First steps in the formulation of praziquantel nanosuspensions for pharmaceutical applications.

Praziquantel (PZQ), a broad spectrum anthelmintic drug, cannot be found in acceptable dosage forms for elderly patients, paediatric patients, and for veterinary use. In fact, very little has been done up to now in the formulation of liquid dosage forms, being they always formulated for parenteral administration. To beat this important challenge, it was accomplished a comprehensive analysis of the influence of two elementary physicochemical aspects, i.e. surface thermodynamic and electrokinetic properties, on the colloidal stability of PZQ nanosuspensions. The hydrophobic character of the drug, intensely determining the flocculation curves, was confirmed by the thermodynamic characterization. The electrophoretic characterization, in combination with the sedimentation and relative absorbance versus time curves, highlighted that the electrical double layer thickness and the surface charge can play an essential role in the stability of the pharmaceutical colloid. Finally, it was demonstrated that controlling the pH values and the incorporation of electrolytes can help in formulating PZQ aqueous nanosuspensions with appropriate stability and redispersibility behaviours for pharmaceutical use.

Primeros estudios de preformulación en el desarrollo de una suspensión farmacéutica de micropartículas de Praziquantel destinada a la vía de administración oral / First preformulation studies in the development of a pharmaceutical suspension of Praziquantel microparticles destined to the oral route of drug administration

Introducción: Uno de los fármacos de primera línea en el tratamiento de la esquistosomiasis es el Praziquantel. Numerosas son las formas farmacéuticas sólidas orales desarrolladas hasta la fecha, siendo éstas poco adecuadas para determinados grupos de población, ej. tercera edad y Pediatría, y Veterinaria. Este trabajo describe los primeros pasos en el desarrollo de un estudio de preformulación dirigido al diseño de una forma farmacéutica líquida de administración oral para este principio activo. Método: Se caracterizó la forma y tamaño de las partículas de Praziquantel con las que se pretendía preparar una suspensión acuosa, mediante microscopía electrónica de barrido. Además, se analizó el efecto que el pH y el tipo de electrolito y su concentración tenían sobre el comportamiento de las suspensiones formuladas, gracias a medidas de electroforesis (potencial zeta) y espectrofotometría ultravioleta-visible (turbidimetría en función del tiempo). Resultados: La población de partículas de fármaco se caracterizó por una forma acicular y un tamaño micrométrico, con una distribución de tamaños heterogénea. Se comprobó cómo controlando la composición del medio de dispersión, en términos de pH y electrolitos, podía definirse la carga eléctrica superficial de las partículas de fármaco y, así su proceso de sedimentación, obteniéndose el sistema más adecuado para la vía de administración oral (sistema floculado). Conclusiones: Se han definido las condiciones iniciales de formulación de suspensiones acuosas de Praziquantel destinadas a la vía oral. Un control adecuado de la composición de la fase externa resulta fundamental en el establecimiento del mejor sistema (floculado) para esta vía de administración

Introduction: One of the first-line drugs against schistosomiasis is Praziquantel. Up to now, numerous oral solid dosage forms have been developed, being they considered of little help to elder patients, pediatrics, and Veterinary. Initial steps in the development of preformulation studies aiming the design of a Praziquantel liquid pharmaceutical dosage form to be administered orally are described. Method: Size and shape of Praziquantel particles were characterized by scanning electron microscopy. Furthermore, it was investigated the effect of pH and type of electrolyte and its concentration in the aqueous dispersion media on the behaviour of the suspensions. To that aim, electrokinetic determinations (zeta potential) and ultraviolet-visible spectrophotometry measurements (turbidimetry as a function of time) were done. Results: Drug particles were characterized by an acicular shape and a micrometer size (heterogeneous size distribution). It was observed that controlling the composition of the aqueous dispersion media, in terms of pH and electrolytes, helped in defining the surface electrical charge of the drug particles and, thus the sedimentation profile, obtaining the more adequate system for the oral route of drug administration (flocculated system). Conclusions: Initial conditions to formulate aqueous Praziquantel suspensions for the oral route have been defined. An appropriate control of the composition of the external phase of the suspension is a key aspect when establishing the best liquid pharmaceutical system (flocculated) for this administration route

Physicochemical Properties and Anti-Opisthorchosis Effect of Mechanochemically Synthesized Solid Compositions of Praziquantel with Glycyrrhizic Acid Disodium Salt.

