Development of a "green process" for the isolation of natural functional extracts with anti-cancer activity - Application of high-pressure technology

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Monotherapy and combined therapy of new potential antitumor compounds: antiproliferative activities and biological targets

Review article Martins, P., Marques, M., Coito, L., Pombeiro, A.J.L., Baptista, P.V., Fernandes, A.R. 2014. Organometallic Compounds in Cancer Therapy: Past Lessons and Future Directions. Anti-cancer Agents in Medicinal Chemistry 14. PMID: 25173559

Development of novel active pharmaceutical ionic liquids and salts based on antibiotics and anti-fungal drugs

Ionic Liquids (ILs) are class of compounds, which have become popular since the mid-1990s. Despite the fact that ILs are defined by one physical property (melting point), many of the potential applications are now related to their biological properties. The use of a drug as a liquid can avoid some problems related to polymorphism which can influence a drug´s solubility and thus its dosages. Also, the arrangement of the anion or cation with a specific drug might be relevant in order to: a) change the correspondent biopharmaceutical drug classification system; b) for the drug formulation process and c) the change the Active Pharmaceutical Ingredients’ (APIs). The main goal of this Thesis is the synthesis and study of physicochemical and biological properties of ILs as APIs from beta-lactam antibiotics (ampicillin, penicillin G and amoxicillin) and from the anti-fungal Amphotericin B. All the APIs used here were neutralized in a buffer appropriate hydroxide cations. The cation hydroxide was obtained on Amberlite resin (in the OH form) in order to exchange halides. The biological studies of these new compounds were made using techniques like the micro dilution and colorimetric methods. Overall a total of 19 new ILs were synthesised (6 ILs based on ampicillin, 4 ILs, based on amoxicillin, 6 ILs based on penicillin G and 4 ILs based on amphotericin B) and characterized by spectroscopic and analytical methods in order to confirm their structure and purity. The study of the biological properties of the synthesised ILs showed that some have antimicrobial activity against bacteria and yeast cells, even in resistant bacteria. Also this work allowed to show that ILs based on ampicillin could be used as anti-tumour agents. This proves that with a careful selection of the organic cation, it is possible to provoke important physico-chemical and biological alteration in the properties of ILs-APIs with great impact, having in mind their applications.

In vitro anti-Candida activity of Glycyrrhiza glabra L.

The severity and frequency of opportunistic fungal infections still growing, concomitantly to the increasing rates of antimicrobial drugs resistance. Natural matrices have been used over years due to its multitude of health benefits, including antifungal potential. Thus, the present work aims to evaluate the anti-Candida potential of the phenolic extract and individual phenolic compounds of Glycyrrhiza glabra L. (licorice), by disc diffusion assay, followed by determination of the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for both planktonic cells and biofilms. Licorice extract evidenced inhibitory potential against the nineteen tested Candida strains, but no pronounced effect was observed by testing the most abundant individual phenolic compounds. Candida tropicalis strains were the most sensible, followed by Candida glabrata, Candida parapsilosis and, then, Candida albicans. Lower MIC and MFC values were achieved to C. glabrata and C. tropicalis, which confirms its susceptibility to licorice extract; however, for C. tropicalis strains a higher variability was observed. Anti-biofilm potential was also achieved, being most evident in some C. glabrata and C. tropicalis strains. In general, a twice concentration of the MIC was necessary for planktonic cells to obtain a similar potential to that one observed for biofilms. Thus, an upcoming approach for new antifungal agents, more effective and safer than the current ones, is stablished; notwithstanding, further studies are necessary in order to understand its mechanism of action, as also to assess kinetic parameters.

In vivo antioxidant activity of phenolic compounds: facts and gaps

Background: Numerous diseases have been related with free radicals overproduction and oxidative stress. Botanical preparations possess a multitude of bioactive properties, including antioxidant potential, which has been mainly related with the presence of phenolic compounds. However, the mechanisms of action of these phytochemicals, in vivo effects, bioavailability and bio-efficacy still need research. Scope and Approach: The present report aims to provide a critical review on the aspects related with the in vivo antioxidant activity of phenolic extracts and compounds from plant origin. Key findings: Biological functions beyond the human metabolism were discussed, comparing in vivo vs. in vitro studies, as also focusing the conditioning factors for phenolic compounds bioavailability and bio-efficacy. Furthermore, an upcoming perspective about the use of phytochemicals as life expectancy promoters and anti-aging factors in human individuals was provided. Conclusions: Overall, and despite all of those advances, the study of the biological potential of numerous natural matrices still remains a hot topic among the scientific community. In fact, the available knowledge about the responsible phytochemicals for the biological potential, their mechanisms of action, the establishment of therapeutic and prophylactic doses, and even the occurrence of biochemical inter-relations, is considerable scarce.

