Biowaiver monographs for immediate-release solid oral dosage forms: Stavudine

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate-release (IR) solid oral dosage forms containing stavudine (d4T) are reviewed. According to Biopharmaceutics Classification System (BCS), d4T can be assigned to BCS class I. No problems with BE of IR d4T formulations containing different excipients and produced by different manufacturing methods have been reported and, hence, the risk of bioinequivalence caused by these factors appears to be low. Furthermore, d4T has a wide therapeutic index. It is concluded that a biowaiver is appropriate for IR solid oral dosage forms containing d4T as the single active pharmaceutical ingredient (API) provided that (a) the test product contains only excipients present in the IR d4T drug products that have been approved in a number of countries for the same dosage form, and (b) both test product and its comparator are either very rapidly dissolving or rapidly dissolving with similarity of dissolution profiles demonstrated at pH 1.2, 4.5, and 6.8. (c) 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:1016, 2012

Spectroscopic Study on the Structural Differences of Thermally Induced Cross-Linking Segments in Emeraldine Salt and Base Forms of Polyaniline

This paper reports the spectroscopic study on the structural differences of thermally induced cross-linking segments in polyaniline in its emeraldine salt (PANI-ES) and base (PANI-EB) forms. Casting films of PANI-ES (ES-film) and PANI-EB (EB-film) were prepared and heated at 150 degrees C under atmospheric air for 30 min. Raman spectra excited at 632.8 nm of heated ES-film presented the characteristic bands of phenazine-like structures at 1638, 1392, and 575 cm(-1), whereas EB-film showed lower relative intensities for these bands. The lower content of phenazine-like segments in heated EB-film is related to residual polaronic segments from preparation procedures, as revealed by Raman. This statement was confirmed by a sequence of thermal and doping experiments in both films. Quantum-chemical calculations by density functional theory (DFT) and time-dependent density functional theory (TD-DFT) showed that the phenazine-like structure presents the intense Raman band at 1350 cm(-1) due to heterocycle breathing mode, and the non-phenazine-like structure (substituted hydrophenazine-type) presents higher energy for HOMO-LUMO transition, indicating the lack of conjugation in the heterocycle compared with the phenazine-like structure. According to experimental and theoretical data reported here, it is proposed that only thermally treated PANI-ES presents phenazine-like rings, whereas PANI-EB presents heterocyclic non-aromatic structures.

DETERMINATION OF DICLOFENAC SODIUM IN RABBIT PLASMA BY HPLC/UV: EVALUATION AND CHARACTERIZATION OF ITS CRYSTALLINE FORMS, ANHYDROUS AND HYDRATE, ON THE ANTIPYRETIC EFFECT

Diclofenac sodium (DS) is a non-steroidal anti-inflammatory drug that is widely prescribed for the treatment of rheumatoid arthritis and post-surgery analgesia. The active pharmaceutical ingredient is the anhydrous form; however, it can also exist in hydrate form. In this context, knowing the properties of the solid state is important and relevant in the pharmaceutical area because they have a significant impact on the solubility, bioavailability, and chemical stability of the drugs. In the present study, data from XRPD, FTIR spectroscopy, and thermal analysis were used for the identification and characterization of DS forms (anhydrous and hydrate). An HPLC method was optimized to evaluate the plasma concentration of DS in rabbits. The optimized method exhibited good linearity over the range 0.1-60 mu g/mL with correlation coefficients of >0.9991. The mean recovery was 100%. Precision and accuracy were determined within acceptable limits. Finally, to compare the pharmacological properties of anhydrous and hydrate DS forms, we investigated their effects in the febrile response induced by lipopolysaccharide from E. coli in rabbits. The results show that the antipyretic effect of anhydrous and hydrate DS forms are similar.

