HIV latency-reversing activity of latex from Euphorbia umbellata (Pax) Bruyns and three diterpenes isolated from this species.

Two unusual phorbol esters, namely 20-deoxyphorbol-3,4,12-triacetate-13-phenylacetate (1) and phorbol-3,4,12,13-tetraacetate-20-phenylacetate (2) plus ingol-3,8,12-triacetate-7-phenylacetate (3) were isolated from the latex of Euphorbia umbellata and identified by HRESIMS and 2D NMR. Compound 1 is herein described for the first time. Assignment of the phenylacetyl group at C-7 in compound 3 was suggested by the HMBC and NOESY spectra obtained in pyridine-d5. In addition to the latex and its distinct terpenoid fractions, the isolated compounds were tested as latent reversal agents against HIV-1-infected J-Lat cells, with reference to phorbol-12-myristate-13-acetate and ingenol-B. Compound 2 reverted 75-80% the viral latency on the GFP-positive cells, resulting EC50 3.70 µg/mL (SI 6.7), while 1 induced 34-40% reactivation at the same concentration range (4-20 µg/mL). The ingol derivative 3 was ineffective. Phorbol esters were confirmed as effective constituents in the latex since the fraction containing them was 2.4-fold more active than the lyophilised latex at the lowest concentration assayed.

Development and Characterization of PLGA Nanoparticles Containing 17-DMAG, an Hsp90 Inhibitor.

Leishmaniasis is a spectrum of neglected tropical diseases and its cutaneous form (CL) is characterized by papillary or ulcerated skin lesions that negatively impact patients' quality of life. Current CL treatments suffer limitations, such as severe side effects and high cost, making the search for new therapeutic alternatives an imperative. In this context, heat shock protein 90 (Hsp90) could present a novel therapeutic target, as evidence suggests that Hsp90 inhibitors, such as 17-Dimethylaminoethylamino-17-Demethoxygeldanamycin (17-DMAG), may represent promising chemotherapeutic agents against CL. As innovative input for formulation development of 17-DMAG, nano-based drug delivery systems could provide controlled release, targeting properties, and reduced drug toxicity. In this work, a double emulsion method was used to develop poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing 17-DMAG. The nanoparticle was developed using two distinct protocols: Protocol 1 (P1) and Protocol 2 (P2), which differed concerning the organic solvent (acetone or dichloromethane, respectively) and procedure used to form double-emulsions (Ultra-Turrax® homogenization or sonication, respectively). The nanoparticles produced by P2 were comparatively smaller (305.5 vs. 489.0 nm) and more homogeneous polydispersion index (PdI) (0.129 vs. 0.33) than the ones made by P1. Afterward, the P2 was optimized and the best composition consisted of 2 mg of 17-DMAG, 100 mg of PLGA, 5% of polyethylene glycol (PEG 8000), 1.5 mL of the internal aqueous phase, 1% of polyvinyl alcohol (PVA), and 4 mL of the organic phase. Optimized P2 nanoparticles had a particle size of 297.2 nm (288.6-304.1) and encapsulation efficacy of 19.35% (15.42-42.18) by the supernatant method and 31.60% (19.9-48.79) by the filter/column method. Release kinetics performed at 37°C indicated that ~16% of the encapsulated 17-DMAG was released about to 72 h. In a separate set of experiments, a cell uptake assay employing confocal fluorescence microscopy revealed the internalization by macrophages of P2-optimized rhodamine B labeled nanoparticles at 30 min, 1, 2, 4, 6, 24, 48, and 72 h. Collectively, our results indicate the superior performance of P2 concerning the parameters used to assess nanoparticle development. Therefore, these findings warrant further research to evaluate optimized 17-DMAG-loaded nanoparticles (NP2-17-DMAG) for toxicity and antileishmanial effects in vitro and in vivo.

Challenging identification of polymorphic mixture: Polymorphs I, II and III in olanzapine raw materials.

Olanzapine (OLZ), a drug for the treatment of schizophrenia, presents in more than 60 crystal forms. Polymorphs I, II and III were reported, however, the preparation conditions for pure II and III have not been reported. Polymorph IV was reported but this form is actually polymorph II described at different temperature. The diversity of solid forms of OLZ, the change in the nomenclature found in the literature and the presence of polymorphic mixture in samples, increase the difficulty for a correct solid state characterization. Therefore, the goal was the polymorphic identification of three OLZ raw materials, highlighting the limitation of conventional techniques (typically used in analytical control) and the necessity to use a combination of advanced ones to solve this challenge. The samples were studied by conventional techniques such as powder X-ray diffraction, thermoanalytical techniques, infrared spectroscopy. In apart from that, synchrotron powder X-ray diffraction (SPXRD) and solid state nuclear magnetic resonance (ss-NMR) were used. All samples were in accordance with the pharmacopoeia criteria. However, the conventional techniques were not specific for the complete polymorphic identification. Therefore, a combination of advanced techniques (SPXRD and ss-NMR) was necessary to identify the mixture of polymorphs (I, II and III) in all samples.

Vibrational spectroscopic and Hirshfeld surface analysis of carvedilol crystal forms.

The drug carvedilol, used to treat cardiovascular conditions, is known to exist in distinct crystalline forms. Polymorphs II and III and the hydrate are characterized by variations in their molecular packing and conformation. This study deals with the spectroscopic (supported by quantum chemistry calculations) characterization of carvedilol structures. Band assignments were performed considering the isolated molecules and periodic calculations. We discuss the correlation between the vibrational modes and the intermolecular forces in the crystalline structures. Towards a better understanding of the intermolecular interactions, Hirshfeld surface was used. Besides band shifts related to stretching vibrations of N-H and O-H groups, differences between other modes have shown the possibility of using infrared spectroscopy to distinguish the crystal forms; technique routinely used in quality control of pharmaceutics. According to the spectroscopic analysis, the N-H groups participate in stronger bonds in the polymorph III, which contributes to its greater stability. With Hirshfeld surface we concluded that the bond with the nitrogen of the aliphatic chain participating as hydrogen acceptor in polymorph II is responsible for the pointy peaks in the fingerprint plot. This can explain why polymorph II shows lower dissolution in acid medium, as described in a previous work of our group.

Efavirenz dissolution enhancement III: Colloid milling, pharmacokinetics and electronic tongue evaluation.

Efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor (NNRTI), is part of first-line therapy for the treatment of human immunodeficiency virus type 1 infection (HIV-1/AIDS). This drug shows relatively low oral absorption and bioavailability, as well as high intra- and inter-subject variability. Several studies have shown that treatment failure and adverse effects are associated with low and high EFV plasma concentrations, respectively. Some studies suggest different EFV formulations to minimize inter-patient variability and improve its solubility and dissolution; however, all of these formulations are complex, using for instance, cyclodextrins, dendrimers and polymeric nanoparticles, rendering them inviable industrially. The aim of this work was to prepare simple and low-cost suspensions of EFV for improvement of solubility and dissolution rate by using colloid mill, spray or freeze-drying, and characterization of the powders obtained. The results demonstrated an increase in the dissolution rate of EFV, using 0.2% of sodium lauryl sulfate (SLS) and 0.2% of hydroxypropylcellulose (HPC) or hydroxypropylmetilcellulose (HPMC) in both freeze and spray dried powders. The pharmacokinetic studies demonstrated improved pharmacokinetic parameters for the formulation containing SLS and HPC. The powders obtained, which present enhanced dissolution properties, can be incorporated in a solid dosage form for treatment of AIDS in paediatric patients with promising results.