors, such as gallic acid, but other mechanisms can also be involved.

Contribution to the Preclinical Safety Assessment of Lannea velutina and Sorindeia juglandifolia Leaves.

Dried leaves of Lannea velutina A. Rich. and Sorindeia juglandifolia (A. Rich.) Planch. ex Oliv. (family Anacardiaceae) are used in African traditional medicine. Although these medicinal plants have widespread use in the treatment of inflammatory diseases, there is no scientific data concerning their preclinical or clinical safety. This work aimed to investigate the phytochemical properties of the leaves of both species using HPLC-UV/DAD, as well as the in vivo oral repeated-dose toxicity of 70% hydroethanolic leaf extract of S. juglandifolia and the in vitro genotoxicity of 70% hydroethanolic leaf extracts of L. velutina and S. juglandifolia. Clinical signs of toxicity, body weight variations, and changes in food consumption, mortality, and blood biochemical parameters were monitored. Genotoxicity was assessed using the bacterial reverse mutation assay (Ames test) with and without metabolic activation, according to OECD guidelines. The obtained results showed the presence of gallic acid and anacardic acid as the main marker constituents in both species. No significant changes in general body weight or food intake were observed; small significant changes with no critical relevance were observed in the blood biochemistry of animals treated with S. juglandifolia hydroethanolic extract (50, 400, and 1000 mg/kg body weight) compared to those in the control group. No genotoxicity was observed in the bacterial reverse mutation assay with S. juglandifolia and L. velutina extracts (up to 5 mg/plate). The safety data obtained in vivo and lack of genotoxic potential in vitro points to the safe medicinal use of S. juglandifolia and L. velutina extracts.

U-turn time and velocity dependence on the wavelength of light: multicellular magnetotactic prokaryotes of different sizes behave differently.

'Candidatus Magnetoglobus multicellularis' is a multicellular magnetotactic prokaryote found in the Araruama lagoon in Rio de Janeiro, Brazil. This microorganism shows a photokinesis that depends on the incident light wavelength, but that dependence can be canceled by the presence of radio-frequency (RF) electromagnetic fields. The present manuscript has as its aim to study the effect of light wavelength and RF fields on the U-turn time of 'Candidatus Magnetoglobus multicellularis', a behavior more related to magnetotaxis. As the experiments were performed during the night, the microorganisms were greater in size than normal, indicating that they were in the process of division. Our results show that when normal in size, the microorganism's U-turn time is modified by the light wavelength (lower for blue light than for green and red light), but RF fields do not affect that U-turn time dependence on the light wavelength. For the microorganism in the process of division, we describe for the first time how the photokinesis and U-turn time dependence on the light wavelength disappear. It is proposed that methyl-accepting chemotaxis proteins are involved in that light wavelength dependence for the U-turn time, but still more studies are necessary to understand how RF fields cancel the photokinesis light wavelength dependence, but do not affect the dependence of the U-turn time.

An evaluation framework for new approach methodologies (NAMs) for human health safety assessment.

The need to develop new tools and increase capacity to test pharmaceuticals and other chemicals for potential adverse impacts on human health and the environment is an active area of development. Much of this activity was sparked by two reports from the US National Research Council (NRC) of the National Academies of Sciences, Toxicity Testing in the Twenty-first Century: A Vision and a Strategy (2007) and Science and Decisions: Advancing Risk Assessment (2009), both of which advocated for "science-informed decision-making" in the field of human health risk assessment. The response to these challenges for a "paradigm shift" toward using new approach methodologies (NAMS) for safety assessment has resulted in an explosion of initiatives by numerous organizations, but, for the most part, these have been carried out independently and are not coordinated in any meaningful way. To help remedy this situation, a framework that presents a consistent set of criteria, universal across initiatives, to evaluate a NAM's fit-for-purpose was developed by a multi-stakeholder group of industry, academic, and regulatory experts. The goal of this framework is to support greater consistency across existing and future initiatives by providing a structure to collect relevant information to build confidence that will accelerate, facilitate and encourage development of new NAMs that can ultimately be used within the appropriate regulatory contexts. In addition, this framework provides a systematic approach to evaluate the currently-available NAMs and determine their suitability for potential regulatory application. This 3-step evaluation framework along with the demonstrated application with case studies, will help build confidence in the scientific understanding of these methods and their value for chemical assessment and regulatory decision-making.