Removal from the plasma of the free and esterified forms of cholesterol and transfer of lipids to HDL in type 2 diabetes mellitus patients

Background: The aim was to investigate new markers for type 2 diabetes (T2DM) dyslipidemia related with LDL and HDL metabolism. Removal from plasma of free and esterified cholesterol transported in LDL and the transfer of lipids to HDL are important aspects of the lipoprotein intravascular metabolism. The plasma kinetics (fractional clearance rate, FCR) and transfers of lipids to HDL were explored in T2DM patients and controls, using as tool a nanoemulsion that mimics LDL lipid structure (LDE). Results: C-14- cholesteryl ester FCR of the nanoemulsion was greater in T2DM than in controls (0.07 +/- 0.02 vs. 0.05 +/- 0.01 h(-1), p = 0.02) indicating that LDE was removed faster, but FCR H-3- cholesterol was equal in both groups. Esterification rates of LDE free-cholesterol were equal. Cholesteryl ester and triglyceride transfer from LDE to HDL was greater in T2DM (4.2 +/- 0.8 vs. 3.5 +/- 0.7%, p = 0.03 and 6.8 +/- 1.6% vs. 5.0 +/- 1.1, p = 0.03, respectively). Phospholipid and free cholesterol transfers were not different. Conclusions: The kinetics of free and esterified cholesterol tended to be independent in T2DM patients and the lipid transfers to HDL were also disturbed. These novel findings may be related with pathophysiological mechanisms of diabetic macrovascular disease.

Clinical, epidemiological and molecular features of the HIV-1 subtype C and recombinant forms that are circulating in the city of São Paulo, Brazil

Abstract Background The city of Sao Paulo has the highest AIDS case rate, with nearly 60% in Brazil. Despite, several studies involving molecular epidemiology, lack of data regarding a large cohort study has not been published from this city. Objectives This study aimed to describe the HIV-1 subtypes, recombinant forms and drug resistance mutations, according to subtype, with emphasis on subtype C and BC recombinants in the city of São Paulo, Brazil. Study design RNA was extracted from the plasma samples of 302 HIV-1-seropositive subjects, of which 211 were drug-naive and 82 were exposed to ART. HIV-1 partial pol region sequences were used in phylogenetic analyses for subtyping and identification of drug resistance mutations. The envelope gene of subtype C and BC samples was also sequenced. Results From partial pol gene analyses, 239 samples (79.1%) were assigned as subtype B, 23 (7.6%) were F1, 16 (5.3%) were subtype C and 24 (8%) were mosaics (3 CRF28/CRF29-like). The subtype C and BC recombinants were mainly identified in drug-naïve patients (72.7%) and the heterosexual risk exposure category (86.3%), whereas for subtype B, these values were 69.9% and 57.3%, respectively (p = 0.97 and p = 0.015, respectively). An increasing trend of subtype C and BC recombinants was observed (p < 0.01). Conclusion The HIV-1 subtype C and CRFs seem to have emerged over the last few years in the city of São Paulo, principally among the heterosexual population. These findings may have an impact on preventive measures and vaccine development in Brazil.

Trypanocidal activity of Brazilian plants against epimastigote forms from Y and Bolivia strains of Trypanosoma cruzi

Chagas disease is one of the main public health problems in Latin America. Since the available treatments for this disease are not effective in providing cure, the screening of potential antiprotozoal agents is essential, mainly of those obtained from natural sources. This study aimed to provide an evaluation of the trypanocidal activity of 92 ethanol extracts from species belonging to the families Annonaceae, Apiaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Moraceae, Nyctaginaceae, and Verbenaceae against the Y and Bolivia strains of Trypanosoma cruzi. Additionally, cytotoxic activity on LLCMK2 fibroblasts was evaluated. Both the trypanocidal activity and cytotoxicity were evaluated using the MTT method, in the following concentrations: 500, 350, 250, and 100 µg/mL. Benznidazole was used for positive control. The best results among the 92 samples evaluated were obtained with ethanol extracts of Ocotea paranapiacabensis (Am93) and Aegiphila lhotzkiana (Am160). Am93 showed trypanocidal activity against epimastigote forms of the Bolivia strain and was moderately toxic to LLCMK2 cells, its Selectivity Index (SI) being 14.56, while Am160 showed moderate trypanocidal activity against the Bolivia strain and moderate toxicicity, its SI being equal to 1.15. The screening of Brazilian plants has indicated the potential effect of ethanol extracts obtained from Ocotea paranapiacabensis and Aegiphila lhotzkiana against Chagas disease.

