The development of nanoformulations aims to overcome the biopharmaceutical limitations associated with conventional drug delivery. Reducing the particle size to the nanometric scale enhances drug solubility, dissolution rate, and bioavailability. In this study, the development and quality control of ritonavir nanocrystals are described by using applied experimental milling approaches. Ritonavir nanosuspensions were initially prepared at a small scale using an Ultra-Turrax Tube Drive, in which 200 and 500 μm beads were identified as the most efficient for particle size reduction. The process was then successfully scaled up by using a bead mill, achieving particle sizes of approximately 300 nm within 30 min, followed by spray-drying. Solid-state characterization by XRD, TGA, DSC, and hot-stage microscopy confirmed that the nanocrystals retained Form II ritonavir throughout processing. The resulting nanocrystals were physically stable and exhibited a marked improvement in dissolution, particularly in a discriminative dissolution medium (0.04 M POE10LE). Process optimization was achieved by balancing bead sizes and steric stabilizers, such as PVP K30 and HPMC. Biodistribution studies using [99mTc] radiolabeling (labeling efficiency >90%) showed uptake in the liver, kidneys, and intestines, with notable differences in the cardiac distribution between nanocrystals and nanosuspensions. Pharmacokinetic analysis indicated similar overall distribution profiles, with a transient 4 h peak in blood levels for the nanocrystals. Biochemical analyses suggested formulation-dependent hepatic stress, reflected by increased GGT for the nanosuspension, and alterations in carbohydrate metabolism, including elevated glucose and amylase levels, for the nanocrystal formulation. Overall, ritonavir nanocrystals significantly improved dissolution, and the optimized milling and spray-drying approach represents a robust and scalable strategy to enhance the performance of Class II or IV drugs.

The COVID-19 pandemic underscored the urgent need for advanced drug delivery systems to enhance the safety and efficacy of existing antiviral therapies. This study presents an inhalable powder formulation of remdesivir (RDV) using polymeric nano-in-microparticles for pulmonary administration. RDV was nanoencapsulated in a polycaprolactone (PCL) matrix via emulsion-diffusion-solvent evaporation and stabilized with DPPC and Pluronic F127, resulting in nanoparticles (RDV-PCL-NP) of 184 +/- 11 nm and 87% encapsulation efficiency. Cytotoxicity assays in Vero E6 cells confirmed the RDV-PCL-NP safety at therapeutic concentrations, with a marked reduction in the SARS-CoV-2 viral load at 5 mu M RDV. The nanoparticles were spray dried with lactose, yielding a dry powder (RDV-PCL-MP) with 63% process yield. Physicochemical characterization (SEM, FTIR, DRX, DSC/TGA, laser diffraction) confirmed uniform particle size and stability (1-5 mu m) of the RDV-PCL-MP inhalable powder. In vitro lung deposition studies showed 40% fine fraction and 39% respirable fraction. These findings support the potential of RDV-loaded nano-in-microparticles as a scalable pulmonary delivery platform to improve COVID-19 treatment.

The primary objective of the current study is to establish and validate for the first time a method to determine and quantify praziquantel (PZQ) and its main degradation products loaded in poly(methyl methacrylate-co-2-(diethylamino)ethyl methacrylate) P(MMA-co-DEAEMA) microparticles. A high-performance liquid chromatography (HPLC) approach was developed and validated in accordance with the United States Pharmacopeia (USP) guidelines, addressing parameters such as accuracy, linearity, solution stability, precision, specificity, robustness, sensitivity, and system suitability. The method employed a gradient mobile phase consisting of ultrapure water and acetonitrile, flowing at a rate of 1 mL/minute over a Phenomenex Kinetex (R) C18 column (5 & micro;m, 100 & Aring;, 250 & times; 4.6 mm) maintained at 35 degrees C. Detection was performed at the wavelength of 210 nm using a DAD/UV detector. Samples of the active pharmaceutical ingredient (API) praziquantel, microencapsulated praziquantel, placebo, and a mixture of related substances (A, B, and C) were prepared with 0.5% formic acid in water/ethanol, 45:55 v/v as the diluent, and injected at 20 degrees C. The method demonstrated a limit of quantification (LOQ) of 0.20 & micro;g/mL for praziquantel and related substances. The method exhibited an excellent linear response, with all correlation coefficients (R-2) values exceeding 0.998, which is well above the recommended specified limit of R-2 > 0.995. Percent recoveries fell within the acceptable range of (95.0-105.0%), and all results indicated a percentage of relative standard deviation (%RSD) <= 2.0, indicating a robust methodology. Thus, the proposed HPLC technique proved to be selective, accurate, sensitive, and consistent in analyzing both the material content and its main degradation products.

