Synthesis, physicochemical properties of allopurinol derivatives and their biological activity against Trypanosoma cruzi.

Chagas disease is caused by Trypanosoma cruzi (T. cruzi) leading to a huge number of infections and deaths per year, because in addition to many sufferers only having limited access to health services only an inefficient chemotherapy is available using drugs such as benznidazole and nifurtimox. Here, C6-alkyl (2a-c) and N1-acyl (3a-c) derivatives of Allopurinol (Allop, compound with activity against T. cruzi) were synthesized in good yields and their structures were unambiguously characterized. Only 2a, 2b and 3c showed inhibitory activity against the proliferative stages of the parasite when tested at 1 µg mL(-1) with the 3c derivative exhibiting an IC50 value similar to that of Allop and not being toxic for mammalian cells. Relevant pharmaceutical physicochemical properties (pKa, stability, solubility, lipophilicity) were also determined as well by using Lipinski's rule, polar surface area and molecular rigidity. Taken together, the results demonstrated that the studied derivatives had optimal properties for bioavailability and oral absorption. For the stability studies, Micellar Liquid Chromatography was used as the analytical method which was fully validated according to the FDA guidelines and shown to be a suitable, sensitive and simple method for routine analysis of these Allop derivatives.

An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool samples.

Strongyloides stercoralis is a nematode that causes severe infections in immunocompromised patients. The low parasitic burden of chronically infected patients makes diagnosis difficult to achieve by conventional methods. Here, an in-house (IH) method for the isolation of parasite DNA from stools and a PCR assay for the molecular diagnosis of S. stercoralis were optimized. DNA yield and purity improved with the IH method which included a step of incubation of stool samples with a glycine-SDS buffer and mechanical disruption prior to DNA extraction. For the PCR assay, the addition of bovine serum albumin was required to neutralize inhibitors present in stool. The analytical sensitivity of the PCR using DNA as template, isolated with the IH method, was superior to the commercial one. This study demonstrates that a combined method that adds the step of glycine-SDS buffer incubation plus mechanical disruption prior to DNA isolation with the commercial kit increased PCR sensitivity to levels of the IH method. Finally, our assay was tested on 17 clinical samples. With the IH method for DNA isolation, a S. stercoralis specific band was detected by PCR in the first stool sample in all patients (17/17), while with the commercial kit, our S. stercoralis-specific band was only observed in 7 samples. The superior efficiency of the IH and combined methods over the commercial kit was demonstrated when applied to clinical samples with low parasitic burden. These results show that the DNA extraction procedure is a key to increase sensitivity of the S. stercoralis PCR assay in stool samples. The method developed here could help to improve the molecular diagnosis of S. stercoralis.

Reduction of parasite levels in blood improves pregnancy outcome during experimental Trypanosoma cruzi infection.

Infection with a myotropic Trypanosoma cruzi clone induces maternal fertility failure. In the current work, we evaluated whether reduction of maternal parasitaemia before mating has beneficial effects on pregnancy outcome. Female mice were subjected to benznidazole (Bz) treatment after infection. On day 30 of therapy, mating was assessed and pregnancy outcome was determined on day 14 of gestation. Fetal resorptions diminished in T. cruzi-infected Bz-treated mice compared with T. cruzi-infected untreated mice. This was in agreement with the reduction in the blood/solid tissue parasite load and with the percentage of necrotic foci in placental samples from T. cruzi-infected Bz-treated females. To study eventual changes in the immune homeostasis of T. cruzi-infected Bz-treated mice, activation of the immune system was evaluated at the end of Bz therapy and before mating. We found specific IgG1 reduction resulting in a predominance of specific IgG2a, reduced numbers of CD69+ CD4+ cells and diminished frequency and numbers of CD44+ T cells. Concanavalin A-stimulated splenocytes from T. cruzi-infected Bz-treated mice produced higher amounts of IFN-gamma than T. cruzi-infected untreated mice. These results indicate that reduction of maternal parasite load improves pregnancy outcome. These findings correlate with a favourable modulation of the immune response.