The mechanochemical preparation of solid compositions of praziquantel with plant saponin (glycyrrhizic acid disodium salt) is described. The study of a number of physicochemical parameters showed that dissolving solid compositions in water is accompanied by the inclusion of praziquantel molecules into micelles, which are formed in the solution of the glycyrrhizic acid disodium salt. Using the opisthorchiasis model caused by Opisthorchis felineus, we found a 4- to 11-fold increase in the anthelmintic activity of praziquantel in the composition as compared to the official praziquantel. According to the pharmacokinetic data, the use of the composition increased the bioavailability of praziquantel 3 times.

Bimodal X-ray and Infrared Imaging of an Organometallic Derivative of Praziquantel in Schistosoma mansoni.

An organometallic derivative of praziquantel was studied directly in worms by using inductively coupled plasma-mass spectrometry (ICP-MS) for quantification and synchrotron-based imaging. X-ray fluorescence (XRF) and IR absorption spectromicroscopy were used for the first time in combination to directly locate this organometallic drug candidate in schistosomes. The detection of both CO (IR) and Cr (XRF) signatures proved that the Cr(CO)3 core remained intact in the worms. Images showed a preferential accumulation at the worm's tegument, consistent with a possible targeting of the calcium channel but not excluding other biological targets inside the worm.

Development of chiral praziquantel analogues as potential drug candidates with activity to juvenile Schistosoma japonicum.

A series of chiral praziquantel analogues were synthesized and evaluated against Schistosoma japonicum both in vitro and in vivo. All compounds exhibited low to considerable good activity in vivo. Remarkably, worm reduction rate of R-3 was 60.0% at a single oral dose of 200mg/kg against juvenile stage of Schistosoma japonicum. The target compounds displayed in vivo antischistosomal activity against both Schistosoma japonicum and Schistosoma mansoni. Furthermore, all R-isomers displayed stronger antischistosomal activity than S-isomers in vivo, indicating R-isomers were the active enantiomers, while S-isomers were less active ones. This structure activity relationship (SAR) could have important implications in further drug development for schistosomiasis.

New praziquantel derivatives containing NO-donor furoxans and related furazans as active agents against Schistosoma mansoni.

A series of NO-donor praziquantel hybrid compounds was obtained by combining praziquantel (PZQ) and furoxan moieties in a single entity. NO-donor properties of the furoxan derivatives were evaluated by detecting nitrite after incubation of the products in 7.4 pH buffered solution in the presence of L-cysteine. Structurally-related furazans, devoid of NO release capacity, were also synthesized for control purposes. All products were studied for their ability to inhibit recombinant Schistosoma mansoni thioredoxin glutathione reductase (TGR). Mobility and death of adult Schistosoma mansoni worms cultured in the presence of the products were evaluated versus PZQ. Analysis of the results showed that some products were endowed with both PZQ and NO-dependent antiparasitic properties. Compounds 6, 7, 18, and 24 emerged as the most interesting balanced hybrids, worthy of additional study on PZQ-resistant parasites.

Design and synthesis of molecular probes for the determination of the target of the anthelmintic drug praziquantel.

Schistosomiasis is a highly prevalent neglected tropical disease caused by blood-dwelling helminths of the genus Schistosoma. Praziquantel (PZQ) is the only drug available widely for the treatment of this disease and is administered in racemic form, even though only the (R)-isomer has significant anthelmintic activity. Progress towards the development of a second generation of anthelmintics is hampered by a lack of understanding of the mechanism of action of PZQ. In this Letter, we report an efficient protocol for the small-scale separation of enantiomers of 2 (hydrolyzed PZQ) using supercritical fluid chromatography (SFC). The enantiopure 2 was then used to develop several molecular probes, which can potentially be used to help identify the protein target of PZQ and study its mode of action.

Synthesis and SAR studies of praziquantel derivatives with activity against Schistosoma japonicum.