BACKGROUND/OBJECTIVES:Cutaneous leishmaniasis (CL) remains a global health challenge, with treatment options often limited by drug resistance and systemic toxicity. Amphotericin B (AmB) represents a promising alternative. but intravenous administration causes severe systemic adverse effects. Despite growing interest in topical therapies, knowledge gaps remain regarding the comparative efficacy of delivery systems, including the influence of treatment timing and potential intrinsic effects. This study aimed to develop and characterize different topical AmB formulations (polymeric nanoparticles (PCL-AmB), a lipid-based (Oil_AmB) formulation, and a gel emulsion) to evaluate their in vivo efficacy against CL in a murine model, considering treatment initiation timing and potential intrinsic effects of the delivery systems. METHODS:Formulations were prepared and characterized in terms of hydrodynamic size, polydispersity index, and AmB content. Antileishmanial activity was assessed in two independent in vivo experiments, with topical monotherapy administered five days per week for four weeks, starting either 10 or 30 days post-infection, representing early and established chronic stages of infection, respectively. RESULTS:All formulations exhibited nanoscale dimensions and high homogeneity, with the lipid system demonstrating superior AmB solubilization. Both PCL-AmB and Oil_AmB reduced parasite load in the footpad, with Oil_AmB also reducing parasite load in draining lymph nodes. CONCLUSIONS:PCL-AmB and Oil_AmB reduced lesions and parasite burden in L. amazonensis-infected mice. Treatment timing was critical, with early Oil_AmB also reducing parasite loads in draining lymph nodes. These findings suggest that topical AmB formulations may provide a promising alternative for CL treatment, though further studies are required to optimize efficacy and administration schedules.

This study aims to develop and characterize poly (lactic-co-glycolic acid) (PLGA) nanoparticles decorated with chitosan (CS) for the encapsulation of dexamethasone (DEX) (NP-DEX-CS), targeting improved efficacy in the treatment of severe acute respiratory syndrome (SARS) associated with COVID-19. The nanoparticles were systematically characterized for size, zeta potential (ZP), morphology, encapsulation efficiency, and in vitro drug release. Incorporation of CS resulted in significant modifications in the nanoparticles' physical properties, notably an increase in size (from 207.3 +/- 6.7 nm to 264.4 +/- 4.4 nm) and a shift in ZP to positive values (from-11.8 +/- 1.4 mV to +30.0 +/- 1,6 mV). The NP-DEX-CS formulation achieved a high encapsulation efficiency (similar to 79 %) and a drug loading capacity of 6.53 +/- 0.02 %.In addition, the in vitro release rate of DEX from NP-DEX-CS was lower compared to undecorated nanoparticles, with a reduction from approximately 64-37 % within 24 h. Microscopy analyses revealed a smoother surface on the CS-decorated nanoparticles. FTIR and XRD analyses confirmed successful chitosan coating and DEX encapsulation. The CS coating enhanced the tolerability of J774.A1 cells to the nanoparticles, particularly evident at the highest concentration (400ug/mL), resulting in a cell viability >= 70 %. Importantly, the NP-DEX-CS significantly reduced levels of nitric oxide and inflammatory cytokines (IL-1, IL-6, IL-12, and TNF-alpha). These findings suggest that CS-decorated PLGA nanoparticles hold promise as an effective dexamethasone delivery system for treating SARS related to COVID-19.