The contribution of phenolic acids to the anti-inflammatory activity of mushrooms: Screening in phenolic extracts, individual parent molecules and synthesized glucuronated and methylated derivatives

In the present study, the ethanolic extracts of fourteen edible mushrooms were investigated for their anti-inflammatory potential in LPS (lipopolysaccharide) activated RAW 264.7 macrophages. Furthermore the extracts were chemically characterized in terms of phenolic acids and related compounds. The identified molecules (p-hydroxybenzoic, p-coumaric and cinnamic acids) and their glucuronated and methylated derivatives obtained by chemical synthesis were also evaluated for the same bioactivity, in order to establish structure-activity relationships and to comprehend the effects of in vivo metabolism reactions in the activity of the compounds. The extracts of Pleurotus ostreatus, Macrolepiota procera, Boletus impolitus and Agaricus bisporus revealed the strongest anti-inflammatory potential (EC50 values 96 ± 1 to 190 ± 6 µg/mL, and also the highest concentration of cinnamic acid (656 to 156 µg/g), which was also the individual compound with the highest anti-inflammatory activity. The derivatives of p-coumaric acid revealed the strongest properties, specially the derivative methylated in the carboxylic group (CoA-M1) that exhibited similar activity to the one showed by dexamethaxone used as anti-inflammatory standard; by contrast, the derivatives of p-hydroxybenzoic revealed the lowest inhibition of NO production. All in all, whereas the conjugation reactions change the chemical structure of phenolic acids and may increase or decrease their activity, the glucuronated and methylated derivatives of the studied compounds are still displaying anti-inflammatory activity.

Anti-inflammatory and antinociceptive activities of Bauhinia monandra leaf lectin

A galactose-specific lectin from Bauhinia monandra leaves (BmoLL) have been purified through ammonium sulphate fractionation followed by guar gel affinity chromatography column. This study aimed to evaluate the potential anti-inflammatory and antinociceptive activity of pure BmoLL in mice. Anti-inflammatory activity was evaluated by 1% carrageenan-induced inflammation in mice treated with BmoLL. Acetic acid-induced abdominal writhing and hot plate methods evaluated antinociceptive activity. BmoLL significantly inhibited the carrageenan-induced paw edema by 47% (30 mg/kg) and 60.5% (60 mg/kg); acetylsalicylic acid (ASA, 100 mg/kg) showed inhibition of 70.5%, in comparison to controls. Leukocyte migration, an immune response to the inflammation process, was significantly reduced in presence of BmoLL; in mice treated with \ASA\ the decrease in leukocyte migration was similar to 15 mg/kg of the lectin. BmoLL at doses of 15, 30 and 60 mg/kg significantly reduced the number of animal contortions by 43.1, 50.1 and 71.3%, respectively.BmoLL leukocyte migration was significantly reduced; in mice treated with \ASA\ the decrease in leukocyte migration was similar to 15 mg/kg of the lectin. BmoLL at doses of 15, 30 and 60 mg/kg significantly reduced the number of animal contortions by 43.1, 50.1 and 71.3%, respectively. The lectin (30 and 60 mg/kg) showed a significant effect in the hot plate assay. BmoLL anti-inflammatory and antinociceptive effects were dose-dependent. The search for new and natural compounds, with minimal side effects, to control pain and inflammation, is constantly increasing. BmoLL has great potential as a natural anti-inflamatory product that can be explored for pharmacological purposes.

High-throughput hydrophilic interaction chromatography coupled to tandem mass spectrometry for the optimized quantification of the anti-Gram-negatives antibiotic colistin A/B and its pro-drug colistimethate.