Recommendations from a global cross-company data sharing initiative on the incorporation of recovery phase animals in safety assessment studies to support first-in-human clinical trials.

An international expert group which includes 30 organisations (pharmaceutical companies, contract research organisations, academic institutions and regulatory bodies) has shared data on the use of recovery animals in the assessment of pharmaceutical safety for early development. These data have been used as an evidence-base to make recommendations on the inclusion of recovery animals in toxicology studies to achieve scientific objectives, while reducing animal use. Recovery animals are used in pharmaceutical development to provide information on the potential for a toxic effect to translate into long-term human risk. They are included on toxicology studies to assess whether effects observed during dosing persist or reverse once treatment ends. The group devised a questionnaire to collect information on the use of recovery animals in general regulatory toxicology studies to support first-in-human studies. Questions focused on study design, the rationale behind inclusion or exclusion and the impact this had on internal and regulatory decisions. Data on 137 compounds (including 53 biologicals and 78 small molecules) from 259 studies showed wide variation in where, when and why recovery animals were included. An analysis of individual study and programme design shows that there are opportunities to reduce the use of recovery animals without impacting drug development.

Scientific considerations for complex drugs in light of established and emerging regulatory guidance.

On March 9, 2012, the New York Academy of Sciences brought together experts representing a variety of perspectives--including academic, industrial, regulatory, as well as those from physicians and consumers--to discuss considerations for the non-biological complex drug (NBCD) regulatory approval pathway, given the emerging regulatory guidelines for biosimilars (follow-on biological complex drugs). Some of the organizers of the conference expressed their belief that NBCDs share a number of characteristic features with biologicals: the structure cannot be fully defined by the available (physicochemical) analytical tests, and quality assurance is based on in-depth knowledge, consistency, and control of the production process. However, their view on NBCDs was not universally accepted among the experts who participated in the conference. Plenary sessions addressed the most recent regulatory developments, experimental design, interchangeability, and immunogenicity issues for follow-on versions of complex drugs from the perspective of key audiences, including industry, regulatory agencies, physicians, and consumers. This report summarizes these various perspectives on NBCDs and the scientific and regulatory considerations associated with complex drug categories.

Towards consensus practices to qualify safety biomarkers for use in early drug development.

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.

Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium.

The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortium's (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

Challenges with advanced therapy medicinal products and how to meet them.

Advanced therapy medicinal products (ATMPs), which include gene therapy medicinal products, somatic cell therapy medicinal products and tissue-engineered products, are at the cutting edge of innovation and offer a major hope for various diseases for which there are limited or no therapeutic options. They have therefore been subject to considerable interest and debate. Following the European regulation on ATMPs, a consolidated regulatory framework for these innovative medicines has recently been established. Central to this framework is the Committee for Advanced Therapies (CAT) at the European Medicines Agency (EMA), comprising a multidisciplinary scientific expert committee, representing all EU member states and European Free Trade Association countries, as well as patient and medical associations. In this article, the CAT discusses some of the typical issues raised by developers of ATMPs, and highlights the opportunities for such companies and research groups to approach the EMA and the CAT as a regulatory advisor during development.

Joint EFPIA/CHMP SWP workshop: the Emerging Use of Omic Technologies for Regulatory Non-Clinical Safety Testing.

A workshop was held on October 26-27, 2004, in Bonn, Germany, to discuss the potential use of omic technologies for regulatory non-clinical safety testing of pharmaceuticals. The meeting was hosted by the European Federation of Pharmaceutical Industries and Associations (EFPIA). The workshop was held in conjunction with the 6th European preclinical assessors meeting, which was organized in Bonn by the German Federal Institute for Drugs and Medical Devices (BfArM) and the Safety Working Party (SWP) of the Committee for Medicinal Products for Human Use (CHMP). Approximately 100 scientists, roughly half from the European pharmaceutical industry and half from European regulatory authorities, attended the workshop. The authors of this report constitute the organizing committee members.