Lopinavir (LPV), a potent HIV protease inhibitor, is frequently co-administered with ritonavir as Kaletra (R). Despite its clinical efficacy, this formulation faces several challenges, including high production costs, potential gastrointestinal irritation due to the presence of ethanol and surfactants, stability concerns, and meal-dependent administration. To overcome these limitations, this study aimed to develop LPV nanocrystals via a scalable and reproductive top-down approach. By reducing particle size to the nanoscale, LPV can be formulated into a more effective and patient-friendly dosage form, potentially enhancing solubility, dissolution rate, and bioavailability. To achieve this goal, a fractional factorial design (26-2) was employed to screen key formulation parameters. The selected formulation, processed using a bead mill, was characterized in terms of particle size, zeta potential (ZP), dispersion stability, thermal properties, crystallographic features, and spectroscopic properties. The findings indicate that the optimized formulation demonstrated a particle size of 489 nm with a PDI of 0.27. Which collectively ensured dispersion stability. Notably, in vitro dissolution studies across three different media demonstrated a significantly enhanced dissolution profile for LPV nanocrystals compared to pure API, suggesting potential enhancement in oral bioavailability. This study aligns with key technological and regulatory requirements by proposing a novel formulation with substantial public health implications.

Solid-state modifications can improve drug performance. Studies have shown that multicomponent crystals, such as cocrystals and eutectic compositions, have successfully improved the performance of certain drugs, including their solubility. Nevirapine (NEV) is an antiretroviral drug with low aqueous solubility, impacting its bioavailability. This work aimed to study different nevirapine/co-former solid eutectic systems, define their phase diagrams and evaluate their dissolution properties. Caffeine (CAF), Theobromine (TEOB), and Theophylline (THEO) were chosen as co-formers due to their functional groups that can interact with NEV. Aiming to determine both temperature and eutectic composition, phase and Tamman diagrams were obtained using the differential scanning calorimetry (DSC) analysis of the mixtures indifferent NEV-co-former compositions (%w/w). Powder Xray diffraction (PXRD) and DSC were used to characterize the eutectic materials. To assess the influence of eutectic systems on dissolution properties, we determined the powder dissolution profiles and intrinsic dissolution rates of anhydrous NEV and eutectic systems NEV-CAF and NEV-THEO using different dissolution media (pH 1.2 and pH 6.8). The eutectic compositions were calculated through the phase diagrams, interpolating the curves obtained by linear regression. Thus, the eutectic composition of the NEV-CAF system was determined to be 36.55 % NEV and eutectic temperature at 201.1 degrees C, while in the NEV-THEO system, the eutectic was obtained in a composition of 71.04 % NEV and eutectic temperature at 217.8 degrees C. Tamman diagrams were generated using the enthalpy values obtained from the DSC curves, and eutectic compositions were calculated using linear regression. The analysis revealed a eutectic composition of 36.51 % of NEV for the NEV-CAF system, and a eutectic composition of 70.94 % of NEV for the NEV-THEO system. It was not possible to determine the eutectic mass fraction of NEV-TEOB. A comparison of dissolution profiles and intrinsic dissolution rates in different dissolution media showed a significant improvement in the NEV dissolution rate in both eutectic systems. In an acidic medium, NEV dissolved 16 times faster in the NEV-CAF sample and 4 times faster in the NEV-THEO sample compared to pure anhydrous NEV. In a neutral medium, the dissolution profile of NEV was even more favorable in the same eutectic systems, showing that the increase in dissolution is relevant in a wide range of pH. In addition, the intrinsic dissolution rate in the two eutectic systems was higher than that of anhydrous NEV in all employed dissolution mediums. These eutectic systems improve the dissolution rate compared to pure NEV, offering the potential for enhancing the dissolution of poorly water-soluble drugs in the future.

