Nuno Vale

Primaquine revisited six decades after its discovery.

Primaquine was firstly synthesized in 1946 in the USA, and is the most representative member of the anti-malarial 8-aminoquinolines. Six decades have passed and primaquine is still the only transmission-blocking anti-malarial clinically available, displaying a marked activity against gametocytes of all species of human malaria, including multi-resistant Plasmodium falciparum strains. Primaquine is also effective against all exoerythrocytic forms of the parasite and is used in conjunction with other anti-malarials for the treatment of vivax and ovale malaria. However, primaquine is often associated with serious adverse effects, in consequence of its toxic metabolites. 5-Hydroxyprimaquine or 6-methoxy-8-aminoquinoline has been considered to be directly responsible for complications such as hemolytic anemia. Primaquine toxicity is aggravated in people deficient of 6-glucose phosphate dehydrogenase or glutathione synthetase. Adverse effects are further amplified by the fact that primaquine must be repeatedly administered at high doses, due to its limited oral bioavailability. Over the last two decades, Medicinal Chemists have battled against primaquines disadvantages, while keeping or even improving its unequalled performance as an anti-malarial. The present text revisits primaquine and its properties on the occasion of its 60th anniversary and aims to give a general overview of what has been the path towards the development of effective and safe primaquine-based anti-malarials. Presently, aablaquine and tafenoquine the two most promising primaquine analogues are already in the final stages of clinical trials against Plasmodium vivax and P. falciparum. Both compounds are a new hope against malaria and other primaquine-sensitive illnesses, such as Pneumocystis Pneumonia or the Chagas disease.

Cyclization-activated Prodrugs

Many drugs suffer from an extensive first-pass metabolism leading to drug inactivation and/or production of toxic metabolites, which makes them attractive targets for prodrug design. The classical prodrug approach, which involves enzyme-sensitive covalent linkage between the parent drug and a carrier moiety, is a well established strategy to overcome bioavailability/toxicity issues. However, the development of prodrugs that can regenerate the parent drug through non-enzymatic pathways has emerged as an alternative approach in which prodrug activation is not influenced by inter- and intraindividual variability that affects enzymatic activity. Cyclization-activated prodrugs have been capturing the attention of medicinal chemists since the middle-1980s, and reached maturity in prodrug design in the late 1990 s. Many different strategies have been exploited in recent years concerning the development of intramoleculary-activated prodrugs spanning from analgesics to anti-HIV therapeutic agents. Intramolecular pathways have also a key role in two-step prodrug activation, where an initial enzymatic cleavage step is followed by a cyclization-elimination reaction that releases the active drug. This work is a brief overview of research on cyclization-activated prodrugs from the last two decades.

Phenolic Profiling of Portuguese Propolis by LC-MS Spectrometry: Uncommon Propolis Rich in Flavonoid Glycosides

INTRODUCTION Propolis is a chemically complex resinous substance collected by honeybees (Apis mellifera) from tree buds, comprising plant exudates, secreted substances from bee metabolism, pollen and waxes. Its chemical composition depends strongly on the plant sources available around the beehive, which have a direct impact in the quality and bioactivity of the propolis. Being as Portugal is a country of botanical diversity, the phenolic characterisation of propolis from the different regions is a priority. OBJECTIVE Extensive characterisation of the phenolic composition of Portuguese propolis from different continental regions and islands. METHOD Forty propolis ethanolic extracts were analysed extensively by liquid chromatography with diode-array detection coupled to electrospray ionisation tandem mass spectrometry (LC-DAD-ESI-MS(n) ). RESULTS Seventy-six polyphenols were detected in the samples and two groups of propolis were established: the common temperate propolis, which contained the typical poplar phenolic compounds such as flavonoids and their methylated/esterified forms, phenylpropanoid acids and their esters, and an uncommon propolis type with an unusual composition in quercetin and kaempferol glycosides - some of them never described in propolis. CONCLUSION The method allowed the establishment of the phenolic profile of Portuguese propolis from different geographical locations, and the possibility to use some phenolic compounds, such as kaempferol-dimethylether, as geographical markers. Data suggest that other botanical species in addition to poplar trees can be important sources of resins for Portuguese propolis.