The role of empirical research in the study of complex forms of governance in agroindustrial systems

The growing complexity of supply chains poses new challenges for Agricultural Research Centers and statistical agencies. The aim of this perspective paper is to discuss the role of empirical research in understanding the complex forms of governance in agribusiness. The authors argue that there are three fundamental levels of analysis: (i) the basic structure of the market, (ii) the formal contractual arrangements that govern relations within the agroindustrial system and (iii) the transactional dimensions governed by non-contractual means. The case of the agrochemical industry in Brazil illustrates how traditional analyses that only address market structure are insufficient to fully explain the agricultural sector and its supply chain. The article concludes by suggesting some indicators which could be collected by statistical agencies to improve understanding of the complex relationships among agribusiness segments. In doing so, the paper seeks to minimize costs and to enable a better formulation of public and private policies.

Human toxocariasis: diagnosis, worldwide seroprevalences and clinical expression of the systemic and ocular forms.

Although human toxocariasis ranks among the most common zoonotic infections worldwide, it remains relatively unknown to the public. The causal agents are the nematode parasites Toxocara canis and T. cati, whose definitive hosts are dogs and cats, respectively. When embryonated eggs are accidentally ingested by humans, larvae hatch in the small intestine, penetrate the intestinal wall and migrate, via the bloodstream, to the liver, lungs, muscles, eye and central nervous system. Although most human infections are asymptomatic, two well-defined clinical syndromes are classically recognised: visceral larva migrans (a systemic disease caused by larval migration through major organs) and ocular larva migrans (a disease limited to the eyes and optic nerves). Two less-severe syndromes have recently been described, one mainly in children (covert toxocariasis) and the other mainly in adults (common toxocariasis). Here, the current laboratory diagnosis, epidemiology and main clinical features of both the systemic and ocular forms of human toxocariasis are reviewed. New developments in serological diagnosis are described, the available seroprevalence data are analysed, and the results of relevant clinical studies that have been published over the last decade are explored, to provide an updated overview of this neglected but highly prevalent human infection.

Understanding the solid forms of 5E-phenylethenylbenzofuroxan with different in vivo anti-T. cruzi activity

5E-Phenylethenylbenzofuroxan (5PhEBfx) was reported as an excellent anti-Chagas drug candidate. However, its oral bioavailability was affected by the crystallization process. Two samples exhibiting variable in vivo activity was investigated: a thin yellow powder (5PhEBfx-Y) and orange needles (5PhEBfx-O). X-ray powder diffraction, differential scanning calorimetry, vibrational spectroscopy, optical and electron scanning microscopies were applied to investigate both solid forms in order to correlate the solid-state properties with the variable bioavailability of 5PhEBfx. It was observed that 5PhEBfx-Y have a better solubility and consequently higher bioavailability when compared with 5PhEBfx-O. This result suggests that the difference of activity between these two 5E-Phenylethenylbenzofuroxanes could be associated with the solid forms, which also cause the coloration variation.

Each one teaches one: characterizing active forms of proteins by single molecule force spectroscopy