Amphotericin B (AmB) is a widely used antifungal drug that is also prescribed to some neglected diseases, such as leishmaniasis. Its usage is limited by its low oral bioavailability and side effects, leading to the exploration of alternative delivery systems. Polymeric nanoparticles (PNPs) have emerged as a promising drug delivery pathway, offering potential benefits such as controlled release and improved drug bioavailability. In this work, AmB-loaded poly-(lactic acid) (PLA) and polycaprolactone (PCL) PNPs were produced by nanoprecipitation and characterized by dynamic light scattering, scanning transmission electron microscopy, and Raman spectroscopy. Subsequently, their stability was tested in static multiple light scattering (SMLS) analysis, and their penetrability was determined in an ex vivo porcine skin model. The obtained results indicated that the PNPs were successfully produced. The PLA + AmB PNPs were able to reach the viable epidermis, while the PCL + AmB PNPs permeated the stratum corneum, suggesting that both may be useful for the topical treatment of fungal infections and cutaneous leishmaniasis.

Praziquantel (PZQ) is an anthelmintic agent used worldwide for the treatment of schistosomiasis. PZQ is used as a racemate, and it is practically insoluble in water. The PZQ racemate is a white to nearly white crystalline powder, and few studies showed a color change from white to pink. However, no special attention has been given to this matter. The present study aimed at a comprehensive understanding of PZQ change in color because a significant impact on its dissolution was observed. We discuss a series of analytical techniques, and we emphasize the importance of understanding solid state properties together with the conventional quality control evaluation. Two batches of PZQ raw material with different colors (white and light pink) were used. The dissolution profiles of the samples and the wettability were significantly different, and in addition, the pink color sample, when it came into contact with an acid medium, turned white. Because of these results, the presence of another phase in the pink sample was investigated and confirmed by powder X-ray diffraction. We were able to isolate the unknown phase for the first time, and with the characterization using Raman, infrared, and nuclear magnetic resonance, we proved that the pink color was related to an impurity with low crystallinity. It was observed through microscopy that this impurity with low crystallinity after the contact with the acid medium crystallizes, causing the pink color to turn white. Also, we showed that depending on the HPLC method, this impurity cannot be detected, which is critical for quality control.

Background: Amphotericin B (AmB) is a commonly utilized antifungal agent, which is also recommended for the treatment of certain neglected tropical diseases, including leishmaniasis. However, its clinical application is constrained because of its poor oral bioavailability and adverse effects, prompting the investigation of alternative drug delivery systems. Polymeric nanoparticles (PNPs) have gained attention as a potential drug delivery vehicle, providing advantages such as sustained release and enhanced bioavailability, and could have potential as AmB carriers. However, concerns persist regarding nanomaterials’ toxicity, requiring more studies. Zebrafish (Danio rerio) embryos were used as a valuable model for toxicity testing, especially because of their genetic similarity to humans and standardized developmental assessments. Methods: In this study, we produced and characterized AmB loaded and non-loaded PNPs by nanoprecipitation, dynamic light scattering, transmission electron microscopy, atomic force microscopy and spectroscopy. Afterwards, we verified their toxicity through in vitro MTT assays in three cell lines (HEK293, HepG2, and J774 A1) and in vivo tests with zebrafish embryos. Results: In both trials, it was noted that nanoencapsulation of the drug led to increased toxicity when compared to non-encapsulated AmB, possibly indicating that they penetrated the embryo’s chorion. Nevertheless, it was demonstrated that the polymers used are safe and they are not the cause of toxicity, neither are the nanostructures per se. Conclusions: Therefore, it is believed that the objective of improving the bioavailability of AmB may have been achieved, and the observed toxicity was probably linked to AmB’s ability to destabilize cell membranes.