Phenylephrine induces endogenous noradrenaline release in the rat vas deferens through nitric oxide synthase pathway.

We have previously observed that in the rat vas deferens nitric oxide synthase pathway potentiated phenylephrine-induced contractility raising the possibility of a facilitatory role on neurotransmission by nitric oxide. To confirm this hypothesis we studied the effect of phenylephrine on the concentration response curves obtained in preparations from reserpine-treated rats in the absence and presence of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). The endogenous noradrenaline released by normal preparations (without reserpine) was measured in the perfusion fluid of preparations stimulated with phenylephrine, in the absence and presence of L-NMMA, L-NMMA + the nitric oxide donor 3-morpholinosydnonimine hydrochloride (SIN-1), the alpha1-adrenoceptor antagonist prazosin and the blocker of noradrenaline carrier desipramine. The phenylephrine-induced noradrenaline release in a calcium-free medium was also measured. L-NMMA decreased the Emax of phenylephrine concentration response curves obtained in preparations from normal (reserpine-untreated) but not from reserpine-treated rats. In the perfusion fluid of preparations incubated with phenylephrine, a concentration-dependent increase of noradrenaline was observed which was reversed by L-NMMA and restored when SIN-1 was added together with the nitric oxide synthase inhibitor. The concentration-dependent phenylephrine-induced noradrenaline increase was not modified by desipramine but was abolished by 10 microM prazosin. In calcium-free medium, phenylephrine failed to increase the noradrenaline concentration. These results suggest that in the rat vas deferens, nitric oxide pathway potentiates the phenylephrine-induced contractility through a mechanism which involves calcium-dependent release of endogenous noradrenaline and seems to depend, at least partially on the activation of alpha1-adrenoceptors.

Alternatives models in carcinogenicity testing--a European perspective.

Transgenic mouse strains offer the prospect of significant benefits in the in vivo assessment of carcinogenic potential. The European Regulatory Authorities have been supportive of their inclusion as one of the second-test options in the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human use (ICH). However, there is a concern regarding premature systematic use of these models. At present, the information from the International Life Sciences Institute (ILSI) project suggests that the transgenic models under study are similarly sensitive to genotoxic pharmaceuticals. There are apparently some false negatives and false positives. For regulatory purposes, it is not yet possible to differentiate the models with respect to hazard identification and risk assessment. The evaluation of the models has reached an interesting but, at certain points, equivocal stage. Based on the weight of evidence gathered thus far, regulatory authorities cannot neglect the outcome of such studies but need to be cautious in their interpretation of data from such models, and the application in risk assessment procedures.

Nitric oxide synthase/guanylate cyclase pathway modulates the rat vas deferens contractility induced by phenylephrine.

The involvement of the nitric oxide synthase/soluble guanylate cyclase pathway on the modulation of phenylephrine-induced contractility in the rat vas deferens was investigated. Phenlylephrine-concentration response curves were obtained in absence and in presence of inhibitors, N(G)-Nitro-L-arginine (L-NOARG), NG-Nitro-L-arginine methyl esther (L-NAME) or N(G)-monomethyl-L-arginine (L-NMMA) or GC inhibitior, 1H-(1,2,4)-oxadiaziol-(4,3-a)quinoxalin-1-one (ODQ) or nitric oxide donor, 3-morpholinosydnonimine hydrochloride (SIN-1) alone or together with L-NMMA or ODQ. Both nitric oxide synthase and GC inhibitors reduced the Phe-Emax. SIN-1 alone did not change phenylephrine-induced responses and it could reverse the L-NMMA effect but not ODQ effect. The reduction of the phenylephrine-induced contractility obtained in consequence of the inhibition of the nitric oxide/GC pathway suggest that, in the rat vas deferens, despite its well identified relaxant properties, nitric oxide potentiates the contractility induced by adrenergic stimulation.