Interfacial behaviour and quantitative analysis of hexadecyl phosphocholine drug at a polarized liquid/liquid interface.

The interfacial behaviour of the amphiphilic drug hexadecyl phosphocholine (HePC, also called miltefosine) was analysed by cyclic voltammetry applied at the water/1,2-dichloroethane interface. HePC is the only oral drug currently approved for the treatment of visceral, mucosal  and cutaneous leishmaniasis. Because of its amphiphilic character, it can interact with biological membranes, solubilizing their compounds and leading to cell disruption. These interactions are responsible for its side effects and toxicity; therefore, HePC quantification in biological fluids and pharmaceutical preparations is extremely important. However, the lack of a chromophore in its structure prevents its spectroscopic determination. For this reason, the main challenge of this work was to propose an electroanalytical method for the quantification of this drug, which constitutes a simpler alternative than liquid chromatography-tandem mass spectrometry already reported. With this aim, in the first part of this work, the mechanism of the electrochemical process occurring after polarizing the interface was studied. By varying the experimental conditions, it was possible to determine that in a first step, at open circuit or at low potential values, HePC spontaneously adsorbed to the interface. Later, as the potential increased, the transfer of the anions present in the organic phase towards the aqueous side of the interface, where the HePC polar head groups were present, occurred thus forming adsorbed "ion pairs" and producing an increase in positive current. Subsequently, in the negative sweep, the "ion pairs" dissociated and desorbed giving rise to a negative peak. In this way, both negative and positive currents were considered useful for quantitative purposes. In the second part of this work, an appropriate experimental procedure was designed and proposed as a quantitative methodology for the HePC determination, which consisted of cleaning the interface and controlling the time at open circuit, followed by the voltammetric analysis. A linear response of both, positive or negative, peak currents with drug concentration was obtained within an acceptable range, providing a simple solution for the HePC quantification problem. Future studies will be carried out to evaluate the quantification and selectivity in real matrices containing polymer micelles working as HePC nanocarriers with the aim of avoiding the adverse effects of HePC when it is orally or intravenously administered.

Deficiency of CD73 activity promotes protective cardiac immunity against Trypanosoma cruzi infection but permissive environment in visceral adipose tissue.

Damaged cells release the pro-inflammatory signal ATP, which is degraded by the ectonucleotidases CD39 and CD73 to the anti-inflammatory mediator adenosine (ADO). The balance between ATP/ADO is known to determine the outcome of inflammation/infection. However, modulation of the local immune response in different tissues due to changes in the balance of purinergic metabolites has yet to be investigated. Here, we explored the contribution of CD73-derived ADO on the acute immune response against Trypanosoma cruzi parasite, which invades and proliferates within different target tissues. Deficiency of CD73 activity led to an enhanced cardiac microbicidal immune response with an augmented frequency of macrophages with inflammatory phenotype and increased CD8+ T cell effector functions. The increment of local inducible nitric oxide (NO) synthase (iNOS)+ macrophages and the consequent rise of myocardial NO production in association with reduced ADO levels induced protection against T. cruzi infection as observed by the diminished cardiac parasite burden compared to their wild-type (WT) counterpart. Unexpectedly, parasitemia was substantially raised in CD73KO mice in comparison with WT mice, suggesting the existence of tissue reservoir/s outside myocardium. Indeed, CD73KO liver and visceral adipose tissue (VAT) showed increased parasite burden associated with a reduced ATP/ADO ratio and the lack of substantial microbicidal immune response. These data reveal that the purinergic system has a tissue-dependent impact on the host immune response against T. cruzi infection.

Extemporaneous Indomethacin Oral Suspension Prepared from Injectable Ampules for Therapy in Premature Infants and Pediatric Patients.

Indomethacin is used for off-label prescription for the treatment of patent ductus arteriosus in premature infants. In Argentina, indomethacin is only available as a suppository, dermic cream, injectable ampules, and delayed-release capsules. Aiming to improve pediatric treatment and minimize the risk associated with improper dosage, this work focused on the development of an extemporaneous 0.2% indomethacin oral suspension, starting from the commercially injectable formulation. Two 150-mL batches of suspension were prepared using Generally Recognized as Safe excipients. The suspensions were stored for 17 days at room temperature. Physical stability, morphological analysis of suspended particles, sedimentation volume, easy re-suspension, and dynamic viscosity were studied. The indomethacin content, dissolution studies, and microbiological attributes of nonsterile pharmaceutical products were also evaluated. After 17 days of storage, the suspension was easily re-dispersed after 15 seconds of the hand-shaking technique. There were no detectable changes in color, odor, and/or flavor. The suspension showed minimal changes in pH, viscosity, shape, and mean size of the suspended indomethacin particles. The content uniformity and drug dissolution remained within the acceptable range during storage. This oral liquid suspension is an interesting alternative to be prepared by hospital pharmacy services for optimizing the pediatric treatment of patent ductus arteriosus.

Preclinical pharmacokinetics of benznidazole-loaded interpolyelectrolyte complex-based delivery systems.