Fungal infections cause 1.7 million deaths annually, which can be attributed not only to fungus-specific factors, such as antifungal resistance and biofilm formation, but also to drug-related challenges. In this study, the potential of Amphotericin (AmB) loaded polymeric nanoparticles (AmB-NPs) combined with murine monoclonal antibodies (mAbs) (i.e., CC5 and DD11) was investigated as a strategy to overcome these challenges. To achieve this goal, AmB-NPs were prepared by nanoprecipitation using different polymers (polycaprolactone (PCL) and poly(D,L-lactide) (PLA)), followed by comprehensive characterization of their physicochemical properties and in vitro biological performance. The results revealed that AmB-loaded NPs exhibited no cytotoxicity toward mammalian cells (baby hamster kidney cells—BHK and human monocyte cells—THP-1). Conversely, both AmB-NPs demonstrated a cytotoxic effect against C. albicans, C. neoformans, and H. capsulatum throughout the entire evaluated range (from 10 µg/mL to 0.1 µg/mL), with a significant MIC of up to 0.031 µg/mL. Moreover, the combination of AmB-NPs with mAbs markedly intensified antifungal activity, resulting in a synergistic effect that was two to four times greater than that of AmB-NPs alone. These findings suggest that the combination of AmB-NPs with mAbs could be a promising new treatment for fungal infections that is potentially more effective and less toxic than current antifungal treatments.

The COVID-19 pandemic has raised concern regarding respiratory system diseases and oral inhalation stands out as an attractive non-invasive route of administration for pulmonary diseases such as chronic bronchitis, cystic fibrosis, COVID-19 and community-acquired pneumonia. In this context, we encapsulated azithromycin in polycaprolactone nanoparticles functionalized with phospholipids rich in dipalmitoylphosphatidylcholine and further produced a fine powder formulation by spray drying with monohydrated lactose. Nanoparticles obtained by the emulsion/solvent diffusion-evaporation technique exhibited a mean hydrodynamic diameter around 195–228 nm with a narrow monomodal size distribution (PdI < 0.2). Nanoparticle dispersions were spray-dried at different inlet temperatures, atomizing air-flow, aspirator air flow, and feed rate, using lactose as a drying aid, resulting in a maximal process yield of 63% and an encapsulation efficiency of 83%. Excipients and the dry powder formulations were characterized in terms of morphology, chemical structure, thermal analyses and particle size by SEM, FTIR, DSC/TGA and laser light diffraction. The results indicated spherical particles with 90% at 4.06 µm or below, an adequate size for pulmonary delivery. Aerosolization performance in a NGI confirmed good aerodynamic properties. Microbiological assays showed that the formulation preserves AZM antimicrobial effect against Staphylococcus aureus and Streptococcus pneumoniae strains, with halos above 18 mm. In addition, no formulation-related cytotoxicity was observed against the human cell lines BEAS-2B (lung epithelial), HUVEC (endothelial) and HFF1 (fibroblasts). Overall, the approach described here allows the production of AZM-PCL nanoparticles incorporated into inhalable microparticles, enabling more efficient pulmonary therapy of lung infections.

Leishmaniasis is a disease caused by Leishmania spp., affecting millions of people around the world. For decades, its treatment has been based on pentavalent antimonials, which notoriously cause toxic side effects in patients. In this study, epoxy-α-lapachone incorporated into an oil-in-water-type microemulsion (ELAP-ME) and meglumine antimoniate (MA) were assayed in monotherapy and in combination (ELAP-ME/MA) in BALB/c mice infected with Leishmania (Leishmania) amazonensis. In general, there was a reduction in paw lesion size (up to 37% reduction) and decreases of parasite loads in the footpad (∼40%) and lymph nodes (∼31%) of animals treated with ELAP-ME/MA, when compared to the non-treated control groups. Analyses of serum biochemical parameters revealed that the ELAP-ME/MA showed lower renal and hepatic toxicity when compared to MA 2-doses/week monotherapy. These findings indicate that the ELAP-ME/MA combination may be a promising approach for the treatment of cutaneous leishmaniasis.