The synthesis and structure-activity relationship (SAR) studies of praziquantel derivatives with activity against adult Schistosoma japonicum are described. Several of them showed better worm killing activity than praziquantel and could serve as leads for further optimization.

Synthesis of fluorescent derivatives of praziquantel: cell-imaging and interaction with Schistosoma japonicum cercariae.

Schistosomiasis is one of the most burdensome of the neglected tropical diseases. Praziquantel is a recommended drug for treatment against all forms of schistosomiasis. To investigate the interaction between praziquantel and Schistosoma japonicum cercariae, two praziquantel derivatives (PZQ-2 and PZQ-3) and one praziquantel fluorescent derivative (PZQ-5) have been synthesized and characterized using nuclear magnetic resonance spectroscopy (NMR) and MS spectra. The cytotoxicity of PZQ-2, PZQ-3 and PZQ-5 was measured by performing the methyl thiazolyl tetrazolium (MTT) assay. The cell viability for them shows that the three compounds exhibit low cytotoxicity to HeLa cells. Cell imaging experiments demonstrate that PZQ-5 is biocompatible and cell-permeable, which indicates that PZQ-5 is suitable for studying their interaction. Confocal fluorescence microscopy revealed that PZQ-5 is mainly located at the cercarial tegument, which leads to the death of cercariae with the increase in time.

Enhancing the usefulness of cross dehydrogenative coupling reactions with a removable protecting group.

A removable protecting group has been identified that allows the products of widely-used cross dehydrogenative couplings to be synthetically elaborated. The method can be used with enantiopure amines with no loss of enantiomeric excess. The methodology is exemplified by a new synthesis of enantiopure praziquantel, the drug used in the treatment of millions of people suffering from the neglected tropical disease, schistosomiasis.

Ferrocenyl derivatives of the anthelmintic praziquantel: design, synthesis, and biological evaluation.

The design, synthesis, and biological evaluation of 18 ferrocenyl derivatives (4A-12A and 4B-12B) of the most well-known drug against schistosomiasis, namely praziquantel (PZQ), are reported. These compounds, which have been all isolated as racemates, were unambiguously characterized by ¹H and ¹³C NMR spectroscopy, mass spectrometry, and elemental analysis as well as by X-ray crystallography for 4A, 5A, and 7A. Cytotoxicity studies revealed that the complexes were moderately toxic toward a cervical cancer cell line (HeLa) and, importantly, significantly less active toward a noncancerous cell line (MRC-5). The in vitro anthelmintic activity of the 18 ferrocenyl PZQ derivatives was tested against adult Schistosoma mansoni, and values in the micromolar range (26-68 µM) were determined for the four most active compounds. It was also demonstrated using two compounds of the series as models (8A and 8B) that the complexes were stable when incubated for 24 h at 37 °C in human plasma.

Praziquantel derivatives exhibit activity against both juvenile and adult Schistosoma japonicum.

A praziquantel analog 10-hydroxy praziquantel and eight praziquantel/peroxide conjugates were synthesized. The biological activity of these compounds was evaluated against juvenile and adult stages of Schistosoma japonicum. Unlike praziquantel, 10-hydroxy praziquantel exhibits activity against both juvenile and adult Schistosoma japonicumin. All hybrid compounds displayed modest to significant worm killing activity. The present study has important significance for the development of hybrid antischistosomal drugs.

Synthesis of new praziquantel analogues: potential candidates for the treatment of schistosomiasis.

An efficient synthesis of antischistosomal drug praziquantel and analogues was achieved and the synthetic route designed was to afford structurally diverse analogues for better structure-activity relationship understanding. Total of nineteen PZQ analogues with structural variations at amide, piperazine and aromatic moieties have been synthesized and fully characterized. Among all the new analogues tested for antischistosomal activity, one dimethoxy tetrahydroisoquinoline analogue and two tetrahydro-ß-carboline analogues exhibited moderate activity against adult Schistosoma mansoni. Tetrahydro-ß-carboline analogues showed moderate activity whereas the presence of p-trifluoromethylbenzoyl and p-toluenesulphonyl moieties resulted in complete suppression of antischistosomal activity.

MCR synthesis of praziquantel derivatives.