Schistosome and liver fluke derived catechol-estrogens and helminth associated cancers

Infection with helminth parasites remains a persistent public health problem in developing countries. Three of these pathogens, the liver flukes Clonorchis sinensis, Opisthorchis viverrini and the blood fluke Schistosoma haematobium, are of particular concern due to their classification as Group 1 carcinogens: infection with these worms is carcinogenic. Using liquid chromatography-mass spectrometry (LC-MS/MS) approaches, we identified steroid hormone like (e.g., oxysterol-like, catechol estrogen quinone-like, etc.) metabolites and related DNA-adducts, apparently of parasite origin, in developmental stages including eggs of S. haematobium, in urine of people with urogenital schistosomiasis, and in the adult stage of O. viverrini. Since these kinds of sterol derivatives are metabolized to active quinones that can modify DNA, which in other contexts can lead to breast and other cancers, helminth parasite associated sterols might induce tumor-like phenotypes in the target cells susceptible to helminth parasite associated cancers, i.e., urothelial cells of the bladder in the case of urogenital schistosomiasis and the bile duct epithelia or cholangiocytes, in the case of O. viverrini and C. sinensis. Indeed we postulate that helminth induced cancers originate from parasite estrogen-host epithelial/urothelial cell chromosomal DNA adducts, and here we review recent findings that support this conjecture.

Mass spectrometry techniques in the survey of steroid metabolites as potential disease biomarkers: A review

Mass spectrometric approaches have been fundamental to the identification of metabolites associated with steroid hormones, yet this topic has not been reviewed in depth in recent years. To this end, and given the increasing relevance of liquid chromatography-mass spectrometry (LC-MS) studies on steroid hormones and their metabolites, the present review addresses this subject. This review provides a timely summary of the use of various mass spectrometry-based analytical techniques during the evaluation of steroidal biomarkers in a range of human disease settings. The sensitivity and specificity of these technologies are clearly providing valuable new insights into breast cancer and cardiovascular disease. We aim to contribute to an enhanced understanding of steroid metabolism and how it can be profiled by LC-MS techniques.

New times, new trends for ethionamide: In vitro evaluation of drug-loaded thermally carbonized porous silicon microparticles

Multidrug-resistant tuberculosis (MDR-TB) has become a worldwide problem and a major public health concern. The mechanisms of resistance are fairly well characterized for most agents, but MDR limits the therapeutic usefulness of both new and classical medicines against TB. Ethionamide (ETA) is a thioamide antibiotic and one of the most widely used drugs as second line agent for the treatment of MDR-TB. Over the years, some studies have emerged to improve the bioavailability of this drug and of its active metabolites. However, inactive metabolites of ETA are still a major drawback in its application against TB. Porous silicon (PSi) materials can be applied to improve the dissolution behavior of poorly water-soluble compounds and to overcome toxicity and other drug-related problems in oral delivery. In the present work, we have loaded ETA into thermally carbonized-PSi (TCPSi) microparticles and studied the solubility, toxicity, permeability, and metabolic profiles of the PSi-loaded drug. The solubility and permeability of ETA was clearly enhanced after loaded into TCPSi particles at different pH-values. ETA was in general toxic at concentrations above 0.50mM to HepG2, Caco-2, and RAW macrophage cells, but the toxicity was drastically reduced when the drug was loaded into the microparticles. ETA showed a fast metabolization process in the presence of the TCPSi particles. In addition, new thiolated metabolites were identified from incubation of ETA-loaded PSi with HepG2 liver cells, which opens new perspectives toward both the understanding of ETA metabolism and the development of novel ETA-based systems with improved efficacy against MDR-TB.