This Ph.D. candidate thesis collects the research work I conducted under the supervision of Prof.Bruno Samor´ı in 2005,2006 and 2007. Some parts of this work included in the Part III have been begun by myself during my undergraduate thesis in the same laboratory and then completed during the initial part of my Ph.D. thesis: the whole results have been included for the sake of understanding and completeness. During my graduate studies I worked on two very different protein systems. The theorical trait d’union between these studies, at the biological level, is the acknowledgement that protein biophysical and structural studies must, in many cases, take into account the dynamical states of protein conformational equilibria and of local physico-chemical conditions where the system studied actually performs its function. This is introducted in the introductory part in Chapter 2. Two different examples of this are presented: the structural significance deriving from the action of mechanical forces in vivo (Chapter 3) and the complexity of conformational equilibria in intrinsically unstructured proteins and amyloid formation (Chapter 4). My experimental work investigated both these examples by using in both cases the single molecule force spectroscopy technique (described in Chapter 5 and Chapter 6). The work conducted on angiostatin focused on the characterization of the relationships between the mechanochemical properties and the mechanism of action of the angiostatin protein, and most importantly their intertwining with the further layer of complexity due to disulfide redox equilibria (Part III). These studies were accompanied concurrently by the elaboration of a theorical model for a novel signalling pathway that may be relevant in the extracellular space, detailed in Chapter 7.2. The work conducted on -synuclein (Part IV) instead brought a whole new twist to the single molecule force spectroscopy methodology, applying it as a structural technique to elucidate the conformational equilibria present in intrinsically unstructured proteins. These equilibria are of utmost interest from a biophysical point of view, but most importantly because of their direct relationship with amyloid aggregation and, consequently, the aetiology of relevant pathologies like Parkinson’s disease. The work characterized, for the first time, conformational equilibria in an intrinsically unstructured protein at the single molecule level and, again for the first time, identified a monomeric folded conformation that is correlated with conditions leading to -synuclein and, ultimately, Parkinson’s disease. Also, during the research work, I found myself in the need of a generalpurpose data analysis application for single molecule force spectroscopy data analysis that could solve some common logistic and data analysis problems that are common in this technique. I developed an application that addresses some of these problems, herein presented (Part V), and that aims to be publicly released soon.

Effects of Paramuricea clavata (Risso, 1826) (Anthozoa: Plexauridae) forests on settlement of benthic species: an experimental approach

Rationale: Coralligenous habitat is considered the second most important subtidal “hot spot” of species diversity in the Mediterranean Sea after the Posidonia oceanica meadows. It can be defined as a typical Mediterranean biogenic hard bottom, mainly produced by the accumulation of calcareous encrusting algae that, together with other builder organisms, form a multidimensional framework with a high micro-spatial variability. The development of this habitat depends on physical factors (i.e. light, hydrodynamism, nutrients, etc.), but also biologic interactions can play a relevant role in structuring the benthic assemblages. This great environmental heterogeneity allows several different assemblages to coexist in a reduced space. One of the most beautiful is that characterised by the Mediterranean gorgonian Paramuricea clavata (Risso, 1826) that can contribute to above 40% of total biomass of the community and brings significant structural complexity into the coralligenous habitat. In sites moderately exposed to waves and currents, P. clavata can form high-density populations (up to 60 colonies m-2) between 20 – 70 m in depth. Being a suspension feeder, where it forms dense populations, P. clavata plays a significant role in transferring energy from planktonic to benthic system. The effects of the branched colonies of P. clavata could be comparable to those of the forests on land. They can affect the micro scale hydrodynamism and light, promoting or inhibiting the growth of other species. Unfortunately, gorgonians are threatened by several anthropogenic disturbance factors (i.e. fishing, pollution, tourism) and by climatic anomalies, linked to the global changes, that are responsible of thermal stress, development of mucilage and enhanced pathogens activity, leading to mass mortality events in last decades. Till now, the possible effects of gorgonian forest loss are largely unknown. Our goal was to analyse the ecological role of these sea fan forests on the coralligenous benthic assemblages. Experimental setup and main results: The influence of P. clavata in the settlement and recruitment of epibenthic organisms was analysed by a field experiment carried out in two randomly selected places: Tavolara island and Portofino promontory. The experiment consisted in recreate the presence and absence of the gorgonian forest on recruitment panels, arranged in four plots per type (forested and non-forested), interspersed each other, and deployed at the same depth. On every forested panel 3 gorgonian colonies about 20 cm height were grafted with the use of Eppendorf tubes and epoxy resin bicomponent simulating a density of 190 sea fans per m-2. This density corresponds to a mean biomass of 825 g DW m-2,3 which is of the same order of magnitude of the natural high-density populations. After about 4 months, the panels were collected and analysed in laboratory in order to estimate the percent cover of all the species that have colonized the substrata. The gorgonian forest effects were tested by multivariate and univariate permutational analyses of the variance (PERMANOVA). Recruited assemblages largely differed between the two study sites, probably due to different environmental conditions including water quality and turbidity. On overall, the presence of P. clavata reduced the settlement and recruitment of several algae: the shadow caused by the gorgonian might reduce light availability and therefore their growth. This effect might be greater in places where the waters are on average more clear, since at Portofino it is less visible and could be masked by the high turbidity of the water. The same pattern was registered for forams, more abundant outside gorgonian forest, probably linked with algal distribution, shadowing effect or alimentary competition. The last one hypothesis could be valid also for serpulids polychaetes that growth mainly on non-forested panels. An opposite trend, was showed by a species of bryozoan and by an hydroid that is facilitated by the presence of P. clavata, probably because it attenuates irradiance level and hydrodynamism. Species diversity was significantly reduced by the presence of P. clavata forests at both sites. This seems in contrast with what we expected, but the result may be influenced by the large algal component on non-forested panels. The analysis confirmed the presence of differences in the species diversity among plots and between sites respectively due to natural high variability of the coralligenous system and to different local environment conditions. The reduction of species diversity due to the presence of gorgonians appeared related to a worst evenness rather than to less species richness. With our experiment it is demonstrated that the presence of P. clavata forests can significantly alter local coralligenous assemblages patterns, promoting or inhibiting the recruitment of some species, modifying trophic relationships and adding heterogeneity and complexity to the habitat. Moreover, P. clavata could have a stabilising effect on the coralligenous assemblages.