The pharmaceutical industry is mostly dedicated to the production of drugs in solid forms such as tablets and capsules with active ingredients and excipients in the same physical state. Regarding the regulatory requirements for the analytical development and implementation of solid-state analyses, internationally recognized pharmaceutical compendia play an important role. However, the information contained in the various general chapters and monographs differs from each other, especially in countries that have national pharmacopeias, such as Brazil. Thus, the main objective of this work is to critically evaluate, based on the technical-scientific literature, the harmonization of the United States, European, British, Japanese and Brazilian pharmacopeias with respect to the following analytical techniques used for solid-state characterization: X-ray powder diffraction, differential scanning calorimetry, thermogravimetric analysis, and infrared and Raman spectroscopies. The working principle of each analytical technique, as well as the methodological parameters that impact the implementation of the analyses, are detailed. The results indicate that, in terms of solid-state characterization analysis, the adoption of unified scientific standards and principles is not yet a reality for all compendia. Additionally, the lack of harmonization between BrazP and the other compendia is especially significant, considering that AN VISA is an ICH member, the main entity responsible for promoting harmonization.

Efavirenz, an antiretroviral drug, is a class 2 according to the biopharmaceutics classification system. Many dissolution enhancement systems have been tried and our group had success using wet milling to decrease particle size and granules were obtained by spray and freeze drying. In this paper we present data related to the upgrade in the process, raising the solids concentration in the suspension from 10 to 50% (m/v) and changing the drying step for a fluid bed granulation. After that, tablets were obtained. Granules and tablets were fully evaluated and a pharmacokinetic study was also performed with the granules. By powder X-ray diffraction (PXRD) and Fourier transform infrared spectroscopy (FTIR) it was possible to prove that there was no phase transition in the sample after milling and drying. Dissolution efficiency of 4 from 5 granules was higher than 90%, considering 83% for the raw material. Tablets were technically approved but the dissolution was impacted and just 2 out of 11 showed results > 80%. A high enhancement in the bioavailability was also observed, around 172%. So, it is possible to conclude that efavirenz microcrystals with enhanced dissolution and bioavailability can be formulated into tablets and are a viable system to develop a new drug product.

This article aims to analyze the development of a rapid and sensitive HPLC–MS/MS method for quantification of efavirenz in rat plasma. The developed method included a liquid-liquid extraction process with methyl-tert-butyl ether solution, where hydrochlorothiazide was used as internal standard. The analyte was separated on an ACE Phenyl C18 column and eluted by a system consisting of mobile phase A (0.2% acetic acid) and mobile phase B (acetonitrile) at a proportion of 35:65 (v/v), pumped with a flow rate of 1.0 mL min-1 (run time < 4 min). Mass spectrometric detection was performed on a triple quadrupole instrument using multiple reaction monitoring. The electrospray ionization source was performed in negative ion mode. The precursor/product ion pairs monitored were m/z 313.644→68.800 and m/z 295.541→268.700 for efavirenz and internal standard, respectively. The limit of quantification was 10.0 ng mL-1 and calibration curves were linear over 10.0–1,500 ng mL-1. Intra-day and inter-day precision at three levels were 1.80–10.49% and 4.72–10.28%, respectively. Accuracy ranged between 93.54% and 105.73%. Finally, the described method was applied to rats administered with efavirenz, demonstrating the suitability for quantification of efavirenz in a pharmacokinetic study. Therefore, it can be used in normal, hemolyzed or lipemic samples for efavirenz quantification.

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-d 5. 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.

Developing biomaterials for tissue regeneration is a promising alternative for the recovery of various tissues, including bone. Atorvastatin (ATV) has a series of beneficial effects which includes bone anabolism, vasodilating, and anti-inflammatory actions. The main objective of this work was to produce and characterize polycaprolactone (PCL) matrices incorporated with ATV. Samples were prepared by the solvent casting technique. Scanning electron microscopy (SEM), X-Ray diffraction (XRD), Fourier transform infrared spectrometry (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and in vitro release studies were performed. FTIR analysis showed that no chemical bonds were formed between PCL and ATV. SEM analysis showed that the amount of ATV affects sample morphology. According to XRD and thermal analysis, the main ATV characteristics were maintained. The studies showed that PCL/ATV samples release the drug in a prolonged way since its release reaches around 50% after 15000 minutes of analysis and the model that showed the best fit for the studied matrices was the Higuchi model, with a correlation coefficient above 0.95.