Hydrogel depots for local co-delivery of osteoinductive peptides and mesenchymal stem cells

The outcome of cell-based therapies can benefit from carefully designed cell carriers. A multifunctional injectable vehicle for the co-delivery of human mesenchymal stem cells (hMSCs) and osteoinductive peptides is proposed, to specifically direct hMSCs osteogenic differentiation. The osteogenic growth peptide (OGP) inspired the design of two peptides, where the bioactive portion of OGP was flanked by a protease-sensitive linker, or its scrambled sequence, to provide faster and slower release rates, respectively. Peptides were fully characterized and chemically grafted to alginate. Both OGP analogs released bioactive fragments in vitro, at different kinetics, which stimulated hMSCs proliferation and osteogenesis. hMSCs-laden OGP-alginate hydrogels were tested at an ectopic site in a xenograft mouse model. After 4weeks, OGP-alginate hydrogels were more degraded and colonized by vascularized connective tissue than the control (without OGP). hMSCs were able to proliferate, migrate outward the hydrogels, produce endogenous extracellular matrix and mineralize it. Moreover, OGP-groups stimulated hMSCs osteogenesis, as compared with the control. Overall, the ability of the proposed platform to direct the fate of transplanted hMSCs in loco was demonstrated, and OGP-releasing hydrogels emerged as a potentially useful system to promote bone regeneration.

Tumour-like phenotypes in urothelial cells after exposure to antigens from eggs of Schistosoma haematobium: an oestrogen-DNA adducts mediated pathway?

Chronic infection with the blood fluke, Schistosoma haematobium, is associated with squamous cell carcinoma of the bladder. Previously, it has been shown that soluble extracts of mixed sex adult S. haematobium worms (SWAP) are tumourigenic, both in vitro and in vivo. In addition, oestrogen-related molecules in SWAP of S. haematobium down-regulate oestrogen receptors (ERs) alpha and beta in oestrogen responsive cells. Moreover, schistosome oestrogens occur in sera of persons with schistosomiasis haematobia and repress transcription of ERs in urothelial cells. Given that eggs of S. haematobium are the developmental stage directly responsible for urogenital disease during schistosomiasis haematobia, we suspected that soluble antigens from S. haematobium eggs exhibit similar or more potent tumorigenic capacity. Here we investigated the tumorigenic potential of soluble egg antigens (Sh-SEA) of S. haematobium and the endocrine system in favouring parasitism by schistosomes. The findings confirmed that 6.25μg/ml of Sh-SEA was enough to stimulate cell proliferation, reduce apoptosis and increase oxidative stress of Sh-SEA-exposed urothelial cells. In addition, genotoxic effects of Sh-SEA on these cells were determined by using alkaline single-cell gel electrophoresis (Comet). Furthermore, Liquid Chromatography Diode Array Detection Electron Spray Ionisation Mass Spectrometry indicated the presence of catechol-oestrogens in S. haematobium SEA. A prospective oestrogen-DNA adduct mediated pathway in S. haematobium egg induced bladder cancer is also discussed.

Schistosoma haematobium: Identification of new estrogenic molecules with estradiol antagonistic activity and ability to inactivate estrogen receptor in mammalian cells

We have previously identified the expression of an estradiol (E2)-related molecule by Schistosoma haematobium total antigen (Sh). We now show that this molecule has an antagonistic effect of estradiol in vitro. Our results are consistent with the existence of an estrogenic molecule that antagonizes the activity of estradiol. We found evidence for this molecule as we identified and characterized by mass spectrometry new estrogenic molecules previously unknown, present in schistosome worm extracts and sera of Schistosoma-infected individuals. We also show that Sh is able to interact in vitro with estrogen receptor (ER), explaining how host endocrine system can favor the establishment of schistosomes. These findings highlight the exploitation of the host endocrine system by schistosomes and represent an additional regulatory component of schistosome development that defines a novel paradigm enabling host-parasite interactions. The identification of these molecules opens new ways for the development of alternative drugs to treat schistosomiasis.

Metabolism of the Antituberculosis Drug Ethionamide

Ethionamide (ETH) is an important second-line antituberculosis drug used for the treatment of patients infected with multidrug-resistant Mycobacterium. Although ETH is a structural analogue of isoniazid (INH), both are pro-drugs that need to be activated by mycobacterial enzymes to exert their antimicrobial activity. ETH mechanism of action is thought to be identical to INH although the pathway of activation is distinct from that of INH. ETH is activated by an EthA enzyme, leading to the formation of an Soxide metabolite that has considerably better activity than the parent drug. This review comprehensively examines the aspects related with the metabolism of ETH since its discovery up to today.