Preparation of new crystal forms via photochemical, mechanochemical and sol-gel methods

This work of thesis involves various aspects of crystal engineering. Chapter 1 focuses on crystals containing crown ether complexes. Aspects such as the possibility of preparing these materials by non-solution methods, i.e. by direct reaction of the solid components, thermal behavior and also isomorphism and interconversion between hydrates are taken into account. In chapter 2 a study is presented aimed to understanding the relationship between hydrogen bonding capability and shape of the building blocks chosen to construct crystals. The focus is on the control exerted by shape on the organization of sandwich cations such as cobalticinium, decamethylcobalticinium and bisbenzenchromium(I) and on the aggregation of monoanions all containing carboxylic and carboxylate groups, into 0-D, 1-D, 2-D and 3-D networks. Reactions conducted in multi-component molecular assemblies or co-crystals have been recognized as a way to control reactivity in the solid state. The [2+2] photodimerization of olefins is a successful demonstration of how templated solid state synthesis can efficiently synthesize unique materials with remarkable stereoselectivity and under environment-friendly conditions. A demonstration of this synthetic strategy is given in chapter 3. The combination of various types of intermolecular linkages, leading to formation of high order aggregation and crystalline materials or to a random aggregation resulting in an amorphous precipitate, may not go to completeness. In such rare cases an aggregation process intermediate between crystalline and amorphous materials is observed, resulting in the formation of a gel, i.e. a viscoelastic solid-like or liquid-like material. In chapter 4 design of new Low Molecular Weight Gelators is presented. Aspects such as the relationships between molecular structure, crystal packing and gelation properties and the application of this kind of gels as a medium for crystal growth of organic molecules, such as APIs, are also discussed.

Role of immunosuppressive and biologic therapy in pediatric IBD

We aimed to evaluate the role of anti-TNF-alpha therapy with infliximab and adalimumab in a cohort of pediatric patients followed by our Center from 2002 to 2012. The cohort of patients examined consisted of 40 patients: 34 with Crohn disease (85%), 5 with ulcerative colitis (12.5%), one with chronic pouchitis after IPAA for ulcerative colitis (2.5%). All patients were treated with the anti-TNF-α biologic agents infliximab and adalimumab. Thirty-six received infliximab therapy: 19/36 received only infliximab, 17/36 received infliximab and then adalimumab due to loss of response to infliximab and steroid dependency; 4 patients received only adalimumab (infliximab-naïve). Anti-TNF treatment was started before 18 years of age in 34 patients: 29 received infliximab and 5 started adalimumab during childhood. Medical charts were reviewed and safety and efficacy of anti-TNF-alpha have been determined in this population.