Colistin is a last resort's antibacterial treatment in critically ill patients with multi-drug resistant Gram-negative infections. As appropriate colistin exposure is the key for maximizing efficacy while minimizing toxicity, individualized dosing optimization guided by therapeutic drug monitoring is a top clinical priority. Objective of the present work was to develop a rapid and robust HPLC-MS/MS assay for quantification of colistin plasma concentrations. This novel methodology validated according to international standards simultaneously quantifies the microbiologically active compounds colistin A and B, plus the pro-drug colistin methanesulfonate (colistimethate, CMS). 96-well micro-Elution SPE on Oasis Hydrophilic-Lipophilic-Balanced (HLB) followed by direct analysis by Hydrophilic Interaction Liquid Chromatography (HILIC) with Ethylene Bridged Hybrid - BEH - Amide phase column coupled to tandem mass spectrometry allows a high-throughput with no significant matrix effect. The technique is highly sensitive (limit of quantification 0.014 and 0.006μg/mL for colistin A and B), precise (intra-/inter-assay CV 0.6-8.4%) and accurate (intra-/inter-assay deviation from nominal concentrations -4.4 to +6.3%) over the clinically relevant analytical range 0.05-20μg/mL. Colistin A and B in plasma and whole blood samples are reliably quantified over 48h at room temperature and at +4°C (<6% deviation from nominal values) and after three freeze-thaw cycles. Colistimethate acidic hydrolysis (1M H2SO4) to colistin A and B in plasma was completed in vitro after 15min of sonication while the pro-drug hydrolyzed spontaneously in plasma ex vivo after 4h at room temperature: this information is of utmost importance for interpretation of analytical results. Quantification is precise and accurate when using serum, citrated or EDTA plasma as biological matrix, while use of heparin plasma is not appropriate. This new analytical technique providing optimized quantification in real-life conditions of the microbiologically active compounds colistin A and B offers a highly efficient tool for routine therapeutic drug monitoring aimed at individualizing drug dosing against life-threatening infections.

Antitumor activity of chemical modified natural compounds

Search of new activity substances starting from chemotherapeutic agents, continously appears in international literature. Perhaps this search has been done more frequently in the field of anti-tumor chemotherapy on account of the unsuccess in saving advanced stage patients. The new point in this matter during the last decade was computer aid in planning more rational drugs. In near future "the accessibility of supercomputers and emergence of computer net systems, willopen new avenues to rational drug design" (Portoghese, P. S. J. Med. Chem. 1989, 32, 1). Unknown pharmacological active compounds synthetized by plants can be found even without this eletronic devices, as tradicional medicine has pointed out in many contries, and give rise to a new drug. These compounds used as found in nature or after chemical modifications have produced successful experimental medicaments as FAA, "flavone acetic acid" with good results as inibitors of slow growing animal tumors currently in preclinical evaluation for human treatment. In this lecture some international contributions in the field of chemical modified compounds as antineoplasic drugs will be examined, particularly those done by Brazilian researches.

Mutation analysis of the different tfd genes for degradation of chloroaromatic compounds in Ralstonia eutropha JMP134.

Ralstonia eutropha JMP134 possesses two sets of similar genes for degradation of chloroaromatic compounds, tfdCDEFB (in short: tfdI cluster) and tfdDII CII EII FII BII (tfdII cluster). The significance of two sets of tfd genes for the organism has long been elusive. Here, each of the tfd genes in the two clusters on the original plasmid pJP4 was replaced by double recombination with a gene fragment in which a kanamycin resistance gene was inserted into the respective tfd gene's reading frame. The insertion mutants were all tested for growth on 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), and 3-chlorobenzoate (3-CBA). None of the tfdDII CII EII FII BII genes appeared to be essential for growth on 2,4-D or on 3-CBA. Mutations in tfdC, tfdD and tfdF also did not abolish but only retarded growth on 2,4-D, indicating that they were redundant to some extent as well. Of all tfd genes tested, only tfdE and tfdB were absolutely essential, and interruption of those two reading frames abolished growth on 2,4-D, 3-CBA ( tfdE only), and MCPA completely. Interestingly, strains with insertion mutations in the tfdI cluster and those in tfdDII, tfdCII, tfdEII and tfdBII were severely effected in their growth on MCPA, compared to the wild-type. This indicated that not only the tfdI cluster but also the tfdII cluster has an essential function for R. eutropha during growth on MCPA. In contrast, insertion mutation of tfdDII resulted in better growth of R. eutropha JMP134 on 3-CBA, which is most likely due to the prevention of toxic metabolite production in the absence of TfdDII activity.