Benznidazole (BZ), first-line drug for Chagas treatment, is available as immediate-release tablets. High frequency of administration, long-term therapy, and side effects of BZ conspire against treatment adherence, and negatively impact in therapeutic success. We have developed BZ-loaded interpolyelectrolyte complexes (IPECs) composed of polymethacrylates (EE-EL-BZ) or polysaccharides (Ch-AA-BZ) for controlled BZ release. This work aimed to evaluate their preclinical pharmacokinetics compared to Abarax® (reference treatment) and to correlate them with the in vitro BZ release. A randomization schedule with a 3 × 2 cross-over design was used. Each healthy dog received a single oral dose of 100 mg of BZ from EE-EL-BZ, Ch-AA-BZ or Abarax®. BZ quantification was performed in plasma by a validated HPLC-UV method. Moreover, in silico simulations using the pharmacokinetic software PK Solutions 2.0™ were calculated for the multiple-dose administration at two dose regimens: 100 mg of BZ administered every 12 and 24 h. Also, the relationship between in vitro dissolution and in vivo plasma BZ concentration profiles in a single step was model for IVIVC analysis. BZ was rapidly absorbed from all formulations. The Cmax value for Ch-AA-BZ was 32% higher than reference (p < 0.05) and an earlier Tmax (4.2 h) was observed as compared to EE-EL-BZ (6.0 h). For both IPECs, the Tmax values were higher (p < 0.05) and the areas under the curve were 25% greater than those of Abarax® (p < 0.01). Despite these variations in pharmacokinetics parameters, simulations of once or twice daily dosing showed that all formulations reached a steady-state range concentration above of the minimum therapeutic dose while avoiding high BZ concentrations related to increased side effects. A linear level A IVIVC model was established using plasma concentration profiles and dissolved data obtained. Thus, BZ-loaded IPECs prolonged drug release and formulated as capsules showed improved in vivo performance, in terms of bioavailability and Tmax values, which were significantly higher compared to Abarax®. These BZ carrier systems would be useful for oral administration in the treatment of Chagas disease.

CD73 Inhibition Shifts Cardiac Macrophage Polarization toward a Microbicidal Phenotype and Ameliorates the Outcome of Experimental Chagas Cardiomyopathy.

Increasing evidence demonstrates that generation of extracellular adenosine from ATP, which is hydrolyzed by the CD39/CD73 enzyme pair, attenuates the inflammatory response and deactivates macrophage antimicrobial mechanisms. Although CD73 is emerging as a critical pathway and therapeutic target in cardiovascular disorders, the involvement of this ectonucleotidase during myocardial infection has not been explored. Using a murine model of infection with Trypanosoma cruzi, the causal agent of Chagas cardiomyopathy, we observed a sudden switch from the classical M1 macrophage (microbicidal) phenotype toward an alternative M2 (repairing/anti-inflammatory) phenotype that occurred within the myocardium very shortly after BALB/c mice infection. The observed shift in M1/M2 rate correlated with the cardiac cytokine milieu. Considering that parasite persistence within myocardium is a necessary and sufficient condition for the development of the chronic myocarditis, we hypothesized that CD73 activity may counteract cardiac macrophage microbicidal polarization, rendering the local immune response less effective. In fact, a transient treatment with a specific CD73 inhibitor (adenosine 5'-α,ß-methylene-diphosphate) enhanced the microbicidal M1 subset predominance, diminished IL-4- and IL-10-producing CD4(+) T cells, promoted a proinflammatory cytokine milieu, and reduced parasite load within the myocardium during the acute phase. As a direct consequence of these events, there was a reduction in serum levels of creatine kinase muscle-brain isoenzyme, a myocardial-specific injury marker, and an improvement in the electrocardiographic characteristics during the chronic phase. Our results demonstrate that this purinergic system drives the myocardial immune response postinfection and harbors a promising potential as a therapeutic target.

Clomipramine and Benznidazole Act Synergistically and Ameliorate the Outcome of Experimental Chagas Disease.

Chagas disease is an important public health problem in Latin America, and its treatment by chemotherapy with benznidazole (BZ) or nifurtimox remains unsatisfactory. In order to design new alternative strategies to improve the current etiological treatments, in the present work, we comprehensively evaluated the in vitro and in vivo anti-Trypanosoma cruzi effects of clomipramine (CMP) (a parasite-trypanothione reductase-specific inhibitor) combined with BZ. In vitro studies, carried out using a checkerboard technique on trypomastigotes (T. cruzi strain Tulahuen), revealed a combination index (CI) of 0.375, indicative of a synergistic effect of the drug combination. This result was correlated with the data obtained in infected BALB/c mice. We observed that during the acute phase (15 days postinfection [dpi]), BZ at 25 mg/kg of body weight/day alone decreased the levels of parasitemia compared with those of the control group, but when BZ was administered with CMP, the drug combination completely suppressed the parasitemia due to the observed synergistic effect. Furthermore, in the chronic phase (90 dpi), mice treated with both drugs showed less heart damage as assessed by the histopathological analysis, index of myocardial inflammation, and levels of heart injury biochemical markers than mice treated with BZ alone at the reference dose (100 mg/kg/day). Collectively, these data support the notion that CMP combined with low doses of BZ diminishes cardiac damage and inflammation during the chronic phase of cardiomyopathy. The synergistic activity of BZ-CMP clearly suggests a potential drug combination for Chagas disease treatment, which would allow a reduction of the effective dose of BZ and an increase in therapeutic safety.