Schistosomiasis, a high volume neglected tropical disease affecting more than 200 million people worldwide, can only be effectively treated by the tetrahydroisoquinoline drug praziquantel (PZQ). Herein, we describe an efficient approach to access PZQ derivatives by the Ugi 4-component reaction followed by the Pictet-Spengler reaction in a two-step, one-pot procedure. 30 novel PZQ derivatives are described based on the Ugi 4-component reaction and an X-ray structure of a novel derivative revealing different conformation compared with PZQ is discussed. Several analogues comparable in activity to the drug PZQ have been identified based on an in vitro Schistosoma mansoni worm viability assay.

Macromolecular bases of antischistosomal therapy.

Schistosomiasis is a widespread tropical parasitic disease, currently treated with Praziquantel, whose precise molecular target is actually unknown. Several other drugs are known to kill the schistosomes in vivo and in vitro, but these are seldom employed because of toxicity, high cost, complex administration or other reasons. The improvement of known drugs or the development of entirely new ones is a desirable goal, in view of the fact that strains of Schistosoma mansoni with reduced sensitivity to Praziquantel have appeared. In this review, we tried to collect the information available on known or putative macromolecular targets of schistosomicidal drugs; thus we focused on the biochemistry of the parasite, rather than the clinical properties of the drugs. The rationale of this approach is that drug design may become realistic if the mechanism of action of each known drug were known at atomic detail, ideally as the 3D structure of each drug in complex with its target. Important macromolecular targets of known drugs reviewed below are: Thioredoxin Glutathione Reductase; Cyclophilin; Acetyl Cholinesterase; Proteases and Purine Nucleoside Phosphorylase. Moreover, a few enzymes of the parasite are known, or thought, to be "druggable", and therefore interesting, even though no specific drugs are available as yet: examples of such enzymes are Glutathione Peroxidase and Peroxiredoxins.

Amino acid derived heterocycles: lewis acid catalyzed and radical cyclizations from peptide acetals.

Bicyclization of peptide acetals via nucleophilic attack of a phenyl group on an endocyclic acyliminium ion 4 was explored as a route to novel amino acid derived heterocycles and peptidomimetic scaffolds. In the presence of protic acid, bridged structures such as 6 are formed readily from phenylalanine derivatives, but the fused-ring analogues 5 could not be obtained in good yield. In contrast, radical cyclization of the bromophenyl dihydropyrazinone 7 provides an effective alternative for the synthesis of 5 (n = 0, 1, 2). Additional versatility in this process was demonstrated by efficient synthesis of a different fused ring system, represented by the antihelmintic praziquantel, 8.

Synthetic and bioorganic investigations of 2-cyclohexylthiocarbonyl( 1,2,3,6,7,11B)hexahydro-4H-pyrazino [2-1a] isoquinoline-4-thione, a new dithione mimic of the universal anthelminthic Praziquantel.

As a continuation for our previous approaches to establish structure-antischistosomal activity relationship (SAR) among some new rationally synthesized analogues of praziquantel, herein a new C-4 and C-12 dithione mimic of the drug namely, 2-cyclohexylthiocarbonyl (1, 2, 3, 6, 7, 11b) hexahydro-4H-pyrazino[2-la]isoquinoline-4-thione (II) was synthesized and antischistosomally investigated (mice infected with S. masoni cercariae). Further, some significant biochemical and toxicological parameters for both the control and the dithione II treated mice, particularly the total serum and liver proteins, liver enzymes, serum total lipids, cholesterol, triglycerides, albumin, globulins and creatinine, were assayed. The determined induced amino acid profile of liver protein hydrolysate could indicate a close similarity of the working biological mechanism for both I and II. Comparable to praziquantel, the dithione II was found, still promisingly antischistosomally active (approximately 70% of I, collective average activity, based on 500 mg II/kg mouse body weight). Equally, generally tolerant toxicity parameters for liver and kidney functions could be attributed. Due to the still absence of quasi-potent praziquantel candidates since its discovery (1975), the dithione II could be considered as an interesting anthelminthic candidate susceptible for further profound studies and structure modulations. In this context, some perspectives were also suggested.