Biomorphological Alterations Induced by an Anti-juvenile Hormonal Compound, 2-(2-EthoxyEthoxy)Ethyl Furfuryl Ether, on Three Species of Triatominae Larvae (Hemiptera, Reduviidae)

Applied topically to larvae of Rhodnius prolixus Stal, Triatoma infestans (Klug) and Panstrongylus herreri Wygodzinsky, vectors of Trypanosoma cruzi, the causative agent of Chagas'disease, a synthetic, furan-containing anti-juvenile hormonal compound, 2-(2-ethoxyethoxy)ethyl furfuryl ether induced a variety of biomorphological alterations, including precocious metamorphosis into small adultoids with adult abdominal cuticle, ocelli, as well as rudimentary adultoid wings. Some adultoids died during ecdysis and were confined within the old cuticle. The extension of these biomorphological responses is discussed in terms of the complexity of the action of anti-juvenile hormonal compounds during the development of triatomines

Synthesis and characterization of a new class of anti-angiogenic agents based on ruthenium clusters.

New triruthenium-carbonyl clusters derivatized with glucose-modified bicyclophosphite ligands have been synthesized. These compounds were found to have cytostatic and cytotoxic activity and depending on the number of bicyclophosphite ligands, and could be tuned for either anti-cancer or specific anti-angiogenic activity. While some compounds had a broad cellular toxicity profile in several cell types others showed endothelial cell specific dose-dependent anti-proliferative and anti-migratory efficacy. A profound inhibition of angiogenesis was also observed in the in vivo chicken chorioallantoic membrane (CAM) model, and consequently, these new compounds have considerable potential in drug design, e.g. for the treatment of cancer.

In Vitro and in Vivo Anti-Trypanosoma cruzi Activity of a Novel Nitro-derivative

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.

Anti-leishmanial activity of alkaloidal extract from Aspidosperma ramiflorum

Infections due to protozoa of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials (SbV), which present renal and cardiac toxicity. Besides, the precise chemical structure and mechanism of action of these drugs are unknown up to date. In order to find new drugs against leishmaniasis, we have been studying extracts of Brazilian trees. In the present study, we have evaluated the effectiveness of an alkaloid extract of Aspidosperma ramiflorum Muell. Arg. (Apocynaceae), against the extracellular forms promastigotes of L. (L.) amazonensis and L. (V.) braziliensis. The alkaloid extract of A. ramiflorum was much more effective against L. (L.) amazonensis (LD50 < 47 µg/ml) than L. (V.) braziliensis. Based on these in vitro results against L. (L.) amazonensis new studies should be made to find the compounds with anti-leishmanial activity.

Selective PDE4 inhibitors as potent anti-inflammatory drugs for the treatment of airway diseases

Phosphodiesterases (PDEs) are responsible for the breakdown of intracellular cyclic nucleotides, from which PDE4 are the major cyclic AMP metabolizing isoenzymes found in inflammatory and immune cells. This generated greatest interest on PDE4 as a potential target to treat lung inflammatory diseases. For example, cigarette smoke-induced neutrophilia in BAL was dose and time dependently reduced by cilomilast. Beside the undesired side effects associated with the first generation of PDE4 inhibitors, the second generation of selective inhibitors such as cilomilast and roflumilast showed clinical efficacy in asthma and chronic obstrutive pulmonary diseases trials, thus re-enhancing the interest on these classes of compounds. However, the ability of PDE4 inhibitors to prevent or modulate the airway remodelling remains relatively unexplored. We demonstrated that selective PDE4 inhibitor RP 73-401 reduced matrix metalloproteinase (MMP)-9 activity and TGF-beta1 release during LPS-induced lung injury in mice and that CI-1044 inhibited the production of MMP-1 and MMP-2 from human lung fibroblasts stimulated by pro-inflammatory cytokines. Since inflammatory diseases of the bronchial airways are associated with destruction of normal tissue structure, our data suggest a therapeutic benefit for PDE4 inhibitors in tissue remodelling associated with chronic lung diseases.